Patent Publication Number: US-10780141-B2

Title: Herbal combinations for treating eczema

Description:
PRIORITY AND RELATED APPLICATIONS 
     This application claims priority to United States provisional patent applications Nos. 62/198,637, filed Jul. 29, 2015 and 62/297,796, filed Feb. 19, 2016. 
     This application is also a continuation-in-part (CIP) which claims priority to U.S. patent application Ser. No. 14/754,266, filed Jun. 29, 2015. 
     This application is related to U.S. patent application Ser. No. 13/900,525, filed May 22, 2013; now U.S. Pat. No. 9,095,606, which is a CIP of U.S. Ser. No. 13/361,978, filed Jan. 31, 2012. 
     This application is also related to U.S. patent application Ser. Nos. 13/900,526, 14/094,535, 13/152,039, and 14/034,423, and to U.S. Pat. Nos. 8,734,859, 8,597,695, 8,541,382, and 9,066,974, and to US published patent application nos. 20140206631 and 20140205685. 
     All of these priority and related patents and patent applications are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates generally to treatments for eczema. More specifically it concerns the use of herbal combinations and combinations of certain herbs, certain herbal extracts and/or certain molecular components of certain herbs, alone or in combination with or supplemental to one or more other known or novel or experimental treatments, formulated as a shampoo, conditioner, cream, ointment or other topical scalp, hair or skin treatment to treat an eczematic condition. 
     2. Description of the Related Art 
     Often, eczema is not detected because it is not visible underneath the hair of the person who is suffering with eczema. Sometimes, head or scalp eczema is confused with dandruff or other head or scalp conditions that are sometimes deemed to be treatable with ordinary shampoos and conditioners. 
     There exist treatments for psoriatic and eczematic scalp conditions which are steroid-based. Steroid-based treatments are sometimes effective, but can also sometimes have serious negative short-term or long-term side effects. Clobex shampoo (0.05%), by Galderma Labs USA, is an example of a steroid, specifically a corticosteroid, based shampoo that was approved by FDA for treatment of scalp psoriasis. CLOBEX® (clobetasol propionate) Shampoo, 0.05%, contains clobetasol propionate, a synthetic fluorinated corticosteroid, for topical dermatologic use. The corticosteroids constitute a class of primarily synthetic steroids used topically as anti-inflammatory and antipruritic agents. Clobetasol propionate is a white to practically white crystalline, odorless powder insoluble in water. While Clobex shampoo has been shown to be effectiveness in treating scalp psoriasis in a significant percentage of test subjects, a large percentage of test subjects were also subjected to serious negative side effects. It is desired to have an effective and safe, non-steroidal medicinal formulation for treating eczema, and/or psoriasis or another skin condition. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates the components of a shampoo in accordance with certain embodiments. 
         FIG. 2  illustrates a hair growth cycle and a hair fall control strategy in accordance with certain embodiments. 
         FIG. 3  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Body Weight. 
         FIG. 4  effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on % Body Weight Change. 
         FIG. 5  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Absolute Ear Thickness. 
         FIG. 6  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Absolute Ear Thickness. 
         FIG. 7  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Ear Thickness Change. 
         FIG. 8  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Ear Thickness Change. 
         FIG. 9  illustrates effects of combinations of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on Ear Punch Biopsy Weight. 
         FIGS. 10A-10G  show photographs of TPA induced inflammation in mice ears that were represented for all the experimental groups which demonstrated the gross effect of test items. 
         FIG. 11  illustrates % Inhibition of Ear Inflammation on topical treatment. 
         FIG. 12  illustrates % Inhibition of Ear Inflammation on oral treatment. 
         FIGS. 13A-13H  illustrate histopathological findings in accordance with certain embodiments. 
         FIG. 14  illustrates effects of topical application of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on epidermal ear thickness. 
         FIG. 15  illustrates effects of oral applications of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on epidermal ear thickness 
         FIG. 16  illustrates effects of topical application of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on MPO activity in TPA induced mice ears. 
         FIG. 17  illustrates effects of oral administration of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin on MPO activity in TPA induced mice ears. 
         FIG. 18  illustrates the % Inhibition of serum Nitric oxide content on of Sheng Di Huang, Da Huang and Jin Yin Hua and of combinations of digoxin and emodin treatment. 
         FIG. 19  illustrates the effect of proliferation of Digoxin, emodin and their combination on MDA 435 cell lines of Melanoma and Breast Cancer. 
         FIG. 20  is a bar chart that illustrates the effects of certain herbs and combinations of herbs on live cancer cells, including (C1) Sheng Di Huang—Rhemannia; as well as (C2) the combination of Jin Yin Hua—Lonicerae, Da Huang— Rhei , Mu Dan Pi—Moutan, and Di Gu Pi—Cortex Lycii, and (C3) the combination of Xian He Cao—Agrimoniae and Chun Gen PI—Ailanthi. 
         FIGS. 21-23  show plots of growth of cancer cells versus dilution factor for each of 18 different herbs. 
         FIG. 24  is a bar chart that illustrates the effects of certain herbs and combinations of herbs on live cancer cells. 
     
    
    
     DETAILED DESCRIPTIONS OF THE EMBODIMENTS 
     Treatment methods and medicinal compositions are provided for treating eczema, and other skin conditions. Certain embodiments involve the use of herbal combinations and combinations of certain herbs, certain herbal extracts and/or certain molecular components of certain herbs, alone or in combination with or supplemental to one or more other known or novel or experimental treatments. Certain embodiments are formulated as a shampoo, conditioner, cream, ointment or other topical scalp or hair treatment. 
     Methods and medicinal compositions are provided to treat eczema, and/or other skin conditions such as psoriasis, psoriatic arthritis, or other inflammatory skin disorders, dandruff including seborrhoeic dandruff, seborrhea, acne, burns, dermatitis including aptoptic dermatitis, warts, keratosis, acne, alopecia, hirsutism or capitis, melanoma or non-melanoma skin cancer, basal cell cancer (BCC), squamous cell cancer (SCC), scleroderma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi&#39;s sarcoma, keratoacanthoma, spindle cell tumors, sebaceous carcinomas, microcystic adnexal carcinoma, Paget&#39;s disease of the breast, atypical fibroxanthoma, leiomyosarcoma, and angiosarcoma, Marjolin&#39;s ulcers, or warts or burns, or another skin ailment, lesion or sore, particularly of the scalp and also for other skin regions affected by an ailment that is susceptible to topical or hair treatment, as well as hair fall or hair loss conditions. 
     In certain embodiments, herbs are advantageously combined as herbal combinations including Da Huang and Sheng Di Huang. Certain embodiments also include Jin Yin Hua. Other embodiments include one or more of Mu Dan Pi, Di Gu Pi, Xian He Cao, and Chun Gen Pi. Certain embodiments include herbal extracts or molecular components such as emodin, digoxin, and/or other molecules such as aucubin, beta-sitosterol, vanillic acid, rhein, rhapontin and carvacrol. Combinations of these and other herbs, herbal extracts and/or molecules described herein are provided in various embodiments. 
     Along with certain combinations of herbs and/or herbal extracts, emotives or molecular components as described herein, a shampoo, conditioner, cream, ointment or other topical eczema treatment in accordance with certain embodiments may include one or more other components including one or more surfactants and/or co-surfactants, thickening agents, pH adjusters, buffers, aesthetic additives, water, hydro-alcoholic hair serum and/or another dispersive agent, solvent, solubilizer or vehicle, hair-fall or hair-loss control actives, conditioners, preservatives and/or moisturizers and/or vitamins, humectants, one or more cationic polymers, silicone, a suspending agent, perfume and/or other additives that may be consistent with a healthy shampoo, conditioner, cream, ointment or other topical eczema treatment. 
     A sulphate free eczema shampoo may be formulated with the following ingredients:
         An active herbal combination of Da Huang, Sheng Di Huang and Jin Yin Hua,   aqua (DM water),   PEG 80 Sorbitan Laurate,   Sodium Lauroyl Sarcosinate,   Disodium EDTA,   Niacinamide,   Citric Acid,   Sodium Benzoate, and   Phenoxy Ethanol.       

     In alternative embodiments, the active herbal combination may include Da Huang and Sheng Di Huang without Jin Yin Hua, or Da Huang and Jin Yin Hua without Sheng Di Huang, or Sheng Di Huang and Jin Yin Hua without Da Huang, or Da Huang alone with Sheng Di Huang nor Jin Yin Hua, or Sheng Di Huang alone without Da Huang nor Jin Yin Hua, or Jin Yin Hua alone without Da Huang nor Sheng Di Huang. 
     In alternative embodiments, a sulphate free eczema shampoo may include PEG-4 Dilaurate, PEG-7 Glyceryl Cocoate, one or more PEG-20 Almond Glycerides, and/or PEG-25 Hydrogenated Castor Oil instead of or in addition to PEG 80 Sorbitan Laurate. 
     In alternative embodiments, an eczema shampoo may include sodium lauryl sulphate instead of or in addition to Sodium Lauroyl Sarcosinate, although an eczema shampoo in accordance with these alternative embodiments would not be sulphate free. 
     In alternative embodiments, a sulphate free eczema shampoo may include ascorbic acid instead of or in addition to citric acid.
         The sulphate free eczema shampoo may further include one or more of the following additional components:   CAPB; or   Glycerine, Diethylene Glycol, Propylene Glycol, and/or one or more Ceramides, Oils and/or Butters; or   PEG 4 rapseedamide, Macrogol 200, and/or Polyoxyethylen(4); or   Perfume allergen free and/or any GRAS natural perfume; or   Poly Quat 10, Poly Quat 4, and/or Poly Quat 44; or   PEG 150 Distearate, Antil 120 Plus, and/or Antil 127; or   Olive Leaf Extract;   Or combinations of two or more of these additional components.       

     A sulphate free eczema shampoo may be formulated with the following ingredients: 
     An active herbal combination of Da Huang, Sheng Di Huang and Jin Yin Hua (6 mg), aqua (DM water), CAPB, PEG 80 Sorbitan Laurate, Sodium Lauroyl Sarcosinate, Glycerine, PEG 4 rapseedamide, Perfume allergen free, Disodium EDTA, Niacinamide, Poly Quat 10, Citric Acid, PEG 150 Distearate, Sodium Benzoate, Phenoxy Ethanol, and Olive Leaf Extract. 
     In alternative embodiments, the active herbal combination may include Da Huang and Sheng Di Huang without Jin Yin Hua, or Da Huang and Jin Yin Hua without Sheng Di Huang, or Sheng Di Huang and Jin Yin Hua without Da Huang, or Da Huang alone with Sheng Di Huang nor Jin Yin Hua, or Sheng Di Huang alone without Da Huang nor Jin Yin Hua, or Jin Yin Hua alone without Da Huang nor Sheng Di Huang. 
     An eczema shampoo may be formulated with the following ingredients: 
     An active herbal combination of Da Huang, Sheng Di Huang and Jin Yin Hua (6 mg), Aqua (D M Water), Sodium Laureth Sulphate 70%, Cocomonoethanolamide, Dimethiconol (and) Triethanolamine-Dodecylbenzenesulfonate, Ethylene Glycol Distearate, Sodium Chloride, D-Panthenol, Polyacrylic Acid 940, Guar Hydroxypropyltrimonium Chloride, Methylchloroisothiazolinone and Methylisothiazolinone, Sodium Hydroxide, Ethylenediaminetetraacetic acid tetrasodium salt, Tocopheryl acetate and Citric Acid. 
     In alternative embodiments, an eczema shampoo may include sodium lauryl sulphate instead of or in addition to sodium laureth sulphate 70%. 
     In alternative embodiments, an eczema shampoo may include SLES, SLS, ALES, and/or ALS instead of or in addition to Cocomonoethanolamide. 
     In alternative embodiments, an eczema shampoo may include glycol stearate and/or glycol stearate SE instead of or in addition to ethylene glycol distearate. 
     In alternative embodiments, an eczema shampoo may include Coconut oil and/or avocado oil instead of or in addition to D-Panthenol. 
     In alternative embodiments, an eczema shampoo may include Polyvinylalcohol (PVA) instead of or in addition to Polyacrylic Acid 940. 
     In alternative embodiments, an eczema shampoo may include Potassium hydroxide instead of or in addition to Sodium Hydroxide. 
     In alternative embodiments, an eczema shampoo may include ascorbic acid instead of or in addition to citric acid. 
     An eczema shampoo may also include perfume, perfume baby splash and/or any GRAS natural perfume. 
     An eczema shampoo may also include Cocoamidopropylbetaine instead of or in addition to Cocomonoethanolamide. 
     A hair lotion for treating eczema may be formulated as follows: 
     DM Water, an active herbal combination of Da Huang, Sheng Di Huang and Jin Yin Hua (6 mg), Propylene Glycol, Glyceryl Monostearate SE, Light Liquid Paraffin, Cetyl alcohol, Stearic acid, Cyclopentasiloxane, Phenoxyethanol &amp;Triethylene glycol, Triethanolamine, Ethylenediaminetetraacetic acid tetrasodium salt and Alkyl Acrylates. 
     In alternative embodiments, an eczema shampoo may include 1,3-Propanediol instead of or in addition to Propylene Glycol. 
     In alternative embodiments, an eczema shampoo may include white soft paraffin instead of or in addition to Light Liquid Paraffin. 
     In alternative embodiments, an eczema shampoo may include cationic guar, one or more silicones, Honeyquat, and/or one or more polyquats instead of or in addition to Cetyl alcohol. 
     In alternative embodiments, an eczema shampoo may include Emulsifying wax, Beeswax instead of or in addition to stearic acid. 
     In alternative embodiments, an eczema shampoo may include C13-16 isoparaffin (and) C12-C14 isoparaffin (and) C13-C15 alkane instead of or in addition to Cyclopentasiloxane. 
     In alternative embodiments, an eczema shampoo may include Biotin, methyparaben, carbomer, cetyl alcohol, and/or propylparaben instead of or in addition to Triethanolamine. 
     A hair lotion for treating eczema may further include Coconut oil, olive oil, butter, bacon grease, peanut oil and/or or macadamia oil. 
     A hair lotion for treating eczema may also include perfume, perfume baby splash and/or any GRAS natural perfume. 
     A method of treating a scalp condition in accordance with certain embodiments includes applying to the scalp a medicinal composition formulated as a shampoo, conditioner, cream, ointment or other topical scalp or hair treatment that comprises a low concentration formula 5-10 grams or more of an active herbal combination of Sheng Di Huang, Da Huang and Jin Yin Hua in an example 16 fluid ounce formulation. A high concentration formula may include 50-150 grams of the active herbal combination in an example 16 fluid ounce formulation. A medium concentration formula may include 10-50 grams of the active herbal combination in an example 16 fluid ounce formulation. 
     In certain embodiments, the medicinal composition may include 1%-20% of the active herbal combination. In certain embodiments, the medicinal composition may include 2.5%-10% of the active herbal combination. In certain embodiments, the medicinal composition may include 2.5%-5.0% of the active herbal combination. In certain embodiments, the medicinal composition may include 3.5 wt. % or more of Da Huang. In certain embodiments, the medicinal composition may include 15.4 wt. % or more of a combination of Da Huang and Sheng Di Huang. 
     In certain embodiments, the herbal combination may include 2-20 grams of Jin Yin Hua, 2-20 grams of Da Huang and 6-60 grams of Sheng Di Huang. In certain embodiments, the medicinal composition may include 0.2 wt. %-4 wt. % of Jin Yin Hua, 0.2 wt. %-4 wt. % of Da Huang, and 0.6 wt. %-12 wt. % of Sheng Di Huang. 
     In certain embodiments, the herbal combination may include 13.3-60 grams of Sheng Di Huang, 3.3-15 grams of Da Huang, and 3.3-15 grams of Jin Yin Hua. 
     In certain embodiments, the combination of Sheng Di Huang, Da Huang and Jin Yin Hua comprises 20 wt. %-80 wt. % of the active herbal combination. For example, the active herbal combination may include 12 wt. %-48 wt. % of Sheng di Huang, 4 wt. %-16 wt. % of Da Huang and 4 wt. %-16 wt. % of Jin Yin Hua. 
     The active herbal combination may include 18 wt. % or more of Sheng Di Huang, 6 wt. % or more of Da Huang and 6 wt. % or more of Jin Yin Hua. 
     A medicinal composition is also provided, in accordance with certain embodiments, that is formulated as a shampoo, conditioner, cream, ointment or other topical scalp or hair treatment, comprising an herbal combination of 13.3 grams or more of Sheng Di Huang and 3.3 grams or more of Da Huang in a 16 fluid ounce formulation. In certain embodiment, the herbal combination also includes 3.3 grams of Jin Yin Hua. 
     The medicinal composition may include 0.5 wt. % or more of Da Huang and 1.5 wt. % or more of Sheng Di Huang. In certain embodiments, the medicinal composition also includes 0.5 wt. % or more of Jin Yin Hua. 
     The medicinal composition may include 0.5 wt. %-1.5 wt. % of Da Huang and 1.5 wt. %-4.5 wt. % of Sheng Di Huang. In certain embodiments, the medicinal composition also includes 0.5 wt. %-1.5 wt. % of Jin Yin Hua. 
     The medicinal composition may include 6 wt. % or less of Da Huang and 18 wt. % or less of Sheng Di Huang. In certain embodiments, the medicinal composition includes 6 wt. % or less Jin Yin Hua. 
     The active herbal combination may include 40 wt. % or more of Sheng Di Huang and 10 wt. % or more of Da Huang. In certain embodiments, the active herbal combination may include 10 wt. % or more Jin Yin Hua. 
     The active herbal combination within the medicinal composition may include 22.4 wt. % or more, 30 wt. % or more or 60 wt. % or more of the combination of Sheng Di Huang, Da Huang and Jin Yin Hua. 
     The active herbal combination may include 15.4 wt. % or more of Sheng Di Huang, 3.5 wt. % or more of Da Huang and 3.5 wt. % or more of Jin Yin Hua. 
     The active herbal combination may include 18 wt. % or more of Sheng Di Huang, 6 wt. % or more of Da Huang and 6 wt. % or more of Jin Yin Hua. 
     Other medicinally active or inactive components may be included in combination with the herbal combinations described herein. The herbal combination may be administered alone or in combination with or supplemental to one or more other medicinal treatments. 
     Referring to  FIG. 1 , an example of a shampoo in accordance with certain embodiments is illustrated as including nine generalized components. These nine components include a surfactant, a thickening agent, a pH adjuster/buffer, an aesthetic additive, water, a conditioner, one or more active herbs or herbal extracts or molecules, a preservative and moisturizers/vitamins. Shampoos in accordance with various alternative formulations may include fewer than all of these nine components and they may include other active or inactive components known to those skilled in the art as having some advantage when included in a shampoo formula or in a medicinal combination for treating eczema, or another skin condition such as psoriasis, melanoma, or dermatitis or hair loss or another head or scalp disorder or ailment. 
     A shampoo in accordance with certain embodiments includes one or more surfactants that may be known or discovered as being advantageous for cleaning hair with a shampoo. A primary surfactant may be included to provide flash foam for cleaning the hair by removing dirt and other impurities. A secondary surfactant may be included to provide stable foam and to reduce the harshness of the primary surfactant. A surfactant may be used that includes a charged, hydrophilic head group and a long, hydrophobic alkyl chain tail. Surfactants are configured to break molecular bonds between dirt and hair and to transport the dirt into an aqueous medium to be rinsed free from the hair and scalp. Examples of surfactants that may be contained in a shampoo in accordance with certain embodiments include sodium laureth sulphate, ammonium laureth sulfate, and sodium cocoyl isethionate. Examples of co-surfactants include cocamide MEA and cocoamidopropyl betaine. 
     A shampoo in accordance with certain embodiments may include a thickening or suspending agent. Examples of thickening or suspending agents that may be contained in accordance with certain embodiments include carbomer and PEG 150 distearate. The thickening agent may be included to stabilize the shampoo during storage and/or to prevent the setting or dumping of pigments and silicone. 
     A pH adjuster or buffer may be included in a shampoo in accordance with certain embodiments. An example of a pH adjuster or buffer includes citric acid, tartaric and sodium hydroxide. The pH adjuster or buffer is configured to cause the shampoo to be gentle to the skin. A lower pH may cause hair to be compact and to shine and to protect the surfactant from hydrolysis, and as such, the pH adjuster or buffer may serve to lower the pH of the shampoo. However, alternative embodiments include pH adjusters that serve to raise the pH of a shampoo that contains an herbal formula that exhibits an exceptionally low pH. 
     An aesthetic additive may be included in a shampoo in accordance with certain embodiments. Examples of aesthetic additives include colorants, opacifiers, UV absorbers, perfumes and natural and artificial fragrances. 
     One or more conditioners may be included in a shampoo in accordance with certain embodiments. The one or more conditioners may include a cationic polymer such as guar hydroxypropyl trimonium chloride. The one or more conditions may include silicone and/or a silicone emulsion such as dimethiconol, dimethicone, or amodimethicone. The silicone and/or silicone emulsion may serve to coat the hair and cause the hair to become soft, smooth and shiny. 
     A shampoo in accordance with certain embodiments includes one or more active herbs or herbal extracts or emotives that are described in several examples herein. These one or more active herbs or herbal extracts serve to promote treatment of eczema, and/or another condition such as psoriasis, dermatitis, melanoma, hair loss and other hair or scalp conditions described herein or understood by those skilled in the art. 
     A preservative may be included in a shampoo in accordance with certain embodiments. The preservative may be configured to prevent microbial growth. Examples of preservatives that may be contained in a shampoo in accordance with certain embodiments include paraben free, formaldehyde donor free and halogenated free. 
     A moisturizer and/or one or more vitamins may be included in a shampoo in accordance with certain embodiments. Examples include combinations of D-Panthenol, vitamin E acetate, sodium PCA, glycerine and one or more amino acids. The moisturizer and/or vitamins may be configured to penetrate into hair shaft, seal cuticles and keep hair moisturized. 
     A shampoo in accordance with certain embodiments may include a hydro-alcoholic hair serum. Referring to  FIG. 2 , a hair growth cycle includes exogen, anagen, catagen and tetogen phases. The anagen phase involves active hair growth, whereby hair follicles regenerate and generate pigmented hair shafts. The telogen phase is a resting phase. The catagen phase involves cessation of hair growth and pigmentation, and release of papilla from the bulb. A hydro-alcoholic hair serum may be configured as a concentrated product that is typically left on the hair for a more extended duration than an ordinary shampoo with a typical shower routine. The hydro-alcoholic hair serum may be configured to be light and non-sticky on the scalp and as a non-irritant, to be light and non-sticky on the hair, to have little or no effect on hair volume, to strengthen scalp and DPC, to provide keratinization and collagen synthesis, to promote hair growth or to control hair fall, or combinations thereof. For example, the attributes of a hydro-alcoholic hair serum in accordance with certain embodiments may assist or promote treatment of eczema. A hydro-alcoholic hair serum in accordance with certain embodiments may include water, alcohol, humectant, solubilizer, water-based polymer, scalp conditioner, niacinamide, caffeine and panthenol. The ratio of alcohol, water and solubilizer may be adjusted depending of the solubilization power of the active herbal treatment composition. 
     A method is provided for treating eczema, and/or another disorder such as psoriasis, dermatitis, melanoma, and/or hair loss. The eczema treatment includes administering to a patient suffering from and/or diagnosed with eczema. The eczema treatment includes combinations of certain herbs, herbal extracts, and/or compounds or molecules extracted from certain herbs or herbal extracts. Among these are herbs are one or more of Da Huang, Sheng Di Huang, and Jin Yin Hua, and/or one or more of Mu Dan Pi, Di Gu Pi, Xian He Cao, and Chun Gen Pi. Particularly studied among combinations of these herbs and others described herein include the three herbs Sheng Di Huang, Da Huang and Jin Yin Hua, three combinations of two of these three herbs, and formulae including one of these three herbs. 
     In one example, a shampoo formulation may include between 2.5 wt. %-5.0 wt. % of a combination of Sheng Di Huang, Da Huang and Jin Yin Hua. 
     In another example, a shampoo formulation may include between 1.0 wt. %-10.0 wt. % of a combination of Sheng Di Huang, Da Huang and Jin Yin Hua. 
     In another example, a shampoo formulation may include between 1.0 wt. %-15.0 wt. % of a combination of Sheng Di Huang, Da Huang and Jin Yin Hua. 
     An eczema regimen may include combinations of applications of shampoo, cream and/or lotion to affected scalp or other skin areas. 
     Administration in a treatment regimen of certain combinations with one or two or more of these herbs serve to treat hair and scalp conditions as provided in accordance with embodiments described herein. Specific embodiments include advantageous combinations of Da Huang and Sheng Di Huang, as indicated below and in U.S. patent application Ser. No. 13/018,435, which is incorporated by reference, as well as with combinations including Jin Yin Hua with Da Huang and/or Sheng Di Huang. Further embodiments include combinations of DaHuang, Sheng Di Huang and/or Jin Yin Hua with one or more of Mu Dan Pi, Di Gu Pi, Xian He Cao, and/or Chun Gen Pi. 
     Further embodiments include combinations of beta-sitosterol or saw palmetto, or both, with Da Huang, Sheng Di Huang and/or Jin Yin Hua, and one or more of Mu Dan Pi, Di Gu Pi, Xian He Cao, and/or Chun Gen Pi and/or one or more other herbs or molecules described herein. Further embodiments include herbal combinations of one or more of Sheng Di Huang, Da Huang and Jin Yin Hua with combinations of one or more of emodin, digoxin, beta-sitosterol, saw palmetto, aucubin, rhein, rhapontin, vanillic acid, carvacrol or other herbs or molecules described herein or as understood by those skilled in the art. Other embodiments include combinations including one or more of more of Mu Dan Pi, Di Gu Pi, Xian He Cao, and/or Chun Gen Pi. 
     Contained within each of the seven herbs are several molecular constituents. Observed reductions of psoriatic inflammation and other studied effects owing to a treatment regimen of periodic shampooing with an herbal formula in accordance with the embodiments can be as a result of various combinations of active molecules contained in Da Huang, Jin Yin Hua and/or Sheng Di Huang, and of combinations of the herbs themselves. 
     A method of treating Melanoma is also provided including administering to a patient diagnosed with Melanoma a treatment regimen that includes periodic application to the scalp of an herbal formula including Sheng Di Huang, Da Huang or Jin Yin Hua, or combinations thereof, along with the administration of Aldesleukin (Proleukin), Dacarbazine, Ipilimumab (Yervoy), or Peginterferon-alpha or combinations thereof. 
     Another method of treating Melanoma is provided including administering to a patient diagnosed with Melanoma a treatment regimen that includes periodic applications to the scalp of an herbal formula including Sheng Di Huang, Da Huang or Jin Yin Hua, or combinations thereof, along with the administration of Paclitaxel, Cisplatin, Carboplatin, Vinblastine, Vincristine or Vindesine, or combinations thereof. The treatment regimen may also include Aldesleukin (Proleukin), Dacarbazine, Ipilimumab (Yervoy) or Peginterferon-alpha, or combinations thereof. 
     Another method of treating Melanoma is provided including administering to a patient diagnosed with Melanoma a treatment regimen that includes periodic application to the scalp of Sheng Di Huang, and Da Huang, or Sheng Di Huang, Da Huang and Jin Yin Hua in combination with or supplemental to periodic doses of Fluorouracil or another pyrimidine analog, Pembrolizumab, methotrexate, Nivolumab, Pidilizumab, one or more monoclonal antibodies, one or more drugs that target PD-1 receptors or PD-1 inhibitors, and/or with radiation treatments such as proton therapies for treating choroidal melanomas, endolymphatic radio-isotope infusion or other radiation therapy such as external beam radiotherapy or brachytherapy, topical chemotherapy, e.g., with imiquimod or 5-fluorouracil, and cryotherapy, photodynamic therapy, electrodesiccation, curettage, Mohs&#39; micrographic surgery. 
     A method of treating eczema is provided including administering to a patient diagnosed with eczema a treatment regimen that includes periodic doses, such as by application to the scalp by shampooing and rinsing or otherwise, of a combination of Sheng Di Huang and Da Huang and/or a combination of Sheng Di Huang, Da Huang and Jin Yin Hua and/or beta-sitosterol, saw palmetto, emodin, digoxin, aucubin, rhein, rhapontin, vanillic acid, carvacrol, or combinations thereof, along with Consentyx (secukinumab) or other IL-17 inhibitor, IL-12, IL-23 or TNF-α inhibitors, Otezla (apremilast) or other phosphodiesterase-4 inhibitor, Humira, Stelara, alefacept, methotrexate, cyclosporine, hydroxycarbamide, fumarates such as dimethyl fumarate, topical retinoids, monoclonal antibodies such as infliximab, adalimumab, golimumab, or certolizumab pegol, a proteasome inhibitor, Donavan&#39;s solution, vitamin A, D, D 3  or related analogs or synthetic forms, paricalcitol, calcipotriol, fluocinonide, dithranol, decubol, psoralen, acitretin, coconut oil, mineral oil, balnoetherapy or baths in the dead sea or in formulations of dead sea or dead sea-like minerals, salts, emollients, or oils, fish oil or gluten-free dieting, UV light therapy, dermabrasion, laser therapy, cryotherapy, or combinations of two or more of these or combinations of these with other treatments or medications or compositions described herein. 
     A method of treating or preventing hair loss is also provided including periodically applying to the hair and scalp such as by shampooing an herbal combination of Sheng Di Huang and Da Huang and/or a combination of Sheng Di Huang, Da Huang and Jin Yin Hua, and/or a combination of one or more of Sheng Di Huang, Da Huang or Jin Yin Hua or other herbs described herein or as understood by those skilled in the art with saw palmetto or beta-sitosterol or a combination of beta-sitosterol and saw palmetto, and/or one or more of emodin, digoxin, aucubin, rhein, rhapontin, vanillic acid, or carvacrol, or combinations thereof. 
     Another method of treating hair loss is provided including periodic application to the hair and scalp of an herbal formula including one or more of Da Huang, Sheng di Huang or Jin Yin Hua formulated with or supplemental to a treatment that includes minoxidil, finasteride, dutasteride, anthralin, or an anabolic steroid or corticosteroid treatment, or an acne treatment, or a hormone replacement therapy, or an oral contraceptives or antiandrogen such as spironolactone or flutamide. 
     A method of treating glioblastoma is provided including periodically applying to the scalp such as by shampooing or application of a cream, or by oral administration or sub-dermal injection or IV, of a combination of Sheng Di Huang and Da Huang and/or a combination of Sheng Di Huang, Da Huang and Jin Yin Hua and/or beta-sitosterol, saw palmetto, emodin, digoxin, aucubin, rhein, rhapontin, vanillic acid, carvacrol, or combinations thereof, along with TEMODAL, methotrexate, betamethasone, temozolomide, or Avastin, or combinations thereof. 
     Methods of relieving side effects of medical treatment for a hair or scalp condition, improving quality of life of a patient undergoing medical treatment, inhibiting certain growth factors or markers, increasing a therapeutic index of therapeutic compounds for treating a hair or scalp disorder, condition or disease, and modulating hematological or immunological activity for the treatment of a disease, are also provided, including administering a combination of Sheng Di Huang, Da Huang and Jin Yin Hua, along with one or more of the following: Vincristine Sulfate, Cyclophosphamide, Doxorubicin Hydrochloride, Methotrexate, Cyclosporine, Clofarabine, Cytarabine, Dasatinib, Daunorubicin Hydrochloride, Gleevec (Imatinib Mesylate), Nelarabine, Oncaspar (Pegaspargase) Doxorubicine, Daunorubicin, Vincristine Paclitaxel, Cisplatin, Carboplatin, Vinblastine, Vincristine, Vindesine, Aldesleukin (Proleukin), Dacarbazine, Ipilimumab (Yervoy), Talidomid, Revlomid, Gleevac, Dasatinib, Sprycel, Nilotinib, Tasigna, Hydroxyurea, Hydrea, Valkade, Peginterferon-alpha, an RCHOP combination, or an ABVD combination, Folfiri q Folfox, Cisplatinum, etoposide, mitomycin C, vindesine Taxotere, Paclitaxel, Docetaxel, Mitoycin C, Doxorubicin, Mitozantrone, vinblastine, Etoposide, Estramustine Phosphate, cyclophosphamide, doxorubicin, Adriamicin, fluorouracil, 5fu, Taxol, Carboplatinum, Erbitux, TEMODAL, temozolomide, or Avastin, or combinations thereof. 
     Advantageous effects of a shampoo or other hair or scalp treatment regimen may be bolstered by further combinations with active ingredients and/or by one or more buffer molecules or one or more molecules serving as some helpful vehicle for the active molecules. The chemistries and pharmacologies of certain herbs and molecules that may be included in a shampoo containing an herbal formula are described in examples in accordance with certain embodiments. 
     In certain embodiments, certain parts of the herbs are used such as the roots, stems, leaves, husks, branches, barks, sap, or kernels or combinations thereof. For example, for Da Huang at least the root may be used. For Sheng Di Huang, at least dried root tuber may be used. For Jin Yin Hua, at least dried flower may be used. For Mu Dan Pi, at least dried root bark may be used. For Di Gu Pi, at least dried root bark may be used. For Xian He Cao, at least dried aerial part of Agrimonia  Pilosa  Ledeb may be used. For Chun Gen Pi, at least dried bark of the root or stem may be used. 
     In certain further embodiments, combinations of Sheng Di Huang and Da Huang, or two or more of the three herbs including Jin Yin Hua, or combinations of the seven herbs including Mu Dan Pi, Di Gu Pi, Xian He Cao and/or Chun Gen Pi with any one or more of the three herbs, may be combined with any one or more of the following additional eleven herbs including Zi Cao, or  radix arnebiae  (arnebia root) or  radix lithospermi  (gromwell root), Xuan Shen, or  radix scrophulariae  (figwort root), Shi Gao or  gypsum fibrosum  ( gypsum ), Bai Shao, or  radix paeoniae  alba (white peony root), Chi Shao or  radix paeoniae rubra  (red peony root), Hong Hua or  flos carthami  (safflower), Da Qing Ye or  folium isatidis  (woad leaf), Qing Dai or  indigo naturalis  (natural  indigo ), Bai Zhu or  rhizoma atractylodis macrocephalae  (largehead atractylodes rhizome), and Shi Wei or  folium pyrrosiae  (shearer&#39;s pyrrosia leaf), Rou Gui or  cortex cinnamomi  ( cinnamomum  bark). An advantageous herbal shampoo formula may also include beta-sitosterol, saw palmetto, emodin, digoxin, aucubin, rhein, rhapontin, vanillic acid, or carvacrol, or combinations thereof, or any of the other molecules described herein or as may be understood to those skilled in the art. 
     Da Huang 
       Radix  Et  Rhizoma Rhei  (Rhubarb) 
     Chemistry 
     Da Huang contains free antraquinones, anthraquinone glycosides, and bianthrones. Among the free antraquinones contained within Da Huang are alizarin, aloe emodin, chrysophanol, citreorosein, emodin, laccaic acid D, physcion, and rhein. Among the anthraquinone glycosides contained within Da Huang are 1,8-dihydroxy-3-methylanthraquinone-1-O-β-D-glucoside (Palmatin), aloe emodin 1′-O-β-D-glucopyranoside, aloe emodin 1-O-β-D-glucopyranoside, chrysophanol 1-O-β-D-glucopyranoside (chrysophanein), chrysophanol D-glucopyranoside, emodin 1-O-β-D-glucopyranoside, emodin 3-O-β-D-glucopyranoside (Glucoemodin), emodin 8-O-β-D-glucopyranoside, physcion 1-O-β-D-glucopyranoside, physcion 8-O-β-D-gentiobioside, physcionin, and rhein 1-O-β-D-glucopyranoside. Among the bianthrones contained in Da Huang are aloe emodin bianthrone, chrysophanol bianthrone, palmidins A-C, rheidins A-C, sennidins A, B and C and sennosides A-F. 
     Da Huang also includes other compounds including 2-(−2-hydroxy-propyl)-5-methyl-7-hydroxy-chromone, 2-(−2-hydroxypropyl)-methyl-7-hydroxy-chromanone, 2,5-dimethyl-7-hydroxychromone, 2-methyl-5-carboxymethyl-7-hydroxychromone, 3 napthalenes, 3,5,4′-trihydroxystilbene 4′-β-D-(2″-O-galloyl)-glucopyranoside, 3,5,4′-trihydroxystilbene 4′-O-β-D-(6″-O-galloyl)-glucopyranoside, 4′-O-methylpiceid, Rhapontin, Rheinosides A-D, Stilbene gakkates 3,5,4′-rtihydroxystilbene 4′-O-β-D-glucopyranoside, Stilbene piceid and Tannins. 
     Pharmacology 
     Purgative Effect of Da Huang 
     Da Huang is well known as a purgative agent. The active constituents are the combined anthraquinones, especially sennosides. The content of sennosides correlates with the purgative activity of rhubarb. 
     Table 1 illustrates the oral purgative ED 50  values of the anthraquinones 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 ED 50    
                   
                 ED 50    
               
               
                 Anthraquinones 
                 (mg/kg) 
                 Anthraquinones 
                 (mg/kg) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Sennoside A 
                 13.5 
                 Aloe-emodin-8-glycoside 
                 71.6 
               
               
                 Sennoside B 
                 13.9 
                 Emodin monoglucoside 
                 103.6 
               
               
                 Sennoside C 
                 13.3 
                 Aloe-emodin 
                 59.6 
               
               
                 Sennoside D 
                 13.8 
                 Rhein 
                 97.5 
               
               
                 Sennoside E 
                 13.5 
                 Emodin 
                 &gt;500 
               
               
                 Sennoside F 
                 16.1 
                 Physcion 
                 &gt;500 
               
               
                 Rhein-8-glucoside 
                 20.0 
                 Chrydophanol 
                 &gt;500 
               
               
                   
               
            
           
         
       
     
     Studies on the mechanisms of action found that sennosides act predominantly on the large intestine. The most potent purgative activity was obtained from the rhubarb extract or Sennoside A by gastrical administration and from sennidin by intravenous route. Inhibition of the intestinal flora in mice with chloramphenicol significantly decreased the activity prosthetic sugar group of the anthraquinone glycosides prevented the anthrone from being oxidized before they are transported into the large intestine and hydrolysed by the bacterial enzyme into free sennidins. It was found that sennosides are hydrolysed by microbial β-glycosidase in a stepwise fashion to the corresponding sennidins via 8-monoglycosides. The resulting metabolites sennidins were further reduced, possibly by a reductase bound to cell membranes of intestinal bacteria, to rheinanthrone as the purgative principle. 
     Ligation of the junction of the large and small intestines failed to prevent anthraquinone glycoside producing a purgative effect in the large intestine. Oral administration of rhubarb started to produce effect 6-8 h later. These results suggest that there is also a large part of anthraquinone glycosides absorbed in the small intestine and transformed in the liver before they act on the pelvic plexus and produce peristalsis and purgation. 
     On the other hand, small doses (0.05-0.3 g by oral administration) rhubarb caused constipation because of its high content of tannins. The constipation effect can be prevented by decocting rhubarb together with Huang Lian ( Rhizoma  Coptidis). This is because the tannins and berberine, the main constituent of Huang Lian, form gelatinous precipitates during decoction. 
     Antimicrobial Effect of Da Huang 
     Tested by mixing virus with dilutions of aloe emodin for 15 min at 37° C., herpes simplex virus type 2 and type 3, varicella-zoster virus, pseudorabies virus, influenza virus were inactivated. Electron microscopic examination of the virus demonstrated that the envelopes were partially disrupted, indicating that it is directly virucidal to enveloped viruses. Emodin and rhein showed antiviral activity against human cytomegalovirus (HCMV) strain AD-169. When tested against a ganciclovir-resistant strain of HCMV, the EC 50  value for rhein was superior to the value obtained for the AD-169 strain. The aqueous extract of  R. palmatum  inhibited hepatitis B virus (HBV) polymerase activity and to bind hepatitis B virus surface antigen (HBsAg). Intravenous dose of 50 mg/kg of the extract to duck hepatitis B virus (DHBV) carrier ducklings showed antiviral activity against DHBV using serum DHBV DNA level and DHBV DNA polymerase activity as antiviral indicators. 
     Rhubarb exhibited inhibition against staphylococci,  Streptococcus hemolyticus, Corynebacterium diphtheriae, Bacillus subtilis, B. brucellosis, B. mycoides, B. smegatis, Mycobacterium graminis, Yersenia pestis, Salmonella tophi, S. paratyphi, Shigella dysenteriae  and  Neisseria gonorrhea. Staphylococci  and  Neisseria gonorrhea  were most sensitive to the herb. The main antibacterial components were the anthraquinone derivatives with the structure of 1,9-dihydroxyanthraquinone. 3-Carboxyrhein, hydroxyaloe-emodin and hydroxyemodin showed the most potent antibacterial activity. The bacteriostatic concentrations of rhein, emodin and aloe-emodin against staphylococci, streptomycin,  Corynebacterium diphtheria, Bacillus subtilis, B. anthracis  and  Salmonella tophy  were mitochondrial respiratory chain of microorganisms. Respiration of  Staphylococcus aureus  was strongly inhibited by emodin, aloe-emodin and rhein. Rhein, emodin and rhein specifically interfered with the redox function NADH dehydrogenase. 
     The aqueous, ethanolic and ether extracts of rhubarb are also antifungal against many pathogenic fungi, including  Achorion schoenleini, Trichopphyton concentricum, T. violaceum, T. gypsum, Nocardia asteroids, Epidermophyton flocosum  and  Sporotrichum schenckii . The decoction of rhubarb exhibited inhibition against the influenza virus. The minimal effective dose in chicken embryo in vitro and semi in vivo was 5 mg per embryo. 
     Antineoplastic and Antimutagenic Effects of Da Huang 
     Intraperitoneal administration of 75 mg/kg of emodin produced a 45% inhibition against the mammary carcinoma of mice. The inhibition rates of 5 mg/kg of rhein and emodin against murine melanoma were 76% and 73%. Rhein, emodin and aloe-emodin inhibited murine leukemia P 388  in vivo, increasing the survival time and decreasing the ascites volume. They also inhibited Ehrlich ascites carcinoma, emodin was a strong inhibitor of respiration in Ehrlich ascites carcinoma cells, with an ED 50  of 20 μg/ml. Cellular respiration in leukemia L 1210  cells was also inhibited. Palmatin, Cysophanein and physcionin also exhibited moderate cytotoxic activity against several types of carcinoma cells. 
     The extract of the herb from  R. palmatum  and emodin induced a dose-dependent decrease in the mutagenicity of benzo(a)pyrene [B(a)P,], 2-amino-3-methylimidazo(4,5-f) quinoline (IQ) and 3-amino-1-methyl-5H-pyrido(4,3-b)indole (trp-P-2) in  Salmonella typhimurium  TA98. It was further found that emodin reduced mutagencity of IQ by direct inhibition of the hepatic microsomal activation and not by interaction with proximate metabolites of IQ and/or by modification of DNA repair processes in the bacterial cell. Emodin also markedly decreased the mutagenicity of 1-nitropyrene (1-NP) in a dose dependant manner in Ames-microsomal test with  S. typhimurium  TA98 and the genotoxicity in SOS chromotest with  E. coli  PQ37. Furthermore, emodin significantly inhibited the formation of 1-NP DNA adducts in  S. typhimurium  TA98. The results suggest that emodin acts as a blocking and/or suppressing agent to reduce the direct-acting mutagenicity of 1-NP. 
     Hemostatic Effect of Da Huang 
     Rhubarb is also in TCM as a hemastatic agent. The hemastatic activity has been proved experimentally and clinically. Rhubarb is effective for both external and internal hemorrhage. It was effective in the treatment and prevention of experimental gastric bleeding and ulcer formation in rats. Significant therapeutic effects of the powdered rhizome of  R. palmatum  in the treatment of gastrointestinal bleeding were also reported. It reduces coagulation time and the permeability and fragility of capillaries. It increases fibrinogen and promotes bone marrow to produce platelets. 
     Immunosuppressive Effect of Da Huang 
     Emodin at 3×10 −7 -3×10 −4 M dose-dependently suppressed the responses of human mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction. It was further found that after exposure to emodin (10 −6  M) the production of interleukin-1 (IL-1) and interleukin-2 (IL-2) and the expression of IL-2 receptor were all decreased, Emodin may be a new template for the development of better immunosuppressive agents for use against transplantation and autoimmune disease. 
     Choleretic Effect of Da Huang 
     Rhubarb can stimulate construction of the gallbladder and relax Oddi&#39;s sphinctercan, thus promoting bile secretion. It also increases the contents of bilirubin and bile acid. 
     Other Effects of Da Huang 
     Oral administration if emodin and rhein provoked marked diuretic, natriuretic and kaliuretic effects in rabbits. Oral administration of rhubarb increased urinary excretion of sodium and potassium, alkalizing urine to a pH value as high as 8.4. Rhubarb also inhibits the activities of pepsase, trypsase, pancreatic amylase and pancreatic lipase. It lowers blood pressure and blood cholesterol levels. Rabbits with fever induced by subcutaneous injection of the pneumococci responded with reduced temperature after oral administration of the decoction of rhubarb. 
     Intraperitoneal administration of 15 mg/kg of emodin exhibited antiinflammatory activity against carrageenin-induced pedal inflammation in rats. In the same dosage it also showed antiulcerative activity against pylorus-ligated, aspirin and immobilization stress-induced gastric ulcer in rats. It decreased acid and pepsin output and augmented mucus secretion in terms of total carbohydrate:protein ration in the gastric juice of aspirin treated pylorusligated rats, indicating that the antiulcerative effect of emodin may be due to this effect on gastric secretion. 
     Mu Dan Pi 
       Cortex moutan  (Paeony Bark) 
     Chemistry 
     Mu Dan Pi contains Apiopaeonoside, Benzoyloxypaeoniflorin, Benzoylpaeoniflorin, Galloyloxypaeoniflorin, Galloypaeoniflorin, Mudanpiosides A-F, Oxypaeoniflorin, Paeconoside, Paeoniflorin, Paeonisuffral, Paeonisuffrone, Paeonol, Paeonolide, Pentagalloyglucose 1, 2, 3, 4, 6, and Suffruticosides A-E 
     Pharmacology 
     Antimicrobial Activity of Mu Dan Pi 
     The decoction of the root bark exhibited a strong antibacterial activity in vitro against the following bacteria:  Bacillus subtilis, Escherichia coli, Salmonella typhi, S. paratyphi, Protues vulgaris, Pseudomonas aeruginosa. Staphylococcus aureus, Strephtococcus hemolyticus, Diplococcus pneumoniae  and  Vibrio cholerae . Paeonol was one of the antibacterial components; its MIC values were 1:2000 against  Staphylococcus aureus,  1:1500 against  Bacillus subtilis  and  Escherichia coli.    
     Anti-Inflammatory Effect of Mu Dan Pi 
     The 70% methanolic extract of the root bark inhibited rat paw swelling induced by carrageenin. Paeonol was found active in inhibiting rat paw swelling induced by dextran, acetic acid or carrageenin. It also inhibited the increase of intra-abdominal capillary permeability of mice and cutaneous capillary permeability of guinea pigs caused by acetic acid or 5-HT. On the other hand, the water soluble fraction free from paeonol as well as the glycoside fraction also exhibited a significant inhibitory action on rat paw edema due to carrageenin. The water-soluble fraction was also effective in either preventing or treating adjuvant-induced arthritis in rats. The methanolic extract, the glycosidic fraction and paeonol inhibited blood platelet aggregation. ADP- or collagen-induced human plateet aggregation was inhibited by paeonol. The formation of thromboxan B 2  was also inhibited but the formation of 12-hydroxy-5,8,10,14-eocpsatetraenoic acid from C-arachidonate was stimulated. Besides, paeonol inhibited the formation of prostanoids such as prostaglandins and thromboxanes from C-arachidonate in rat peritoneal macrophages. Thus the anti-inflammatory action of the root bark was related to the inhibitory effects of paeonol on prostanoid synthesis. 
     Hypotensive Effect of Mu Dan Pi 
     The blood pressure of dogs with essential or renal hypertension was significantly reduced after oral administration of 5 g/kg of the decoction of the root bark for 5 days and 10 g/kg for two more days. The blood pressure of dogs with renal hypertension was also lowered after oral of 10 g/kg of the decoction free paeonol for 10 days. Oral administration of 0.5-1.0 g/kg of paeonol also produced hypotensive action renal hypertensive dogs and rats. 
     Effect on the CNS of Mu Dan Pi 
     Intraperitoneal or oral administration of paeonol decreased the spontaneous activity of mice, antagonized caffeine-induced hyperactivity and prolonged cyclobarital-induced sleep. At higher doses, paeonol caused disappearance of the righting reflex in mice. It also antagonized convulsions due to cardiazol, strychnine, nicotine and electric shock. Furthermore, paeonol was found to have antipyretic and analgesic activities. Paeonol decreased the body temperature of normal mice and the mice with typhoid and paratyphoid vaccine-induced fever. Oral administration of paeonol produced an analgesic effect against acetic acid-caused writhing and tail pain by pressing in mice. 
     Effect on Obesity of Mu Dan Pi 
     The aqueous extract of the herb was given as drinking water at the concentration of 0.5% to (SLN×C 3 H/He) F 1  obese mice between 3 and 32 weeks of age. The treatment resulted in a significant decline, particularly in males, in food intake and in the Lee index, An index of obesity, and furthermore an increase in glucose tolerance. No significant difference was observed between the experimental and the control groups in the serum free fatty acid levels. There was little difference between groups in the weights of heart, liver, lung, spleen and major endocrine organs in both sexes and in the pattern of oestrous cycles in females, There results indicate that the herb protects against obesity, especially in males, at least partly by a decrease in food intake and an increase in glucose metabolism. 
     Other Effects of Mu Dan Pi 
     Paeonol exhibited anticholinergic and antihistaminic actions on isolated ileum of mice and guinea pigs. It also prevented stress ulcer in mice and inhibited gastric secretion in rats and spontaneous motility of rat uterus in situ. The extract of the root bark and paeonol were also of antimutagenic activity. They decreased the frequency of mutations induced by 4-nitroquinoline 1-oxide in  Escherichia coli  WP2s. 
     Sheng Di Huang 
       Radix rehmanniae  (Chinese Foxglove Root) 
     Chemistry 
     Sheng Di Huang contains 4-(α-L-rhamnopyranosyloxy)-3-methoxybenzoylajugol, Aceutoside, Ajugol, Aucubin, Campesterol, Castanosides A and F, Catalpol, digoxin Echinacoside, E-feruloylajugol, Isoacetoside, Jioglutoside A and B, Jionosides A&amp;B, Leonuride, Mannitol, Melittoside, p-courmaroylagujol, p-hydorxybenzoylajugol, Purpureaside C, Rehmaglutins A, Rehmaionosides A-C, Rehmanniosides A-D, Rehmapicroside, Vanilloylajugol, Z-feruloylajugol, and β-sitosterol, 
     Pharmacology 
     Effects on Adrenocortical Function and Cortisol Metabolism of Sheng Di Huang 
     The herb was able to stop the decrease of plasma corticosterone concentration due to administration of dexamethasone and prevent the adrenal cortex from atrophy. The corticosterone level in rabbits receiving dexamethasone was increased at week 4 and week 6 when the herb was concurrently applied. No morphological changes were observed in the pituitary gland and adrenal cortex of rabbits receiving concurrent treatment of dexamethasone and the herb. A single large dose (3 g/kg) of the root or together with two other herbs Zhi Mu ( Rhizoma  Anemarrhenae) and Licorice (0.9 g/kg each) given orally antagonized the inhibitory effect of dexamethasone on the pituitary-adrenal system of rabbits, thereby increasing plasma corticosterone level. 
     This mixture also antagonized the inhibitory action of dexamethason on the early morning cortisol secretion peak in 12 normal subjects as tested in diurnal dexamethason suppression test. The crude extract (8 mg) of the root, when incubated with the liver sections of rabbits, protected cortisol from being reduced on the double bond between C 4  and C 5 , and the ketone at C 3  and being degraded of the hydroxyl groups at C 17  and C 21 , and the ketone at C 20 , thus delaying the metabolism of cortisol in the liver. When the herb was used simultaneously with exogenous adrenocortical hormones, plasma cortisol could still be kept at a nearly normal level. The mechanism is believed to be a kind of competitive effect which influenced the binding of cortical hormone to the receptors and affected the uptake of corticosteroid hormone by the liver cells, thereby slowing down the catabolism of cortisol. 
     Cardiovascular and Diuretic Effects of Sheng Di Huang 
     The effects of the herb on the heart were largely dependent on doses. There was no obvious cordial activity at 0.1 or 0.5% concentration. At 1% concentration, cordial effect was observed in the isolated perfused frog heart. This action was more obvious in weak heart. When concentrations were increased to 2-5%, the heart was inhibited. Its effects on blood pressure was also dose-dependent. In an experiment with perfused vessels, 1-3% of the extract caused vasoconstriction while at 5% vasodilation. 
     Intravenous injection of 2.5 ml of the root extract produced a diuretic effect in anesthetized dogs. This action may be related to the cordial and renal vasodilation activities. 
     Effect on Blood Glucose of Sheng Di Huang 
     Results in animal experiments have been inconsistent on the effect of the herb on blood glucose. Hypoglycemic effects of alcoholic extract of the root at a subcutaneous dose of 2 g/kg or an oral dose of 4 g/kg in rabbits were reported by early researchers. The result obtained from the subcutaneous injection was more significant: the blood sugar was decreased to the lowest level 4 h after medication. Subcutaneous administration of the alcoholic extract to rabbits also inhibited the prolonged hyperglycemic effect elicited by carbohydrates from the root of  Codonopsis pilosula  (Dang Shen). Intramuscular administration of 20 g of the same extract also suppressed and prevented epinephrine-induced hyperglycemia in rabbits. Other studies, however, reported that the aqueous or alcoholic extract could only reduce the blood glucose of normal rabbits and was not effective in hyperglycemia due to epinephrine. But there were also reports that the herb had no effect on the normal blood glucose level of rabbits. The decoction or the ethanolic extract at 6 g/kg had no effect on the normal blood glucose measurements of rabbits within 6 h of medication. Subcutaneous administration of 20 g/kg of the same agents also failed to antagonize epinephrine-induced hyperglycemia in rats. More recently, a weak hypoglycemic activity of rehmannioside D in spontaneous diabetic mice was reported. 
     Antiinflammatory and Immunosuppressive Effects of Sheng Di Huang 
     Formaldehyde-induced edema of rat paws subsided after oral administration of the decoction or alcoholic extract at the daily dose of 10 g/kg for 5 days. However, another report claimed that only the decoction and not the alcoholic extract had a significant anti-inflammatory activity. At the oral dose of 100 mg/kg, jionoside B and acetoside produced 36% and 18% suppression of hemolytic plaque forming cells in the spleens of mice. In the same test conditions, intraperitoneal dose of 30 mg/kg of cyclophosphamide had a 52.5% suppression. 
     Hemostatic Effect of Sheng Di Huang 
     The coagulation time in rabbits was reduced after giving the yellow needle crystal obtained from the ethanolic extract of the root. Intraperitoneal administration of 10 g/kg of the decoction or ethanolic extract, or oral administration of the charred herb shortened the bleeding time in mice with tail wounds. 
     Effect on Hemorheology of Sheng Di Huang 
     The effects of the herb on the hemorheology of inflammatory, thromosic and intact animals were examined. Oral administration of 200 mg/kg of the 50% ethanolic extract of the herb inhibited the reduction of fibrinolytic activity erythrocyte deformability, the decrease in erythrocyte counts and the increase in connective tissue of the thoracic artery in a chronic inflammatory model, adjuvant-induced arthritis. However, it was ineffective on the development of edema in the arthritic rats and on acute and chronic inflammation. In addition, the extract inhibited the reduction of erythrocyte deformability but not the decrease of coagulative factors in a thrombosic model, endotoxin-induced disseminated intravascular coagulation (DIC). It also exhibited a promoting effect on erythrocyte deformability and fibrinolytic activity in intact rats. There results suggests that oral administration of the extract can prevent an inducement of impediment in the peripheral microcirculation of various chronic diseases through the improvement of hemorheology. 
     Other Effects of Sheng Di Huang 
     Antiradiation, antifungal and antihepatotoxic activities have also been observed with extract of the root in animals. The 100% injection solution of the root given intraperitoneally at 1 ml daily for 6 days mitigated platelet damage in rats caused by 600 rad of γ-irradiation. The aqueous extract of the root inhibited intro fungi mentagrophyton.  Microsporum gypseum  and  M. audouini . The decoction of the root showed protective effect in mice against CCI-caused liver intoxication. Oral or intraperitoneal administration of 10 g/kg of the decoction or the alcoholic extract potentiated the hypnotic effect of pentobarbital sodium. Intraperitoneal dose of 20 g/kg of the decoction or the alcoholic extract protected mice from hypobaric hypoxia. 
     Jin Yin Hua 
       Flos lonicerae  (Honeysuckle Flower) 
     Chemistry 
     Jin Yin Hua contains 2,6,6-trimethyl-2-vinyl-5-hydroxytetrahydropyran, Benzyl-alcohol, Carvacrol, Cis and trans-2-methyl-2-vinyl-5-(α-hydroxyisopropyl)-tetrahydrofuran, Epivogeloside, Eugenol, Geraniol, Hex-1-ene, Hex-3-en-1-ol, Isochlogogenic acid b+c (two isomers of 3,4-dicaffeoyl quinic acid), Isochlorogenic acid a (3,5-dicaffeoyl quinic acid), Linalool, Loganin, Lonicerin, Lonicerin, Luteolin, Methylcaffeate, Pinene, Saponins with oleanolic acid, Secologanin dimethylacetal, Secoxyloganin, sweroside, Vanillic acid, Venoterpin (gentialutine), Vogeloside, α-terpineol, and β-phenylethyl alcohol 
     Pharmacology 
     Antimicrobial Activities of Jin Yin Hua 
     The extracts of both the flower and vine inhibited in vitro the following bacteria  Staphylococcus aureus, Streptococcus hemolyticus, Escherichia coly, Shigella dysenteriae, Vibrio cholera, Salmonella typhi, S. oaratyphi, Diplococcus pneumoniae, Neisseria meningitides, Pseudomonas aeruginosa  and  Mycobacterium tuberculosis . It also potentiated the action of penicillin against the drug-resistant  Staphylococcus aureus . Chlorogenic acid and isochlorogenic acid are believed to be the chief antibacterial components of the flower. Luteolin also showed an antibacterial activity. More than half of the mice receiving the LD dose of  Pseudomonas aeruginosa  or its endotoxin survived after given 7.5 g/kg of the injection solution of the flower by intraperitoneal administration. Intravenous administration of 6 g/kg of the distillate of the flower was also therapeutically effective in rabbits poisoned by the endotoxin of  Pseudomonas aeruginosa.    
     Antifungal activity was observed with the aqueous extract of the flower against  Microsporum ferrugineum  and  Nocardia  asteroids. In the monolayer primary culture of the epithelial cells of human embryonic kidney, the decoction of the flower inhibited influenza virus, ECHO virus and herpes virus. 
     Anti-Inflammatory Effect of Jin Yin Hua 
     Intraperitoneal administration of 0.25 g/kg of the flower inhibited carrageenin-induced paw edema in rats. Given twice a day at 8 g/kg for 6 days by Intraperitoneal injection, the extract of the flower showed antiexudative and antihyperplastic effects on croton oil-induced granuloma. Intraperitoneal administration of the injection solution increased the phagocytic activity of the inflammatory cells in mice. The decoction diluted to 1:1280 concentration was still able to promote leukocytic phagocytosis. 
     Central Stimulant Effect of Jin Yin Hua 
     Oral administration of chlorogenic acid produced central stimulation in mice and rats in experiments using electric shock and revolving cage; the potency of the central stimulation was ⅙ that of caffeine. No addictive or synergistic action was observed when they were used together. 
     Antilipemic Effect of Jin Yin Hua 
     Oral administration of 2.5 g/kg of the flower reduced the intestinal absorption of cholesterol and the plasma cholesterol level. In vitro experiments showed that the flower conjugated with cholesterol. 
     Other Effects of Jin Yin Hua 
     Intraperitoneal administration of an aqueous-ethanolic extract of the flower of  L. japonica  to mice on day 8 after mating decreased pregnancy in the test animals dose-dependently. Intrauterine and intra-amniotic administration of the extract killed the fetuses in dogs and caused abortion in monkeys, respectively. The extract of the flower exhibited a mild prophylactic effect against experimental gastric ulcer in rats when given orally. Large oral doses of chlorogenic acid increased gastrointestinal peristalsis and promoted gastric and bile secretion. Chlorogenic acid had a stimulant effect on the isolated rat uterus. 
     Di Gu Pi 
       Cortex lycii radicis  (Wolfberry Bark) 
     Chemistry 
     Di Gu Pi contains 5α-stigmastan-3 6-dione, Betaine, Cinnamic acid, Kukoamine A, Linoleic acid, Lyciumamide, Melissic acid, Sugiol, and β-sitosterol 
     Pharmacology 
     Antipyretic Effect of Di Gu Pi 
     The aqueous or alcoholic extract of the herb, given orally or by injection, produced a significant antipyretic effect in rabbits with fever induced by pyrogen. Betaine was also active. A strong antipyretic effect was also exhibited by the aqueous fraction of the alcoholic extract at doses ranging from 0.75 to 7.5 g/kg equivalent of the crude drug. The precipitates of the extract from lead salt also showed comparable antipyretic activity to synthetic antipyretic analgesics. 
     Hypoglycemic Effect of Di Gu Pi 
     Oral administration of the decoction of the herb decreased blood glucose level in rabbits by 14% in average; this action lasted for 7-8 h. The peak action was observed 3 to 4 h after administration. It was also less effective when give subcutaneously. Subcutaneous dose of 6 g/kg of the extract elicited a mean reduction of 14% of the blood glucose of rabbits after 1 h. 
     Hypotensive and Anticholesterolemic Effect of Di Gu Pi 
     The decoction, macerate, tincture and injection solution of the herb produced a significant hypotensive effect in anesthetized dogs, cats, rabbits by intravenous or intramuscular administration and in anaesthetized rats by oral administration. Repeated intravenous administration at lower doses did not induce rapid tolerance. Intravenous injection of 0.375 g/kg of the injection solution resulted in sudden drop of blood pressure and death of anesthetized dogs. Bradycardia, prolongation of PR interval and depressed T wave in the ECG were observed. Kukoamine A induced hypotension in rats when given intravenously at a dose of 5 mg/kg. 
     Daily oral administration of 10 g/kg of the extract of the herb for 3 weeks decreased the serum cholesterol in rabbits by 36.9% with little effect on triglyceride. 
     Antimicrobial Activity of Di Gu Pi 
     In the sensitivity test using the paper disc method, the decoction of the herb strongly inhibited  Bacillus typhosus, Salmonella  paratyphi A, and  Shigella flexneri  but was inactive against  Staphylococcus aureus . It was a weak bacteriostatic against  Mycobacterium tuberculosis. In the primary monolayer tissue culture of the embryonic renal cells, the decoction prevented the pathogenic changes in the cells due to Asian influenza virus A JK strain.    
     Effect on the Uterus of Di Gu Pi 
     The 100% injection solution of the bark showed stimulation effects on normal rat uterus and isolated mouse uterus. The activity of 1 ml of the solution was comparable to that of 0.054 unit of pituitrin. 
     Xian He Cao 
       Herba agrimoniae  (Hairyvein Agrimonia Herb) 
     Chemistry 
     Xian He Cao contains Agrimols A, B, C, D and E, Agrimoniin, 
     Agrimonolide, Agrimorphol, Apigenin-7-glucoside, Caffeic acid, Ellagic acid, Gallic acid, Luteolin-7-glucoside, Pendinculagin, Potentillin, and Quercetin 
     Pharmacology 
     Teniacidal Effect of Xian He Cao 
     The winter sprout of the herb is used in folk medicine to expel tenia and the active principle was found to be agrimophol. Agrimophol acts directly on the parasite. It inhibits the glycogenolysis, aerobic and anaerobic metabolism in the parasite. 
     The herb and agrimophol are also lethal to some other parasites, such as  Trichomonas vaginalis , blood fluke and roundworm. 
     Antibacterial Activities of Xian He Cao 
     Six compounds, luteolin-7-glucoside, apigenin-7glucoside. Quercetin, ellagic acid, caffeic acid and gallic acid, isolated from the herb were active against bacillary dysentery. Combination use of luteolin-7-glucoside and ellagic acid, apigenin-7-glucoside and apigenin-7-glucoside was more effective than the respective individual compounds. 
     Antitumor Effect of Xian He Cao 
     Agrimoniin had antitumour activity when given as a pre- or posttreatment. A single dose of 10-30 mg/kg prolonged the life span of mice bearing MM 2  tumors or yielded cures when given intravenously or orally prior to or after tumor inoculation. Agrimoniin also inhibited the growth of MH-134 and Meth-A solid tumors in mice. It was strongly cytotoxic to MM 2  cells in vitro, but the activity was almost completely abolished by the addition of fetal calf serum to the culture. Intraperitoneal administration of agrimoniin increased the number of peripheral white blood cells and the proportion of monocytes. The antitumor activity of agrimoniin appeared to be due to its enhancement of the immune response. 
     Cardiovascular Effect of Xian He Cao 
     Intravenous administration of the alcoholic extract of the herb increased blood pressure and stimulated respiration in anesthetized rabbits and dogs, but the alcohol-soluble fraction of the aqueous extract lowered blood pressure in rabbits. Perfused into the blood vessels of rabbit ear and frog hind limb, it caused vasoconstriction at low concentrations and vasodilation at high concentrations. The extract and agrimoniin increased the heart rate and cardiac contractility of frogs and toads. On the other hand, the alcohol-soluble fraction of the aqueous extract inhibited the isolated frog heart. 
     Chun Gen Pi 
       Cortex ailanthi  (Tree-of-Heaven Bark) 
     Chemistry 
     Chun Gen Pi contains 1-(1′,2′-dihydroxyethyl)-4-methoxy-β-caboline, 1-(2′-hydroxyethyl)-4-methoxy-β-carboline, 1-(2-hydroxy-1-methoxy)-ethyl-4-methoxy-β-carboline, 13(21)-dehydro-glaucarubinone, 13(21)-dehydroglaucarubolone, 1-acetyl-4-methoxy-β-carboline, 1-carbamoyl-β-carboline, 1-carbomethoxy-β-carboline, 1-hydroxycanthin-6-one, 1-methoxycanthin-6-one, 1-methoxy-canthin-6-one-3-oxide, 5-hydroxymethylcanthin-6-one, 6-methoxy-β-carboline-1-carboxylic methyl ester, Ailanthone, Ailantinols A and B, Amarolide, Amarolide 11-acetate, Canthin-6-one, Canthin-6-one-3-oxide, Chaparrinone, Chaparrolide, Glaucarubinone Quassinoids Δ 13(18) -dehydro-glaucarubinone, Δ 13(18) -dehydroglaucarubolone, Shinjudilactone, Shinjulactones A-N, and β-carboline-1-propionic acid. 
     Pharmacology 
     Glaucarubinone and ailanthone showed amebicidal activity in vitro against the parasite  Entamoeba histolytica . Some quassinoids markedly inhibited the growth of chloroquine-resistant  Plamodium falciparum . Glaucarubinone produced complete inhibition at 0.0006 μg/ml. Ailanthone also showed potent antiulcer activity. 
       FIG. 19  illustrates the effect on live cells of three combinations of herbs each in three concentrations 1:40, 1:20 and 1:10. C1 includes Sheng Di Huang, C2 includes Jin Yin Hua, Da Huang, Mu Dan Pi and Di Gu Pi, and C3 includes Xian He Cao and Chun Gen Pi.  FIG. 19  illustrates that each of these three example combinations provides a reduction of the live cell numbers particularly in higher concentrations, while C1 and C2 appear to be more effective than C3. Each of C1 and C2 includes one or two of the three herbs Sheng Di Huang, Jin Yin Hua and Da Huang, while C3 does not. In addition, the combination of four herbs in C2 appears to be more effective than the single herb in C1. 
       FIGS. 20-23  illustrate plots for each of the eighteen herbs of growth as a percentage of control versus dilution factor.  FIGS. 16-18  illustrate that the three herbs Sheng Di Huang, Da Huang and Jin Yin Hua are most effective, while the four herbs Mu Dan Pi, Di Gu Pi, Xian He Cao and Chun Gen Pi are effective, and the eleven herbs Zi Cao, Xuan Shen, Shi Gao, Bai Shao, Chi Shao, Hong Hua, Da Qing Ye, Qing Dai, Bai Zhu, Shi Wei and Rou Gui are somewhat less effective. 
       FIG. 24  illustrates the effect on live cells of five combinations of herbs each in two concentrations 1:40 and 1:80. Ca1 includes a combination of all eighteen herbs, C2 includes a combination of Jin Yin Hua, Da Huang, Mu Dan Pi and Di Gu Pi, C3 includes a combination of Da Huang, Sheng Di Huang and Jin Yin Hua, C4 and C5 include combinations of the other eleven herbs (Zi Cao, Xuan Shen, Shi Gao, Bai Shao, Chi Shao, Hong Hua, Da Qing Ye, Qing Dai, Bai Zhu, Shi Wei and Rou Gui) of the eighteen herbs, i.e., not in combination with any of the seven herbs (Sheng Di Huang, Da Huang, Jin Yin Hua, Mu Dan Pi, Di Gu Pi, Xian He Cao and Chun Gen Pi). Specifically, C4 includes Shi Gao, Shi Wei, Bai Zhu, Rou Gui, Hong Hua and C5 includes Zi Cao, Xuan Shen, Chi Shao, Bai Shao, Da Qin Ye, and Qing Dai.  FIG. 24  illustrates that combinations of all eighteen herbs, as well as combinations of the three herbs (Sheng Di Huang, Da Huang and Jin Yin Hua) are most effective, while the combination C2 was less effective, and the combinations C4 and C5 were still less effective. 
     Herbal Ingredients 
     Aloe Emodin 
     Aloe emodin has a molecular weight around 270.24 g/mol. As to its anti-cancer activity, aloe emodin exhibits cytotoxicity in SCC of tongue, cervix cancer cells; and apoptoticity through MAPK-JNK cascade in hepatoma cells. Aloe emodin also tends to induce P53 and apoptosis. In addition, the cytotoxicity of aloe emodin induces effects in melanoma, and gastric carcinoma. As to its anti-inflammatory activity, aloe emodin exhibits anti TNF and anti virality in enveloped viruses—HSV, PSV, VSV, and INF. Aloe emodin also decreases COX2 and INOS expression in inflammation, and increases IFN in JEV and EV71. Aloe emodin may also be ingested for its properties as a laxative. Use of aloe emodin can induce nausia, and intestinal contraction causing abdominal pain. Long use of anthraquinones can lead to kidney and liver damage. The half-life of aloe emodin is about 78 min. 
     Chrysophanol 
     Chrysophanol has a molecular weight around 254.24 g/mol. As to its anti-cancer activity, chrysophanol exhibits necrosis in hepatic cancer cells, including ATP change and ROS cascade. Chrysophanic acid inhibits EGFR in colon cancer cells. As to its anti-inflammatory activity, chrysophanol can serve as an antiseptic, bactericide, candidicide, and/or cathartic. In addition, chrysophanol can be used as an anti- staph aureus  and an anti- Bacillus subtilis . Chrysophanol can be used to suppress the activation of NF-kB. and caspase-1 in LPS-stimulated macrophages. Chrysophanol is also an anti polio virus compound. Chrysophanol an be used as a hemostat and as a pesticide, and can be used in the treatment of menorrhagia, including bleeding following abortion, epistaxis, functional uterine bleeding and thrombocytopenia. Chrysophanol acts as a purgative, and can be used to converts aloe emodin through P450 in the liver. Emodin and chrysophanol may be ingested in combination as an anti cancer treatment agent. Chrysophanol has a half life of about 2.75 hours. 
     Emodin 
     Also known as  Rheum emodi , emodin has a molecular weight around 270.24 g/mol. As to its anti-cancer activities, emodin exhibits pro apoptoticity in prostate cancer cells through P53, and P21. Emodin increases ROS, and improves chemotherapy effect in prostate cells which are drug resistant. Emodin exhibits pro apoptoticity through inhibition of IL6 in myltiple myaloma. Emodin exhibits anti-matastaticity0 through integrins effect. Emodin decreases HER2 in breast cancer and improves chemotherapoitic effect. Emodin induces cytotoxis in tongue carcinoma, and inhibits NFkB and other pro inflammatory cytokines. As to its anti-inflammatory activities, emodin may be used as an anti ulcer agent through  H pylori  destruction and change in gastric fluid. Emodin induces a stabilizing effect on atherosclerotic plaque in vessels, and improves insulin and glucose changes in type 2 diabetes. Emodin promotes anti  plasmodium  against malaria, and may be used as an immunosuppressive, pesticide, purgative, spasmolytic, styptic, vasoelaxant, and/or viricide. Emodin has a half-life of approximately 227 min and converts to two active metabolites through P450 in the liver. 
     Rhein 
     Also know as cassic acid, Rhein has a molecular weight around 284.22 g/mol. As to its anti-cancer activity, rhein acts as an anti proliferative in hepatic carcinoma, breast cancer, SCC of lungs, and cervical cancer. Rhein improves taxol effect in breast cancer, and inhibits nasopharengeal carcinoma and EGFR. Rhein serves as a sinergist with mitomycin. Rhein exhibits cytotoxicity in tongue carcinoma. Rhein may be used for anti angiogenesis. As to its anti-inflammatory activity, rhein exhibits anti fibroticity, and promotes anti proliferation of hepatic cells through inhibition of TGFb1. Rhein may be used as an anti oxidant. Rhein decreases IL1B, and IL18 proinflammatory cytokines. Rhein is also anti bacterial, and may be used against  staph. Aureus . Rhein also increases sensitivity to ADH (drug). Rhein has a half-life of about 205 min (approx). Rhein is hydrophobic, and may be combined with lysin in order to be hydrophilic, as rhein lysinate. 
     Chrysophanein 
     Chrysophanein exhibits significant in-vitro cytotoxic activity in cancer cell lines 
     Rhapontin 
     Rhapontin has a molecular weight of around 420.41 g/mol. As to its anti-cancer activities, rhapontin induces apoptosis and suppresses KATO III cell-growth in stomach cancer. Rhapontin also provides protective effects on LDL and erythrocytes against oxidative damage. Rhapontin has a half life of about 23.5 minutes. 
     Stilbene Piceid 
     Stilbene piceid has a molecular weight around 390.2 g/mol. As to its anti-cancer activities, stilbene piceid inhibits DNA synthesis in LLC cells. Stilbene piceid also has an inhibitory effect on lipoxygenase. Stilbene also exhibits antioxidant activity and inhibits alpha-glucosidase. Stilbene piceid also inhibits lipid peroxidation induced by ADP and NADPH in liver microsomes. Stilbene acts directly on smooth muscle to promote pulmonary artery relaxation. 
     Tannin 
     Tannin has a molecular weight around 500-3000 g/mol. As to its anti-cancer activity, tannin suppresses the growth of MCF-7 breast cancer cells. Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis. Tannin exhibits different antiproliferative effects against cervical and colon cancer cells grown in vitro. In pomegranate, tannin modulates inflammatory cell signaling in colon cancer cells. Tannin promotes apoptosis through induction of p53 non-small cell lung cancer cells. As to its anti-inflammatory activity, tannin in tomato suppresses COX-2 expression. Tannin can be used as an in vitro antioxidant and/or antiplatelet and also as an anti-inflammatory due to its free radical scavenging effects. Tannin may be used for its antiviral, antibacterial and/or antiparasitic effects. Tannin can be used in the treatment of HFE hereditary hemochromatosis. Tannin is capable of reversing 6-hydroxydopamine induced toxicity. Tannin has a dental use, as well, as tannin-fluoride preparation reduces gingival inflammation around abutment teeth. A large intake of tannins may cause bowel irritation, kidney irritation, liver damage, irritation of the stomach and/or gastrointestinal pain. A correlation has been made between esophogeal or nasal cancer in humans and regular consumption of certain herbs with high tannin concentrations. Tannins inhibit the absorption of minerals such as iron which may, if prolonged, lead to anemia. Tannins are present in soil, plants, water, tea, wine, and fruit. Tannin has a half life of about 3.15 hours. 
     Carvacrol 
     Carvacrol, also known as cymophenol, has a molecular weight around 150.217 g/mol. As to its anti-cancer activities, carvacrol promotes anti-tumor effects on human metastatic breast cancer cells, including MDA-MB 231. Carvacrol is a potent inhibitor of cell growth in Human Non-Small Cell Lung cancer. Carvacrol also inhibits growth of myoblast cells even after activation of mutated N-ras oncogene. As to its anti-inflammatory activities, carvacrol activates PPAR and suppresses COX-2 inflammation. Carvacrol may be used for its antiproliferative and antiplatelet properties. Carvacrol is present in the essential oil of  Origanum vulgare , oil of thyme, oil obtained from pepperwort, and wild bergamot. Carvacrol inhibits the growth of several bacteria strains, e.g.  Escherichia coli  and  Bacillus cereus , and in  Pseudomonas aeruginosa , carvacrol disrupts the bacteria membrane. Carvacrol may be used for its antioxidant activity. Carvacrol has a half life of about 1.29 hours. 
     Eugenol 
     Eugenol has a molecular weight of about 164.2 g/mol. Eugenol induces apoptosis in human colon cancer cells, and inhibits invasion and angiogenesis of gastric carcinogenesis induced by MNNG. Eugenol in honey significantly inhibits the growth of Ehrlich ascites carcinoma. Eugenol-related biphenyl (S)-6,6′-dibromo-dehydrodieugenol elicits specific antiproliferative activity on neuroectodermal tumour cells partially triggering apoptosis. Eugenol causes melanoma growth suppression through inhibition of E2F1 transcriptional activity. Eugenol may also be used as an antiseptic. Eugenol also inhibits platelet aggregation induced by agonists, including collagen, ADP and calcium ionophore. Eugenol may be extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, basil and bay leaf. Eugenol is also used in perfumeries, flavorings, essential oils and in medicine as a local antiseptic and anesthetic. Eugenol may be used for its antioxidative properties. Eugenol exhibits hepatotoxicity. An overdose of eugenol may induce convulsions, diarrhea, nausea, unconsciousness, dizziness, and/or rapid heartbeat. Eugenol can be allergenic. Eugenol may express carcinogenicity through oxidative DNA damage by its metabolite. Eugenol has a half-life of 1.975 hours, and under certain conditions, may have a half life up to 4 hours or even 18 hours. 
     Geraniol 
     Geraniol has a molecular weight of about 154.25 g/mol. As to its anti-cancer activities, Geraniol promotes an antiproliferative mechanism in human pancreatic adenocarcinoma cells. Geraniol also promotes anti-proliferative and cell cycle regulatory effects in human breast cancer cells. Geraniol can cause a 2-fold reduction of thymidylate synthase and thymidine kinase expression in colon cancer cells. Geraniol, as a component of plant essential oils, sensitizes human colon cancer cells to 5-fluorouracil treatment. Geraniol suppresses pancreatic tumor growth without significantly affecting blood cholesterol levels. As to its anti-inflammatory activities, Geraniol diminishes the levels of inflammatory markers induced by pamidronate stimuli in vitro and in vivo. Geraniol also promotes inhibitory effects on nitric oxide and prostaglandin E 2  production in macrophages. Geraniol is a component of rose oil, palmarosa oil, and citronella oil, and small quantities of geraniol are in geranium and lemon, has a rose-like odor and is commonly used in perfumes. Geraniol can be used as effective plant-based mosquito repellent. Geraniol is found in cigarettes. As to biologic use, ion-exchange iontophoresis combined with geraniol is a highly effective transdermal delivery system. Geraniol suppresses  Candida  cell growth in the vagina and its local inflammation when combined with vaginal washing. Gernaiol is also allergenic. Geraniol has a half life of about 0.713 hours. 
     Luteolin 
     Luteolin has a molecular weight of 286.24 g/mol. As to its anti-cancer activity, luteolin, particularly in combination with standard anticancer drugs such as cisplatin, serves as a HDAC inhibitor, e.g., for the treatment of lung cancer. Luteolin promotes synergistic/additive growth inhibitory effects and may be effective in chemoprevention treatment of head and neck and lung cancers. Luteolin induces G1 arrest in human nasopharyngeal carcinoma cells. Luteolin not only protects DNA from oxidative damage, but also increases repair activity in Caco-2 cells. A low concentration of Luteolin has little toxic effect on cancer cells, but such low concentrations can sensitize chemotherapeutic drugs in various cancer cell lines. Luteolin selectively inhibits chymotrypsin-like and trypsin-like proteasome catalytic activities in tumor cells. Luteolin inhibits invasion of prostate cancer PC3 cells through E-cadherin. 
     Luteolin is a PDE4 inhibitor and a general phosphodiesterase inhibitor, and an Interleukin 6 inhibitor. Luteolin inhibits inflammatory response and improves insulin sensitivity in the endothelium. Luteolin prevents LPS-induced TNF-α expression in cardiac myocytes through inhibiting NF-κB signaling pathway. Luteolin inhibits myelin basic protein-induced human mast cell activation and mast cell-dependent stimulation of Jurkat T cells. Luteolin inhibits cyclooxygenase-2 expression and scavenges reactive oxygen species. 
     Luteolin is found in leaves, but it is also seen in celery, thyme, dandelion, rinds, barks, clover blossom and ragweed pollen. Luteolin is useful in the prevention and treatment of skin photoaging. Luteolin inhibits microglia and alters hippocampal-dependent spatial working memory. Luteolin enhances insulin sensitivity via activation of PPARγ transcriptional activity in adipocytes. Luteolin can induce nausea, vomiting and gastric hypersecretion. Luteolin has a half life of about 1.2 hours. 
     Saponins 
     As to the anti-cancer activities of saponins with oleanolic acid, achyranthoside H methyl ester, a novel oleanolic acid saponin derivative from  Achyranthes fauriei  roots, induces apoptosis in human breast cancer MCF-7 and MDA-MB-453 cells via a caspase activation pathway. Saponion with oleanolic acid exhibit insecticidal activity against the Mexican bean beetle larvae ( Epilachna varivestis ). 
     Vanillic Acid 
     Vanillic acid has a molecular weight of 168.14672 g/mol. Vanillic acid suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo. Vanillic acid enhances the activity of human lymphocyte proliferation and secretion of IFN-gamma. Vanillic acid has a beneficial effect on DSS-induced ulcerative colitis, thereby manifesting its usefulness in the regulation of chronic intestinal inflammation. Phenolic compounds in mushroom  Lentinula edodes  (shiitake) are hepatoprotective through their suppression of immune-mediated liver inflammation. Vanillic acid is found in the root of  Angelica sinensis , and in olive oil. Vanillic acid promotes reduced cellular tyrosinase activity, DOPA oxidase and melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins 2 (TRP-2) and TRP-1. Vanillic acid contributes to the prevention of the development of diabetic neuropathy by blocking the methylglyoxal-mediated intracellular glycation system. There exist an oxidized form of vanillin. Vanillic acids has a half life of about 10.552 hours. 
     α-Terpineol 
     The anti-cancer activities of α-terpineol, or alpha-terpineol, are partly mediated through the suppression of NF-kappaB activation. α-terpineol exhibits antiproliferative effects on erythroleukemic K562 cells. α-terpineol inhibits gene expression of the IL-6 receptor. α-terpineol suppresses fMLP-, LPS- and PMA-stimulated superoxide production. α-terpineol is found in cajuput oil, pine oil, and petitgrain oil, and is a common ingredient in perfumes, cosmetics, and flavors and tea. α-terpineol demonstrates different degrees of growth inhibition against 15 different genera of oral bacteria. α-terpineol can cause postural hypotension in pine oil, and can cause eye irritation. There are three isomers, alpha-, beta-, and gamma-terpineol. Alpha-terpineol has a half life of about 1.245 hours. 
     Aucubin 
     
         
         
           
             Aucubin has a molecular weight of 346.32978 g/mol. 
           
         
       
    
     1. Antiproliferative activity is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells. 
     2. research—DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which aucubin have shown antitumoral activity. 
     3. research—cytotoxic activity against MCF7-breast adenocarcinoma, HeLa-cervix adenocarcinoma, A431-skin carcinoma of epithelial origin. 
     4. can obstruct H(2)O(2)-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation. 
     Aucubin enhance the activity of human lymphocyte proliferation and secretion of IFN-gamma. Aucubin is found in the leaves of  Aucuba japonica  (Cornaceae),  Eucommia ulmoides  (Eucommiaceae), and  Plantago asiatic  (Plantaginaceae). Aucubin protects against liver damage induced by carbon tetrachloride or alpha-amanitin, particularly when dosed intra-peritoneally. Aucubin provides neuroprotection in primary diabetic encephalopathy. Aucubin treatment can lower blood glucose. Aucubin can produce an increase in the level of lipid peroxidation and a decrease in activities of antioxidant enzymes in liver and kidneys. Aucubin has a half life of about 42.5 minutes. 
     Digoxin 
     Digoxin, also known as  digitalis , has a molecular weight of 780.938 g/mol. Digoxin treatment can inhibit HIF-1alpha synthesis and block tumor growth. Digoxin induces apoptosis in a human acute T-cell lymphoblastic leukemia cell line. Digoxin can serve as a specific neuroblastoma growth inhibitor and an unspecific inhibitor of angiogenesis. 
     Digoxin is widely used in the treatment of various heart conditions, namely atrial fibrillation, atrial flutter and sometimes heart failure that generally cannot be controlled by other medication. Digoxin increases myocardial contractility, such that the heart rate is decreased, while blood pressure increases as stroke volume is increased, leading to increased tissue perfusion. Digoxin improves myocardial efficiency due to improved hemodynamics, and improves the ventricular function curve. Digoxin affects the kidney by increased renal blood flow and increased GFR. A mild diuretic effect is seen typically only in heart failure. Digoxin may cause AV junctional rhythm and ectopic beats (bigeminy) resulting in ventricular tachycardia and fibrillation. 
     Digoxin can induce loss of appetite, nausea, vomiting and diarrhea as the gastrointestinal motility increases. Other common effects of Digoxin are blurred vision, visual disturbances (yellow-green halos and problems with color perception), confusion, drowsiness, dizziness, insomnia, nightmares, agitation, and depression, as well as a higher acute sense of sensual activities. Less frequent adverse effects of digoxin (0.1%-1%) include: acute psychosis, delirium, amnesia, convulsions, shortened QRS complex, atrial or ventricular extrasystoles, paroxysmal atrial tachycardia with AV block, ventricular tachycardia or fibrillation, and heart block. Dangerous interactions can occur between digoxin and verapamil, amiodarone, erythromycin, and epinephrine. The efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin. Digoxin treatment increases the risk of invasive breast cancer among postmenopausal women. Digoxin has a half-life around 36 hours. 
     Isoacetoside 
     Isoacetoside, as an extract from  Tecoma stans , exhibits a cytotoxic effect on human hepatocarcinoma cells (Hep-G2). Isoacetoside has a half life of about 3.7-6.4 hours. 
     β-Sitosterol 
     β-sitosterol, or beta-sitosterol, has a molecular weight around 414.71 g/mol. β-sitosterol may be used to treat prostatic carcinoma and breast cancer. β-sitosterol may have chemopreventive potential by virtue of its radical quenching ability in vitro, with minimal toxicity to normal cells. β-sitosterol also attenuates beta-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis. β-sitosterol significantly inhibits the growth, and induces the apoptosis, of SGC-7901 human stomach cancer cells in vitro. The decrease of the bcl-2/bax ratio and DNA damage may produce apoptosis induced by beta-sitosterol in SGC-7901 human stomach cancer cells. β-sitosterol enhances tamoxifen effectiveness on breast cancer cells by affecting ceramide metabolism. β-sitosterol has a proapoptotic effect that is mediated through the activation of ERK and the block of the PI3K/Akt signal pathway in MCA-102 cells. Therefore, beta-sitosterol has a strong potential as a therapeutic agent for preventing cancers such as fibrosarcoma. Beta-sitosterol is an effective apoptosis-promoting agent and that incorporation of more phytosterols in the diet may serve a preventive measure for breast cancer. An anti-microtubule characteristic of beta-sitosterol may contribute to the proliferation inhibition of SiHa cells in cervical cancer. β-sitosterol activates the sphingomyelin cycle and induces apoptosis in LNCaP human prostate cancer cells. β-sitosterol promotes anti-asthmatic actions that may be mediated by inhibiting the cellular responses and subsequent release/synthesis of Th2 cytokines. β-sitosterol may have therapeutic potential in allergic asthma. 
     β-sitosterol is found in  Nigella sativa , pecans,  Serenoa repens  (saw palmetto), avocados,  Curcurbita pepo  (pumpkin seed),  Pygeum africanum , cashew fruit, rice bran, wheat germ, corn oils, soybeans, sea-buckthorn, wolfberries, and  Wrightia tinctoria.    
     β-sitosterol reduces blood levels of cholesterol, and is sometimes used in treating hypercholesterolemia. β-sitosterol may produce a positive effect on male hair loss in combination with Saw palmetto. β-sitosterol may play a major role in herbal therapy, especially in the treatment of benign prostatic hyperplasia. Beta-sitosterol is a naturally occurring phytosterol that may be used to cure atherosclerosis, diabetes, cancer, and inflammation and is also an antioxidant. β-sitosterol has a half life of about 0.966 hours. 
     By taking more than the recommended dose of β-sitosterol, people may suffer from stomach upset, nausea, diarrhea, gas or constipation, impotence (also known as erectile dysfunction or ED), decreased sex drive. Beta-Sitosterol should be avoided during pregnancy and breast-feeding, since it is not proven to be benign with regard to potential effects on unborn and newborn children. β-Sitosterol is also not recommended for individuals with sitosterolemia, a rare inherited fat storage disease, because people with this condition have too much β-sitosterol and related fats in their system, taking β-sitosterol will only worsen this condition. High levels of β-sitosterol concentrations in blood have been correlated with increased severity of heart disease in men having previously suffered from heart attacks, and may cause allergy. 
     Half-lives have been indicated for certain molecules. The half-lives of molecules can vary from these, e.g. generally based on the herb growing and/or preparation conditions, how it is combined with other herbs of molecules in treatment, or based on patient characteristics and behaviors such as eating and drinking and physical activity, or on potency or other factors. Doses and dose periods may be determined based in part on the half lives. Typically, however, doses and dose periods will be determined based on characteristics of the patient, the patient&#39;s condition and the patient&#39;s history, as well as on the expertise and experience of the attending physician. 
     The treatment method may include periodic shampooing with a formula including one or more of the herbs described herein in combination with approximately 5 ug/ml or 10 ug/ml or more of emodin, alone or in combination with respectively 0.05 ug/ml or 0.10 ug/ml or more of digoxin. Other combinations may be used in the treatment, including combining 5 ug/ml or more of emodin alone or with at least approximately 0.10 ug/ml digoxin, or at least approximately 10 ug/ml emodin alone or with at least approximately 0.10 ug/ml digoxin, or more than 5 ug/ml of emodin alone or with at least approximately 0.05 ug/ml digoxin, or at least approximately 10 ug/ml emodin alone or with at least approximately 0.05 ug/ml digoxin. Other combinations may be used and prescribed by physicians depending on factors such variances in weight, age, gender, family or patient history, or other characteristics specific to patients. 
     The shampoo regimen may include once or twice daily shampoo treatments, or two or more weekly doses weekly or otherwise. Doses may be taken more than once or twice a day, while the amounts of each dose would be determined according to the periodicity of the treatments. 
     Methods of preparing shampoo as a medicinal treatment are also provided, including preparing a shampoo formula that includes Sheng Di Huang, Da Huang or Jin Yin Hua, or combination thereof, alone or in combination with another herb or molecule described herein or as understood by those skilled in the art. 
     A hair or scalp shampoo is also provided, including one or more of Sheng Di Huang, Da Huang or Jin Yin Hua and/another herb or molecule described herein or as understood by those skilled in the art. 
       FIG. 19  illustrates the effect of proliferation of digoxin, emodin and their combination on MDA 435 are cell lines of Melanoma and Breast Cancer. In this case, concentration of 1/100 had no effect (same as Control) ⅓ had good effect and 1/10 killed all cells. Normal cells were affected 50-90% less than the rate of pathological cells. Treatments described herein may be prepared for topical use for treatment of melanoma, BCC (basal cyr carcinoma) and inflammatory skin diseases like Eczema. For example, combinations of herbs and/or herbal extracts as described herein may be prepared as a cream to apply onto the skin or as a shampoo, conditioner or other hair and scalp cleaning or rinsing agent. Herb or herb extract combinations described herein also may be injected to infected areas of the scalp or other skin region of a patient using a syringe, or they may be taken orally or intravenously. An example method for preparation of an external cream in accordance with certain embodiments is provided below. 
     Preparation of External Cream/Ointment 
     First, one of more of the herbs may be cooked, for example, as described elsewhere herein or as may be understood or determined by those skilled in the art. Da Huang may be cooked for 5 or 10 minutes or more. A purgative property of Da Huang may be reduced by cooking for 15 minutes or more. The herbs may be filtered through a 0.2 micron filter. The herbs may be centrifuged. A cream is then prepared that may be somewhat more or less than half herbs and half cream, e.g., a 30% liquid of herbs in 1:1 ratio and 70% cream may be used. The herbs can also be prepared as a tincture, e.g., soaking the herbs in alcohol for a period of time such as 2 weeks in a ratio of 1:3, for example. This herbal liquid can then be mixed with the cream in the same way as described above. 
     Shampoo treatments described herein may also be effective against immunodeficiency diseases such as HIV and AIDS, as well as other conditions affecting the immune system and affecting the hair or scalp. 
     EXAMPLES 
     The embodiments are directed to advantageous medicines prepared as a shampoo or other hair or scalp eczema treatment and methods of treatment for eczema and preparation of eczema medicines and treatments for eczema, and/or other conditions such as psoriasis, melanoma or another skin condition formulated as a shampoo, conditioner, ointment, spray, or other topical scalp or hair treatment including an herbal combination with certain herbs such as Da Huang and Sheng Di Huang, or Da Huang, Sheng Di Huang and Jin Yin Hua, and/or Mu Dan Pi, Di Gu Pi, Xian He Cao and/or Chun Gen Pi, and/or any one or more of the eleven additional herbs including Zi Cao, or  radix arnebiae  (arnebia root) or  radix lithospermi  (gromwell root), Xuan Shen, or  radix scrophulariae  (figwort root), Shi Gao or  gypsum fibrosum  ( gypsum ), Bai Shao, or  radix paeoniae alba  (white peony root), Chi Shao or  radix paeoniae rubra  (red peony root), Hong Hua or  flos carthami  (safflower), Da Qing Ye or  folium isatidis  (woad leaf), Qing Dai or  indigo naturalis  (natural  indigo ), Bai Zhu or  rhizoma atractylodis macrocephalae  (largehead atractylodes rhizome), Shi Wei or  folium pyrrosiae  (shearer&#39;s pyrrosia leaf), and/or Rou Gui or  cortex cinnamomi  ( cinnamomum  bark), and/or combinations of certain herbal ingredients, and/or one or more herbal ingredients described herein including Emodin, Rhein, and/or Rhapontin of Da Huang, Carvacrol, Vanillic acid, and/or Sitosterol of Jin Yin Hua, and/or Aucubin, Digoxin, and/or beta-sitosterol of Sheng Di Huang and/or other ingredients present within the two, the three, the seven and/or even the eighteen herbs, may be included in a shampoo treatment in accordance with certain embodiments. 
     In one embodiment, a shampoo treatment may include applying a dose, such as in a process of shampooing the hair and scalp, of active herbal ingredients, extracts or molecules to the scalp and hair from an example 16 fluid ounce formulation that includes between 13.3 grams to 60 grams of Sheng Di Huang, or 3.3 grams to 30 grams of Da Huang, or both, alone or in combination with one of more other herbs or molecules to be administered daily or 2-3 times daily or up to 10× daily or less than daily even as little once or twice a week. In other embodiments, the shampoo formulation further includes 3.3 grams to 30 grams of Jin Yin Hua. In other embodiments, two of these three are included in the shampoo regimen. In further embodiments, one, two, three or all four of the herbs Mu Dan Pi, Xian He Cao, Chun Gen Pi and Di Gu Pi is/are combined with one, two or all of the three herbs Sheng Di Huang, Da Huang and Jin Yin Hua within a 16 ounce example shampoo or hair lotion formulation, including 3.3 grams to 15 grams of any of the other four of the seven herbs, e.g., 3.3 grams to 15 grams of Mu Dan Pi, 3.3-10 grams to 15 grams of Xian He Cao, 3.3-10 grams to 15 grams of Chun Gen Pi, and/or 3.3-5 grams to 15 grams of Di Gu Pi. 
     In one example, an example 16 fluid ounce shampoo formulation includes 40 grams of Sheng Di Huang, 15 grams of Da Huang and 15 grams of Jin Yin Hua with multiple other ingredients described in examples herein to be administered as a shampoo regimen. The combination of herbs may be cooked once or twice, and the herb:shampoo/lotion ratio may be 1:20 or 1:40 or otherwise. The dose may be 1-10 days or even multiple times daily including 2-6 times daily, and even as often as 10 times daily. 
     In another example, a shampoo regimen includes 5 grams of Mu Dan Pi, 20 grams of Sheng Di Huang, 5 grams of Xian He Cao, 5 grams of Chun Gen Pi, 5 grams of Di Gu Pi, 10 grams of Da Huang and 10 grams of Jin Yin Hua in a 16 fluid ounce shampoo or hair lotion formulation. The combination may be cooked once or twice, and the herb:shampoo/lotion ratio may be 1:10, 1:20, 1:40 or otherwise. The dose may be 1-10 days or even multiple times daily including 2-6 times daily, and even as often as 10 times daily. 
     In another example, a shampoo regimen is supplemented by a low dose herbal formula including 3.3 grams of two or more of Da Huang, Sheng Di Huang and Jin Yin Hua, and/or one or more of the following seven herbs: 3.3 grams of Da Huang, 13.3 grams of Sheng Di Huang, 3.3 grams of Jin Yin Hua, 3.3 grams of Mu Dan Pi, 5 grams of Di Gu Pi, 10 grams of Xian He Cao, and 10 grams of Chun Gen Pi in water or in a dry or a lipidized formulation or otherwise configured for topical, oral, intravenous or sub-dermal injectable administration. 
     In another example, a shampoo regimen includes a high dose herbal formula including 30 grams or more of Da Huang, and/or with one or more of the following seven herbs: 60 grams of Sheng Di Huang, 30 grams of Jin Yin Hua, 15 grams of Mu Dan Pi, 15 grams of Di Gu Pi, 15 grams of Xian He Cao, and 15 grams of Chun Gen Pi, in an example 16 fluid ounce formulation, including other shampoo ingredients as may be described in example embodiments herein, or the herbal formula may be dissolved in water or prepared in a dry or a lipidized formulation or otherwise configured for topical, oral, intravenous or sub-dermal injectable administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a formula that includes the three herbs including 3.3-20 grams of Da Huang, 10-60 grams of Sheng Di Huang, and 3.3-20 grams of Jin Yin Hua together with multiple other ingredients in an example 16 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a formula including 3.3-20 grams of Da Huang and 10-60 grams of Sheng Di Huang in a 16 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a formula including 3.3-20 grams of Jin Yin Hua and 10-60 grams of Sheng Di Huang in a 16 fluid ounce bottle. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a formula including 3.3-20 grams of Da Huang and 3.3-20 grams of Jin Yin Hua in a 16 fluid ounce bottle. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo formula including one active herb, e.g., 3.3-30 grams of Da Huang, in a 16 ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo formula including one active herb, e.g., 3.3-30 grams of Jin Yin Hua, in a 16 ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including one active herb, e.g., 10-90 grams of Sheng Di Huang, in a 16 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including three active herbs including 10-100 grams of Da Huang, 25-250 grams of Sheng Di Huang, and 10-100 grams of Jin Yin Hua in a 16, 32 or 64 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including 10-100 grams of Da Huang and 25-250 grams of Sheng Di Huang in a 16, 32 or 64 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including 10-100 grams of Jin Yin Hua and 25-250 grams of Sheng Di Huang, in a 16, 32 or 64 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including 10-100 grams of Da Huang and 10-100 grams of Jin Yin Hua in a 16, 32 or 64 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including one herb, e.g., 10-100 grams of Da Huang, in a 16, 32 or 64 fluid ounce shampoo or hair lotion formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula including one herb, e.g., 10-100 grams of Jin Yin Hua, in a 16, 32, or 64 fluid ounce shampoo or hair lotion formula. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion including 25-250 grams of Sheng Di Huang in a 16, 32 or 64 fluid ounce shampoo or hair lotion formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion that includes 30-300 grams of Da Huang, 60-600 grams of Sheng Di Huang, and 30-300 grams of Jin Yin Hua in a 32-640 fluid ounce shampoo or hair lotion formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 30-300 grams of Da Huang, and 60-600 grams of Sheng Di Huang in a 32-640 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formulation that includes 30-300 grams of Da Huang and 30-300 grams of Jin Yin Hua in a 32-640 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 60-600 grams of Sheng Di Huang and 30-300 grams of Jin Yin Hua in a 32-640 fluid ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 30-300 grams of Da Huang in a 32-640 ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 30-300 grams of Jin Yin Hua in a 32-640 ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 60-600 grams of Sheng Di Huang in a 32-640 ounce formulation. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 2-10 grams of Da Huang in a 10 fluid ounce shampoo or hair lotion formula, or the Da Huang may be formulated in dry or lipidized form for oral, topical, sub-dermal or IV administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 5-25 grams of Sheng Di Huang in a 10 fluid ounce shampoo or hair lotion formula, or the Sheng Di Huang may be formulated in dry or lipidized form for oral, topical, sub-dermal or IV administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 2-10 grams of Jin Yin Hua in a 10 fluid ounce shampoo or hair lotion formula, or the Jin Yin Hua may be formulated in dry or lipidized form for oral, topical, sub-dermal or IV administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 10-20 grams of Da Huang, 20-40 grams of Sheng Di Huang, and 10-20 grams of Jin Yin Hua in a 32-64 fluid ounce shampoo or hair lotion formulation, or the active herbal combination may be formulation in dry or lipidized form for oral, topical, sub-dermal or IV administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 1.5-15 grams of Da Huang, 3.5-35 grams of Sheng Di Huang, and 1.5-15 grams of Jin Yin Hua in about 300 grams of shampoo or hair lotion product, or the herbal combination may be formulated in dry or lipidized form for oral, topical, sub-dermal or IV administration. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo formula that include 25-75 grams of Da Huang, 45-135 grams of Sheng Di Huang, and 25-75 grams of Jin Yin Hua in about 250-2000 grams of shampoo product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo formula that includes 3.3-10 grams of Da Huang, and 13.3-40 grams of Sheng Di Huang in about 100-300 grams of shampoo product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo formula that includes 30-100 grams of Da Huang and 60-200 grams of Sheng Di Huang in about 600-6000 grams of shampoo or hair lotion product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formulation that includes 10-50 grams of Da Huang and 30-150 grams of Sheng Di Huang in about 400-2000 grams of shampoo or hair lotion product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 20-50 grams of Da Huang and 40-100 grams of Sheng Di Huang in about 600-5000 grams of shampoo or hair lotion product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion product that includes 15-100 grams of Da Huang and 45-300 grams of Sheng Di Huang in about 600-4000 grams of shampoo or hair lotion product solution. 
     In another example, a shampoo regimen includes periodic applications to the hair and scalp of a shampoo or hair lotion formula that includes 25-75 grams of Da Huang and 50-150 grams of Sheng Di Huang in about 800 grams of shampoo or hair lotion product solution. 
     Examples of low, medium and high concentration herbal shampoo and/or hair lotion products or oral, IV, dermal adhesive or sub-dermal formulations may include one or more of the herbs described herein, such as any combination of the herbs contained in the seven herb and eighteen herb combinations described herein, that are administered in doses that are applied to the hair and scalp as a shampoo or hair lotion and that are the same as or similar to or commensurate with the low, medium and high dose example formulas for the one, two and three herb combinations described above, including corresponding percentage changes when four or more herbs are included in the supplemental herbal formula that is administered before during or after a shampoo dose or regimen. Many other combinations may be used depending on characteristics of the patient such as age and weight, the condition of the patient, and the patient&#39;s history. 
     Doses in between the low dose and high dose examples for the formulas that include one, two, or three or more herbs, or the seven herb formula, or the eighteen herb formula are also within the scope of further examples with per herb doses and/or total herb doses that are within the ranges provided in the examples above. Under certain conditions, doses above the high dose formula or below the low dose formula may be used as determined by a physician using his or her expertise and experience both generally in the field and with specific patients. In addition, other combinations of two, three, four, five, or more of these 18 herbs, and/or including one or more other herbs as understood by those skilled in the art, may be used in further examples of formulas wherein the dose ranges described in the above examples or otherwise as determined by a physician may be used. Further herbs not described herein may also be included in formulas including combinations of the described herbs and/or molecules, molecular extracts or molecular compounds described herein or understood by those skilled in the art. 
     In the examples, certain doses of herb combinations have been described in certain amounts of shampoo or hair lotion product solution. Mixing the herbs in water and then into one of the example shampoo formulae described herein is an example of a way to prepare the herbal shampoo or hair lotion products. Another liquid such as DMSO or an oil may be used instead of or in addition to water. The herbs may be integrated with the other components of the shampoo formula in a variety of ways including mixing with one of the components before integration with the rest of the shampoo. The herbs may be formed into powders or dissolvable solids or the herbs may be dissolved into solution and poured or a syringe or fluid transport tubing may be used. Any of the herbal combinations may be formed into a shampoo treatment or rinse, or as cream and rubbed onto the skin or hair, or dissolved into a solution such that a syringe may be used to inject a patient sub-dermally with an herbal combination. 
     In another example, the effect of CXCR4 antagonists on the survival of NB4, HL60, Jurkat leukemic cells and HT29 colon cancer and prostate tumor cells was examined. Digoxin significantly inhibited the growth of leukemic cells at concentrations between (0.05- to 1 microgram/ml). Emodin by itself inhibited the growth of leukemic cells only at concentrations of more than 5 microgram/ml. Combinations of digoxin at concentrations of 0.1 microgram/ml and either 5 or 10 microgram/ml of emodin increased significantly the tumor killing ability of both compounds. Digoxin, Emodin, and their combination also add partial however significant effect on HT29 tumor cells. 
     The effect of the proliferation of digoxin, emodin, and their combination on the viability of cancer cell lines from different origin was studied. Harvested non adherent human hematopoietic cancer cell lines NB4, HL60 and Jurkat, cells were seeded at 2×105 viable cells/1 ml per well into a 24-well plate in triplicates in a medium supplemented with 10% FCS and incubated with different concentrations of digoxin, emodin, and their combination for 24 hours. Following the incubation, the cells were stained with propidium iodide (PI) (Sigma, St. Louis, Mo.) and percent of viable PI-negative cells in culture was determined by FACScalibur analysis (Becton Dickinson Immunocytometry Systems), using CellQuest software. Adherent prostate cancer PC3 cells and colon cancer HT29 cells were seeded at 1×10 −5  viable cells/1 ml per well into a 24-well plate under conditions described above, and following a 24-hour exposure to digoxin, emodin, and their combination the cells were harvested, washed with PBS and stained with PI and counted as described for hematopoietic cells. 
     The therapeutic index of digoxin is understood to be between 0.125 m″g to 0.5 m″g, while the LD50 values of emodin in mice are understood to be 0.56 g/k″g. 
     When combining D and E in accordance with certain embodiments, and/or combining digoxin or emodin, or both, with one or more of the herbs, there is a surprising and unexpected symbiotic effect. 
     Certain treatments may be prepared as a mixture of herbs that are known to have separately digoxin “D” and emodin “E”. Aloe-emodin is a natural anthraquinone compound that is present in some traditional medicinal plants such as  Rhei Rhizoma  and  Rheum palmatum . Interestingly, aloe-emodin has been found to have lesser cytotoxicity towards normal human cells. In-vitro tests with synthesized D&amp;E molecules have been performed on a cell-line. The in-vitro with the plants was similar to the one with the isolated molecules. 
     Emodin may be extracted from traditional medicinal plants such as  Rhei Rhizoma  and  Rheum Palmatum . In one embodiment, the source of Emodin used is Da Huang—Chinese name, or Rhubarb Root—English name, or  Rheum Plamatum —Botanical name, or  Radix Rhisoma Rhei —Pharmaceutical name. Emodin may be extracted from Rhubarb, Buckthorn and/or Japanese Knotweed ( Fallopia Japonica ). Aloe-emodin may be used which is a variety of emodin found in Socotrine, Barbados, and Zanzibar aloes. 
     A medicinal cancer treatment using Emodin is prepared in certain embodiments by mixing the herb, Da Huang, in water at a ratio of approximately 10:1. The herb may be ground to a fine powder. The water may be added to the fine powdered herb, and the pot covered. After boiling, the heat is lowered in certain embodiments to about 70 degrees centigrade. The aqueous mixture is cooked for another hour. The liquid is then strained into a container. In some cases, this may be done twice. In the second cooking, the ratio may be reduced to 7.5:1. The second cooking may take about 45 minutes including the boiling. 
     Single herbs or combinations of two or more herbs alone or with any one or more of the described molecules may be prepared in a process involving the following or a subset or variation thereof: grinding the herbs to a fine texture in the mixer for around 2-3 min until it looked fine powder; weighing the powder (e.g., 25 gm) and transferring to a beaker (e.g., 2000 ml); adding distilled water (RT) to powdered herbs in a ratio of 1:20 (gm of herbs: ml of water) and soaking the herbs for 15-20 min; boiling the mixture to 85-90° C.; cooling the temperature of the mixture down to 70-75° C. after removing it from the hot plate; covering the beaker properly with aluminum paper and cooking the mixture at 70° C. on hot plate—the total time of cooking of the mixture may be approximately 60 min which includes boiling, cooling it down and cooking; straining the mixture with the help of a manual strainer; after filtration, centrifuging the extract at 5000 rpm for 15 min and collecting the supernatant; filter sterilizing the supernatant by passing through 0.2 μm syringe filter; storing the clear filtrate at 4° C.-subsequent dilutions may be prepared. 
     The source of Digoxin in certain embodiments is Sheng Di Huang-Chinese name, or Foxglove root—English name, or  Radix Rhemania —Pharmaceutical name. The preparation of the Digoxin may be the same as for the Emodin. In certain embodiments, the herbs from which the Emodin and Digoxin are extracted are cooked together. Digoxin may be extracted from  Digitalis Purpurea  or Purple Foxglove. 
     In certain embodiments, the prepared treatment is not pure. For example, certain treatments may involve a “vegetable soup” type regiment made of more than one and even two, three, seven or eighteen herbal ingredients per specific examples provided herein, or any combinations of the herbs described herein, or combinations of the herbs described herein with other herbs not mentioned herein. In certain embodiments, herbal ingredients are mixed in solution and a patient may drink the liquid. While freeze-drying the herb in powder form may be possible, the above-described process appears to be more effective. 
     Harvested non adherent human hematopoietic cancer cell lines NB4 (AML), HL60 and Jurkat were seeded at 2×10 5  viable cells/1 ml per well into a 24-well plate in triplicates in a medium supplemented with 10% FCS and incubated with different concentrations of digoxin, Emodin, and their combination for 24 hours. Following the incubation, the cells were stained with propidium iodide (PI) (Sigma, St. Louis, Mo.) and percent of viable PI-negative cells in culture was determined by FACScalibur analysis (Becton Dickinson Immunocytometry Systems), using CellQuest software. Adherent prostate cancer PC3 cells and colon cancer HT29 cells were seeded at 1×10 5  viable cells/1 ml per well into a 24-well plate under conditions described above, and following 24-hour exposure to digoxin, Emodin, and their combination, the cells were harvested, washed with PBS and stained with PI and counted as described for hematopoietic cells. 
     A scalp, hair or other skin condition treatment formulated as a shampoo, conditioner, cream, ointment, spray or other topical scalp, hair or skin treatment including an herbal combination of Da Huang and Sheng Di Huang, or Da Huang, Sheng Di Huang and Jin Yin Hua, or other combinations of the herbs and/or molecules described herein may also be combined with other treatments described herein and/or as may be understood by those skilled in the art and/or as may be described in literature such as the following which are hereby incorporated by reference as disclosing alternative embodiments and compounds that may be combined with Da Huang, Sheng Di Huang or Jin Yin Hua, or combinations thereof, in a cocktail or otherwise effective shampoo therapy:
     Shraibom, N. (2015). Molecular and herbal combinations for treating psoriasis. U.S. Pat. No. 9,095,606 B1.   Abstract: p1573-3HX—A new drug developed based on Traditional Chinese Medicine. 23rd EADV Congress, 2014.   Deng, S., May, B. H., Zhang, A. L., Lu, C., Xue, C. C., 2013. Br. J. Dermatol. 169, 769-782.   Jeong, S. J., Lim, H. S., Seo, C. S., Kim, J. H., Jin, S. E., Yoo, S. R., Shin, H. K., 2015. Phytomedicine 22, 326-332.   Jin, S. E., Lim, H. S., Kim, Y., Seo, C. S., Yoo, S. R., Shin, H. K., Jeong, S. J., 2015. Evid. Based Complement. Alternat. Med. Article ID 728380, 12 pages.   Koo, J., Arain, S., 1998. Arch. Dermatol. 134, 1388-1393.   Parker, S., Zhang, A. L., Zhang, C. S., Goodman, G., Wen, Z., Lu, C., Xue, C. C., 2014. Trials 15, 495.   Chronic Lymphocytic Leukemia by the Leukemia &amp; Lymphoma Society;   Medifocus.com Medifocus Guide on Chronic Lymphocytic Leukemia;   Ranjit Thomas, et al., Spontaneous Clinical Regression in Chronic Lymphocytic Leukemia, British Journal of Haematology, 2002, 116, 341-345;   Dragomir Marisavljevic, et al., Spontaneous Clinical Remission of Chronic Lymphocytic Leukemia, Haema 2003; 6(3): 394-397;   Upshaw J D Jr, et al., Spontaneous Remission of B cell Chronic Lymphocytic Leukemia associated with T Lymphocytic Hyperplasia in bone marrow, South Med J. 22 Jun. 1995(6): 647-9;   Wiernik P H, Second neoplasms in patients with chronic lymphocytic leukemia, Current Treat Options Oncol. June 2004; 5(3):215-23;   Luis Fayad M D and Susan O&#39;Brien M D, Chronic Lymphocytic Leukemia and Associated Disorders, Medical Oncology: A Comprehensive Review, 1995;   Michael J. Keating, et al., Biology and Treatment of Chronic Lymphocytic Leukemia, American Society of Hematology, Hematology 2003, 153-175;   GE Marti, et al, Descriptive Epidemiology of Chronic Lymphocytic Leukemia (CLL);   Sarfaraz K. Niazi, Handbook of Pharmaceutical Manufacturing Formulations, Culinary &amp; Hospitality Industry Publications Services; and   U.S. Pat. Nos. 5,872,103; 6,197,754; 6,740,665; 6,812,255; 7,268,162; 7,358,222; 7,381,535; 7,393,656; 7,563,584; 7,643,609; 7,695,926; 7,790,905; 8,541,382; 8,734,859; and   

     United States published applications no. 20030211180; 20050026849; 20050196473; 20060205679; 20070191262; 20080152700; 20080220441; 20090018088; 20090143279; 20090215042; 20090269772; 20100068198; 20100092585; 20100144647; 20100167286; 20120122807; 20050008664; and
     PCT published applications no. WO01/66123A2; WO2004/052294A2; WO2006/053049A2; WO2007/130124A1; and WO2012/063134A2.   

     A treatment regimen for eczema, and/or another skin and/or scalp condition such as dandruff, melanoma or another scalp condition includes in certain embodiments an herbal combination of Sheng Di Huang and Da Huang, otherwise referred to herein as the three herb combination or “2-HX.” Another treatment regimen for eczema, and/or another disorder such as psoriasis, dandruff, melanoma or another scalp condition includes an herbal combination of Jin Yin Hua, Sheng Di Huang and Da Huang, otherwise referred to herein as the three herb combination or “3-HX.” Another treatment regimen of periodic doses of an advantageous combination of emodin and digoxin is provided to treat eczema. Another treatment regimen for eczema includes a combination of emodin (E) and digoxin (D) with Jin Yin Hua, Sheng Di Huang and Da Huang, otherwise referred to herein as the three herb combination or “3-HX.”. Another treatment regimen for eczema includes a combination of emodin and digoxin, or D&amp;E, and Jin Yin Hua, Sheng Di Huang and Da Huang or 3-HX, with Mu Dan Pi, Di Gu Pi, Xian He Cao and Chun Gen Pi, which forms a combination of D&amp;E and the seven herb combination referred to herein. 
     The herbal combination may be formulated as a shampoo, conditioner, cream, ointment, spray or other topical scalp or hair treatment. The herbal combination and/or the treatment regimen may be administered topically, orally, subcutaneously or intravenously. The treatment regimen may include both the herbal combination and another medicinal treatment such as any of the other treatments described herein or another known or experimental treatment. The herbal combination can serve to enhance the potency of another scalp treatment and/or to reduce toxic side effects and/or dosage of another scalp treatment. 
     Anti-psoriatic activity of 3-HX, and of D&amp;E was determined in studies using 12-O-Tetradecanoylphorbol-13-Acetate (TPA) induced ear inflammation in male C57BL/6 mice. All the animals were randomized based upon the body weight and allotted to seven groups with 6 animals in each group. Groups G1 and G2 were treated topically with test item 3HX at the dose levels of 1:4 and 1:8 dilutions respectively. Groups G3 and G4 were treated orally with test item 3HX at the dose levels of 500 and 1000 mg/kg respectively. Group G5 and G6 were treated topically and orally with D and E respectively. Group G7 served as both TPA control (right ear) and solvent control (left ear) and was treated orally with Na-CMC. 
     Among the various experimental groups topical application of 3-HX (1:4) and D&amp;E and oral treatment of low dose of 3-HX (500 mg/kg) and D &amp; E exhibited maximum reduction in ear thickness. Similar trend of test items response was observed in TPA-induced ear thickness change. Topical application of test items exerted optimum % inhibitory activity against TPA induced ear inflammation in mice whereas oral treatment exhibited marginal anti-inflammatory activity. In the punch biopsy weight investigation maximum reduction was obtained in topically D &amp; E and 3-HX (1:4) treated groups whereas marginal reduction was obtained on oral treatment with test items. The epidermal ear thickness results obtained from histopathological photographs were in accordance with punch biopsy weight results where topical application of test items was found to exert better effect in comparison with oral treatment. Among the evaluation of test items over inflammatory biomarkers viz., enzyme myeloperoxidase (MPO) and nitric oxide levels, topical application of D &amp; E and 3-HX (1:4) resulted in marked suppression of enzyme MPO activity whereas marginal inhibition of nitric oxide content generation was obtained on oral administration of test items respectively. Thus, based upon the present experiment findings it is suggested that topical application of 3-HX at the ratio of 1:4 and D &amp; E as well as oral treatment of low dose of 3-HX (500 mg/kg) may act as promising drug candidate for the treatment of eczema. 
     Test Item Information 
     
       
         
           
               
             
               
                   
               
             
            
               
                 TEST ITEM-1 
               
            
           
           
               
               
            
               
                 Name of the test item/code 
                 3-HX 
               
               
                 Lot No. 
                 NA 
               
               
                 Pack Size 
                 NA 
               
               
                 Physical Description 
                 NA 
               
               
                 Storage condition 
                 Room Temperature 
               
               
                 Test Item Analysis 
                 Analysis for the identity and purity of the 
               
               
                   
                 test item was not conducted as part of this 
               
               
                   
                 study, and is the responsibility of the 
               
               
                   
                 sponsor 
               
            
           
           
               
            
               
                 TEST ITEM-2 
               
            
           
           
               
               
            
               
                 Name of the test item/code 
                 Digoxin (D) 
               
               
                 Lot No. 
                 051M1374V 
               
               
                 Pack Size 
                 NA 
               
               
                 Physical Description 
                 White to off white powder 
               
               
                 Storage condition 
                 At Room Temperature 
               
               
                 Test Item Analysis 
                 Analysis for the identity and purity of the 
               
               
                   
                 test item was not conducted as part of this 
               
               
                   
                 study, and is the responsibility of the 
               
               
                   
                 Sponsor 
               
               
                 Precautions in handling 
                 Avoid contact with skin and eyes 
               
            
           
           
               
            
               
                 TEST ITEM-3 
               
            
           
           
               
               
            
               
                 Name of the test item/code 
                 Emodin (E) 
               
               
                 Lot No. 
                 M3074 
               
               
                 Pack Size 
                 NA 
               
               
                 Physical Description 
                 Orange powder 
               
               
                 Storage condition 
                 Stored at +4° C. 
               
               
                 Test Item Analysis 
                 Analysis for the identity and purity of the 
               
               
                   
                 test item was not conducted as part of this 
               
               
                   
                 study, and is the responsibility of the 
               
               
                   
                 sponsor 
               
               
                 Precautions in handling 
                 Avoid contact with skin and eyes 
               
               
                   
               
            
           
         
       
     
     Vehicle Control 
     The vehicle control is 2% DMSO in methanol. 
     Test System 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Species 
                 
                   Mus musculus 
                 
               
               
                 Strain 
                 C57BL/6 
               
               
                 Source 
                 Animal Facility, Dabur Research 
               
               
                   
                 Foundation 
               
               
                 Sex 
                 Male 
               
               
                 Age 
                 6-8 Weeks 
               
               
                 Body weight range 
                 16.47-36.51 g 
               
               
                 Acclimatization 
                 12 Days 
               
               
                 Randomization 
                 Animals were randomized based on their 
               
               
                   
                 body weight 
               
               
                 Identification of animals 
                 By cage labeling and tail marking 
               
               
                 Total no of animals 
                 42 
               
               
                 Total no of groups 
                 7 
               
               
                 Total no of animals per group 
                 6 
               
               
                 Total no of animals per cage 
                 6 
               
               
                   
               
            
           
         
       
     
     Animal Husbandry Conditions 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Room temperature 
                 21.8 to 24.2° C. 
               
               
                   
                 Relative humidity 
                 56 to 59% 
               
               
                   
                 Light/dark cycle 
                 12-hourly 
               
               
                   
                 Feed 
                 Conventional feed purchased from a 
               
               
                   
                   
                 Golden feed, Mehrauli was provided ad 
               
               
                   
                   
                 libitum to the animals. 
               
               
                   
                 Water 
                 Filtered drinking water was provided ad 
               
               
                   
                   
                 libitum. 
               
               
                   
                   
               
            
           
         
       
     
     Justification for Selection of Test System 
     Male C57BL/6 mice were selected as the test system, as they were commonly reported in literature to evaluate the effect of test item for anti-psoriatic potential on TPA induced ear inflammation model. 
     Chemicals 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 Catalogue No. 
                   
                 Lot No. 
               
               
                 Name of the Chemical 
                 or CAS No. 
                 Company 
                 or Batch No. 
               
               
                   
               
             
            
               
                 Phorbol 12-myristate 13-acetate 
                 P8139-1MG 
                 Sigma life sciences, 
                 SLBC5412V 
               
               
                 (TPA) 
                   
                 USA 
                   
               
               
                 Dimethyl sulfoxide (DMSO) 
                 67-68-5 
                 Merck Specialties Pvt 
                 80291205001730 
               
               
                   
                   
                 Ltd., Mumbai, India 
                   
               
               
                 Methanol 
                 M0275 
                 RANKEM Fine 
                 R106M10 
               
               
                   
                   
                 Chemicals Ltd. New 
                   
               
               
                   
                   
                 Delhi, India 
                   
               
               
                 Sodium dihydrogen phosphate 
                 17845 
                 Merck Specialties Pvt 
                 DL1DR511556 
               
               
                 2-hydrate cryst. Pure 
                   
                 Ltd., Mumbai, India 
                   
               
               
                 Hexadecyltrimethyl-ammonium 
                 H5882 
                 Sigma life sciences, 
                 120M0141V 
               
               
                 bromide 
                   
                 USA 
                   
               
               
                 o-Dianisidine Dihydrochloride 
                 195136 
                 MO Biomedicals LLC 
                 M1271A 
               
               
                 di-Sodium hydrogen phosphate 
                 7558-79-4 
                 Merck Specialties Pvt 
                 MF7M571743 
               
               
                 anhydrous Purified 
                   
                 Ltd., Mumbai, India 
                   
               
               
                 Hydrogen Peroxide Solution 
                 7722-84-1 
                 Fisher Scientific 
                 1889 7202-5 
               
               
                 Orthophosphoric acid 
                 7664-38-2 
                 Fisher Scientific 
                 1309 7100-5 
               
               
                 Sulphanilamide 
                 63-74-1 
                 Central Drug House 
                 930308 
               
               
                   
                   
                 Pvt Ltd. New Delhi 
                   
               
               
                 Glycine 98% 
                 56-40-6 
                 Acros Organics, USA 
                 A013597501 
               
               
                 Sodium chloride 
                 7647-14-5 
                 Merck Specialties Pvt 
                 MJ0M602975 
               
               
                   
                   
                 Ltd., Mumbai, India 
                   
               
               
                 Hydrochloric acid min. 35% 
                 7647-01-0 
                 Merck Specialties Pvt 
                 HD7H570285 
               
               
                 GR 
                   
                 Ltd., Mumbai, India 
               
               
                   
               
            
           
         
       
     
     Method 
     Principle 
     Excessive proliferation of keratinocytes is a characteristic of eczema, and this cell type is a well reported target of therapy for this disease. In the present study, chronic skin inflammation was induced by repeated topical application of TPA, which is recognized by prolonged skin reaction &amp; epidermal hyperplasia. In this model the potential of three test items—3-HX, Digoxin (D) and Emodin (E) were evaluated for reduction of epidermal hyperplasia reflected by change in ear thickness &amp; keratinocyte proliferation in C57BL/6 mice. 
     Experimental Procedure 
     The study was conducted on healthy, adult, male C57BL/6 mice. All the animals were acclimatized to laboratory condition prior to experiment initiation. 
     5 mg/ml stock of TPA was prepared in DMSO and aliquot of the same was diluted to 1:50 with methanol to achieve final TPA concentration of 100 μg/ml for topical application. For oral application of 3-HX, a stock solution of 100 mg/ml was prepared in distilled water and was further diluted to 50 mg/mL, in-order to administer the dose of 500 mg/kg and 1000 mg/kg. For topical application, required amount of 3HX was dissolved in DMSO to obtain the final dilution of 1:4 and 1:8. For oral application of test item D, 10 mg/ml was prepared in DMSO and was further formulated with 0.25% Na-CMC to obtain final strength of 0.1 mg/ml. For topical application, required amount of D was dissolved in DMSO and methanol and stored at −20° C. Similarly, for oral application of test item E, 10 mg/ml stock was prepared in 0.25% Na-CMC and 0.1% Tween 80. For topical application, required amount of E was dissolved in DMSO and diluted with methanol. 
     On Day 0, all the animals were randomized based on body weight and allotted to seven groups containing 6 animals per group. Groups G1 and G2 were treated topically with test item 3HX at the dose levels of 1:4 and 1:8 dilutions respectively. Groups G3 and G4 were treated orally with test item 3HX at the dose levels of 500 and 1000 mg/kg respectively. Group G5 was treated topically with test item D and E, in combination, at the concentration of 1 μg and 100 μg respectively. Group G6 was treated orally with test item D at 1 mg/kg. After 30 min. of D administration, test item E was dosed orally at 100 mg/kg. Group G7 served as both TPA control (right ear) and solvent control (left ear) and was treated orally with Na-CMC. 
     20 μL of TPA solution containing 2 μg of TPA in vehicle (2% DMSO and 98% methanol) was applied topically on both ventral and dorsal side of the right ear of all the groups of animals on day 0, 2, 4, 7 and 9. However, the left ear of Group G7 animals was treated topically with 20 μL of solvent (2% Dimethylsulfoxide+98% methanol) on day 0, 2, 4, 7 and 9. 
     All the test item formulations was administered orally to Groups-G3, G4, and G6 at the dose volume of 10 ml/kg from day 0 to day 9. For topical application, 20 μL of test items (3HX and D+E) was dissolved appropriately in the TPA solvent i.e. methanol and applied daily from Day 0 to Day 9 to Groups-G1, G2 and G5. 
     The ear thickness was measured daily using digital caliper. On Day 10, animal&#39;s blood was withdrawn by retro-orbital plexus. Blood serum was separated for the estimation of nitric oxide level by Griess method. Further, all the animals were humanely sacrificed and ear punch biopsies were collected, weighed and subjected for histopathological analysis, immunohistochemical Ki-67 staining and Myeloperoxidase (MPO) activity. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Allocation of Animals 
               
            
           
           
               
               
               
               
               
            
               
                 Groups 
                 Treatment 
                 Volume &amp; Dose 
                 Route 
                 No. of Animals 
               
               
                   
               
               
                 G1 
                 TPA + 3HX 
                 20 μL + 20 μL of 1:4 
                 Topical 
                 6 
               
               
                 G2 
                 TPA + 3HX 
                 20 μL + 20 μL of 1:8 
                 Topical 
                 6 
               
               
                 G3 
                 TPA + 3HX 
                 20 μL + 500 mg/kg 
                 Oral 
                 6 
               
               
                 G4 
                 TPA + 3HX 
                 20 μL + 1000 mg/kg 
                 Oral 
                 6 
               
               
                 G5 
                 TPA + D&amp;E 
                 20 μL + 20 μL of stock 
                 Topical 
                 6 
               
               
                   
                   
                 (1 μg D + 100 μg E) 
                   
                   
               
               
                 G6 
                 TPA + D&amp;E 
                 20 μL + 1 mg/kg D 
                 Oral 
                 6 
               
               
                   
                   
                 and 100 mg/kg E 
                   
                   
               
               
                 G7 
                 TPA Control 
                 20 μL TPA + 
                 Oral 
                 6 
               
               
                   
                   
                 0.25% Na—CMC 
               
               
                   
               
            
           
         
       
     
     Observations/Calculations 
     Body Weight 
     Body weight of all the animals was recorded from day 0 to day 10. The % change in body weight for each animal was calculated using the given formula:
 
% Body Weight Change=Body Weight at ‘ X ’ day-Body weight at day ‘0’×100 Body weight at day ‘0’
 
     “x”: Day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 
     Ear Thickness Measurement 
     Upper, middle and lower ear thickness of both right and left ear of Group G1 and right ear of Group G2 to G7 was measured and recorded daily from Day 0 to Day 9 using digital caliper (MITUTOYO) and their average value was considered for further calculations. 
     Mean absolute ear thickness (in mm) are shown in  FIGS. 24-25 . 
     Ear Thickness Change (Etc) 
     Mean ear thickness change or ear edema was calculated based on the absolute ear thickness values.
 
Ear thickness change (mm) ( x )={Absolute ear thickness (mm) at “ x ” Time Point}−{Absolute ear thickness (mm) at day 0}
 
     “x”: day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 
     Ear thickness change are shown in  FIGS. 24-25 . 
     Photograph &amp; Ear Biopsy Weight 
     Photographs of ear was taken for each animal and presented. Mean ear biopsy weight (4 mm punch biopsy) was calculated and represented in tabulated and graphical form. 
     Percentage Inhibition of Ear Inflammation 
     Percentage inhibition of ear inflammation was calculated for individual animal using the formula:
 
=Control ETC (mm)−Test ETC (mm)/Control ETC (mm)×100%
 
     Inhibition was tabulated and represented in graphical form. 
     Histopathology and Immunohistochemistry 
     Hematoxylin &amp; Eosin dye stained ear histopathological photographs were subjected for epidermal ear thickness measurement using UTHSCSA Image tool, Version 3.0. The software was calibrated by Motic calibrated slide for 10× magnification and ear thickness was selected randomly at four points each for both upper and lower epidermis. Data was calculated as mean and SEM for each group and represented in tabulated and graphical form 
     Myeloperoxidase (MPO) Activity 
     MPO activity was performed by colorimetric method as described by Rajp et al. (2007). Briefly, three punch biopsy ear (4 mm) tissue samples in each group were pooled, weighed and homogenized (100 mg/3 mL) in 50 mM/L phosphate buffer containing 0.5% CTAB. Homogenized samples were sonicated for 10s, freezed and thawed at 20-30° C. for three times, and centrifuged at 12000 rpm and 4° C. for 25 min. 250 μL of supernatant was be mixed with 625 μL of phosphate buffer (50 mmol/1, pH 6) containing 0.167 mg/ml o-dianisidine dihydrochloride and 125 μL hydrogen peroxide (0.0005%). MPO activity was calculated by using the formulae and represented in graphical and tabular form.
 
IU/mL=Δ A ×final volume in cuvette/8.3×volume of sample added (μL)×dilution factor.
 
     Where ΔA is the average of change in absorbance per minute. 
     IU/gm tissue=(IU/mL)/(gm tissue/mL) 
     Serum Nitric Oxide Level 
     Nitric oxide levels was also measured by colorimetric assay using Griess reagent. Briefly, equal volume of serum was mixed with Griess reagent and incubated for 15 min at 37° C. Absorbance was measured at 546 nm and percentage inhibition of nitric oxide level was calculated using the formula:
 
=Control Abs−Test Abs/Control Abs×100%
 
     Inhibition was tabulated and represented in graphical form. 
     Statistical Analysis 
     All data were expressed in Mean±SEM and suitably analyzed by Two way ANOVA and One-way ANOVA followed by Posthoc Bonferroni and Dunett&#39;s test respectively for statistical significance. *P&lt;0.05, **P&lt;0.01 and ***P&lt;0.001 for TPA control Vs vehicle or treated group. 
     Study Deviation 
     The method adopted for MPO estimation was changed as stated in the section on MYELOPEROXIDASE (MPO) ACTIVITY. Statistical analysis was done by Two way and One way ANOVA followed by Posthoc-Bonferroni and Dunnet&#39;s test respectively, as it was appropriate for study data analysis. 
     Results 
     Effect of Treatments on Body Weight Change 
     In the present study, the body weight was recorded daily for 10 consecutive days (i.e day 0 to day 9) in all the experimental groups. No significant changes in body weight were observed in any of the experimental group (Table 3;  FIG. 3 ). % body weight change was also calculated for all the experimental groups. Marginal reduction in % body weight change was observed on oral administration of 3-HX at high dose of 1000 mg/kg and D &amp; E treated animals (Table 4;  FIG. 4 ). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Effect of 3-HX and D &amp; E treatment on Body Weight 
               
               
                 (Unit: gm; Mean ± SEM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Treatment 
                 Day 0 
                 Day 1 
                 Day 2 
                 Day 3 
                 Day 4 
                 Day 5 
                 Day 6 
                 Day 7 
                 Day 8 
                 Day 9 
                 Day 10 
               
               
                   
               
               
                 3-HX (1:4); 
                 27.4 ± 2.7 
                  28.5 ± 2.4 
                  28.2 ± 2.4 
                 27.8 ± 2.4 
                 27.9 ± 2.3 
                 27.8 ± 2.2 
                 27.8 ± 2.3 
                 28.2 ± 2.3 
                 28.3 ± 2.3 
                 28.3 ± 2.3 
                 27.7 ± 2.4 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX (1:8); 
                 27.5 ± 2.0 
                  28.4 ± 1.6 
                  28.0 ± 1.6 
                 28.0 ± 1.6 
                 27.9 ± 1.6 
                 27.8 ± 1.5 
                 28.3 ± 1.6 
                 28.4 ± 1.6 
                 28.9 ± 1.6 
                 28.1 ± 1.4 
                 28.9 ± 1.6 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX 
                 27.6 ± 1.6 
                  27.7 ± 1.6 
                  27.3 ± 1.6 
                 27.4 ± 1.6 
                 27.5 ± 1.6 
                 27.2 ± 1.6 
                 27.3 ± 1.6 
                 27.6 ± 1.6 
                 28.0 ± 1.5 
                 28.1 ± 1.5 
                 28.1 ± 1.4 
               
               
                 (500 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX 
                 27.5 ± 1.7 
                 27.97 ± 1.6 
                  27.6 ± 1.5 
                 27.8 ± 1.6 
                 27.9 ± 1.5 
                 27.8 ± 1.5 
                 27.2 ± 1.8 
                 27.0 ± 1.7 
                 26.4 ± 2.0 
                 26.1 ± 2.2 
                 26.6 ± 2.2 
               
               
                 (1000 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D and E; 
                 27.7 ± 1.6 
                  28.1 ± 1.4 
                  28.2 ± 1.3 
                 28.3 ± 1.3 
                 28.3 ± 1.3 
                 28.3 ± 1.2 
                 28.6 ± 1.2 
                 28.8 ± 1.2 
                 28.9 ± 1.2 
                 29.2 ± 1.2 
                 29.7 ± 1.2 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D and E; 
                 28.0 ± 1.5 
                  26.1 ± 1.3 
                 25.64 ± 1.3 
                 25.9 ± 1.2 
                 26.7 ± 1.2 
                 26.9 ± 1.3 
                 25.9 ± 1.4 
                 25.5 ± 1.5 
                 25.1 ± 1.6 
                 24.8 ± 1.6 
                 24.3 ± 1.7 
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Na-CMC 
                 28.1 ± 1.4 
                  27.5 ± 1.2 
                  27.4 ± 1.1 
                 27.8 ± 1.2 
                 27.5 ± 1.1 
                 27.6 ± 1.0 
                 27.5 ± 0.9 
                 28.3 ± 0.8 
                 28.7 ± 0.8 
                 28.4 ± 0.8 
                 28.8 ± 0.9 
               
               
                   
               
               
                 Each column represents mean ± SEM of n = 6 mice. Data was statistically analyzed by Two way ANOVA followed by Posthoc Bonferrini&#39;s test. No statistical significance was obtained in any of the experimental group. 
               
            
           
         
       
     
       FIG. 3  illustrates effects of 3-HX and D &amp; E treatment on Body weight change. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Effect of 3-HX and D &amp; E treatment on % Body Weight Change 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Treatment 
                 Day-1 
                 Day-2 
                 Day-3 
                 Day-4 
                 Day-5 
                 Day-6 
                 Day-7 
                 Day-8 
                 Day-9 
                 Day-10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 3-HX (1:4); 
                 4.6 
                 3.6 
                 2.3 
                 2.9 
                 2.6 
                 2.6 
                 3.9 
                 4.2 
                 4.1 
                 2.4 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX (1:8); 
                 4.1 
                 2.4 
                 2.4 
                 2.1 
                 1.9 
                 3.8 
                 3.9 
                 5.9 
                 3.2 
                 6.1 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX 
                 0.5 
                 0.5 
                 −0.8 
                 −0.3 
                 −1.3 
                 −1 
                 0 
                 1.7 
                 2.1 
                 2.3 
               
               
                 (500 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3-HX 
                 2 
                 0.8 
                 1.4 
                 1.9 
                 1.5 
                 −1.3 
                 −1.6 
                 −4.6 
                 −5.7 
                 −3.9 
               
               
                 (1000 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D and E; 
                 1.7 
                 1.9 
                 2.5 
                 2.6 
                 2.7 
                 3.6 
                 4.6 
                 5.1 
                 6 
                 7.9 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D and E; 
                 −6.9 
                 −8.4 
                 −7.4 
                 −4.4 
                 −3.6 
                 −7.3 
                 −8.5 
                 −10 
                 −11.3 
                 −13.2 
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Vehicle 
                 −1.8 
                 −2.2 
                 −0.8 
                 −2 
                 −1.6 
                 −1.6 
                 1.2 
                 2.6 
                 1.5 
                 3 
               
               
                   
               
               
                 Each column represents mean ± SEM of n = 6 mice. 
               
            
           
         
       
     
       FIG. 4  illustrates effects of 3-HX and D &amp; E treatment on % Body Weight Change. 
     Effect of Treatments on Absolute Ear Thickness 
     In the present study, the absolute ear thickness was calculated daily for 10 consecutive days (i.e day 0 to day 9) in all the experimental groups (Table 5). Significant increase (P&lt;0.001) in ear thickness was observed in the TPA control (Group7/Right ear) when compared with the vehicle control (Group7/Left ear) in entire treatment schedule. 
     Topical application of 3-HX at both the tested ratios of 1:4 and 1:8 significantly reduced the absolute ear thickness when compared with TPA control. Similarly, topical application of D &amp; E also significantly reduced the absolute ear thickness when compared with TPA control (P&lt;0.001) ( FIG. 5 ). 
     Oral administration of 3-HX at dose of 500 mg/kg and 1000 mg/kg was found to significantly reduce TPA induced absolute ear thickness. D &amp; E treatment also markedly reduced absolute ear thickness on oral administration for 10 days (P&lt;0.001) ( FIG. 6 ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Effect of 3-HX and D &amp; E treatment on Ear Thickness 
               
               
                 (Unit: mm Mean ± SEM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Treatment 
                 Day-0 
                 Day-1 
                 Day-2 
                 Day-3 
                 Day-4 
                 Day-5 
                 Day-6 
                 Day-7 
                 Day-8 
                 Day-9 
               
               
                   
               
               
                 3HX, 1:4 
                  0.3 ± 0.01 
                 0.54 ± 
                 0.54 ± 
                 0.72 ± 
                 0.78 ± 
                 0.68 ± 
                 0.75 ± 
                 0.68 ± 
                 0.58 ± 
                 0.57 ± 
               
               
                 Topical 
                   
                 0.04*** 
                 0.04*** 
                 0.02*** 
                 0.05*** 
                 0.04*** 
                 0.06** 
                 0.05*** 
                 0.03*** 
                 0.04*** 
               
               
                 3HX, 1:8 
                 0.31 ± 0 
                 0.59 ± 
                 0.54 ± 
                  0.7 ± 
                 0.87 ± 
                 0.67 ± 
                 0.73 ± 
                 0.67 ± 
                 0.63 ± 
                 0.52 ± 
               
               
                 Topical 
                   
                 0.04*** 
                 0.03*** 
                 0.01*** 
                 0.03** 
                 0.02*** 
                 0.05*** 
                 0.07*** 
                 0.04*** 
                 0.02*** 
               
               
                 3HX 
                 0.31 ± 0 
                 0.56 ± 
                 0.55 ± 
                 0.67 ± 
                 0.81 ± 
                 0.72 ± 
                 0.77 ± 
                 0.7 ± 
                 0.68 ± 
                 0.67 ± 
               
               
                 (500 mg/k g); 
                   
                 0.05*** 
                 0.04*** 
                 0.04*** 
                 0.04*** 
                 0.03*** 
                 0.05 
                 0.04*** 
                 0.03 
                 0.03 
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3HX 
                 0.31 ± 0 
                 0.64 ± 
                 0.61 ± 
                 0.75 ± 
                 0.92 ± 
                 0.73 ± 
                 0.74 ± 
                 0.68 ± 
                 0.71 ± 
                  0.8 ± 
               
               
                 (1000 mg/ kg); 
                   
                 0.03 
                 0.02*** 
                 0.04*** 
                 0.03 
                 0.02*** 
                 0.03** 
                 0.02*** 
                 0.02 
                 0.04** 
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D&amp;E, 
                 0.31 ± 0 
                 0.56 ± 
                 0.64 ± 
                 0.66 ± 
                 0.82 ± 
                  0.8 ± 
                 0.67 ± 
                 0.51 ± 
                 0.52 ± 
                 0.49 ± 
               
               
                 Topical 
                   
                 0.03*** 
                 0.05*** 
                 0.04*** 
                 0.01*** 
                 0.03*** 
                 0.03*** 
                 0.02*** 
                 0.02*** 
                 0.02*** 
               
               
                 D&amp;E, Oral 
                  0.3 ± 0 
                 0.66 ± 
                 0.64 ± 
                 0.68 ± 
                  0.8 ± 
                 0.79 ± 
                 0.85 ± 
                 0.72 ± 
                 0.82 ± 
                 0.69 ± 
               
               
                   
                   
                 0.03 
                 0.04*** 
                 0.05*** 
                 0.05*** 
                 0.05*** 
                 0.06 
                 0.08** 
                 0.16** 
                 0.13 
               
               
                 TPA 
                  0.3 ± 0 
                  0.7 ± 
                 0.88 ± 
                 0.91 ± 
                 0.95 ± 
                  0.9 ± 
                 0.83 ± 
                  0.8 ± 
                 0.73 ± 
                 0.72 ± 
               
               
                 Control 
                   
                 0.03*** 
                 0.01*** 
                 0.02*** 
                 0.04*** 
                 0.02*** 
                 0.02*** 
                 0.03*** 
                 0.03*** 
                 0.05*** 
               
               
                 Vehicle 
                  0.3 ± 0 
                 0.31 ± 0 
                 0.32 ± 0 
                 0.32 ± 0 
                 0.33 ± 0 
                 0.34 ± 0.01 
                 0.34 ± 0.01 
                 0.35 ± 0.01 
                 0.33 ± 0.1 
                 0.33 ± 0.1 
               
               
                 Control 
               
               
                   
               
               
                 Each column represents mean ± SEM of n = 6 mice. Data was statistically analyzed by Two way ANOVA followed by Posthoc Bonferroni test. ***P &lt; 0.001, **P &lt; 0.01 and *P &lt; 0.05 for vehicle control Vs TPA control; Treatment Vs TPA control. 
               
            
           
         
       
     
       FIG. 5  illustrates effects of topical application of 3-HX and D &amp; E on Absolute Ear Thickness in TPA induced mouse. 
       FIG. 6  illustrates effects of oral application of 3-HX and D &amp; E on Absolute Ear Thickness in TPA induced mouse. 
     Effect of Treatment on Ear Thickness Change 
     The change in ear thickness was also calculated from the absolute ear thickness for all the experimental groups to find the edema caused by inflammation. In the TPA control group significant elevation in ear thickness was observed when compared with vehicle (Solvent) control (P&lt;0.001) (Table 6). 
     Topical application of 3-HX at the tested ratios significantly decreased the ear edema up to Day 5 in comparison with TPA control (P&lt;0.001). However, from Day 6 onwards marginal decrease in ear thickness change was observed. Similarly, significant decrease in ear thickness change was also observed on D &amp; E topical application for entire treatment schedule except for Day 5 and Day 6 (P&lt;0.001) ( FIG. 7 ). 
     Oral application of 3-HX at the two tested concentration significantly decreased the ear edema up to Day 5 in comparison with TPA control (P&lt;0.001). However, from Day 6 onwards marginal decrease in ear thickness change was observed. Similarly, significant decrease in ear thickness change was also observed on oral administration of D &amp; E up to Day 3 only when compared with TPA control (P&lt;0.001) However, marginal decrease in ear thickness change was observed on further D &amp; E treatment ( FIG. 8 ). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Effect of 3-HX and D &amp; E treatment on Ear Thickness Change 
               
               
                 (Unit: mm; Mean ± SEM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Treatment 
                 Day-1 
                 Day-2 
                 Day-3 
                 Day-4 
                 Day-5 
                 Day-6 
                 Day-7 
                 Day-8 
                 Day-9 
               
               
                   
               
               
                 3HX, 1:4 
                 0.24 ± 
                 0.24 ± 
                 0.42 ± 
                 0.48 ± 
                 0.38 ± 
                 0.45 ± 
                 0.42 ± 
                 0.28 ± 
                 0.27 ± 
               
               
                 Topical 
                 0.01 
                 0.04*** 
                 0.04* 
                 0.02 
                 0.05** 
                 0.04 
                 0.06  
                 0.05 
                 0.03 
               
               
                 3HX, 1:8 
                 0.28 ± 
                 0.23 ± 
                 0.39 ± 
                 0.56 ± 
                 0.36 ± 
                 0.42 ± 
                 0.37 ± 
                 0.33 ± 
                 0.22 ± 
               
               
                 Topical 
                 0.00 
                 0.04*** 
                 0.03** 
                 0.01 
                 0.03** 
                 0.02 
                 0.05 
                 0.07 
                 0.04* 
               
               
                 3HX, 
                 0.26 ± 
                 0.25 ± 
                 0.37 ± 
                 0.51 ± 
                 0.41 ± 
                 0.47 ± 
                 0.39 ± 
                 0.38 ± 
                 0.36 ± 
               
               
                 500 mpk, Oral 
                 0.00 
                 0.05*** 
                 0.04*** 
                 0.04 
                 0.04* 
                 0.03 
                 0.05 
                 0.04 
                 0.03 
               
               
                 3HX, 
                 0.32 ± 
                  0.3 ±  
                 0.44 ± 
                  0.6 ± 
                 0.42 ± 
                 0.43 ± 
                 0.37 ± 
                  0.4 ± 
                 0.49 ± 
               
               
                 1000 mpk, Oral 
                 0.00 
                 0.03*** 
                 0.02* 
                 0.04 
                 0.03* 
                 0.02 
                 0.03 
                 0.02 
                 0.02 
               
               
                 D&amp;E, Topical 
                 0.25 ± 
                 0.33 ± 
                 0.35 ± 
                 0.51 ± 
                 0.49 ± 
                 0.36 ± 
                  0.2 ± 
                 0.22 ± 
                 0.18 ± 
               
               
                   
                 0.00 
                 0.03*** 
                 0.05*** 
                 0.04 
                 0.01 
                 0.03* 
                 0.03*** 
                 0.02** 
                 0.02*** 
               
               
                 D&amp;E, Oral 
                 0.35 ± 
                 0.33 ± 
                 0.37 ± 
                  0.5 ± 
                 0.48 ± 
                 0.55 ± 
                 0.42 ± 
                 0.52 ± 
                 0.39 ± 
               
               
                   
                 0.00 
                 0.03*** 
                 0.04*** 
                 0.05 
                 0.05 
                 0.05  
                 0.06 
                 0.08 
                 0.16 
               
               
                 TPA Control 
                 0.39 ± 
                 0.58 ± 
                 0.61 ± 
                 0.64 ± 
                 0.59 ± 
                 0.53 ± 
                  0.5 ± 
                 0.43 ± 
                 0.42 ± 
               
               
                   
                 0.00*** 
                 0.03*** 
                 0.01*** 
                 0.02*** 
                 0.04*** 
                 0.02*** 
                 0.02***  
                 0.03*** 
                 0.03*** 
               
               
                 Vehicle 
                 0.01 ± 0 
                 0.02 ± 0 
                 0.02 ± 0 
                 0.03 ± 0 
                 0.04 ± 
                 0.04 ± 
                 0.04 ± 
                 0.03 ± 
                 0.03 ± 
               
               
                 Control 
                   
                   
                   
                   
                 0.00 
                 0.01 
                 0.01 
                 0.01 
                 0.01 
               
               
                   
               
               
                 Each column represents mean ± SEM of n = 6 mice. Data was statistically analyzed by Two way ANOVA followed by Posthoc Bonferroni test. ***P &lt; 0.001,**P &lt; 0.01 and *P &lt; 0.05 for vehicle control Vs TPA control; and for treatment Vs TPA control. 
               
            
           
         
       
     
       FIG. 7  illustrates effects of oral application of 3-HX and D &amp; E on Ear Thickness Change (Effect of 3-HX and D &amp; E oral treatment on thickness change in TPA induced mouse ear). 
       FIG. 8  illustrates effects of oral application of 3-HX and D &amp; E on Ear Thickness Change (Effect of 3-HX and D &amp; E oral treatment on thickness change in TPA induced mouse ear). 
     Effects of Treatments on Ear Punch Biopsy Weight 
     On Day 10, all the experimental animals were euthanized and a standard 4 mm ear punch biopsy was collected and weighed. Significant increase in biopsy weight was observed in TPA control (P&lt;0.01) when compared with vehicle control as depicted in Table 7. Ear punch biopsy weight of 3-HX (1:4; topical) treated mice was found to be significantly reduced in comparison to TPA control (P&lt;0.05). However, marginal reduction in ear punch biopsy weight was observed on topical treatment of 3-HX at the ratio of 1:8. Based upon punch biopsy weight, the % activity of 3-HX at both the tested ratios of 1:4 and 1:8 were calculated and was found to be 29.34% and 17.58% respectively. Significant reduction in punch biopsy weight was also observed in D &amp; E topical treatment group (P&lt;0.05) when compared with TPA control. Topical treatment with D &amp; E exhibited 31.40% activity against TPA induced inflammation. 
     A dose-dependent reduction in ear biopsy weight was observed in 3-HX oral administration when compared with TPA control ( FIG. 9 ). 3-HX at concentration of 500 mg/kg and 1000 mg/kg exhibited 11.40% and 14.90% activity respectively; however no statistical difference was obtained among the 3-HX oral treated and TPA control group. D &amp; E oral administration also reduced ear punch biopsy thickness when compared with TPA control and exhibited 21.93% activity. However, no statistical difference was obtained among the D &amp; E treated animals and TPA control group. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Effect of 3-HX and D &amp; E treatment on Ear Punch Biopsy Weight 
               
               
                 (Unit: mg; Mean ± SEM) 
               
            
           
           
               
               
               
            
               
                   
                   
                 Biopsy Weight 
               
               
                   
                 Treatment 
                 (mg) 
               
               
                   
                   
               
               
                   
                 3-HX (1:4); Topical 
                  7.25 ± 0.33* 
               
               
                   
                 3-HX (1:8); Topical 
                 8.46 ± 1.05 
               
               
                   
                 3HX-500 mg/kg; Oral 
                 9.09 ± 1.14 
               
               
                   
                 3HX-1000 mg/kg; Oral 
                 8.73 ± 0.34 
               
               
                   
                 D &amp; E; Topical 
                  7.04 ± 0.29* 
               
               
                   
                 D &amp; E; Oral 
                 8.01 ± 0.58 
               
               
                   
                 TPA control 
                  10.26 ± 0.82** 
               
               
                   
                 Vehicle control 
                 3.08 ± 0.21 
               
               
                   
                   
               
               
                   
                 Each point represents the mean ± SEM of n = 6 mice per group. 
               
               
                   
                 **P &lt; 0.01 for Vehicle Control Vs TPA control and 
               
               
                   
                 *P &lt; 0.05 for treatment Vs TPA control. 
               
            
           
         
       
     
       FIG. 9  illustrates effects of 3-HX and D &amp; E treatment on Ear Punch Biopsy Weight (mg). 
     Photographs of Mice Ears 
     Photographs of TPA induced inflammation in mice ear were represented for all the experimental groups which demonstrated the gross effect of test items. 
       FIGS. 10A-10G  illustrates Photographs of ear, including: 
     Group 1: 3-HX (1:4); Topical: G1A1-G1A6 
     Group 2: 3-HX (1:8); Topical: G2A1-G2A6 
     Group 3: 3-HX (500 mg/kg); Oral: G3A1-G3A6 
     Group 4: 3-HX (1000 mg/kg); Oral: G4A1-G4A6 
     Group 5: D &amp; E; Topical: G5A1-G5A6 
     Group 6: D &amp; E; Oral: G6A1-G6A6 
     Group 7: TPA Control (Right Ear) &amp; Vehicle Control (Left Ear): G7A1-G7A6 
     Note: G denotes Group Number and A denotes Animal number 
     Percentage Inhibition 
     In order to calculate percentage inhibition of ear inflammation on test item treatment, the basal ear thickness change represented by vehicle control was subtracted from each group. The percentage inhibition of ear inflammation by test items was calculated as tabulated in table 8. 
       FIG. 11  depicts the % inhibition of ear inflammation on topical application of 3-HX and D &amp; E. 3-HX at the ratio of 1:4 exerted 35.96% activity on Day 8 which sustained up to Day 9. 3-HX at the ratio of 1:8 exerted maximum inhibition activity of 52.44% on Day 9 treatment. Similarly, topical application of D &amp; E exerted 64.94% inhibitory activity from Day 7 which sustained up to Day 9 treatment. 
       FIG. 12  depicted the % inhibition of ear inflammation on oral administration of 3-HX and D &amp; E. 3-HX at both the doses of 500 mg/kg and 1000 mg/kg exhibited approximately 32% inhibitory activity on Day 5 which subsequently got decreased on further treatment up to Day 9. Similarly, D &amp; E oral administration exerted marginal inhibitory activity against TPA induced inflammation. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 % Inhibitory Activity of Ear Inflammation 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Treatment 
                 Day-1 
                 Day-2 
                 Day-3 
                 Day-4 
                 Day-5 
                 Day-6 
                 Day-7 
                 Day-8 
                 Day-9 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 3HX (1:4); 
                 39.86 
                 61.42 
                 32.25 
                 27.16 
                 38.41 
                 16.90 
                 17.34 
                 35.96 
                 37.68 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3HX (1:8); 
                 28.99 
                 62.02 
                 37.01 
                 13.44 
                 41.63 
                 21.72 
                 28.54 
                 24.72 
                 52.44 
               
               
                 Topical 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3HX  
                 35.36 
                 59.55 
                 40.91 
                 22.25 
                 32.46 
                 12.41 
                 22.76 
                 12.92 
                 15.19 
               
               
                 (500 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3HX 
                 17.68 
                 50.94 
                 28.35 
                 6.45 
                 32.16 
                 21.03 
                 28.17 
                 6.60 
                 −17.05 
               
               
                 (1000 mg/kg); 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Oral 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 D&amp;E; Topical 
                 36.81 
                 44.91 
                 43.10 
                 21.44 
                 18.75 
                 34.02 
                 64.94 
                 52.81 
                 60.74 
               
               
                 D&amp;E; Oral 
                 9.71 
                 44.21 
                 39.96 
                 23.43 
                 19.76 
                 −3.91 
                 17.47 
                 −24.02 
                 8.74 
               
               
                   
               
            
           
         
       
     
       FIG. 11  shows % Inhibition of Ear Inflammation on topical treatment (% Inhibitory activity of 3-HX and D &amp; E on topical application). 
       FIG. 12  shows % Inhibition of Ear Inflammation on oral treatment (% Inhibitory activity of 3-HX and D &amp; E on oral application). 
     Histopathological and Immunohistochemistry Findings 
     The H&amp;E-stained ear sections of all the experimental groups are represented in  FIG. 13 . Edema and inflammatory cell infiltration are scored as nil (−), mild (+), moderate (++) and severe (+++). Moreover, the epidermal thickness was also calculated for all the experimental groups. Repeated TPA application resulted in a marked increase in ear thickness with almost nil hyperkeratosis and moderate edema. Moreover, severe inflammatory cell infiltration in the dermis was also observed. A significant increase in epidermal ear thickness (P&lt;0.01) was also observed in TPA control group when compared with vehicle control (Table 9). 
     Topical administration of 3-HX at the ratio of 1:4 showed almost nil hyperkeratosis and moderate edema. However, severe inflammatory cell infiltration was observed upon 10 days topical application. Similarly, topical administration of 3-HX at the ratio of 1:8 showed mild hyperkeratosis and edema with severe inflammatory cell infiltration. D &amp; E topical application for 10 consecutive days resulted into moderate hyperkeratosis with moderate edema and infiltration of inflammatory cells. The epidermal thickness was also observed to be significantly reduced on 3-HX topical treatment at both the ratios when compared with TPA control (P&lt;0.05). However, on D &amp; E topical application no significant results were obtained when compared with TPA control ( FIG. 14 ). 
     Oral administration of 3-HX at the low dose of 500 mg/kg resulted into mild hyperkeratosis and edema with moderate to severe infiltration of inflammatory cells. 3-HX at the high dose of 1000 mg/kg resulted into mild to moderate hyperkeratosis and edema with severe infiltration of inflammatory cells. Similarly, oral administration of D &amp; E leads to mild hyperkeratosis with moderate edema and severe inflammatory cell infiltration. The epidermal ear thickness was also found to be significantly reduced on 3-HX treatment at higher dose of 1000 mg/kg alone (P&lt;0.05) ( FIG. 15 ). However, in other oral treatment groups no significant differences were obtained in comparison with TPA control. 
     FIGS.  30   a - 30   h : Histopathological Findings 
     Group 1: 3-HX (1:4); Topical. Photographs of H&amp;E-stained mouse ear cross-sections of 3-HX (1:4; topical) treated mice in the TPA inflammation model. Reduction in hyperkeratosis and edema was observed (10× magnification). 
     Group 2: 3-HX (1:8); Topical. Photographs of H&amp;E-stained mouse ear cross-sections of 3-HX (1:8; topical) treated mice in the TPA inflammation model. Reduction in hyperkeratosis and edema was observed (10× magnification). 
     Group 3: 3-HX (500 mg/kg); Oral. Photographs of H&amp;E-stained mouse ear cross-sections of 3-HX (500 mg/kg; oral) treated mice in the TPA inflammation model. Reduction in hyperkeratosis and edema was observed (10× magnification). 
     Group 4: 3-HX (1000 mg/kg); Oral. Photographs of H&amp;E-stained mouse ear cross-sections of 3-HX (1000 mg/kg; oral) treated mice in the TPA inflammation model. Reduction in hyperkeratosis and edema was observed (10× magnification). 
     Group 5: D &amp; E; Topical. Photographs of H&amp;E-stained mouse ear cross-sections of D &amp; E topically treated mice in the TPA inflammation model. Reduction in hyperkeratosis, edema and inflammatory cell infiltration was observed (10× magnification). 
     Group 6: D &amp; E; Oral. Photographs of H&amp;E-stained mouse ear cross-sections of D &amp; E orally treated mice in the TPA inflammation model. Reduction in hyperkeratosis and edema was observed (10× magnification). 
     Group 7: TPA Control (Right Ear). Photographs of H&amp;E-stained mouse ear cross-sections in the TPA induced inflammation model. Severe infiltration of inflammatory cells with moderate edema in ears was observed (10× magnification). 
     Group 7: Vehicle control (Left Ear). Photographs of H&amp;E-stained mouse ear cross-sections of vehicle control group (10× magnification). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Effect of Test items Treatment on Epidermal ear thickness 
               
               
                 (Unit: μm; Mean ± SEM) 
               
            
           
           
               
               
               
            
               
                   
                 Treatment 
                 Epidermal Ear Thickness 
               
               
                   
                   
               
               
                   
                 Vehicle Control 
                 12.15 ± 3.08 
               
               
                   
                 TPA Control 
                  54.42 ± 7.76** 
               
               
                   
                 3-HX (1:4); Topical 
                  36.41 ± 5.16* 
               
               
                   
                 3-HX (1:8); Topical 
                  34.06 ± 2.46* 
               
               
                   
                 3HX-500 mg/kg; Oral 
                 43.26 ± 3.36 
               
               
                   
                 3HX-1000 mg/kg; Oral 
                  34.49 ± 3.81* 
               
               
                   
                 D &amp; E; Topical 
                 37.88 ± 4.85 
               
               
                   
                 D &amp; E; Oral 
                 37.48 ± 3.11 
               
               
                   
                   
               
               
                   
                 **P &lt; 0.01 for vehicle control compared to TPA Control; 
               
               
                   
                 *P &lt; 0.05 for TPA Control compared to 3-HX treatment group. 
               
            
           
         
       
     
       FIG. 14  illustrates effects of topical application of 3-HX and D &amp; E on epidermal ear thickness. 
       FIG. 15  illustrates effects of oral treatment of 3-HX and D&amp;E on epidermal ear thickness. 
     Myeloperoxidase (MPO) Activity 
     Table 10 illustrated the effect of test items over enzyme myeloperoxidase, released by neutrophils and macrophages during inflammation process. In the present study, the enzyme activity was found to be markedly enhanced in TPA control group when compared with vehicle control. Topical application of 3-HX at both the ratios profoundly inhibited the MPO activity on 10 days of consecutive application in comparison with TPA control. Topical application of standard D &amp; E showed maximum inhibition of MPO activity among all the topical treatment groups ( FIG. 16 ). 
     Oral administration of 3-HX showed dose-dependent inhibition of enzyme MPO against TPA induced mice ear inflammation. D &amp; E oral treatment also observed to inhibit the MPO levels when compared with TPA control ( FIG. 17 ). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Effect of Test items Treatment on MPO activity in TPA induced 
               
               
                 mice ear 
               
               
                 (Unit: μm; Mean ± SEM) 
               
            
           
           
               
               
            
               
                 Treatment 
                 IU/gm tissue (Mean ± SEM) 
               
               
                   
               
               
                 Vehicle Control 
                 0.094 ± 0.052 
               
               
                 TPA Control 
                 0.487 ± 0.184 
               
               
                 3-HX (1:4); Topical 
                 0.238 ± 0.044 
               
               
                 3-HX (1:8); Topical 
                 0.244 ± 0.037 
               
               
                 3HX-500 mg/kg; Oral 
                 0.380 ± 0.051 
               
               
                 3HX-1000 mg/kg; Oral 
                 0.339 ± 0.038 
               
               
                 D &amp; E; Topical 
                 0.196 ± 0.046 
               
               
                 D &amp; E; Oral 
                 0.258 ± 0.068 
               
               
                   
               
            
           
         
       
     
       FIG. 16  illustrates effects of topical application of 3-HX and D &amp; E on MPO activity in TPA induced mice ear. 
       FIG. 17  illustrates effects of oral administration of 3-HX and D&amp;E on MPO activity in TPA induced mice ear. 
     Nitric Oxide (NO) Activity 
     Table 11 illustrated the % inhibition of nitric oxide levels in serum of TPA induced inflammation on treatment of test items. Topical application of 3-HX at both the ratios exerted poor inhibitory effect over nitric oxide levels on 10 days of consecutive application. Similarly, topical application of standard D &amp; E also exhibited poor inhibitory response over serum NO levels. 
     Oral administration of 3-HX at the dose of 500 mg/kg and 1000 mg/kg exhibited 32.53% and 30.62% inhibitory activity respectively over serum nitric oxide content against TPA induced inflammation. D &amp; E oral treatment also observed to exert 25.10% inhibitory activity ( FIG. 18 ). 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Effect of Test items Treatment on % Inhibition of Nitric oxide  
               
               
                 level in serum of TPA induced inflammation 
               
            
           
           
               
               
               
            
               
                   
                 Treatment 
                 % Inhibition 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 3-HX (1:4); Topical (n = 6) 
                 5.35 
               
               
                   
                 3-HX (1:8); Topical (n = 3) 
                 7.10 
               
               
                   
                 3-HX (500 mg); Oral (n = 4) 
                 32.53 
               
               
                   
                 3-HX (1000 mg); Oral (n = 4) 
                 30.62 
               
               
                   
                 D &amp; E; Topical (n = 4) 
                 6.02 
               
               
                   
                 D &amp; E; Oral (n = 4) 
                 25.10 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 n represents plasma samples size analyzed for NO activity 
               
            
           
         
       
     
       FIG. 18  shows % Inhibition of serum Nitric oxide content on 3-HX and D&amp;E treatment (Effect of 3-HX and D&amp;E on serum nitric oxide level). 
     These examples demonstrate anti-psoriatic potential of 3-HX and D &amp; E on both topical and oral treatment in TPA induced ear inflammation in C57BL/6 mice. The results demonstrated no significant changes in body weight among all the experimental groups. However, in % body weight change, a marginal reduction was observed on oral administration of D &amp; E and high dose of 3-HX, although both ranged within the tolerable range. 
     In the present study results marked induction of ear inflammation on repeated TPA application was observed as indicated by significant increase in ear thickness. Topical application of TPA is reported to induce cutaneous inflammation and epidermal hyperplasia (Clark et al., 1985). The TPA application resulted in a series of events of numerous cellular, biochemical, and molecular changes that eventually lead to the pathological alterations of the mouse skin (Kensler et al., 1987; Nakamura et al., 1998, 2000). Among the topically treated groups, high concentration of 3-HX (1:4) and D&amp;E was found to exert maximum anti-inflammatory activity as indicated by significant reduction in ear thickness. Among the orally administered experimental groups, low dose of 3-HX (500 mg/kg) exhibited maximum reduction in ear thickness followed by D &amp; E and high dose of 3-HX (1000 mg/kg). 
     The effect of topical and oral treatment of test items was also examined over TPA-induced ear thickness change which also represents inflammatory edema. Topical application of 3-HX (1:4) exerted maximum reduction in ear thickness change followed by D &amp; E and low concentration of 3-HX (1:8). Among the orally administered groups, low dose of 3-HX (500 mg/kg) rendered maximum reduction in ear thickness change followed by D &amp; E and high dose of 3-HX (1000 mg/kg). The overall % inhibitory activity of test items based upon the ear thickness and ear thickness change demonstrated that topical application of D &amp; E resulted in maximum inhibition of ear thickness followed by 3-HX (1:8) and 3-HX (1:4). On the other side, oral application of all the test items exerted marginal inhibitory activity against TPA induced ear inflammation in mice. 
     The effect of test items was further evaluated by measuring the standard 4 mm punch biopsy weight after completion of treatment schedule. 10 consecutive day TPA application resulted in profound increase in punch biopsy weight of mice ear suggesting the chronic inflammatory response. Topical application of D &amp; E exerted maximum reduction in ear punch biopsy weight followed by 3-HX (1:4) and 3-HX (1:8). Oral administration of 3-HX and D &amp; E resulted in marginal reduction in ear punch biopsy weight suggesting the beneficial effect of test items on topical application in comparison with oral treatment. 
     To further explore the anti-psoriatic potential of test items, the epidermal ear thickness was evaluated in histopathological photographs. The histopathogical findings were in accordance with punch biopsy weight results where topical application of test items was found to exert better effect against TPA induced increase in epidermal ear thickness ear in comparison with orally treated experimental groups. Moreover, marked reduction in hyperkeratosis, ear edema and inflammatory cell infiltration was also observed among all the treatment groups when compared with TPA control. 
     In the present study, the effect of test items was also investigated over inflammatory biomarkers viz., enzyme myeloperoxidase and nitric oxide in TPA model of inflammation. In the present study marked increase in MPO activity was observed in TPA control mice ear homogenate which is in accordance with the previous reports (Lee et al. 2009). MPO is commonly used as an index of granulocyte infiltration and the enzyme inhibition is indicative of anti-inflammatory activity in the chronic inflammation model (Ajuebor et al. 2000). Topical application of D &amp; E resulted in maximum suppression of enzyme activity followed by 3-HX (1:4) and 3-HX (1:8). Among the orally treated experimental groups, D &amp; E exerted marked suppression of enzyme activity whereas 3-HX exhibited marginal activity. 
     Similarly, marked increase in nitric oxide levels were observed in TPA control group. In the present study, oral administration of test items partially inhibited the generation of nitric oxide content against TPA induced mice inflammation. 
     The embodiments have been described as demonstrated anti-psoriatic potential of 3-HX and D &amp; E on both topical and oral application in TPA-induced ear inflammation model using C57BL/6 mice. Based upon the present findings, it is suggested that topical application of D &amp; E and 3-HX may act as potential therapeutic intervention for the treatment of inflammatory skin diseases like eczema through inhibition of myeloperoxidase activity. Future investigations are recommended in-order to find out the mechanism of action which is required for the development of novel therapeutics for the treatment of eczema. 
     The following are incorporated by reference:
     Rajp A, Adu D and Savage C O (2007). Meta-analysis of myeloperoxidase G-463/A polymorphism in anti-neutrophil cytoplasmic autoantibody-positive vasculitis. Clinical and Experimental Immunology. 149: 251-256.   Clark S D, Wilhelm S M, Stricklin G P, Welgus H G (1985) Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetatein human skin fibroblasts. Arch. Biochem. Biophys. 241, 36-44.   Kensler T W, Egner P A, Moore K G, Taffe B G, Twerdok L E, Trush M A (1987) Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin. Toxicol. Appl. Pharmacol. 90, 337-346.   Nakamura Y, Murakami A, Ohto Y, Torikai K, Tanaka T, Ohigashi H (1998) Suppression of tumor promoter induced oxidative stress and inflammatory responses in mouse skin by superoxide generation inhibitor 10-acetoxychavicol acetate. Cancer Res. 58, 4832-4839.   Nakamura Y, Torikai K T, Ohto Y, Murakami A, Tanaka T, Ohigashi H (2000) A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose- and timing-dependent enhancement and involvement of bioactivation by tyrosinase. Carcinogenesis 21, 1899-1907.   Lee D Y, Choi G, Yoon T, Cheon M S, Choo B K, Kim H K (2009) Anti-inflammatory activity of  Chrysanthemum indicum  extract in acute and chronic cutaneous inflammation. Journal of Ethnopharmacology 123:149-154.   Ajuebor M N, Singh A, Wallace J L (2000) Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis. American Journal of Physiology Gastrointestinal and Liver Physiology 279, G238-G244.   

     List of Tables 
     
       
         
           
               
               
             
               
                   
               
               
                 Table No. 
                 Heading/Title 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 1. 
                 Oral purgative ED 50  values of the anthraquinones. 
               
               
                 2. 
                 Allocation of animals 
               
               
                 3. 
                 Effect of 3-HX and D &amp; E treatment on Body Weight 
               
               
                 4. 
                 Effect of 3-HX and D &amp; E treatment on % Body  
               
               
                   
                 Weight Change 
               
               
                 5. 
                 Effect of 3-HX and D &amp; E treatment on Ear Thickness 
               
               
                 6. 
                 Effect of 3-HX and D &amp; E treatment on Ear Thickness  
               
               
                   
                 Change 
               
               
                 7. 
                 Effect of 3-HX and D &amp; E treatment on Ear Punch  
               
               
                   
                 Biopsy Weight 
               
               
                 8. 
                 % Inhibitory Activity of Ear Inflammation 
               
               
                 9. 
                 Effect of Test items Treatment on Epidermal ear thickness 
               
               
                 10. 
                 Effect of Test items Treatment on MPO activity in  
               
               
                   
                 TPA induced mice ear 
               
               
                 11. 
                 Effect of Test items Treatment on % Inhibition of Nitric  
               
               
                   
                 oxide level in serum of TPA induced inflammation 
               
               
                   
               
            
           
         
       
     
     Abbreviations Used 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Abbreviation 
                 Explanation 
               
               
                   
                   
               
             
            
               
                   
                 ° C. 
                 Degree Centigrade 
               
               
                   
                 % 
                 Percentage 
               
               
                   
                 μl 
                 Microliter 
               
               
                   
                 μm 
                 Micrometer 
               
               
                   
                 ANOVA 
                 Analysis of variance 
               
               
                   
                 CPCSEA 
                 Committee for the Purpose of Control and  
               
               
                   
                   
                 Supervision of Experiments on Animals 
               
               
                   
                 DMSO 
                 Dimethylsulfoxide 
               
               
                   
                 DRF 
                 Dabur Research Foundation 
               
               
                   
                 ETC 
                 Ear Thickness Change 
               
               
                   
                 M 
                 Molar 
               
               
                   
                 mg 
                 milligram 
               
               
                   
                 ml 
                 milliliter 
               
               
                   
                 ml/kg 
                 milliliter/ kilogram 
               
               
                   
                 mm 
                 millimeter 
               
               
                   
                 mM 
                 millimolar 
               
               
                   
                 SEM 
                 Standard Error Mean 
               
               
                   
                 SD 
                 Standard Deviation 
               
               
                   
                 TPA 
                 12-O-Tetradecanoylphorbol-13-Acetate 
               
               
                   
                 IHC 
                 Immunohistochemistry 
               
               
                   
                 IU 
                 International Unit 
               
               
                   
                   
               
            
           
         
       
     
       FIG. 20  illustrates the effect on live cells of three combinations of herbs each in three concentrations 1:40, 1:20 and 1:10. C1 includes Sheng Di Huang, C2 includes Jin Yin Hua, Da Huang, Mu Dan Pi and Di Gu Pi, and C3 includes Xian He Cao and Chun Gen Pi.  FIG. 20  illustrates that each of these three example combinations provides a reduction of the live cell numbers particularly in higher concentrations, while C1 and C2 appear to be more effective than C3. Each of C1 and C2 includes one or two of the three herbs Sheng Di Huang, Jin Yin Hua and Da Huang, while C3 does not. In addition, the combination of four herbs in C2 appears to be more effective than the single herb in C1. 
       FIGS. 21-23  illustrate plots for each of the eighteen herbs of growth as a percentage of control versus dilution factor.  FIGS. 21-23  illustrate that the three herbs Sheng Di Huang, Da Huang and Jin Yin Hua are most effective, while the four herbs Mu Dan Pi, Di Gu Pi, Xian He Cao and Chun Gen Pi are effective, and the eleven herbs Zi Cao, Xuan Shen, Shi Gao, Bai Shao, Chi Shao, Hong Hua, Da Qing Ye, Qing Dai, Bai Zhu, Shi Wei and Rou Gui are somewhat less effective. 
       FIG. 24  illustrates the effect on live cells of five combinations of herbs each in two concentrations 1:40 and 1:80. Ca1 includes a combination of all eighteen herbs, C2 includes a combination of Jin Yin Hua, Da Huang, Mu Dan Pi and Di Gu Pi, C3 includes a combination of Da Huang, Sheng Di Huang and Jin Yin Hua, C4 and C5 include combinations of the other eleven herbs (Zi Cao, Xuan Shen, Shi Gao, Bai Shao, Chi Shao, Hong Hua, Da Qing Ye, Qing Dai, Bai Zhu, Shi Wei and Rou Gui) of the eighteen herbs, i.e., not in combination with any of the seven herbs (Sheng Di Huang, Da Huang, Jin Yin Hua, Mu Dan Pi, Di Gu Pi, Xian He Cao and Chun Gen Pi). Specifically, C4 includes Shi Gao, Shi Wei, Bai Zhu, Rou Gui, Hong Hua and C5 includes Zi Cao, Xuan Shen, Chi Shao, Bai Shao, Da Qin Ye, and Qing Dai.  FIG. 24  illustrates that combinations of all eighteen herbs, as well as combinations of the three herbs (Sheng Di Huang, Da Huang and Jin Yin Hua) are most effective, while the combination C2 was less effective, and the combinations C4 and C5 were still less effective. 
     It is contemplated, as people with ordinary skill in the art would do, that the newly separated compounds may be each individually or in combination used as an ingredient to prepare a pharmaceutical composition for a particular treatment purpose. As it is the status of the art in the pharmaceutical industry, once substantially pure preparations of a compound are obtained, various pharmaceutical compositions or formulations can be prepared from the substantially pure compound using conventional processes or future developed processes in the industry. Specific processes of making pharmaceutical formulations and dosage forms (including, but not limited to, tablet, capsule, injection, syrup) from chemical compounds are not part of the invention and people of ordinary skill in the art of the pharmaceutical industry are capable of applying one or more processes established in the industry to the practice of the present invention. Alternatively, people of ordinary skill in the art may modify the existing conventional processes to better suit the compounds of the present invention. For example, the patent or patent application databases provided at USPTO official website contain rich resources concerning making pharmaceutical formulations and products from effective chemical compounds. Another useful source of information is Handbook of Pharmaceutical Manufacturing Formulations, edited by Sarfaraz K. Niazi and sold by Culinary &amp; Hospitality Industry Publications Services, which is incorporated by reference. 
     While the invention has been described in terms of several embodiments, those skilled in the art will recognize that the invention is not limited to the embodiments described, but can be practiced with modification and alteration within the spirit and scope of the appended claims. The description is thus to be regarded as illustrative instead of limiting of the invention as set forth in the appended claims including structural and functional equivalents thereof.