Abstract:
Disclosed herein an herbal acne control compositions comprising extracts of  Swietenia mahagoni, Salix tetrasperma, Aloe vera  and  Curcuma longa  and pharmaceutically acceptable excipients. Also disclosed are the methods of making the composition, formulating the same into different delivery systems and use thereof for the prevention of acne related skin disorders in human beings.

Description:
FIELD OF THE INVENTION 
     This invention, in general, relates to an acne control composition. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention provides a herbal acne control composition comprising extracts of  Salix tetrasperma, Swietenia mahagoni, Aloe Vera , and  Curcuma longa  and method of manufacturing the same and use thereof. 
     BACKGROUND OF THE INVENTION 
       Acne vulgaris  or acne is the most common skin disease affecting nearly 80% of persons between ages of 11 and 35 years. Acne affects all races and ethnicities with equal significance. Approximately 17 millions Americans are affected by this most common cutaneous disorder in United States. 
     Acne is a disease that is caused by the obstruction of sebaceous follicles, which are primarily located on the face and trunk due to excessive production of sebum in the follicles. The obstruction causes the formation of a microcomedo that may evolve into comedones such as a whitehead or a black head or an inflammatory lesion. 
     A blackhead occurs when the trapped sebum and bacteria partially open to the surface and turn black due to melanin, the skin&#39;s pigment. Blackheads can last for a long time because the contents very slowly drain to the surface. 
     The  Propionibacterium  acnes, a resident anaerobic organism, proliferate in the environment created by the mixture of excessive sebum and follicular cells and produces chemotactic factors and pro-inflammatory mediators that may lead to the inflammation. 
     There are many treatments available for  Acne vulgaris  depends on the type of clinical lesions. In case of comedonal acne, topical retinoids, hormonal therapies or oral isotretinoin are mostly available treatment. Benzoyl peroxide, topical antibiotics such as Clindamycin, Erythromycin, Tetracyclin and oral Isotretinoin or combination of all these medications are available for mild to moderate inflammatory acne. 
     To address the above problems of  Acne vulgaris , major research activities have been directed towards developing a acne control composition that can effective against the four processes of Acne viz. sebum production, hyperkeratinization, infection with  propionibacterium  acnes and inflammation. The present invention is an outcome of years of research to identify an herbal remedy possess anti inflammatory, anti kerolytic, sebum control and anti bacterial agent and methods of developing the same as cosmeceutical product for topical application for control and treatment of  Acne vulgaris.    
     PRIOR ART 
     United States Patent Application No. 20070003582 by Heng et al. teaches the preparation of topical medication comprises curcumin, water, alcohol, cellulose, carbomer, diazolinyl urea and anti inflammatory agent and other excipients for treatment of acne and for reversing of ageing and sun damage. 
     United States Patent Application No. 20070048234 by Waugh et al. teaches the preparation of anti acne kit comprises vasoconstrictor, anti acne agent, skin cleanser, an astringent and a sunscreen for treating severe cases of acne. 
     SUMMARY OF THE INVENTION 
     It is a principal object of the present invention to provide an herbal acne control composition comprising extracts of  Salix tetrasperma, Swietenia mahagoni, Aloe vera , and  Curcuma longa  and pharmaceutically acceptable excipients. 
     Another object of the present invention is to provide an herbal acne control composition comprises extracts of  Salix tetrasperma, Swietenia mahagoni, Aloe vera , and  Curcuma longa , wherein said composition is also effective for any other skin related disorder. 
     Another object of the present invention is to provide a process for preparing herbal acne control composition comprising extracts of  Salix tetrasperma, Swietenia mahagoni, Aloe vera , and  Curcuma longa , wherein the process provides selection of solvents to obtain extracts of said herbs having rich bioactive constituents. 
     Yet another object of the present invention is to provide an acne control composition, wherein said composition is effective for anti inflammatory, anti bacterial, anti kerolytic and anti-seborrhatic activities. 
     The above and the other objects of the present invention are attend according to following preferred embodiments of the present invention, however the scope of the invention is not restricted to the particular embodiment. 
     In accordance with one preferred embodiment of the present invention, there is provided a herbal acne control composition, wherein the composition comprising the extracts of  Aloe vera, Salix tetrasperma, Swietenia mahagoni  and  Curcuma longa  and pharmaceutically acceptable excipients selected from binder, diluent, surfactant, perfume and preservative as a carrier or otherwise. 
     In accordance with another preferred embodiment of the present invention, there is provided a herbal acne control composition, wherein the extract is obtained from all or any specific part/s of said herbs, preferably leaves of  Aloe vera , bark of  Swietenia mahagoni  and  Salix tetrasperma  and rhizomes of  Curcuma longa.    
     In yet another preferred embodiment, there is provided a process for preparation of herbal acne control composition, wherein, the process comprises of extracting the bark of  Salix tetrasperma  employing any suitable extraction technique, preferably a hot-soxhlation or percolation technique and wherein a selective solvent or solvent system are used to yield high extractive value and rich content of stable bioactive salicylic acid. Said solvent is selected from a group of organic solvents and water, preferably non polar long chain hydrocarbon, an ester, most preferably selected from n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol alone or in combination thereof. 
     In accordance with another preferred embodiment of the present invention, there is provided a method for extraction of bark of  Swietenia mahagoni , wherein said extraction method is performed using any suitable extraction technique, preferably a hot-soxhlation or percolation technique and wherein a selective solvent or solvent system are used to yield high extractive value and rich content of stable bioactive ingredients. Said solvent is selected from a group of organic solvents and water, preferably lower chain alcohol/s and water, most preferably water. 
     In accordance with another embodiment of the present invention, there is provided a method for extraction of leaves of  Aloe vera , wherein said extraction method is performed using any suitable extraction technique, preferably a hot-soxhlation or percolation technique and wherein a selective solvent or solvent system are used to yield high extractive value and rich content of stable bioactive ingredients including but are not limited to mono and di saccharides. Said solvent is selected from a group of organic solvents and water, preferably lower chain alcohol/s and water, most preferably methanol. 
     In accordance with another embodiment of the present invention, there is provided a method for extraction of rhizomes of  Curcuma longa , wherein said extraction method is performed using any suitable extraction technique, preferably a hot-soxhlation or percolation technique and wherein a selective solvent or solvent system are used to yield high extractive value and rich content of stable bioactive ingredients including but are not limited to tetrahydrocurcuminoids. Said solvent is selected from a group of organic solvents and water, preferably halo hydrocarbons and lower chain alcohol, most preferably selected from dichloromethane and methanol alone or in combination thereof. 
     In still another preferred embodiment of the present invention, there is provided a process for preparation of acne control composition, wherein, the process comprises of extracting the herbs by percolation method employing suitable solvent, filtering the resultant herbs extract, concentrating the extract to dryness on a rotatory evaporator or on a steam bath at optimum temperature and producing a herbal composition comprising the said dry extract and pharmaceutically acceptable excipients. 
     In still another preferred embodiment of the present invention, there is provided a process for preparation of acne control composition, wherein, the process comprises of extracting the herbs by hot-soxhlation method employing suitable solvent, filtering the plant extract, concentrating the plant extract to dryness on a rotatory evaporator or on a steam bath at optimum temperature and producing a herbal composition comprising the said dry extract and pharmaceutically acceptable excipients. 
     In accordance with another preferred embodiment of the present invention, there is provided a method of screening of  Swietenia mahagoni  water extract for antibacterial activity against  Propionibacterium  acnes. 
     In accordance with another embodiment of the present invention, there is provided a herbal acne control composition, wherein said composition is formulated in various delivery system preferably in the form of cream, face wash, face gel, face scrub or face pack. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       Further objects of the present invention together with additional features contributing thereto and advantages accruing there from will be apparent from the following description of preferred embodiments of the invention which are shown in the accompanying drawings and figures, wherein: 
         FIG. 1 . HPLC chromatogram of methanolic extract of  Aloe vera    
         FIG. 2 . HPLC chromatograms of solvent extracts of  Salix tetrasperma    
         FIG. 3 . HPLC chromatogram of water extract of  Swietenia mahagoni    
         FIG. 4-11 . Representative photomicrographs of Rabbit ear skin obtained from different groups (Histopathology) 
         FIGS. 4 . &amp;  5 . Shows normal skin architecture of rabbit car (Group-1) 
         FIGS. 6 . &amp;  7 . Showing intense epithelial and sebaceous gland proliferation and also infiltration of Inflammatory cells in untreated positive control (Group-2). The changes are indicated by arrows. 
         FIGS. 8 . &amp;  9 . Showing reversal of epithelial and sebaceous gland proliferation and also infiltration of inflammatory cells in Tretenoin (0.025%) cream treatment group (Group-3) 
         FIGS. 10 . &amp;  11 . Showing higher degree of reversal of epithelial and sebaceous gland proliferation and also infiltration of inflammatory cells in FD/OCC/10 cream application group (Group-4) 
         FIG. 12 . Topical anti-inflammatory activity of Tetrahydro curcuminoids (THC-09) and  Curcuma longa  extract (CU-18) in Croton oil induced ear edema. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     While this specification concludes with claims particularly pointing out and distinctly claiming that, which is regarded as the invention, it is anticipated that the invention can be more readily understood through reading the following detailed description of the invention and study of the included examples. 
     The present invention discloses an herbal acne control composition, wherein the composition comprising the extracts of  Aloe vera, Salix tetrasperma, Swietenia mahagoni  and  Curcuma longa  and pharmaceutically acceptable excipients selected from but are not limited to binder, diluent, surfactant, perfume and preservative as a carrier or otherwise. 
     The disclosed acne control composition is effectively used for the any skin related problem, preferably acne control. Said composition provides protection to the user from mild to severe pimples, black heads, white heads, papules, nodules, pustules, inflammatory lesions and cysts and free from any toxicity, toxic residues and irritation if used. 
     The herbs extracts according to the present invention are screened to characterize the therapeutic effectivity, such as for anti-inflammatory activity of  Curcuma longa  extract, anti-seborrhatic activity of  Aloe vera  extract, anti-bacterial activity of  Swietenia mahagoni  and anti-kerolytic activity of  Salix tetrasperma.    
     The solvent extract of  Salix tetrasperma  is standardized against salicylic acid by High Performance Liquid Chromatography technique. In the same line, extracts of  Aloe vera, Curcuma longa , and  Swietenia mahagoni  are also standardized employing their respective phytochemical marker such as carbohydrates and curcuminoids respectively. 
     The present invention involves the selection and identification of the herbs and obtaining the extract by subjecting the same to solvent extraction. The bioassay guided fractionation of extracts to identify the active compounds and to develop a herbal composition comprising extracts and fractions of  Aloe vera, Curcuma longa, Salix tetrasperma, Swietenia mahagoni  and a safe cosmeceutical composition for treatment and control of Acne and other skin related diseases in human beings. 
     The examples that follow are not intended to limit the scope of the invention as defined hereinabove or as claimed below. 
     EXAMPLE 1 
     Preparation of Extract from the Leaves of  Aloe vera  by Percolation Method 
     The shade dried material of leaves of  Aloe vera  is pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. 
     EXAMPLE 2 
     Preparation of Extract from the Leaves of  Aloe vera  by Hot-soxlation Method 
     The coarse powdered material of leaves of  Aloe vera  is subjected to hot-soxhalation using solvents n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at optimum temperature and recycled until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above described respective solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. 
     EXAMPLE 3 
     Preparation of Extract from the Bark of  Salix tetrasperma  by Percolation Method 
     The shade dried material of bark of  Salix tetrasperma  is pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol, water, n-hexane:chloroform (1:1), n-hexane:ethyl acetate (1:1), n-hexane:acetone (1:1), chloroform:acetone (1:1), ethyl acetate:methanol (1:1), acetone:water (1:1) and methanol:water (1:1) at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. 
     EXAMPLE 4 
     Preparation of Extract from the Bark of  Salix tetrasperma  by Hot-soxlation Method 
     The coarse powdered material of bark of  Salix tetrasperma  is subjected to hot-soxhalation using solvents n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol, water, n-hexane:chloroform (1:1), n-hexane:ethyl acetate (1:1), n-hexane:acetone (1:1), chloroform:acetone (1:1), ethyl acetate:methanol (1:1), acetone:water (1:1) and methanol:water (1:1) at optimum temperature and recycled until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above described respective solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. The yield of each extract is summarized in table-1. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 S. 
                   
                   
                 Yield 
                   
               
               
                 No 
                 Code No. 
                 Mode of extraction 
                 (%) 
                 Nature of extract 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ERS-1 
                 Hexane 
                 1.00 
                 Green colour sticky 
               
               
                 2 
                 ERS-2 
                 Solvent ether 
                 1.50 
                 Green colour sticky 
               
               
                 3 
                 ERS-3 
                 Chloroform 
                 2.00 
                 Green colour sticky 
               
               
                 4 
                 ERS-4 
                 Dichloromethane 
                 2.20 
                 Green colour sticky 
               
               
                 5 
                 ERS-5 
                 Hexane:chloroform(1:1) 
                 0.90 
                 Green colour sticky 
               
               
                 6 
                 ERS-6 
                 Ethylacetate 
                 1.80 
                 Green colour sticky 
               
               
                 7 
                 ERS-7 
                 Methanol 
                 6.00 
                 Brown powder 
               
               
                 8 
                 ERS-8 
                 Ethylalcohol 
                 5.50 
                 Brown powder 
               
               
                 9 
                 ERS-9 
                 Hexane:ethylacetate (1:1) 
                 2.00 
                 Green colour sticky 
               
               
                 10 
                 ERS-10 
                 Hexane:Acetone(1:1) 
                 1.80 
                 Green colour sticky 
               
               
                 11 
                 ERS-11 
                 Chloroform:acetone (1:1) 
                 2.00 
                 Green colour sticky 
               
               
                 12 
                 ERS-12 
                 Acetone 
                 3.00 
                 Brown powder 
               
               
                 13 
                 ERS-13 
                 Ethylacetate:methanol (1:1) 
                 5.00 
                 Brown powder 
               
               
                 14 
                 ERS-14 
                 Acetone:water (1:1) 
                 15.00 
                 Brown powder 
               
               
                 15 
                 ERS-15 
                 Methanol:water (1:1) 
                 10.00 
                 Brown powder 
               
               
                 16 
                 ERS-16 
                 Water 
                 6.00 
                 Brown powder 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 5 
     Preparation of Extract from the Bark of  Swietenia mahagoni  by Percolation Method 
     The shade dried material of bark of  Swietenia mahagoni  is pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at room temperature for 24 h to 48 h, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. 
     EXAMPLE 6 
     Preparation of Extract from the Bark of  Swietenia mahagoni  by Hot-soxlation Method 
     The coarse powdered material of bark of  Swietenia mahagoni  is subjected to hot-soxhalation using solvents n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at optimum temperature and recycled until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above corresponding solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. 
     EXAMPLE 7 
     Preparation of Extract from the Rhizomes of  Curcuma longa  by Percolation Method 
     The shade dried material of rhizomes of  Curcuma longa  is pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. 
     EXAMPLE 8 
     Preparation of Extract from the Rhizomes of  Curcuma longa  by Hot-soxlation Method 
     The coarse powdered material of rhizomes of  Curcuma longa  is subjected to hot-soxhalation using solvents n-hexane, diethyl ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and water at optimum temperature and recycled until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above corresponding solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. 
     EXAMPLE 9 
     Standardization of Methanolic Extract of  Aloe vera  by HPLC 
     Sample preparation: Accurately 100 mg of methanolic extract was weighed in 100 ml of volumetric flask and dissolved in Water:Methanol (1:1) solvent mixture and made upto the volume of 100 ml to get 1 mg/ml concentration. 
     HPLC conditions: Accurately 20 uL of the sample was injected over reverse phase column and run with mobile phase of 0.1% of Phosphoric acid and water in the ratio of 90:10 at flow rate of 1 ml/min. and detected at 210 nm. The chromatogram of the same is given as  FIG. 1 . 
     EXAMPLE-10 
     Standardization of Extracts of  Salix tetrasperma  by HPLC 
     Standard preparation: Accurately weighed 10 mg of Salicylic acid is taken into 10 ml of volumetric flask and dissolved in HPLC grade methanol and sonicate for 5 min. This standard solution is further diluted with methanol to get 100 ug/ml as working standard. 
     Sample preparation: Accurately 100 mg of each extract was weighed in 100 ml of volumetric flask and dissolved in HPLC grade methanol and made upto the volume of 100 ml to get 1 mg/ml concentration. 
     HPLC conditions: Accurately 20 uL of the sample is injected over reverse phase column and run with mobile phase of 0.1% acetic acid in water and methanol in the ratio of 60:40 at flow rate of 1 ml/min and detected at 270 and 254 nm. 
     The chromatogram of each extract is given in  FIG. 2  and percentage composition of salicylic acid is given in table.2. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Yield (%) of 
               
               
                 S. No. 
                 Code No 
                 Salicylic acid 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ERS-1 
                 Not detected 
               
               
                 2 
                 ERS-2 
                 6.48 
               
               
                 3 
                 ERS-3 
                 1.98 
               
               
                 4 
                 ERS-4 
                 2.48 
               
               
                 5 
                 ERS-5 
                 0.58 
               
               
                 6 
                 ERS-6 
                 4.40 
               
               
                 7 
                 ERS-7 
                 4.53 
               
               
                 8 
                 ERS-8 
                 4.34 
               
               
                 9 
                 ERS-9 
                 7.54 
               
               
                 10 
                 ERS-10 
                 8.91 
               
               
                 11 
                 ERS-11 
                 6.44 
               
               
                 12 
                 ERS-12 
                 4.62 
               
               
                 13 
                 ERS-13 
                 5.40 
               
               
                 14 
                 ERS-14 
                 2.66 
               
               
                 15 
                 ERS-15 
                 2.79 
               
               
                 16 
                 ERS-16 
                 0.74 
               
               
                   
               
             
          
         
       
     
     EXAMPLE-11 
     Fractionation and Column Chromatography of Water Extract of  Swietenia mahagoni    
     About 400 g of the water extract is fractionated into methanol soluble fraction and methanol insoluble fractions. The yield of the methanol soluble fraction is 48%. The methanol soluble fraction is subjected to column chromatography using silica gel as adsorbent and eluted in the column with n-hexane, ethyl acetate and methanol. The details are given in Table. 3. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 S. No 
                 Fraction No 
                 Solvent used for elution 
                 Yield 
                 Nature of fractions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AV-32/2 
                 Hexane 
                 100 
                 mg 
                 Colourless oily 
               
               
                 2 
                 AV-32/3 
                 Hexane:ethylacetate(95:5) 
                 100 
                 mg 
                 Colourless oily 
               
               
                 3 
                 AV-32/4 
                 Hexane:ethylacetate(90:10) 
                 350 
                 mg 
                 Yellow oily 
               
               
                 4 
                 AV-32/5 
                 Hexane:ethylacetate(85:15) 
                 200 
                 mg 
                 Yellow waxy 
               
               
                 5 
                 AV-32/6 
                 Hexane:ethylacetate(80:20) 
                 500 
                 mg 
                 Yellow waxy 
               
               
                 6 
                 AV-32/7 
                 Hexane:ethylacetate(75:25) 
                 250 
                 mg 
                 Brown oily 
               
               
                 7 
                 AV-32/8 
                 Hexane:ethylacetate(70:30) 
                 200 
                 mg 
                 Brown oily 
               
               
                 8 
                 AV-32/9 
                 Hexane:ethylacetate(60:40) 
                 230 
                 mg 
                 Light brown syrupy 
               
               
                 9 
                 AV-32/10 
                 Hexane:ethylacetate(50:50) 
                 300 
                 mg 
                 Light brown syrupy 
               
               
                 10 
                 AV-32/11 
                 Hexane:ethylacetate(25:75) 
                 400 
                 mg 
                 Light brown syrupy 
               
               
                 11 
                 AV-32/12 
                 Ethylacetate 
                 18 
                 g 
                 brown syrupy 
               
               
                 12 
                 AV-32/13 
                 Ethylacetate:methanol (95:5) 
               
               
                 13 
                 AV-32/14 
                 Ethylacetate:methanol (90:10) 
               
               
                 14 
                 AV-32/15 
                 Ethylacetate:methanol (85:15) 
                 2 
                 g 
                 Dark brown syrupy 
               
               
                 15 
                 AV-32/16 
                 Ethylacetate:methanol (80:20) 
                 1 
                 g 
                 Dark brown syrupy 
               
               
                 16 
                 AV-32/17 
                 Ethylacetate:methanol (70:30) 
                 2 
                 g 
                 Dark brown syrupy 
               
               
                 17 
                 AV-32/18 
                 Ethylacetate:methanol (60:40) 
                 1.3 
                 g 
                 Dark brown syrupy 
               
               
                 18 
                 AV-32/19 
                 Ethylacetate:methanol (50:50) 
                 1.0 
                 g 
                 Brown syrupy 
               
               
                 19 
                 AV-32/20 
                 Ethylacetate:methanol (25:75) 
                 2.5 
                 g 
                 Brown syrupy 
               
               
                 20 
                 AV-32/21 
                 Methanol 
                 51 
                 g 
                 Dark brown pasty 
               
               
                   
               
             
          
         
       
     
     EXAMPLE-12 
     Standardization of  S. Mahagoni  Water Extract by HPLC 
     Sample preparation: The water extract (AV-32) is accurately weighed for 100 mg in volumetric flask, dissolved in water and sonicated for 5 minutes to make up the volume to 100 ml to get the concentration of 1 mg/ml sample solution and filtered through 0.2 micron syringe filter. 
     Mobile Phase Preparation: Accurately 1.88 g of Hexane Sulfonic Acid Sodium salt is added to 10 ml of glacial Acetic acid and 1.3 ml of Triethylamine in 1 L volumetric flask and volume is made upto the mark with double distilled water. The mobile phase is sonicated for 10 minutes and filtered through 0.45 u filter paper. 
     HPLC conditions: Accurately 20 uL of samples is injected over reverse phase column and ran with the mobile phase at 1 ml/min and detected at 254 nm. HPLC chromatograms are summarized in  FIG. 3 . 
     EXAMPLE-13 
     Antibacterial Activity of Extracts of  S. mahagoni  Against  Propionibacterium  Acnes 
     Plant Extract Preparation: The stock solutions of the plant extracts are prepared in Dimethyl Sulphoxide (DMSO) or in double distilled water as per the solvent specifications. The working solution (100 mg/ml) is prepared accordingly. 
     Bacterial strains— Propionibacterium acnes : The bacterial strain (MTCC No. 1951) is procured from Microbial Type Culture Collection Chandigarh. The strain is re-cultured, maintained in Rabbit Blood Agar plates (Blood Agar with 5% Sterile rabbit blood) as well as Fluid Thioglycollate medium. The cultures are stained by Grams staining procedure and are observed for their characteristics. 
     In-vitro Anti-bacterial Activity Evaluation 
     Anti-bacterial activity is evaluated by Agar well diffusion assay &amp; by Minimal Inhibitory concentration (MIC) evaluation. 
     Agar Well Diffusion Assay 
     The Rabbit Blood Agar plates are inoculated with the broth culture of (Density equivalent to 1/100 th  dilution of 0.5 McFarland turbidometer)  P. acnes  with a sterile cotton swab. 6 mm wells are punched on the agar and filled with the plant extract (100 mg/ml). Plates are kept in anaerobic jar incubated in incubator set at 37° C. for 48 hrs. Zones of inhibition are measured &amp; recorded in millimetre (mm). 
     Minimal Inhibitor Concentration (MIC) Evaluation 
     The Rabbit Blood Agar is prepared &amp; kept molten at 55° C. Different dilution of plant extracts &amp; solvents are taken in a sterile 50 mm petriplates. The extracts are diluted with molten agar to make up the volume to 4 ml. After solidification of the media, the plates are inoculated with 20 μl of broth culture of (Density equivalent to 1/100 th  dilution of 0.5 McFarland turbidometer)  P. acnes . Plates are incubated for 48 hrs at 37° C. MIC is determined as the minimum concentration of the drug showing maximum inhibition. Results are recorded in Table-4. 
     
       
         
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Zone of 
                   
               
               
                 Organism Tested 
                 Extract 
                 Inhibition 
                 MIC 
               
               
                   
               
             
             
               
                 
                   P. acnes 
                 
                   S. Mahagoni  water extract 
                 31 mm 
                 2 mg 
               
               
                   
                   S. Mahagoni  methanol 
                 34 mm 
                 4 mg 
               
               
                   
                 extract 
               
               
                   
               
             
          
         
       
     
     EXAMPLE-14 
     Comparative Evaluation of  Aloe vera  Extracts with Tretenoin for Anti-seborrhatic Activity in Rabbits 
     Material and Methods: Laboratory bred New Zealand white rabbits weighing between 2.2-2.7 kgs were used for the study. The animals were housed and acclimatised to a constant temperature of 22±2° C. and will be exposed to 12 hrs day &amp; night cycle. The animals were fed with synthetic diet and water ad libitum. 
     Procedure: Twenty-four rabbits were divided in to 4 groups of six each. UV irradiated oleic acid is applied to the ventral surface of the rabbit ears, near the auditory canal, using camlin brush for 28 days. Group I rabbits did not receive the comedogen and serves as control. Rabbits from group II were applied with comedogen (UV irradiated oleic acid) for 28 days and served as positive control. Rabbits from group III and IV were applied with comedogen. After two hours of the comedogen application anti-acne creams Tretenoin and plant extracts were applied respectively for 28 days. Ear tissue was collected on 28 th  day for histopathological evaluation. The tissues were fixed in 10% neutral buffered formalin (NBF), the formalin fixed tissues were then processed by paraffin technique, and sections of 5-μm thickness cut and stained by the routine H &amp; E method. The histopathological changes were recorded semi-quantitatively by an experienced and qualified histopathologist. 
     Group Classification: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                 No. of 
                   
               
               
                 Group 
                 animals 
                 Treatment 
               
               
                   
               
             
             
               
                 1 
                 6 
                 Normal control 
               
               
                 2 
                 6 
                 Positive control (oleic acid application alone) 
               
               
                 3 
                 6 
                 Oleic acid + Tretenoin (0.025%) Cream 
               
               
                 4 
                 6 
                 Oleic acid +  Aloe vera  extract 
               
               
                   
               
             
          
         
       
     
     UVA irradiation of Oleic acid: Oleic acid is poured into a glass petriplates to form a thin layer and exposed to UVA irradiation from 300 W sun lamp for 18 hrs. 
     Results: Summary of lesions observed in all groups is given in table-5 and histopathological pictures are shown in  FIGS. 4-11 . 
     
       
         
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
             
             
               
                   
                   
               
               
                   
                 Groups 
               
             
          
           
               
                   
                   
                 1 
                 2 
                 3 
                 4 
               
               
                 Lesions 
                 Intensity 
                 (n = 6) 
                 (n = 6) 
                 (n = 6) 
                 (n = 6) 
               
               
                   
               
             
          
           
               
                 Epithelial 
                 ++ 
                 0 
                 0 
                 0 
                 100 
               
               
                 proliferation 
                 +++ 
                 0 
                 0 
                 100 
                 0 
               
               
                   
                 ++++ 
                 0 
                 100 
                 0 
                 0 
               
               
                 Sebaceous 
                 + 
                 0 
                 0 
                 0 
                 50 
               
               
                 glandular 
                 ++ 
                 0 
                 0 
                 50 
                 50 
               
               
                 proliferation 
                 ++++ 
                 0 
                 100 
                 50 
                 0 
               
               
                 Inflammatory 
                 +++ 
                 0 
                 50 
                 50 
                 100 
               
               
                 cell collection 
                 ++++ 
                 0 
                 50 
                 50 
                 0 
               
               
                   
               
             
          
         
       
     
     EXAMPLE-15 
     Screening of Plant Extracts for Topical Anti-inflammatory Activity Using Croton Oil Induced Model of Ear Edema in Rats 
     The objective of this study is to evaluate the topical anti-inflammatory properties of Tetrahydrocurcuminoids (THC-09) and Curcuminoids from  Curcuma longa  extract (CU-18) by croton oil induced ear edema model in rats and compare its activity against standard reference drugs Hydrocortisone and Diclofenac. 
     Animals: Around 40 male rats of Wistar strain weighing between 150 to 200 gms were used for the study. The animals were procured from in-house animal house and quarantined for 7 days before the start of the experiment and subjected for routine veterinary examination. Local animal ethics committee approved the protocol of the study. Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air supply (12-15 air changes/hr). Environment: temperature 19-25° C., relative humidity 30-70%, with a 12 hr light and 12 hr dark cycle. Temperature and humidity were recorded. 
     Animals were housed in groups of 4 per cage in standard polypropylene cages (size approximately: L 410×B 220×H140 mm), with stainless steel top grill having facilities for pelleted food and drinking water in bottle; bedding: steam sterilized clean paddy husk was used and changed at least thrice a week. Rats/Mice (pellet) Feed manufactured by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India were provided. Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aqua guard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai, India was provided. 
     Based on the body weight stratification rats were randomized into 5 groups of 8 animals each, and each animal was coded and identified by their unique accession number. 
     Chemicals and Reagents 
     All the chemicals and reagents used were of analytical grade. 
     Croton Oil Preparation: 
     4 parts croton oil, 10 parts ethanol, 20 parts pyridine, 66 parts ethyl ether 200 μl croton oil, 500 μl ethanol,1000 μl pyridine, 3300 μl ethyl ether and 5000 μl of the total mixture will be kept in 5 aliquots of 1 ml each and this mixture was stored in a place away from light. The test compounds were solubilized in this preparation just before the topical application. 
     Test Compounds and Concentration:
         1. THC-09 (Tetrahydrocurcuminoids), 10 mg/ml of croton oil preparation.   2. CU-18 ( Curcuma longa  extract), 10 mg/ml of croton oil preparation.   3. Hydrocortisone, 1 mg/ml of croton oil preparation.   4. Diclofenac, 2 mg/ml of croton oil preparation.       

     Procedure: 
     For testing in rats the above-mentioned croton oil mixture was prepared in aliquots of 1 ml just before use. The reference and the test compounds were also dissolved in the same solution. The animals were divided in to 5 groups of 8 animals each. Group-I animals received 20 μl of croton oil preparation topically on both sides (inner and outer surface) of left ear for induction of inflammation. Group-II, III, IV and V animals were treated with THC-09, CU-18, Hydrocortisone and Diclofenac (all solubilized in croton oil preparation) in a similar manner as that of group I respectively, the concentration of drugs was as described above. 
     The animals were observed for erythema and edema of the ear and thickness of same was measured after second and third hour after the croton oil application. A visual scoring was also recorded unequivivocally by two independent observers. The scoring pattern is as follows; Low=1, Medium=2, and High=3, on the basis of erythema and thickness of the ear compare to right normal ear. 
     Statistics: All the data were analyzed by one-way ANOVA followed by Dunnet&#39;s post multiple comparison test. 
     Result: It was found that, the application of croton oil preparation to rat ear&#39;s, greatly increased the erythema and edema coupled with increase in thickness of ear. All the tested drugs significantly reversed these changes, when tested in above mentioned manner and dose in both 2 and 3-hour post administration. Results are summarized in  FIG. 12 . 
     EXAMPLE-16 
     Manufacturing Process of Acne Control Face Cream 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Phase 
                 Name of the Ingredients 
                 % Use 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 Octyl Stearate 
                 2.00 
               
               
                   
                 Capric/Caprylic Triglyceride 
                 2.00 
               
               
                   
                 Steareth 2 
                 2.0 
               
               
                   
                 Steareth 21 
                 1.5 
               
               
                   
                 Cetostearyl alcohol 
                 1.5 
               
               
                   
                 Stearic Acid 
                 2.0 
               
               
                   
                 Cetyl Palmitate 
                 1.0 
               
               
                   
                 Propyl Paraben 
                 0.1 
               
               
                   
                 Extract of  Salix tetrasperma   
                 0.1 
               
               
                   
                 DC-200 
                 0.5 
               
               
                 B 
                 DM Water 
                 Qs 
               
               
                   
                 Glycerine 
                 2.0 
               
               
                   
                 Xanthan Gum 
                 0.2 
               
               
                   
                 Acrylates/C10-30 alkyl acrylate Crosspolymer 
                 0.2 
               
               
                   
                 Di Sodium EDTA 
                 0.05 
               
               
                   
                 Extract of  Aloe vera   
                 0.2 
               
               
                   
                 Extract of  Swietenia mahagoni   
                 0.2 
               
               
                   
                 Extract of  Curcuma longa   
                 0.1 
               
               
                   
                 Propylene Glycol 
                 5.0 
               
               
                   
                 Methyl paraben 
                 0.2 
               
               
                 C 
                 Phenoxy ethanol 
                 0.3 
               
               
                   
                 Vit E Acetate 
                 0.2 
               
               
                 D 
                 Perfume 
                 Qs 
               
               
                   
               
             
          
         
       
     
     Manufacturing Procedure
         1. Heat Phase A to 80° C.   2. Dissolve Di sodium EDTA in Water. Disperse Gum and polymer in water. Dissolve active in the water phase. Add all the remaining ingredients in the phase. Heat to 80° C.   3. Add Phase A into Phase B at 80° C. Mix for 15 min.   4. Start Cooling   5. Add Phase C at 50° C., mix well   6. Add Phase D at 45° C., mix well       

     EXAMPLE-17 
     Manufacturing Process of Acne Control Face Wash 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 % 
               
               
                 S. No. 
                 Ingredients 
                 Ingredients 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Extract of  Aloe vera   
                 0.05 
               
               
                 2. 
                 Extract of  Swietenia mahagoni   
                 0.05 
               
               
                 3. 
                 Extract of  Salix tetrasperma   
                 0.10 
               
               
                 4. 
                 Extract of  Curcuma longa   
                 0.10 
               
               
                 5. 
                 Glycolic acid 
                 0.55 
               
               
                 6. 
                 Disodium EDTA 
                 0.05 
               
               
                 7. 
                 PEG-7 glyceryl cococate 
                 2.00 
               
               
                 8. 
                 Plantacare 818 UP (Coco glucoside) 
                 30.00 
               
               
                 9. 
                 Plantacare 2000 UP (Coco glucoside) 
                 15.00 
               
               
                 10. 
                 Amaze XT (Dehydro xanthangum) 
                 1.50 
               
               
                 11. 
                 Glycacil 2000 (IPBC) 
                 0.30 
               
               
                 12. 
                 Neolone PE (MIC &amp; Phenoxyethanol) 
                 0.60 
               
               
                 13. 
                 Perfume 
                 0.40 
               
               
                 14. 
                 DM Water 
                 49.30 
               
               
                 15. 
                 Total 
                 100.00 
               
               
                   
               
             
          
         
       
     
     Manufacturing Method 
     The two-plantacare ingredients Plantacare 818 UP and Plantacare 2000 UP are weighed in main vessel as per formula and its pH is adjusted by glycolic acid solution. Disodium EDTA is dissolved in a portion of water in a vessel with propeller stirrer. Amaze XT is sprinkled added into the agitated water and mixing without any lumps. This amaze XT mixture is slowly transferred into the main vessel and mixed slowly. Other ingredients are added one by one into the main vessel with slow mixing with similar intervals. 
     EXAMPLE-18 
     Manufacturing Process of Acne Control Face Scrub 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                   
                 % 
               
               
                   
                 Ingredients 
                 ingredients 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Extract of  Aloe vera   
                 0.05 
               
               
                   
                 Extract of  Swietenia mahagoni   
                 0.05 
               
               
                   
                 Extract of  Salix tetrasperma   
                 0.10 
               
               
                   
                 Extract of  Curcuma longa   
                 0.10 
               
               
                   
                 Glycolic acid 
                 0.10 
               
               
                   
                 Disodium EDTA 
                 0.05 
               
               
                   
                 PEG-7 glyceryl cococate 
                 2.00 
               
               
                   
                 Plantacare 2000 UP (Coco glucoside) 
                 10.00 
               
               
                   
                 Amaze XT (Dehydro xanthangum) 
                 2.50 
               
               
                   
                 Polyethylene Beads 
                 1.00 
               
               
                   
                 Flora Beads 
                 0.25 
               
               
                   
                 Glycacil 2000 (IPBC) 
                 0.30 
               
               
                   
                 Neolone PE (MIC &amp; Phenoxyethanol) 
                 0.60 
               
               
                   
                 Perfume 
                 0.40 
               
               
                   
                 DM Water 
                 82.50 
               
               
                   
                 Total 
                 100.00 
               
               
                   
                   
               
             
          
         
       
     
     Manufacturing Method 
     A portion of water is taken in a vessel with propeller stirrer. EDTA DS is dissolved in the water. Carbomer is sprinkle into the water slowly. The propeller is allowed to stir till the carbomer becomes homogenous. The carbomer liquid is carefully neutralized by Triethanol amine with slow mixing. SLES is transferred into the main vessel with slow mixing; avoid excess foaming in the product. Other ingredients are added one by one to the product with slow mixing. 
     EXAMPLE-19 
     Manufacturing Process of Acne Control Pore Cleansing Face Mask 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                   
                 % 
               
               
                   
                 Name of ingredients 
                 Ingredients 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Xanthan gum 
                 0.75 
               
               
                   
                 Glycerin 
                 3.00 
               
               
                   
                 Di sodium EDTA 
                 0.05 
               
               
                   
                 Extract of  Aloe vera   
                 0.25 
               
               
                   
                 Extract of  Salix tetrasperma   
                 0.05 
               
               
                   
                 Extract of  Curcuma longa   
                 0.10 
               
               
                   
                 Extract of  Swietenia mahagonia   
                 0.125 
               
               
                   
                 Propylene glycol 
                 3.00 
               
               
                   
                 Methyl paraben 
                 0.25 
               
               
                   
                 Propyl paraben 
                 0.10 
               
               
                   
                 Glycerin 
                 3.00 
               
               
                   
                 Stearic acid 
                 2.00 
               
               
                   
                 Ceto stearyl alcohol I.P 
                 0.50 
               
               
                   
                 Iso propyl myristate B.P 
                 2.00 
               
               
                   
                 Glyceryl mono state SE 
                 0.50 
               
               
                   
                 Light Kaolin 
                 26.00 
               
               
                   
                 Bentonite imp 
                 8.00 
               
               
                   
                 Sodium lauryl sulfate 
                 0.20 
               
               
                   
                 Imidazolidinyl urea 
                 0.20 
               
               
                   
                 Fragrance 
                 0.50 
               
               
                   
                 DM Water 
                 49.425 
               
               
                   
                 Total 
                 100.00 
               
               
                   
                   
               
             
          
         
       
     
     Manufacturing Procedure 
     
         
         
           
             1. A Phase—Weigh Xanthan gum and Glycerine as per formula and disperse Xanthan gum in glycerine. Keep it at RT, Immediately transfer this to Phase B. 
             2. B Phase—Weigh DM water and EDTA in main Mixer and weigh extract of  Aloe vera  keep it separate. 
             3. C Phase—Weigh propylene glycol and extract of  Swietenia mahagoni  keep it separate. 
             4. D Phase—Weigh Methyl paraben and propyl paraben keep it separate. 
             5. E Phase—Weigh Stearic Acid, Ceto stearyl alcohol, Isopropyl myristate, Glyceryl mono stearate SE, extracts of  Salix tetrasperma  and  Curcuma longa  in the side kettle. 
             6. F Phase—Weigh Light Kaolin pure, Bentonite powder in a clean poly bag and mix well. 
             7. G Phase—Weigh SLS powder in a separate vessel &amp; Imid urea in a separate vessel and dissolve in the water completely. 
             8. H Phase—Weigh perfume and keep it separate. 
           
         
       
    
     Manufacturing Process 
     
         
         
           
             1. Mix ‘Phase B’ ingredients in the main mixer filter through muslin cloth and add ‘Phase A’ to Phase B. Heat it to 80° C. and maintain the temperature. 
             2. Heat ‘Phase C’ ingredients to 80° C. in the melting kettle. 
             3. Transfer ‘Phase C’ into ‘Phase A&amp; B’ in the main mixer at 80° C., mix for 10 minutes. 
             4. Add ‘Phase D’ slowly with continuous mixing to ‘Phase ABC’. Mix ‘Phase ABCD’ till there is no lumps. 
             5. In oil phase kettle take all the Phase E ingredients melt it by heating to 80° C. 
             6. Transfer Phase E to Phase ABCD.Start Homogenize for 20 minutes. 
             7. To the above mixer add phase F ingredients slowly by continuously mixing, final product should be uniformly. Cool it. 
             8. At 55° C., Add ‘Phase G’ to the main mixer containing ‘Phase ABCDEF’ mixture and mix well. 
             9. Add ‘Phase H’ at 45° C. to the main mixer containing ‘Phase ABCDEFG’ and mix well. 
             10. Ensure the product is free of lumps. If required pass through colloidal mill. 
           
         
       
    
     EXAMPLE 20 
     Clinical Trial Evaluation of Acne-n-Pimple Total Care Package for the Treatment of  Acne - vulgaris    
     An open Clinical trial on Acne Control was conducted in 25 patients with a mean age of 21.0±4.18 years with 9 of male and 16 female patients. The trial was aimed to evaluate the efficacy of Acne-n-Pimple total care package in  Acne - vulgaris , which includes Anti-acne face wash, Anti-acne cream and anti-acne scrub. 
     Study Design: This was an open clinical trial. Twenty five patients satisfying the inclusion criteria were selected for the study after obtaining their informed consent. The volunteers were requested to use the acne package as per the direction of the investigator (Anti-acne face wash: twice a day, Anti-acne cream: twice daily on affected area after washing face with above face wash, Anti-acne-scrub: twice a week). 
     The patients were evaluated on Day 14 and on Day-28 of the study for black heads, inflamed papules, inflamed pustules, cysts, nodules, white heads and blemishes. The parameters were reviewed at initial and at the end of 14 and 28 days. At the end of the study, the results were statistically analyzed. Apart from above parameters the package was also evaluated for the cosmetic effect such as; exfoliation, moisturizing effect, smoothening effect, soothing effect and healing without scar formation etc. 
     The patients and the investigators were asked to rate the outcome of the treatment separately and the severity score was recorded and analyzed on all the days. The local/systemic adverse effects, if any, were noted during follow-up of the study. 
     Statistical analysis: Statistical analysis was carried out using Fischer&#39;s exact test to find out the level of significance. The severity score for acne was analysed using Friedman test followed by Dunnett&#39;s multiple comparison posthoc test. The severity score was expressed as mean±SD. The minimum level of significance was fixed at p&lt;0.05. Statistical analysis was carried out using GraphPad Prism software Version 4.01. 
     Results: The results and the statistical significance for various parameters employed during the Acne Control treatment are given in Table-10 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 10 
               
             
             
               
                   
                   
               
               
                   
                 Treatment Duration 
               
             
          
           
               
                 No. 
                 Parameters 
                 Initial 
                 Day 14 
                 p value 
                 Initial 
                 Day 28 
                 p value 
               
               
                   
               
             
          
           
               
                 1. 
                 Open Comedones 
                 25 
                 19 
                 p &lt; 0.05 
                 25 
                 1 
                 p &lt; 0.001 
               
               
                 2. 
                 Closed Comedones 
                 25 
                 19 
                 p &lt; 0.05 
                 25 
                 0 
                 p &lt; 0.001 
               
               
                 3. 
                 Inflamed Papules 
                 20 
                 7 
                 p &lt; 0.05 
                 20 
                 0 
                 p &lt; 0.001 
               
               
                 4. 
                 Pustules 
                 11 
                 4 
                 NS 
                 11 
                 1 
                 p &lt; 0.05  
               
               
                 5. 
                 Blemishes 
                 18 
                 15 
                 NS 
                 18 
                 5 
                 p &lt; 0.001 
               
               
                 6. 
                 Erythema 
                 16 
                 8 
                 p &lt; 0.05 
                 16 
                 3 
                 p &lt; 0.001 
               
               
                 7. 
                 Hypopigmentation/ 
                 13 
                 4 
                 p &lt; 0.05 
                 13 
                 3 
                 p &lt; 0.05  
               
               
                   
                 Hyperpigmentation 
               
               
                   
               
               
                 Note: 
               
               
                 NS: Not significant 
               
             
          
         
       
     
     The severity score for acne was 1.80±0.86, 1.32±0.90 and 0.00±0.00 initial, day 14 and on day 28, respectively. The improvement in severity score for acne on day 28 vs initial was significant at p&lt;0.001 and day 28 vs day 14 was p&lt;0.01 as compared to the initial values. The patient&#39;s remarks on the overall response to the treatment showed a complete cure in 84% and marked improvement in 8%. The investigators remarks on the overall response to the treatment showed a complete cure in 92% and marked improvement in 8%. Two patients developed signs of irritation and no adverse reactions were reported in all the remaining 23 patients. The test package was found to have good cosmetic acceptability and compliance of all the patients involved in the study. 
     While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those skilled in the art without departing from the scope and spirit of this invention.