Abstract:
Provided is a tear-free formulation for suppressing or reducing ocular irritancy of a pre-made cosmetic or therapeutic formulation or an ophthalmic formulation suitable for application to an eye of a subject or to a skin region surrounding an eye of a subject.

Description:
TECHNOLOGICAL FIELD 
       [0001]    The invention generally relates to natural preparations for suppressing or attenuating ocular irritancy. 
       BACKGROUND 
       [0002]    Formulations such as cleansing formulations (e.g. shampoos, facial soaps, etc.) generally cause irritation to the ocular membrane when coming in contact with the eyes. This imposes an unpleasant feeling to the user, which results in stinging or tearing, and in certain cases may even be harmful. This problem is of particular importance in shampoos designated for use by infants and children. 
         [0003]    Cleansing formulations are irritating mainly because they comprise surfactants, especially ionic surfactants, which interact with the mucous membrane, and because the pH of such formulation differs from the normal pH of the eye being 7.0 to 7.3. 
         [0004]    Recent studies conducted by the U.S. environmental protection agency (EPA) revealed that human blood and lipid tissues contain up to 400 hazardous chemicals originating from cosmetics and food products. Those chemicals may cause various side effects, such as headaches, eye irritation, skin irritation, and long term effects such as the development of various cancer diseases. 
         [0005]    Such studies have encouraged the development of “natural” cosmetic compositions; however, to date, none of the available compositions are 100% natural body soaps/shampoos. Several of the products known today contain mainly synthetic ingredients, with the addition of a very small amount of natural ingredients. Other known products contain a higher percentage of natural ingredients; however, they are mainly composed of synthetic materials. The most natural known products today contain chemically modified natural ingredients. 
         [0006]    Products composed of natural ingredients may also contain irritating substances that cause tear, itching and redness in the eye when coming in contact with the ocular membrane. Thus, problems associated with the development of a product that is wholly natural become vastly more complex where the product must also demonstrate no irritancy to the user&#39;s eyes. This development is particularly important in products intended for use in babies and young children. 
         [0007]    WO2012/077120 discloses natural formulations for skin or hair for providing cleansing, moisturizing, minimizing skin imperfections, reducing skin oiliness, providing fragrance to the hair or skin and reliving skin dryness and signs of aging. 
         [0008]    FR2810550 discloses solutions for use in medicine containing homogeneous extracts of the white and yolk of quail eggs for the treatment of allergies such as eye irritation. 
         [0009]    ES2333574 discloses natural compositions for treating skin irritation containing chicken egg yolk. 
       General Description 
       [0010]    The inventors of the present invention have developed a class of products comprising a combination or a mixture of naturally occurring ingredients which has a suppressing or attenuating effect on the ocular irritancy of formulations which come into contact with ocular membranes; such formulations were found to be useful on cosmetic formulations as well as on therapeutic formulations such as, for example, shampoos, conditioners, soaps, make-up removers, eye-drops, and others. 
         [0011]    The preparations of the invention have been found to be efficient in suppressing or attenuating ocular or skin irritancy, per se, and also endow other formulations which are known or expected to have a degree of irritancy with reduced or diminished irritating effects. 
         [0012]    Thus, in accordance with a first aspect of the invention, there is provided a formulation comprising a combination of lecithin and egg yolk, at least one natural oil, and optionally hyaluronic acid and/or hyaluronate, said formulation being capable of reducing, diminishing or ameliorating irritation to an eye region or to a skin region surrounding the eye. 
         [0013]    In another aspect, the invention provides a formulation comprising a combination of lecithin and egg yolk, at least one natural oil, and optionally hyaluronic acid and/or hyaluronate, said formulation being capable of reducing, diminishing or ameliorating irritation to an eye. 
         [0014]    In a further aspect, the invention provides a formulation comprising 
         [0015]    1—at least one of lecithin and egg yolk, 
         [0016]    2—at least one natural oil, and 
         [0017]    3—optionally hyaluronic acid and/or hyaluronate, 
         [0000]    said formulation being adapted for adding to a skin care formulation or hair care formulation exhibiting irritation to an eye region or to a skin region surrounding the eye, the formulation capable of reducing, diminishing or ameliorating irritation caused by the skin care formulation coming in contact with the eye region or the skin region surrounding the eye. 
         [0018]    The low irritancy formulation is thus herein classified as a minimally or non-irritating formulation. As known in the field of the invention, such formulations are typically known as “tear-free” formulations. 
         [0019]    The low irritancy formulation may thus be used as a baby shampoo, baby bath, mild skin cleanser, facial skin cleanser, sensitive skin cleanser and other cleansing formulations suitable for use on infant skin. 
         [0020]    The formulations of the invention may similarly be utilized on human infant and adult skin regions, eyes, or skin regions surrounding the eyes. 
         [0021]    In another aspect, the formulation comprising at least one of lecithin and egg yolk, at least one natural oil, and optionally hyaluronic acid and/or hyaluronate may be used for any purpose in which low irritancy to the skin and/or eyes is desirable. The formulation may for example be utilized as an additive (add on) to formulations which are irritating to the (human or non-human) eyes or skin; thereby rendering such irritating formulations with a reduced or diminished irritancy. 
         [0022]    In some embodiments, the formulation is selected from a baby shampoo, baby bath, mild skin cleanser, facial skin cleanser, sensitive skin cleanser and cleansing formulations suitable for use on human infant skin; or adult shampoo, adult bath, mild skin cleanser, facial skin cleanser, sensitive skin cleanser and cleansing formulations suitable for use on human adult skin. 
         [0023]    In a further aspect, the invention pertains to the use of any one of the above minimally or non-irritating formulations for the preparation of an additive to an existing formulation, for reducing, diminishing or ameliorating irritation to an eye or a skin region surrounding the eye. 
         [0024]    According to the invention, the formulation comprising the naturally occurring ingredients recited hereinabove or below, is adapted to produce the herein referred to non-irritating additive or additive. According to some embodiments, the additive may be stored before it is added to an eye-irritating formulation. 
         [0025]    As may be clear to the skilled in the art, the additive of the invention does not substantially alter any one existing characteristics of an irritating formulation, apart from rendering it less irritating or not irritating at all. In other words, properties such as cleaning properties, foaming e.g., of a shampoo formulation, ease of removal by washing, feeling and after-feeling effects are not significantly reduced or attenuated by the addition of a formulation of the invention. 
         [0026]    The invention also pertains to a method for suppressing or reducing ocular irritancy of a cosmetic or therapeutic formulation suitable for application to an eye of a subject or to a skin region surrounding an eye of a subject, the formulation being capable of causing irritation to an ocular membrane, the method comprising: mixing prior to application of said formulation to the eye or a skin region surrounding said eye an effective amount of an additive, as defined herein. 
         [0027]    The formulations and additives of the invention are capable of suppressing, reducing, delaying, or diminishing any one or more irritating feeling in the eyes of a subject; said irritating feeling being associated with one or more component or ingredient present in a cosmetic or therapeutic formulation to be applied to the eye or a skin region surrounding the eye. 
         [0028]    The additive may be applied to the eye prior to, concomitant with or after a cosmetic or therapeutic formulation has been applied to the eye or a skin region surrounding the eye. The additive is typically applied to the cornea of the eye. In some embodiments, the additive is added and mixed into the cosmetic or therapeutic formulation during the manufacture process of said cosmetic or therapeutic formulation; thus rendering said cosmetic or therapeutic formulation tear-free. 
         [0029]    Thus, the additive may be in the form of an add-on formulation which may be added to a ready-made cosmetic or therapeutic formulation or may be the cosmetic or therapeutic formulation itself. 
         [0030]    It is thus a further object of the invention to provide a non-skin-irritating or non-eye-irritating formulation, either formulation being selected from a cosmetic formulation for application onto a skin region of a subject, a cleansing formulation, a therapeutic topical formulation, an ophthalmic formulation, a solid form formulation or a liquid form formulation, and a cleansing, or therapeutic formulations suitable for infants and children. 
         [0031]    As stated above, the formulations of the invention are all natural formulations, namely comprising components and ingredients originating or extracted or obtained or derived from nature. No synthetic ingredients are included in formulations of the invention. 
         [0032]    The “lecithin” comprised in formulations of the invention is typically derived from animal or plant sources such as soy beans, eggs, milk, marine sources, rapeseed, cottonseed, and sunflower. In some embodiments, the lecithin is derivable from soy beans and/or egg yolk. 
         [0033]    The “natural oils” are typically selected from almond oil, aragan oil, avocado oil, calendula oil, castor oil, coconut oil, grape seed oil, jojoba oil, linseed oil, macadamia oil, meadowfoam oil, olive oil, palm oil, sesame seed oil, sun flower oil, vegetable squalane and squalane, oil from eggs or yolk, jojoba wax and lanolin. 
         [0034]    In some embodiments, the natural oils are selected from castor oil, jojoba oil, jojoba wax, meadowfoam oil, vegetable squalane and squalane, oil from eggs or yolk, lanolin, bees wax, carnauba wax, candelilla wax, cocoa and animals wax/butter. 
         [0035]    In some embodiments, the formulations according to the invention comprise at least one additional additive selected amongst phospholipids (lecithins), polysaccharides, polyols, saponins, salicilates, preservatives, perfumes, natural coloring agents, emollients, non-aqueous or aqueous solvents or mixtures, and other active or non-active components. 
         [0036]    In some embodiments, a formulation according to the invention comprises one or more phospholipids and/or one or more polysaccharide. 
         [0037]    In some embodiments, the formulation further comprises at least one emollient and/or at least one polyol. The polyol is selected from mannitol, glycerol, zemea, trehalose, fructose, mannose, sucrose, glucose, dextrose, trehalose, mannitol, lactose, rhamnose, sorbitol, honey extracts, glycol, diols and polyols such as vegetable glycerin, 1,2-butanediol, propylene glycol, ethanol. Other polyols included are betaine, natural urea, lactic acid and other alpha hydroxy acids. 
         [0038]    In some embodiments, the polyol is selected from mannitol, glycerol, zemea and trehalose. 
         [0039]    In some embodiments, the phospholipid is soy bean lecithin. 
         [0040]    In some embodiments, the polysaccharide is selected, in a non-limiting fashion, from xanthan, Tragacanth gum, Carrageenan gum, Alginates, Konjac gum Agar-Agar, gum Arabic, Guar gum, Starch, Gellan gum, Pectin, Cellulose, Welan and Dituan gum, Locust bean gum, Dammar gum, Kauri gum, Spruce gum, Fenugreek gum and gum anima,  sclerotium  gum and poly glutamic acid. 
         [0041]    In some embodiments, the polysaccharide is selected from sodium hyaluronate, xanthan gum, Carrageenan, Konjac gum, Tragacanth gum, Alginates, Guar gum, Locust bean gum,  sclerotium  gum, and poly glutamic acid 
         [0042]    In some embodiments, the emollient is a natural oil selected from jojoba oil and squalene oil. 
         [0043]    In some embodiments, the formulation comprises at least one phospholipid, at least one polysaccharide and at least one emollient. 
         [0044]    In some embodiments, the formulation according to the invention is the formulation shown in Table 1: 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 A formulation according to the invention 
               
             
          
           
               
                   
                 Ingredient 
                 Chemical Family 
               
               
                   
                   
               
               
                   
                 Soy-bean 
                 Phospholipids 
               
               
                   
                 Lecithin 
               
               
                   
                 Jojoba oil 
                 Emollient 
               
               
                   
                 Squalene oil 
                 Emollient 
               
               
                   
                 Egg Yolk 
                 Phospholipids + cholesterol + 
               
               
                   
                   
                 Emollient 
               
               
                   
                 Sodium 
                 Polysaccharides 
               
               
                   
                 Hyaluronate 
               
               
                   
                   
               
             
          
         
       
     
         [0045]    In some embodiments, the sodium hyaluronate may be replaced or combined with at least one other polysaccharide, as exemplified in Table 2. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 A formulation according to the invention 
               
             
          
           
               
                   
                 Ingredient 
                 Chemical Family 
               
               
                   
                   
               
               
                   
                 Soy-bean Lecithin 
                 Phospholipids 
               
               
                   
                 Jojoba oil 
                 Emollient 
               
               
                   
                 Squalene oil 
                 Emollient 
               
               
                   
                 Egg Yolk 
                 Phospholipids + cholesterol + 
               
               
                   
                   
                 Emollient 
               
               
                   
                 Sodium 
                 Polysaccharides 
               
               
                   
                 Hyaluronate 
               
               
                   
                 and/or 
               
               
                   
                 Xanthan gum 
               
               
                   
                 and/or 
               
               
                   
                 Carrageenan 
               
               
                   
                   
               
             
          
         
       
     
         [0046]    In some embodiments, the formulation may comprise one or more polyol. In some embodiments, the formulation comprises at least one phospholipid, at least one polysaccharide, at least one polyol and optionally at least one emollient. 
         [0047]    In some embodiments, the formulation may comprise at least one preservative. 
         [0048]    In other embodiments, the formulation may comprise at least one saponin. In such embodiments, the formulation may comprise at least one phospholipid, at least one polysaccharide, at least one saponin and optionally at least one emollient and/or at least one polyol. 
         [0049]    Exemplary formulations are depicted in Table 3 and Table 4. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 A formulation according to the invention 
               
             
          
           
               
                   
                 Ingredient 
                 Chemical Family 
               
               
                   
                   
               
               
                   
                 Soy-bean 
                 Phospholipids 
               
               
                   
                 Lecithin 
               
               
                   
                 Jojoba oil 
                 Emollient 
               
               
                   
                 Squalene oil 
                 Emollient 
               
               
                   
                 Egg Yolk 
                 Phospholipids + cholesterol + 
               
               
                   
                   
                 Emollient 
               
               
                   
                 Mannitol and/or 
                 Poly-ol 
               
               
                   
                 Glycerol and/or 
               
               
                   
                 Zemea and/or 
               
               
                   
                 Trehalose 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 A formulation according to the invention 
               
             
          
           
               
                   
                 Ingredient 
                 Chemical Family 
               
               
                   
                   
               
               
                   
                 Soy-bean Lecithin 
                 Phospholipids 
               
               
                   
                 Jojoba oil 
                 Emollient 
               
               
                   
                 Squalene oil 
                 Emollient 
               
               
                   
                 Egg Yolk 
                 Phospholipids + cholesterol + 
               
               
                   
                   
                 Emollient 
               
               
                   
                 Sodium 
                 Polysaccharides 
               
               
                   
                 Hyaluronate and/or 
               
               
                   
                 Xanthan gum 
               
               
                   
                 and/or Carrageenan 
               
               
                   
                 Mannitol and/or 
                 Poly-ol 
               
               
                   
                 Glycerol and/or 
               
               
                   
                 Zemea and/or 
               
               
                   
                 Trehalose 
               
               
                   
                   
               
             
          
         
       
     
         [0050]    In some embodiments, each of the formulations of the invention, e.g., those depicted in Tables 1-4 comprises at least one saponin. 
         [0051]    As used herein, the at least one “saponin” is at least one naturally obtained saponin compound or saponin material, as known in the art. When isolated from a natural source, the at least one saponin may be used in its substantially pure form (namely at least 85%, 87%, 92%, 95%, or 98% purity), or may be used as a saponin-containing extract isolated by a method known in the art or by a method of the invention, as disclosed herein. 
         [0052]    In some embodiments, the saponin material is a purified compound of the saponin family. 
         [0053]    In other embodiments, the saponin material is a saponin-containing extract. In accordance with the present invention, the saponin-containing extract (herein referred to for the purpose of brevity as “saponin extract”) contains at least between 0.2% and 95 wt % saponins, out of the total weight of the dry content of the extract. In some embodiments, the extract used in accordance with the present invention comprises between 0.2% and 99 wt % saponins out of the total weight of the dry content of the extract. 
         [0054]    In some embodiments, the extract used in accordance with the present invention comprises between 10% and 80 wt % saponins out of the total weight of the dry content of the extract. In other embodiments, the extract used in accordance with the present invention comprises between 10% and 60 wt % saponins out of the total weight of the dry content of the extract. In further embodiments, the extract used in accordance with the present invention comprises between 10% and 50 wt % saponins out of the total weight of the dry content of the extract. In additional embodiments, the extract used in accordance with the present invention comprises between 10% and 40 wt % saponins out of the total weight of the dry content of the extract. In still additional embodiments, the extract used in accordance with the present invention comprises between 10% and 30 wt % saponins out of the total weight of the dry content of the extract. 
         [0055]    In some embodiments, the extract used in accordance with the present invention comprises between 10% and 20 wt % saponins out of the total weight of the dry content of the extract. 
         [0056]    In some embodiments, the extract used in accordance with the present invention comprises between 0.2% and 10 wt % saponins out of the total weight of the dry content of the extract. 
         [0057]    The saponin-containing extract may be obtained from any natural source known to comprise saponins. Such natural source may be a plant source, some of which are detailed infra, and also from non-plant sources such as animal sources and marine organisms, such as starfish and sea cucumbers. In some embodiments of the invention, the saponins are extracted from a plant source, naturally grown or genetically modified to have high saponin content. 
         [0058]    In some embodiments of the invention, the saponin material is obtained by extraction from a plant source by employing water, alcohol or a water/alcohol solution. In some embodiments, the alcohol is ethanol or methanol. 
         [0059]    In some embodiments, the extraction is achieved by employing a water/alcohol solution. In some embodiments, the water/alcohol solution has a water:alcohol ratio of between 80:20 to 20:80. In further embodiments, the water/alcohol solution has a water:alcohol ratio of between 60:40 to 40:60. In further embodiments, the water/alcohol solution has a water:alcohol ratio of between 70:30 to 30:70. In further embodiments, the water/alcohol solution is 80:20 water/alcohol, 60:40 water/alcohol, 50:50 water/alcohol, 40:60 water/alcohol ratio, 20:80 water/alcohol, 70:30 water/ethanol or 30:70 water ethanol. 
         [0060]    The extraction time may vary without limitation from 2 to 8 hours, at or above room temperature (20° C.-30° C.), e.g., above 30° C., 40° C., 50° C. or 60° C. In some embodiments, the extraction is carried out at a temperature between 30° C. and 70° C. 
         [0061]    In some embodiments, the saponin material is obtained from a plant source. The plant source may be selected from shikakai, soy beans, beans, peas ( Pisum sativum ), lucerne, tea, spinach, sugar beet,  quinoa , liquorice, sunflower, horse chestnut,  ginseng , oats,  capsicum  peppers, aubergine, tomato seed, alliums, asparagus, yam, fenugreek,  yucca  and  ginseng , lucerne, mung beans,  Bupleurum falcatum, Camellia oleifera, Camellia sinensis Desmodium adscendens, Gypsophila, Panax quinqufolius, Panax japonicas, Quillaja saponaria, Sapindus delavayi, Sapindus mukorossi, Sapindus marginatus, Sapindus saponaria, Sapindus trifoliatus, Saponaria officinalis, Styrax japonica , and  Yucca schidigera  or any mixture thereof. Any part of the plant may be used for extracting the saponin material, including leaves, stems, roots, bulbs, blossom and fruit (including the skin, flesh and seed of the fruit). 
         [0062]    In some embodiments, the saponin material is an extract of  Camellia oleifera, Saponaria officinalis , or  Sapindus mukorossi  or a mixture thereof. 
         [0063]    In other embodiments, the saponin material is an extract of  Camellia oleifera , or  Sapindus mukorossi  or a mixture thereof. 
         [0064]    In other embodiments, the saponin material is an extract of  Sapindus mukorossi.    
         [0065]    The saponin material obtained from a plant source, e.g.,  Camellia oleifera , and/or  Sapindus mukorossi , may be extracted by treating the plant source in a water/alcohol solution under conditions permitting extraction of the saponin material into the solution. The so-extracted saponin containing material may optionally thereafter be purified by any means known in the art, including: filtration, centrifugation, re-crystallization, distillation, adsorption, chromatographic methods, fractionation, etc. 
         [0066]    In some embodiments, the plant source is first dried and ground before being treated in the water/alcohol solution. 
         [0067]    In some embodiments, the saponin material is extracted from a plant source following a method comprising:
       1. Treating the plant source in a 40:60 to 60:40 water:alcohol solution for a period of time and under conditions permitting extraction of the saponin material from said plant source into said solution, as defined hereinabove;   2. optionally, drying said saponin-containing solution to obtain a saponin-containing solid material; and   3. optionally, purifying said saponin-containing solid material.       
 
         [0071]    In some embodiments, the water:alcohol solution employed is about 50:50. 
         [0072]    In some embodiments, the plant source is one or both of  Camellia oleifera  and  Sapindus mukorossi . In some embodiments, the plant source is  Sapindus mukorossi  and the saponin material is extracted from the nut shell. In other embodiments, the plant source is  Camellia oleifera  and in some embodiments the saponin material is extracted from the defatted seed meal of  Camellia oleifera.    
         [0073]    In some embodiments, the formulation of the invention is a cosmetic formulation comprising an additive according to the invention. 
         [0074]    In other embodiments, the formulation of the invention is a therapeutic formulation comprising an additive according to the invention. 
         [0075]    In some embodiments, the additive is selected from the formulations listed in Table 1, Table 2, Table 3 or Table 4 above. 
         [0076]    In some embodiments, and for the purpose of demonstrating a cosmetic formulation according to the invention, in Table 5, a shampoo formulation is provided: 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 A shampoo formulation according to the invention. 
               
             
          
           
               
                   
                 Ingredient 
                 Chemical Family 
               
               
                   
                   
               
               
                   
                 
                   Sapindus Mukorossi 
                 
                 Saponins 
               
               
                   
                 extract and/or 
               
               
                   
                 
                   Camellia oleifera 
                 
               
               
                   
                 extract and/or 
               
               
                   
                   Quillaja  extract 
               
               
                   
                 Aspen bark ( Populus   
                 Salicaceae 
               
               
                   
                   tremuloides ) extract 
               
               
                   
                 Soy-bean Lecithin 
                 Phospholipids 
               
               
                   
                 Jojoba oil and/or 
                 Emollient 
               
               
                   
                 Squalene oil 
               
               
                   
                 Egg Yolk 
                 Phospholipids + cholesterol + 
               
               
                   
                   
                 Emollient 
               
               
                   
                 Sodium Hyaluronate 
                 Poly saccharides 
               
               
                   
                 and/or Xanthan gum 
               
               
                   
                 and/or Carrageenan 
               
               
                   
                 Mannitol and/or 
                 Poly-ol 
               
               
                   
                 Trehalose and/or 
               
               
                   
                 Zemea and/or 
               
               
                   
                 Glycerol 
               
               
                   
                   
               
             
          
         
       
     
         [0077]    In some embodiments, an exemplary shampoo of the invention is provided in Table 6: 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 A shampoo formulation according to the invention 
               
             
          
           
               
                   
                   
                 Conc. Range 
               
               
                 Ingredient 
                 Chemical Family 
                 wt % 
               
               
                   
               
               
                 
                   Sapindus Mukorossi 
                 
                 Saponins 
                 0.5-6   
               
               
                 extract 
               
               
                 
                   Camellia oleifera 
                 
                 Saponins 
                 0-6 
               
               
                 extract 
               
               
                   Quillaja  extract 
                 Saponins 
                 0-6 
               
               
                 Aspen bark 
                 Salicaceae 
                 0.1-0.8 
               
               
                 ( Populus   
               
               
                   tremuloides ) extract 
               
               
                 Soy-bean Lecithin 
                 Phospholipids 
                 0.1-5   
               
               
                 Jojoba oil 
                 Emollient 
                 0.2-4   
               
               
                 Squalene oil 
                 Emollient 
                 0.2-4   
               
               
                 Egg Yolk 
                 Phospholipids + cholesterol + 
                 0.2-5   
               
               
                   
                 Emollient 
               
               
                 Sodium Hyaluronate 
                 Poly saccharides 
                   0-0.8 
               
               
                 Xanthan gum 
                 Poly saccharides 
                   0-2.5 
               
               
                 Carrageenan 
                 Poly saccharides 
                   0-2.5 
               
               
                 Mannitol 
                 Poly-ol 
                   0-2.5 
               
               
                 Glycerol 
                 Poly-ol 
                  0-15 
               
               
                 Zemea 
                 Poly-ol 
                  0-15 
               
               
                   
                   
                 optional 
               
               
                 Trehalose 
                 Poly-ol 
                   0-2.5 
               
               
                   
                   
                 optional 
               
               
                 Perfume 
                   
                   0-1.5 
               
               
                   
               
             
          
         
       
     
         [0078]    A shampoo formulation of Table 6 is hereinbelow referred to as Shampoo no. 1 (see experimental details below). 
         [0079]    In some embodiments, the formulations of the invention comprise a selection of natural ingredients selected from at least one saponin material; at least one phospholipid; at least one emollient; at least one poly saccharide; and at least one alcohol or polyol. Depending on the nature of the formulation, it may further comprise at least one therapeutic agent; at least one coloring agent; at least one solvent, excipient or diluents. 
         [0080]    The formulation may be in the form of a solid or liquid ready for use formulation. 
         [0081]    Any additive formulation according to the invention may be used as a formulation for reducing irritation of a ready-made formulation for application onto a skin or membrane tissue region of a subject. Thus, the formulations of the invention are also applicable to reduce or diminish irritations of a skin region by a formulation suitable for application on such a region. The formulations of the invention are not solely applicable for reduction of irritation in the eye. 
     
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     Methods 
       [0082]    Evaluation of the Suppression or Attenuation of Ocular Irritancy 
         [0083]    The suppression of the ocular irritancy of a formulation which comes in contact with an ocular membrane by the addition of the additive of the invention to the formulation can be tested using one of several commonly used methods, including the Draize eye test, Bovine Cornea Opacity Test (BCOP), Isolated chicken eye test (ICE), hens egg chorioallantoic membrane (HET-CAM) assay, epithelial model cultivated from human corneal cells. A combination of several tests other than the Draize eye test is considered to provide reliable replacement for the animal Draize eye test. 
         [0084]    Bovine Cornea Opacity Test (BCOP) 
         [0085]    The BCOP test can be performed by following the procedure in the OECD Guidelines for the Testing of Chemicals, Section 4 test no. 437 or Pierre Gautheron et al.  Fundamental and Applied Toxicology  1992 18, 442-449. The BCOP test uses isolated corneas from the eyes obtained as a by-product from abattoirs. Each treatment group (test substance, negative/positive controls) consists of a minimum of three eyes where the cornea has been excised and mounted to a holder. The critical factor while applying the formulations which comprise the no-tear additive (or the control formulations) is ensuring that the test solution adequately covers the epithelial surface. Irritancy effects to the cornea are measured as permeability to fluorescein and opacity, which when combined gives an In Vitro Irritancy Score (IVIS) for each treatment group. Permeability is determined by measuring the amount of fluorescein dye that penetrates through the cornea. Opacity is determined by measuring the amount of light transmitted through the cornea. A substance that induces an IVIS equal to or higher than 55.1 is defined as a corrosive or severe irritant. 
         [0086]    Isolated Chicken Eye Test (ICE) 
         [0087]    The ICE test can be performed by following the procedure in the OECD Guidelines for the Testing of Chemicals, Section 4 test No. 438. Similarly to the BCOP test, The ICE method uses eyes collected from chickens obtained from slaughterhouses where they are killed for human consumption. The eye is enucleated and mounted in an eye holder with the cornea positioned horizontally. The test substance and negative/positive controls are applied to the cornea. Toxic effects to the cornea are measured by a qualitative assessment of opacity, a qualitative assessment of damage to epithelium based on fluorescein retention, a quantitative measurement of increased thickness (swelling), and a qualitative evaluation of macroscopic morphological damage to the surface. The endpoints are evaluated separately to generate an ICE class for each endpoint, which are then combined to generate an Irritancy Classification for each test substance. 
         [0088]    Hens Egg Chorioallantoic Membrane (HET-CAM) Assay 
         [0089]    Hen eggs are placed in commercial incubators. On day 10 of development, the eggs are removed from the incubator and candled to determine the viability of the embryo. A rectangular window is removed from the shell directly over the air cell and the egg membrane is carefully moistened with 2-3 ml 0.9% saline and returned to the incubator. 
         [0090]    The eggs are then dosed and observed continuously for 5 minutes for the appearance of lysis, hemorrhaging and/or coagulation which is documented. 
         [0091]    In addition, the eggs are scored for severity at 1 and 5 minutes. The severity of each reaction after 1 and 5 minutes is recorded. 
         [0092]    Human Corneal Epithelial Cell Line 
         [0093]    An immortalized human corneal epithelial (HCE) cell line was purchased from ATCC Company. HCE cells were established by infecting primary human corneal epithelial cells with a recombinant SV40-adenovirus vector and by cloning three times to obtain a continuously growing cell line. Initially, HCE cells were grown in a culture medium containing 1 vol of Dulbecco&#39;s modified Eagle&#39;s medium and 1 vol of Ham&#39;s nutrient mixture F-12 supplemented with 15% (vol/vol) fetal bovine serum, 1% (vol/vol) antibiotic, antimycotic solution (penicillin 10,000 U/ml, streptomycin 10,000 mg/ml and amphotericin B 25 mg/ml), 2 mM L-glutamine, 5 mg/ml insulin, 10 ng/ml human epithelial growth factor, 0.5% (vol/vol) dimethylsulfoxide (DMSO, Sigma). For cytotoxicity testing, HCE cells were grown in the same medium and plated at a density of 15,000-30,000 cells/well in a 96-well microtiter plate. The cells were exposed to the test compound-containing medium 24 hours after plating (before the cell culture started to form multilayers and became confluent). 
         [0094]    Human cells were incubated in an appropriate medium with the diluted shampoo samples (1:75) for 10 min, and were compared with negative (SLES) and positive controls (saline). After 24 hour incubation at 37° C. under 75% humidity and 5% CO 2  atmosphere, the cells were examined for viability by the MTT method (see Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.  J. Immunol Methods.,  1983 65: 55-63). To this end (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide from Sigma Israel was added to the medium. The calorimetric measurement was performed by an ELISA reader. 
       Example 1 
       Sapindus Mukorossi  Extract Concentration 
       [0095]    HCE cell line was exposed to three shampoo formulations: the shampoo formulation of Table 6 (herein referred to as Shampoo No. 1) having an amount  Sapindus mukorossi  extract of 2.25 weight %, and two other Shampoo formulations, Shampoo No. 2 and Shampoo No. 3 which deviate from Shampoo No. 1 in the concentration of  Sapindus mukorossi  extract by having 1.5 and 3.0 wt %, respectively. The HCE cell line was exposed to of Shampoo No. 1 at room temperature such that 50% of the ECH cell line remained viable. The HCE cell line was then exposed to each one of Shampoo No. 2 and Shampoo No. 3, respectively at the same conditions which it was exposed to Shampoo No. 1 and the viability percentage of the HCE cell line was measured, and the result described in Table 7. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 % Viability of HCEC as a function of  Sapindus mukorossi   
               
               
                 extract concentration in shampoo (the shampoo&#39;s composition 
               
               
                 as described in table 1, except the %  Sapindus mukorossi  extract) 
               
             
          
           
               
                 Shampoo 
                 
                   Sapindus mukorossi 
                 
                 Viability of HCEC (%) 
               
               
                 No. 
                 extract (wt %) 
                 (±10%) 
               
               
                   
               
             
          
           
               
                 2 
                 1.5 
                 100% 
               
               
                 1 
                 2.25 
                 50% 
               
               
                 3 
                 3 
                 22% 
               
               
                   
               
             
          
         
       
     
         [0096]    As one can see, the highest viability was obtained when  Sapindus Mukorossi  extract concentration was the lowest and it may thus be determined that  Sapindus Mukorossi  extract is an irritant agent 
       Example 2 
     Chamomile Extract 
       [0097]    Chamomile extract is known for treating inflammation associated with hemorrhoids when applied topically. According to the prior art, chemical components of chamomile extract demonstrated anti-inflammatory, anti-hyperglycemic, anti-genotoxic, and anti-cancer properties. Hence we studied its ability to reduce eye irritation. However, the addition of chamomile extract (1 wt %) to Shampoo No. 3 under the same exposure conditions to Shampoo Nos. 1 to 3, did not change the viability percentage of HCEC, which remained about 22%. 
       Example 3 
     Film Creation on Top of the Eye 
       [0098]    Lecithins Composition and Content 
         [0099]    The viability percentage of HCEC as a function of lecithin type (egg or soy-bean) and their concentration in the shampoo were studied. The result described in Table 8. 
         [0100]    The lecithins listed in Table 8, were added to the shampoo described in Table 6 (i.e., 0.8% soy-bean lecithin+0.5 egg yolk or 1% egg yolk+0.3% soy-bean lecithin). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 % Viability of HCEC as a function lecithins type in the shampoo (the 
               
               
                 shampoo&#39;s composition as described in Table 6, 3 wt %  Sapindus  extract) 
               
             
          
           
               
                   
                   
                 Viability of HCEC 
               
               
                   
                 Lecithin type and percentage 
                 (%) (±10%) 
               
               
                   
                   
               
               
                   
                 0.5 wt % soy-bean lecithin (addition) 
                 20% 
               
               
                   
                 0.5 wt % egg yolk (addition) 
                 60% 
               
               
                   
                 No addition 
                 22% 
               
               
                   
                   
               
             
          
         
       
     
         [0101]    As the results indicate, the addition of 0.5% of soy-ban lecithin did not change the viability percentage of HCEC. However, the addition of 0.5% of egg yolk improved the viability percentage significantly. 
       Example 4 
     Comparative Example 
     Shampoo Formulations Including 
       [0102]      
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Shampoo formulations 
               
             
          
           
               
                   
                 Chemical 
                 Formulation 1 
                 Formulation 2 
                 Formulation 3 
                 Formulation 4 
               
               
                 Ingredient 
                 Family 
                 Conc. wt % 
                 Conc. wt % 
                 Conc. wt % 
                 Conc. wt % 
               
               
                   
               
             
          
           
               
                 
                   Sapindus Mukorossi 
                 
                 Saponins 
                   
               
               
                 extract 
               
               
                   Camellia oleifera  extract 
                 Saponins 
               
               
                   Quillaja  extract 
                 Saponins 
               
               
                 Aspen bark ( Populus   
                 Salicaceae 
               
               
                   tremuloides ) extract 
               
               
                 Soy-bean Lecithin 
                 Phospholipids 
               
               
                 Jojoba oil 
                 Emollient 
               
               
                 Squalene oil 
                 Emollient 
               
               
                 Egg Yolk 
                 Phospholipids + 
               
               
                   
                 cholesterol + 
               
               
                   
                 Emollient 
               
               
                 Sodium Hyaluronate 
                 Poly 
               
               
                   
                 saccharides 
               
               
                 Xanthan gum 
                 Poly 
               
               
                   
                 saccharides 
               
               
                 Carrageenan 
                 Poly 
               
               
                   
                 saccharides 
               
               
                 Mannitol 
                 Poly-ol 
               
               
                 Glycerol 
                 Poly-ol 
               
               
                 Zemea 
                 Poly-ol 
               
               
                 Trehalose 
                 Poly-ol 
               
               
                 Perfume 
               
               
                   
               
               
                 Formulation 1 - Egg + Soy (according to the invention) 
               
               
                 Formulation 2 - Egg only 
               
               
                 Formulation 3 - Soy only 
               
               
                 Formulation 4 - Other lechitin (not soy or egg) 
               
             
          
         
       
     
       Example 5 
     Reduction of Oils and Lecithins 
       [0103]    The effect of oils and lecithin on the viability percentage of HCEC was tested in the shampoo described in Table 6, but in each test, one of the following ingredients was missed out (replaced by water). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 % Viability of HCEC as function of oil and lecithin absence in the 
               
               
                 shampoo (the shampoo composition as described in Table 6, 
               
               
                 except 2.25%  Sapindus  extract) 
               
             
          
           
               
                   
                   
                 Viability of HCEC 
               
               
                   
                 missed out component 
                 (%) (±10%) 
               
               
                   
                   
               
               
                   
                 oil 
                 20% 
               
               
                   
                 soya lecithin 
                 40% 
               
               
                   
                 Whole shampoo 
                 50% 
               
               
                   
                   
               
             
          
         
       
     
         [0104]    The results presented in Table 10 demonstrate that when oil or soy-bean lecithin was absent, the viability was reduced. The higher reduction obtained while the oil was absent. 
       Example 6 
     Hydrogel Creation 
       [0105]    Hydrogels of sodium hyaluronate have been used for many years in ophthalmic surgery in Europe and in the U.S. to maintain the shape of the eye, to cover surgical instruments and to protect the corneal endothelium from damage, thus we test its influence. 
         [0106]    Reduction of Sodium Hyaluronate 
         [0107]    The effect of sodium hyaluronate absence on the viability percentage of HCEC was tested on the shampoo described in Table 6, but the sodium hyaluronate replaced by water. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 % Viability of HCEC as function of sodium hyaluronate absence in 
               
               
                 the shampoo (the shampoo composition as described in Table 7, 
               
               
                 except 2.25%  Sapindus  extract) 
               
             
          
           
               
                   
                   
                 Viability of HCEC 
               
               
                   
                 missed out component 
                 (%) (±10%) 
               
               
                   
                   
               
               
                   
                 Without sodium hyaluronate 
                 35% 
               
               
                   
                 Whole shampoo 
                 50% 
               
               
                   
                   
               
             
          
         
       
     
         [0108]    The results presented in Table 11 demonstrate that when sodium hyaluronate was absent, the viability was reduced. 
       Example 7 
     The Influence of Saponin Type 
       [0109]      Camellia oleifera  Seed Extract (Tea) 
         [0110]      Quillaja Saponaria  Extract 
         [0111]      Saponaria officinalis  Extract 
         [0112]    In order to compare the effect of the saponin extracts from different plants, the  Quillaja  extract (Table 6) was replaced once with  Camellia  extract, and seconds with  Saponaria  extract (All shampoos contained 2.25 wt %  Sapindus  extract). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 % Viability of HCEC as a function of saponins type in the shampoo 
               
               
                 (the shampoo composition as described in Table 6, 
               
               
                 except 2.25%  Sapindus  extract) 
               
             
          
           
               
                   
                 Saponin type 
                 Viability of HCEC (%) (±10%) 
               
               
                   
                   
               
             
          
           
               
                   
                   Quillaja  extract 
                 50% 
               
               
                   
                   Camellia  extract 
                 100% 
               
               
                   
                   Saponaria  extract 
                 100% 
               
               
                   
                   
               
             
          
         
       
     
         [0113]    As the results indicate, the viability percentage increased significantly when  Quillaja  replaced by  Camellia  or  Saponaria  extracts. 
         [0114]    In Vivo Methods 
         [0115]    One drop of diluted shampoo; 6 wt. % by saline, was instilled into one volunteers&#39; eye, and one drop of saline his other eye. Each shampoo&#39;s composition was examined on 10 volunteers. The evaluations of the eye irritation criteria were as follows: 
         [0116]    The intensity of eye irritation: burning, stinging and/or itching according to the following scale:
       None   Slight prickling, tingling and/or slight burning and/or stinging and/or slight itching   Moderate burning and/or stinging and/or moderate itching   Severe burning and/or stinging and/or strong itching       
 
         [0121]    The evaluations of the eye irritation were done immediately after the after 8-10 min, 15 min, and 1 hour. The scores were collected, and average of the scores is appearing in the following tables. 
         [0122]    The study was accompanied by an ophthalmologist. 
         [0123]    Egg Yolk Concentration 
         [0124]    The eye irritation of shampoos containing different egg yolk concentration was studied, and the result described in Table 13. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 irritation average score as a function of egg yolk concentration (shampoo 
               
               
                 composition as described in Table 6) 
               
             
          
           
               
                   
                 Egg yolk concentration 
                   
               
               
                   
                 (wt %) 
                 Average Score 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 2.5 (±0.4)   
               
               
                   
                 0.5 
                 1.1 (±0.3)   
               
               
                   
                 1.5 
                 0.2 (±0.3)   
               
               
                   
                   
               
             
          
         
       
     
         [0125]    As one can see in Table 13, significant reduction in eye irritation was obtained while increasing the egg yolk concentration. 
         [0126]    Chamomile Extract 
         [0127]    The effect of chamomile extract (1 wt % addition) on eye irritation was tested in the shampoo described in Table 6. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 irritation average score as a function of chamomile addition (shampoo 
               
               
                 composition as described in Table 6) 
               
             
          
           
               
                   
                   
                 Average 
               
               
                   
                 Chamomile addition 
                 Score 
               
               
                   
                   
               
               
                   
                 No addition 
                 1.1 (±0.3)   
               
               
                   
                 1 wt % chamomile addition 
                 1.3 (±0.3)   
               
               
                   
                   
               
             
          
         
       
     
         [0128]    The addition of chamomile extract to shampoo didn&#39;t improve the eye irritation as was demonstrated also at the in-vitro test. 
         [0129]    Sodium Hyaluronate 
         [0130]    The effect of sodium hylorunate on eye irritation was tested in the shampoo described in Table 6, with or without sodium hylorunate. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 irritation average score with or without sodium hylorunate (shampoo 
               
               
                 composition as described in Table 7, 5 wt % egg yolk) 
               
             
          
           
               
                   
                   
                 Average 
               
               
                   
                 Sodium hyaluronate addition 
                 Score 
               
               
                   
                   
               
               
                   
                 With 0.05 wt % sodium 
                 0.2 (±0.3)   
               
               
                   
                 hylorunate 
               
               
                   
                 Without sodium hylorunate 
                   1 (±0.3)   
               
               
                   
                   
               
             
          
         
       
     
         [0131]    The eye irritation was decreased significantly when the shampoo content 0.05 wt % of sodium hylorunate. 
       Example 8 
     Haemolysis Test 
       [0132]    Haemolysis test (which serves as a model for eye irritation) is conducted in order to determine the safety of the products of the invention in comparison to the commercial surfactant SLES (sodium lauryl ether sulfate). 
         [0133]    Preparation of the erythrocyte suspension: erythrocytes of sheep blood is separated by centrifugation at 1250 g, for 15 minutes at room temperature, wash three times with phosphate-buffered saline solution (PBS, pH 7.4), and centrifuge twice under the same condition. The blood volume is completed with PBS. This suspension is maintained at 4 degrees centigrade for up to three days. 
         [0134]    The assay procedure: 20 μL of each sample are diluted up to ml of the suspension, and are incubated with for 30 minutes in ice. The incubation is terminated by a rapid, high-speed (1800 g) centrifugation for 30 minutes. The extent of haemolysis is determined in spectrophotometrically at 540 nm against a blank (PBS). The extent of haemolysis, expressed as a percentage, is calculated as the absorbance of an erythrocyte suspension incubated with each product, relative to that of a completely haemolysed control (100 percent, at distilled water) at 540 nm. The Hm50 (50 percent haemolysis) is determined from concentration-response curves. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 28 
               
             
             
               
                   
               
               
                 Hm 5o  of  Sapindus  extract, the sampoo describe in 
               
               
                 table 6 with or without lecithin 
               
             
          
           
               
                   
                 Hm 50   
               
               
                   
                   
               
             
          
           
               
                   
                   Sapindus Mukorossi  extract 
                   
               
               
                   
                   Sapindus Mukorossi  extract and egg 
               
               
                   
                 yolk lecithin 
               
               
                   
                   Sapindus Mukorossi  extract and soy- 
               
               
                   
                 bean lecithin 
               
               
                   
                   Sapindus Mukorossi  extract and 
               
               
                   
                 lecithin (soy been and egg yolk) 
               
               
                   
                 formulation 1 described in Table 9 
               
               
                   
                 formulation 2 described in Table 9 
               
               
                   
                 formulation 3 described in Table 9