NZ614287B2 - Bioactive botanical cosmetic compositions and processes for their production - Google Patents

Abstract

Disclosed is a bioactive botanical cosmetic composition comprising: a cell serum fraction filtrate derived from cell juice extracted from a fresh plant biomass, the cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation activities, wherein the fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucifera) and red clover (Trifolium pratense); and a stabilising agent, wherein the cell growth stimulation activity is due to stimulation of proliferation of at least one type of cell, wherein the cell serum fraction filtrate is derived from the cell juice according to the following steps: providing the plant cell juice, the plant cell juice having been extracted from the fresh plant biomass; treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; and refining the cell serum fraction under conditions effective to yield the cell serum fraction filtrate, wherein the refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate. stimulation activities, wherein the fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucifera) and red clover (Trifolium pratense); and a stabilising agent, wherein the cell growth stimulation activity is due to stimulation of proliferation of at least one type of cell, wherein the cell serum fraction filtrate is derived from the cell juice according to the following steps: providing the plant cell juice, the plant cell juice having been extracted from the fresh plant biomass; treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; and refining the cell serum fraction under conditions effective to yield the cell serum fraction filtrate, wherein the refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate.

Description

BIOACTIVE BOTANICAL COSMETIC COMPOSITIONS AND PROCESSES FOR THEIR PRODUCTION CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part ofUS. Patent Application Serial No. 12/116,924, filed May 7, 2008, which is a divisional of US. Patent Application Serial No. /351,910, filed January 24, 2003, now US. Patent No. 7,442,391, issued October 28, 2008, which claims the priority benefit ofUS. Provisional Patent Application Serial No. ,886, filed January 25, 2002, the sures of which are hereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION {0002] The present invention relates to bioactive botanical cosmetic compositions and processes for their production and their use.

BACKGROUND OF THE INVENTION Over the past several decades, the cosmetic industry has embraced the use of plants and plant products in a variety of ic formulations and products. Although this trend is expected to continue, there is a need for more refined and higher quality botanical ingredients that consistently exhibit characteristics that are appealing to the cosmetic industry and consumers Some ofthese appealing bioactive teristics include anti-inflammatory and antioxidant activity. Coloration, safety, compatibility, and increased shelf life are also valuable characteristics of cosmetic formulations d from botanical ingredients.

[0004] The cosmetic industry as a whole has increased its support of efforts to develop and market “natural” cosmetic formulations using a host of single and blended botanical ingredients that are currently available to the industry. This approach differs from the synthetic ient—based approach that has allowed the ic industry to develop cosmetics with consistent product integrity, performance, and shelf life ofraw al ingredients. One ofthe major deterrents toward the use of botanical ingredients is the inconsistency of the performance and stability of the ingredients, ally with regard to bio active botanical ingredients. Many of the bioactive botanical cosmetic ingredients used as ients in cosmetic formulations exhibit lost potency, odor ions, unwanted darkening in coloration, and undesirable sedimentation. These negative utes increase the risk of microbiological ination and proliferation, instability, and safety ns with regard to the final products made from the bioactive botanical ingredients.

In order to ensure quality, safety, and consistency, the cosmetic industry has developed and ented s rd operating procedures and strict specification controls for all incoming raw materials for use in cosmetic formulations. Most, if not all, of the current botanical extracts fail to comply with the increasing controls and consistency parameters of the cosmetic industry. Current plant extraction methods limit product specification parameters leaving many windows of variability for quality, performance, and compatibility. In addition, current extraction methods fail to deliver the full spectrum of activities that exist within plant cells. Thus, the full ial of botanical-based cosmetic formulations is not being realized due to the inadequacy of the extraction methods for bioactive botanical cosmetic ingredients.

Many of the t s for ting bioactive components from plants involve techniques that are l to the plant tissue or the bioactive components of interest contained in that tissue, or both. Further, many of the current extraction and separation methods yield crude botanical extracts that contain biological or chemical contaminants that can cause a loss of bioactivity potency, increased cytotoxicity, and decreased shelf life.

Further, in order to yield more refined botanical extracts, current extraction methods often e the use of harsh chemical solvents.

[0007] Thus, there is a need for a method of extracting bioactive botanical compositions that preserves the bioactivity of the ition and that yield tent results from lot-to-lot. Further, botanical compositions that are able to meet the industry standards with respect to shelf life, cytotoxicity, quality, and performance are needed in the cosmetic industry.

[0008] The present invention is directed to ming these ncies in the art.

SUMMARY OF THE INVENTION In one aspect of the present invention, there is provided a bioactive botanical cosmetic composition comprising: a cell serum fraction filtrate derived from cell juice extracted from a flesh plant biomass, said cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation activities, wherein said fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucz’fera) and red clover (Trifolz'um pratense); and _ 2 _ [followed by page 2a] a stabilizing agent, wherein said cell growth stimulation activity is due to stimulation of proliferation of at least one type of cell, n said cell serum fraction filtrate is derived from the cell juice according to the ing steps: providing the plant cell juice, said plant cell juice having been extracted from the fresh plant biomass; ng the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; sing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; and g the cell serum fraction under conditions effective to yield the cell serum fraction filtrate, wherein said refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum on filtrate. [0009a] In a further aspect of the present ion, there is provided a method for preparing a ive botanical cosmetic composition, said method comprising: ing a plant cell juice, said plant cell juice having been extracted from a fresh plant biomass, wherein said fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucifera) and red clover (Trz’folz‘um pratense); treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; refining the cell serum fraction under conditions ive to yield a cell serum fraction filtrate having antioxidant activity, cell growth stimulation ty, and/or both antioxidant and cell growth stimulation activities, wherein said refining comprises subjecting the cell serum fraction to a ature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate; and stabilizing the cell serum fraction filtrate under conditions effective to yield a stable bioactive botanical cosmetic composition exhibiting said antioxidant activity, cell growth stimulation activity, or both antioxidant and cell growth stimulation activities. .231. [followed by page 2b] [0009b] The present invention relates to a bioactive cal cosmetic composition including (1) a membrane fraction derived from cell juice extracted from a fresh plant biomass and (2) a stabilizing agent. The membrane fraction has antiproteolytic activity, cell growth inhibition activity, and/or both antiproteolytic and cell growth inhibition activities.

The oteolytic activity is due to inhibition of at least one proteinase and the cell growth inhibition activity is due to inhibition of proliferation of at least one type of cell. -21)- [followed by page 3] W0 2012/148527 PCT/U52012/025899 The present invention also s to a bioactive botanical cosmetic ation suitable for topical application to a mammal. The bioactive botanical ic formulation includes a cosmetically acceptable carrier and a cosmetically effective amount of the bioactive cal cosmetic composition described above.

The present invention also relates to a method for inhibiting anti—inflammatory activity in skin tissue of a mammal. This method involves applying to the skin tissue the above described bioactive botanical cosmetic composition in an amount effective to enhance the antiproteolytic activity in the skin tissue.

The t invention also relates to a method for normalization of cell disorders in skin tissue of a mammal. This method involves applying to the skin tissue the above~described bioactive botanical ic composition in an amount effective to inhibit unwanted hyper-proliferation of skin cells.

The present invention also relates to a method for preparing a bioactive botanical ic composition, which involves providing a plant cell juice that has been extracted from a fresh plant s. The plant cell juice is then treated under conditions effective to separate it into a membrane on and a cell juice supernatant. The membrane fraction is transformed under conditions effective to yield a stable bioactive botanical cosmetic composition exhibiting antiproteolytic, cell growth inhibition activity, and/or both antiproteolytic and cell growth inhibition ties, where the antiproteolytic activity is due to tion of at least one nase and the cell growth inhibition activity is due to inhibition of cell growth of at least one type of cell.

The present invention also relates to a bio active botanical cosmetic composition made by the method described immediately above.

The present invention also relates to a bioactive botanical cosmetic formulation suitable for topical application to a mammal, The formulation includes a cosmetically acceptable carrier and a cosmetically ive amount ofthe ive botanical cosmetic composition described immediately above.

The present invention also relates to a method for inhibiting anti-inflammatory activity in skin tissue of a mammal by ng to the skin tissue thc ive botanical cosmetic composition described above in an amount effective to enhance the antiproteolytic activity in the skin tissue.

The present invention firrther relates to a method for normalization of cell disorders in skin tissue of a mammal, involving applying to the skin tissue the bioactive PCT/U52012/025899 botanical cosmetic composition having cell growth inhibition activity in an amount effective to inhibit unwanted proliferation of skin cells.

The t invention also relates to a bioactive botanical cosmetic composition including the membrane on made by the method described above.

The present invention also relates to a bioactive botanical cosmetic composition including (1) a cell serum fraction derived from cell juice extracted from a fresh plant biomass, where the cell serum fraction has antioxidant activity, cell growth ation activity, and/or both antioxidant and cell growth stimulation activities, and (2) a stabilizing agent. The cell growth stimulation ty is due to stimulation of proliferation of at least one type of cell.

The present ion also relates to a bioactive botanical ic formulation suitable for topical application to a mammal, including a cosmetically acceptable r and a cosmetically effective amount ofthe bioactive botanical cosmetic ition described immediately above.

[0021] The present invention fiirther relates to a method for enhancing the antioxidant activity in skin tissue of a mammal, involving applying to the skin tissue of the mammal the bioactive botanical cosmetic formulation described above in an amount effective to increase the antioxidant activity in the skin tissue.

The present invention also relates to a method for stimulation of cell proliferation in skin tissue of the mammal, involving applying to the skin tissue the bioactive botanical cosmetic formulation described above in an amount effective to stimulate fibroblast proliferation in the skin tissue.

The present ion also relates to a method for preparing a bioactive botanical cosmetic composition, which involves providing a plant cell juice that has been extracted from a fresh plant biomass. The plant cell juice is then treated under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant.

The cell juice supernatant is processed under conditions ive to separate the cell juice supernatant into a cytoplasm fraction and a cell serum fraction. The cell serum fraction is refined under conditions effective to yield a cell serum fraction te. The cell scrum fraction filtrate is stabilized under conditions ive to yield a stable bioactive botanical cosmetic composition ting antioxidant activity, cell growth stimulation activity, or both antioxidant and cell growth ation activities.

The present invention also relates to a stable bioactive botanical cosmetic composition made by the method bed immediately above.

W0 2012/148527 2012/025899 The present invention also relates to a bioactive botanical ic formulation suitable for topical application to a mammal, including a cosmetically acceptable carrier and a cosmetically effective amount ofthe ive botanical cosmetic composition made by the process bed above.

The present invention also relates to a method for enhancing the antioxidant activity in skin tissue of a mammal. This method involves applying to the skin tissue the bioactive botanical cosmetic composition described above in an amount effective to increase the antioxidant activity in the skin tissue.

The t invention further relates to a method of stimulation of cell eration in skin tissue of a mammal. This method involves applying to the skin tissue the bioactive botanical cosmetic composition described above in an amount effective to stimulate fibroblast proliferation in the skin .

The method for preparing bioactive botanical cosmetic compositions is advantageous over the methods currently available in that it yields plant extracts that capture the filll spectrum of activity ned in the plant cells. These extracts can then be separated into either cell serum or membrane components, while still maintaining the bioactivity contained within each component. Further, the compositions produced according to the method of the present invention have xicity s that are demonstrably safer for skin than other conventional plant extracts. In addition, the compositions of the present invention meet the microbial requirements of the cosmetic industry. Thus, due to the consistency, quality, safety, shelf life, and significant ivity potency with regard to anti- inflammatory and antioxidant capabilities, the bioactive botanical cosmetic compositions of the present ion are significant improvements over the botanical cosmetic ingredients available currently.

[0029] The bioactive botanical cosmetic itions of the present invention exhibit the anti-inflammatory and antioxidant ties that are valuable to the cosmetic industry.

Further, the eytotoxicity profiles of the bioactive botanical cosmetic compositions are within the industry standards for ic ingredients and exhibit cell pro liferative stimulatory activity and certain cell growth tory activity at levels that are advantageous as topical skin cosmetics. The method for ing the bioactive botanical cosmetic ingredients of the present invention may be used on a wide variety of plants to yield consistent, stable, and quality bioactive botanical cosmetic compositions.

The bioactive botanical cosmetic compositions of the present invention meet the industry standards with respect to the microbial requirements of cosmetic raw material ingredients. The industry standard requires that all active and inactive ingredients (i.e., all exeipients of the cosmetic formulations) not be such that they contribute to the finished formulation composition of a cosmetic product. Typically, these finished formulation compositions have preservative systems that prevent microbial contamination that could risk the integrity ofthe product. In one standard use by the ry to test the protective strength of a preservative system, a product is subjected to a 28-day challenge test during which time microorganisms are inoculated into a product to see if it withstands these treatments without becoming contaminated. In addition, in the cosmetic ry, each ingredient is also scrutinized to make sure that the level of microorganisms is not so high as to result in subsequent contamination of a product or pose a risk on the shelf life ofthe product if it is not optimally ved.

Specifically, the industry standard statistic that “the microbiological requirements for active ingredients in the cosmetic area state that a total microbial count of a maximum 100 microorganisms per gram or per ml may be tolerated. The sample (10 g) must furthermore be free from Escherichia coli, Candida albicans, Pseudomonas sp., and Staphylococcus aureus” (G. A. Nowak, “Cosmetic ations,” fizr Chem. , Augsburg, 1:126 (1985), the entire disclosure ofwhich is incorporated herein by nce).

The bioactive botanical cosmetic compositions of the present invention satisfy the above requirements and therefore pose no risk to finished cosmetic formulation compositions.

[0032] The bioactive botanical cosmetic compositions of the present invention have highly valuable bioactive attributes with t to the skin, including, for example, antiinflammatory and antioxidant activities, as well as cell proliferative stimulatory characteristics. It is generally known that there is the balance between newly born and dead skin cells. m attributes of skin are found in young and healthy skin (i.e., usually found in people under the age of 25). Before this age, skin cells are in a regulated state and are in a alanced system ofrenewal; born at the deepest basal layers and ally proliferating (i.e., rising from the deep skin) to the top (i.e., the layer which we visually appreciate). This balance of cells being shed is part of an equilibrium of l. This equilibrium is lost as a result of adult aging, and there is a slow down in the proliferation rate after new cells are born. The concept of increasing or stimulating cell proliferation is based on restoring the optimum equilibrium that is found in “younger” skin. This has led to an interest in cell proliferation stimulators such as ids and AHA (alpha hydroxy acids). Such ingredients lly increase the rate ofproliferation through an irritation mode of action, leading to smoother, younger-looking skin due to rated cell proliferation. The bioactive botanical W0 2012/148527 PCT/U52012/025899 cosmetic compositions of the t invention, in particular those derived fi‘om the cell serum fractions, exhibit y to stimulate cell proliferation.

In a more comprehensive manner, increased skin proliferation is a key to wound healing and dermatological conditions. There are certain dermatological conditions whereby skin proliferation tends to be in a hyper—proliferative state. These conditions border and cross e states manifesting themselves on the skin. Conditions such as psoriasis, eczema, and dandruff are all hyper—proliferative conditions, Skin cell growth inhibitors are then the obvious and ted approach to slowing down the rate ofproliferation to normalize the rate. Various of the bioactive botanical cosmetic compositions ofthe present invention, in particular those derived from the membrane traction, exhibit such cell inhibition attributes. ation occurs for many reasons on the skin. Usually ated with , today s are beginning to understand the cascading effects o- inflammation. This micro~inflammation ofthe skin can result from irritating ingredients such as soaps and cytotoxic ingredients, ordinary UV light such as minimal sunlight, and in a more drastic manner from intense exposure to the sun. Recently, the role of inflammation on skin aging has been more clearly understood and suggested to be an indirect route to formation of free-radicals, which have been clearly ated for their role in membrane lipid oxidation.

Thus, anti-inflammatory agents are important cosmetic ingredients, but the regulatory restrictions limit their use as drugs. However, the bioactive botanical cosmetic compositions ofthe t invention, particularly those derived from the membrane fraction, trate anti-inflammatory attributes. {0035] The role of antioxidants has become increasingly important for nutrition and cosmetic products. Antioxidants retard, protect against, and help repair the adverse effects of oxidative degradation. In the plant world, nature has provided natural antioxidants that protect against many oxidative factors. The bioactive botanical ic compositions of the present invention, particularly those derived from the cell serum fraction, demonstrate such idant activity.

BRIEF DESCRIPTION OF DRAWINGS FIG, I is a schematic drawing demonstrating one embodiment of the process for preparing the bioactive botanical cosmetic compositions of the present invention.

PCT/U52012/025899 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to bioactive botanical cosmetic compositions derived from either the membrane fraction or the cell serum fraction of plants. As used herein, the term “Membrane-Derived Cosmetic ition” generally refers to a bioactive botanical cosmetic composition ofthe present invention that is derived from the membrane fraction of a plant. The term “Serum-Derived ic ition” generally refers to a bioactive botanical cosmetic composition of the present invention that is derived from the cell serum on ofa plant.

The present invention also relates to processes for ing the ive botanical ic compositions of the present invention, as well as methods for using the compositions.

Membrane-Derived Cosmetic Compositions The Membrane—Derived Cosmetic Compositions ofthe present ion include (1) a membrane fraction derived from cell juice extracted from a fresh plant biomass and (2) a stabilizing agent. The membrane fraction has antiproteolytic activity, cell growth inhibition activity, and/or both antiprotcolytic and cell growth inhibition activities. The antiproteolytic activity is due to inhibition of at least one proteinase and the cell growth inhibition activity is due to inhibition of proliferation of at least one type of cell. Examples of stabilizing agents that are suitable for use in the present ion include fiers, preservatives, antioxidants, polymers, and mixtures thereof.

In one aspect of the t invention, the Membrane-Derived Cosmetic Composition has antiprotcolytic activity against proteinase groups such as serinc protcinascs and matrix metalloproteinases. Examples of the serine proteinase include neutrophil elastase and trypsin inhibitor. An example of a matrix metalloproteinase is gelatinase B. In another aspect of the present invention, the inhibition of the proteinase is reversible. Since serinc proteinases have certain positive physiological roles when present at lled levels, use of reversible inhibitors will not impact these normal enzymatic functions. The reversible inhibition would not cause undesirable long term cations to defense and repair mechanisms which can be ed by irreversible inhibitors.

[0041] The Membrane-Derived Cosmetic Composition of the t invention has an antiproteolytic potency ranging from an ICSO value een about 0.1 and about 25.0 ug dry matter/ml. As used in the present application, the term “IC50 value” represents the PCT/U82012/025899 tration ofdry matter contained in the membrane fraction required to achieve 50 percent inhibition of the proteinase.

The Membrane-Derived ic Composition of the present invention has a cell growth inhibition ty potency ranging from an NRUsO value ofbetween about 25 and 500 ug dry matter/ml. As used in the present application, the term “NRU50 value” represents the concentration of dry matter in the membrane fraction required to reduce the Viability of the type ofcell to 50 percent. An example ofa type of cell that is inhibited from proliferating due to the Membrane—Derived Cosmetic Composition is a fibroblast.

The ne-Derived Cosmetic Composition ofthe present invention be derived from membrane fractions of all types of plants. Examples of suitable plants that may be used as sources of fresh plant biomass in the present invention include plants from the following families: Asteraceae, Fabaceae, eae, and Poaceae. In ular, examples of specific plants that have been tested and found riate as fresh plant s sources include, without limitation, Trz'folz'um pratense, Nelumbo ra, Calendula qflicz‘nalis, Medicago sativa, Lavarzdula angustifolia, Salvia aficinalz's, and Hordeum vulgare. The ne-Derived Cosmetic Composition may be derived from flower tissue (e.g., Trz‘folium pratense, Nelumbo nucifera, Calendula ofiicz‘nalz's) and/or from leaf and stem tissue (e. g., Trlfolium pratense, Nelumbo nuczfera. Salvia officinalis) ofplants.

In one embodiment, the membrane on d from the plant cell juice makes up between about 0.5 and about 95 weight percent ofthe Membrane-Derived Cosmetic Composition.

The Membrane-Derived ic Composition ofthe present invention can have the following specific physico—chemical values: (1) a non—volatile residue value of between about 0.1 and 30 percent; (2) a specific gravity value ofbetween about 0.5 and 2.0 g/cm3 ; (3) a viscosity value of between about 300 and 50,000 cps; and (4) a pH value of between about 2.5 and 9.5.

The present invention also relates to a bioactive botanical ic formulation suitable for topical application to a mammal, including to humans, where the formulation includes a cosmetically acceptable carrier and a cosmetically effective amount of the ne-Derived Cosmetic Composition. Examples of suitable cosmetically acceptable carriers for use in the present invention include a hydrophilic cream base, a hydrophilic lotion base, a hydrophilic surfactant base, a hydrophobic cream base, a hydrophobic lotion base, and a hydrophobic surfactant base. In one embodiment of the formulation, the Membrane- W0 2012/148527 PCT/U52012/025899 Derived Cosmetic Composition is present in an amount ranging from between about 0.001 percent and about 90 percent ofthe total weight of the formulation.

The present invention also relates to a method for inhibiting anti-inflammatory activity in skin tissue of a mammal, which method involves ng to the skin tissue the Membrane—Derived Cosmetic Composition in an amount effective to enhance the antiproteolytic ty in the skin tissue.

The present invention also s to a method for normalization of cell disorders in skin tissue of a mammal. This method involves applying to the skin tissue the Membrane—Derived Cosmetic Composition in an amount effective to inhibit ed hyper- proliferation of skin, cells.

Serum-Derived Cosmetic Compositions The Serum-Derived Cosmetic Compositions of the present invention include ( 1) a cell serum fraction derived fiom cell juice extracted from a fresh plant biomass, where the cell serum fiaction has antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation ties, and (2) a stabilizing agent. The cell growth stimulation activity is due to stimulation of eration ofat least one type of cell. Examples of stabilizing agents le for use in the present invention e a preservative and an antioxidant. Suitable preservatives for use in the present invention include potassium sorbate, sodium bcnzoatc, sodium methyl parabcn, and citric acid. An example ofa suitable antioxidant for use in the present invention is sodium metabisulfite.

In one embodiment, the antioxidant activity ofthe Derived Cosmetic Composition includes superoxidc scavenging activity and neutrophil respiratory burst inhibitory activity. The Serum-Derived Cosmetic Composition has a superoxide scavenging potency ranging from an lCRso value ofbetween about 50 and 190 ug of dry matter/ml. As used in the present application, the term “ICR50 value” represents the concentration of dry matter contained in the cell serum fraction required to inhibit 50 percent of cytochrome c ion. The cell serum-derived cosmetic ingredient has a cell growth stimulation potency ranging from between about 1.0 and 125 ug of dry matter/ml and an NRU value ofbetween about 110 and 190 t, where the “NRU value” represents cell viability. The Serum- Derived Cosmetic Composition inhibits the respiratory bursts at n about 1.0 and 5.0 dry material/ml and stimulates the respiratory bursts at between about 120 and 180 ug dry material/ml. The Serum—Derived Cosmetic ition has the y to cause biphasic modulation of atory bursts from phorbol myristate e—stimulated neutrophils.

W0 20] 27 PCT/U52012/025899 An example of a type of cell that is stimulated to proliferate due to the Serum- Derived Cosmetic Composition es a fibroblast.

The Serum-Derived Cosmetic Composition ofthe present invention may be derived from cell serum fractions fi'om all types of plants. Examples of suitable plants that may be used as sources of fresh plant biomass in the present invention include plants from the following families: Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular, examples of specific plants that have been tested and found appropriate as fresh plant biomass sources include, without limitation, Trz’folz’um pratense, Nelumbo nucz‘fem, Calendula ofiicinalz's, Aledz'cago sativa, ula angustz'folz'a, Salvia oflicz'nalz's, and Hardeum vulgare. The Serum-Derived Cosmetic Composition may be derived from flower tissue (e.g., olium pratense, Nelumbo nucz’fera, Calendula oflz‘cinalis) and/or from leaf and stem tissue (e.g., Trz'folz'um pratense, Nelumbo nucz‘fera, Horedeum vulgare, Lavandula angusnfolia, Medicago sativa, and Salvia ali‘s).

In one ment, the cell serum fraction derived from the plant cell juice makes up between about 1 and 10 weight percent of the Serum—Derived Cosmetic Composition.

The present invention also relates to a ive botanical cosmetic formulation suitable for topical application to a mammal, ing a cosmetically acceptable carrier and a cosmetically ive amount ofthe Derived Cosmetic Composition.

Examples of le cosmetically acceptable rs include, without limitation, a hydrophilic cream base, a hydrophilic lotion base, a hydrophilic surfactant base, a hydrophobic cream base, a hydrophobic lotion base, and a hydrophobic surfactant base. In one embodiment, the Serum—Derived Cosmetic Composition is present in an amount ranging from between about 0.001 percent and 95 t ofthe total weight of the cosmetic formulation.

The present ion further relates to a method for enhancing the antioxidant activity in skin tissue ofa mammal, involving applying to the skin tissue the Serum-Derived Cosmetic ition in an amount effective to increase the antioxidant activity in the skin tissue.

[0056] The present invention also relates to a method for stimulation of cell proliferation in skin tissue of a mammal, involving applying to the skin tissue the Serum- d Cosmetic Composition in an amount effective to stimulate cell proliferation in the skin tissue.

W0 2012/148527 Overall Process for Preparing ive Botanical Cosmetic Compositions By way of e, the overall process for ing the ive botanical cosmetic compositions of the present invention is described below in reference to As depicted in fresh plants are harvested, collected, and washed 2 to yield fresh plant biomass. This fi'esh plant biomass is subjected to grinding, maceration, and pressing 4 to yield plant cell juice 6 and press-cake 8. Plant cell juice 6 is then filtered h nylon mesh to yield filtered plant cell juice 12. Filtered plant cell juice 12 is exposed to microwave treatment 14 in order to ate plant cell juice 12. The coagulated plant cell juice is cooled 16 and then subjected to centrifugation 18 in order to yield membrane fraction 20 and plant cell juice supernatant 30. ne on 20 is used to prepare membrane-derived bioactive cal cosmetic composition 28 (i.e., the Membrane-Derived ic Composition), as described below. Plant cell juice supernatant 30 is used to prepare cell serum—derived bioactive botanical cosmetic composition 52 (i.e., the Serum—Derived Cosmetic Composition), as bed below.

[0058] To produce bioactive botanical cosmetic composition 28, membrane fraction is incorporated into polymer matrix 22 and stabilized with prepared polymers, preservatives, and antioxidants 24. The stabilized membrane fraction is then neutralized 26 to yield the membrane-derived bioactive botanical cosmetic composition 28.

To produce bioactive botanical cosmetic composition 52, plant cell juice supernatant 30 is subjected to isoelectric precipitation 32 to yield a mixture containing asm fraction 36 and cell serum fraction 38. In order to separate cell serum fraction 38 from cytoplasm fraction 36, the mixture is subjected to eentrifugation 34. Cell serum fraction 38 is then subjected to microwave treatment to cause coagulation 42. Depending on the plant source, prior to microwave ent, cell serum on 38 is first pH-adjusted. Afier coagulation 42, the mixture is then cooled 44, followed by filtration 46 to yield cell serum filtrate 48. Cell serum filtrate 48 is stabilized with preservatives and antioxidants 50 to yield cell serum—derived bioactive botanical cosmetic composition 52.

Process for Preparing the Membrane-Derived Cosmetic Compositions In one embodiment, the s for preparing the Membrane-Derived Cosmetic Compositions is as follows. This method involves providing plant cell juice that has been extracted from a fresh plant biomass. The plant cell juice is then treated under conditions effective to separate it into a membrane fraction and a cell juice supernatant. The W0 2012/148527 PCT/U52012/025899 resulting membrane fraction has antiproteolytic activity, cell growth inhibition activity, or both antiproteolytic and cell growth inhibition activities. The membrane fraction is then converted under conditions effective to yield a stable ive cal cosmetic composition exhibiting antiproteolytic, cell growth inhibition ty, or both antiproteolytic and cell growth inhibition activities, where the oteolytic ty is due to inhibition of at least one proteinase and the cell growth inhibition activity is due to inhibition of cell growth of at least one type of cell.

The plant cell juice may be extracted from all types ofplants. Examples of suitable plants that may be used as s of fresh plant biomass in the present include, without limitation, plants from the following families: Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular, examples of specific plants that have been tested and found appropriate as flesh plant biomass sources include, without limitation, Trz'folium pratense, Nelumbo nucz‘fera, Calendu/a oflicinalis, Medicago sativa, ula angustz‘folia, Salvz'a oflicz‘nalz‘s, and Hordeum e. Various parts of the plants may be used. For example, the stems and leaf tissue may be used for many types of plants. For other plants, the flowers be used as sources of plant cell juice for use in the present invention. For example, one embodiment ofthe present invention uses flower tissue of Trzfolz’um pratense, Nelumbo nucz‘fe/‘a, or Calendula oflicinalis for the extraction of the plant cell juice. In another embodiment, the leaf and stem tissue of Trzfoiz'um pratense, Nelumbo nucz‘fera, or Salvia ofiicz'nalis is used.

The plant cell juice may be extracted using various extraction techniques.

However, the extraction technique should result in plant cell juice that preserves the bioactive components ofthe plant.

An exemplary method ofpreparing the plant biomass for use in extraction of plant cell juice involves harvesting, collecting, and washing of the fresh plants. Suitable steps to follow for preparing the fresh plant biomass include, for e, the ing: (1) preservation of the inherent moisture content of the plant cells; (2) optimization ofthe height of cut used during harvesting of above-ground plant tissue; (3) reservation t integrity during harvesting (e.g., during cutting ofthc ground plant tissue); (4) minimization of environmental impact and time factors of biological degradation ofthe plant biomass; and (5) cleaning ofthe plant biomass prior to processing (e.g., prior to grinding and maceration).

Each ofthese steps is discussed below.

PCT/U52012/025899 Preservation of nt Moisture Content: The g should be done to avoid wilting due to moisture loss. Optimal conditions are those where natural moisture content is maintained and preserved.

Optimal and Preferred Height of Cut: The plants should be cut at least several centimeters above the ground to limit the amount of soil and other debris in the collected biomass. For example, all useable leaf and stem biomass of any given plant source (e.g., alfalfa, barley, lavender, or sage) may be cut at a height of r than or equal to 5 centimeters above . If flower tissue is used as the plant biomass source, the flowers are separated from the whole plant prior to extraction ofthe plant cell juice.

[0066] Preservation of Plant Integrity During Harvesting: Harvesting ofthe plant biomass may be by cutting the above ground stem and leaftissue ofthe plant. The cutting is ted in a manner that avoids or minimizes the chopping, mashing, crushing, or other type of injury ofthe plant. For large-scale industrial ting, where it may not be possible to avoid chopping due to the type of equipment required, care is taken to minimize injury that could lead to microbial growth, moisture loss, intensification of oxidation, polymerization, isomerization, and hydrolysis ses (i.e., unwanted catabolic processes) in collected plants. For example, in one embodiment of the present invention, lavender and sage are cut and collected by hand as whole plants. In another ment, alfalfa and barley tissue are cut using harvesting equipment. In that case, the minimum ng height above ground for each plant is greater than or equal to 5 centimeters. r, particular attention is made to minimize injury during and alter cutting. In r embodiment, marigold whole plants are collected by hand and the flowers are then separated for fiirther processing.

Minimization of Environmental Impact and Time Factors of Degradation: Delivery time of cut plant material to the processing ty and exposure ofbiomass to sun, high temperature, and other negative environmental factors, should be minimized to prevent the impact nted degradation processes as described above. For example, in one embodiment of the present invention, the delivery time for alfalfa and barley for r processing does not exceed 30 minutes from the time of cutting. In another embodiment, plants that undergo long distance transport are treated to a post-cutting procedure involving immediately placing the plant biomass into Styrofoam coolers containing bags of frozen gel packs to help maintain freshness and natural moisture content during overnight ry to the processing facility. These procedures were conducted for plant biomass from lavender, marigold, and sage. Other post-cutting procedures that e the results described above may be used as well. -14_ PCT/U82012/025899 Cleaning Step Prior to Grinding and Maceration: A washing step to remove the soil particles and other debris from plants prior to r processing is performed once the plant tissue is harvested. The washing is achieved using a low-pressure rinse for a short on under conditions to prevent the initiation ofthe release of the cell juice from biomass, to cause injury, or to remove valuable components. For example, in one ment ofthe present invention, the washing of the plant biomass was accomplished in less than or equal to 5 s with a water pressure of less than or equal to l kg/cmz.

Residual water wash did not contain any green or yellow pigments, which indicates the e ofsubsequent injury. The excess water is removed from washed biomass in order to keep the dry matter content close to natural level.

After the plant tissue s is harvested, as described above, further processing of the plant tissue biomass is performed to yield plant cell juice. In one ment, the harvested plant tissue biomass is subjected to grinding, maceration, and pressing to extract the ellular content, i.e., the cell juice, and to te it fiom the I5 nriched cake containing predominantly cell walls.

An example of a suitable processing protocol involves the steps described below. A hammer mill may be used to grind plants to yield plant tissue particles of a small size in a short time and without significant increase ofbiomass temperature. In one embodiment, a modified hammer mill is used to produce the maximum size ofmacerated plant particles less than or equal to 0.5 centimeters during less than or equal to 10 s of treatment, where the increase ofbiomass temperature is less than or equal to 5°C.

Exposure of ground and macerated plant biomass is minimized to prevent the impact ofunwanted catabolic processes, as described above. The extraction ofthe plant cell juice and its tion from the press-cake is commenced as soon as possible after grinding and maceration ofthe plant biomass. The plant biomass is processed in a short time and without significant increase in temperature. In one embodiment, immediately after grinding and maceration, the plant biomass is pressed using a horizontal, continuous screw press (Compact Press “CP-6”, t Corporation, FL). The pressure on the cone is maintained at level 24 kg/cmz, screw speed is at 12 rpm, and the temperature incrcasc is lcss than or equal to 5°C.

The initial cell juice usually contains small fiber particles, which can absorb valuable cell juice components and also block the hoses and pumps. The above particles should be removed by filtration or low-speed centrifugation. For example, the initial cell W0 2012/148527 PCT/U82012/025899 juices produced after the pressing step are filtered h four layers of nylon fabric prior to using the plant cell juice in the methods of the present invention.

Once plant cell juice is extracted, the plant cell juice is then treated to a processes involving (1) performing a “membrane fraction coagulation step” to yield a coagulated cell juice mixture and (2) performing a “membrane fraction separation step” on the coagulated cell juice mixture to yield a membrane fraction and a cell juice supernatant. In one embodiment, the membrane fraction coagulation step includes destabilizing the cell juice to yield a ated cell juice e. The destabilizing may be achieved using a variety of ilization techniques, including, for example, temperature treatment, o-membrane treatment, and chemical treatment. Suitable temperature treatment for use in the present invention may include (1) heating the cell juice extract to a treatment temperature required to induce coagulation of the membrane fraction (e.g., to a temperature ofbetween about 45 and 70 degrees Celsius) and (2) cooling the cell juice to a temperature effective to allow fithher quantitative separation of said membrane fraction from said cell juice supernatant (e. g., to a temperature ofbetween about 30 and 45 degrees Celsius). After destabilization is achieved, a membrane fraction separation step is performed. This step includes, for example, separating the coagulated cell juice mixture into the ne on and the cell juice supernatant using separating techniques ing filtration and centrifugation.

The freshly obtained ne fraction commonly referred to in the art, as “protein-vitamin concentrate,” is a paste having intensive color and specific odor that is plant raw material source specific. The membrane fraction is represented predominantly by chloroplasts present in the green parts of plant or mostly by plasts present in flowers.

The composition of the membrane fraction includes predominantly phospholipids, membrane ns, chlorophyll, and carotenoids. The drying of membrane fraction results in irreversible loses of many valuable properties ed for the exploration of membrane fraction as a cosmetic ient. Without drying, the unstable ne fraction is quickly transformed into the dark color un~dispersible and insoluble conglomerates having strong and non-characteristic odor. As result, such material cannot be used as a cosmetic ient. The described procedure that follows allows for transformation of y obtained membrane fractions into stable and active cosmetic ingredients.

Once the membrane fiaction is separated from the cell juice supernatant, the membrane fraction is then subjected to a formulation process prior to aggregation of the membrane fraction, including the ing steps: (1) performing a “stabilization step” to yield a stabilized membrane fiaction component; (2) performing a “polymer matrix -l6- W0 2012/148527 oration step” on the ized membrane fraction component to yield a membrane fraction ; and (3) ming a “neutralization step” on the membrane fraction matrix to yield the ne-Derived Cosmetic Composition of the present invention.

In one embodiment, the stabilization step involves mixing a non-ionic emulsifier and at least one antioxidant with the membrane fraction to yield a stabilized membrane fraction component. In the r matrix incorporation step, the stabilized membrane Fraction component is incorporated into a r matrix to yield a membrane fraction matrix. Suitable rs for use in the present invention include, for example, at least one polymeric emulsifier and at least one preservative. The ne fiaction matrix is then subjected to the neutralization step, which step involves adjusting the pH ofthe membrane fraction matrix to a range ofbetween 2.5 and 6.5, yielding the Membrane—Derived Cosmetic Composition described in the present application.

In another ment, stabilization of the Membrane—Derived Cosmetic Composition to yield approximately 100 grams of the composition is performed as follows: (a) Stabilization of membrane on includes its mixing with non—ionic emulsifier Polysorbate 80 (Tween 80) and antioxidants (Tenox 4). As an example, 20 grams fresh membrane on are mixed vigorously until homogeneous with 3.5 grams ofTween 80 and 0.1 gram ofTenox 4 (solution of Butylated Hydroxyaniso 1e and Butylated Hydroxytoluene in oil) while avoiding aeration during mixing. (b) Preparation ofthe sion ofpolymeric emulsifier, aerylates/CIO- C30 aerylate crosspolymer: As an example, 0.9 gram Pemulen TR-2 was sed in 69.2 grams warm deionized water and mixed until uniform using moderate agitation, avoiding aeration. In parallel, 5 grams of Glycerin and 1 gram of Phenonip (mixture of Phenoxyethanol (and) Methylparaben (and) Butylparaben (and) Ethylparaben (and) Propylparaben) are combined in separate vessel and mixed until uniform. With moderate agitation, phases containing Pemulen and Glycerin with Phenonip are combined and mixed until uniform. (c) Incorporation of membrane fraction into polymer matrix: As an example, the phase containing membrane fraction, Tween 80 and Tenox 4 is added to the phase containing Pemulen, Glycerin and Phenonip and mixed with vigorous agitation while avoiding aeration. (d) Neutralization ofthe product: As an example, the batch containing membrane fraction and other components is neutralized with 18% aqueous solution of _17_ PCT/U82012/025899 Sodium Hydroxide (NaOH) and mixed vigorously to produce m system having pH=5.0i0.4.

The resulting multiphase products are opaque gels trating properties that fiilly satisfy all requirements to the cosmetic ingredients. it was found that m composition of preservatives and anti—oxidants in multiphase cosmetic ingredients is very similar for all plant s and different combinations ofPemulens and also Carbopols can be effectively used. Stability studies indicate that cosmetic ingredients produced from membrane fractions via methods described are stable at 8°C. for at least 4-6 months ining physico-ehemical integrity and activities.

Process for Preparing Serum-Derived Cosmetic Compositions The t invention also relates to a method for preparing the Scrum- Derived Cosmetic Composition ting antioxidant activity, cell growth stimulation activity, or both antioxidant and cell growth stimulation activities. The method involves providing a plant cell juice that has been extracted from a fresh plant biomass, as already described above with respect to the Membrane-Derived Cosmetic Composition. The plant cell juice is then treated under conditions effective to te the plant cell juice into a membrane fraction and a cell juice supernatant. This step is also the same as described above with respect to the Membrane—Derived Cosmetic Composition. The cell juice atant is processed under conditions effective to separate the cell juice supernatant into a asm fraction and a cell serum fraction. The cell serum fraction is refined under conditions effective to yield a cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, or both idant and cell growth stimulation activities. The cell scrum fraction filtrate is stabilized under conditions effective to yield a stable bioactive botanical cosmetic composition exhibiting antioxidant activity, cell growth stimulation activity, or both antioxidant and cell growth stimulation ties.

The plant cell juice may be extracted from all types ofplants. Examples of suitable plants that may be used as sources of fresh plant biomass in the present include, without limitation, plants from the following families: Asteraceae, Fabaeeae, Lamiaceae, and Poaeeae. In particular, examples of ic plants that have been tested and found appropriate as fresh plant biomass sources include, without limitation, Trz’fblz'um se, Nelumbo nucifera, Calendula Qflicinalz’s, Medicago saliva, Lavandula angustg‘foiz'a, Salvz'a ofiz‘cz'nalz's, and Hordeum vulgare. ~18- PCT/U82012/025899 As described above, once the plant cell juice is separated into a membrane fraction and a cell juice supernatant, the cell juice supernatant is subjected to a processing step. In one embodiment, the processing step involves (1) performing a “cytoplasmic fraction precipitation step” to yield a cytoplasm/cell serum mixture including the cytoplasmic fraction and the cell serum fraction, and (2) performing a “cell serum separation step” to separate the cytoplasmic fraction fiom the cell serum fraction. The cytoplasmic fi‘action includes inantly white soluble proteins; in C3 plants, these ns largely consist ofthe enzyme ribulo se biphosphate carboxilase. The cell serum ns low molecular weight soluble components.

IO [0082] The cytoplasmic on precipitation step may include inducing precipitation ofthe cytoplasmic fraction within the cell juice supernatant using a suitable precipitation technique, including, for example, isoelectric titration and electrodialysis. In one embodiment, the isoelectric titration es adjusting the pH ofthe cell juice supernatant to between about 2.5 and 6.5. The cytoplasm/cell serum mixture is induced to separate into a asmic fraction and a cell serum fraction using a suitable separation teclmique, including, for example, such techniques as filtration and centrifugation. As an example, the precipitation was induced by a titration method utilizing by 5.0N Hydrochloric Acid (HCl) to pH=4.0.

The quantitative criteria to evaluate the complete separation of cytoplasm on is the absence of detectable levels of high molecular weight proteins and/or the absence of ribulose biphosphate carboxilase in subsequent filtrate or supernatant. As an e, the precipitated cell juice supernatants may be separated in a erated centrifuge for greater than or equal to 20 minutes at greater than or equal to 3,000 g, and an absence of the proteins having molecular weight of greater than or equal to 10,000 in cell serum having pH=4.0 were achieved.

The cell serum is commonly referred to as “brown juice,” although initially this clear liquid has a slight yellow color and slight characteristic odor. In several hours, the unstable cell serum is rsibly transformed into dark brown color suspension containing heavy precipitate and strong non-characteristic odor. As a result, “brown juice” cannot be used as a ic ingredient. The bed procedure that s allows for the refinement of cell serum (brown juice) to yield a stable and active cosmetic ingredients. This is accomplished by removing from the cell serum the major components responsible for the irreversible ormations that lead to generation ofunwanted itate and deterioration ofcolor and odor. This procedure includes: pH adjustment, heat treatment, cooling, vacuum PCT/U82012/025899 filtration, and stabilization. Some specific regiment procedures may vary according to plant source cell serum. It should be noted that this procedure must be used immediately after separation of cell serum from cytoplasm fraction is completed.

Once the cell serum fraction is ed, it is the subjected to a g process. This refining process includes (1) performing a “temperature ent step” to yield a coagulated cell serum fraction, and (2) performing a ication step to yield a cell serum fraction filtrate. A suitable temperature treatment step for use in the present invention involves (1) heating the cell serum fraction to a heating temperature required to induce ation within the cell serum fraction, and (2) immediately g the cell selum fiaction to a temperature effective to allow fithher quantitative separation of said cell serum fraction filtrate. In one embodiment, the heating temperature is between about 80 and about 95 degrees Celsius, and the cooling ofthe heated cell seium fraction is to a temperature of at least as low as about 15 degrees Celsius. A suitable clarification step for use in the present invention involves clarifying the coagulated cell serum fraction to yield a cell serum fraction filtrate, where the clarifying involves clarification techniques such as filtration and eentrifiigation. In one embodiment, the filtration may involve vacuum filtrating the coagulated cell serum fraction to yield the cell serum fraction e. In another embodiment, prior to the ature treatment step, the cell serum fraction is adjusted to a pH ofbetween about 3.0 and 4.0, as riate.

[0086] After the cell serum filtrate is produced, it is then subjected to the stabilizing Step mentioned above to yield the Serum-Derived Cosmetic Composition. In one embodiment, the stabilizing step involves incubating the cell serum fraction filtrate in a mixture of at least one vative and at least one antioxidant to yield a ized cell serum fraction filtrate. Suitable preservatives for use in the present invention include, for example, potassium sorbate, sodium te, sodium methyl paraben, and citric acid. An example ofa suitable antioxidant for use in the present invention is sodium metabisulfite.

In one embodiment, stabilization of the cell serum may be performed as follows: (a) As an example, the pH adjustment is performed for cell serum ed from sage and marigold flowers induced by a titration method utilizing by 5.0N hloric Acid (HCl) to pH=3.0. Such ment is not necessary for cell serum obtained from alfalfa, barley and lavender. (b) Heat treatment is performed for cell sera obtained from all useable plant sources. As an example, cell sera are exposed to microwave treatment under the PCT/U52012/025899 temperature probe control. This treatment is continued until the temperature reaches 90°C.

The temperature probe indicates the point required to induce the complete coagulation of the unwanted ents. Once coagulation is induced, the treated cell juice is immediately cooled to lO°C. (e) The ated cell sera can be clarified by filtration or eentrifugation.

As an example, the coagulated cell sera may be vacuum filtrated through double layers of Whatman No. 2 filters. The precipitates are discarded, and es are used for fiirther processing. (d) Stabilization of the filtrates included addition of specific preservatives and anti-oxidants and incubation of the mixtures until their complete lization is achieved (usually r than or equal to 30 minutes of extensive mixing is required).

Stabilized cell serum filtrates demonstrate properties which fully satisfy all requirements ofcosmetic ingredients. Stability studies indicate that cosmetic ingredients produced from cell serum via these methods are stable at room temperature for at least 10-12 months (i.e., they maintain physieo-chemical integrity and activities).

EXAMPLES Example 1 Preparation of Cosmetic Botanical Ingredient 101 Derived from Alfalfa (Medicago 2O sativa) Cell Serum Fractions.

Biomass Preparation. ent amounts of fresh alfalfa (Meclz'cago sativa) plant biomass (i.e., stem and leaf tissue) were harvested to yield approximately 100 kg of dry matter. The level of dry matter in the fresh alfalfa plant s was calculated to be 15.75 percent, requiring harvesting of approximately 635 kg of fresh alfalfa plant biomass to yield 100 kg ofdry matter. Care was taken to preserve the inherent moisture content of the plant biomass and to avoid wilting due to moisture loss. The plants were cut at least 5 centimeters above the ground to limit the amount of soil and other debris in the ted plant biomass.

The cutting was conducted in such a manner as to avoid or minimize chopping, mashing, and crushing of the plants. The harvested plants were red for processing not more than 60 minutes after cutting. This was done to minimize exposure ofthe plant biomass to sun, high temperature, and other negative environmental factors. A washing step was performed to remove soil particles and other debris fi'orn the plants prior to further processing. This washing was lished by washing the ted plants for §5 minutes in El kg/crn2 water re. The residual water wash did not contain any green pigments, indicating W0 2012/148527 PCT/U52012/025899 proper water pressure and washing duration. The excess water was removed from the washed plant biomass.

Grinding, Maceration, and ng of Plant Biomass. After harvesting, collecting, and washing the plant biomass, the plants then underwent grinding, maceration, and pressing to extract the ellular content (i.e., the plant cell juice) and to separate it from the nriched press-cake. A hammer mill was used to grind the alfalfa biomass to yield plant tissue particles of ly small size in a short amount of time and without significant increase of biomass temperature. The hammer mill was set to produce the maximum size of macerated plant particles of £05 centimeters during £10 seconds of treatment. This resulted in only an increase of §5° C. biomass temperature. A horizontal continuous screw press (Compact Press “CF—6”, Vincent ation, FL) was used to extract the plant cell juice from the plant biomass. The re on the cone of the screw press was maintained at a level of 24 kg/cmz, with a screw speed of 12 rpm and only a temperature increase of E50 C. This treatment yielded the press-cake and the plant cell juice.

The initial plant cell juice contained small fiber particles, which were removed by filtration through four layers ofnylon fabric or by using low-speed centrifugation.

Separation of the Membrane on from the Cell Juice. The filtered plant cell juice was exposed to microwave treatment using a temperature probe control. This treatment continued until the temperature ofthe cell juice reached 60° C. Once ation was induced, the treated cell juice was immediately cooled to 40° C. Separation of the membrane Fraction from the coagulated cell juice was achieved using centrifugation at greater than or equal to 3,000 g for greater than or equal to 20 minutes. This yielded a ne fraction (precipitate) and a cell juice supernatant, which ned a cytoplasm traction and a cell serum fraction (i.e., low lar weight soluble components). The cell juice supernatant was used for further processing to yield Cosmetic Botanical Ingredient 101. The membrane fraction was preserved for use in preparing a rpart Membrane~Derived Cosmetic Composition.

Separation ofthe Cfloplasm Fraction from the Cell Juice Supernatant. In order to separate out the asm fraction, the cell juice supernatant was subjected to isoelectric precipitation. Precipitation ofthe cytoplasm fraction was induced using a ion method utilizing 5.0 N hydrochloric acid (HCl) to bring the pH of the cell juice supernatant to 4.0.

The separation ofprecipitated asm fraction from the cell serum was achieved by centrifugation at greater than or equal to 3,000 g for greater than or equal to 20 minutes. This W0 2012/148527 PCT/U82012/025899 resulted in a cell serum (supernatant) that could be r refined to yield Botanical Cosmetic Ingredient 101.

Treatment of the Cell Serum to e Cosmetic Botanical Ingredient 101.

The refinement ofthe cell serum involved the following steps: heat treatment, cooling, filtration, and stabilization. Refinement was performed immediately after separation of the cell serum from the cytoplasm fraction. The cell serum was d to microwave ent using a temperature probe control. This treatment continued until the temperature of the cell serum reached 90° C. Once coagulation was induced, the treated cell serum was immediately cooled to 10° C. The coagulated cell serum was vacuum filtrated through double layers of n No. 2 filters. The precipitate was discarded and the resulting cell serum filtrate used for further processing (i.e., stabilization). ization of the cell serum filtrate achieved by adding preservatives and antioxidants and incubating the e until complete solubilization was achieved. The preservatives and antioxidants used included the following: 0.1% potassium sorbate, 0.1% sodium benzoate, 0.1% sodium methyl paraben, and 0.2% sodium metabisulfite. This preparation resulted in the production of 18.1 kg ofDry Matter yield (or approximately 340 Liters) of the ic Botanical Ingredient 101 , which was used for characterization of its physico-ehemical and bioactive qualities. The recommended storage conditions for Cosmetic Botanical ient 101 include storage in a closed container protected from light at a temperature ofbetween 15 and 25° C.

Example 2 Product Specifications of Cosmetic Botanical Ingredient 101 Derived from Alfalfa (Medicago sativa) Cell Serum Fractions.

[0094] Cosmetic Botanical Ingredient 101 was prepared according to the process described above in Example 1. es of Cosmetic Botanical Ingredient 101 were conducted to ine its various physico-chemical, microbial, xicity, and bioactivity teristics, as described below. ic Botanical Ingredient 101 is a clear liquid, which has a light-yellow color and a light-characteristic odor. No solvent (ie. glycol, oil, water) was added to the carrier medium.

Table 1 summarizes the Physical and Chemical data of Cosmetic Botanical Ingredient 101.

W0 2012/148527 PCT/U52012/025899 Tabie 1 m-i’hysimf amt Charisma}: Data 0....“ Enroll/[IV \‘ it’m' ______ vita Commit, a. SN: Exam :3 2:5, Refemtmas: {i=3 Hawaiian}; istzy anti .E-‘E‘siyaitas, 80“ Edition, C'RC Fm“! uwmonMa; :23 iE-Earidim‘k ut‘teaamisay and Physics. saw 13:33am (:RC Press, '1 ”HE’UWR Sat 'wi'siuh aw: harefiyyiacmfigmmwa by awe/mama in {hair .1 antiwar, Table 2 describes the UV-Spectra data regarding Cosmetic Botanical Ingredient 101. \‘t\ a i ' " . i t .. i N; NW“ N“ k u i E ‘ " 1 t \ i ~N~s\s\‘\\\\\\\\n‘-§--- My .1 E .

Table 3 summarizes the microbial analysis data for Cosmetic cal Ingredient 101. This data demonstrates that Cosmetic Botanical Ingredient 101 es the cosmetic industry requirements regarding colony forming units and absence ofpathogens.

W0 2012/148527 PCT/U82012/025899 mate LLaa—Mitflb-ifi Manet; twin“... , §Coimry g if ‘ item) - ' Cosmetic cal Ingredient 101 was determined to be stable (i.e., maintaining physical and chemical integrity) for at least 12—18 months while stored at a ature een 15 and 25° C. in a closed container protected fiom light. N0 toxic effect was detected. In a controlled clinical evaluation, Cosmetic Botanical Ingredient 101 did not demonstrate 50% inhibition of neutral red uptake (NRUSQ) by 3T3 asts in the concentration range 0-2,500 pg dry matter/ml. The NRUSO ofpositive l (epidermal growth factor) >2,500 ug/ml. Cosmetic Botanical Ingredient 101 demonstrated superoxide scavenging ability. In a controlled clinical evaluation, Cosmetic Botanical Ingredient 10] demonstrated a 50% inhibition of cytochrome 0 reduction (ICRso) at a concentration 149 dry matter/ml. The ICRSO ofpositive control rinic acid)=26.5 . Cosmetic Botanical Ingredient 101 is a biodegradable product.

Example 3 Preparation of Cosmetic Botanical Ingredient 201 Derived from Barley (Hordeum vulgare) Cell Serum Fractions.

The process for preparing Cosmetic Botanical Ingredient 201 was identical to the process described in Example 1 with regard to Cosmetic Botanical Ingredient 101, with the variations noted below. Fresh stem and leaf tissue of barley (Hordezmz e) was used as the plant biomass starting material. The level of dry matter in the fresh barley plant biomass was calculated to be 13.67 percent, requiring harvesting of imately 732 kg of fresh barley plant s to yield 100 kg of dry matter. The preparation resulted in the production of 15 .1 kg of Dry Mattcr yicld (or approximately 433 litcrs) of Cosmetic Botanical Ingredient 201.

PCT/U52012/025899 Example 4 t Specifications of Cosmetic Botanical Ingredient 201 Derived from Barley (Hordeum vulgare) Cell Serum Fractions.

Cosmetic Botanical Ingredient 20] was prepared ing to the process described above in Example 3. Analyses of Cosmetic Botanical Ingredient 201 were conducted to determine its various o—chemical, microbial, cytotoxicity, and bioactivity characteristics, as bed below. Cosmetic Botanical Ingredient 201 is a clear liquid, which has a light—yellow color and a light—characteristic odor. No solvent (i.e., glycol, oil, water) was added to the carrier medium.

Table 4 summarizes the Physical and al data of Cosmetic Botanical Ingredient 201.

Tame. 4.—»—Fiusiit:ai 31:11:11 {liwtniml {him paws-net“ E gggthfqi R1151: its 9.151111 (“1331mm $11 31% 1: 212113111: 25, 3.53 ~ “Waited“"‘ .. ..

Reflmitams: El I Haskiimtflc. 1.1% C111:1; ”and Physit‘t 3G“;Léstwn,WC 3:831? 19§§1a3£l£30 $431}; [2] Rantibmk of.flhmziaéry and Higgins: 86'“ Haitian, {3313 9:313; mm) 1%2% which, are hereby inenmwamfi by reitemme in their mtiflrtyx W0 2012/148527 PCT/U82012/025899 Table 5 summarizes the UV-Speetra data for Cosmetic Botanical Ingredient 201. "fable fist—éifi-‘VSpma-a Ape-x. 1m“: #3 Bad, mi tieigtsgam M\\\\\~“\\\\\\vfi\-‘u3,.~..»u.u —----"...........---u-- 3: \ t Aims“ Abs X1111)' . ‘ _ awn-x. (”mun-J ial analyses trated that Cosmetic Botanical Ingredient 201 satisfies the cosmetic industry requirements for cosmetic ingredients with regard to CFUs and absence ofpathogens (see Table 3, above, for methods).

[00104] Cosmetic Botanical Ingredient 201 was determined to be stable (i.e,, maintaining physical and chemical integrity) for at least 12-18 months while stored at a temperature ofbetween 15 and 25° C. in a closed container protected from light. No toxic effect was detected. In a controlled clinical evaluation, Cosmetic Botanical ient 201 did not demonstrate 50% inhibition ofneutral red uptake (NRUso) by 3T3 asts in the concentration range 0-2,500 pig dry matter/ml. The NRUso ofpositive control (epidermal growth ) >2,500 tig/ml. Cosmetic Botanical Ingredient 201 demonstrated superoxide scavenging ability. In a controlled al evaluation, Cosmetic Botanical Ingredient 201 trated a 50% inhibition of cytochrome c reduction (ICRso) at a concentration 160 dry matter/ml. The ICR 50 of positive control rinic aeid)=2.65 ug/ml. Cosmetic Botanical Ingredient 201 is a biodegradable product. _27- W0 2012/148527 PCT/U82012/025899 Preparation of Cosmetic Botanical Ingredient 301 Derived from Lavender (Lavandula angustifoliat) Cell Serum Fractions.

The process for preparing ic Botanical Ingredient 301 was identical to the process described in e 1 with regard to Cosmetic cal ient 101 , with the variations noted below. Fresh stem and leaf tissue of lavender (Lavandula angustifolz'a) was used as the plant biomass starting material. The level of dry matter in the fresh lavender plant biomass was calculated to be 13.24 percent, requiring harvesting of imately 755 kg of fresh lavender plant biomass to yield 100 kg of dry matter. Also, the preservative and antioxidant mixture contained the following: 0.1% potassium sorbate, 0.1% sodium te, 0.1% sodium methyl paraben, 0.1% citric acid, and 0.2% sodium metabisulfite. The preparation resulted in the tion of 18.5 kg ofDry Matter yield (or approximately 444 ) of Cosmetic Botanical Ingredient 301.

Example 6 Product Specifications of Cosmetic Botanical Ingredient 301 Derived from Lavender (Lavandula angustifolia) Cell Serum Fractions.

[00106] Cosmetic Botanical Ingredient 301 was prepared according to the process described above in Example 5. Analyses of ic Botanical Ingredient 301 were conducted to determine its various physico-chemical, microbial, cytotoxicity, and bioactivity characteristics, as described below. Cosmetic Botanical Ingredient 301 is a clear liquid, which has a brown—yellow color and a characteristic odor. No solvent (i.e., glycol, oil, water) was added to the carrier medium.

Table 6 summarizes the Physical and Chemical data of ic Botanical Ingredient 301.

W0 2012/148527 2012/025899 Tania fiwu'fiyS‘mfii and Gamma; 33313 “3%:th . .asasp’te 23¢ “Mattias; t” «insists: giants: . ~ ~ v.ww.~.v.'.\\~.\\\\\\\\\s\\\s\u~ ' u ~~~- - ~-~ - ~ ~ Bali's; ))>Wm\\\.\\\\\\ \\\\K we. ind ‘1“ \‘\\“\\\.,,W(rlflffNJ'N'M \«x w.-.

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Table 7 describes the UV—Spectra data regarding Cosmetic cal Ingredient 301.

Yawn: “in—EN Spectre Parameter ; Sam um _ M~M\hfl=‘.\u‘. -.-- «past, mm \\\\ Microbial analyses demonstrated that Cosmetic Botanical Ingredient 301 satisfies the ic ry requirements for cosmetic ingredients with regard to CFUs and absence ofpathogens (see Table 3, above, for methods).

] Cosmetic Botanical Ingredient 301 was determined to be stable (i.e., maintaining physical and chemical integrity) for at least 12-18 months while stored at a temperature ofbetween 15 and 25° C. in a closed container protected from light. No toxic effect was ed. In a controlled clinical evaluation, ic Botanical Ingredient 301 did not demonstrate 50% inhibition ofneutral red uptake (NRUso) by 3T3 fibroblasts in the concentration range 0-400 ng dry matter/ml. The NRUso ofpositive control (epidermal growth factor) >2,500 ng/ml. Cosmetic Botanical Ingredient 301 demonstrated elastase inhibitory activity, gelatinase B inhibitory activity, and superoxide scavenging ability.

] Table 8, below, describes the bioactivity results regarding Cosmetic Botanical Ingredient 301. new. fi——B§mfiivity Iflemfis ofCesmefie ignianieai ingredient: 30:15 ,__........ ' ___§...i * 3 Method u VVVVVVV i Kinsmen Ehkihimny i * z: ‘- \ memt

[00112] In a controlled clinical evaluation, ic Botanical Ingredient 301 demonstrated a 50% inhibition of cytochrome 0 reduction (ICRSO) at a concentration 158 dry matter/ml. The ICRso ofpositive control (rosmarinic aeid)=26.5 ug/ml. Cosmetic Botanical Ingredient 301 is a biodegradable product. e 7 Preparation of ic Botanical ient 401 Derived from Marigold Flower dula alis) Cell Serum Fractions.

The process for ing Cosmetic Botanical Ingredient 401 was identical to the process described in Example 1 with regard to Cosmetic Botanical Ingredient 101, with the variations noted below. Fresh flower tissue ofmarigold (Calendula oflicz'nalis) was used as the plant biomass starting material. The level of dry matter in the fresh marigold flower plant biomass was ated to be 7.80 percent, requiring harvesting of approximately 1,282 kg of fresh marigo 1d flower plant biomass to yield 100 kg of dry matter. The flowers were separated from the whole plants after cutting the plant and prior to washing. The processing ofthe flowers (i.e., beginning with the washing step and prior to grinding) started not more than 3 to 4 hours afler cutting of the plant. Also, prior to ave treatment ofthe cell serum fraction, the pH ofthe cell serum was first adjusted to a pH of 3.0, using a titration method utilizing 0.5 N hydrochloric acid (HCl). The preparation resulted in the production of 27.1 kg ofDry Matter yield (or approximately 704 liters) of Cosmetic Botanical Ingredient 401.

W0 2012/148527 PCT/U82012/025899 Example 8 Product Specifications of Cosmetic Botanical Ingredient 401 Derived from Marigold Flower (Calendula offcmalzs) Cell Serum Fractions. ic Botanical Ingredient 401 was prepared according to the process bed above in Example 7. Analyses of Cosmetic Botanical Ingredient 401 were conducted to determine its various physico—chemical, ial, cytotoxicity, and bioactivity characteristics, as described below. Cosmetic Botanical Ingredient 401 is a clear liquid, which has a yellow color and a light—characteristic color. No solvent (i.e., glycol, oil, water) has been added to the carrier medium.

Table 9 describes the Physical and Chemical data of Cosmetic Botanical Ingredient 401. 13231:: 9.“Pfivsiesai and Chemical flats )a\\“\\\\v.~.~.>.\v.-. ' > > > ‘ . . ,- > > > > > w. ow. 1 See ”E«empire 32.1) E "Xiehnd 3" i See ay, {21%;} NM.»\\\\\\\\\\\\\\\\\\\\\\\ Rimming 111 [Hammett of (3118;111:3111; 11116911315135, 80$ Kiddie-ma (“RC Press 199.9—am“ 5-911; {'11.]Ha:a.<§imek i raga Mammy 33‘3’ 116113015, mt: Pmé‘iék‘ 10 82? which are have); inmrpomted by mtereme in their entirety, Table 10 summarizes the ctra data for Cosmetic Botanical Ingredient 401. ram 16.»-~~vU\iuSpeetim , 1 1 . : 1 - .. \ . t ' V“ t l x x t .

. S t . 1 ‘ . i ‘-“If ,-_,_.,._ ".11..." mg . r ‘ , » . 1 . .. \ . \ . l l \ . - » ~ i 1 .

S . \ \ 1‘ \ 1 . . t.. .- \ \ .......a “Bu.\~_\~_\w“wmw,~u.m«\m~~~~~~——*fi‘mfiw~fi W0 48527 Microbial analyses demonstrated that Cosmetic Botanical Ingredient 401 es the cosmetic industry ements for cosmetic ingredients with regard to CFUs and absence ofpathogens (see Table 3, above, for methods).

Cosmetic Botanical ient 401 was determined to be stable (i.e., maintaining physical and chemical integrity) for at least 12—18 months while stored at a temperature ofbetween 15 and 25° C. in a closed container protected from light. No toxic effect was detected. In a controlled clinical evaluation, Cosmetic Botanical Ingredient 401 did not demonstrate 50% inhibition of neutral red uptake (NRU50) by 3T3 fibroblasts in the concentration range 0—2,500 ug dry matter/ml. The NRUso ofpositive control (epidermal growth factor) >2,500 pig/ml. Cosmetic Botanical Ingredient 401 demonstrated stimulation effect on cell proliferation and superoxide scavenging ability. In a controlled clinical evaluation, Cosmetic Botanical Ingredient 401 stimulated 3T3 fibroblasts proliferation. This -15% ation was observed over a range from 5 to 100 pg dry matter/ml. The stimulation by positive control (epidermal growth factor)=20—30%. In a controlled clinical evaluation, Cosmetic cal Ingredient 401 demonstrated superoxide ging activity resulting in 50% tion of rome c reduction (ICRso) at a concentration 153 pg dry matter/ml. The ICR 50 of positive control (rosmarinic acid)=26.5 rig/ml. Cosmetic Botanical Ingredient 401 is a biodegradable product.

Example 9 Preparation of Cosmetic Botanical ient 501 Derived from Sage (Salvia officinalis) Cell Serum ons.

The process for preparing Cosmetic Botanical Ingredient 501 was identical to the process described in Example 1 with regard to Cosmetic Botanical Ingredient 101, with the variations noted below. Fresh stem and leaf tissue of sage (Salvz‘a officinalis) was used as the plant biomass starting material. The level of dry matter in the fresh sage plant biomass was calculated to be 10.64 percent, requiring harvesting of approximately 940 kg of fresh sage plant biomass to yield 100 kg of dry matter. Prior to ave treatment of the cell scrum fraction, the pH ofthe cell serum was first adjustcd to a pH of 3.0, using a titration method with 0.5 N hydrochloric acid (HCl). Also, the preservative and antioxidant mixture contained the following: 0.1% potassium e, 0.1% sodium benzoate, 0.1% sodium methyl n, 0.1% citric acid, and 0.2% sodium metabisulfite. The preparation resulted in the production of 14.9 kg ofDry Matter yield (or approximately 370 liters) of Cosmetic cal Ingredient 501.

W0 2012/148527 Example 10 Product Specifications of Cosmetic cal Ingredient 501 Derived from Sage (Salvia ofi‘icinalis) Cell Serum Fractions.

] Cosmetic Botanical Ingredient 501 was ed according to the process described above in Example 9. Analyses ofCosmetic Botanical Ingredient 501 were conducted to ine its s physico-chemical, microbial, cytotoxicity, and bioactivity characteristics, as described below. Cosmetic Botanical Ingredient 501 is a clear liquid, which has a brown-yellow color and a characteristic odor. No solvent (i.e., , oil, water) was added to the carrier medium.

Table 11 describes the Physical and Chemical data of Cosmetic Botanical Ingredient 501.

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PCT/U52012/025899 Table 12 summarizes the UV-Spectra data for Cosmetic Botanical Ingredient 501. 73735113 Imw'tfvangctm 1/, '1 Apex, mar 32. ”14 “nayI.

Sinai, nm ‘ f ‘ Apes, rim Microbial analyses demonstrated that Cosmetic Botanical Ingredient 501 satisfies the cosmetic industry requirements for cosmetic ingredients with regard to CFUs and absence ofpathogens (see Table 3, above, for methods). ic Botanical ient 501 was determined to be stable (i.e., IO maintaining physical and chemical integrity) for at least 12~18 months while stored at a temperature ofbetween 15 and 25° C. in a closed container protected from light. No toxic effect was detected. In a controlled clinical tion, Cosmetic Botanical Ingredient 501 did not demonstrate 50% ofneutral red uptake (NRUso) by 3T3 fibroblasts in the tration range 0-2,430 ug dry matter/ml. The NRUso of positive control (epidermal growth factor) >2,500 ug/ml. ic Botanical Ingredient 501 demonstrated elastase inhibitory activity, gelatinase B inhibitory activity, and superoxide scavenging ability. (See Table 13, below.) fable t3»«-»B§(mnt§¥ity Realms {if thematic: Immune-a! ingredient 503 Annuity Malawi ‘ . i91M .' q s s‘ - I} meeinhfisitar}? § See e 13:5 ‘ S Q ‘ asei? Inhibitory Elna e‘ . S ; “swam: a” In a controlled clinical evaluation, Cosmetic Botanical Ingredient 50] demonstrated superoxide scavenging activity, resulting in 50% inhibition of cytochrome c W0 2012/148527 PCT/U52012/025899 reduction (ICRSO) at a concentration >160 ug dry matter/m1. The ICRso of positive control (rosmarinie acid)=26.5 rig/ml. Cosmetic Botanical Ingredient 501 is a biodegradable product.

Example 11 Preparation of Cosmetic Botanical ient 402 Derived from Marigold Flower (Calendula officinalis) Membrane Fractions.

The process for ing Cosmetic Botanical Ingredient 402 was cal to the process described in Example 7 with regard to Cosmetic Botanical ient 401, with the variations noted below. Once the membrane fraction (precipitate) was separated from the d cell juice, the process described in Example 7 was no longer followed. d, the membrane fraction was treated to yield Cosmetic Botanical Ingredient 402, as described below.

] Treatment ofMembrane Fraction to Produce Cosmetic Botanical Ingredient fl); The membrane on was stabilized and incorporated into a polymer matrix. This was performed ately after separation of the ne fraction from cell juice. To prepare approximately 100 grams ofCosmetic Botanical Ingredient 402, the cell membrane fraction was stabilized by mixing it with non-ionic emulsifier Polysorbate 80 (Tween 80) and antioxidants (Tenox 4). cally, 20 grams of fresh membrane fraction was mixed vigorously with 3.5 grams ofTween 80 and 0.1 gram of Tenox 4 (solution ofButylated Hydroxyanisole and Butylated Hydroxytoluene in oil) until homogeneous, while ng aeration during .

Once stabilized, the membrane fraction was incorporated into a polymer matrix (i.e., a dispersion of polymeric emulsifier, acrylates/ClO—C30 acrylate crosspolymer).

The polymer matrix was prepared by dispersing 0.9 grams of Pemulen TR—Z in 69.2 grams of warm deionized water and mixing until uniform using moderate agitation, while avoiding aeration. In parallel, 5 grams of Glycerin and 1.0 gram of Phenonip (mixture of Phenoxyethanol (and) Methylparaben (and) Butylparaben (and) Ethylparaben (and) Propylparaben) were combined in a separate vessel and mixed until uniform. With moderate agitation, the phases containing Pemulen and Glycerin with Phenonip were ed and mixed until uniform. To incorporate the membrane fraction into the polymer matrix, the phase containing the membrane fi'action, Tween 80, and Tenox 4 was added to the phase containing the Pemulen, Glycerin, and Phenonip, and then mixed with vigorous agitation while avoiding aeration. Stabilization ofthe membrane fraction mixture was ed by neutralizing it with 18% aqueous solution ofsodium hydroxide (NaOH) and mixed 2012/025899 vigorously to produce a uniform system having a pH of 5.03204. This preparation, which d from 100 kg of fresh marigold flower plant biomass (approximately 1,282 kg of fresh marigold flower biomass having 7.80 percent dry matter), resulted in the tion of 9.5 kg ofDry Matter yield (or approximately 205 liters) of Cosmetic Botanical Ingredient 402, which was used for characterization of its physico -chemical and bioactive qualities. The recommended storage conditions for Cosmetic Botanical Ingredient 402 include storage in a closed ner protected from light at a temperature n 2 and 8° C Example 12 Product Specifications of Cosmetic Botanical Ingredient 402 Derived from Marigold Flower (Calendula oflicinalis) Membrane Fractions.

] Cosmetic Botanical Ingredient 402 was prepared according to the process described above in Example 1 1. Analyses ofCosmetic Botanical Ingredient 402 were ted to determine its various physico—chemical, microbial, cytotoxicity, and bioactivity teristics, as described below. Cosmetic Botanical Ingredient 402 is an opaque gel, which has an orange color and light-characteristic odor. Cosmetic Botanical Ingredient 402 was formulated utilizing the natural cell juice constituents gelled with a polymer to assure the highest level of purity uniformity, compatibility, stability, safety and efficacy.

[00130] Table 14 bes the Physical and Chemical data of Cosmetic Botanical Ingredient 402.

Traffic 1$.»x~m}’l:}=si_¢$i§ 351$ Chem-Sen] 333ml fiftitzti Cert. V ona t , § ....i.i...c..c..imafmmmimmmun-~ é Luiem, E25) NYE. m.Eixamnie 33.

“Method at . » v.sauna-v.\\\‘m\\\\\-N“\———- ] Table 15 summarizes the L"‘a"‘b"< values data ing Cosmetic Botanical Ingredient 402.

WC 2012/148527 Table. ifi "mt.*s’ifli“ Valnes Paramfl 1 s 1 1 3.. \*»\-.\~. vs“ 15* § “1......W....."fie—mm 131‘” § Microbial analyses demonstrated that Cosmetic Botanical Ingredient 402 satisfies the cosmetic industry requirements for cosmetic ingredients with regard to CFUs and absence ogens (see Table 3, above, for s).

Cosmetic Botanical Ingredient 402 was determined to be stable (i.e., maintaining physical and chemical integrity) for at least 12-18 months while stored at a temperature ofbetween 2 and 8° C. in a closed container protected from light. Cosmetic Botanical Ingredient 402 is a biodegradable t. No toxic effect was detected. In IO controlled al evaluation, ic Botanical Ingredient 402 did not demonstrate 50% inhibition ofneutral red uptake ) by 3T3 fibroblasts in the concentration range 0—354 ug dry matter/ml. The NRUso of positive control (epidermal growth factor) >2,500 rig/ml. ic Botanical Ingredient 402 demonstrates elastase inhibitory activity and trypsin inhibitory activity. Table 16 summarizes certain bioactivity results for Cosmetic Botanical Ingredient 402. l Mama-st: inhibitory ¢~\\\ WY- § “um Exampl‘c 333': S “\aiether} 2‘1”" ...,......i:.....1.... i i $1313., 1I~alnb1mrv 131112111111131.1193 (NV-m“ \W\ 1111111113111 {1111111511111" 111111111 5.11j itw-Siattth 111111311111. lfifiifi, v. 51313 {331‘ 111.11, iaifiehis 1331 inflm‘z‘gmfmii’dw21:,“ mfamfie 1:: its 11:11am.

W0 2012/148527 PCT/U82012/025899 Example 13 Preparation of ic Botanical Ingredient 502 Derived from Sage (Salvia alis) Membrane ons.

] The process for preparing Cosmetic Botanical Ingredient 502 was identical to the process described in Example 11 with regard to Cosmetic Botanical Ingredient 402, with the variations noted below. Fresh stem and leaf tissue of sage (Salvz'a oflicinalz's) was used as the plant biomass starting material. The level of dry matter in the fresh sage plant biomass was calculated to be 10.64 percent, requiring harvesting of approximately 940 kg of fresh sage plant biomass to yield 100 kg of dry matter. This preparation ed in the production of 6.7 kg ofDry Matter yield (or approximately 124 liters) of Cosmetic Botanical Ingredient 502.

Example 14 t Specifications of Cosmetic Botanical Ingredient 502 Derived from Sage (Salvia ofi‘icinalis) Membrane Fractions.

Cosmetic Botanical Ingredient 502 was prepared according to the process described above in Example 13. Analyses of Cosmetic Botanical Ingredient 502 were ted to ine its various physico-chemical, microbial, cytotoxicity, and ivity characteristics, as described below. Cosmetic Botanical Ingredient 502 is an opaque gel, which has a green color and characteristic odor. Cosmetic Botanical Ingredient 502 has been formulated utilizing the natural cell juice constituents gelled with a polymer to assure the highest level of purity uniformity, compatibility, stability, safety and efficacy.

[00136] Table 17 describes the Physical and al data of Cosmetic cal Ingredient 502.

Tame IKPI’m-‘aieni and {Ifhmzfiesd Data “islld‘ir’i‘zlfiiiia Residue, Sac Example . 3 \ :‘ “admirers 25' 3 sea, is: {ii'ittflti‘fi germ use «as: t Viscosity. are s “w.— PCT/U82012/025899 Table 18 bes the L*a*b* values for Cosmetic Botanical Ingredient 502.

Table: 38Er‘tflh" Fame-s ”‘tramete:W“‘ ‘iw Resets. E ..... m ‘ eE-xarupée 2-5 "wetland 3’ Microbial analyses demonstrated that Cosmetic Botanical ient 502 satisfies the cosmetic industry requirements for ic ingredients with regard to CFUs and absence ogens (see Table 3, above, for methods).

Cosmetic Botanical Ingredient 502 was determined to be stable (i.e., maintaining physical and chemical integrity) for at least 12-18 months while stored at a temperature ofbetween 2 and 8° C. in a closed container protected from light. Cosmetic IO Botanical Ingredient 502 is a biodegradable t. No toxic effect was detected. Cosmetic Botanical Ingredient 502 demonstrates elastase inhibitory activity and gelatinase B inhibitory activity. (See Table 19, below.) Table 1‘33»Biomtwtt} Regatta far {annex}: Batmileai hsgxcdiant 502l §' W? ‘ 2'2“th W i 10;; g K («55.3133 R ‘ E ”14/” v r w“ [See Example“a1‘s. “waliuzfl ‘\{eihiki 3" Cie’taténase'a inhibitor—5 l 53;; Farm: 3355‘; «2:36 Norma ‘ “Niefimd it” i ................. __“__,L_A___,_ ....................m“; WNW.

Example 15 Distribution of Dry Matter Regarding Preparation of ic Botanical Ingredients From a, Barley, er, Marigold Flowers, and Sage ] Various fractions collected during the production of Cosmetic Botanical Ingredients 101 , 201, 301, 401, 402, 501, and 502 were analyzed and compared for dry matter distribution.

Table 20 shows the distribution of 100 kg dry mater between the cell juices and press-cakes 0f the various processes. It was determined that the process of the present invention permits extracted yield sion into plant cell juices in the range of from about 20 to 40 percent of initial biomass dry matter.

W0 2012/148527 PCT/U52012/025899 “table it}; w-‘A-ii’éistribution of13:31 kgihy M’s-Stw‘ Between {Taxi} }: and Pm»Cake-s . m\\\\\\\\w\\x‘l\\\‘tittii t \‘-\\\\m\\w~\v~u . ..W-vid Product ‘ Table 21 shows that the yield ofmembrane fi'actions' dry matter was in the range fiom 6% to 13% of initial biomass dry matter and from 25% to 45% of cellJuice dry matter. Based on high dry matter yield, membrane fractions were selected as a prospective source for preparation of hase cosmetic ingredients.

Tame: 2'1 .Im ifiistrfbuttnn of my Matter n Memhmae Fracfimks and Cat! Seine firtamsmaianbr \\\\\\\\W\W\M\5a““5““Exxmnxmw~xm-~xchxw\\\u\\\x\\m‘\\\\\"aw>M!\\t\\\\\wm«\\K\\\\\\§x\\\( .......

Printers: F13)“ 1‘54 . . m~m\\\\\\\\\x\\\\\\ Let} 3mm ngcmmnt t T“: ,,.,.....\“w“‘“m «ugwV «w \MQCummmMmuwfi 'uxxx“\“nxmn‘««\v‘w‘v‘ ..\ The process of the present invention permitted the following distribution of dry matter between cytoplasm ons and cell serum (see Table 22).

Tutti: 22w“ mtiim ofDry s: between {53%pin-sm inas and {toil Serum - www>>>>>xw-~“ “W‘s“\«sauna..-\\\x\\\m\\\\\\\\~vw\\\\\\‘\\\ma‘ \\W\\\\\M\\\\\'.\\\\\V\\WM\\\V\V~\'\ grin Shawn) ran-rem Table 22 shows that the yield ofcytoplasm ons dry matter did not exceed 2.5% of initial biomasses dry matter and subsequently 11% of cell juice supernatant dry matter. Most of cell juice supernatant dry matter was concentrated in cell sera: 88.8% (alfalfa), 97.4% (barley), 95.9% (lavender), 96.8% (marigo 1d flowers), and 98.7% (sage).

Based on high dry matter yield, cell sera were selected as a prospective source for preparation ofsoluble cosmetic ingredients.

PCT/U52012/025899 Example 16 Optimum Composition of Preservatives and idants for Cosmetic Botanical Ingredients From Alfalfa, Barley, Lavender, Marigold Flowers, and Sage The optimum composition ervatives and idant was determined to be very similar for all plant sources (i.e., from alfalfa, barley, lavender, marigold flowers, and sage (see Table 23).

Tame. 23,.“ (immune Cumyositiea 6f vatives am; Aafiaxiémt {$45) Refit-lime! fur Staitijiizmfims oi‘Cei} Serum filtrates “xxx\'\‘l\‘\‘(('\’\'\\\'(Vii-fi-Z-I-P'émx“ \WV“\\\N'L\’~.‘.\s\\\'\\\\\\\\\\‘.\\‘?\\ “1“‘2;an}:§§v§:§;}§§::::\““““n“Ni: ~ \\ ‘ Conga-men: {anemia Madison? Sage F'gfikvfifiwmm “xxx-x \\\\"\‘~.V.->§.V-T-S \\\\\\§\:\§\‘A\\\\\\<\< x'c-:-.\.\.~.\xx\\\\\ :0 A ‘ ‘ -:-:\::~:::“\:.\“ ”rm-W“‘sssi new”?“u..uu.‘ -i.«u}...—. “mammmwiawimu . mmsmswzw Example 17 ison of Various Characteristics of Cosmetic Botanical Ingredients From Alfalfa, Barley, Lavender, Marigold Flowers, and Sage

[00146] The o—chemical, spectral, microbial, logical, performance and efficacy data related to cosmetic botanical ients are presented in Tables 24 and 25.

U Taisk: 14M migraine! and {mosaics} hummus of (Jasmciic Ingreéianm Produced {ram Cali Settings \\\\m\m\n\\\M"§i&<\\\\\\'W\\\'\\\\‘\\ “\~m~\“\mm“m¢»»»x “\\~.\\\\\\\\~2{~, nautical it: '- idiens swim n2 Scarce «xW, “xxx“ . paw».

Faramefietc 33'; 3m $33 583 'f ’5 migoid Sage \\\\m\w~\\\\x_ M\\\\\\\\ 5W$\\\‘\\ Selim {Tcmmnfl/> «medial, mv W0 2012/148527 PCT/U52012/025899 TablefaZSn-n— {Iv—Synetrni Pmmflies {if {lasim‘fifl ingredients ymfiumfl item {fail Samms w. xs{A\\\\\\\W\A\\\\\\\\\\\\\\‘Z "mswm‘nxa:\v\.~.\.\\\\‘.\W\\ " 3 E {animate Entaninal Ingretiiem mi} “Mtf‘ho‘h fiflk § 5E .\\\.\\\\\V~\ eter\\\\\\\\.\\\&\>\}}\\> m (ll/MFl-G’oflfi’flfly/¢ ’ H Star;\;_\\m~3,\\»\~m E“m\\\\\"«\ The data presented in Table 24 and Table 25 demonstrates that although five plant source raw materials were used (even belonging to different plant families), the properties of the cosmetic botanical ingredients produced from a variety of cell sera are very similar. This similarity can be very le for manufacturing of highly standardized natural products based on the cosmetic botanical ingredients described above.

The data presented in Table 26 trate that all tested cosmetic botanical ingredients satisfy the cosmetic industry requirement to the CPU level (colony forming . The absence ofpathogens also satisfied the industry requirements to the safety of cosmetic ingredients.

Tahiti 364m Mmmbial new at{Midis immflenm 3"de from K4:1! Serums IVA/AU \\\\\\W““\§:~\A\x~m‘w~\-.\ Sam and Firm; Snare Pmmmm m \“\«WW\\\\\\\\\\\~\\\AV\MAW\tit ______________________ § . A 3% (“Shifts Lavender Meagan! -\ 4414411111 In on, the ic botanical ingredients demonstrated absence of cytotoxicity in the Wide concentration . As an example, a test involving neutral red W0 2012/148527 PCT/U52012/025899 uptake (NRU) by 3T3 fibroblasts, which is commonly used for determination of cytotoxic ity, was ed to assess cytotoxicity of cosmetic botanical ingredients. The NRU value is proportional to the number of viable cells in this in Vitro test population. It was found that 50% inhibition of neutral red uptake (NRUSO) by 3T3 fibroblasts was not reached even at very high trations of tested cosmetic ingredients. As a comparison with respect to the safety profile, most ofthe cosmetic botanical ingredients produced by the described methods were close to Epidermal Growth Factor (EGF) as used as a positive control (see Table 27).

Table: Elm Cytatisxic‘fisy’ [mm at“ aesthetic: ingmdirnas Ifimfiucm {mm {Tait Serums and mzi (3me Farms) {ESE Pac.......““.“\“u“u,-m» ~ ~. :m xx ‘ u... .— 05::gr:was Parameter n/«a * NCM: Narnia}: Cali hierphoingy; M132 Madame Lam‘s iiy.

The cosmetic botanical ingredients produced from cell sera have cantly higher safety profile when compared with water extract isolated by conventional method fiom the same batch of dried raw material or commercial extract obtained from the same plant source. As an e, the data related to marigold flowers ted in the table below (see Table 28). “rabidEfivwv {Vinttsxmx} {bar's 9! Casmfir. Manning! Ingredient 4M “inter Extract anti (Lammgreifl Extract Initiated from Briaf‘iyflid Waivers , '3‘ "CM: 3’22":ma! (“Teri Mmpimiogy; 18LT: Rig?) invert ttxieity‘.

All cosmetic botanical ingredients produced from cell sera demonstrated anti- oxidant properties or more specifically superoxide scavenging ability. As an example, the trations required to inhibit 50% of cytochrome c reduction (ICRso) were determined and rosmarinic acid (RA) was used as a positive control (see Table 29).

W0 2012/148527 ram 39.? Summit-me Semengtssg sanity- as? Cnsmetia ingredients Madness! {mm 423:3 Serums amt Rnsnwrinifi Aeifd eianmalingredients and Nant- finalise:s<c<<.\\\\“\ “““w--»-4~‘.~:_....-...i.t I: grit”: 3e; ini““““§“5m s flashy § Lavender ld Sage uvlzv/rrl/‘rvll/zrlwl/ first}; pgifiry‘ :49 use :58 ' ' . 3v"? Example 18 Characterization of Cosmetic Ingredients Produced from Membrane Fraction.

The o-chemical, optical, microbial, toxicological, performance and efficacy data related to selected cosmetic ingredients produced from membrane fiactions are ted in Tables 30 and 31.

Tabm 3i},~—~ eofilkemimi t’mwrtm of ic Ingredients Faximmd {mm Membrane Fractions u«~un,~,~,»a.~,-.-.

“Pattie 3'1.£*3*§§* s af {Sussex-air: Ingresfiema hemmed from sfigmbmne Fanatical: “\\“\\\~.-.~.~.v.- a s 1' (In!) ~. v:1; - 4m,» ‘ . $ y, (I a - Q‘- S 5a 7 rn”2II The data presented in Table 30 and Table 31 demonstrate cant differences between properties of cosmetic ingredients produced from membrane fractions obtained from leaf-and-stalk biomass (sage) and from flowers (marigo 1d). Generally, the above reflects the difference between chloroplasts, which are predominantly concentrated in sage membrane fraction, and chromoplasts, which are predominantly concentrated in marigold flowers.

W0 48527 PCT/U82012/025899 Microbial analyses demonstrated that ic Botanical Ingredients 402 and 502 satisfy the cosmetic industry requirements for cosmetic ingredients with regard to CFUs and absence ofpathogens (see Table 3, above, for methods).

Example 19 nflammatory and Antioxidant is of Cosmetic Botanical Ingredients: Objectives and Rationale for Selected Experimental Models Cosmetic Botanical Ingredients 101, 102, 201, 202, 301, 302, 401, 402, 501, and 502, as well as others, were analyzed for their anti-inflammatory and antioxidant qualities. The results of these analyses are summarized in this Example 19, below. The procedures and results are explained in Examples 20~24, below.

The bed procedure pertains to the distribution of concentrated serum- derived and membrane—derived cosmetic botanical ingredients. These ients demonstrated two important activities (antiproteolytic activity and antioxidant activity) towards reducing connective tissue damage associated with inflammation. The pattern of distribution for antiproteolytic activity is ively in the membrane fractions and subsequently in hase botanical cosmetic ingredients, where as the distribution pattern for idant activity is selectively in the serum fraction and subsequently soluble in cosmetic ingredients. Membrane—derived cosmetic ingredients contain ents which inhibit both of the two major classes of ctive proteinases, i.e., serine proteinases exemplified by neutrophil elastase and matrix metalloproteinases exemplified by nase B. The potential ofthe membrane—derived cosmetic ingredients to achieve inhibition ofthe synergistic lytie ties of inflammatory cells merits consideration of their use in topical applications for anti—inflammatory formulations. The mode of inhibition of these cosmetic ingredients suggests that their effects are reversible, and, they would not cause undesirable long term modifications to defense or repair isms.

The selective distribution of antioxidant activities into the serum—derived cosmetic ingredients presents an additional direction to incorporate an important biological activity which reduces damage caused by the reactive oxygen species generated by inflammatory cells. The serum-derived cosmetic ingredients obtained from multiple botanical s s potent modulatory activities which diminish the capacity ofthe inflammatory cells to te reactive oxygen species rather than simply neutralizing the oxidants. The described method employed in generation ofthe serum-derived cosmetic ingredients result in the preservation ofthis modulatory activity along with scavenging activity. The conventional WO 48527 PCT/U82012/025899 procedures for obtaining aqueous extracts simply achieve only some distribution of scavenging activity alone, The selective distribution of one type ogical activity into the membrane- derived cosmetic ingredient and another activity into the derived cosmetic ingredient obtained from the same botanical sources represents an opportunity to employ novel topical formulations in which two phases are maintained in stable composition.

Cosmetic Botanical Ingredients 101, 20l , 30l, 401, 402, 501, and 502 (collectively referred to herein as the “Cosmetic Botanical Ingredients”) were evaluated for their anti-inflammatory and antioxidant activities. There are multiple mechanisms for injury IO to connective tissue that may arise as a consequence of the inflammatory s. The one final common pathway leading to inflammatory tissue injury involves destruction of the components ofthe stroma by white blood cell-derived proteolytic enzymes. Accordingly, assays were employed to te the capacity of the different Cosmetic Botanical Ingredients to inhibit these inflammatory proteinases. In the tion, two proteinases were used: neutrophil elastase and neutrophil gelatinase. These two enzymes degrade the components ofthe extracellular matrix of human connective tissue in a istic manner.

Moreover, neutrophil elastase can vate the body's own tory defenses against phil gelatinase while conversely, the gelatinase can inactivate the body's own antielastase defenses. Thus, cosmetic botanical ingredients which can inhibit these two s provide significant protection against inflammatory injury. The assays selected permit quantitation ofthe inhibitory ty ofthese cosmetic botanical ingredients and provide information regarding some basic features of the mode of inhibition.

In addition to degradative proteinases, inflammatory processes are often ated with release of reactive oxygen species from the activated cells. These reactive species include xide anions, hydroxyl radicals, hydrogen peroxide, and hypochlorous acid. The biological effects of these oxidants can lead to inactivation of important endogenous antiproteolytic defenses in the human tissue. Assays were employed which measure the capacity of the cosmetic botanical ingredients to lower the levels of reactive oxygen s released by activated inflammatory cells. Additionally, assays were used to quantitate the capacity ofthe cosmetic botanical ingredients to neutralize ve oxygen species of endogenous and exogenous origins.

W0 2012/148527 PCT/U52012/025899 Example 20 Evaluation of Anti-Elastase Activity.

During the inflammatory process, elastase activity is directly related to the actions of multiple enzymes, but neutrophil elastase is ted at the t concentrations and is the most active proteinase t the widest variety of connective tissue components, including elastin. In the evaluation assay, inhibition of this enzyme employed a synthetic e peptide ate (Methoxysuccinyl—Ala-Ala-Pro~Val—p-Nitroanilide) that is specific for neutrophil elastase. The source of neutrophil elastase was a purified enzyme preparation derived from the airway secretions of patients with cystic s. Analysis ofthe concentration dependence of inhibition leads to the quantitation ofpotency of the inhibitory activity. This activity is expressed as that concentration of dry matter within each cosmetic botanical ingredient required to achieve 50% inhibition (IC50). In addition, the value ofthe inhibition constant, K,, was determined. Graphical analysis of the inhibition data also provides the information related to the mode of inhibition (reversible or irreversible). Since neutrophil elastase has positive physiological roles when present at controlled levels, riminate use of irreversible inhibitors may compromise these normal functions ofthe enzyme.

] Table 32 describes the results ofthe in vitro elastase inhibition studies of the serum-derived and membrane-derived cosmetic ingredients.

Table 32m Elastase Inhibition is‘lvsduafikm s‘if {Extremism Botanical. enis ' Q32 {Sign Membrane E’metitt 3’1 i’a'riétisstéfmmfii_;:....

Elastase tion activity has been fied predominantly in cosmetic ingredients obtained from membrane fractions. Cosmetic ingredients produced from a, barley and marigold flowers serum fractions did not display any elastase tion. Cosmetic ingredients obtained from lavender and sage serum fractions did show much lower inhibitory -47_ W0 2012/148527 PCT/U82012/025899 activity compared with corresponding cosmetic ingredients obtained from membrane fractions. The above pattern of distribution of elastase inhibition activity between the membrane-derived and serum-derived cosmetic botanical ingredients was found for all tested raw material s.

Selected cosmetic ingredients obtained from membrane fractions demonstrate elastase inhibition activity which is comparable in magnitude with the activity of a specific elastase inhibitor used as a positive control.

Cosmetic ingredients obtained from membrane fiactions demonstrated properties consistent with “classical” simple competitive and reversible elastase tors, while the positive l has a complex inhibitory behavior (including some irreversible inhibitory activity).

The inhibitory properties of cosmetic ingredients produced from membrane fractions toward the most destructive inflammatory proteinase (neutrophil elastase) qualifies these ingredients as valuable ents of topical products for use as anti—inflammatory agents. e 21 se Inhibition Evaluation of the Marigold Products.

[00167] The in vitro se inhibition evaluation of marigold ts are described below and summarized in Table 33.

Table 339% Etta-time Inhibitim Ewaiumitm at“ Minriggtsiti meme Extract is .1 ‘ \\\\ Cosmetic ient 402, obtained from ld flowers membrane fraction, demonstrated the highest elastase inhibition activity, but the cosmetic ingredient 401 obtained from serum fraction, which was derived from the same raw material and separated from membrane fraction during cell juice fractionation s, has no detectable inhibition activity. -48— W0 2012/148527 PCT/U82012/025899 The water extract which was produced by tional extraction methods was obtained from the same batch ofraw material and did not display elastase inhibition activity.

The commercial extract, which was derived from same raw material, displayed only minimal anti-elastase activity.

The cosmetic ingredient 402 derived fi‘om same raw material displayed anti- elastase activity by about two orders of magnitude over that of the commercial extract.

Example 22 Evaluation of Anti-Gelatinase Activity of the Cosmetic Botanical Ingredients.

Neutrophils n two major enzymes from the class ofmatrix metalloproteinases, which collectively are implicated in extensive connective tissue destruction: neutrophil collagenase (MMP-8) and nase B (MMP—Q). Because elastase has poor activity against native collagen, and neutrophil collagenase alone cannot solubilize the connective tissue protein by , gelatinase B is considered as a major contributor to inflammatory injury to the extracellular matrix. A c assay for this enzyme was used to te the potential of cosmetic botanical ingredients to inhibit degradation ofthe extracellular matrix mediated by inflammatory cell-derived matrix metalloproteinases.

Gelatinase B activity was detected by ysis of a low molecular weight synthetic substrate (APMA). tors of nase B diminish the accelerating rate of enzyme reaction product formation in a dose-dependent fashion. Such enzyme inhibition was found when tested cosmetic ingredients were added to the reaction mixture.

The anti~gelatinase B data regarding the Cosmetic Botanical Ingredients is described below and summarized in Table 34.

Tab!» firm I}: FEM? nase it Kuhihiiiaii Evaiuamm of{fosmfic- Bfiififlifiaf Ingredients.

Mk "—. mm} 2% Ifiiii‘hiiitm {immune {mama‘s iiigsedient e} § _ ma.... {18?} ggm‘ii Hub \ « All derived cosmetic botanical ingredients have trated modest gelatinase B inhibition activity.

PCT/U52012/025899 ] Cosmetic Botanical Ingredient 502 obtained from the sage membrane fraction demonstrated significant nase B tion activity, which exceeded by at least three times the corresponding activity of the serum-derived cosmetic ingredient. The pattern of distribution of gelatinase B inhibitory activity between membrane-derived and serum-derived cosmetic ingredients was similar to the bution pattern found for anti—elastase activity.

Cosmetic Botanical Ingredient obtained from sage membrane fraction trated potent gelatinase B inhibition activity (1C50=24.9 pig/ml) comparable to that of a positive l (rosmarinic acid) having 1C50=30 rig/ml.

The inhibitory properties of membrane-derived Cosmetic Botanical Ingredients toward gelatinase B indicate that this ingredient has value as an active component of anti—inflammatory topical products.

Example 23 In Vitro Evaluation of Suproxide ging ty for the Cosmetic Botanical ients.

Reactive oxygen species generated by activated inflammatory cells (endogenous or exogenous) create specific oxidant which was used to provide the measurements roxide ging activity. The assay used for evaluation ofsuperoxide scavenging activity relates to the one form of antioxidant activity, which is of benefit in neutralizing the damage ated with oxidation. To generate superoxide anions in high yield and in a controlled fashion, an enzymatic system (xanthine oxidase) was used. The conversion of xanthine to hypoxanthine by this enzyme generates amounts of superoxide anions, which are stoichiometric with the amount of substrate provided. The assay used was based on the reduction of cytochrome c from its ferric to ferrous form as a sensitive measure of superoxide levels. The advantage of using cytochrome 0 reduction to detect superoxide anions generated by the action of xanthine oxidase on xanthine is that the same measure be ed to detect the e of superoxide anions by activated inflammatory cells undergoing a “respiratory burst.” Cosmetic Botanical Ingredients which decrease the magnitude ofthe respiratory burst but do not scavenge enzymatically generated superoxide anions are ably inhibiting some aspect of cell function rather than acting as scavengers ofthe reactive oxygen species generated by the cells.

The superoxide scavenging data is described below and Tables 35 and 36.

W0 2012/148527 ‘i'abie 35am Evaluation cf fiupararsitic Seswmging activity of (ifififilfiifi flotanieni ingrtzdimmt wsws\§\\w““m\\\ 1.l/ Qumrsétic Btstani ‘ mismmmsmtfi If}?{Alfiiifa 3am: Ems;rim}? 71/1;/tI/lll/ s\\\~«\\\\\\«\s\\\t 103 a anrmfictt} $51 {Mars gait: c mgg’tggggi‘ac ”wry/1 Superoxide scavenging y is fully trated in serum-derived cosmetic botanical ingredients. Membrane-derived cosmetic botanical ingredients did not demonstrate any xide scavenging ability. The above bution pattern of superoxide scavenging ability was found for all tested raw material sources. derived cosmetic botanical ingredients demonstrated approximately % ofthe superoxide scavenging ability of the positive control (rosmarinic acid).

The superoxide ging ability of the serum fractions suggest that these cosmetic botanical ingredients have value as prospective components to act as topical antioxidant and UV-protectant products.

Tame 36am E—vaiami-Rm affingmroxida Sam-staging Activity cf ikf Prfisiuzcts My¢4aalullydlullzllflv a..-.“\“~““““““““\mw...-.~.u.“w..u“w“. \Wx\ gnaw ld flowers serum-derived Cosmetic Botanical Ingredient demonstrated significant superoxide scavenging ability, but the membrane-derived Cosmetic Botanical Ingredient (derived from the same raw al) has no detectable inhibition ability.

Commercial extract, which was derived from the same raw material, displayed superoxide scavenging ability which was comparable to that of the serum—derived Cosmetic Botanical Ingredient.

W0 2012/148527 The water extract isolated by tional methods from the same batch of raw material demonstrated higher superoxide scavenging ability than the serum-derived Cosmetic Botanical Ingredient or the commercial extract.

Example 24 Evaluation of the Effect of Cosmetic Botanical Ingredients on Neutrophil Respiratory Burst.

The superoxide scavenging activity of different cosmetic botanical ingredients described in the previous Example 23 was ed with an in vitro enzymatically generated source of superoxide anions, and with an in Vivo reactive oxygen s generated by activated inflammatory cells (i.e., neutrophils). Neutrophils are ally important sources ofreactive oxygen species, because they are ed in greatest numbers to sites of local ation and because they convert some of the species, such as superoxide anions and hydrogen peroxide to an antioxidant such as hypochlorous acid. Detection ofthe superoxide anions which are released into the extracellular environment by neutrophils is a sensitive measure ofthe overall levels of activity of these cells to te multiple reactive oxygen species, collectively referred to as the “respiratory burst.” The same reagent was employed to detect the extracellular superoxide derived from neutrophils as used to detect superoxide formed enzymatically, i.e., ferricytochrorne c. Because this molecule is a protein and cannot enter the neutrophil, it does not detect ellular reactive oxygen species.

] Phorbol myristate acetate (PMA), which is known to mimic the signals for at least two independent pathways for neutrophil activation, was used as a stimulant of respiratory burst in vivo. The rate of cytochrome 0 reduction by the PMA—activated neutrophils is proportional to the ude ofthe respiratory burst in these cells. Results of dose-dependent inhibition were expressed in terms of the maximal rate of cytochrome 0 ion observed afier a 150 second lag phase following addition ofPMA.

A review ofthe data regarding the tion of neutrophil atory burst studies are presented in Table 37. _52_ PCT/U52012/025899 ‘i‘alfir. 3?...” Net:(mantis Respfiramry Beret: 3:miaatian elf Cosmefie Botanicaé‘ Ingredients i 3......»Au\\\\\\\\\\\\\\\\\ a as T an Esau l t«gm? psg‘rtsi ggfmj ‘3C(3.mmtisz. Botanimi mix—9.2.1.7. . § ygfmi s higerlieat theta“3 \ w. z uxm»\\~¥\-.-»~— . ma“ ... \tmruiatmn ’ _ :::::: ' {Tint}: inhibit-km “13“.“ w“.c“‘.~t.vw‘_~“ i Canigeinmiilbmrm Both tested Cosmetic Botanical ient 401 and 501 demonstrated ic modulation ofthe respiratory burst from PMA—stimulated neutrophils. At low concentrations, the serum-derived cosmetic botanical ingredients ted strong inhibitory activity, but at high concentrations this inhibitory ty was replaced by net modest ation ofthe atory burst above that of neutrophils stimulated with PMA alone. Therefore, serum- derived cosmetic botanical ingredients contain components having a stimulatory effect neutrophils, but these components have only moderate potency, as stimulation is observed only at high concentrations. In addition, the serum-derived cosmetic botanical ingredients contain other components which inhibit the respiratory burst at very low concentrations. The inhibition ofthe neutrophil respiratory burst at these low concentrations (~25 ug dry material/ml) cannot be due simply to superoxide ging activity, which required much higher concentrations of dry material (~150 rig/ml) to be detected.

Commercial extract, which was derived from marigold flowers, did not display any atory effect and demonstrated only inhibition of atory burst activity.

Water extract isolated by conventional methods from the same batch of marigold flowers as the derived cosmetic botanical ingredient, did not display stimulatory activity. The conventional extract did not retain any ofthe extremely potent inhibitory activity towards the neutrophil respiratory burst seen at very low (2.5 [.tg/ml) concentrations of the serum—derived cosmetic cal ingredients.

PCT/U82012/025899 Example 25 Protocols Used for Determining n Characteristics of Cosmetic Botanical Ingredients 101, 201, 301, 401, 402, 501, and 502 The following are s methods used for determining certain characteristics of Cosmetic Botanical Ingredients 101, 201, 301, 401, 402, 501, and 502. These methods are referenced throughout the above Examples. References below to the “tested products” or the “test samples” refer to Cosmetic Botanical Ingredients 101, 201, 301, 401, 402, 501, and 502.

Method 1: Method for Determination of Solid Content. The procedure for determination of solid content included evaporation of the tested t in the water bath at 100° C. until complete evaporation of water, oven storage ofthe sample at 105° C. for 3 hours, cooling to room temperature, and immediate determination of the weight of the container with solid matter.

Method 2: Method for Determination ofNon—Volatile Residue. The procedure for determination of non—volatile residue included oven storage of the tested product at 105° C. for 5 hours, cooling, and immediate determination of the weight of the container with solid matter.

Method 3: Method for Determination b* Values. The ure for determination ofL*a*b* values utilized Hunter Labscan fixed geometry calorimeter with ing geometry of 0°/45°. rd illuminant D 65 with viewing window facing upward was used. The container with tested product was placed on viewing window and measured through the bottom. The following CIELAB equations were used: C*=(a *2 +1) *2)1/2 DE*=[(DL)2 +(Da*)2 +(Db*)2] “2 DH=[(DE*)2 —(DL*)2 —(DC*)2 11” Method 4: Method for Determination l noids Content and Lutein CW. The tested samples were extracted with acetone. After homogenization and vacuum filtration, all extracts were fied with 30% potassium hydroxide in methanol. The carotenoids were successively extracted with petroleum ether. Afier additional treatment and rc-solubilization in ethanol, all s were measured at 446 ] In order to determine the lutein content, an additional dried sample from each sample extraction was used for HPLC analysis. The dried sample was re-solubilized in MTBE and methanol. The e phase HPLC system with (250><4.60 mm ID.) 5 um C13 column (“Vydac”) was used. The identity of lutein was conformed by the co-chromatography PCT/U82012/025899 ofan authentic standard. The molar absorptivity coefficient for lutein in ethanol is 144,800 em‘] mo 1-].

] Method 5: Method for Determination of Elastase Inhibitog ty. The elastase tory activity oftested fi'actions was determined using the assay, which employs neutrophil elastase (a purified enzyme preparation produced by “Elastin Products”) and synthetic peptide soluble substrate Methoxysuccinyl-Ala—Ala-Pro-Val-p-Nitroanilide produced by ”. Enzymatic cleavage of the ate s in generation of sing yellow color over time (405 nm); the rate of color generation is diminished by increasing concentrations oftested fractions ning inhibitory activity. is ofthe concentration dependence of inhibition permits quantitation of the y of the inhibitory activity, expressed as that concentration of dry matter within each tested fraction required to achieve 50% tion (ICso), but also provides information relating to the mode of inhibition.

For the determination ofICSO, the concentration of elastase was 2.5 rig/ml and concentration of substrate was 150 [.LM. For the determination of K1, the concentrations of ate were 100 nM and 200 nM.

Method 6: Method for Determination of Gelatinase B Inhibitory Activity. The commercially distributed assay (MMP—9 Activity ELISA produced by “Amersham Pharmacia”), which captures Gelatinase B specifically onto multiwell microplates by immune recognition, was used afier other proteinases were washed away. The tic activity was detected at 405 nm by hydrolysis of a low molecular weight synthetic substrate for Gelatinase B: APMA. Analysis ofthe concentration dependence of inhibition was used to determine the y oftested product dry .

Method 7: Method for Determination of Supfloxide Scavenging Activity. The enzymatic system, which uses xanthine oxidase (a purified enzyme preparation produced by “Sigma”), was used to generate superoxide anions in high yield and in a controlled fashion.

The conversion ofxanthine to hydroxanthine by this enzyme generates amounts of superoxide anions and reduction of ferricytochrome c to ferrocytoehrome c was used as a ive measure of superoxide levels. The measurements of ferrocytochrome 0 level (550 nm), when tested fractions were added to the reaction system, allow for determination oftheir superoxide scavenging activity. The final concentrations per well were for cytochrome c 75 [,tM, xanthine 425 inn/L, and xanthine oxidase 10 mU/ml.

Method 8: Method for Determination ofInhibition of the Neutrophil Respiratom Burst. The phorbol ate acetate (PMA produced by Alexis Corporation, San Diego, Calif.) was used as a trigger of the respiratory burst activity demonstrated by W0 2012/148527 PCT/U82012/025899 neutrophils. The detection of superoxide anions, which are released into the extracellular environment by neutrophils, was achieved Via measurements of ferrocytochrome c level. The rate of cytochrome c reduction by PMA—activated neutrophils is proportional to the magnitude ofthe respiratory burst in these cells. s ofdose-dependent inhibition were expressed in terms ofmaximal rate of cytochrome 0 reduction observed at 550 nm after 150 seconds lag phase following addition of PMA.

Example 26 Protocols Used for Determining n Characteristics of Various Cosmetic Botanical Ingredients of the Present Invention, Including Cosmetic Botanical Ingredients 601 and The following are various methods used for determining certain characteristics ofvarious cosmetic cal ingredients of the present invention, ing, without tion, ic Botanical Ingredients 601 and 701. These methods are referenced throughout the Examples. In particular instances, references below to the “tested products” or the “test samples” refer to ic Botanical Ingredients 601 and/or 701.

Osmolality

[00204] Osmolality is the measure of solute concentration, defined as the number of osmoles ofsolute per kg of solution. Osmolality measures the number ofosmoles of solute particles per unit mass ofsolution. lity is distinct from molarity e it measures moles ofsolute particles rather than moles of so lute. The distinction arises e some compounds can dissociate in solution, whereas others cannot. Ionic compounds, such as salts, can dissociate in solution into their constituent ions, so there is not a one—to-one relationship between the molality and the osmolality of a solution. For example, sodium chloride (NaCl) dissociates into Na+ and Cl- ions. Thus, for every 1 mole ofNaCl in solution, there are 2 s ofsolute particles (i.e., a 1 M NaCl solution is a 2 Osm NaCl solution). Both sodium and chloride ions affect the c pressure of the solution. Nonionic compounds do not dissociate, and form only 1 osmole of solute per 1 mole of solute. For e, a 1 M solution of glucose is l Osm (Widmaier, Eric P.; Hershel Raff, Kevin T. Strang (2008). ‘s Human Physiology, llth Ed. McGraw-Hill. pp. 108-112). Osmometer, model 3250 (Advanced Instruments, Inc) was used to ine osmolalities of the ingredients. This instrument utilizes freezing point depression as measuring ple. Freezing point is colligative property that is dependent on the presence of dissolved particles and their number, PCT/U82012/025899 but not their identity. The freezing point sion happens both when the solute is electrolyte, such as various salts, and a ectrolyte, such as carbohydrates.

Dry matter Dry matter reflects the concentration ofnon volatile components in the ingredients. Dry matter levels were determined by comparing the weight of liquid sample with weight of residual dry matter afier liquid components have been evaporated. Disposable aluminum weighing dishes (VWR 25433—016), Ohaus Explorer E00640 balance (Ohaus ation) and Shel Lab model 140013 oven (VWR) set at 105 C were utilized. Dry matter tage is calculated as (‘tare+dry’-‘tare’) / (‘tare+wet’-‘tare’) * 100.

Color (Gardner Scale) The Gardner Color scale as specified in ASTM D1544 is a single number colour scale for grading light transmitting samples with color characteristics ranging fiom light yellow to brownish red. The scale is defined by the chromaticities of glass standards numbered from 1 for the lightest to 18 for the darkest. The Gardner Color of samples determined on the Lovibond r Comparator 3000 (The Tintometer Limited of Salisbury, UK), a 3-field instrument for visually determining the Gardner Color of samples by direct comparison with colored glass standards.

Refractive Index, nD tive Index was determined by measuring on Arias 500 refractometer from Reichert ical Instruments (Depew, NY) with ature regulation provided at model 12108-10 temperature controller from Cole-Farmer (Vernon Hills, Illinois).

Procedure is based on the instruction manual for Arias 500 refractometer, sections 6.0, 4.1 and 4.4-4.5. pH Determination pH is defined as minus the decimal logarithm of thc hydrogen ion activity in a solution and used to determine pH is a measure of the acidity or basicity of a solution. pH levels were determined on a pH meter Model 250 pH / ISE / tivity meter from Denver Instrument Company ia, NY) with pH / ATC electrode number 3007291 (Denver Instrument Company). Procedure is based on Denver Instrument Company 301127.1 Rev. D manual, pages ii and 9 - 12.

W0 2012/148527 PCT/U52012/025899 Lambda max, nm Lambda max, nm was determined on Ultrospee 4300 pro UV / Visible spectrophotometer from Bioehrom Ltd (Cambridge, UK), formerly under GE Healtheare, formerly known as Amersham Bioseienees, with water heated cell holder (Amersham part #80—2106-08). The procedure is based on sections 2 and 4 from Amersham manual number 8025 entitled SWIFT [l Applications Software [JV/Visible ophotometers, and pages 7 and 15 from Amersham manual number 80—2111—79 entitled pee 4300 pro ible Spectrophotometer User Manual. Instrument control was ed by SWIFT H sofiware suite (Bioehrom Ltd) and temperature regulation by C1320 Mini Circulator from Torrey Pines Scientific (Carlsbad, CA).

Determination of Protein The Kjeldahl method was used to measure the protein nitrogen content.

Microbiological Limits Microbial content and limits: Total Plate Count, CPU/g; Mold and Yeast, CPU/g; E. coli; Salmonella .81).; Staphylococcus aureus; Pseudomonas 5]). were determined according to US Pharmacopoeia XXX, NF25, <61>, Microbiological Limit Tests.

Trypsin tion Trypsin is a proteolytie enzyme that is involved in in vivo epidermal eration and inflammation. Trypsin inhibition ty was ined by a kinetic colorimetric assay designed for use with 96-well iter plates (mieroplates) and computer-controlled microplate reader. Enzymatic activity in cleaving the substrate was indicated by a development ofyellow color measured as se in absorbance at 405nm ngth. The mean ofmaximum rates of absorbance increase for negative control wells was considered as 100% of enzyme activity, and IC50 was calculated as concentration of sample in the well necessary to reduce the enzyme activity to 50%. Lower ICso values indicate higher trypsin inhibition activity. L—BAPA (Nu-Benzoyl-L-arginine 4-nitroanilide hydrochloride) substrate, trypsin, and solvent reagents were obtained from Sigma-Aldrich. pH 8.2 Tris-CaClz buffer was used for preparing g solutions of n and L-BAPA substrate. Deionized water was used as solvent for buffer reagents, as negative control, and ~58— W0 2012/148527 PCT/U82012/025899 the diluents for preparing serial dilutions ofthe samples. Reaction volume in each well 200 pl, with concentration oftrypsin equal to 6011M and substrate equal to 0.5mM.

Tyrosinase inhibition Tyrosinase is a copper—containing monooxygenase zing the o— hydroxylation of monophenols to the corresponding catechols (monophenolase or cresolase ty), and the oxidation of monophenols to the corresponding o-quinones (diphenolase or olase activity). These functions of tyrosinase play an important role in the formation of melanin pigments during melanogenesis. Melanin production is principally sible for skin color and plays an ant role in prevention of sun-induced skin .

However, abnormal accumulation of melanin products in skin is responsible for hyperpigmentations including a, chloasma, freckles, and senile lentigines, which can lead to an undesired aesthetic appearance (Jeon et a1. (2005) Bull. Korean Chem. Soc, Vol. 26: 1135-1 137).

Tyrosinase is an important enzyme in the biosynthesis of melanin. Tyrosinase inhibition assay was used to search for ingredients that can interfere with the y of mushroom tyrosinase enzyme to convert L-tyrosine to droxyphenylalanine (L-DOPA).

Tyrosinase inhibition activity was determined by a kinetic colorimetric assay designed for use with 96-well microtiter plates plates) and computer-controlled microplate reader. Enzymatic activity in converting the L—tyrosine substrate to L—DOPA (L- 3,4—dihydroxyphenylalanine) was indicated by a development n color measured as increase in absorbance at 475nm wavelength. The mean ofmaximum rate of absorbance increase for negative control wells was ered as 100% of enzyme activity, and 1C50 was calculated as concentration of sample in the well necessary to reduce the enzyme activity to 50%. Lower ICso values indicate higher tyrosinase inhibition activity. L-tyrosine substrate and mushroom tyrosinase were obtained from Sigma. 1X pH 7.4 PBS (Phosphate Buffered Saline) buffer solution was obtained from Gibco. PBS was used for preparing working solutions of mushroom tyrosinase and L-tyrosine substrate. Deionized water was used as negative control and as diluent for preparing serial dilutions of the samples. on volume in each well was 200mL, with concentration ofmushroom tyrosinasc equal to 13 units / mL and L-tyrosine substrate equal to 0.5mM.

LDH (lactate dehydrogenase) release cytotoxicity method LDH (lactate ogenase) e cytotoxicity determination method is based on the fact that n cell component substances are typically sequestered inside the W0 48527 2012/025899 cells and scarce in ellular medium. Loss of cell viability leads to loss of cell membrane integrity and release of such substances. ellular LDH concentration can be measured colorimetrically with use of a dye which is converted by LDH into colored form. Cytotoxicity of a test article can therefore be determined by adding the test article to growth medium of cell culture, measuring LDH concentration in the medium after proper incubation time, and comparing the results with those obtained fiom growth medium tive control cell e of healthy untreated cells, as well as those from positive control cell culture treated with a known cytotoxic agent.

MatTek MelanoDerm Assay ] MatTek Melanoderm Assay determines effect of a test article on melanin— related skin pigmentation (such as for whitening / lightening applications). The assay is based on macroscopic and microscopic observations and endpoint melanin content ination in a co—culture ofnormal human keratinocytes and melanocytes that models human epidermis and its color development. The Melanoderm cell cultures are maintained and observed for the duration ofthe study, with untreated negative controls compared to positive controls which have known melanogenesis—affecting substances added to growth medium, as well as cultures which have the test article added to growth medium. At the end of the study, the cell cultures are harvested and processed to measure melanin content using a colorimetric assay. idant activity idant is an agent that reduces the damage caused by oxidation.

Antioxidant activity was determined by ORAC testing using an adaptation of the method described in “Performing Oxygen Radical Absorbance Capacity (ORAC) Assays with Synergy HT Multi—Detection late Reader” Application Note from BioTek available at (www.biotek.com/resources/docs/ORAC_Assay_Application_Note.pdf) for use with Synergy 2 microplate reader from BioTek Instruments Inc (Winooski, VT). In this assay, AAPH (2, 2'- azobis 2-amino-propane) generates reactive oxygen species which damage the fluorescent probe (sodium ccin). Antioxidants such as (R) x methyl ether prevent or slow this damage, and their effects can be quantified by fluorescence measurements. Fluorescence readings were taken with excitation wavelength set at 485 nm and emission wavelength set at 528 nm, with reaction volume of 200 pl, AAPI-I concentration of 55 mM, sodium fluorescein concentration of 1.33 uM, and (R)—Trolox methyl ether concentration range between 80 uM and 2 uM. Sodium fluorescein (Fluka 46960), AAPH (Sigma ) and (R)-Trolox methyl WC 2012/148527 ether (Fluka 93509) were obtained from Sigma-Aldrich (St. Louis, MO). AUC (Area Under Curve) values were calculated as sum of tions nt fluorescence g for the well divided by first fluorescence reading for the well). Average of AUC values ofwells with deionized water was subtracted From AUC ofwells with (R)-Trolox methyl ether and wells with test articles to obtain AUC corresponding to preservation of fluorescence by antioxidants. A calibration curve was ted as function of a wells’ antioxidant-related AUC showing (R)-Trolox methyl ether weight-equivalent ORAC activity. ORAC activity for test articles was then calculated as units weight test article necessary to achieve antioxidant effect equal to one produced by 1 unit weight (R)-Trolox methyl ether, with lower numbers indicating higher ORAC activity.

DPPH (2,2-Diphenyl-l-Picrylhydrazyl) free radical scavenging activity Free radical scavenger is an ingredient that reacts with free radicals in biological system, reduces free radical—induced damage, and protects against the effects of free radicals. Free radical scavenging activity, i.e. DPPH (2,2 —Diphenyl—1-Picrylhydrazyl) free radical scavenging activity, was determined by a c colorimetric assay d for use with glass-coated polypropylene 96-well microtiter plates (catalog number 400 062) from SUN-SRi (Rockwood, TN) and Synergy 2 microplate reader from BioTek Instruments Inc ski, VT). Absorbance was measured at 515 nm ngth. Reaction volume in each microplate well was 200 ul, with initial concentration ofDPPH equal to 114 uM. L—ascorbic acid was used as positive control. DPPH (Sigma D9132) and USP L-ascorbic acid (Sigma A— 2218) were obtained from Aldrich (St. Louis, MO). Stoichiometry of the reaction was calculated and expressed as units weight test article necessary to quench 1 unit weight DPPH, with lower numbers indicating higher activity. This method was d from procedure described in (“Use of a free radical method to evaluate antioxidant ty” by W. Brand- Williams et al, published in LWT - Food Science and Technology, Volume 28, Issue 1, 1995, pp 25~30).

Superoxide scavenging assay

[00221] xide scavenging assay protocol was adapted from “Rapid Microplate Assay for Superoxide Scavenging Efficiency” in Journal ofNeuroscience Methods 97 (2000) pp. 139-144. -61— W0 2012/148527 PCT/U52012/025899 Example 27 Preparation of Cosmetic Botanical Ingredient 601 derived from red clover (Trifolium pratense) Cell Serum Fractions.

[00222] The process for preparing Cosmetic Botanical Ingredient 601 was identical to the process described in Example 1 with regard to Cosmetic Botanical Ingredient 10] with the variations noted below. Fresh stem, flower and leaftissue of red clover lz'wn pratense) was used as the plant biomass starting material. The level of dry matter in the Fresh red clover plant s was ated to be 15.16 percent, requiring ting of approximately 560 kg of fresh red clover plant biomass to yield 100 kg of dry matter. The ation resulted in the tion of 15.36 kg of Dry Matter yield (or approximately 300 liters) of Cosmetic Botanical Ingredient 601.

Example 28 Product Specifications of Cosmetic Botanical Ingredient 601 d from red clover (Trifolium prafense) Cell Serum Fractions.

Cosmetic Botanical Ingredient 601 was prepared according to the process described above in Example 27.

[00224] Analyses of Cosmetic Botanical Ingredient 601 were ted to determine its various physico-chemical, microbial, and bioactivity characteristics, as described below.

Cosmetic Botanical Ingredient 601 is a clear liquid, which has a yellow-reddish color and a characteristic odor. No solvent (i.e., , oil, or water) was added to the carrier medium.

Table 38 summarizes the Physical, chemical and organolaeptic characteristics of Cosmetic Botanical Ingredient 601.

W0 48527 Table 38: Physical, chemical and organolaeptic characteristics of Cosmetic Botanical Ingredient 601 Characteristics Description/Range —a_ ammo litymwater 4.5-6.1 tive index (nD) 1.3440 — 1.3460 pl-l 3.842 Osmolality (mOsm / kg) 780-910 UV max, mm +— 350—358 —i Total Plate Count (CFU / gm) ‘1 < 10 Mold / Yeast (CFU / gm) < 10 E. coli (CFU/ gm) _J Negative/ 10 gm Salmonella sp. (CFU / gm) Negative/ 10 gm Staphylococcus aureus (CFU / gm) Negative/ 10 gm

[00226] ic Botanical Ingredient 601 contained 0101-0104 % of nitrogen determined by Kjeldahl method that indicates that it is substantially protein-free Proteins, including those in plants, can cause protein contact dermatitis in sensitive individuals. Shortly after contact with the causative proteinacous material, such individuals can experience symptoms such as acute urticarial or vesicular eruption on the skin, often accompanied by pruritus, burning, and/or stinging. (V. Janssens, et al., “Protein contact dermatitis: myth or reality?”, h Journal atology 1995; 132: 1—6).

Thus, it is highly desirable that skin care materials contain as little proteins as possible. As used herein, “substantially free of ns” means less than 1% (from 0% to 1%) total protein content using the Kjeldahl method.

] Microbial analyses demonstrated that ic Botanical Ingredient 601 maintaining physical and chemical integrity) for at least 12-18 months while stored at a temperature ofbetween 15 and 25° C. in a closed container protected fiom light.

Cosmetic Botanical Ingredient 601 is a biodegradable ingredient.

W0 2012/148527 PCT/U52012/025899 Example 29 Summary of activity and assay data regarding ic Botanical Ingredient 601 Red Clover (Trifolium pratense) Cell serum fractions.

According to mushroom tyrosinase inhibitor efficacy testing Red Clover Serum Fraction can t tyrosinase, achieving IC5o values about 0.02% w/v.

According to superoxide scavenging assay col adapted from “Rapid Microplate Assay for Superoxide ging ncy” in Journal oscience Methods 97 (2000) pp. 139—144) Red Clover Serum Fraction is capable ofscavenging superoxide, ing IC50 ofabout 0.03% w/v.

According to DPPH firee radical ging assay (protocol based on “Use of a free radical method to evaluate antioxidant activity” in LWT - Food Science and Technology, Volume 28, Issue 1, 1995, pp 25-30) Red Clover Serum Fraction is capable of quenching DPPH, with about 9.5 units dry weight of the material required to completely quench 1 unit weight DPPH.

] According to Oxygen Radical Absorbance Capacity assay (protocol based on “Performing Oxygen Radical Absorbance Capacity (ORAC) Assays with Synergy HT Multi- Detection Microplate Reader” Application Note from BioTek Instruments) Red Clover Serum Fraction can serve as an antioxidant, with about 4.4 units dry weight of the material required to provide ORAC effect equal to 1 unit weight of (R)-Trolox methyl ether.

Example 30 Preparation of Cosmetic Botanical Ingredient 701 derived from Lotus (Nelumbo nucifera) Cell Serum Fractions The process for preparing Cosmetic Botanical Ingredient 701 was identical to the process described in Example 1 with regard to Cosmetic Botanical ient 101, with the variations noted below. Fresh stem, flower and leaftissue ofLotus (Nelumbo nucgfera) was used as the plant biomass starting material. The level of dry matter in the fresh Lotus (Nelumbo nucifera) plant biomass was calculated to be 20.7 percent, requiring ting of imately 483 kg ofLotus (Nelumbo nuczfera) plant biomass to yield 100 kg of dry matter. The preparation resulted in the production of approximately 13 kg Dry Matter yield (or imately 262 liters) of Cosmetic Botanical Ingredient 701.

W0 2012/148527 PCT/U52012/025899 Example 3] Product Specifications of Cosmetic Botanical Ingredient 701 d from Lotus (Nelumbo nucifera) Cell Serum Fractions.

Cosmetic Botanical Ingredient 701 was prepared according to the process described above in Example 30.

Analyses of Cosmetic Botanical Ingredient 701 were conducted to determine its various physico-chemical, microbial, and bioactivity characteristics, as described below.

Cosmetic Botanical Ingredient 701 is a clear liquid, which has a yellow color and a characteristic odor. No solvent (i.e., glycol, oil, or water) was added to the carrier medium.

Table 39 summarizes the Physical, chemical and organolaeptic characteristics ofCosmetic Botanical Ingredient701.

Table 39: Physical, al and organolaeptic characteristics of Cosmetic Botanical Ingredient 701 Characteristics Description/Range Appearance Clear Yellow Liquid Odor Characteristic Solubility in water Soluble in any ratio Color (Gardner scale) 5-6 13370—13450 pH 3.9-4.4 lity (mOsm / kg) 460—610 UV max, nm 261—269 Total Plate Count (CFU / gm) < 10 Mold / Yeast (CFU / gm) < 10 E. coli (CFU / gm) Negative/ 10 gm Salmonella sp. (CPU / gm) Negative/ 10 gm Staphylococcus aureus (CFU / gm) Negative/ 10 gm monas sp. (CFU / gm) Negative/ 10 gm ] Cosmetic Botanical Ingredient 701 contains . 167 % ofnitrogen determined by Kjeldahl method that tes that it is ntially protein—free.

[00239] Proteins, including those in plants, can cause protein contact dermatitis in ive individuals. Shortly after contact with the causative nacous material, such individuals can experience symptoms such as acute urticarial or vesicular on on the skin, often accompanied by us, burning, and/or stinging. (V. Janssens, et al., “Protein contact dermatitis: myth or reality‘?”, British Journal ofDermatology 1995; 132: 1—6).

PCT/U82012/025899 Thus, it is highly desirable that skin care materials contain as little proteins as possible. As used herein, antially free of proteins” means less than 1% (from 0% to 1%) total protein content using the Kjeldahl method.

Microbial analyses demonstrated that Cosmetic cal Ingredient 701 satisfies the cosmetic industry requirements for cosmetic ingredients with regard to CFUs and e ofpathogens Cosmetic Botanical ient 701 was determined to be stable (i.e., ining physical and chemical integrity) for at least 12~18 months while stored at a temperature een 15 and 25° C. in a closed ner protected from light.

Cosmetic Botanical Ingredient 701 is a biodegradable ingredient.

Example 32 Summary of activity and assay data regarding Lotus (Nelumbo nucifera) serum ons.

[00243] According to LDH release cytotoxicity method (MB ch protocol 701— 01) study completed by MB Research, Lotus Serum Fraction does not show cytotoxicity in concentration ranges up to 10% oftest article by volume in cell culture medium.

According to MatTek MelanoDerm Assay (MB Research protocol 750-01) Lotus Serum Fraction at concentration 5% v/v or 0.19% w/V in cell culture medium produce a lightening / whitening effect in keratinocyte /melanocyte cell culture model as determined by approximately 30% decrease in melanin levels below ve control and e ning of the tissue noticeable with a naked eye. ing to trypsin inhibitor efficacy testing performed Lotus Serum Fraction is capable of inhibiting trypsin, with IC50 calculated as about 0.04% w/V.

[00246] According to superoxide scavenging assay (protocol adapted fi‘om “Rapid Microplate Assay for Superoxide Scavenging Efficiency” in Journal oscience Methods 97 (2000) pp. 139-144) g Lotus Serum Fraction is capable ofscavenging superoxide, achieving ICSO of about 0.016% W/V.

According to DPPH free radical scavenging assay (protocol based on “Use of a flee radical method to evaluate antioxidant activity” in LWT - Food Science and Technology, Volume 28, Issue 1, 1995, pp 25—30) Lotus Serum Fraction is capable of quenching DPPH, with about 2.5 units dry weight of the material required to completely quench 1 unit weight DPPH.

According to Oxygen Radical Absorbance Capacity assay (protocol based on “Performing Oxygen Radical Absorbance Capacity (ORAC) Assays with Synergy HT Multi— Detection Microplate Reader” Application Note from BioTek Instruments) Lotus Serum Fraction can serve as an antioxidant, with about 1.7 units dry weight of the material required to provide ORAC effect equal to 1 unit weight of (R )-Trolox methyl ether.

Although preferred embodiments have been ed and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the range and scope of the invention and these are therefore ered to be within the scope of the ion as defined in the claims which follow.

Claims (42)

WHAT IS CLAIMED IS:

1. A bioactive botanical cosmetic composition comprising: a cell serum fraction filtrate derived from cell juice extracted from a fresh plant biomass, said cell serum fraction e having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth ation activities, wherein said fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nuczfera) and red clover (Trifolium pratense); and a stabilizing agent, wherein said cell growth stimulation activity is due to stimulation of eration of at least one type of cell, 10 wherein said cell serum fraction filtrate is d from the cell juice according to the following steps: providing the plant cell juice, said plant cell juice having been extracted from the fresh plant biomass; treating the plant cell juice under conditions effective to te the plant cell juice 15 into a membrane fraction and a cell juice atant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; and refining the cell serum fraction under conditions effective to yield the cell serum fraction filtrate, wherein said g comprises ting the cell serum fraction to a 20 temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate.

2. The bioactive botanical cosmetic composition according to claim 1, wherein said stabilizing agent is ed from the group consisting of a preservative and an 25 antioxidant.

3. The bioactive botanical cosmetic composition according to claim 2, wherein said preservative is selected from the group consisting of potassium e, sodium benzoate, sodium methyl paraben, and citric acid.

4. The bioactive cal cosmetic composition according to claim 2 or 3, wherein said antioxidant is sodium metabisulfite. —68-

5. The bioactive cal cosmetic composition according to claim 2 or 3, wherein said antioxidant ty is selected from the group consisting of superoxide scavenging activity and neutrophil respiratory burst inhibitory activity.

6. The bioactive botanical ic composition according to any one of the preceding claims, wherein said cell serum fraction filtrate comprises between about 1 and about 10 weight percent of said bioactive botanical ic composition.

7. The bioactive botanical cosmetic composition according to claim 6, wherein 10 said bioactive botanical cosmetic composition has a superoxide scavenging potency range from an ICR50 value of between about 50 and about 190 pg of dry matter/ml, wherein said ICRSO value represents the concentration of dry matter ned in the cell serum fraction e required to inhibit 50 percent of cytochrome c reduction. 15

8. The bioactive botanical ic composition according to any one of the preceding claims, wherein said cell serum fraction filtrate has a cell growth stimulation potency ranging from between about 1.0 and 125 pg of dry matter/m1.

9. The bioactive botanical cosmetic composition ing to claim 8, wherein 20 said cell serum fraction filtrate has an NRU value ofbetween about 110 and 190 percent, wherein said NRU value represents cell Viability.

10. The ive botanical cosmetic composition according to any one of the preceding claims, wherein said bioactive botanical cosmetic ition has an ability to 25 cause biphasic modulation of respiratory bursts from phorbol myristate e-stimulated neutrophils, in that said composition inhibits said respiratory bursts at between about 1.0 and

5.0 ,ug dry material/ml and stimulates said respiratory bursts at between about 20 and 180 ug dry material/m1. 30 11. The bioactive botanical cosmetic composition ing to any one of the preceding claims, wherein said ive botanical cosmetic composition comprises characteristics as set forth in Table 38 or Table 39.

12. A bioactive botanical cosmetic ation, suitable for topical ation to a mammal, comprising a cosmetically able carrier and a cosmetically effective amount of the bioactive botanical cosmetic composition according to any one of claims 1 to 11.

13. The cosmetic formulation ing to claim 12, n the cosmetically acceptable carrier is selected from the group consisting of a hydrophilic cream base, a hydrophilic lotion base, a hydrophilic surfactant base, a hydrophobic cream base, a hydrophobic lotion base, and a hydrophobic surfactant base. 10

14. The ic formulation according to claim 12 or 13, wherein said bioactive botanical cosmetic composition is present in an amount ranging from between about 0.001 percent and about 95 t of the total weight of the cosmetic formulation.

15. The bioactive botanical cosmetic composition according to any one of claims 15 1 to 11, wherein said plant source is lotus (Nelumbo ra).

16. The bioactive botanical coSmetic composition according to any one of claims 1 to 11, wherein said plant source is red clover (Trz'folz'um pratense). 20

17. The cosmetic ation according to any one of claims 12 to 14, wherein said plant source is lotus (Nelumbo ra).

18. The cosmetic formulation according to any one of claims 12 to 14, wherein said plant source is red clover (Trz'folz‘um pratense).

19. A method for preparing a bioactive botanical cosmetic composition, said method comprising: providing a plant cell juice, said plant cell juice having been extracted from a fresh plant biomass, n said fresh plant biomass is from a plant source selected from the 30 group consisting of lotus (Nelumbo nucz‘fera) and red clover (Trifolz‘um pratense); treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; refining the cell serum on under conditions effective to yield a cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation activities, wherein said refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction e; and stabilizing the cell serum fraction filtrate under conditions effective to yield a stable bioactive botanical cosmetic ition exhibiting said antioxidant activity, cell growth stimulation ty, or both antioxidant and cell growth stimulation ties.

20. The method according to claim 19, n said treating comprises: coagulating the plant cell juice to yield a coagulated cell juice mixture, and separating the coagulated cell juice to a membrane fraction and a cell juice supernatant.

21. The method according to claim 20, wherein said coagulating comprises: destabilizing the plant cell juice to yield a coagulated cell juice mixture, n said destabilizing is achieved by heat treatment, electro-membrane treatment, chemical treatment, and/or their combination.

22. The method according to claim 21, wherein said heat treatment comprises: heating the plant cell juice to a heat ent temperature required to induce coagulation of the membrane fraction, and cooling the heated cell juice to a temperature effective to permit quantitative 25 separation of said membrane fraction from said cell juice atant.

23. The method according to claim 22, wherein said heating is carried out at 45 to 70 degrees Celsius. 30

24. The method according to claim 22 or 23, wherein said cooling is carried out at 30 to 45 degrees Celsius.

25. The method according to claim 20, wherein said ting is carried out by filtration or centrifugation.

26. The method according to any one of claims 19 to 25, wherein said sing comprises: subjecting the cell juice supernatant to a cytoplasmic fraction precipitation step to yield a cytoplasm/cell serum e comprising the cytoplasmic fraction and the cell serum fraction, and separating the cytoplasmic fraction from the cell serum fraction.

27. The method according to claim 26, wherein said asmic fraction 10 precipitation step is carried out by isoelectric titration, electrodialysis, and/or their combination.

28. The method according to claim 27, wherein said isoelectric titration comprises: 15 adjusting the pH of the cell juice supernatant to between about 2.5 and 6.5.

29. The method according to any one of claims 26 to 28, wherein said separating is carried out by filtration or centrifugation. 20

30. The method ing to any one of claims 19 to 29, wherein said temperature treatment step comprises: heating the cell serum fraction to a heating temperature required to induce ation within the cell serum fraction, and cooling the cell serum fraction to a temperature effective to allow fiirther quantitative 25 separation of said cell serum fraction filtrate.

31. The method according to claim 30, wherein said g temperature is at 80 to 95 s Celsius. 30

32. The method according to claim 30 or 31, wherein said cooling is to a temperature of at least as low as about 15 degrees Celsius.

33. The method according to any one of claims 19 to 32, wherein said clarifying is carried out by filtration or centrifugation.

34. The method according to claim 33, wherein said ion comprises: vacuum filtrating the coagulated cell serum fraction to yield said cell serum fraction filtrate.

35. The method according to any one of claims 19 to 34, further comprising: adjusting the pH of the cell serum fraction to about 3.0 to 4.0 immediately prior to said refining. 10

36. The method according to any one of claims 19 to 35, wherein said stabilizing comprises: incubating said cell serum fraction filtrate in a mixture of at least one preservative and at least one antioxidant to yield the stable bioactive botanical ic composition. 15

37. The method according to claim 36, wherein said preservative is selected from the group consisting of potassium sorbate, sodium benzoate, sodium methyl paraben, and citric acid.

38. The method according to claim 36 or 37, wherein said antioxidant is sodium 20 sulfite.

39. The method according to any one of claims 19 to 38, wherein said plant source is lotus (Nelumbo ra). 25

40. The method according to any one of claims 19 to 38, wherein said plant source is red clover (Trifolium pratense).

41. A stable bioactive cal cosmetic composition made by the method according to any one of claims 19 to 40.

42. The bioactive botanical cosmetic composition according to claim 1, substantially as herein described with reference to the