METHOD OF REGULATING A CONDITION OF MAMMALIAN KERATINOUS TISSUE

FIELD

The present invention relates generally to regulating a condition of mammalian keratinous tissue with achachariu. In some instances, achachariu serum fractions and/or extracts are used to regulate a skin or hair condition, for example, by incorporating the fraction or extract into a suitable personal care composition.

BACKGROUND

Mammalian keratinous tissue, particularly human skin and hair, is constantly subjected to a variety of insults from both extrinsic and intrinsic sources. Extrinsic sources include ultraviolet radiation, environmental pollution, wind, heat, infrared radiation, low humidity, harsh surfactants, abrasives, and the like, whereas intrinsic sources include chronological aging and other biochemical changes from within the body. Whether from an extrinsic or intrinsic source, these insults can lead to visible signs of damage to skin and hair. In skin, this damage may manifest as undesirable thinning, fine lines, wrinkling, hyperpigmentation, sallowness, sagging, dark under-eye circles, puffy eyes, enlarged pores, diminished rate of skin cell turnover, flaking, scaling, dryness, and/or roughness. For hair, these insults can contribute to, for example, hair bleaching, split ends, fragility, roughness, hair loss, reduction in hair growth rate. Currently, there are a number of personal care products available to consumers for improving the health and physical appearance of keratinous tissues, the majority of which are directed to delaying, minimizing or even reversing the changes typically associated with aging or environmental damage. However, there is a continuing need for products and methods that seek to remedy these undesirable keratinous tissue conditions.

One potential source of bioactive ingredients for regulating conditions in mammalian keratinous tissue is achachariu. Achachariu (Garcinia humilis) is a plant belonging to the Clusiaceae (or Guttiferae) family and is widely distributed in the region of Santa Cruz, Bolivia. The achachariu plant and/or fruit is used in Bolivian folk medicine for its healing, digestive, and laxative properties. In Brazil, achachariu is popularly known as "achacha" and is used in folk medicine to treat rheumatism, inflammation, pain and gastric disorders (Alves TMA, Silva AF, Brandao M, Grandi TSM, Smania EF et al. (2000) "Biological Screening of Brazilian Medicinal Plants." Mem Inst Oswaldo Cruz 95: 367-373 and Barbosa W, Chagas EA, Martins L, Pio R, Tucci ML et al. (2008) "Germinacao de sementes e desenvolvimento inicial de plantulas de achachariu." Rev Bras Frutic 30: 263-266). Some studies suggest that achachariu, like many plants, contains unwanted components of concern, such as benzophenones (e.g., guttiferone A) and proteins. See, e.g., Acuna UM, et al., (2009) "Polyisoprenylated benzophenones from Clusiaceae: potential drugs and lead compounds." Curr Top Med Chem 9: 1560-1580). Benzophenones are known to exhibit various biological activities such as cytotoxic, genotoxic antimicrobial, antiviral and antioxidant activity. See, e.g., Terrazas PM, et al., (2013) "Benzophenone guttiferone A from Garcinia achachairu Rusby (Clusiaceae) Presents Genotoxic Effects in Different Cells of Mice." PLoS ONE 8(11): e76485). And proteins are known to cause allergic reactions when contacted with skin. See, e.g., V. Janssens, et al., (2015) "Protein contact dermatitis: myth or reality?" British Journal of Dermatology; 132: 1-6). Thus, in some instances it may be desirable to remove these unwanted components or reduce their concentrations substantially when providing an achachariu ingredient for incorporation into a personal care composition.

SUMMARY

Disclosed herein is a method of regulating a condition of mammalian keratinous tissue, comprising: identifying a target portion of keratinous tissue in need of treatment or where treatment is desired; and applying a personal care composition comprising an effective amount of an achachariu serum fraction to the target portion of keratinous tissue. The method may include one or more of the following features in any combination: the achachariu serum fraction is obtained from achachariu fruit; the achachariu serum fraction is obtained from the peel of the achachariu fruit; the achachariu serum fraction is made by a process comprising filtering achachariu cell juice to remove particulates; the achachariu serum fraction has a dry matter content of less than 25%; the personal care composition is applied to the target portion of keratinous tissue at least once per day during a treatment period; the personal care composition is a rinse-off type composition, and at least some of the personal care composition is removed from the target portion of keratinous tissue after application; rinsing at least some of the personal care composition off the target portion of keratinous tissue with water; removing at least some of the personal care composition from the target portion of keratinous tissue with an implement; the achachariu serum fraction is present in the personal care composition at an amount of from about 0.01% to about 15%.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of a cross-sectional view of an achachariu fruit. FIG. 2 is a schematic of a process for preparing bioactive serum fractions and bioactive fraction extracts derived from achachariu.

FIGS. 3 A, 3B, 4 A, 4B are graphs illustrating absorbance spectra of achacha serum fractions and extracts.

FIGS. 5 - 8 illustrate the respective amino acid losses for serine, glutamine/glutamic acid, valine, and proline observed in a protein loss assay.

FIG. 9 illustrates the total amino acid loss observed in a protein loss assay.

DETAILED DESCRIPTION

It has now been discovered that achachariu (Garcinia humilis), especially bioactive extracts and serum fractions obtained from particular portions of the achachariu plant, possess properties that may be beneficial for regulating conditions of mammalian keratinous tissue. In particular, bioactive achachariu fruit extracts and serum fractions exhibit surprising properties related to anti-oxidation, inhibiting melanin production, inhibiting lipogenesis, and reducing hair protein loss, which can be exploited to provide a wide variety of different skin and/or hair health benefits. Thus, it is now possible to regulate a particular skin and/or hair condition by applying an effective amount of achachariu to a target portion of keratinous tissue where treatment is needed or desired.

It may also be desirable to provide achachariu serum fractions and/or extracts that are free or substantially free of benzonphenones (e.g., guttiferone A) and/or proteins. As used herein, "substantially free of proteins" means less than 0.15% total protein content determined by hydrolyzed and un-hydrolyzed amino acid analysis conducted on an Hitachi L-8900 amino acid analyzer, and "substantially free of benzophenones" means less than 0.1% total benzophenones, including guttiferone A, as determined by the method described in Dal Molin MM, et al. (2012) Phytochemical analysis and antinociceptive properties of Garcinia achachariu Rusby (Clusiaceae) seeds. Arch Pharm Res 35: 623-631.

As used in the description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All percentages disclosed herein are by weight of the total composition, unless specifically stated otherwise. All ratios are weight ratios, unless specifically stated otherwise. The number of significant digits conveys neither a limitation on the indicated amounts nor on the accuracy of the measurements. All numerical amounts are understood to be modified by the word "about" unless otherwise specifically indicated. All measurements are understood to be made at 25°C and at ambient conditions, where "ambient conditions" means conditions under about one atmosphere of pressure and at about 50% relative humidity. All such weights as they pertain to listed ingredients are based on the active level and do not include carriers or byproducts that may be included in commercially available materials, unless otherwise specified. All numeric ranges are inclusive of narrower ranges and combinable; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated.

The compositions of the present invention can comprise, consist essentially of, or consist of, the essential components as well as optional ingredients described herein. As used herein, "consisting essentially of means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

Definitions

"About," as used herein, modifies a particular value, by referring to a range equal to the particular value, plus or minus twenty percent (+/- 20%) or less (e.g., less thanb 15%, 10%, or even less than 5%).

"Actives" means compounds that, when applied to keratinous tissue and/or a target portion of keratinous tissue, provide a benefit or improvement to the keratinous tissue. The actives herein can be skin care actives, hair care actives, or a combination thereof.

"Apply" or "application", as used in reference to a composition, means to apply or spread the compositions of the present invention onto a human skin surface such as the epidermis.

"Derivative" means a molecule similar to that of another one, but differing from it with respect to a certain functional moiety (e.g., esters, ethers, amides, amines, carboxylic acids, hydroxyls, acetyls, thiols, halogens, thiols, and/or salt derivatives of the relevant molecule).

"Dermatologically acceptable" means that the compositions or components described are suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

"Effective amount" means an amount of a compound or composition sufficient to significantly induce a positive benefit to keratinous tissue, such as a health, appearance, and/or feel benefit, including, independently or in combination, the benefits disclosed herein, but low enough to avoid serious side effects (i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan). An "effective amount of achachariu" is an amount of achachariu sufficient to regulate a desired condition of mammalian keratinous tissue when topically applied thereto in a personal care composition.

"Exogenous solvent" means any solvent placed in contact with the plant material for the purpose of separating compounds from the plant material. It may be a solvent originally present in plant material but added in higher amounts (e.g., in case of water extracts), or a solvent not inherently present in plant material. Plant material herein can include any solid or liquid portion of the AchachariuAchachariu plant.

"Extract," when referring to achachariu, means a material (e.g., one or more phytochemicals) obtained from a portion of achachariu plant material (e.g., peel, fruit, seed, stem, bark, leaves, roots, and/or a combination of these), which may be fresh, dried, or partially dried, by contacting the plant portion(s) with an exogenous solvent and separating the desired material from the solvent using a conventional extraction process. Conventional extraction processes for obtaining plant extracts are well known in the art.

"Hair care composition" refers to a composition for regulating and/or improving a hair condition. Some nonlimiting examples of hair-care compositions include shampoos and hair conditioners.

"Hair conditioning agent" includes cationic surfactants, high melting point fatty compounds, a silicone compounds, and mixtures thereof that provide a conditioning benefit to hair. Hair care compositions that include a hair conditioning agent may be referred to as "hair conditioners." However, it is to be appreciated that shampoos may also include a hair conditioning agent.

"Keratinous tissue" refers to keratin-containing layers positioned as the outermost protective covering of mammals which includes, but is not limited to, skin, hair, nails, cuticles, etc.

"Regulating the condition of mammalian keratinous tissue," as used herein, means improving the appearance and/or feel of keratinous tissue.

"Salts" refer to ionic forms of a given compound combined with counterions deemed acceptable for a given application (e.g., food, topical, pharmaceutical). Examples include but are not limited to sodium, potassium, calcium, ammonium, manganese, copper, and/or magnesium salts of a given compound.

"Shampoo" refers to a hair care composition that includes a combination of a detersive surfactant and a dermatologically acceptable carrier. "Shave prep composition" refers to a topical personal care composition that is applied before, during, and/or after shaving. "Shaving" refers to the removal of facial or body hair with a razor or similar device. Examples of shave prep compositions include shaving creams, gels, and foams.

"Topical" refers to a composition that is intended to be applied to a bodily surface such as skin or hair.

Personal Care Composition

The personal care compositions suitable for use in the methods herein include a safe and effective amount of achachariu (Garcinia humilis). The achachariu may be in form of a serum fraction, an extract, a powder, a juice, or a combination thereof. The achachariu may be obtained from any part of the Garcinia humilis plant, as desired, but it may be particularly desirable to use the achachariu fruit or a portion thereof (e.g., peel, pulp, and/or seed). Some testing suggests that the achachariu fruit, especially the peel, may contain higher concentrations of biologically active compounds (e.g., carbohydrates, citric acid, flavonoids, xanthones and tannins) than other parts of the achachariu plant for regulating particular conditions of mammalian keratinous tissue. The achachariu extract and/or serum fraction may be obtained according to the methods described in more detail below and combined with a dermatologically acceptable carrier, along with any optional igredients, using conventional methods of making personal care compositions of the type. The personal care composition may then be packaged for commercial distribution.

FIG. 1 illustrates the distinct layers and parts of the achachariu fruit, as shown by a section passing through a long axis of the fruit. Whole fruit, a particular part of fruit, or any combination of parts of fruit may be used to provide a serum fraction or extract herein. However, this is not meant to exclude further distinctions of fruit parts than described here, or leaves that may be retained during harvesting. As shown in FIG. 1 , the outermost layer of the achachariu fruit is the "Peel", which is comparatively thick and tough. The next inward layer is the "Flesh", which is comparatively soft and yielding. The flesh surrounds a comparatively large "Seed". Especially large fruit may have more than one seed.

The amount of achachariu that is "effective" can differ from one particular source (e.g., manufacturer) to another, and can be determined by the skilled artisan based upon a particular product's level of activity (e.g., level of active components present). The concentration of active components in the particular achachariu product to be used will depend on factors such as the final dilution volume of the product, the particular method employed, the natural range of variation among individual plants, and other common factors known to those skilled in the art. In some instances, an effective amount of achachariu may range from 0.01% to 15% (e.g., from 0.1% to 10%, 0.2% to 7%, 0.5% to 5%, or even 1% to 3%).

The personal care compositions herein are intended for topical application to mammalian keratinous tissue. The present compositions may be leave-on-type compositions such as moisturizing lotions, serums, and creams, or rinse-off-type compositions such as body washes, shampoos, hair conditioners, shower gels, skin cleansers, cleansing milks, and shave prep compositions. Rinse-off-type compositions are applied topically to the skin or hair and then subsequently removed (e.g., via washing and/or with an implement such as a towel or razor), usually within minutes of application. The personal care compositions herein may be provided in a variety of forms, including, but not limited to, emulsions, lotions, milks, liquids, solids, creams, gels, mousses, ointments, pastes, serums, sticks, sprays, tonics, aerosols, foams, and/or pencils. In some instances, the present compositions may be in a form suitable for use with an implement such as, for example, a cosmetic applicator (powered or unpowered); a reusable or disposable wipe, tissue, towel, or diaper; a razor or other shaving device; a personal cleansing implement such as a mesh shower sponge, conventional sponge, or wash cloth; a swab; and/or a pen.

FIG. 2 illustrates an exmplary method for providing bioactive achachriu ingredients. It is to be appreciated that portions of the process referring to "fruit" can include any portion of the achachariu plant, as desired. The individual steps illustrated in FIG. 2 are described in more detail below.

"Cleaning" involves the removal of debris from the harvested achachariu plant material, prior to further processing, in a way that avoids injury to the fruit or removal of juice or other desirable components. For example, cleaning can be performed by low-pressure rinsing with potable water, under conditions where residual water wash would not noticeably contain plant pigments.

"Selection of material" involves separating various parts of the achachariu plant material to further process a particular part alone, or in combination with other parts. This includes, but is not limited to selecting whole fruit, peel, flesh, seed and/or leaves for further processing.

"Maceration" is an optional step for rendering the plant material and/or selected parts of the plant material into smaller particles and otherwise disrupting the integrity of the fruit to ease the following expelling of liquid juice. If the selected plant material is especially yielding (such as the flesh of the fruit) or otherwise deemed suitable for conditions and equipment of the pressing step, maceration may be omitted. Examples of suitable maceration implements include, but are not limited to, devices such as a crusher, a grinder, or a mill (e.g., knife mill or hammer mill). To prevent temperature-induced degradation of plant material, maceration can include temperature monitoring and selection of maceration parameters which minimize the risk of temperature increase during this step.

"Pressing" involves the application of mechanical force to the selected, and optionally macerated, achachariu plant material to separate at least some of the liquid component from the rest of the plant material. The applied mechanical force may be provided by any suitable means known in the art (e.g., ambient gravity, centrifugal force from a rotary expeller, pressure from the piston of a hydraulic press, or rollers or a screw of appropriate type of press).

"Mechanical separation" involves the removal of relatively large achachariu particles (e.g., pulp or agglomerated/aggregated cloud particles) from the liquid component of the achachariu plant material.

"Juice" refers to the liquid material expelled from achachariu plant material as a result of pressing. Juice can contain solid particles, semi-solid particles, and/or droplets of water- immiscible liquids of a variety of sizes (collectively referred to as "achacaha particles") in an aqueous serum. Achachariu particles, based on size and ease of removal, can be qualitatively described as either "pulp" or "cloud". While details depend on properties of the involved materials (e.g. whole fruit versus selected parts) and exact processing parameters, an example of a size boundary between "pulp" and "cloud" could lie between about 1 and about 100 microns.

"Pulp" refers to relatively large achachariu particles present in juice and those same achachariu particles removed from the juice by mechanical separation. It is not uncommon to see and distinguish individual pulp particles with a naked eye. Pulp particles suspended in the juice are amenable to removal by mechanical separation, including, but not limited to, sedimentation by ambient gravity, skimming, passing through a mesh or a filter, or centrifugation.

"Cloud" refers to relatively small achachariu particles present in the achachariu juice, and may even include dissolved compounds (e.g., high molecular weight compounds such as proteins and polysaccharides), which can be readily induced (e.g., by coagulation or temperature change) to form particles. Cloud particles are typically visible as turbidity of the achachariu juice, and individual cloud particles are generally indistinguishable to the naked eye. Cloud particles are generally dispersed in the achachariu juice and such dispersions tends to remain stable for a longer period time than a pulp suspension, especially colloidal components of the cloud. Removal of cloud particles by mechanical means such as those used for pulp removal can be difficult.

"Destabilizing treatment" generally involves exposing a material to electromagnetic waves in order to modify one or more physical properties of the material (e.g., the real component of low-frequency dielectric constant). With regard to achachariu juice, destabilization treatment degrades stability of the particle dispersion in the juice by causing agglomeration and/or aggregation of particles (especially cloud particles) into assemblies that are sufficiently large and stable to enable and/or improve their subsequent removal by mechanical separation techniques such as those described above for pulp removal.

"Serum" refers to liquid portion of achachariu juice remaining after pulp and cloud, as well as possible contaminants of concern, has been removed. Achachariu serum is free or substantially free of achachariu particles.

"Finished ingredient" refers to serum, extract, and/or particle-free extract that is in a form suitable for subsequent sale and/or incorporation into a personal care product. The finished ingredient may include preservatives that protect the serum, extract, particle-free extract, and/or compositions containing the same against environmental challenges such as temperature, atmosphere (e.g., oxygen), light, and microorganisms. It is to be appreciated that an ingredient free of preservatives is also contemplated herein. Surprisingly, it has been found that the processes described herein produce finished ingredients with multifunctional biological activities that are either free of or substantially free of benzophenones and proteins.

"Post-destabilization precipitate" refers to achachariu particles removed from the juice via mechanical separation following destabilizing treatment.

"Solvent extraction" in FIG. 2 involves conducting an extraction on achachariu particles removed from the achachariu juice with one or more solvents (e.g., dipropylene glycol).

The extract shown in FIG. 2 refers to the solution resulting from the solvent extraction of the achachariu pulp and post-destablization precipitate. In some instances, any achachariu particles remaining in the extract may be separated (e.g., mechanically) to yield a particle-free extract. The extract and/or particle-free extract may serve as a finished ingredient, or as a base for a finished ingredient via addition of preservatives.

"Post-extraction precipitate" refers to achachariu particles separated from the extract in

FIG. 2. Achachariu Serum Fraction

The compositions herein may include an achachariu serum fraction and/or salt, isomer, or derivative thereof. Serum fraction techniques involve separating the fresh cell juice found in plant material from the rest of the plant matter and processing the juice to provide a stable, refined serum. Examples of achachariu serum fractions that may be suitable for use in the compositions and methods herein include Recentia ^® GH (whole fruit) and Recentia ^® GH-P (peel only) from Ashland Specialty Ingredients. Achachariu peel serum fraction has the INCI designation "Garcinia Humilis Peel Extract" and the CAS No. 1622986-60-0.

The achachariu serum fractions herein may be preparerd from fresh achachariu using a method that helps maintain the integrity of the bioactive components present in the achachariu plant material. Care should be taken to preserve the achachariu plant material integrity during harvesting and transport, so as to minimize environmental factors such as moisture loss and biological degradation. All steps of the serum fraction making process should be completed in the shortest possible period of time to minimize exposure of the fresh achachariu plant material to sun, high temperature, and other undesirable environmental factors. Harvesting (e.g., by hand or mechanical cutting) should be conducted in a manner that avoids or minimizes the chopping, mashing, crushing, or other type of injury to the achachariu plant material to be used (e.g., fruit). Harvest and transport of achachariu plant material should be conducted in a manner to avoid moisture loss or spoilage. After harvesting, the achachariu plant material (e.g., fruit, leaves) is cleaned to remove debris prior to further processing. Cleaning is performed under conditions to prevent the initiation of the release of the juice from the fruit, to cause injury, or to remove valuable components. For example, the harvested achachariu plant material may be washed with water at low-pressure (e.g., 1 kg/cm ² or less) for a short duration (e.g., 5 minutes or less). Excess water is removed from washed fruit biomass before processing.

The washed plant material is processed (e.g., macerated and pressed) to yield juice. In some instances, the whole fruit or a portion thereof (e.g., peel, fruit flesh, and/or seed) is macerated (e.g., by grinding) and then pressed to separate the juice from the rest of the plant matter. For example, a hammer mill may be used to grind the whole fruit to yield plant material particles of a desired size (e.g., less than or equal to 0.5 cm) in a short time (e.g., 10 seconds or less) and without significant increase of biomass temperature (e.g., 5° C or less). The separation of juice from the achachariu plant material is commenced as soon as possible after maceration of the achachariu plant to avoid a significant increase in temperature of the juice and/or pulp. For example, macerated achachariu leaves can be pressed using a horizontal, continuous screw press, wherein the pressure on the cone is maintained at 24 kg/cm ² , screw speed is at 12 rpm, and biomass temperature increase is 5°C or less.

The achachariu juice can be subjected to further processing immediately, or the juice may be frozen at about -30° C and thawed and processed later. Achachariu juice typically contains a variety of achachariu particles (i.e., pulp and cloud) in an aqueous serum. Thus, in order to obtain the desired serum fraction, all or substantially all of the achachariu particles should be removed from the juice. In some instances, it may be desirable to provide a serum fraction that includes less than 25% dry matter (e.g., less than 20%, 15%, 12%, 10% or even less than 9% dry matter). The pulp may be removed from the achachariu juice using a suitable mechanical separation technique such as, for example, straining, filtration (including filtration utilizing a pressure gradient), skimming, sedimentation by ambient gravity, decanting, centrifugation, and/or a combination of these to yield pulp-free or substantially pulp-free juice. The cloud may be removed by exposing the juice to electromagnetic waves to destabilize the cloud dispersion in the juice. The frequency of electromagnetic waves may range between 300 MHz and 50 GHz. Suitable devices for generating such electromagnetic waves include, but are not limited to magnetrons, power grid tubes, klystrons, klystrodes, crossed-field amplifiers, travelling wave tubes, and gyrotrons. In some instances, a magnetron operating at a frequency of 915 MHz, 2.45 GHz, and 5.8 GHz can allow the value of real component of low- frequency dielectric constant (εό) to be decreased during the treatment by between 10 and 40 compared to its value prior to treatment. The value of real component of low-frequency dielectric constant (εό) can be determined using broadband dielectric spectroscopy data obtained via equipment and software from Agilent Technologies: PNA-L Network Analyzer N5230C with 85070E dielectric probe kit, N4693-60001 electronic calibration module, and 85070 software. The calculation is performed according to method described in the article Cole, K. S., & Cole, R. H. (1941). Dispersion and absorption in dielectrics I. Alternating current characteristics. The Journal of Chemical Physics, 9(4), 341-351.

Other non-limiting examples of techniques for providing serum fractions are disclosed in U.S. Patent Nos. 7,442,391 and 7,537,791 to Koganov and U.S. Pub. No. 2012/0201768, filed bySwanson, et al.

Achachariu Extract

The compositions and methods herein may employ an achachariu extract. An achachariu extract is obtained by separating compounds from achachariu plant matter with an exogenous solvent. Consistent with the general principle of "like dissolves like," the choice of extraction solvent largely determines the type and number of compounds that will result from any particular extraction technique. For instance, polar compounds are typically extracted out by using a polar solvent, while non-polar compounds are extracted out by using a non-polar solvent. The correlation between solvent polarity and the types of materials isolated using traditional solvent extraction is described in Houghton & Raman, Laboratory Handbook for the Fractionation of Natural Extracts (1998).

The achachariu extracts herein may be obtained using any suitable extraction technique known in the art. In some instances, the achachariu extract may be obtained by the following procedure: (i) place the desired portion of dried plant material (e.g., whole fruit, fruit pulp, peel, seeds, stem, bark, leaves) in a conical glass percolator; (ii) add the indicated percentage of extraction solvent in a w/w ratio of 1 part plant material to 2 parts extraction solvent (when the indicated percentage of extraction solvent is less than 100%, the remaining solvent is water (e.g., 95% ethanol with 5% water, 50% ethanol with 50% water)); (iii) allow the extraction to proceed for 16 to 24 hours; (iv) collect the percolate, and repeat the above process until the resulting percolate is substantially free from plant additional extract; (v) combine the percolates, evaporate to dryness under reduced pressure, and store the resulting extract under nitrogen at less than 4 degrees Celsius. Extracts may be used without any further modification or may be modified (e.g., ethoxylated, esterified) to form a derivative material.

Achachariu Powder or Juice

The personal care compositions for use with the methods herein may include an achachariu powder or juice. Achachariu juice may be obtained by macerating and process achachariu plant material, for example, as described hereinabove. Achachariu powder may be obtained by drying achachariu plant material and processing the dried material into particles. Additionally or alternatively, the achachariu plant material may be processed into particles and then dried. Processing the achachariu into particles may include one or more grinding steps performed before and/or after drying. The achachariu biomass may be dried and processed into particles using any suitable method known in the art. The achachariu powder may have a weight average particle size of from 50 to 750 microns. Once the achachariu is processed into particles of the desired size, the powder can be incorporated into a personal care composition using conventional methods for making such compositions. Dermatologically Acceptable Carrier

The personal care compositions for use with the methods herein include a dermatologically acceptable carrier at an amount of 20% to 99.99% (e.g., 50% to 99%, 60% to 98%, 70% to 95%, or even 60% to 80%) by weight of the composition. The carrier may be aqueous or anhydrous. The form of the carrier is not particularly limited, and can be any suitable form known in the art for the application desired (e.g., solutions, dispersions, emulsions and combinations thereof). "Emulsions" refer to compositions having an aqueous phase and an oil phase. Emulsion carriers include, but are not limited to oil-in-water, water-in-oil and water-in- oil-in-water emulsions. Emulsion carriers herein may include from 0.01% to 10% (e.g., 0.1% to 5%) of an emulsifier (e.g., nonionic, anionic, cationic emulsifier, or a combination thereof). Suitable emulsifiers are disclosed in, for example, U.S. Patent 3,755,560, U.S. Patent 4,421,769, and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986).

Optional Ingredients

The present compositions may contain a variety of optional ingredients that are conventionally used in the particular product type provided, as long as they do not undesirably alter product stability, aesthetics, or performance. The optional ingredients, when incorporated into the composition, should be suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like within the scope of sound judgment. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes a wide variety of nonlimiting cosmetic and pharmaceutical ingredients. The compositions herein may include 0.0001% to 50% (e.g., 0.001 % to 20% or even 0.01% to 10%), by weight of the composition, of optional ingredients. Some non-limiting examples of optional ingredients include abrasives, absorbents, aesthetic components such as pigments, opacifying agents, colorings/colorants, particles, essential oils, anti-caking agents, foaming agents, anti-foaming agents, binders, biological additives, vitamins, minerals, moisturizers, emollients, humectants, skin tone agents, lubricating agents, sensates (e.g., menthol), fragrances, anti-dandruff agents, buffering agents, bulking agents, chelating agents, chemical additives, biocides, denaturants, astringents, external analgesics, anti-inflammatory agents, sunscreen agents, film formers and/or polymers for aiding film-forming properties and substantivity of the composition, pH adjusters, propellants, reducing agents, sequestrants, conditioning agents, thickeners, and combinations of these. Some of these and other optional ingredients are described in more detail below.

The personal care compositions herein may include 0.1% to 10% (e.g., 0.5% to 7%) of a conditioning agent to provide a particular conditioning benefit to hair and/or skin. For example, some suitable hair conditioning agents include silicones (e.g., silicone oils, cationic silicones, silicone gums, high refractive silicones, and silicone resins), organic conditioning oils (e.g., hydrocarbon oils, polyolefins, and fatty esters), and combinations thereof. Some non-limiting examples of suitable silicone conditioning agents, and optional suspending agents for the silicone, are described in U.S. Reissue Pat. No. 34,584; and U.S. Pat. Nos. 4,152,416, 4,364,837, 5,104,646, 5,106,609, 5,674,478, 5,750,122, 7,465,439, 7,041,767, and 7,217,777. Some non- limiting examples of skin conditioning agents include vitamins, minerals, peptides and peptide derivatives, sugar amines, sunscreens, oil control agents, particulates, flavonoid compounds, hair growth regulators, anti-oxidants and/or anti-oxidant precursors, preservatives, protease inhibitors, tyrosinase inhibitors, anti-inflammatory agents, moisturizing agents, exfoliating agents, skin lightening agents, sunless tanning agents, lubricants, anti-acne actives, anti-cellulite actives, chelating agents, anti-wrinkle actives, anti-atrophy actives, phytosterols and/or plant hormones, N-acyl amino acid compounds, antimicrobials, and antifungals. Other non-limiting examples of skin conditioning agents can be found in U.S. Pub. Nos. 2010/0272667 and 2008/0206373. In addition to providing a skin health and/or appearance benefit, skin conditioning agents for use in shave prep compositions may also help to reduce the coefficient of friction for the shave prep composition, thereby decreasing the potential for skin irritation related to shaving.

The personal care compositions herein may include 0.05% to 5% (e.g., 0.1% to 4% or even 0.25% to 3%) of a thickening agent. Suitable classes of thickening agents include, but are not limited to carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, gums and mixtures thereof. Non-limiting examples of suitable thickening agents are described in the CTFA International Cosmetic Ingredient Dictionary, 10 ^th Ed. (2004), pp. 2294 - 96.

The personal care composition herein may include from 5% to 50% of an anionic, zwitterionic, and/or amphoteric detersive surfactant. The concentration of the detersive surfactant in the composition should be sufficient to provide the desired cleaning and lather performance. Some non-limiting examples of anionic detersive surfactants include disodium N- octadecylsulfosuccinnate; disodium lauryl sulfosuccinate; diammonium lauryl sulfosuccinate; tetrasodium N-(l,2-dicarboxyethyl)-N-octadecylsulfosuccinnate; diamyl ester of sodium sulfosuccinic acid; dihexyl ester of sodium sulfosuccinic acid; dioctyl esters of sodium sulfosuccinic acid; and olefin sulfonates. Some particularly suitable examples of anionic surfactants are alkyl and alkyl ether sulfates. Other non-limiting examples of anionic detersive surfactants are described in U.S. Pat. Nos. 2,486,921 ; 2,486,922; 2,396,278; and 3,332,880.

The personal care compositions herein may include a cationic surfactant system that includes one or more cationic surfactants. If present, the cationic surfactant system may be incorporated into the composition at a level of 0.1% to 10% (e.g., 0.5% to 8%, 1% to 5%, or even 1.4% to 4%). In certain hair care compositions, the cationic surfactant may be balanced to provide desirable ease-to-rinse, feel, rheology, and wet conditioning benefits.

The personal care compositions herein may include a high melting point fatty compound. The high melting point fatty compounds useful herein typically have a melting point of 25 °C or higher, and may be selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof. It is to be appreciated that the compounds disclosed in this section of the specification can in some instances fall into more than one classification, e.g., some fatty alcohol derivatives can also be classified as fatty acid derivatives. However, a given classification is not intended to be a limitation on that particular compound, but is done so for convenience of classification and nomenclature. Further, it is to be appreciated that, depending on the number and position of double bonds, and length and position of the branches, certain compounds having certain required carbon atoms may have a melting point of less than 25°C. Such compounds of low melting point are not intended to be included in this section. Nonlimiting examples of the high melting point compounds are found in International Cosmetic Ingredient Dictionary, Fifth Edition, 1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The personal care compositions herein may include a cationic and/or a non-ionic polymer at an amount of 0.05% to 3% (e.g., 0.075% to 2.0%, or even 0.1% to 1.0%). Suitable cationic polymers will have cationic charge densities of at least 0.5 meq/gm (e.g., at least 0.9 meq/gm, 1.2 meq/gm, or even at least 1.5 meq/gm), but typically less than 7 meq/gm or even less than 5 meq/gm, at the pH of intended use of the composition, which may range from pH 3 to pH 9. Herein, "cationic charge density" of a polymer refers to the ratio of the number of positive charges on the polymer to the molecular weight of the polymer. The average molecular weight of such suitable cationic polymers will generally be between 10,000 and 10 million, (e.g., between 50,000 and 5 million or even between 100,000 and 3 million). Some non-limiting examples of suitable cationic polymers include copolymers of vinyl monomers having cationic protonated amine or quaternary ammonium functionalities with water soluble spacer monomers such as acrylamide, methacrylamide, alkyl and dialkyl acrylamides, alkyl and dialkyl methacrylamides, alkyl acrylate, alkyl methacrylate, vinyl caprolactone, vinyl pyrrolidone, and cationic guar gum derivatives such as guar hydroxypropyltrimonium chloride. Some non- limiting examples of non-ionic polymers are polyalkylene glycols having a molecular weight of more than about 1000. Other non- limiting examples of polymers are disclosed in the CTFA Cosmetic Ingredient Dictionary, 3rd edition, edited by Estrin, Crosley, and Haynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C. (1982)).

The personal care compositions herein may include a suspending agent at concentrations effective for suspending water-insoluble material in dispersed form in the compositions or for modifying the viscosity of the composition. Such concentrations range from 0.1% to 10%, or even from 0.3% to 5.0%. Suspending agents useful herein include anionic polymers, nonionic polymers, and vinyl polymers.

The personal care compositions herein may include particles. The particles may be dispersed water-insoluble particles. The particles may be inorganic, synthetic, or semisynthetic. The particles may have a mean average particle size of between 1 μιη and 1 mm (e.g., between 10 μιη and 800 μιη, 50 μιη and 500 μιη, or even between 100 μιη and 400 μιη.

In some instances, the above cationic surfactants, together with high melting point fatty compounds and an aqueous carrier, may be combined to form a gel matrix in a hair care composition. A gel matrix may be suitable for providing certain hair conditioning benefits such as slippery feel during the application to wet hair and softness and moisturized feel on dry hair. In view of providing the above gel matrix, the cationic surfactant and the high melting point fatty compound are contained at a level such that the weight ratio of the cationic surfactant to the high melting point fatty compound is in the range of, for example, 1:1 to 1:10, or even 1:1 to 1:6.

Methods of Regulating Keratinous Tissue Condition

Personal care compositions comprising an effective amount of achachariu can be used to regulate a condition of mammalian keratinous tissue by topically applying the personal care composition herein to mammalian keratinous tissue. The present method may include identifying a target portion of keratinous tissue in need of treatment and/or where treatment is desired, and then applying the personal care composition to the target portion of keratinous tissue. Identifying a target portion of keratinous tissue may be based on, for example, the presence of a visible condition (e.g., dryness, brittleness, split ends, undesirable feel, redness, inflammation, discoloration, or wrinkles). In some instances, the target portion of keratinous tissue may not exhibit visible signs of a condition, but a user may still wish to target such an area if it is one that is known to develop an undesirable condition (e.g., skin or hair surfaces that are typically not covered by clothing and areas of skin prone to shaving irritation).

Personal care compositions containing an effective amount of achachariu may be applied once a day, twice a day, or on a more frequent daily basis, during a treatment period. The treatment period is ideally of sufficient time for the achachariu to provide the desired benefit. For example, the treatment period may be of sufficient time for the achachariu to provide a noticable and/or measurable improvement in a hair or skin condition. The treatment period may last for at least 1 week (e.g., about 2 weeks, 4 weeks, 8 weeks, or even 12 weeks). In some instances, the treatment period will extend over multiple months (i.e., 3-12 months) or multiple years. In some instances, a cosmetic composition containing an effective amount of achachariu may be applied most days of the week (e.g., at least 4, 5 or 6 days a week), at least once a day or even twice a day during a treatment period of at least 2 weeks, 4 weeks, 8 weeks, or 12 weeks.

The personal care compositions herein may be applied locally or generally. In reference to application of the composition, the terms "localized", "local", or "locally" mean that the composition is delivered to the targeted area while minimizing delivery to keratinous surfaces where treatment is not needed or desired. While certain embodiments herein contemplate applying a composition locally to an area, it will be appreciated that the compositions herein can be applied more generally or broadly to one or more keratinous surfaces. In certain embodiments, the compositions herein may be used as part of a multi-step beauty regimen, wherein the present composition may be applied before and/or after one or more other compositions.

Using the personal care compositions herein according to the present methods can provide a health or appearance benefit to the treated keratinous tissue. Some non-limiting examples of skin benefits include reducing the appearance of wrinkles, deep lines, fine lines, crevices, bumps, large pores; increasing the convolution of the dermal-epidermal border; skin lightening; increasing elasticity, decreasing sagging, reducing cellulite; reducing the appearance of under-eye circles, reducing the appearance of discoloration, reducing hyperpigmentation, increasing skin luminosity, and combinations thereof. Some non-limiting examples of hair benefits include shine, softness, combability, antistatic properties, wet-handling, damage, anti- dandruff, manageability, body, and greasiness.

The personal care compositions may be applied by any suitable means known for applying such products, including rubbing, wiping or dabbing with hands, fingers and/or an implement. Non-limiting examples of implements include a sponge or sponge-tipped applicator, a swab (for example, a cotton-tipped swab), a pen optionally comprising a foam or sponge applicator, a loofa, a brush, a wipe, a cloth, and combinations thereof. The composition may be pre-applied to the applicator and, for example, delivered to the user pre-packaged as such, or the user may be instructed to apply the composition to the applicator prior to use. In some instances, the composition may be stored in an implement, for example, in a separate storage area for the composition. In such an example, the composition may be transferred to the applicator from the storage area, for example, by squeezing, breaking, and/or other suitable means. The composition may be applied to the keratinous tissue by contacting the applicator and composition to the skin. Contact may include, for example, light pressure, dabbing, rubbing, wiping, and the like. When targeted application is desired, the composition may be applied only to the target area of keratinous tissue.

Methods of Determining the Effectiveness of Achachariu

Irritation and inflammation in human skin can lead to visible signs of skin aging (e.g. discoloration and wrinkles), decreased mechanical strength, decreased protective functions, and lessened ability to recover from stress and injuries. Ubiquitous stressors such as sunlight and surfactants can be especially problematic. Mitigating irritation and inflammation of the skin, particularly those caused by common stressors, is important and desirable.

The adverse effects of light, most commonly sunlight (though artificial sources are included), on human skin are well known. Overly high exposure to sunlight may cause acute adverse reaction involving irritation and inflammation, such as sunburn. Exposures insufficient to cause acute reactions can still trigger inflammation-related processes. Accumulated inflammatory damage from sunlight exposure causes degradation of skin resilience and development of an undesirable appearance (i.e., photoaging).

Surfactants are also known to cause adverse reactions on human skin. Surfactants are used in a variety of personal care and cleansing products to allow or improve processes of cleansing, foaming, emulsifying, solubilizing, and dispersing. Repetitive contact with surfactant- containing products has been shown to cause damage of the skin barrier due to surface or interface activities of the surfactants, which can be perceived by consumers as dryness, itchiness, swelling, redness, and/or pain. The weakened barrier subsequently leads to deeper penetration of the surfactants into skin and induced irritation and inflammation. As surfactants are widely used in hand soaps, facial and body washes, shampoos and conditioners, as well as dish, laundry and housecleaning detergents, human skin contact with surfactants is common. Irritation and inflammation are commonly viewed as a "cascade" proceeding from necessary release of a signaling compound Interleukin (IL) 1 -alpha (or IL-la) to induction of other downstream cytokines and chemokines such as interleukins IL-6 and IL-8 or other signaling molecules (Weiss T, Basketter DA, Schroder KR. In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicol In Vitro 2004; 18 (3): 231- 43). However, previously published data suggest that "cascade" view might not be a comprehensive model.

It has been shown that sodium dodecyl sulfate (SDS), a single compound commonly used as a benchmark source of surfactant stress in both in vitro and in vivo studies, can trigger different portions of the irritation and inflammation process without significantly affecting release of a primary cytokine such as IL-la, depending on concentration. The complexity of irritation and inflammation response of skin cells implies that signaling "network" model is a more adequate analogy than a signaling "cascade" model. This indicates that mitigation of such a complex signaling process must affect more than one pathway, such as by using a multifunctional bioactive ingredient.

One of the methodologies for studying and quantifying irritation and inflammation includes culturing cells of the tissue most likely to come in contact with stress sources, such as viable epidermal keratinocytes from human skin. Human epidermal keratinocytes (HEK) have become the focus of attention in irritant-induced skin inflammation by virtue of their epidermal location, importance in maintaining the integrity of the stratum corneum barrier, and the ability to produce a variety of inflammatory mediators. Keratinocytes can release a variety of signalling substances (e.g., interleukins and arachidonic acid metabolites) in response to a range of irritants, including surfactants and sunlight. The amounts of these signalling substances present in a tissue sample can be measured via techniques such as Enzyme-Linked Immunosorbent Assay (ELISA). Bioactive substances that reduce the release of inflammatory signalling substances may help control the signs of irritation and inflammation in human skin.

Besides signalling substances, other very important compounds in processes of irritation and inflammation are those that directly cause the damage. Especially notable inflammatory damage substances are free radicals (especially reactive oxygen species) and protein-degrading enzymes (proteases). It is possible to detect the presence or measure the activity of such damaging substances by incubating them with a substrate they can alter or degrade. The alteration or degradation of the substrate can be measured directly (e.g., by loss or development of color or fluorescence) or indirectly (e.g., by ELISA). In some cases, the presence or activity of damaging substances can be measured directly or indirectly, for example, with spectroscopic techniques (e.g., Electron Paramagnetic Resonance techniques and the use of chromogenic artificial stable free radicals like DPPH).

The tests selected for determining the effects of serum fractions and extracts of achachariu, and personal care compositions containing the same, include inhibition of proteases (damaging compounds) in non-cell-based bioassays, inhibition of chemokines, cytokines and prostaglandins (signalling compounds) in cultured human skin cells, inhibition of a compound capable of both signalling and causing damage in cultured human cells, and measuring release or lack thereof of substances indicating cell damage or sensitization. These tests illustrate safety (e.g., via a lack of cytotoxicity and sensitization potential) and the efficacy and potency of achachariu in inhibiting signalling and damaging compounds involved in skin irritation, inflammation, and aging processes. The cells in the assays described herein are normal human adult epidermal keratinocytes (HEK), unless specifically stated otherwise.

EXAMPLES The following examples are provided for the purpose of illustration only and should in no way be construed as being limiting, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1

Preparation of a Serum Fraction from Achachariu Whole Fruit

In this example, achachariu (Garcinia humilis) whole fruit are used to produce a serum fraction. The achachariu fruit are harvested fresh from trees, inspected, and cleaned prior to processing. Unsound fruit is not used. Selected whole fruit are ground, pressed, and mechanically separated to provide juice and pulp. The yield of juice from achachariu whole fruit after grinding, pressing and mechanical separation is about 60 to 69% weight/weight; the pH of juice from achachariu whole fruit is from 3.0 to 3.3. The juice is immediately subjected to destabilizing treatment by electromagnetic waves in a continuous flow system that includes magnetrons operating at a frequency of 5.8 GHz. The parameters of the electromagnetic waves in the destabilization step are set to achieve the decrease in value of real component of low- frequency dielectric constant (εό) during the treatment by about 20 Farads per meter (F/m) compared to its value prior to treatment. The de- stabilized juice is immediately pumped through a continuous flow centrifuge to yield a serum and post-destabilization precipitate. The serum is substantially free of benzophenones and protein. Example 2

Preparation of Serum Fraction from Achachariu Peel

In this example, achachariu fruit peel is used to produce a serum fraction. The achachariu fruit are harvested fresh from trees, inspected, and cleaned prior to processing. Unsound fruit is not used. The fruit peel is separated from whole fruits and then ground, pressed, and mechanically separated to produce juice and pulp. The yield of juice from achachariu fruit peel after grinding (maceration), pressing and mechanical separation is about 25 to 35% weight/weight; the pH of juice from achachariu fruit peel is 2.7 to 3.0. The juice from fruit peel is immediately subjected to destabilizing treatment by electromagnetic waves in a continuous flow system that includes magnetrons operating at a frequency of 2.45 GHz. The parameters of the electromagnetic waves in the destabilization step are set to achieve the decrease in value of real component of low-frequency dielectric constant (εό) during the treatment by about 30 Farads per meter (F/m) compared to its value prior to treatment. The de- stabilized juice is immediately pumped through a continuous flow centrifuge to yield a serum and post-destabilization precipitate. The serum is free or substantially free of benzophenones and protein.

Example 3

Preparation of Serum Fraction from Achachariu Fruit Flesh and Seeds

In this example, achachariu fruit flesh and seeds are used to produce a serum fraction. The achachariu fruit are harvested fresh from trees, inspected, and cleaned prior to processing. Unsound fruit is not used. The fruit flesh and seed are separated from whole fruits and then ground, pressed and mechanically separated to produce juice and pulp. The yield of juice from Achachariu (Garcinia humilis) fruit flesh and seeds after grinding, pressing and mechanical separation is about 30 to 40% weight/weight; the pH of juice from achacha fruit flesh and seeds is 3.8 to 4.2. The juice is immediately subjected to destabilizing treatment by electromagnetic waves in a continuous flow system that includes magnetrons operating at a frequency of 2.45 GHz. The parameters of the electromagnetic waves in the destabilization step are set to achieve the decrease in value of real component of low-frequency dielectric constant (εό) during the processing by about 25 Farads per meter (F/m) compared to its value prior to treatment. The de-stabilized juice is immediately pumped through a continuous flow centrifuge to yield a serum and post-destabilization precipitate. The serum is free or substantially free from benzophenones and protein.

Example 4 Preparation of Extracts from Achachariu Fruit by Solvent Extraction

Solvent extractions were conducted with pulp particles, with post-destabilization precipitate particles, including cloud, or with their combination using one or more solvents under particular extraction conditions: type of solvent(s), ratio of solvent to plant material (pulp, or post-destabilization precipitate, or their combinations), extraction temperatures, agitation, time of exposure, etc., in order to extract materials that are soluble in the particular solvents. The ratio of pulp and/or post-destabilization precipitate to solvent can range from 1:2 to 1 : 1000. Extraction temperature can range from 4 ^Q C to 100 °C. Time of the exposure may vary from about 15 minutes to about 96 hrs.

Example 5 - Personal Care Composition Formulations Exemplary personal care compositions comprising achachariu are provided in Tables 1, 2, and 3 below.

Table 1 - Shampoo

1. Mirapol AT-1, Copolymer of Acrylamide(AM) and TRIQUAT, MW=1,000,000; CD=

1.6 meq./gram; 10% active; Rhodia

2. Jaguar C500, MW - 500,000, CD=0.7; Rhodia

3. Mirapol 100S, 31.5% active; Rhodia

4. Sodium Laureth Sulfate, 28% active; P&G

5. Sodium Lauryl Sulfate, 29% active; P&G

6. Glycidol Silicone VC2231-193C

7. Tegobetaine F-B, 30% active; Goldschmidt Chemicals Monamid CMA, 85% active; Goldschmidt Chemical

Ethylene Glycol Distearate, EGDS Pure; Goldschmidt Chemical

Sodium Chloride USP (food grade); Morton; note that salt is an adjustable ingredient, higher or lower levels may be added to achieve target viscosity.

Recentia ^® GH; AkzoNobel

Table 2 - Hair Conditioner

Glycidol Silicone VC2231-193C

Glycidol Silicone VC2231-193F

Glycidol Silicone VC2231-193A

SF1202; Momentive Performance Chemicals

Genamin TM KMP; Clariant

Konol TM; Shin Nihon Rika

Konol TM; Shin Nihon Rika

Kathon TM CG (Methylchloroisothiazolinone/Methylisothiazolinone); Rohm & Haas Panthenol; Roche

Panthenyl ethyl ether; Roche

Recentia ^® GH-P; AkzoNobel

Table 3 - Shave Prep Composition

Natrosol 250 H H R (H EC) 0.80 0.80 0.80

Glycerin - - 0.50

Brij 35 (Laureth-23, 100%

Active) 2.00 2.00 2.00

Salicylic Acid - - -

Arlamol E (PPG-15 Stearyl

2.00 -- 2.00

Ether)

Achachariu Serum Fraction ⁶ 0.5 2.0 5.0

Acusol OP301 Opacifier (40%

solids) (Water and -- -- -- Styrene/Acrylic Copolymer)

DC 1503 - 2.00 -

Expancel 920-WE40 D24 - - -

Disod ium EDTA ( EDETA BD) 0.10 0.10 0.10

Perfume 1 0.15 0.15 0.15

Perfume 2 - - -

Perfume 3 - - -

Perfume 4 - - -

Glydant Plus 0.20 0.20 0.20

Example 6 - Preservatives

Various preservatives were tested for use with the bioactive serum fractions and bioactive extracts of achacha herein. Table 4 shows an example of combination/concentrations of preservatives that may be particularly suitable for preparing finished achacha ingredients (e.g., prepared from the serum fractions described in Examples 1, 2, and 3). It has been found that the preservatives listed in Table 4 help prevent the formation of precipitates in a finished product.

Table 4

Example 7 - Physico-Chemical Characteristics

Table 5 illustrates methods for testing and evaluating certain physico-chemical characteristics of achacaha serum fractions and extracts. It is to be appreciated that the methods are not limited to the specific instruments or techniques shown, and equivalent instruments and techniques may be used to achieve substantially the same results, as known by those skilled in the art.

Table 5

Property Test Method Units

Appearance Determined organoleptically. N/A

Odor Determined organoleptically. N/A

Color Determined on Lovibond Comparator 3000 Gardner Scale. Gardner

Turn on comparator lamp. Measure 8mL of sample into sample scale tube. Insert tube into comparator. Rotate the knobs until two color standards nearest in color to the sample have been

located. Record the value of the sample color accordingly. If the color of the sample is substantially similar to both, rather than a single standard, then record it as a value between the values of the two standards.

Dry Matter Dry matter is determined by comparing the weights of liquid %

sample with residual dry matter after water has been

evaporated. Procedure is based on standard laboratory

practices commensurate with available equipment. Select a suitable aluminum weighing dish (e.g., VWR 25433-016) and weight the dish. Add approximately 4mL of liquid sample to the dish with the dish on the scale. Determine the weight of the

liquid sample by subtracting the dish weight from the total weight. Repeat this procedure with two additional weighing dishes. Place the dishes in a ThermoScientific "Lindberg Blue

M" Gravity oven at 105 degrees Celsius for 24 hours. After 24

hours, remove the dishes and allow them to cool for

approximately 5 minutes at room temperature. Weigh each dish. Determine the weight of the dry sample. Calclulate dry matter percentage by dividing dry sample weight by liquid sample weight. Dry matter percentage for the sample is the average of the dry matter percentages for the three dishes.

Refractive index Determined using a Reichert Arias™ 500 brand refractometer nD

according to instruction manual sections 6.0, 4.1 and 4.4-4.5.

Temperature regulation is provided by Cole-Parmer Polystat temperature controller, model number 12108-10 set at 20 C.

Automatic Reading Method is enabled. Place 0.5mL of

deionized water on the surface of the lower measuring prism, taking care to avoid bubble formation. Close the cell and turn the shadowline adjustment knob to bring the shadowline within

the crosshairs. Wait for temperature at refractometer measuring

cell to stabilize, then push Read button. Repeat until refractive

index of deionized water is determined as 1.333 at least three times in a row. Rinse the lower and upper surfaces of the measuring cell with deionized water and blot dry with lint-free Property Test Method Units wipe. Place 0.5mL of sample on the surface of the lower measuring prism. Close the cell and turn the shadowline adjustment knob to bring the shadowline within the crosshairs.

Wait for temperature at refractometer measuring cell to stabilize, then push Read button. Repeat until stable readings

have been obtained for sample material at least three times in a row. Record this value as the Refractive Index.

Density Determined with Densito 30PX densitometer from Mettler g/cm ³

Toledo. Procedure is based on Operating Instructions for Densito 30PX, sections 4 and 6. Set the instrument display to

g/cm ³ . Calibrate the instrument with 4 cm ³ of deionized water, avoiding bubble intake or formation. If the density of the calibration sample deviates by more than 0.05% from the expected density of water at ambient temperature, recalibrate

the densitometer as per Operating Instructions. Eject the calibration sample and fill the sample loops with 4 cm ³ of

sample, avoiding bubble intake or formation. Record the reading. Eject the sample and repeat steps above for additional readings, until receiving three matching readings in a row.

Record the value of density (specific gravity) for the sample.

pH Determined by measuring on a Denver Instrument Model 250 N/A pH / ISE / conductivity meter with pH / ATC electrode number

300729.1. Procedure is based on manufacturer's 301127.1 Rev.

D manual, pages ii and 9 through 12. Use pH 4.01 and pH 7.00 buffers to calibrate the pH meter.

Total Plate Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU / Count Microbiological Limit Tests gm

Mold / Yeast Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU /

Microbiological Limit Tests gm

E. coli Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU /

Microbiological Limit Tests gm

Salmonella sp. Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU /

Microbiological Limit Tests gm

Staphylococcus Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU / aureus Microbiological Limit Tests gm

Pseudomonas Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU / sp. Microbiological Limit Tests gm

Benzophenones, Dal Molin MM, Silva S, Alves DR, Quintao NLM, Delle %

Monache F et al.(2012) Phytochemical analysis and

antinociceptive properties of Garcinia achachariu Rusby (Clusiaceae) seeds. Arch Pharm Res 35: 623-631.

Protein Amino acid analysis conducted on Hitachi L-8900 amino acid %

analyzer according the manufacturer's instructions. Some physico-chemical characteristics of the present achacha serum fractions and extracts are illustrated in Tables 6, 7, and 8 below. The serum fractions and extracts are prepared according to the methods described in the corresponding example(s) above. The achacha serum fraction included in the finished ingredient Recentia® GH-P (CAS RN# 1622986-60-0), which is illustrated in Table 8 below, is prepared according to the process described in Example 2.

Table 6 - Serum Fractions

Table 7 - Extracts

Table 8 - Recentia® GH-P

Test Parameter Result

Appearance Clear Orange Liquid Test Parameter Result

Odor Characteristic

Solubility in water Soluble in any ratio

Color (Gardner scale) 5-12

Dry matter (%) 9.0-12.1

pH 2.9-3.8

Refractive index (nD) 1.349-1.355

Total Plate Count (CFU / g) < 100

Mold / Yeast (CFU / g) < 100

E. coli (CFU / g) Negative / 10 g

Salmonella sp. (CFU / g) Negative / 10 g

Staphylococcus aureus (CFU / g) Negative / 10 g

Pseudomonas sp. (CFU / g) Negative / 10 g

Example 8 - Absorbance Spectra

Absorbance spectra (in wavelength ranges 200-400 nm and 400-1000 nm) were obtained from 100 microliter samples of serum fractions and extracts of achachariu using a Synergy 2 multi-mode microplate reader (BioTek Instruments, Inc) with 96-well black quartz microplate (Hellma Analytics GmbH). All dilutions were done as volume/volume. Spectra of 100 microliter aliquots of respective solvents (ultrapure deionized water and fragrance grade dipropylene glycol) were subtracted from sample spectra. A summary of the absorbance spectra results are illustrated in FIGS. 3A, 3B, 4A, and 4B. FIGS. 3A and 4A illustrate absorbance spectra of a wavelength range of 200-400 nm. The samples of achacha serum fractions and extracts analyzed in FIGS 3A and 3B included GH 0785, GH 1082, and GH 1083. The samples of achacha serum fractions and extracts analyzed in FIGS 4A and 4B included GH 0837, GH 0838, GH 0841, and GH 0842.

Example 9 - Biological Activity

Serums and finished ingredients prepared according to various embodiments herein were evaluated for biological activity of interest. The evaluations included in this example are: cytotoxicity; skin sensitization potential; inhibition of trypsin activity; inhibition of elastase activity; inhibition of Kallikrein 5; inhibition of IL-6, IL-8, and/or PGE2; ARE activation; melanin synthesis inhibition; lipogenesis inhibition; and protein loss inhibition in hair. The test methods and results of these evaluations are discussed in more detail below. Evaluation of Cytotoxicity

Lactate Dehydrogenase (LDH) is a key cytoplasmic enzyme. Presence of LDH outside the cells at levels above normal background leakage is an indicator of cell damage or cell death. Assays quantifying LDH in cell culture medium are commonly employed to assess potential cytotoxicity. Observation of cells under a microscope that identify cell rupture or changes in cell morphology can also contribute to the assessment of cytotoxicity.

Normal human adult epidermal keratinocytes (HEK) and all cell culture supplies in the cytotoxicity evaluation were obtained from Life Technologies Co. (Carlsbad, CA, USA). The cells were grown and then maintained in keratinocyte basal medium 154 (Ml 54) with added human keratinocyte growth supplements (HKGS) at 37 °C in an atmosphere of 5% CO ₂ and used between passages 2 to 4. For the experiments, HEK cells were trypsinized, seeded in 96-well plates, and grown to -80% confluence. HEK were then exposed, or not, to a stress factor, and incubated for 16 hours with or without test articles at various concentrations. After incubation, HEK cell supernatant medium samples were collected, and levels of LDH were evaluated using Cytoscan™ LDH Assay kit (Catalog # 786-210, produced by G-Biosciences, St. Louis, MO, USA). Untreated, unstressed HEK cells were lysed using kit-supplied lysis buffer as a positive control, with lysate used as assay positive control and a measure of maximum LDH release. Lower induced LDH release, when confirmed by microscopy, indicates lower cytotoxicity.

Evaluation of Skin Sensitization Potential

It is possible for a material to not be cytotoxic, and yet be unsafe due to provoking an allergic reaction upon skin contact. Typically, initial exposure to an allergen sensitizes the immune system, and following exposures cause an allergic response. Recent progress in understanding the mechanisms of skin sensitization identified interleukin-18 (IL-18) production in normal human epidermal keratinocytes (HEK) as a useful biomarker for skin contact sensitization (Corsini, et al., "Use of IL-18 Production In a Human Keratinocyte Cell Line to Discriminate Contact Sensitizers from Irritants and Low Molecular Weight Respiratory Allergens." Toxicol In Vitro. 2009 Aug; 23(5):789-96; Teunis, et al., "Transfer of a Two-tiered Keratinocyte Assay: IL-18 Production by NCTC2544 to Determine the Skin Sensitizing Capacity and Epidermal Equivalent Assay to Determine Sensitizer Potency." Toxicol In Vitro.2013 April; 27(3): 1135-50). IL-18 is considered a suitable in vitro alternative to animal skin sensitization testing methods such as the Local Lymph Node Assay. Thus, IL-18 production in HEK was evaluated to determine sensitization potential. Normal human adult epidermal keratinocytes (HEK) were cultured as described above for the cytoxicity evaluation. After incubation with test articles or controls for 16 hours, the HEK cells were lysed with 100 μΐ/well of 0.5% Triton X-100 in pH 7.4 Phosphate Buffered Saline (PBS). The cell lysates were collected, and IL-18 was quantified using Human IL-18 ELISA Kit (Catalog # 7620, produced by MBL International Co., Woburn, MA, USA). A known skin sensitizer, p r -phenylenediamine (/?PD) used as a positive control, significantly induced IL-18 compared to vehicle control in HEK cultures. Fold changes of IL-18 levels between test articles and respective vehicle controls were calculated and compared to pPD (positive control). Lower induction of IL-18 indicates lower sensitization potential.

Inhibition of Trypsin Activity

Collagen fibers provide mechanical strength and support for the skin. A ubiquitous protease, trypsin, is associated with damage and inflammation. Trypsin breaks down collagen, potentially leading to decreased mechanical strength of the skin, as well as wrinkles and darkening after stress or injury (Burns T, Breathnach S, Cox N, Griffiths C. Rook's Textbook of Dermatology. Eighth Edition. Wiley-Blackwell, 2010. Vol. 1 Sections 8.21 to 8.27. Vol. 2 Section 29.7).

Trypsin inhibition was determined via an EnzChek kit utilizing casein substrate with intra- molecularly quenched fluorescent label moieties (Catalog # E6638, produced by Life Technologies). Testing was conducted according to manufacturer instructions. Digestion buffer concentrate was diluted in deionized water. Substrate and bovine trypsin (Sigma catalog number T9201) were dissolved and diluted in the digestion buffer. Test articles were dissolved and diluted in digestion buffer. Calibration curve was constructed with amounts of trypsin ranging from 1000 nanograms to about 1.4 nanograms in reaction volume. Soybean trypsin inhibitor, type I-S (Sigma) was used as a positive control.

Amount of trypsin in wells with test articles and controls was fixed at 1000 nanograms.

IC ₅₀ was calculated as concentration of test article in the reaction volume (e.g. microtiter plate well) necessary to reduce the trypsin activity to 50%. Lower IC ₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of Elastase Activity

Elastin is a protein essential to elastic fiber network contained in connective tissues which depend on elasticity for their function, such as skin. Excessive elastase activity, commonly related to inflammation, degrades elastin and decreases strength and resilience of the skin. During inflammatory processes, elastase can be found in areas beyond those where it is produced or secreted. Human neutrophil elastase inhibition by test articles was determined in kinetic colorimetric assay described by Elastin Products Company, Inc. (Elastin Products Company. Assay with N-MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) as substrate. Elastin Products Company, Inc. Research Biochemicals Catalogue. 2004. p.84) and modified for its use with 96-well microtiter plates (Corning 3641) from Corning, Inc. (and Synergy 2 microplate reader from BioTek Instruments, Inc.. The N-Methoxysuccinyl-Ala-Ala-Pro-Val- pNA substrate (EPC, Catalog No: FH237), and elastase (EPC SE563) were from Elastin Products Company (Owensville, MI, USA). Working solution of elastase was prepared with 0.15 M pH 7.5 Tris-HCl buffer containing 50 mM NaCl. Working solution of substrate was prepared in 0.15 M pH 5.0 acetate buffer containing 100 mM NaCl, with an aliquot of 2 % by volume of final buffer of l-methyl-2-pyrrolidone used for initial dissolution of the substrate. Deionized water was used to dissolve buffer components. Reaction volume in each well was 224 μΐ; concentration of elastase was 0.87 units/ml, and substrate, 363 μΜ.

Enzymatic activity in cleaving the substrate was indicated by a development of yellow color measured as increase in absorbance at 410 nm wavelength. The mean of maximum rate of absorbance increase in negative control wells was considered as 100% of enzyme activity. IC ₅₀ was calculated as concentration of test article in the well which reduced the elastase activity to 50%. Lower IC ₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of Kallikrein 5

Kallikrein 5 (KLK5), also known as stratum corneum tryptic enzyme, is a trypsin- like serine protease. Recent in vitro and in vivo evidence implicates increased levels of KLK5 in augmented inflammatory response such as rosacea (Two AM, Del Rosso JQ, Kallikrein 5- mediated inflammation in rosacea: clinically relevant correlations with acute and chronic manifestations in rosacea and how individual treatments may provide therapeutic benefit. J Clin Aesthet Dermatol. 2014 Jan; 7(1): 20-5) and in induction of atopic dermatitis-like lesions (Briot A. et al., Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome. J Exp Med. 2009 May 11 ;206(5): 1135-47). Normal human adult epidermal keratinocytes (HEK) were cultured as described above for the cytoxicity evaluation. After incubation with test articles or controls for 16 hours, HEK cell culture supernatants were collected. KLK5 was quantified using a human KLK5 immunoassay Quantikine ELISA kit (Catalog # DKK500, produced by R&D Systems, Minneapolis, MN). The changes of KLK5 concentrations between test articles and vehicle controls were calculated and compared. IC ₅₀ (concentration of test article necessary to reduce KLK5 levels to 50% compared to samples from untreated cells) values were calculated by sigmoidal curve fitting with SigmaPlot 10.0 (Systat Software). Lower IC ₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of IL-6 and/or IL-8 Induced by SDS

Normal human adult epidermal keratinocytes (HEK) were cultured as described above for the cytoxicity evaluation. The cells were then incubated with test articles and/or controls for 16 hours. Presence of sodium dodecyl sulfate (SDS) in cell cultivation medium at specific concentrations was used for induction of chemokines and cytokines. IL-8 was induced by 6 μg/mL SDS, IL-6 by 12.5 μg/mL SDS. After incubation, HEK cell supematants were collected. Quantikine® ELISA kits (R&D Systems Inc, Minneapolis, MN) were used to quantify these interleukins in the supematants. IL-8 was quantified by Human CXCL/IL-8 Immunoassay kit (Catalog # D8000C), and IL-6 was quantified by Human IL-6 Immunoassay kit (Catalog # D6050). IC ₅₀ (concentration of test article necessary to reduce interleukin levels to 50%, with samples from untreated cells considered as 0% and samples treated solely with respective inducing quantity of SDS as 100%) values were calculated by sigmoidal curve fitting with SigmaPlot 10.0 (Systat Software). Lower IC ₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of IL-6, IL-8, and PGE2 Induced by Full-Spectrum Sunlight from Artificial Source Normal human adult epidermal keratinocytes (HEK) were cultured as described above for the cytoxicity evaluation. The cells were washed once, and M154 was replaced with PBS. Both the washing and the replacement were done with PBS to remove light-absorbing components of M154. The 96-well plate containing HEK was then covered with UV-transparent

1 mm quartz sheet, placed on white underlay atop controlled Peltier-cooled surface maintaining room temperature, and irradiated with a dose of 20 J/cm ² of artificially produced full spectrum sunlight at dose rate of about 1100 W/m ² , as measured via pyranometer through same quartz cover. PBS was then removed and replaced with M154, and cells were incubated with test articles and/or controls for 16 hours. Identical manipulations, with exception of presence of sunlight, were carried out with HEK serving as unstressed controls. Irradiation equipment was obtained from Solar Light Company, Glenside, PA and included Solar Simulator LS1000-6R-

002 in Airmass 1.5 configuration using plain mirror; XPS1000 precision current source, and PMA2144 Pyranometer. After incubation, HEK cell supematants were collected. Quantikine® ELISA kits (R&D Systems Inc, Minneapolis, MN) were used to quantify interleukins in the supernatants. IL-8 was quantified by Human CXCL/IL-8 Immunoassay kit (Catalog # D8000C), IL-6 was quantified by Human IL-6 Immunoassay kit (Catalog # D6050); and PGE2 was quantified using Parameter™ Prostaglandin E2 Assay (Catalog # KGE004B). IC5 ₀ (concentration of test article necessary to reduce interleukin or prostaglandin levels to 50%, with samples from non-irradiated cells considered as 0% and from irradiated cells considered as 100%) values were calculated by sigmoidal curve fitting with SigmaPlot 10.0 (Systat Software). Lower IC5 ₀ values indicate higher potency and a degree of efficacy.

Table 9 summarizes the results of evaluating inhibition of IL8 and PGE2 activity induced by full-spectrum sunlight from an artificial source and cytotoxicity (LDH/microscopy).

Table 9

Table 10 summarizes the results of evaluating inhibition of IL8 induced by SDS; KLK5 inhibition; IL18 inhibition; trypsin inhibition; elastase inhibition; and cytotoxicity.

Table 10

Material IL8 KLK5 IL18 Cytotoxicity Trypsin Elastase inhibition (LDH/ Inhibition Inhibition (SDS) microscopy)

Achachariu Not tested IC ₅₀ Maximum No IC ₅₀ IC ₅₀ (Garcinia 0.18% 1.46 fold cytotoxicity 0.37% 0.26% humilis) Fruit (benchmark at 0.1%

Peel Serum sensitizer Material IL8 KLK5 IL18 Cytotoxicity Trypsin Elastase inhibition (LDH/ Inhibition Inhibition (SDS) microscopy)

Fraction, 7.8 fold)

Lot 0822

Achachariu About 65% ic ₅₀ Maximum No IC ₅₀ IC ₅₀

(Garcinia inhibition 0.2% 1.8 fold cytotoxicity 0.32% 0.28% humilis) at 0.1% (benchmark at 0.1%

Whole Fruit sensitizer

Serum 7.8 fold)

Fraction,

Lot 0786

In summary, biological activity results suggest that preparations of achachariu: (i) are not skin sensitizers; (ii) are not cytotoxic at selected concentrations that could be relevant to their concentrations in finished product formulation; and (iii) potential usefulness for mitigating signs of skin aging caused by inflammation and related process (e.g., those triggered by stresses to the skin, including environmental stress such as full-spectrum sun exposure). More specifically, the test results suggested the following: (i) against PGE2, achachariu whole fruit extract is slightly more effective than fruit peel extract; (ii) against PGE2, achachariu fruit peel serum fraction is more effective than whole fruit serum fraction, which is tentatively better than fruit flesh+ fruit seed serum fraction; and (iii) achachariu whole fruit serum fraction is effective for inhibiting SDS-induced IL-8 activity, but not against sun-induced IL8, which is surprising. Also surprising was: (i) the potent inhibition demonstrated by fruit peel extract against sun-induced IL8 and fruit peel serum fraction against sun-induced PGE2; (ii) the lack of any notable activity by fruit flesh + fruit seed serum fraction; (iii) fruit peel serum fraction and whole fruit serum fraction are more cytotoxic than fruit flesh + fruit seed serum fraction; and (iv) whole fruit extract is more cytotoxic than fruit peel extract.

Anti- Oxidant Response Element Activation

The ARE is the so-called "master switch" believed to control the antioxidant defense system of most cells. When the ARE is activated in response to oxidative stress, the corresponding genes signal the cell to begin producing reduction/oxidation regulators and/or reactive oxygen species ("ROS") quenching proteins and enzymes. ROS are highly reactive molecules formed naturally within cells as a natural byproduct of the normal metabolism of oxygen and play a role in cell signaling and homeostasis. However, when a cell is exposed to a stressor such as heat or UV radiation, ROS levels can increase, and in some instances dramatically. As the damage caused by ROS accumulates over time, it causes more and more oxidative stress at the cellular level that ultimately may lead to tissue damage and/or organ dysfunction. Thus, without being limited by theory, it is believed that if achachariu can demonstrate the ability to activate the ARE, then applying an effective amount of achachariu to keratinous tissue may help fight cellular damage associated with oxidative stress.

ARE activation was quantitated using the ARE-32 reporter cell line available from CXR- Biosciences as described below. ARE-32 is a stable MCF7 cell line containing pGL8x-ARE (8 copies of the rat GST ARE linked to the luciferase gene) and pCDNA3.1, which contains the neomycin selectable marker. Selection was performed in the presence of G418 and resistant clones were isolated. Clones were screened for induction of luciferase in response to tBHQ.

The ARE-32 cells are maintained routinely in Dulbecco's Modified Eagle Medium (phenol red free) ("DMEM") containing: 10% fetal bovine serum ("FBS"), 50 units/ml penicillin & 50 μg/ml streptomycin, 0.8 mg/ml G418. Cells are subcultured every 3-4 days. If needed, cells can be frozen in medium that contains 90% FBS and 10% DMSO.

ARE Method

In a 96 well-plate, lxlO ⁴ cells/well are seeded in 100 μΐ DMEM containing 50 units/ml penicillin, 50 μg/ml streptomycin, 0.8 mg/ml G418 and 10% FBS. Next, the cells are incubated at 37° C in a 5% CO ₂ incubator for 24 hrs, and then the medium is replaced with 100 μΐ fresh media. The test samples are treated with achachariu serum fractions at the concentration listed in Table 11 (1 ul per well), the positive control is 25 uM TBHQ. (10 mM tBHQ of stock solution freshly prepared in DMSO). 100 ul of media is added after treatment for a final assay volume of 200 uL. The cells are incubated at 37° C in CO ₂ incubator for another 24 hrs. The test samples are then assayed for luciferase activity with Steady-glo™ brand assay system according to the manufacturer's instruction.

The results of the test are summarized in Table 11. At concentrations of 0.167% to 1.5%, both the whole fruit and peel serum fractions appear to provide more ARE activation than than the control. The whole fruit also demonstrated a directional increase at 0.056% versus the control.

Table 11 - ARE Activation

v/v% achachariu Whole fruit ¹ p- value vs. Peel ² p- value vs.

% increase control % increase control vs control vs control

1.5 929 0.000087 1133 0.000046

0.5 489 0.0023 614 0.00086

0.167 197 0.0076 209 0.0043

0.056 134 .0.091 113 0.67 0.0185 103 0.96 92 0.79

0.006173 112 0.83 99 0.99

0.002058 93 0.89 92 0.94

0.000686 120 0.78 93 0.94

Recentia ^® GH-P from AkzoNobel

Melanin Synthesis Inhibition - B16 Assay

Overproduction of melanin is generally associated with a variety of skin pigmentation conditions (e.g., age spots, vitiligo, solar lentigines, and melasma). Thus, without being limited by theory, it is believed that if achachariu can demonstrate the ability to inhibit melanin production, then applying an effective amount of achachariu to skin may help improve the appearance of skin pigmentation conditions.

A commercially available B16-F1 mouse melanoma cell line from American Tissue

Culture Collection, Virginia, USA was employed in a conventional melanin synthesis inhibition assay. The cell culture medium used in the assay is 500 mL of Dulbecco's Modified Eagle's Medium ("DMEM"), 50 mL Fetal Bovine Serum ("FBS"), and 5 mL of penicillin- streptomycin liquid. B16-F1 cells that are cultured in this medium and grown to greater than 90% confluency will synthesize melanin. While not intending to be bound by theory, it is hypothesized that melanin synthesis is stimulated by the culture medium and/or stress induced by growth to a high confluency. The DMEM and FBS can be obtained from American Tissue Culture Collection and the penicillin-streptomycin liquid can be obtained from Invitrogen, Inc., California, USA. Equipment used in the assay include a CO ₂ incubator (e.g., a Forma Series Model 3110 by Therma Scientific, Massachusetts, USA or equivalent); a Hemocytometer (e.g., Bright Line model by Hauser Scientific, Pennsylvania, USA or equivalent); and a UV- Visible Spectrum Plate Reader (e.g., SpectraMax250 from Molecular Devices, California, USA or equivalent).

Day 0: To begin the assay, the cell culture medium is heated to 37 °C and 29 mL of the medium is placed into a T-150 flask. Approximately 1 x 10 ⁶ of B16-F1 passage 1 mouse cells are added to the T-150 flask and incubated for 3 days at 37 °C, 5% CO ₂ , 90% relative humidity, until -80% confluency.

Day 3: The cells from the T-150 flask are trypsinized, and the number of cells is determined using the Hemocytometer. Initiate a 96 well plate with 2,500 cells per well in 100 μΐ _^ of cell culture medium. Incubate the plate at 37°C, 5% CO ₂ , 90% relative humidity for 2 days until at least 20% to 40% confluent. Day 5: Remove the cell culture medium from the plate and replace with fresh culture medium (100 uL per well). Add 1 uL of test compound diluted in a water solvent. Multiple dilution ratios may be tested in order to generate a dose response curve, wherein preferably three wells are treated with each dilution ratio. Positive and negative controls may include wells having the cell culture medium, B16-F1 cells, and the solvent (negative control), and wells comprising the cell culture medium, B16-F1 cells and and a known melanin inhibitor (e.g., deoxyarbutin or kojic acid).

Day 7: Cells should have greater than -90% confluency. If not, this data point is not used. Add 100 uL of a 0.75% sodium hydroxide solution to each well. Read the 96-well plate using the UV-Vis Plate Reader at 410 nm to optically measure the amount of melanin produced between wells that are treated with the fava bean extract and control wells that are not. Wells in which melanin is produced appear brownish in color. Wells in which little melanin is produced appear clear to light purple in color. Percentage of melanin synthesis inhibition is calculated by the following equation:

100 - [OD410 Test Compound - OD410 Control #2] X 100

(OD410 Control #1 - OD410 Control #2)

Where OD410 is the Optical Density at 410 nm as measured by the UV-Vis Spectrum Plate Reader.

When Control #3 is used, the formula for percentage melanin synthesis inhibition is:

100 - [OD410 Test Compound - OD410 Control #3] X 100

(OD410 Control #1 - OD410 Control #2)

The concentration of test agent needed to provide the IC 50 is recorded.

The results of the test are summarized in Table 12, which shows that achachariu inhibits melanin synthesis, and thus is expected to provide a skin lightening benefit.

Table 12 - Melanin Synthesis Inhibition

1. Recentia ^® GH from AkzoNobel

2. Recentia ^® GH-P from AkzoNobel Lipogenesis inhibition

This example demonstrates the ability of achachariu to inhibit lipogenesis in human pre- adipocytes. Lipogeneis involves the synthesis of commonly known lipids such as fatty acids and triglycerides, and is one of primary ways mammals store energy. However, lipogenesis also involves the synthesis of lipids such as sebum. Sebum is a lipid produced by sebocytes, which are a type of skin cell found primarily in the sebaceous glands of mammalian skin. Sebum is produced by the body to lubricate and waterproof the skin and hair of mammals. However, overproduction of sebum can result in oily appearing skin and/or skin that appears to have poor texture. Thus, without being limited by theory, it is believed that if achachariu can demonstrate the ability to inhibit lipogenesis, then applying an effective amount of achachariu to keratinous tissue may help regulate conditions associated with sebum overproduction.

Method

Human pre-adipocytes were selected for use in this example. Because of the known difficulty associated with culturing and testing sebocytes, pre-adipocytes are commonly used as a surrogate for sebocytes to determine the potential of a test agent to inhibit sebum production.

Human subcutaneous pre-adipocytes purchased from Zen-Bio, Inc (Cat. # SP-F-SL) were cultured in PM-1 media (available from Zen-Bio, Inc as Cat# PM-1 (plus 5ng/ml EGF)) to 80 - 90% confluency. The cells were transferred to 96-well clear bottom white plates to provide approximately 40,625 cells/cm ² in the well (-12,500 cells) and 150 μΐ of PM-1 media per well, and then cultured for 24 - 48 hours in a 5% CO ₂ incubator at 37 °C. The PM-1 media was then replaced with differentiation medium (Zen-Bio, Inc. Cat# DM-1), and the cells were incubated for another 6 days. After incubating in the differentiation medium, 90 μΐ of the differentiation medium was carefully replaced with 140 μΐ of human subcutaneous adipocyte medium ((Zen- Bio, Inc. Cat# AM-1). Care was taken not to touch or disturb the cells at the bottom of the well. 2 μΐ of achachariu (Recentia ^® GH-P for AkzoNoble) or control composition (100 μΜ Genistein (Cat# G6649) from Sigma) was added to each well daily for 9 days (total incubation of 15 days). On Day 15, 5 μΐ. of AdipoRed reagent (Lonza; Cat. Number: PT-7009) was slowly added directly to cells in the treatment medium, and the plate was gently mixed after each row addition. The plate was incubated for 15 minutes at room temperature. Lipogenesis was quantitated using an En Vision ^® brand Fluorescent spectrophotometer Plate Reader according to the AdipoRed protocol. The plates were scanned from the bottom using the 451 mirror and (excitation 485 nm; emission 535) filter. Each well was scanned in a Z pattern (7 reads across from left to right, 7 reads diagonally from right to left and 7 reads across from left to right for a total of 21 end points). Percent inhibition was calculated as: Average Control RFU - Sample RFU X 100

Average Control RFU

The cells were assayed and normalized to the control by using a FluoReporter ^® Blue Fluorometric brand dsDNA Quantitation Kit. Immediately after the screen the AdipoRed containing cell media was gently aspirated, cells were rinsed with lOOul IX PBS taking care not to dislodge them from the bottom and ΙΟΟμΙ distilled water was added/well. The plates were frozen at -80°C to lyse the cells and assayed according to the kit instructions at a later date.

The results of the test are summarized in Table 13, which shows that achachariu inhibits lipogenesis, and thus is expected to help regulate conditions associated with the overproduction of sebum.

Table 13 - Lipogenesis Inhibition

Inhibiting the Cell's Inflammation Response to a Stressor - NF-kappa-beta ("NF-kB") assay.

NF-kB (i.e., nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that belongs to the category of "rapid-acting" primary transcription factors (i.e., transcription factors that are present in cells in an inactive state and do not require new protein synthesis in order to become activated), which allows NF-kB to be a first responder to harmful cellular stimuli such as ROS and other stressors. NF-kB is found in almost all animal cell types and is known to be involved in the cellular inflammation pathway. Cellular inflammation is associated with a variety of skin conditions, and thus inhibiting NF-kB activation vis-a-vis cellular inflammation with an effective amount of achachariu may help treat these types of skin conditions.

Method

CellSensor™ NF-κΒ -bla HEK 293T cells (Invitrogen, Cat. # K1165) were plated in assay medium (DMEM with high glucose (Gibco, Cat. #11965) plus 10% dialyzed FBS). The cells were cultured and seeded at 10,000/well in 96-well plates (black-sided Poly-D-Lysine coated plates, BD # 356692), and then incubated at 37 °C and 95% RH for the 72 hours prior to testing. Recombinant human TNFa (available from R&D systems) was used to stimulate NF-kB activation in the cells. Fisetin (3, 7, 3', 4'-tetrahydroxyflavone) was used to inhibit stimulation of the cells by TNFa. A ToxBLAzer™ DualScreen brand screening kit (Invitrogen, Cat. #K1138) was used as the fluorescent substrate according to the manufacturer's instructions.

The following controls were run on each plate in the assay:

High control (Stimulated) = cells + TNFa + 1% DMSO

Blank (Unstimulated) = cells + 1% DMSO

Standard (Positive control inhibitor) = cells + TNFa + Fisetin + 1% DMSO

Negative Control (no cells) = assay medium

NF-KB % inhibition is calculated as: High Control - Sample x 100

High Control - Blank

The results of this test are summarized in Table 14 below. As illustrated in Table 14, the achachariu whole fruit serum fractions and achachariu peel serum fractions provided an IC 50 for NF-kB inhibition of 2%. Thus, applying an effective amount of achachariu to a target portion of keratinous tissue may help regulate conditions related to cellular inflammation.

Table 14

Inhibiting the Cell's Inflammation Response to a Stressor - Prostaglandin E2 ("PGE2") assay.

PGE2 is a hormone-like substance that is known to participate in modulation of inflammation. Cellular inflammation is associated with a variety of skin conditions, and thus inhibiting PGE2 activation vis-a-vis cellular inflammation may help treat these types of skin conditions.

Method

Tert keratinocytes ("tKC") were plated at 40,000 cells/well into 24-well plates in 1 ml/well volume. EpiLife Medium (Life Technologies cat # MEPICFPRF500) supplemented with keratinocyte growth supplement (Life technologies cat #S-001-5) was used as the assay media. The cells were grown to confluence/near confluence, and then subjected to 15 mJ/cm ² UVB- stress. The test compositions (achachariu and vehicle control) (diluted 1:1000) were added, and the plates were incubated for 18-24 hours. The supernatant was removed from each well, and the cells were rinsed with 2 ml/well medium (without supplements). A Cell Titer-Glo assay (measures ATP activity) was conducted on the cells for normalization. The supernatant was tested in a PGE2 assay (Prostaglandin E2 Assay kit from Cisbio Bioassays cat# 62P2APEB) according to the manufacturer's instructions. The PGE2 results were normalized to ATP activity.

The results of the PGE2 assay are summarized in Table 15. The achachariu whole fruit serum fraction and achachariu peel serum fractions both demonstrated the ability to inhibit release of PGE2 from keratinocytes exposed to 15 mJ/cm2 UVB radiation, which illustrates antiinflammatory activity of achachariu. Thus, a personal care composition comprising achachariu may be useful for regulating a condition of mammalian keratinous tissue related to cellular inflammation.

Table 15

Protein Loss Inhibition in Hair

Hair is a protein filament formed primarily of keratin that grows from follicles found in the dermis of mammalian skin. The proteins (i.e., keratin) in hair can be damaged by a variety of endogenous and exogenous stressors (e.g., UV radiation), which can lead to, e.g., dry, brittle, and/or dull looking hair. Thus, it would be desirable to inhibit, prevent or even reverse undesirable protein loss in hair.

It is believed, without being limited by theory, that changes in the level of certain amino acids found in keratin correspond to the overall change in protein level in hair. In this test, serine, proline, glutamic acid, and valine were used as surrogates for overall protein level, since these amino acid residues represent approximately 40% of the protein concentration.

Method.

Achachariu whole fruit serum extract (Recentia® GH) was formulated at 2% (w/v) in a 50:50 ethanokwater chassis with 2% SEPIMAX ZEN brand thickener (Seppic, France) to a final pH of 6.0 to provide a test composition. The same composition, except without any achachariu serum fraction, was used as a control. The test composition and control composition were applied to light-brown, non-chemically-treated, 2g, flat hair switches (6 switches per leg) at 0.2 g/g, and thoroughly distributed through-out the hair switch. Twenty-four hours after treatment, the switches were washed with Pantene® Volume brand shampoo (from the Procter & Gamble Co.). This treatment/wash cycle was repeated ten times, but the hair switches were not washed after the tenth treatment. After the tenth treatment, half the switches from the control and test group were tested for protein loss (0 hours), and the other half were exposed to 40 hours of ultraviolet radiation in an Atlas™ Ci3000+ brand weather-o-meter and then tested for protein loss (40 hours). An internal and outer quartz filter was used to simulate broad- spectrum, outdoor daylight with a specific irradiance of 1.48 W/m ² at 420 nm. Temperature and relative humidity were kept constant at 35 °C and 80% RH. After the UV exposure, the hair switches were tested for protein loss.

Protein levels are determined using the following method. 0.2 - 0.3 g hair samples (2 inches length) are collected from each hair switch and are added to glass scintillation vials. Distilled water is added at a ratio of 10:1 (mL water to g hair). Samples are shaken for 1 h at 2500 rpm on a DVX-2500 Multi-tube Vortexer platform. Amino acid concentration is determined after acid hydrolysis using High-Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS/MS). The analytes (Serine, Proline, Valine, Glutamic acid) and their corresponding internal standards are subjected to hydrophilic interaction chromatography (HILIC) analysis on a ZIC-HILIC column (2.1 x 150 mm, 5 micron particles). Detection and quantitation is by tandem mass spectrometry operating under multiple reaction monitoring (MRM) MS/MS conditions. The amino acid concentrations are determined by back-calculation using a weighted quadratic regression of a calibration curve generated from neat standards.

The results of the test are summarized in Table 16 and illustrated in FIGS. 5 to 9. Amino acid levels are shown in μg/g. P- values of 0.1 or less are considered statistically significant. P- values of greater than 0.1 but less than 0.2 are considered directional. As illustrated in Table 17 and FIGS. 5 to 9, the amount of amino acid loss observed when the samples were exposed to UV radiation for 40 hours increased relative to the corresponding 0 hour samples. However, the achachariu-treated samples exhibited less protein loss at 0 hours and after 40 hours of UV exposure compared to their respective non-achachariu treated counterparts. In addition, the samples treated with achachariu did not exhibit a statistically significant difference in protein loss after 40 hours of UV exposure when compared to the untreated samples at 0 hours. Thus, treatment with achachariu appears to reduce UV-induced protein loss in hair, and the reduction in protein loss at 0 hours suggests that treatment with achachariu may also reduce protein loss in hair due to non-UV stressors.

Table 17

1. Difference between Control level at 0 hours and Treated level at 0 hours.

2. Difference between Control level at 0 hours and Control level at 40 hours.

3. Difference between Control level at 0 hours and Treated level at 40 hours

4. Difference between Treated level at 40 hours and Treated level at 0 hours.

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm".

All documents cited in the Detailed Description of the Invention are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. Whereas particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.