CN111944870B

CN111944870B - Phyllanthus emblica extract fermentation product and preparation and application thereof

Info

Publication number: CN111944870B
Application number: CN202010410047.0A
Authority: CN
Inventors: 林咏翔; 庄伟秀; 林焕祐
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-05-17
Filing date: 2020-05-14
Publication date: 2022-01-28
Anticipated expiration: 2040-05-14
Also published as: TWI747280B; TW202102242A; CN111944870A

Abstract

A composition containing fructus Phyllanthi extract fermented product, application of the fermented product and its active ingredient, and preparation method of the fermented product are provided. Wherein the fermentation product of the emblic leafflower fruit extract comprises the following formula (I) To at least one compound of formula (XIV):

Description

Phyllanthus emblica extract fermentation product and preparation and application thereof

Technical Field

The present invention relates to a composition comprising an extract fermentation product of emblic leafflower fruit containing at least one of the following compounds of formulae (I) to (XIV), and a method for preparing the extract fermentation product of emblic leafflower fruit:

the invention also relates to the application of at least one of the compounds shown in the formulas (I) to (XIV) and the extracted fermentation product of emblic leafflower fruit, in particular to the application of the compounds which can not easily form body fat for individuals, whiten skin, resist skin aging, inhibit the adipogenesis capability of fat cells, promote the fat degradation capability of fat cells, promote weight loss, help metabolism, promote the activity of mitochondria, inhibit the generation of melanin, inhibit the generation of ROS, inhibit the generation of AGEs, promote the expression of SOD2 gene, promote the expression of MPG gene and promote the expression of ERCC1 gene.

Background

Obesity is a state of excess accumulation of body fat. Obesity has become a worldwide health problem due to western dietetic conversion of high oil, high sugar and high fat, general lack of exercise and genetic factors of modern people, and the like. Body Mass Index (BMI) or waist circumference is internationally commonly used as an index for assessing obesity. In taiwan, a BMI value greater than or equal to 24 and less than 27 indicates overweight, while a BMI value greater than or equal to 27 indicates obesity.

Currently common methods of suppressing obesity include dietary management, exercise, lifestyle modification, pharmacotherapy and surgery. Except for severely obese patients, diet control and exercise are generally recommended for fat reduction in clinical settings. However, there is still a need for other more convenient solutions for modern people who are busy and often take exopathic foods.

In addition to fat reduction and weight loss, modern people tend to pay attention to skin whitening and skin aging resistance, and cosmetic products on the market are gradually diversified, for example, vitamin C derivatives for skin application, glutathione for oral administration, whitening needles for injection and the like. However, administration of vitamin C derivatives or glutathione by oral administration or application does not allow effective absorption by the human body, and the actual supplement effect is limited; the effect of the injection whitening needle can be maintained only temporarily, and the injection is required to be performed regularly, so that the cost is high, and anaphylactic reaction can be caused. Therefore, the industry is still engaged in the research on skin whitening and related products for resisting skin aging.

The research of the inventor of the patent finds that the emblic leafflower fruit extract fermentation product can effectively reduce fat accumulation, achieve the effects of whitening and resisting skin aging, and can meet the requirements on fat reduction, weight reduction, whitening and aging resistance.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a method for preparing an extract fermentation product of emblic leafflower fruit, comprising the steps of: (a) extracting emblic leafflower fruit to provide an emblic leafflower fruit extract; (b) fermenting the extract with Saccharomyces cerevisiae and Lactobacillus plantarum to obtain an intermediate fermented product; and (c) fermenting the intermediate fermentation product with an acetic acid bacterium to obtain an extract fermentation product of Emblica officinalis. Preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae BCRC 20271; the lactobacillus plantarum is lactobacillus plantarum BCRC 910760; the acetic acid bacteria is acetic acid bacteria BCRC 11688.

Another object of the present invention is to provide a composition comprising an extract fermentation product of emblic leafflower fruit, wherein the extract fermentation product of emblic leafflower fruit is provided by the above method. Preferably, the emblic extract fermentate contains at least one of the following compounds of formulae (I) to (XIV):

preferably, the composition is a pharmaceutical composition, a food composition or a cosmetic composition.

It is still another object of the present invention to provide a use of an active ingredient for making an individual less prone to form body fat, whitening skin, and/or anti-aging skin, wherein the active ingredient is at least one of the compounds of formulae (I) to (XIV) described above, and the fermentation product of emblica officinalis extract provided by the above method. Preferably, the active ingredient is used in the form of a food composition or a cosmetic composition.

It is still another object of the present invention to provide a use of an active ingredient for preparing a pharmaceutical composition, wherein the pharmaceutical composition is at least one of inhibiting adipogenic ability of adipocytes, promoting adipogenic degradation ability of adipocytes, promoting slimming, aiding metabolism, increasing mitochondrial activity, inhibiting melanin production, inhibiting ROS production, inhibiting AGEs production, increasing SOD2 gene expression, increasing MPG gene expression, and increasing ERCC1 gene expression, and wherein the active ingredient is at least one of the compounds of formulae (I) to (XIV) described above, and the emblic leafflower fruit extract fermentation product provided by the above method.

Drawings

FIGS. 1A to 1N show NMR spectra of compounds of formulae (I) to (XIV), respectively;

FIG. 2 shows the results of microscopic examination of adipocytes stained with oil red O stain, including "control group" adipocytes cultured in a medium without Emblica officinalis extract and Emblica officinalis extract fermentation product, "extract group" adipocytes cultured in a medium with Emblica officinalis extract, and "fermentation product group" adipocytes cultured in a medium with Emblica officinalis extract;

fig. 3 shows the relative fat content of adipocytes in the "extract group" and the "fermentation group" based on the "control group" (indicated by p value <0.001 compared to the control group);

fig. 4 shows JC-1 aggregate ratios of skeletal muscle cells of "control group", which were cultured in a medium containing no emblic leafflower fruit extract and emblic leafflower fruit extract fermentations, "extract group", which were cultured in a medium containing emblic leafflower fruit extract fermentations, and "fermentate group", which were cultured in a medium containing emblic leafflower fruit extract fermentations (indicating a p value <0.01 in comparison with the control group; indicating a p value <0.001 in comparison with the control group), representing mitochondrial activities of the skeletal muscle cells of each group. (ii) a

FIG. 5 shows the relative melanin content of melanoma cells of "control group", "UVA group", "positive control group", "extract group" and "fermentation group", wherein melanoma cells of "control group" are non-irradiated UVA and cultured in a medium not containing emblic leafflower fruit extract and emblic leafflower fruit extract fermentation, melanoma cells of "UVA group" are irradiated UVA but cultured in a medium not containing emblic leafflower fruit extract and emblic leafflower fruit extract fermentation, melanoma cells of "positive control group" are irradiated UVA but cultured in a medium containing kojic acid (kojic acid), "extract group" are irradiated UVA but cultured in a medium containing emblic leafflower fruit extract, and melanoma cells of "fermentation group" are irradiated UVA but cultured in a medium containing emblic leafflower fruit extract fermentation (p value is < 0.01;, p value is p value compared to UVA group < 0.001; # indicates p value <0.001 compared to control group; a means p value <0.05 compared to the extract group);

fig. 6 shows the relative SOD2 expression of human skin fibroblasts of "control group", cultured in a medium without any emblic leafflower fruit extract and emblic leafflower fruit extract fermentation product before UVA irradiation, "extract group", cultured in a medium with any emblic leafflower fruit extract before UVA irradiation, "ferment group", cultured in a medium with any emblic leafflower fruit extract fermentation product before UVA irradiation ("indicates p value < 0.001" compared to control group);

fig. 7 shows relative MPG and ERCC1 expression of human dermal fibroblasts of "control group", cultured in a medium without any emblic leafflower fruit extract and emblic leafflower fruit extract fermentation product before UVA irradiation, "extract group", cultured in a medium with any emblic leafflower fruit extract before UVA irradiation, "ferment group", cultured in a medium with any emblic leafflower fruit extract fermentation product before UVA irradiation, ("indicates p value <0.001 compared to the control group);

fig. 8 shows the relative ROS content of human skin fibroblasts for the "extract group" containing the extract of crambe when used to culture human skin fibroblasts, and for the "ferment group" containing the extracted fermentation of crambe when used to culture human skin fibroblasts (× indicates a p-value <0.05 when compared to the extract group);

FIG. 9 shows the relative AGEs (final glycosylation end products) production for "control" reaction solution without the fermentation product of Emblica officinalis extract and "experimental" reaction solution with the fermentation product of Emblica officinalis extract;

fig. 10 shows the relative fat content of adipocytes of "control group", which are cultured in a medium containing no emblic extract fermentate, EAF, BUF and WF fractions, the "EAF group", which are cultured in a medium containing the ethyl acetate soluble fraction of the emblic extract fermentate, the "BUF group", which is cultured in a medium containing the n-butanol soluble fraction of the emblic extract fermentate, and the "WF group", which is cultured in a medium containing the water soluble fraction of the emblic extract fermentate (the value of p indicates <0.001), compared to the control group);

fig. 11 shows the relative lipolytic capacity of adipocytes of the above-mentioned "control group", "fermentate group", "EAF group", "BUF group" and "WF group" (indicated p-value <0.05 compared to control group);

FIG. 12 shows the relative skeletal muscle mitochondrial activity of skeletal muscle cells of "control group", which was cultured in a medium containing no extract fermentation product of Emblica officinalis, EAF, BUF and WF fractions, of "BUF group", which was cultured in a medium containing an ethyl acetate soluble fraction of the extract fermentation product of Emblica officinalis, of "BUF group", which was cultured in a medium containing an n-butanol soluble fraction of the extract fermentation product of Emblica officinalis, and of "WF group", which was cultured in a medium containing a water soluble fraction of the extract fermentation product of Emblica officinalis (p value <0.05) as compared with the control group ";

FIG. 13 shows the results of microscopic examination of adipocytes stained with oil Red O stain, including "control group" and "experimental group", in which adipocytes were cultured in a medium without the compound of the present invention, and adipocytes were cultured in a medium containing the compounds of formula (I), formula (II), formula (VI), and formula (X) to formula (XIV), respectively;

FIG. 14 shows the relative fat content of adipocytes in the "experimental group" based on the "control group" cultured in a medium not containing the compound of the present invention (indicating that p-value < 0.05;. indicates p-value < 0.01;. indicates p-value < 0.001. compared to the control group);

FIG. 15 shows the relative lipolytic capacity of adipocytes "of the control group" cultured in a medium not containing the compound of the present invention and "experimental group" cultured in a medium containing the compounds of formula (II), (III) and (VII), respectively (p < 0.05;. p <0.01 relative to the control group);

FIG. 16 shows the relative skeletal myomitochondrial activity of skeletal muscle cells of "control group" cultured in media not containing the compounds of the present invention and "experimental group" cultured in media containing the compounds of formulae (I), (IV), and (VI) to (XIV), respectively (p is < 0.01; "p is <0.001) compared to the control group);

fig. 17 shows the relative melanin content of melanoma cells of the "control group", which were not irradiated with UVA and cultured in medium not containing the compound of the present invention, the "UVA group", which were irradiated with UVA, and the "experimental group", which were irradiated with UVA, and cultured in medium not containing the compound of the present invention, the "experimental group", which were irradiated with UVA, and cultured in medium containing the compounds of formulae (II) to (XIV), respectively (p value < 0.05;. p value < 0.01. c > compared to the control group; p value < 0.001. c compared to the control group);

fig. 18 shows HPLC fingerprint spectra of "emblic extract" and "emblic extract fermented product", in which the upper panel shows the results of the emblic extract, and the lower panel shows the results of the emblic extract fermented product.

Detailed Description

Some specific embodiments according to the present invention will be described below; the invention may, however, be embodied in many different forms without departing from the spirit thereof, and the scope of the invention should not be construed as limited to the specific details set forth in the specification.

As used in this specification (and particularly in the claims), unless the context requires otherwise, the words "a," "an," "the," and similar referents are to be construed to cover both the singular and the plural; by "individual" is meant a human or non-human mammal (e.g., dog, cat).

Phyllanthus emblica (Phyllanthus emblica) belongs to Phyllanthus of Phyllanthus family and Phyllanthus genus, and its fruit is spherical and edible.

The terms "saccharomyces cerevisiae", "lactobacillus plantarum" and "acetic acid bacteria" as used herein can be any suitable bacteria for use in the present invention, including commercially available saccharomyces cerevisiae, lactobacillus plantarum and acetic acid bacteria strains (e.g., available from domestic or foreign depository institutions), and saccharomyces cerevisiae, lactobacillus plantarum and acetic acid bacteria strains isolated from natural sources by microbial isolation methods commonly used in the art.

It is known that the increase of expression level of SOD2 gene (superoxide dismutase producing gene) is helpful for enhancing cell antioxidant ability, while the decrease or deletion of SOD2 gene is related to skin aging. The foregoing can be seen, for example: SOD2 removal proteins imaging pharmaceuticals in mouse skin. aging. 4 (2012), 116-118(2012), the entire disclosure of which is incorporated herein by reference. Therefore, if the expression of SOD2 can be effectively improved, the purpose of anti-skin aging (such as anti-skin photoaging) can be achieved.

Furthermore, it is known that increased expression of the MPG gene in a cell contributes to the enhancement of the ability of the cell to repair single strand breaks of DNA, while increased expression of the ERCC1 gene contributes to the enhancement of the ability of the cell to repair structural damage of DNA. The foregoing can be seen, for example: nat Educ 3.9 (2010):26, which is incorporated herein by reference in its entirety. Therefore, if the expression of MPG and/or ERCC1 gene can be effectively improved, the purpose of resisting skin aging (for example, resisting skin photoaging) can be achieved.

The research of the inventor of the patent finds that the emblic leafflower fruit extract can effectively inhibit the adipogenesis capability of fat cells, improve the activity of mitochondria, inhibit the generation of melanin, inhibit the generation of ROS, inhibit the generation of AGEs, improve the expression of SOD2 gene, improve the expression of MPG gene and improve the expression of ERCC1 gene; in addition, the fermentation product provided by fermenting the emblic leafflower fruit extract in the presence of saccharomyces cerevisiae, lactobacillus plantarum and acetic acid bacteria has better capabilities of inhibiting adipogenesis of fat cells, improving mitochondrial activity, inhibiting melanochrome generation, inhibiting ROS generation, inhibiting AGEs generation, improving SOD2 gene expression, improving MPG gene expression and improving ERCC1 gene expression. Therefore, the fermentation product provided by the emblic leafflower fruit extract according to the present invention, i.e., the emblic leafflower fruit extract fermentation product, can be used for promoting weight loss, helping metabolism, making an individual less prone to form body fat, whitening, and resisting skin aging.

The present inventors further obtained the following fourteen active ingredients (i.e., compounds of formulae (I) to (XIV)) from the above-mentioned extract fermentation product of emblica officinalis, all of which have the ability to inhibit adipogenesis by adipocytes, the ability to promote lipolysis by adipocytes, the ability to enhance skeletal muscle mitochondrial activity, and the ability to inhibit melanogenesis:

accordingly, the present invention relates to a method for preparing an extract fermentation product of Emblica officinalis, a composition comprising at least one of the compounds of formulae (I) to (XIV) and an extract fermentation product of Emblica officinalis provided by the method, a use of at least one of the compounds of formulae (I) to (XIV) and the extract fermentation product of Emblica officinalis to make an individual less prone to form body fat, whiten and/or resist skin aging, and a use of at least one of the compounds of formulae (I) to (XIV) and the extract fermentation product of Emblica officinalis to prepare a pharmaceutical composition for inhibiting adipogenic ability of adipocytes, promoting adipogenic degradation ability of adipocytes, promoting weight loss, aiding metabolism, promoting mitochondrial activity, inhibiting melanogenesis, inhibiting production of ROS, inhibiting production of AGEs, and a method for preparing an extract fermentation product of Emblica officinalis, At least one of SOD2 gene expression improvement, MPG gene expression improvement and ERCC1 gene expression improvement.

The method for preparing the emblic extract fermented product according to the present invention comprises the steps of: (a) extracting the emblic leafflower fruit to provide an emblic leafflower fruit extract; (b) fermenting the extract with Saccharomyces cerevisiae and Lactobacillus plantarum to obtain an intermediate fermented product; and (c) fermenting the intermediate fermentation product by using an acetic acid bacterium to obtain an emblic leafflower fruit extract fermentation product.

In step (a), the extraction may be performed using a polar solvent, preferably water, an alcohol (e.g., a C1-C4 alcohol), or a combination thereof. In some embodiments of the invention, water is used as the extraction solvent. In general, the amount of the extraction solvent may be adjusted as necessary, and is not particularly limited as long as the raw material can be uniformly dispersed. For example, a weight ratio of emblic leafflower fruit to extraction solvent of 1: 5 to 1: 20, the dosage of the composition. In addition, the extraction time and the extraction temperature may be suitably selected depending on the extraction solvent used. Pure water is used as an extraction solvent, and the following components are adopted: the weight ratio of the extraction solvent is 1: 5 to 1: for example, the extraction is usually carried out at 50 to 100 ℃ for 0.5 to 3 hours.

In the step (b), saccharomyces cerevisiae and lactobacillus plantarum are added to the extract provided in the step (a) to perform a first fermentation reaction. In some embodiments of the invention, the extract provided in step (a) is fermented with Saccharomyces cerevisiae BCRC 20271 and Lactobacillus plantarum BCRC 910760. In addition, the fermentation time and the fermentation temperature can be selected according to the adopted saccharomyces cerevisiae and lactobacillus plantarum. Taking Saccharomyces cerevisiae BCRC 20271 and Lactobacillus plantarum BCRC 910760 as examples, fermentation is usually carried out at 25-35 deg.C for 1-5 days. In general, the addition amount of Saccharomyces cerevisiae and Lactobacillus plantarum is not particularly limited. For example, in some embodiments of the invention, the brewer's yeast may be added in an amount of 0.01 to 0.5% by weight of the extract and the lactobacillus plantarum may be added in an amount of 0.01 to 0.25% by weight of the extract.

In step (c), acetic acid bacteria are added to the intermediate fermented product provided in step (b) to perform a second fermentation reaction. In some embodiments of the invention, the intermediate fermentation product provided in step (b) is fermented with acetic acid bacteria BCRC 11688. In addition, the fermentation time and fermentation temperature may be suitably selected depending on the acetic acid bacterium to be used. Taking acetic acid bacteria BCRC 11688 as an example, the fermentation is usually carried out for 5 to 15 days at 25 to 35 ℃. In general, the amount of acetic acid bacteria added is not particularly limited. For example, in some embodiments of the invention, acetic acid bacteria may be added in an amount of 1 to 15% by weight of the intermediate fermentation.

After the above steps are completed, operations such as reduced pressure concentration, filtration, and sterilization may be performed as necessary to improve the convenience of using the fermented product extracted from emblic leafflower fruit. For example, the extracted fermentation product of the emblic leafflower fruit may be concentrated under reduced pressure at 40 ℃ to 70 ℃ to provide a concentrated fermentation product; alternatively, the emblic extract fermented product or the concentrated fermented product may be filtered through a 200 to 400 mesh screen to remove residual solids. On the other hand, isomalto-oligosaccharide can be added to the extract fermented product of emblic leafflower fruit in an amount of 40 to 70% (weight/weight) and sterilized at 90 to 120 ℃ for 70 to 90 minutes to provide a drink.

The pharmaceutical composition provided by the present invention can be used for systemic or local administration, and can be delivered by various Drug Delivery Systems (DDS), including oral drug delivery systems (oral drug delivery systems), transdermal drug delivery systems (transdermal drug delivery systems), injectable drug delivery systems (injectable drug delivery systems), and the like. For example, but not limited thereto, the pharmaceutical composition provided by the present invention can be delivered by liposome (liposome), microcapsule (microcapsule), nanoparticle (nanoparticle), microneedle (microneedle), etc. to achieve the purposes of improving bioavailability, controlling drug release rate, precise drug administration to a lesion, reducing drug side effects, etc.

The pharmaceutical composition provided according to the present invention may be in any suitable form without particular limitation, and the corresponding suitable dosage form is adopted according to the intended use; for example, but not limited thereto, the pharmaceutical composition can be administered to a subject in need thereof by oral, intravenous (including drip infusion and bolus injection), intramuscular, subcutaneous, arterial, intraperitoneal, transdermal (e.g., patch, ointment, etc.) administration. Depending on the form of use and the application, a pharmaceutically acceptable carrier may be used to provide the pharmaceutical composition, wherein the carrier is well known to those skilled in the art and includes excipients, diluents, adjuvants, stabilizers, absorption promoters, disintegrants, solubilizers, emulsifiers, antioxidants, binders, tackifiers, dispersants, suspending agents, lubricants, moisture absorbents, and the like.

For example, the pharmaceutical composition can be provided in a form suitable for oral administration by any suitable method, including liquid forms suitable for oral administration including syrups, oral liquids, suspensions, elixirs and the like, and solid forms suitable for oral administration including powders, granules, buccal tablets, dragees, enteric tablets, chewable tablets, effervescent tablets, film-coated tablets, capsules, sustained-release tablets and the like. The pharmaceutical composition provided according to the present invention may contain any pharmaceutically acceptable carrier that does not adversely affect the intended benefits of the active ingredient, i.e., at least one of the compounds of formulae (I) to (XIV) and the extract fermentation product of emblica officinalis. By way of illustration and not limitation, examples of pharmaceutically acceptable carriers for the aforementioned liquid dosage forms include: water, saline solution, dextrose (dextrose), glycerol, ethanol or the like, oils (e.g., olive oil, castor oil, cottonseed oil, peanut oil, corn oil, and germ oil), glycerol, polyethylene glycol, and combinations of the foregoing; examples of pharmaceutically acceptable carriers for such solid dosage forms include: cellulose, starch, kaolin (kaolin), bentonite (bentonite), sodium citrate, gelatin, agar, carboxymethylcellulose, gum arabic, algin, glycerol monostearate, calcium stearate, and combinations thereof.

Any pharmaceutically acceptable carrier that does not adversely affect the desired benefit of the active ingredient (i.e., at least one of the compounds of formulae (I) through (XIV) and the emblic extract fermentate) may also be included in a dosage form suitable for transdermal administration, such as: water, mineral oil, propylene glycol, polyethylene oxide, liquid paraffin, sorbitan monostearate, and polysorbate 60. The pharmaceutical composition can be provided in a form suitable for transdermal administration by any suitable method, such as, but not limited to, emulsions, creams, oils, gels (e.g., hydrogels), pastes (e.g., dispersion pastes, ointments), lotions, sprays, and patches (e.g., microneedle patches).

As for injections or intravenous drip injections, the pharmaceutical composition provided by the present invention may contain one or more components such as isotonic solution, saline buffer (e.g., phosphate buffer or citrate buffer), solubilizer, emulsifier, 5% sugar solution, and other carriers, and may be provided in the form of intravenous infusion solution, emulsion intravenous infusion solution, dry powder injection, suspension injection, or dry powder suspension injection. Alternatively, the pharmaceutical composition may be prepared as a pre-injection solid and the pre-injection solid dissolved in another solution or suspension or emulsified prior to administration to a subject in need thereof to provide the desired injection.

The food composition provided according to the present invention may be a beverage, a solid food, or a semi-solid food, and may be provided in the form of a health food, a functional food, a nutritional supplement, or a special nutritional food. By way of illustration and not limitation, the food composition can be dairy products, meat processed products, breads, wheats, biscuits, ice products, lozenges, capsules, juices, teas, sparkling water, alcoholic beverages, sports beverages, nutritional beverages, infant formula, and the like. Preferably, the food composition is provided in the form of a health food or health food.

In addition, any suitable dietary supplement may be included in the food composition provided according to the present invention, depending on the form of use and the requirements. Examples include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, color retention agents, bulking agents, nutritional additives, coloring agents, flavoring agents (e.g., sweeteners), viscosity agents, caking agents, food industry chemicals, emulsifiers, and quality improving, brewing, and food manufacturing agents.

The recommended usage amount, the standard and the condition of use, or the recommended matters taken together with other foods or medicines can be marked on the external package of the health food, the functional food, the nutrition supplement food or the special nutrition food provided by the invention, so that the user can conveniently take the food by himself without the guidance of doctors, pharmacists or related medical practitioners without safety concerns.

The cosmetic compositions provided according to the present invention may be formulated into any suitable product form. For example, but not limited thereto, the cosmetic composition may be a skin softening lotion, an astringent lotion, a nutritional cream, a massage cream, an essence, an eye cream, an eye essence, a mask, a patch, a spray, a body lotion, a body cream, a body oil, a body essence, and other care products; cosmetic products such as pre-make-up cream, foundation make-up, honey powder, blush, eye shadow, eyebrow powder, mascara, lipstick, etc.; or cleaning products such as skin cleaning oil, skin cleaning cream, skin cleaning water, eye and lip makeup remover, soap, bath lotion and the like. The cosmetic composition may be used in the form of a foam (foam) or an aerosol (aerosol) containing a compressed propellant.

Depending on the form and requirements of the product, any cosmetically or dermatologically acceptable vehicle that does not adversely affect the desired benefits of the active ingredient (i.e., at least one of the compounds of formulae (I) through (XIV) and the extract fermentate of emblica officinalis) may be included in the cosmetic composition provided according to the present invention. For example, but not limited thereto, when the cosmetic composition is an emulsion, cream or gel product, it can be formulated with wax, paraffin, starch, tragacanth, cellulose derivatives, animal oil, vegetable oil, mineral oil, polyethylene glycol, bentonite, silica, talc, zinc oxide, etc. as vehicle; when the cosmetic composition is a powder or aerosol product, components such as lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide, etc. can be used as vehicles; when the cosmetic composition is a solution or emulsion, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, polyethylene glycol, and the like can be used as a vehicle.

When the cosmetic composition is provided in the form of a cleaning product, it may further comprise any surfactant as a vehicle that does not adversely affect the desired benefits of the active ingredient (i.e., at least one of the compounds of formulae (I) through (XIV) and the extract fermentation product of emblica officinalis). Examples of such surfactants include, but are not limited to: fatty acid salts, Alkyl Benzene Sulfonates (ABS), Fatty Alcohol Sulfates (FAS), fatty alcohol polyoxyethylene sulfates (AES), fatty alcohol polyoxyethylene carboxylates (AEC), fatty acid Methyl Ester Sulfonates (MES), fatty alcohol polyoxyethylene ethers (AE), alkyl polyglycolates (AE), alkyl glycosides (APG), Betaine-type surfactants (Betaine-type surfactants), and combinations of the foregoing.

The pharmaceutical composition, food composition or care composition provided according to the present invention may further contain an additive in an appropriate amount, if necessary, such as a toner, a colorant or the like for improving the feeling of the composition in use, and a buffer, a preservative, an antiseptic, an antibacterial agent, an antifungal agent or the like for improving the stability and storability of the composition.

The pharmaceutical, food or cosmetic composition provided according to the present invention may optionally further comprise one or more other active ingredients (e.g., vitamin C, kojic acid, arbutin, tranexamic acid, glutathione, chlorogenic acid, dietary fiber, catechin, beta-polydextrose) to further enhance the efficacy of the composition or to increase the flexibility and formulation of the formulation, as long as the other active ingredients do not adversely affect the efficacy of the active ingredients of the present invention (i.e., at least one of the compounds of formulae (I) to (XIV) and the fermentation product of the extraction of emblic leafflower fruit).

The pharmaceutical, food or cosmetic composition provided according to the invention comprises at least about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 65, 60, 70, 80, or 85% by weight of the total weight of the active ingredient of the composition(s) selected from any one or two of the fermented compounds of formula I: about 0.0001 wt% to about 90 wt%, about 0.001 wt% to about 25 wt%, about 0.01 wt% to about 10 wt%, about 0.01 wt% to about 5 wt%, about 0.05 wt% to about 1 wt%, and about 0.05 wt% to about 0.5 wt%.

The pharmaceutical composition, food composition or care composition provided by the present invention can be administered at different frequencies such as once a day, several times a day, or once a few days, depending on the needs, age, weight, and health condition of the subject to be administered and the purpose of administration. The content of the fermentation product of the extract of Phyllanthus emblica and the compounds of formulae (I) to (XIV) in the pharmaceutical composition, the food composition or the cosmetic composition provided according to the present invention may also be adjusted according to practical requirements, for example: adjusting to the dosage which should be taken or externally applied every day. Generally, when used in the form of a food composition, it is recommended to take 0.8 g (or 8 ml) of the fermented product of emblic extract per day.

The invention will now be further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the invention is indicated in the claims.

Examples

[ preparation examples ]

In the following preparation examples, the materials and equipment used were as follows:

1. nuclear Magnetic Resonance spectroscopy (NMR), 1D and 2D spectra used an Ascend 400MHz (Bruker co., Germany) with chemical shift (chemical shift) expressed in δ.

2. Mass Spectrometer (Mass Spectrometer, MS) tandem Mass spectrometry-two-dimensional ion trap tandem fourier transform Mass spectrometry and ESI-MS/MS: measured using a Bruker amaZon SL system in m/z.

3. Medium Pressure Liquid Chromatography (MPLC) from Medium pressure chromatography

Rf+,Teledyne ISCO,Lincoln,NE。

4. High Performance Liquid Chromatography (High Performance Liquid Chromatography, HPLC; available from Agilent Germany): the high performance liquid chromatography is an Agilent 1200 series; the degasser is an Agilent vacuum degasser 1322A; the flush solvent delivery was an Agilent quaternary pump G1311A; the variable Wavelength Detector (MWD) is Agilent G1314B; the photodiode Array Detector (DAD) is Agilent 1260Infinity DAD VL G1315D, and has detection wavelengths of 210nm, 280nm, 320nm and 365 nm.

5. Analyzing the tubular column:

5μm C18(2)

(250X 10mm, available from Phenomenex, USA).

6. Column Chromatography (Column Chromatography) packing material:

Sephadex LH-20(Pharmacia,Piscataway,NJ,USA.)；

Diaion HP-20(Mitsubishi Chemical Co.,Japan)；

Merck Kieselgel 60(40-63μm,Art.9385)；

Merck

RP-18(40-63μm,Art.0250)。

7. thin Layer Chromatography (Thin-Layer Chromatography)

TLC aluminium sheets(Silica gel 60F₂₅₄0.25mm, available from Merck, Germany);

TLC aluminium sheets(RP-18F₂₅₄-S,0.25mm, available from Merck, Germany).

8. Ultraviolet Lamp (UV Lamp): UVP UVGL-25 with wavelength of 254nm and 365 nm.

9. Solvent (purchased from taiwan merck): n-hexane (n-hexane), ethyl acetate (ethyl acetate), acetone (acetone), methanol (methanol), ethanol (ethanol), acetonitrile (acetonitrile), chloroform-d₁(resolution hierarchy 99.5%), methanol-d₄(99.5%) and heavy water (deuterium oxide, deuterion hierarchy)>99.8%), dimethyl sulfoxide-d₆(Dimethyl sulfoxide-d₆，deuteration degree>99.9％)。

A. Preparation of emblic leafflower fruit extract fermentation product

A-1, taking fruit (cleaned) of fructus Phyllanthi (source: China), and mixing the fruit with the weight ratio of 1: 10 (emblic leafflower fruit: water) emblic leafflower fruit is mixed with water in a weight ratio to provide a mixture. The mixture was subjected to extraction at 95 ℃ for 1 hour to provide an extract of emblic leafflower fruit. Cooling the emblic extract to room temperature for the subsequent fermentation step.

A-2, adding Saccharomyces cerevisiae BCRC 20271 and Lactobacillus plantarum BCRC 910760 into the extract of fructus Phyllanthi provided in A-1, and fermenting at 25-35 deg.C for 72 hr to provide an intermediate fermented product. Wherein the addition amount of the saccharomyces cerevisiae is 0.1 wt% of the weight of the emblic leafflower fruit extract, and the addition amount of the lactobacillus plantarum is 0.05 wt% of the weight of the emblic leafflower fruit extract.

And A-3, taking the intermediate fermented product provided by the A-2, adding acetic acid bacteria BCRC 11688 into the intermediate fermented product, and fermenting for 96 hours at the temperature of between 25 and 35 ℃ to provide an emblic leafflower fruit extract fermented product. Wherein the addition amount of the acetic acid bacteria is 5 wt% of the intermediate fermentation product.

And A-4, taking the emblic leafflower fruit extract fermentation product provided by the A-3, and placing at 60 ℃ for reduced pressure concentration to provide a concentrated fermentation product. Subsequently, the concentrated fermented product was filtered through a 200-mesh screen to remove residual solids. Finally, 60% (weight/weight) isomalto-oligosaccharide was added to the fermented product subjected to the aforementioned treatment and sterilized at 100 ℃ for 70 minutes to provide a beverage comprising the emblic extract fermented product.

B. Preparation of the Compounds of formulae (I) to (XIV)

B-1, 10L of the fermentation product of the extraction of emblic leafflower fruit provided by A-3 is taken, ethyl acetate and n-butanol are taken as solvents, and then liquid-liquid phase partition extraction is carried out, so that 26.3 g of ethyl acetate soluble part (EAF, 12.4%), 53.8 g of n-butanol soluble part (BUF, 24.4%) and 131.3 g of water soluble part (WF, 63.2%) are obtained.

B-2, the ethyl acetate soluble fraction (EAF) provided in B-1 was subjected to Sephadex column chromatography (Sephadex LH-20column chromatography) using methanol as an eluent. Subsequently, analysis was performed by thin layer chromatography and eluates of similar results were combined to give 8 fractions (EAF1 to EAF 8). Taking EAF1 to EAF7, respectively carrying out the following purification and separation:

EAF1 was purified by reverse phase-HPLC (methanol with water as solvent, methanol: water ═ 1: 9) to give the compound of formula (III). By hydrogen-nuclear magnetic resonance spectroscopy (¹H-NMR) and electrospray ionization mass spectrometry (ESIMS) were performed to confirm that the compound of formula (III) was glucoroniside (glucoallin).

EAF2 was purified by reverse-HPLC (methanol with water as solvent, methanol: water 1: 9) to give compound of formula (VII), which was purified by reverse-HPLC¹After H-NMR and ESIMS analysis of the chemical structure, it was confirmed that the compound of the formula (VII) was Gallic acid (Gallic acid).

EAF3 was purified by reverse-HPLC (methanol and water as solvents, A)Alcohol: water 1: 4) to obtain the compound of formula (IV) and the compound of formula (VI)¹After H-NMR and ESIMS analysis of the chemical structure, it was confirmed that the compound of formula (IV) was Methyl gallate (Methyl gallate) and the compound of formula (VI) was Tyrosol (Tyrosol).

EAF4 was purified by reverse-HPLC (methanol with water as solvent, methanol: water ═ 1: 3) to give compound of formula (I) and compound of formula (XIII) by¹After analyzing the chemical structure by H-NMR and ESIMS, the compound shown in the formula (I) is determined to be myrobalan tannic acid (Chebulagic acid); the compound of formula (XIII) is 1, 6-digallacyl glucose (1,6-Di-O-galloyl glucose).

EAF5 was purified by reverse-HPLC (methanol with water as solvent, methanol: water ═ 3: 7) to give compound of formula (II), compound of formula (XII), and compound of formula (XIV), by¹After analyzing the chemical structure by H-NMR and ESIMS, the compound of formula (II) was identified as Corilagin (Corilagin), the compound of formula (XII) was identified as 1, 4-bis-oxo-galloyl-3, 6-hexahydrobenzodioxoglucose (1,4-Di-O-galloyl-3, 6-hexahydrodihydroxydiphenylglucose), and the compound of formula (XIV) was identified as 1, 2-bis-oxo-galloyl-3, 6-hexahydrodihydroxydiphenylglucose (1,2-Di-O-galloyl-3, 6-hexahydrodihydroxydiphenylglucose).

EAF6 was purified by reverse-HPLC (methanol with water as solvent, methanol: water ═ 2: 3) to give compound of formula (IX) and compound of formula (X) by¹After analyzing the chemical structure by H-NMR and ESIMS, the compound shown in the formula (IX) is identified to be Quercetin-7-rhamnoside (Quercetin-7-O-rhamnoside); the compound of formula (X) is Quercitrin (Quercitrin).

EAF7 was purified by reverse-HPLC (methanol: water 1: 1 with methanol and water as solvents) to give compound of formula (V), compound of formula (VIII), and compound of formula (XI) by reverse-HPLC¹After analyzing the chemical structure by H-NMR and ESIMS, it was confirmed that the compound of formula (V) was Ellagic acid (Ellagic acid), the compound of formula (VIII) was Quercetin (Quercetin), and the compound of formula (XI) was 3,4,8,9,10-Pentahydroxy-dibenzopyran-6-one (3,4,8,9, 10-Pentahydroxy-dibenzopyran-6-one).

The chemical structural formulas of the compounds of formulae (I) to (XIV) are shown in the following Table 1, and their NMR spectra are shown in FIGS. 1A to 1N, respectively:

TABLE 1

Example 1: effect test of Phyllanthus emblica extract fermented product on inhibiting adipogenesis ability of adipocytes

In this embodiment, the materials and equipment used are as follows:

1. cell lines: OP9 (from

CRL-2749^TM)。

2. Preadipocyte Expansion Medium (Pre-adipocyte Expansion Medium):

90% MEM-Alpha Medium (Minimum Essential Medium Alpha Medium, purchased from Gibco);

20% Fetal Bovine Serum (Fetal Bovine Serum, from Gibco);

1% Penicillin-streptomycin (Penicilin-streptomycin, from Gibco).

3. Adipocyte Differentiation Medium (Differentiation Medium):

20% Fetal Bovine Serum (Fetal Bovine Serum, from Gibco);

1% Penicillin-streptomycin (Penicilin-streptomycin, from Gibco).

4. Phosphate Buffered Saline (PBS): purchased from Gibco

5. Oil red O stain (Oil-red O staining reagent, purchased from Sigma): storing the solution: 3 mg/ml in 100% isopropanol; working solution (Working solution): 60% stock solution in ddH₂And preparing O.

6, ELISA disk reader: purchased from BioTek.

7.10% formaldehyde: purchased from ECHO.

8.100% isopropyl alcohol: purchased from ECHO.

DPBS (Dulbecco's phosphate buffered saline): purchased from Gibco.

10. Microscope: available from ZEISS.

OP9 cells (8X 10)⁴Individual cells/well) were seeded in a 24-well culture plate, each well containing 500 microliters of preadipocyte expansion medium. The aforementioned culture dish was cultured at 37 ℃ for 7 days, and the medium was renewed every 3 days during the culture, wherein the medium used for renewal was adipocyte differentiation medium. After 7 days, the cells were observed under a microscope to confirm the formation of intracellular oil droplets (indicating that OP9 cells had differentiated into adipocytes).

The adipocytes were placed in 24-well plates, and after dividing into three groups (6 wells each), they were cultured in the following media, respectively, at 37 ℃ for 7 days, during which the media was renewed every 3 days:

1. control group: adipocyte differentiation medium (total 500 microliters).

2. Extract group: an adipocyte differentiation medium (total 500. mu.L) containing 2.5 mg/ml of the extract of Emblica officinalis provided in preparation example A-1; and

3. and (3) fermentation product group: adipocyte differentiation medium (total 500. mu.l) containing 2.5 mg/ml of the fermentation product of the extract of Phyllanthus emblica as provided in preparation example A-3 ".

Next, the cells provided in each of the above groups were subjected to the following treatments: the medium was removed and the cells in each well were rinsed twice with 1 ml PBS, followed by fixing the cells in each well with 1 ml 10% formaldehyde for 30 minutes at room temperature. After removing formaldehyde, each well cell was rinsed twice with 1 ml PBS. To each well, 1 ml of 60% isopropyl alcohol was added and allowed to stand for 1 minute. Thereafter, the isopropanol was removed and 1 ml of oil red O stain was added to each well for staining for 1 hour. After removing the oil red O dye, 1 ml of 60% isopropanol was added to each well to destage for 5 seconds. Finally, the oil droplet condition of the fat cells was observed under a microscope and recorded by photographing, and the result is shown in fig. 2.

After the above microscopic observation and photographing were completed, 100% isopropyl alcohol was added to each well of the 24-well culture plate, and the 24-well culture plate was placed in a shaker to be cultured for 10 minutes, so that the intracellular stain was dissolved out. Then, 100. mu.l of the isopropanol solution containing the dye was transferred to a 96-well plate, and the absorbance at a wavelength of 510 nm was measured by an ELISA plate reader to quantify the amount of the dissolved dye, thereby detecting the fat content of each cell group. Finally, relative fat contents of the other groups were calculated based on the results of the "control group" (i.e., the fat content of the control group was set to 100%). The data were statistically analyzed using Excel software to test whether statistical significance was achieved with student t (student's t-test). The results are shown in FIG. 3.

As can be seen from fig. 2, the fat cells of the "fermented product group" were significantly reduced in the area stained with oil red O stain (representing significant reduction of oil droplets) compared to the "control group" or "extract group". In addition, as can be seen from fig. 3, the fat content of the adipocytes of the "extract group" was reduced by about 40%, and the fat content of the adipocytes of the "fermented product group" was reduced by about 55%, as compared to the "control group". The results show that the emblic leafflower fruit extract fermentation product can effectively reduce the fat content in fat cells, and achieves the purposes of inhibiting the adipogenesis capability of the fat cells, making individuals difficult to form body fat and promoting weight loss.

Example 2: experiment for improving activity of mitochondria by extracting fermentation product from phyllanthus emblica

Mitochondria are the main site of energy generation in cells and are involved in cell information transfer and cell activity information, and the higher the activity of mitochondria is, the more the physiological activity of cells as a whole is promoted. It is known that mitochondrial activity can be reflected by the measurement of Mitochondrial Membrane Potential (MMP). To understand the effect of the fermentation product of Phyllanthus emblica of the present invention on increasing the activity of mitochondria, the present embodiment utilizes JC-1 mitochondrial dye to detect the mitochondrial membrane potential, wherein when the mitochondrial membrane potential increases, the JC-1 mitochondrial dye changes from a monomer (monomer) originally having a green fluorescence signal to a polymer (dimer) having a red fluorescence signal. The ratio of monomers to polymers in the cell is detected by a flow cytometer, and the mitochondrial activity of the cell is known.

In this embodiment, the materials and equipment used are as follows:

1. cell lines: skeletal muscle cell C2C12 (purchased from

CRL-1772^TM)。

2. Skeletal muscle cell culture medium: DMEM medium (Dulbecco's modified Eagle's medium) containing 1% penicillin-streptomycin (purchased from Gibco) and 10% fetal bovine serum (purchased from Gibco).

PBS: purchased from Gibco.

4. Working solution: on one hand, a 10x detection solution (assay buffer) is placed at 37 ℃ for preheating, and is diluted into a 1x detection solution by sterilized 1x PBS, and is stored at 37 ℃ after being uniformly stirred; in another aspect, 130. mu.L DMSO was added to lyophilized JC-1 mitochondrial dye to provide a stock of JC-1 mitochondrial dye (which stock can be stored at-20 ℃ for 6 months); the JC-1 mitochondrial dye stock provided above was mixed with the 1 × detector (JC-1 mitochondrial dye stock: 1 × detector: 1: 100) to provide a working solution.

5. Flow cytometry mitochondrial membrane potential detection kit (Flow cytometry mitochrondral membrane potential detection kit): purchased from BD.

6. Flow cytometry: purchased from Beckman.

C2C12 cells (1X 10)⁵Individual cells/well) were inoculated in a 6-well culture plate, and after dividing into three groups (2 wells each), the culture was performed in the following medium (37 ℃ for 24 hours):

1. control group: skeletal muscle cell culture medium (total 2 ml).

2. Extract group: skeletal muscle cell culture medium (total 2 ml) containing 0.156 mg/ml of the extract of emblica officinalis provided in "preparation example a-1"; and

3. and (3) fermentation product group: skeletal muscle cell culture medium (2 ml total) containing 0.156 mg/ml of the emblic extract fermentation provided in preparation example a-3 ".

Next, the cells provided in each of the above groups were subjected to the following treatments: after removing the medium and rinsing each well cell twice with 1xPBS, each well cell was treated with trypsin to collect each well cell in a 1.5 ml microcentrifuge tube, respectively. After centrifugation at 400Xg for 5 minutes, the supernatant was removed and 100. mu.l of the working solution was added to each well, followed by vortex mixing and standing in the dark for 15 minutes. Next, the mixture was centrifuged at 400Xg for 5 minutes. After rinsing each tube with 1 ml of 1 × rinse (wash buffer), it was centrifuged at 400 × g for 5 min (twice more). Finally, each tube cell was resuspended in 500 microliters of 1xPBS containing 2% FBS and analyzed by flow cytometry. Statistical analysis of the data was performed using Excel software and student's t (student's t-test) was used to test whether statistical significance was achieved. The results are shown in FIG. 4.

As can be seen from FIG. 4, the JC-1 aggregate proportion of skeletal muscle cells was significantly higher in the "fermented product group" than in the "control group" or "extract group". The results show that the phyllanthus emblica extract fermentation product can effectively improve the activity of mitochondria and help metabolism.

Example 3: test of the Effect of Phyllanthus emblica extract fermentation product on inhibiting melanogenesis

Ultraviolet rays (320nm to 400nm) in sunlight can directly pass through the epidermal layer to reach melanocytes in the basal layer of the epidermis, causing an increase in melanin, which blackens the skin. To understand the effect of the fermentation product of the present invention on inhibiting melanin production, the following experiment was performed.

In this embodiment, the materials and equipment used are as follows:

1. cell lines: melanoma cells B16F10 (CRL: 6475, available from ATCC).

2. Melanoma cell culture medium: DMEM medium (Dulbecco's modified Eagle's medium) containing 1% penicillin-streptomycin (purchased from Gibco) and 10% fetal bovine serum (purchased from Gibco).

PBS: purchased from Gibco.

4.1N NaOH: by ddH₂And (4) preparing.

ELISA disk reader: purchased from BioTek.

6. Ultraviolet ray chamber (Ultraviolet radiation chamber): purchased from Vilber.

B16F10 cells (1.5X 10)⁵One cell/well) were inoculated in a 6-well culture plate and cultured at 37 ℃ for 24 hours. Next, the cells were divided into five groups, and each group was cultured in a medium (37 ℃ C. for 48 hours) in which the control group cells were not irradiated with UVA during the culture and four groups of cells other than the control group cells were placed in an ultraviolet chamber at 10J/cm during the culture²UVA irradiation of intensity):

1. control group: melanoma cell culture medium (total 3 ml).

UVA group: melanoma cell culture medium (total 3 ml).

3. Positive control group: medium containing 250 μ g/ml kojic acid (total 3 ml);

4. extract group: melanoma cell culture medium (total 3 ml) containing 2.5 mg/ml "emblica extract provided in preparative example a-1"; and

5. and (3) fermentation product group: melanoma cell culture medium (total 3 ml) containing 2.5 mg/ml of the emblica extract fermentation provided in preparation example a-3 ".

Thereafter, the cells provided in each of the above groups were subjected to the following treatments: the medium was removed and the cells were rinsed twice with 1 xPBS. Cells were trypsinized for 3 minutes and each set of cells was collected in a 15 ml centrifuge tube and centrifuged at 400Xg for 5 minutes to pellet the cells. Subsequently, the cells were rinsed twice with 1xPBS, and the cell pellet was resuspended in 200. mu.l of 1 xPBS. After the cell solution having completed the aforementioned treatment was stored in liquid nitrogen for 10 minutes, it was left at room temperature for 30 minutes to thaw the cell solution.

After the cell solution was completely thawed, it was centrifuged at 12,000Xg for 30 minutes. After removal of the supernatant, 120. mu.l of 1N NaOH were added to each group. After mixing well, the mixture was placed in a dry bath at 60 ℃ for 1 hour. 100 μ l each of the cell solutions from each group was placed in a 96-well plate and read for OD at 450 nm. Finally, the relative melanin content of the remaining groups was calculated with the control group as a reference (i.e., the black pigment content of the control group was set to 100%). Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved. The results are shown in FIG. 5.

As can be seen from fig. 5, the melanin content in the "UVA group" cells was significantly higher than that in the "control group", indicating that UVA indeed induced melanin production. On the other hand, the melanin content in the cells of the positive control group, the extract group and the fermentation product group is lower than that of the UVA group, wherein the effect of the fermentation product group is better than that of the positive control group and even better than that of the extract group. The results show that the emblic leafflower fruit extract fermentation product can effectively inhibit melanin generation and can be used for whitening.

Example 4: experiment on effect of extracting fermentation product from emblic leafflower fruit for improving SOD2, MPG and ERCC1 gene expression

In this embodiment, the materials and equipment used are as follows:

RNA Extraction Kit (RNA Extraction Kit): purchased from Geneaid.

2. Reverse transcriptase (A)

III Reverse Transcriptase): purchased from Invitrogen.

3. A primer: GAPDH was used as an internal control as shown in the following table.

KAPA CYBR FAST qPCR Kits (2 ×): purchased from KAPA Biosystems.

Step One Plus: purchased from ABI.

Human dermal fibroblasts were cultured in MEM medium for 24 hours, and then, the cells were divided into three groups and subjected to the following treatments:

1. control group: culturing the cells in MEM medium for 24 hours, and then irradiating the cells with UVA (5J/cm) for 1 hour;

2. extract group: culturing the cells in MEM medium (2 ml in total) containing 2.5 mg/ml of the extract of Emblica officinalis provided in preparation example A-1 for 24 hours, and irradiating the cells with UVA (5J/cm) for 1 hour;

3. and (3) fermentation product group: the cells were cultured in MEM medium (2 ml in total) containing 2.5 mg/ml of the extract fermentation product of Emblica officinalis as provided in preparation example A-3 "for 24 hours, and then irradiated with UVA (5J/cm) for 1 hour.

Next, the cells provided in each of the above groups were subjected to the following treatments: RNA extraction was performed using an RNA extraction kit, and the RNA was reverse-transcribed into cDNA using reverse transcriptase. Subsequently, qPCR (quantitative polymerase chain reaction) was performed on the cDNA provided above using an ABI Step One Plus instrument and KAPA SYBR FAST qPCR kit to detect gene expression levels of SOD2, MPG and ERCC1 in each group of cells. Finally, the relative expression amounts of the genes of the remaining groups were calculated with the control group as a reference (i.e., the gene expression of the control group was set to 1-fold). Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved. The results are shown in FIGS. 6 and 7.

As can be seen from fig. 6 to 7, the expression levels of SOD2, MPG and ERCC1 genes in the cells of the "extract group" and the "fermented product group" were higher than those of the "control group", wherein the expression levels of SOD2, MPG and ERCC1 genes in the cells of the "fermented product group" were higher than those of the "extract group". The above results show that the fermentation product of the present invention has the effects of improving the oxidation resistance of cells and improving the DNA repair ability of cells, and can be used for resisting skin aging (for example, resisting skin photoaging).

Example 5: antioxidant effect test of phyllanthus emblica extract fermentation product

It is known that when the content of Reactive Oxygen Species (ROS) in skin cells is too high, cell tissue is damaged and DNA is damaged, resulting in skin aging. To understand the effect of the fermentation product of emblic leafflower fruit extract of the present invention in inhibiting the production of ROS in skin cells, the following experiment was performed.

In this embodiment, the materials and equipment used are as follows:

1. cell lines: human dermal fibroblasts (Human skin fibroblast, CCD-966sk, BCRC No. 60153).

2. Culture medium: MEM medium (minimal essential medium (Eagle) in Earle's BSS) in 90% Earle's balanced salt solution containing 0.1mM non-essential amino acids (non-essential amino acids), 1.5 g/l sodium bicarbonate and 1mM sodium pyruvate, and 10% fetal bovine serum; purchased from Gibco.

PBS: purchased from Gibco.

DCFH-DA: purchased from Sigma, model SI-D6883-50MG, stock solution: 5 mg/ml, dissolved in DMSO.

5.H₂O₂: purchased from Sigma.

6. Flow cytometry: purchased from BD Accuri.

Human skin fibroblasts (2X 10)⁵One cell/well) were seeded in 6-well culture plates and cultured at 37 ℃ for 24 hours. Next, the medium was removed and the cells were divided into three groups (2 wells each) and cultured in the following medium for 1 hour:

1. control group: containing 1mM H₂O₂The MEM medium of (4).

2. Extract group: containing 1mM H₂O₂And 5 mg/ml "MEM medium of the extract of Phyllanthus emblica (total 2 ml) provided in preparation example A-1".

3. And (3) fermentation product group: containing 1mM H₂O₂And 5 mg/ml "MEM medium (2 ml in total) of the fermentation product of the extract of Phyllanthus emblica as provided in preparation example A-3".

Thereafter, the cells provided in each of the above groups were subjected to the following treatments: after treatment with 5. mu.g/ml DCFH-DA stain at 37 ℃ for 15 minutes, it was treated with H again at 37 ℃₂O₂The treatment was carried out for 1 hour. Rinse with 1 ml of 1xPBSCells were run twice, then 200. mu.l of trypsin was added to each well cell and reacted in the dark for 5 minutes. The groups of cells and medium were collected in 1.5 ml centrifuge tubes and centrifuged at 400Xg for 10 minutes. After removing the supernatant, the cells were rinsed once with 1 xPBS. Then, after centrifugation at 400Xg for 10 minutes and removal of the supernatant, the cell pellet of each group was resuspended in 1 ml of 1 xPBS. Finally, the fluorescence intensities of the excitation light wavelength of 450-490nm and the emission light wavelength of 510-550nm are detected by a flow cytometer. Wherein, the measured fluorescence intensity can represent the ROS content in the cells because ROS can convert DCFH-DA (without fluorescence) into DCF (with fluorescence), and the higher the fluorescence intensity is, the higher the ROS content in the cells is. Finally, the ROS content in the other groups of cells was calculated based on the "control" results (i.e., the ROS content of the control was set to 1-fold). The data were statistically analyzed using Excel software to test whether statistical significance was achieved with student t (student's t-test). The results are shown in FIG. 8.

As can be seen from fig. 8, the relative ROS content of both the "extract group" and the "fermented product group" was less than 1-fold, and the "fermented product group" was lower than the "extract group". The results show that the emblic leafflower fruit extract fermentation product has excellent effect of inhibiting the generation of ROS in skin cells and can effectively resist skin aging.

Example 6: test of Effect of fermented product extracted from Emblica officinalis on inhibiting AGEs production

Glycosylation (glycosylation) refers to the chemical reaction of glucose attached to proteins, which results in the production of the final glycosylated end product (AGEs). The final saccharide end product accumulated in skin cells not only easily causes protein denaturation, leading to skin wrinkle generation, skin relaxation and other aging phenomena, but also causes generation of active oxides, causes oxidation pressure, causes skin cell DNA damage, influences skin cell DNA normal functions, and even induces skin diseases. In order to confirm the effect of the fermentation product of the emblic leafflower fruit extract of the present invention in inhibiting the production of AGEs, the following experiment was performed.

In this embodiment, the materials and equipment used are as follows:

1. disodium hydrogen phosphate (Na)₂HPO₄)

2. Sodium dihydrogen phosphate (NaH)₂PO₄)

3. Bovine serum albumin (Bovine serum albumin, BSA)

D- (-) -Fructose (D- (-) -Fructose, C₆H₁₂O₆)

5. Sodium azide (Sodium azide, NaN)₃)

6.200mM phosphate buffer (Sodium phosphate buffer, pH 7.4)

7. Containing 0.06% NaN in 200mM phosphate buffer ₃60 mg/ml Bovine Serum Albumin (BSA)

8. 1.5M D-fructose in 200mM phosphate buffer

Experimental groups: 0.25 ml of the fermentation product of the extract of Emblica officinalis provided in "preparation example A-3" was uniformly mixed with 0.25 ml of Bovine Serum Albumin (BSA) solution and 0.25 ml of fructose solution to provide a mixed solution. 0.1 ml of the mixed solution is taken, and the fluorescence intensity at the excitation wavelength of 360nm and the emission wavelength of 460nm is measured (this is referred to as "fluorescence value at 0 hour in experimental group"). Then, the mixed solution is placed at 50 ℃ to react for 24 hours, so that the protein in the solution is subjected to glycosylation reaction, and a glycosylation reaction solution is provided. 0.1 ml of the above-mentioned glycosylation reaction solution was taken, and the fluorescence intensity at an excitation wavelength of 360nm and an emission wavelength of 460nm was measured (this is referred to as "fluorescence value for 24 hours in the experimental group").

Control group: 0.25 ml of water was uniformly mixed with 0.25 ml of Bovine Serum Albumin (BSA) solution and 0.25 ml of fructose solution to provide a mixed solution. 0.1 ml of the mixed solution was taken, and the fluorescence intensity at an excitation wavelength of 360nm and an emission wavelength of 460nm was measured (this is referred to as "fluorescence value at 0 hour in control group"). Then, the mixed solution is placed at 50 ℃ to react for 24 hours, so that the protein in the solution is subjected to glycosylation reaction, and a glycosylation reaction solution is provided. 0.1 ml of the above-mentioned glycosylation reaction solution was taken, and the fluorescence intensity at the excitation wavelength of 360nm and the emission wavelength of 460nm (referred to as "fluorescence value for control group for 24 hours") was detected.

Finally, the amount (%) of AGEs produced in the control group and the experimental group was calculated as follows (wherein, lower the amount of AGEs produced indicates higher the anti-glycosylation activity). Finally, based on the results of the "control group" (i.e., setting the AGEs production amount of the control group to 100%), the AGEs production amount of the experimental group was calculated, and the results are shown in fig. 9.

As can be seen from fig. 9, the experimental group has lower AGEs production than the control group, which indicates that the fermentation product of emblic leafflower fruit extract of the present invention can effectively resist glycosylation (inhibit protein glycosylation reaction), and can achieve the effect of anti-skin aging.

Example 7: effect test of Each fraction of fermented product extracted from Emblica officinalis on inhibiting adipogenesis ability of adipocytes

In this embodiment, the materials and equipment used are as follows:

1. cell lines: OP9 (from

CRL-2749^TM)。

2. Preadipocyte Expansion Medium (Pre-adipocyte Expansion Medium):

20% Fetal Bovine Serum (Fetal Bovine Serum, from Gibco);

1% Penicillin-streptomycin (Penicilin-streptomycin, from Gibco).

3. Adipocyte Differentiation Medium (Differentiation Medium):

20% Fetal Bovine Serum (Fetal Bovine Serum, from Gibco);

1% Penicillin-streptomycin (Penicilin-streptomycin, from Gibco).

4. Cell Glycerol-based assay kit (Glycerol cell-based assay kit): purchased from Cayman.

ELISA disk reader: purchased from BioTek.

OP9 cells (8X 10)⁴Individual cells/well) were seeded in 24-well culture plates, each well containing 500 microliters of preadipocyte expansion medium. The culture dish is placed at 37 ℃ for 7 days, and the culture medium is renewed every 3 days during the culture period, wherein the culture medium for renewal is an adipocyte differentiation medium. After 7 days, the cells were observed under a microscope to confirm the formation of intracellular oil droplets (indicating that OP9 cells had differentiated into adipocytes).

The adipocytes were placed in 24-well culture plates, which were divided into four groups, and then cultured in the following media (37 ℃ C., for 7 days, during which the media was renewed every 3 days):

1. "control group": adipocyte differentiation medium (total 500 microliters).

"EAF group": adipocyte differentiation medium (total 500. mu.l) containing 62.5. mu.g/ml of ethyl acetate soluble fraction as provided in preparation example B-1 ".

"BUF group": adipocyte differentiation medium (total 500. mu.l) containing 62.5. mu.g/ml of the n-butanol-soluble fraction provided in preparation example B-1 ".

"WF group": adipocyte differentiation medium (total 500. mu.l) containing 62.5. mu.g/ml of the water-soluble fraction provided in preparation example B-1 ".

In order to understand the effect of each fraction (including ethyl acetate-soluble fraction, n-butanol-soluble fraction, and water-soluble fraction) of the fermented product of emblic leafflower fruit of the present invention on the ability to inhibit adipogenesis by adipocytes, the cells provided in each of the above groups were subjected to the following treatments: the medium was removed and the cells in each well were rinsed twice with 1 ml PBS, followed by fixing the cells in each well with 1 ml 10% formaldehyde for 30 minutes at room temperature. After removing formaldehyde, each well cell was rinsed twice with 1 ml PBS. To each well, 1 ml of 60% isopropyl alcohol was added and allowed to stand for 1 minute. Thereafter, the isopropanol was removed and 1 ml of oil red O stain was added to each well for staining for 1 hour. After removing the oil red O stain, 100% isopropanol was added to each well of the 24-well culture plate, and the 24-well culture plate was placed in a shaker for 10 minutes to dissolve out the intracellular stain. Then, 100. mu.l of an isopropanol solution containing a dye was transferred to a 96-well plate, and the absorbance at a wavelength of 510 nm was measured by an ELISA plate reader to quantify the amount of the dissolved dye, thereby detecting the fat content of each group of cells. Finally, relative fat content of the other groups was calculated based on the results of the "control group". Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved. The results are shown in FIG. 10.

As can be seen from fig. 10, the fat content of the cells of only the "EAF group" was significantly reduced (by 22.9%) compared to the "control group". The above results show that the effect of suppressing adipogenesis of the fermentation product of the emblic leafflower fruit extract of the present invention is mainly derived from the ethyl acetate soluble fraction (EAF).

Example 8: effect test of Each fraction of Phyllanthus emblica extract fermented product for promoting fat degradation ability of adipocytes

To understand the effect of each fraction (including ethyl acetate-soluble fraction, n-butanol-soluble fraction, and water-soluble fraction) of the fermented product of emblic leafflower fruit of the present invention in promoting fat degradation of adipocytes, after completion of culturing of each group of cells in example 7, each group of supernatants was subjected to the following treatments: from each well, 25 microliters of cell culture supernatant was taken and placed in a new 96-well plate. After adding 100. mu.l of a recombinant Free Glycerol Assay Reagent (recombinant Free Glycerol Assay Reagent) to each well, the wells were incubated at room temperature for 15 minutes. Then, absorbance at 540 nm was measured using an ELISA reader. Finally, relative lipolysis amounts of other groups were calculated based on the results of the "control group", and the results are shown in FIG. 11. Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved.

As can be seen from fig. 11, the lipolysis levels of the cells of the "EAF group" and the cells of the "BUF group" were significantly increased (4.0% and 5.1% respectively) compared to the control group. The above results show that the fat degradation promoting effect of the fermentation product of the emblic leafflower fruit extract of the present invention is mainly from an ethyl acetate soluble fraction (EAF) and an n-butanol soluble fraction (BUF).

Example 9: effect test of increasing mitochondrial activity of various levels of phyllanthus emblica extract fermentation

To improve the activity of mitochondria by the fermentation product of the emblic leafflower fruit of the present invention, the present example is conducted with reference to the method steps, materials and equipment of example 2, wherein only the grouping of cells and the treatment of cells are different from example 2. In this example, C2C12 cells were divided into four groups and cultured at 37 ℃ for 24 hours in the following culture medium, respectively, to examine and calculate the effect of each fraction (including ethyl acetate-soluble fraction, n-butanol-soluble fraction, and water-soluble fraction) of fermented Phyllanthus emblica on the enhancement of mitochondrial activity:

1. "control group": skeletal muscle cell culture medium (total 2 ml).

"EAF group": skeletal muscle cell culture medium (2 ml total) containing 31.25. mu.g/ml of the ethyl acetate-soluble fraction provided in preparation example B-1.

"BUF group": skeletal muscle cell culture medium (2 ml total) containing 31.25. mu.g/ml of the n-butanol soluble fraction provided in preparation example B-1.

"WF group": skeletal muscle cell culture medium (2 ml total) containing 31.25. mu.g/ml of the water-soluble fraction provided in preparation example B-1.

Relative mitochondrial activity was calculated for each of the other groups based on the results of the "control group". Statistical analysis of the data was performed using Excel software to test whether statistical significance was achieved by student t (student's t-test). The results are shown in FIG. 12.

As can be seen from fig. 12, compared to the control group, the mitochondrial activities of the "BUF group" and the "WF group" were lower, and the mitochondrial activity of the cells of the "EAF group" was increased by 14.9%, indicating that the effect of the crassula argentea extract fermentation product of the present invention on increasing the mitochondrial activity is mainly from the ethyl acetate soluble fraction.

The results of the above examples 7 to 9 show that the ethyl acetate soluble part of the fermentation product of the emblic leafflower fruit extract of the present invention has the effects of inhibiting lipogenesis, promoting lipolysis, promoting mitochondrial activity, etc., so the following examples will test the effects of the compounds of formulae (I) to (XIV) isolated from the ethyl acetate soluble part.

Example 10: effect test of the Compound of the present invention on inhibition of adipogenic Activity of adipocytes

To understand the effect of the compounds of the present invention in inhibiting adipogenesis, this example was conducted with reference to the method steps, materials and equipment of example 1, wherein only the grouping and treatment of adipocytes were different from example 1. The adipocytes differentiated from OP9 cells were divided into fifteen groups and cultured in the following media (7 days at 37 ℃ C. with the media being refreshed every 3 days) respectively to examine and calculate the effect of each compound on the adipogenic ability of the adipocytes:

1. control group: adipocyte differentiation medium (total 500 microliters).

2. Experimental groups (fourteen groups in total): adipocyte differentiation medium (total 500. mu.l) containing 20. mu.g/ml of the compounds of formulae (I) to (XIV) provided in preparation example B-2 ", respectively.

The fat cells provided in each group were stained with oil red O, and the oil drop condition of the fat cells was observed under a microscope and recorded by photographing, and the results are shown in fig. 13. In addition, relative fat contents of the other groups were calculated based on the results of the "control group", and the results are shown in fig. 14. Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved.

As can be seen from fig. 13 and 14, the areas stained by the oil red stain of the cells treated with the compounds of the present invention of formula (I), formula (II), formula (VI), and formulae (X) to (XIV) were significantly reduced, and the fat content was significantly reduced (by 55.8%, 36.3%, 16.2%, 12.0%, 25.7%, 27.9%, 22.4%, and 23.5%, respectively), as compared to the control group. The foregoing results show that the compounds of formula (I), formula (II), formula (VI) and formulae (X) to (XIV) of the present invention are effective in reducing fat content of adipocytes, and thus are useful in inhibiting adipogenic ability of adipocytes, making it difficult for individuals to form body fat, and promoting weight loss.

Example 11: effect test of the Compound of the present invention on promoting fat degradation ability of adipocytes

To understand the effect of the compounds of the present invention in promoting the fat degradation of adipocytes, after the completion of the culture of each group of cells in example 10, the following treatments were performed on each group of supernatants: from each well, 25. mu.l of cell culture supernatant was taken and placed in a new 96-well plate. After adding 100. mu.l of a recombinant Free Glycerol Assay Reagent (recombinant Free Glycerol Assay Reagent) to each well, the wells were incubated at room temperature for 15 minutes. Then, the absorbance at 540 nm was measured using an ELISA reader. Finally, relative lipolysis amounts of other groups were calculated based on the results of the "control group", and the results are shown in FIG. 15. Statistical analysis of the data was performed using Excel software, and student's t (student's t-test) was used to test whether statistical significance was achieved.

As can be seen from fig. 15, the lipolysis of adipocytes treated with the compounds of formula (II), (III) and (VII) of the present invention was increased by 14.2%, 20.4% and 34.5% compared to the "control group". The above results show that the compounds of formula (II), (III) and (VII) of the present invention are effective in promoting fat decomposition by adipocytes, and thus can be used to promote the fat degradation ability of adipocytes, make it difficult for individuals to form body fat, and promote weight loss.

Example 12: effect test of the Compound of the present invention for improving the Activity of mitochondria

To understand the effect of the compounds of the present invention in promoting mitochondrial activity, this example refers to the method steps, materials and equipment of example 2, wherein only the grouping of cells and the treatment of cells are different from example 2. In this example, C2C12 cells were divided into fifteen groups and cultured at 37 ℃ for 24 hours in the following culture medium, respectively, to examine and calculate the effect of each compound on the enhancement of mitochondrial activity:

1. "control group": skeletal muscle cell culture medium (total 2 ml).

"experimental groups" (fourteen groups in total): skeletal muscle cell culture media (total 2 ml) containing 10. mu.g/ml of each of the compounds of formulae (I) to (XIV) provided in preparation example B-2 ".

Relative mitochondrial activity was calculated for each of the other groups based on the results of the "control group". Statistical analysis of the data was performed using Excel software to test whether statistical significance was achieved by student t (student's t-test). The results are shown in FIG. 16.

As can be seen from fig. 16, the mitochondrial activities of skeletal muscle cells treated with the compounds of formulae (I), (IV), and (VI) to (XIV) of the present invention were increased by 27.1%, 40.5%, 53.3%, 45.1%, 76.8%, 27.2%, 44.1%, 27.2%, 57.3%, 45.0%, and 41.8%, respectively, as compared to the "control group". The above results show that the compounds of formula (I), formula (IV), and formulae (VI) to (XIV) of the present invention are effective in increasing mitochondrial activity and aiding metabolism.

Example 13: test of Effect of the Compound of the present invention on inhibiting melanogenesis

To understand the effect of the compounds of the present invention in inhibiting melanin production, this example refers to the method steps, materials and equipment of example 3, wherein only the grouping of cells and the treatment of cells are different from example 3. In this example, sixteen groups of cells were separated and cultured in the following medium (37 ℃ for 48 hours), wherein the control group cells were not irradiated with UVA during the culture, and fifteen groups of cells other than the control group cells were cultured in the UV chamber at 10J/cm during the culture²UVA irradiation of intensity):

1. "control group": melanoma cell culture medium (total 3 ml).

"UVA group": melanoma cell culture medium (total 3 ml).

"experimental group" (fourteen groups in total): melanoma cell cultures (total 3 ml) each containing 10. mu.g/ml of a compound of formulae (I) to (XIV) as provided in preparation example B-2 ".

Relative melanin content was calculated for each of the other groups based on the results of the "control group". Statistical analysis of the data was performed using Excel software to test whether statistical significance was achieved by student t (student's t-test). The results are shown in FIG. 17.

As can be seen in fig. 17, the melanin content in the "UVA group" cells was significantly higher than in the "control group", indicating that UVA indeed induced melanin production. On the other hand, the melanin contents of the cells treated by the compounds of formulae (II) to (XIV) of the present invention are lower than those of "UVA group", which shows that the compounds of formulae (II) to (XIV) of the present invention are effective in inhibiting melanin production and can be used for whitening.

Example 14: the content of the compound of the present invention varies before and after fermentation of the extract of Phyllanthus emblica

The extract of emblic leafflower fruit provided in "preparation example a-1" and the extract fermented product of emblic leafflower fruit provided in "preparation example a-3" were prepared into sample solutions having a concentration of 10 mg/ml, respectively. Subsequently, 10. mu.l of each sample solution was taken and analyzed for its composition by HPLC. Wherein, the analytical column used in HPLC is Mightysil RP-18GP 250(250x 10mm, 5 μm), the detection wavelength is 250 nm, the elution is performed at a flow rate of 1.0 ml/min, and the composition of the elution is shown in the following Table 2:

table 2:

a: 0.1% formic acid in methanol; b: 0.1% formic acid in water.

The HPLC finger print obtained by the above analysis is shown in FIG. 18, wherein the top graph shows the HPLC finger print of the extract of Emblica officinalis, and the bottom graph shows the HPLC finger print of the fermented product of Emblica officinalis. As can be seen from fig. 18, the fermentation product of the emblic extract obtained by the fermentation method of the present invention has higher contents of the compounds of formula (I) and formula (II) (25% and 31% respectively higher) than the extract of emblic. The foregoing results show that the method for preparing an extract fermented product of emblic leafflower fruit of the present invention can indeed effectively increase the content of active ingredients in the extract of emblic leafflower fruit.

As shown in the above examples, the fermentation product of emblic leafflower fruit extract and the compounds of formulae (I) to (XIV) of the present invention are indeed effective in inhibiting adipogenic ability of adipocytes, promoting adipogenic degradation ability of adipocytes, enhancing mitochondrial activity, inhibiting melanogenesis, inhibiting ROS production, inhibiting AGEs production, enhancing SOD2 gene expression, enhancing MPG gene expression, and enhancing ERCC1 gene expression, and thus can be used for making individuals less likely to form adipose, promoting weight loss, aiding metabolism, whitening, and resisting skin aging. In addition, the method for preparing the emblic extract fermentation product can effectively improve the content of the active ingredients in the emblic extract.

<110> Dajiang biomedical corporation Ltd

<120> phyllanthus emblica extract fermentation product, preparation and application thereof

<130> do not

<150> US 62/849,187

<151> 2019-05-17

<160> 8

<170> PatentIn version 3.5

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Claims

1. A method for preparing an extract fermentation of emblic leafflower fruit, characterized by comprising the steps of:

(a) mixing emblic leafflower fruit with water to provide a mixture, and extracting the mixture to provide an emblic leafflower fruit extract;

(b) fermenting the extract with a saccharomyces cerevisiae and a lactobacillus plantarum at 25 ℃ to 35 ℃ for 1 day to 5 days to obtain an intermediate fermentation product; and

(c) fermenting the intermediate fermentation product at 25-35 deg.C for 96 hr-15 days with acetic acid bacteria to obtain an extract fermentation product of fructus Phyllanthi.

2. The method of claim 1, wherein: the Saccharomyces cerevisiae is Saccharomyces cerevisiae BCRC 20271.

3. The method of claim 1, wherein: the Lactobacillus plantarum is Lactobacillus plantarum BCRC 910760.

4. The method of any of claims 1 to 3, wherein: the acetic acid bacteria is acetic acid bacteria BCRC 11688.

5. A composition characterized by: comprising an emblic extract fermentate provided by the method of any one of claims 1 to 4.

6. The composition of claim 5, wherein: the emblic extract fermentation product contains at least one of the following compounds of the formulas (I) to (XIV):

7. the composition of claim 5, wherein: is a pharmaceutical composition, a food composition or a cosmetic composition.

8. Use of an active ingredient for the preparation of a composition characterized by: the composition is used for making an individual less prone to form body fat, whitening, and/or resisting skin aging, and wherein the active ingredient is the emblic leafflower fruit extract fermentation product provided by the method of any one of claims 1 to 4.

9. Use according to claim 8, characterized in that: the composition is a pharmaceutical composition, a food composition or a cosmetic composition.

10. Use of an active ingredient for the preparation of a pharmaceutical composition, characterized in that: the pharmaceutical composition is at least one of inhibiting adipogenesis ability of adipocytes, promoting adipocyte fat degradation ability, promoting slimming, aiding metabolism, increasing mitochondrial activity, inhibiting melanogenesis, inhibiting ROS production, inhibiting AGEs production, increasing SOD2 gene expression, increasing MPG gene expression, and increasing ERCC1 gene expression, and wherein the active ingredient is the emblic leafflower fruit extract fermentation product provided by the method of any one of claims 1 to 4.