KR102378489B1 - Composition for skin-lightening using an extract of Persicaria filiformis or compounds isolated from the same - Google Patents

Abstract

The present invention discloses a composition for skin whitening using an extract or a compound isolated therefrom having tyrosinase inhibitory activity.

Description

Composition for skin-lightening using an extract of Persicaria filiformis or compounds isolated from the same}

The present invention relates to a composition for skin whitening using an extract of Persicaria filiformis or a compound isolated therefrom.

With the development of medical technology and the improvement of living standards, people's desire for beauty is getting stronger and the perception that appearance itself is a social competitiveness is increasing. Korean Soc. Food Sci. Nutr., 42(3):355 (2013)).

The skin occupies about 16% of the human body and is in direct contact with the external environment, so it plays an important role in protecting the human body from external harmful factors such as temperature, humidity and ultraviolet rays. The skin is composed of the epidermis, dermis, and subcutaneous tissue under the keratin. The epidermis is a thin protective layer composed of keratinocytes, which are mainly made of keratin, melanocytes that produce and secrete melanin, immune cells such as Langerhans cells, and sensory related cells (Merkel's copuscles). It prevents external stimuli and invasion of pathogens, regulates body temperature, and maintains moisture and lipid components.

Melanin is a phenolic polymer natural pigment widely present in living organisms. In the human body, it is synthesized from melanosomes in melanocytes in the basal layer of the epidermis, and metastasized to surrounding keratinocytes to give the color of human skin. When melanin is abnormally low, skin lesions such as vitiligo are induced. Conversely, when melanin is excessively produced, acquired hyperpigmentation such as melasma, postinflammatory melanoderma, and solar lentigo. It can cause a variety of skin diseases, including

The synthesis of melanin is made by making melanosomes from melanocytes present in the basal layer of the skin, and the series of processes in which melanin is synthesized is collectively called the melanogenesis pathway (melanogenesis). Melanin production is based on one of the amino acids, tyrosine, as a substrate, and DOPA (3,4- After conversion to DOPA quinone through dihydroxyphenylalanine), melanin is synthesized by a polymerization reaction with amino acids or proteins after a non-enzymatic reaction and spontaneous oxidation process (Korean J. FOOD SCI. TECHNOL., 2000. 32(3):736; Journal of Life Science, 2013. 23(12):1445; Pigment Cell Res. 1999. 12(1):4-12.). Recently, studies on the inhibition of melanin synthesis using proteins such as tyrosinase, TRP-1, and TRP-2 as well as microphtalmia associated transcription factor (MITF) have been conducted. The major intracellular signaling pathway is the cAMP/PKA (cyclic monophosphate/protein kinase A) pathway. cAMP promotes the expression of MITF via PKA and CREB1 (cAMP responsive element binding protein 1), and MITF is important in the melanin synthesis process. It promotes transcription of tyrosinase, TRP-1, and TRP-2 as transcriptional regulators (Pharmazie. 2015 Oct;70(10):646-9.; Int J Mol Sci. 2015 Oct 13;16(10):24219- 42; Curr Pharm Biotechnol. 2015;16(12):1111-9.).

Because tyrosinase plays the most important role as the first step in producing melanin, studies on whitening agents mainly focus on tyrosinase activity inhibitors or antagonists of tyrosinase substrates to regulate tyrosinase activity. Research has been progressing in the direction of developing (Kor. J. Pharmacogn., 44(3):220, 2013).

The present invention discloses the tyrosinase inhibitory activity of the extract

It is an object of the present invention to provide a composition for skin whitening using an extract or a compound isolated therefrom.

Other objects or specific objects of the present invention will be set forth below.

The present invention has been completed by confirming that, as confirmed in the Examples and Experimental Examples below, the E. serrata extract or the compounds of the following Chemical Formulas 1 to 7 isolated therefrom have tyrosinase inhibitory activity.

In consideration of the above, the present invention can be identified as a composition for skin whitening comprising an active ingredient in the extract of A.

<Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

<Formula 5>

<Formula 6>

<Formula 7>

<Formula 8>

In the present specification, the term "extract" refers to the extraction target of the stalk, stem, above-ground part, rhizome, root, underground part, or a mixture thereof with water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene , acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof refers to an extract obtained by leaching, an extract obtained by using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method is cooling, refluxing, considering the polarity of the active material, the degree of extraction, and the degree of preservation. , heating, ultrasonic radiation, supercritical extraction, etc. arbitrary methods can be applied. In the case of the fractionated extract, a fraction obtained by suspending the extract in a specific solvent and mixing and standing still with a solvent having a different polarity, and adsorbing the crude extract to a column filled with silica gel, etc. It is meant to include the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent is removed by methods such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably, an extract obtained using a mixed solvent of water and ethanol as an extraction solvent, preferably an extract obtained using 70% ethanol as an extraction solvent, and fractionation of the extract with water and hexane, water and methylene chloride or water and ethyl acetate It means an extract obtained by sequentially fractionating the extract of any one fractionation solvent layer obtained by doing this, or suspending the extract in water, and then sequentially fractioning it using hexane, methylene chloride and ethyl acetate. Here, "sequential fractionation" means that the fractions of the water layer are used continuously after fractionation to fractionate with the solvents in the order listed above.

In addition, as used herein, the term "active ingredient" refers to a component that alone exhibits the desired activity or can exhibit activity together with a carrier that has no activity by itself.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit a skin whitening effect, etc. according to the use, formulation, purpose of blending, etc., but a typical effective amount is based on the total weight of the composition will be determined within the range of 0.001 wt % to 15 wt %. As used herein, the term "effective amount" refers to intended medical and cosmetic effects such as skin whitening effect when the composition of the present invention is administered to a mammal, preferably a human subject, during the administration period as suggested by a medical professional. It refers to the amount of the active ingredient included in the composition of the present invention. Such an effective amount can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In a specific aspect, the composition of this invention can be grasped|ascertained as a food composition.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated drinks, and ion drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, confectionery, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and health functional food preparations such as bars can be manufactured.

In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it conforms to the enforcement laws at the time of manufacture and distribution. For example, it is a health functional food according to Korea's "Health Functional Food Act", or confectionery, beans, tea, and beverages according to each food type in the Food Ordinance of Korea "Food Sanitation Act" (Ministry of Food and Drug Safety Notification "Food Standards and Specifications") , food for special use, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added and mixed or infiltrated into food in manufacturing, processing, or preserving food. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance in accordance with the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” announced by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is divided into agents, preservatives, emulsifiers, acidulants, thickeners, etc.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural product, the purpose of nutritional enhancement in addition to flavor may be concurrently used. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or obtained from green tea leaves, horseradish leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. are mentioned, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used as an acidulant. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As a thickening agent, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, etc. can be used.

The food composition of the present invention may contain, in addition to the food additives described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Minerals include calcium preparations such as calcium citrate, magnesium stearate Magnesium preparations, such as iron preparations, such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are mentioned.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the product type.

In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Ordinance of each country or the Food Additives Code.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment. When the composition for skin whitening of the present invention is identified as a pharmaceutical composition, its use may be understood as a composition for improving skin hyperpigmentation.

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of the combination of two or more routes is a case in which two or more formulations of drugs according to the route of administration are combined. For example, one drug is first administered by an intravenous route and the other drug is secondarily administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art together with a suitable carrier It can be prepared in a formulation such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol etc. are mentioned. In the case of formulation activity, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as lubricant. sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and the disintegrating agent is starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or isotonic solution such as 5% dextrose may be used. . When formulated for transdermal administration, it can be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and the route of administration. It can be in the range /kg. Administration may be performed once or divided into several times a day. These dosages should not be construed as limiting the scope of the invention in any respect.

In another specific embodiment, the composition of the present invention can be identified as a cosmetic composition. In particular, when the anti-inflammatory composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for suppressing inflammatory skin troubles, alleviating inflammatory skin irritation, and the like.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product in terms of its use or by law, and specifically functional cosmetics, non-functional products having uses such as improvement of skin troubles and improvement of atopic dermatitis It may be general cosmetics and the like. In terms of product form, it may take any product form, and specifically, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax It can take the form of products such as foundation and spray. In a specific product form, it may be in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

The cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the active ingredient, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and fragrances, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

The cosmetic composition of the present invention can be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except that it contains an active ingredient that exhibits skin whitening activity and the like.

As described above, according to the present invention, it is possible to provide a composition for skin whitening using an extract or a compound isolated therefrom. The composition of the present invention may be commercialized as food, drug or cosmetic.

1 is a schematic diagram showing the process of isolating compounds 1 to 8 from the extract
2 to 17 are NMR analysis results of compounds 1 to 8 ( FIGS. 2, 4, 6, 8, 10, 12. 14 and 16 are ¹ H NMR analysis results of compounds 1 to 8, respectively, and FIGS. 3, 5, 7, 9, 11, 13, 15 and 17 are the results of ¹³ C NMR analysis of compounds 1 to 8, respectively).
18 is a result of structural identification of compounds 1 to 8.
19 to 21 are results of evaluation of the tyrosinase inhibitory activity of the extracts, fractions, and compounds 1 to 8 of the serrata.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Isolation and identification of whitening active ingredients from the extract

After cooling the dry powder (1.4 kg) with 70% ethanol twice at room temperature, the extract was concentrated under reduced pressure to obtain 192.5 g of a powdery 70% ethanol extract.

After the extract was suspended in distilled water, equal amounts of n-hexane, methylene chloride, ethyl acetate, and n-butanol were added thereto to sequentially fractionate.

Among them, silica gel column using chloroform and methanol mixed solvent (5:1, 4:1, 3:1, 2:1, 1:1, v/v) for the ethyl acetate fraction having the best tyrosinase inhibitory activity. Chromatography (silica gel column chromatography) was performed to obtain 5 subfractions according to sequential elution (G83-3-1 to G83-3-5).

Prep HPLC (YMC-ODS, 0-100% MeOH, 10ml/min) was performed for the second fraction (G83-3-2) to obtain compound 1 (G83-5-1).

In addition, prep HPLC (YMC-ODS, 0-100% MeOH, 10ml/min) was performed on the 3rd sub-fraction (G83-3-3) to compound 2 (G83-6-1), 3 (G83-6- 2) and 4 (G83-6-3) were obtained.

Also, for the 4th sub-fraction (G83-3-4), C-18 using a mixed solvent of water and methanol (5:1, 4:1, 3:1, 2:1, 1:1, v/v) Eight small fractions were obtained by performing column chromatography (C-18 column chromatography) (G83-7-1 to G83-7-8).

Prep HPLC (YMC-ODS, 0-100% MeOH, 10ml/min) was performed on the 5th subfraction (G83-7-5) to compound 5 (G83-11-1) and 6 (G83-11- 2) was obtained.

In addition, prep HPLC (YMC-ODS, 0-100% MeOH, 10ml/min) was performed for the 6th small fraction (G83-7-6) to obtain compound 7 (G83-10-4).

In addition, prep HPLC (YMC-ODS, 0-100% MeOH, 10ml/min) was performed for the 7th small fraction (G83-7-7) to obtain compound 8 (G83-12-3).

A schematic diagram of the separation process is shown in FIG. 1 .

Nuclear magnetic resonance (NMR, Bruker Co, Ascend III 700 MHz) was performed to identify the structures of compounds Nos. 1 to 8. The compound was dissolved in DMSO-d6 in Merck Co for NMR, put into a 5 mm NMR tube, ¹ H NMR and ¹³ C NMR analysis were performed, and the results are shown in FIGS. 2 to 17 .

As a result of ¹ H NMR and ¹³ C NMR analysis, compound 1 is Quercetin of Formula 1, and compound 2 is 3,4,5-trihydroxybenzoic acid of Formula 2 , compound 3 is (-)-catechin ((-)-Catechin) of Formula 2, compound 4 is (-)-epicatechin gallate of Formula 4 ((-)-Epicatechin gallate), compound 5 is Quercetin 3-D-glucoside of Formula 5, and compound 6 is Quercetin 3-β-galactoside-2"-galactoside of Formula 6 (Quercetin 3-β-galactoside) -2"-gallate), compound No. 7 is Quercetin 3-O-rhamnoside of Formula 7, and Compound No. 8 is Quercitrin 2"-O-gallate of Formula 8. 2"-O-gallate).

The results of this identification are shown in FIG. 18 .

<Experimental Example> Skin whitening activity - Tyrosinase inhibitory activity test

After adding the sample diluted by concentration to 0.1M potassium phosphate buffer (pH 6.5) and 1250 unit/ml mushroom tyrosinase, the substrate 1.5 mM L-tyrosine was added, mixed well, and reacted at 37°C for 20 minutes. made it Absorbance was measured at 490 nm using a microplate reader, and tyrosinase inhibitory activity (%) was obtained compared to the sample untreated group. While the results are shown in FIGS. 19 to 21 , the IC ₅₀ , which is a 50% inhibitory concentration of tyrosinase, is shown in Table 1 below. The results are the results of three repeated experiments for each concentration.

Tyrosinase inhibitory activity (IC ₅₀ , μg/ml) division Tyrosinase inhibition assay Ethanol extract (70% EtOH) >2000 μg/ml Hexane fraction (Hexane) >1000 μg/ml Methylene chloride fraction (MC) 546.67 μg/ml Ethyl acetate fraction (EA) 255.65 μg/ml Butanol fraction (BuOH) 80.90 μg/ml Residual water layer (DW) >1000 μg/ml compound 1 100< uM compound 2 >1000 uM compound 3 >1000 uM compound 4 >1000 uM compound 5 >1000 uM compound 6 >1000 uM compound 7 >1000 uM compound 8 579.61 uM Arbutin 130.36 μg/ml

As can be seen in FIGS. 19 to 21 and the above table, among the fractions, the butanol fraction, and among the separated compounds, Compound 1 had the most excellent tyrosinase inhibitory activity. The IC ₅₀ of arbutin, an existing skin lightening agent used as a positive control, was 130.36 μg/ml, respectively.

Claims (5)

A food composition for skin whitening comprising Quercitrin 2"-O-gallate as an active ingredient.
A cosmetic composition for skin whitening comprising Quercitrin 2"-O-gallate as an active ingredient.
A composition for preventing or treating skin hyperpigmentation comprising Quercitrin 2"-O-gallate as an active ingredient.
A food composition for skin whitening comprising an active ingredient in any one of the fractions of (a) to (d) below:
(a) an ethyl acetate fraction obtained by suspending a 70% ethanol extract of an oleifera plant in water and fractionating it with ethyl acetate;
(b) a butanol fraction obtained by suspending a 70% ethanol extract of an oleifera outpost in water and fractionating it with butanol;
(c) an ethyl acetate fraction obtained by suspending a 70% ethanol extract of a sagebrush in water and sequentially fractionating it with hexane, dichloromethane, and ethyl acetate as a fractionation solvent; and
(d) A butanol fraction obtained by suspending a 70% ethanol extract of an oleifera plant in water and sequentially fractionating it with hexane, dichloromethane, ethyl acetate and butanol as a fractionation solvent.
A cosmetic composition for skin whitening comprising an active ingredient in any one of the fractions of (a) to (d) below:
(a) an ethyl acetate fraction obtained by suspending a 70% ethanol extract of an oleifera plant in water and fractionating it with ethyl acetate;
(b) a butanol fraction obtained by suspending a 70% ethanol extract of an oleifera outpost in water and fractionating it with butanol;
(c) an ethyl acetate fraction obtained by suspending a 70% ethanol extract of a sagebrush in water and sequentially fractionating it with hexane, dichloromethane, and ethyl acetate as a fractionation solvent; and
(d) A butanol fraction obtained by suspending a 70% ethanol extract of an oleifera plant in water and sequentially fractionating it with hexane, dichloromethane, ethyl acetate and butanol as a fractionation solvent.

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