KR101348471B1 - A composition comprising Larrea cuneifolia Cav. for the prevention and treatment of menopausal symptom - Google Patents

Abstract

The present invention relates to a new plant extract that can act as a phytoestrogen, and in particular, to a composition for preventing and treating menopausal syndrome with Laria cunefolia extract and the active ingredient. In the present invention, a variety of in vitro and in vivo experiments, including estrogen receptor binding, cell proliferation, transcription analysis through the estrogen response gene and uterine proliferation analysis, containing the extract of Larrea cuneifolia Cav. As an active ingredient It is confirmed that the extract acts as an antagonist or partial agonist against the estrogen receptor and is a selective estrogen receptor modulator (SERM). The composition of the present invention containing a Laria Cunefolia extract as an active ingredient can be usefully used for the prevention and treatment of estrogen-related female diseases such as menopause syndrome.

Description

A composition for preventing and treating menopausal syndrome using the extract of Laria cunefolia as an active ingredient {A composition comprising Larrea cuneifolia Cav. for the prevention and treatment of menopausal symptom}

The present invention relates to a new plant extract that can act as a phytoestrogen, and in particular, to a composition for preventing and treating menopausal syndrome with Laria cunefolia extract and the active ingredient.

Menopausal syndrome in women refers to a number of symptoms caused by decreased secretion of female hormone (estrogen) before or after menopause. Symptoms of menopausal syndrome include hot flushes, sudden heartbeat changes, sleep disorders, neurosis, irritability, poor concentration, depressed mood, anxiety, loss of confidence and sexual desire, osteoporosis, and atherosclerosis. . Hormone replacement therapy (HRT) for artificially administering estrogen is widely used as a method for the treatment of menopausal syndrome.

In hormone replacement therapy, the administration of synthetic estrogen hormones is the most common. However, the administration of such synthetic hormones is effective in improving menopausal symptoms, but there is a controversy over the use of long-term use, which may cause uterine cancer, breast cancer, venous thrombosis, and gallbladder disease. Therefore, attempts have been made to utilize plant-derived natural estrogens with hormone-like therapies with similar activities to synthetic estrogens and fewer side effects.

Such plant-derived natural estrogens are called phytoestrogens. Phytoestrogens are plant-derived non-steroidal compounds similar in function and structure to estrogens, and are found in the stems, roots, flowers and seeds of plants. Isoflavones, flavonoids, and lignans, which are found in soybeans and other plants, have phytoestrogen properties. These phytoestrogens, which have similarities to estrogens in their molecular structure, exhibit selective binding to estrogen receptors, In recent years, it has been reported that these phytoestrogens can prevent the production of breast cancer by inhibiting cell proliferation and angiogenesis (Mense SM et al. Environ Health Perspect. 2008; 116: 426-33). However, phytoestrogens, which have been in the spotlight as estrogen replacement therapy, have also recently raised side effects such as uterine cancer and breast cancer. Therefore, there is a need for the development of plant-derived phytoestrogens with higher tissue selectivity and improved side effects.

Larrea cuneifolia Cav. Is a plant of perennial and evergreen shrubs, mainly in Argentina. In South America, it is known that folk remedies are mainly used for dyspepsia, body odor suppression, menstrual control, but the efficacy has not yet been scientifically proven. Plants of the genus Larrea have been reported to have anti-inflammatory, antibacterial and antiviral effects of L. tridentata or L. divaricata shrubs (Heinrich). M. Curr Topics Med Chem 2003; 3: 141 ?? 154; Gagnier JJ et al. Am J Med 2006; 119: 1 ?? 11). In addition, there have been reports of inhibiting the tumor cell colonization formation and inhibiting the cancer cell proliferation of Larix spp. (Garreau B et al. Biochim Biophys Acta 1991; 1094: 339-45; Anesini C et al. Phytomedicine 2001; 8: -7). The Larix plants studied so far contain a large amount of lignan components and also contain cinnamic acid and its derivatives.

A representative component of the known Larix sp. Is the lignan nordihydroguaiaretic acid (NDGA). NDGA is a powerful antioxidant and various physiological activities are being studied. It has been reported that it inhibits tumor cell proliferation through accumulation of PKC (protein kinase C) and cAMP (cyclic adenosine monophosphate) accumulation and inhibition of calcium influx (Erashi M et al., Oncology 1995; 52: 150-155; Pavani M et al., Biochemical Pharmacology 1994; 48: 1935-1942). In addition, anticancer effects of receptor tyrosine kinase (RTK) and insulin-like growth factor receptor (IGF-1R) inhibitors have been reported (Youngren JF et al., Breast Cancer Res Treat 2005; 94: 37-46). NDGA has also been shown to have estrogen receptor binding and transcriptional activity in estrogenic activity evaluation (Fujimoto N et al. Life Sci 2004; 74: 1417-25). However, NDGA content and related biological activities have been confirmed only in some plants of the genus Laria, and various biological activities and related active substances in various plants of the genus Laria have not been identified.

[references]

1. Mense SM et al. (2008). "Phytoestrogens and breast cancer prevention: possible mechanisms of action." Environ Health Perspect. 116 (4): 426-33

2. Heinrich M. (2003). "Ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures?" Curr Topics Med Chem. 3 (2): 141-54

3. Gagnier JJ et al. (2006). "Quality of reporting of randomized controlled trials of herbal medicine interventions." Am J Med. 119 (9): e1-11

4. Garreau B et al. (1991). "Phytoestrogens: new ligands for rat and human alpha-fetoprotein." Biochim Biophys Acta. 1094 (3): 339-45

5. Anesini C et al. (2001). "Different intracellular signals coupled to the antiproliferative action of aqueous crude extract from Larrea divaricata Cav. And nor-dihydroguaiaretic acid on a lymphoma cell line." Phytomedicine. 8 (1): 1-7

6. Erashi M et al. (1995). "Effects of eicosanoid synthesis inhibitors on the in vitro growth and prostaglandin E and leukotriene B secretion of a human breast cancer cell line." Oncology. 52 (2): 150-155

7. Pavani M et al. (1994) "Inhibition of tumoral cell respiration and growth by nordihydroguaiaretic acid." Biochemical Pharmacology. 48 (10): 1935-1942

8. Youngren JF et al. (2005) "Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-erbB2 / HER2 / neu receptors and suppresses growth in breast cancer cells." Breast Cancer Res Treat. 94 (1): 37-46

9. Fujimoto N et al. (2004) "Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA)." Life Sci. 74 (1): 1417-25

The present invention is to provide a new natural estrogen derived from plants that can effectively prevent, treat, and improve the menopausal syndrome of women. It is an object of the present invention to provide a pharmaceutical composition and a dietary supplement for the prevention and treatment of female menopausal syndrome, comprising as an active ingredient Laria cunefolia extract showing different properties from the plants of the genus Laria in relation to estrogen activity. It is done.

In the present invention, as a result of evaluating the significant estrogen effect in L. cunepolia, it was confirmed through experiments that L. cunepolia acts as an antagonist or a partial agonist for the estrogen receptor and shows female hormone in vivo.

Based on the experimental results, in the present invention,

It provides a pharmaceutical composition for the prevention and treatment of menopausal syndrome containing Larrea cuneifolia Cav. Extract as an active ingredient. The extract is preferably extracted with water, an organic solvent or a mixed solvent thereof. The menopausal syndrome includes symptoms of any one of hot flashes, osteoporosis, venous thrombosis and atrophic vaginitis.

Further, in the present invention,

Provided is a dietary supplement for the prevention and improvement of menopausal syndrome containing Laria Cunefolia extract as an active ingredient. The health functional food may have any one form of tablets, capsules, powders, granules, liquids, and pills. In addition, the health functional food may be a food form in which L. cunefolia extract is added to foods, beverages, gums, teas, vitamin complexes, health functional foods, and the like.

In the present invention, a variety of in vitro and in vivo experiments, including estrogen receptor binding, cell proliferation, transcription analysis through the estrogen response gene and uterine proliferation analysis, containing the extract of Larrea cuneifolia Cav. As an active ingredient It was found that the extract acts as an antagonist or partial agonist against the estrogen receptor and is a selective estrogen receptor modulator (SERM). The composition of the present invention containing a Laria Cunefolia extract as an active ingredient can be usefully used for the prevention and treatment of estrogen-related female diseases such as menopause syndrome.

1A shows the effect of 17β-ES (•) and L. cunepolia (◆) on hERα binding of [ ³ H] 17β-ES.
FIG. 1B shows the effect of 17β-ES (•) and L. cunepolia (◆) on hERβ binding of [ ³ H] 17β-ES.
2 shows 17β-ES, ICI 182,780 and L. cunefolia effects on proliferation of MCF-7 cells.
Figure 3 shows the transcriptional effects of 17β-ES, ICI 182,780 and L. cunepolia on the estrogen response genes of MCF-7 cells.
Figure 4 shows the effect of rat uterine proliferation upon administration of L. cunepolia to rats.

Laria cunefolia in the present invention used leaves and stems. The leaves and stems of Laria cunefolia were extracted and used with water, an organic solvent or a mixed solvent thereof. In the present invention, "Laria Cunefolia" means the leaves and / or stems of Laria Cunefolia, unless otherwise specified.

In the present invention, the estrogen action was confirmed through various in vitro and in vivo experiments of L. cunefolia extract, including estrogen receptor binding, cell proliferation, transcriptional analysis through estrogen response genes, and uterine proliferation assay. Experimental results of the estrogen effect of L. cunefolia extract showed binding to estrogen alpha and beta receptors (hERα; IC ₅₀ = 1.23 × 10 ⁻⁷ g / ml, hERβ; IC ₅₀ = 9.49 x 10 ^-8 g / ml). In particular, it showed a 2.8-fold higher affinity for hERβ compared to the binding capacity for hERα. This means that the extract of Laria cunefolia of the present invention has stronger estrogen receptor binding capacity than NDGA, compared to 30 μM of NDGA's hER binding capacity (IC ₅₀ ), which is a major component of the plant of the genus Laria (Fujimoto). N et al. Life Sci 2004; 74: 1417-25). Cell proliferation experiments using estrogen dependent MCF-7 cell line could measure the weak cell proliferation effect of L. cunefolia. Transcription assays with estrogen-responsive genes also showed a weak increase in transcription and were evaluated to have estrogen partial agonism. The co-administration of L. cunefolia and estrogen inhibited the cell proliferation effect of 17β-ES, and the gene transcriptional effect did not change compared with the effect of 17β-ES. However, the combination of the estrogen receptor blockers ICI and L. cunepolia blocked both cell proliferation and estrogen gene transcription. Subcutaneous injection of L. cunefolia into immature rats resulted in uterine proliferation experiments. L. cunefolia extracts inhibited uterine growth in female rats. This suppression of uterine growth is the opposite result of NDGA's uterine growth promoting action. Taken together, the L. cunefolia of the present invention has stronger binding capacity to the estrogen receptor than NDGA, and has also shown an opposite effect on living uterine proliferation. These results indicate that the estrogenic action of L. cunefolia is due to an active ingredient other than NDGA.

From these experimental results, L. cunefolia acts as an antagonist or partial agonist for the estrogen receptor and proves to be a selective estrogen receptor modulator (SERM). This means that L. cunepolia can be used as a therapeutic agent for estrogen related female diseases. Currently, hormone replacement therapy (HRT) for the administration of estrogen is widely used in estrogen-related female diseases, which improves symptoms such as menopausal symptoms and osteoporosis in postmenopausal women. However, there are many cases of avoiding HRT due to various side effects such as estrogen-dependent breast cancer, long-term hormonal therapy and other female cancers or weight gain. Therefore, recently, methods for reducing side effects while having characteristics of hormone replacement therapy have been studied. The results identified for L. cunefolia in the present invention suggest that L. cunefolia can be used for the prevention, treatment and amelioration of estrogen related female diseases such as menopausal syndrome by replacing estrogen for this purpose.

L. cunefolia is a herb that has long been used as a folk remedy for treating indigestion in South America and is known to have no special toxicity or side effects.

A pharmaceutical composition for the prevention and treatment of menopausal syndrome, comprising L. cunefolia extract of the present invention, comprises 0.1 to 50% by weight of the extract, based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, or in any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Witepsol, macrogol, Tween 61, cacao paper, laurin, glycerogelatin and the like may be used as a base for suppositories.

The preferred dosage of the extract of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 10000 mg / kg per day. The administration may be carried out once a day or divided into several times. The above dosage does not limit the scope of the present invention in any aspect.

Extracts of the invention can be administered by a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.

The term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. of the present invention are those conventional meanings known in the art, and include the same or similar in function thereof.

As used herein, the term " health functional food "means food prepared and processed using raw materials or ingredients having useful functions in the human body. The term" functional " And the like, for the purpose of obtaining a beneficial effect for health use such as exercise. The dietary supplement for the prevention and improvement of estrogen-related female diseases of the present invention comprises the extract in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition. The health functional food of the present invention can be manufactured and processed into health functional foods in the form of tablets, capsules, powders, granules, liquids, pills and the like. In addition, the health functional food of the present invention can be prepared and processed in the form of adding L. cunefolia extract to food or beverage. Examples of foods to which L. cunefolia extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

When the health functional food of the present invention is in the form of a beverage (health functional beverage composition), preferably, L. cunefolia extract may be added 0.02 to 50 g based on 100 ml of the beverage. The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the samples of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the sample of the present invention.

[Example]

Hereinafter, the present invention will be described in more detail by Examples, Experimental Examples and Preparation Examples. However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

Example 1 Preparation of L. Cunefolia Extract

Laria cunefolia used leaves and stems. The crude powder L. cuneifolia was obtained by extracting with 10% volume of 70% methanol for 2 weeks. Methanol extract of L. cuneifolia obtained at this time was evaporated in vacuo and lyophilized. In the following Experimental Examples and Preparation Examples, L. cunefolia extract obtained in this Example was used.

Experimental Example 1 [ ³ Competitive Inhibition of L. Cunefolia Extracts on Estrogen Receptor (hER) Binding of H] 17β-ES

In vitro, it was confirmed that L. cunefolia extract competitively binds to [ ³ H] 17β-ES against human estrogen receptor (hER). The binding capacity of L. cunepolia to hER was indirectly determined by measuring the degree of binding separation of [ ³ H] 17β-ES after adding L. cunepolia extract to the complex where ER and [ ³ H] 17β-ES are bound to each other. This can be seen. The results are shown in FIGS. 1A and 1B and Table 1 below. In competitive binding assays, the IC _{50 values} for 17β-ES (●) were 1.16 × 10 ^-9 g / ml (hERα) and 2.49 × 10 ^-9 g / ml (hERβ), respectively. In contrast, the IC ₅₀ of L. cunefolia (◆) was 1.23 × 10 ⁻⁷ g / mL (hERα) and 9.49 × 10 ⁻⁸ g / mL (hERβ), and concentration-dependent [ ³ H] 17β- It has been shown to bind ES.

< Experimental Example  2> L. Cunepolia MCF -7 cell proliferation effect

The estrogen action of L. cunefolia extract was tested based on its ability to increase the proliferation of estrogen receptor dependent MCF-7 breast cancer cells. The results are shown in Fig. MCF-7 cells increased 4.3-fold when treated with only 17β-ES, a positive control, compared to vehicle treated with 0.1% ethanol only when treated with L. cunefolia at a concentration of 10 ^-5 g / ml. 1.2 times increase. However, treatment with L. cunefolia and 17β-ES at concentrations of 10 ⁻⁷ to 10 ⁻⁵ g / mL inhibited cell proliferation up to 1.3, 1.5 and 2.7 times compared to 17β-ES at each concentration. In addition, treatment with the estrogen receptor antagonist ICI 182,780 together completely blocked the cell proliferation of L. cunefolia. Based on the experimental results blocked by the estrogen receptor antagonist ICI 182,780, L. cunepolia binds to the estrogen receptor and can be evaluated to antagonize the receptor.

Experimental Example 3 Analysis of MCF-7 Intracellular Transcription Through Estrogen Response Gene

In MCF-7 cells transfected with an estrogen responsive element (ERE), it was tested whether L. cunepolia changes the activity of the estrogen responsive gene. The results are shown in FIG. 3. Treatment with L. cunepolia alone increased the ERE gene activity by a factor of two compared to the vehicle group at a concentration of 10 ^-6 g / ml, and L. cunefolia (10 ^-8 -10 ^-6 g / ml) was 17β. Treatment with -ES showed high gene activity similar to that of 17β-ES alone. However, the activity of L. cunepolia was blocked when co-administered with estrogen receptor antagonist ICI, suggesting that there was no interaction between L. cunepolia extract and 17β-ES during gene transcription.

Experimental Example 4 Uterine Growth Effect of L. Cunefolia

Uterine proliferation was tested using female rats before and after 20 days of age of immature birth without endogenous estrogen secretion. When corn oil was administered as a control group, the uterine weight ratio (weight ratio) was 4.44, and 17β-ES (3 ㎍ / ㎏) was subcutaneously injected as a positive control, and the uterine weight ratio was 8.18, 1.8 times higher than that of the control group. It was confirmed to increase. When L. cunefolia extract was administered subcutaneously at 100, 500 mg / kg in immature rats, the uterine weight ratio was reduced to 1.64, and the uterine weight ratio was 0.36-fold lower than that of the control group treated with corn oil only. Could. The uterine growth experiments confirmed that L. cunefolia extract inhibits uterine tissue growth. Specific results are shown in Table 2 and FIG. 4.

Experimental Example 5 Acute Toxicity Test

To determine the toxicity of L. cunefolia extract, L. cunefolia extract was administered to rats at 50, 100, 250, and 500 mg / kg for 3 days subcutaneous injection. 50, 100, 250 mg / kg and the maximum dose of 500 mg / kg were repeatedly administered for 3 days and then observed for 4 days. As a result, no abnormal symptoms were observed even at the maximum dose of 500 mg / kg, and no animals died. Therefore, the LD ₅₀ of L. cunefolia is evaluated as a fairly safe drug of 500 mg / kg or more. Specific results are shown in Table 3 below.

Hereinafter, the preparation examples of the pharmaceutical composition comprising the extract of the present invention. In the following Formulation L. Cunefolia extract is the extract obtained in Example 1.

Production Example 1 Preparation of Powder

L. cunefolia extract 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight container to prepare powders.

&Lt; Preparation Example 2 > Preparation of tablet

L. cunefolia extract 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to the usual preparation method of tablets.

Production Example 3 Preparation of Capsule

L. cunefolia extract 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Preparation Example 4 Preparation of Injection

L. cunefolia extract 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient was prepared per ampoule (2 ml).

Production Example 5 Preparation of Liquid

L. cunefolia extract 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and lemon flavor is added, the above components are mixed, and then, purified water is added to adjust the total amount to 100 ml, and then sterilized by filling into a brown bottle. It was.

Claims (7)

A pharmaceutical composition for the prevention and treatment of menopausal symptoms due to reduced estrogen secretion, containing Larrea cuneifolia Cav. Extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is extracted with water, an organic solvent or a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the menopausal symptom comprises one of hot flashes, osteoporosis, and atrophic vaginitis. A dietary supplement for the prevention and improvement of menopausal symptoms due to reduced estrogen secretion, containing Larrea cuneifolia Cav. Extract as an active ingredient. The health functional food according to claim 4, wherein the health functional food has any one form of tablet, capsule, powder, granule, liquid, and pill. The health functional food according to claim 4, wherein the Laria Cunefolia extract is added to the food. The health functional food according to claim 6, wherein the food is any one of foods, beverages, gums, teas, vitamin complexes, and health functional foods.

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