KR101404468B1 - Pharmaceutical composition for anticancer comprising compounds isolated from extract of Citrus unshiu leaf as effective component - Google Patents

Abstract

The present invention relates to a compound selected from the group consisting of 2,4-Di-tert-butylphenol, campesterol and stigmasterol, A salt thereof, or a fraction of a citrus leaf extract containing the compound as an active ingredient.

Description

TECHNICAL FIELD The present invention relates to an anticancer pharmaceutical composition comprising a compound isolated from citrus leaf extract as an active ingredient,

The present invention relates to an anticancer pharmaceutical composition containing a compound isolated from citrus leaf extract as an active ingredient. More specifically, the present invention relates to 2,4-di-tert-butylphenol, A pharmacologically acceptable salt thereof, or a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a fraction of a citrus leaf extract containing the compound, wherein the compound is selected from the group consisting of campesterol and stigmasterol Compositions and foods.

Wenzhou citrus fruit is a big part of production and consumption than other citrus fruits cultivated in Jeju. Wenzhou citric acid contains various flavonoids, and these substances have antioxidant, antibacterial and anticancer effects. However, the efficacy of these potatoes is limited to perilla and flesh, and there is little research on the activity of potato leaves.

Apoptosis is the most well-known programmed cell death, characterized by cell membrane blebbing, activation of caspase proteins, chromatin condensation, and nuclear fragmentation. .

Autophagy is a mechanism of digesting old proteins and damaged organelles themselves, allowing the cells to survive by decomposing and recycling the organelles under stress conditions. However, when the level of self-digestion is high, it causes apoptosis, which is called type II apoptosis or autoprotein apoptosis.

The extracts of Wanju citrus leaf used in the present invention and their fractions showed cell proliferation inhibitory activity against AGS, which is a gastric cancer cell line, and this activity was caused by autophagy rather than apoptosis. Therefore, this study is the first report on the cell death mechanism of the citrus leaf extract and its fractions in Jeju, and it is thought to be useful for the development of physiologically active food using citrus leaves because there is no apoptosis effect on normal cells .

Korean Patent No. 0524373 discloses a pharmaceutical composition for an anticancer agent containing Saururus chinensis extract and a method for producing the same. Korean Patent Publication No. 2008-0088784 discloses an anticancer pharmaceutical composition Composition, but is different from an anticancer pharmaceutical composition containing the citrus leaf extract or its fraction as an active ingredient of the present invention.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for producing a cancer cell extract, which comprises extracting methanol extract of a Chinese cabbage leaf with methanol to obtain a fraction, The inventors of the present invention have completed the present invention by developing a pharmaceutical composition and food for cancer prevention or treatment containing a novel compound having anticancer activity and fractions of the extract of Chrysanthemum morbus Nile leaf containing the compound as an active ingredient .

In order to solve the above problems, the present invention provides a compound selected from the group consisting of 2,4-Di-tert-butylphenol, campesterol and stigmasterol , A pharmaceutically acceptable salt thereof, or a fraction of citrus leaf extract containing the compound as an active ingredient.

The present invention also relates to a compound selected from the group consisting of 2,4-Di-tert-butylphenol, campesterol and stigmasterol, A food for cancer prevention or improvement comprising an acceptable salt or a fraction of citrus leaf extract containing the above compound as an active ingredient.

According to the present invention, the compound of the present invention and the fraction of citrus leaf extract containing the compound can be used safely as a pharmaceutical composition for prevention and treatment of cancer, . In addition, the compounds of the present invention and fractions of citrus leaf extract containing the compounds can be usefully used as functional foods in foods, tea, and the like, thereby revealing various usefulness of the citrus leaves of Wenzhou and improving the productivity and value Can be expected.

FIG. 1 is a graph comparing cell survival rates after treatment with three kinds of cancer cells of AGS, MCF-2, and HeLa for 4 hours, respectively.
FIG. 2 is a graph comparing cell survival rates after treatment with AGS cells for 24 hrs.
FIG. 3 is a graph comparing cell viability after treatment with AGS cells for 48 hrs.
FIG. 4 shows the results of treatment of 25, 50, and 100 μg / mL of Wanju citrus leaf extract with AGS cells for 24 hours, staining nuclei with Hoechst 33342 dye, and observing them with a microscope.
Control: Wenzhou citrus leaf extract without treatment
Apoptosis positive control (PC): Treatment of Guava leaf with 80% MeOH extract at 100 μg / mL
FIG. 5 is a graph showing the ratio of cell populations according to the cell cycle after treatment with AGS cells for 24 hrs.
FIG. 6 shows the results of analysis of flowering cytotoxicity assay by staining with annexin V and PI for 24 hrs.
Control: Wenzhou citrus leaf extract without treatment
FIG. 7 is a graph showing the amount of cleaved PARP and Bcl-2 measured by western blot analysis using a lysate of AGS cells treated for 24 hours with different concentrations of Wanju citrus leaf extract.
C: No treatment of Wanju citrus leaf extract
PC: Treatment of Guava leaf with 80% MeOH extract at 100 μg / mL
Figs. 8 and 9 show that the extracts of Wanju citrus leaf having different concentrations were treated for 24 hours and then stained with acridine orange, and the self-digestion phenomenon was observed through FACS analysis (Fig. 8) and fluorescence microscope (Fig. 9) will be.
Control: Wenzhou citrus leaf extract without treatment
FIG. 10 shows the results of FACS analysis after staining with MDC dye after treating the extract of Wanju citrus leaf with different concentrations for 24 hours.
Control: Wenzhou citrus leaf extract without treatment
FIG. 11 shows Western blot analysis of AGS cells treated for 24 hours with different concentrations of Wanju citrus leaf extract to observe the amounts of the autophagy proteins Beclin-1 and Atg5.
C: No treatment of Wanju citrus leaf extract
PC: Treatment of Guava leaf with 80% MeOH extract at 100 μg / mL
FIG. 12 shows the results of transfection of GFP-LC3, followed by 24 hours of treatment with Citrus japonicus leaf extract and further incubation for 24 hours followed by fluorescence microscopy.
Control: Wenzhou citrus leaf extract without treatment
FIG. 13 shows the results of MTT assay for the apoptosis effect of pretreatment of 1 mM 3-MA for 1 hour, followed by treatment of the extract of Wanju citrus leaf having different concentrations for 24 hours.
FIG. 14 shows the results of FACS analysis of cells stained with JC-1 kit after treatment with citron leaf extract with different concentrations for 6 hours.
Control: Wenzhou citrus leaf extract without treatment
FIG. 15 shows the results of MTT assay for cell activity after culturing for 48 hours in a cell line, fibroblast, which is a normal cell line.
FIG. 16 is a graph comparing cell survival rates after 24 hours and 48 hours treatment of AGS cells with hexane, chloroform, butanol, ethyl acetate and water fractions of a methanol extract of Wanju citrus leaf.
17 is a graph comparing the cell viability after treating the active ingredients in the chloroform fraction of the methanol extract of Chrysanthemum morifolium Leucocyte with AGS cells for 24 hours.
Figure 18 shows that 2,4-Di-tert-butylphenol, campesterol and stigmasterol were treated with AGS cells for 24 hours and then treated with Hoechst 33342 dye Show photos of dyed.
FIG. 19 shows the results obtained by treating 2,4-di-tert-butylphenol, campesterol and stigmasterol with AGS cells for 24 hours and then injecting acridine orange Show photos dyed with dyes.
FIG. 20 shows photographs in which 2,4-Di-tert-butylphenol was treated with AGS cells for 24 hours and stained with propidium iodide.

In order to accomplish the object of the present invention, the present invention provides a process for the preparation of 2,4-di-tert-butylphenol, camesterol and stigmasterol, Or a pharmaceutically acceptable salt thereof, or a fraction of a citrus leaf extract containing the above-mentioned compound as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

In the pharmaceutical composition of the present invention, the citrus may be, but is not limited to, citrus peel.

Also, in the pharmaceutical composition of the present invention, the fraction of the citrus leaf extract may be a fraction of methanol extract of citrus leaf, wherein the fraction of chloroform of the citrus leaf methanol extract is prepared by pulverizing the freeze- % Methanol in a volume ratio of 0.8-1.2: 24-36, sonicating for 40-50 minutes, concentrating and lyophilizing the extract, and extracting the citrus leaf extract with chloroform, preferably lyophilized The powder obtained by crushing the Chinese cabbage leaves and the mixture of 80% methanol at a volume ratio of 1: 30, ultrasonically extracted for 45 minutes, concentrated and lyophilized, and fractionated with chloroform.

In the pharmaceutical composition of the present invention, the cancer may be gastric cancer, breast cancer, cervical cancer, lymphoma, hepatoblastoma, colon cancer or lung cancer, preferably gastric cancer, breast cancer or cervical cancer, But is not limited thereto.

The pharmaceutical composition for preventing or treating cancer of the present invention may comprise 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the compound or the extract containing the compound, based on the total weight of the pharmaceutical composition.

The pharmaceutical composition comprising the compound of the present invention or an extract comprising the compound may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the compounds of the present invention or the extracts containing such compounds may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmacologically active compounds as well as in suitable aggregates .

The pharmaceutical composition comprising the compound according to the present invention or the extract containing the compound can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, And sterile injectable solutions. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition containing the compound or the extract containing the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Various compounds including gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, Or mixtures thereof. 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 ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dose of the compound of the present invention or the extract containing the compound depends on the condition and body weight of the patient, the degree of disease, the drug form, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention or the extract containing the compound is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The compound of the present invention or an extract containing the compound can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention is also directed to a compound selected from the group consisting of 2,4-Di-tert-butylphenol, campesterol and stigmasterol, A food for cancer prevention or improvement comprising an acceptable salt or a fraction of citrus leaf extract containing the above compound as an active ingredient.

The food is not particularly limited as long as it can be ingested to increase anticancer activity.

When the compound of the present invention or an extract containing the compound is used as a food additive, the compound or the extract containing the compound may be directly added or used together with other food or food ingredients, Lt; / RTI > The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the compound of the present invention or the extract containing the compound is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of the food to which the compound or the extract containing the compound can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , Beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention.

In addition to the above, the compound of the present invention or the extract containing the compound can be used as a nutritional supplement, a vitamin, an electrolyte, a flavor, a colorant, a pectic acid and its salt, an alginic acid and its salt, an organic acid, a protective colloid thickener, , Preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the compounds of the present invention or extracts containing the compounds may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

1. Cell culture and transfection ( transfection )

AGS and MCF-7 cells were cultured in an incubator maintained at 37 ° C and 5% CO ₂ , and RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% antibiotic was used. Fibroblasts and HeLa cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% antibiotic. GFP-LC3 was transiently transfected with Lipofectamine (Invitrogen, Paisley, UK) and used in the experiment.

2. Reagents

RPMI 1640, DMEM medium, trypsin / EDTA, fetal bovine serum (FBS), antibiotics, and Hoechst 33342 dye were purchased from Invitrogen and were purchased from Propidium iodide, acridine orange dye, 3-MA Were purchased from Sigma. Annexin V kit and JC-1 kit were purchased from BD Biosciences.

3. Extracts and Fraction  Produce

After lyophilization, the extracts were extracted with 80% methanol at a ratio of 1: 1 (v: v) for 45 minutes. The extract thus obtained was concentrated, lyophilized, dissolved in DMSO, and used in the experiment.

Further, hexane, chloroform, butanol, ethyl acetate and water were sequentially added to the thus-prepared methanol extract of citrus peony leaf, and hexane, chloroform, butanol, ethyl acetate and water fractions were obtained.

4. Cells Survival  And morphology analysis

The cytotoxicity of AGS, a gastric cancer cell line, and fibroblasts, a normal cell, were measured by MTT assay. After the cells were cultured for a certain period of time, the MTT solution of 5 mg / mL was treated for 4 hours. After the medium was removed, the formazan crystal was dissolved in DMSO, and the absorbance was measured at 570 nm.

For the morphological analysis, the AGS cells were cultured in a 60 mm cell culture dish at a concentration of 3 × 10 ⁴ cells / mL for one day, and the cells were further treated for 24 hours. After 24 hours, 10 占 의 of Hoechst 33342 or acridine orange dye was added and stained at 37 占 폚 for 10 minutes, and cells were observed under a microscope.

5. Flow cell  analysis( Flow cytometry 분석 )

AGS cells were added to 100 mm cell culture dishes at 5 × 10 ⁴ cells / mL, cultured for one day, treated with the samples, and cultured for 24 hours. After 24 hours, the cells were collected, fixed with 70% ethanol, and stained with 40 μg / mL propidium iodide at 37 ° C. for 30 minutes to measure the cell cycle.

To observe the autophagy phenomenon, the cells treated with the sample for 24 hours were stained with acridine orange or MDC dye for 20 minutes, and the cells were collected and measured by FACS. Cells treated with the same method were stained with JC-1 kit and the mitochondrial membrane potential was measured by FACS.

6. Western Blot  analysis( Western blot 분석 )

Cells were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3 , 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 μg / mL aprotinin, and 25 μg / mL leupeptin). The lysed cells were kept on ice for 30 minutes and then centrifuged at 13,000 rpm for 30 minutes at 4 ° C. Protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, Ill., USA). Proteins were separated by 12-15% SDS-PAGE and transferred to a buffer containing 192 mM glycine, 25 mM Tris- 20% methanol) to a PVDF membrane (Bio-RAD, HC, USA). After blocking the membrane with 5% skim milk powder, the primary antibody was attached (4 ° C, overnight). The cut PARP, Bcl-2, Beclin-1, and Atg5 antibodies (cell signaling) were diluted 1: 1,000 and used as horseradish peroxidase-conjugated goat anti-rabbit antibody anti-rabbit antibody) was diluted to 1: 5,000. Protein bands were analyzed using the WEST-ZOL plus Western Blot Detection System (iNtRON, Gyeonggi-do, Korea).

Example  One: Wenzhou citrus  Of leaf extract AGS  In cells, non-apoptosis ( non -apoptotic) induction of apoptosis

MTT was performed on three cancer cells of human gastric cancer cells, MCF-7 and human lavage cancer cells, and the highest proliferation inhibitory effect was observed in AGS cells (Fig. 1). Therefore, the cell line to be used in the next experiment was defined as AGS, and the cell proliferation inhibitory effect was also shown in a concentration-dependent manner even when the AGS was treated for 24 or 48 hours (FIGS. 2 to 3).

In order to determine whether the inhibition of cell proliferation is caused by apoptosis, the cells were stained with Hoechst 33342 stain and observed with a microscope. The formation of apoptotic bodies was observed even after treatment with high concentration of Wenzhou citron leaf extract (Fig. 4). In addition, an increase in sub-G1 phase was not observed in FACS (Fig. 5), and the annexin V / PI staining showed staining of PI (shifted upward) rather than annexin V (Fig. 6). The cleavage of PARP, an anti-apoptotic protein, and the increase in the expression level of Bcl-2 (FIG. 7) confirmed that the extract of Wanju citrus leaf induces non-apoptotic cell death in AGS cells.

Example  2: Wenzhou citrus  Of leaf extract AGS  Induce self-digestion in cell lines

Since it was confirmed that the inhibitory effect on cell proliferation of Wanju citrus leaf was not due to apoptosis, autophagy was observed by staining cells with acridine orange or MDC. Autocrine orange staining and MDC staining were performed by FACS analysis. As a result, autophagy phenomenon was observed in AGS cells treated with Wanju citron leaf extract and microscopic observation showed that acidic vesicular organelle (Fig. 8 to Fig. 10). In addition, the expression levels of the autophagy proteins Beclin-1 and Atg5 were increased (FIG. 11), and the punctuation of GFP-LC3 was observed by treating the extract of the Chinese cabbage (Fig. 12).

In order to identify prosurvival autophagy and death autophagy, 3-MA, an autophagy inhibitor, was pretreated and cell activity was measured, indicating that apoptosis was restored (Fig. 13).

When the mitochondrial membrane potential of the cells treated with the citrus leaf extract of Wenzhou was measured, the membrane potential was decreased after 6 hours of treatment. As a result, the mitochondrial membrane potential decreased first, and the self-digestion (Fig. 14).

Example  3: Wenzhou citrus  Effect of Leaf Extract on Human Normal Cells

The extract of Wanju citrus leaf showed a high cell proliferation inhibitory effect on AGS cells of gastric cancer cell line, but did not show cytotoxicity in normal cell line fibroblast (Fig. 15).

Example  4: Wenzhou citrus  Of leaf extract Fraction  Stomach cancer cells AGS Anticancer activity in

To elucidate the anticancer activity of the methanol extracts of Wanju citrus leaf, hexane, chloroform, butanol, ethyl acetate and water fractions were prepared by systematic extraction of methanol extracts. Each solvent fraction was added to AGS cells for 24 hours, 48 hours Lt; RTI ID = 0.0 > MTT < / RTI > assay. As a result, the chloroform fractions were found to be the most active in inhibiting gastric cancer cells by comparing the activity of the solvent fractions. The chloroform fraction having high anticancer activity was analyzed by GC / MS as shown in Table 1 below.

GC / MS analysis of chloroform fractions Constituent Area (%) 2,4- Di - tert - butylphenol 1.31 4- (1,1,3,3-Tetramethylbutyl) phenol 0.45 Coniferol 0.44 Pentadecanoic acid 0.18 6,7-Dimethoxycoumarin 0.14 Phytol 0.9 (Z) -9-Octadecenamide 0.19 Tetracosane 0.38 beta-Tocopherol 3.69 Cholest -5- en -3. beta - yl - methyl ether 32.14 alpha - tocopherol 13.42 Lupeyl acetate 5.05 Campesterol 6.22 Stigmasterol 6.00

Include the candidate materials would be expected to exhibit anti-cancer activity in chloroform fractions, 2,4-di -tert- butyl-phenol (2,4-Di-tert-butylphenol ), alpha-tocopherol (alpha -Tocopherol), campesterol (campesterol) And stigmasterol and the like. The cytotoxic effect of AGS on the five candidate substances contained in the chloroform fraction was measured by concentration.

As a result, 2,4-Di-tert-butylphenol was the most effective candidate for the cell survival rate among the compounds obtained by the GC analysis. Campesterol and stigmasterol ), And the remaining candidate substances did not affect cell viability (Fig. 17).

Example  5: Hoechst  33342 Active ingredient cell suicide by staining apoptosis )

The nuclei were stained with Hoechst 33342. As a result, it was confirmed that apoptosis by 2,4-Di-tert-butylphenol was not apoptosis. In addition, stigmasterol-treated nuclei were stained and observed to form an apoptotic body at 20 μM. After the campesterol treatment, the nuclei were observed for staining, and the formation of apoptotic bodies was weakly observed at 20 μM (FIG. 18). As a result, 2,4-Di-tert-butylphenol induces gastric cancer cell death through mechanisms other than apoptosis, and the chloroform fraction of Ganoderma lucidum extract Inhibitory activity can be expected as the main substance.

Example  6: Acridine  Orange( acridine orange Self - extinguishing action by cell staining using autophagy )

The treatment of 2,4-Di-tert-butylphenol increased the formation of the acidic vacuole (AV), a marker of autophagy, . Stigmasterol treatment showed almost no autophagy, and campesterol treatment showed almost no self-digestion (Fig. 19). The nuclei were stained with PI and observed under a microscope. As a result, it was confirmed that the cells were stained in a concentration-dependent manner in 5 [mu] M-treated cells of 2,4-di-tert-butylphenol ).

As a result, the cell apoptosis caused by the cell self-extinguishing action was observed when 2,4-Di-tert-butylphenol was observed more clearly than campesterol and stigmasterol .

Claims (6)

A pharmaceutical composition for the prophylaxis or treatment of gastric cancer, breast cancer or cervical cancer containing the chloroform fraction of methanol extract of citrus leaf as an active ingredient. The pharmaceutical composition according to claim 1, wherein the citrus is citrus peel. delete [Claim 2] The method according to claim 1, wherein the chloroform fraction of the citrus leaf methanol extract is prepared by mixing the powder obtained by freeze-drying the citrus leaves with methanol at a ratio of 0.8 to 1.2: 24 to 36 at 70 to 90 volume% And extracting the citrus leaf extract prepared by concentration and lyophilization with chloroform. delete Food for preventing or improving gastric cancer, breast cancer or cervical cancer containing the chloroform fraction of methanol extract of citrus leaf as an active ingredient.

Images