Description

PHARMACEUTICAL COMPOSITION FOR ENHANCING SEXUAL ACTIVITY COMPRISING EXTRACTS FROM RHUS

VERNICIFLUA

Technical Field

[1] The present invention relates to a pharmaceutical composition for enhancing male sexual function and treating male climacteric disorders comprising a lacquer tree (Rhus verniciflua) flavonoid extract and/or anhydrous gallic acid separated therefrom as active ingredients. More specifically, the present invention relates to a pharmaceutical composition for enhancing male sexual function comprising, as active ingredients, flavonoids prepared by immersing Rhus verniciflua in a C lower alcohol or a spirituous alcohol or extracting Rhus verniciflua with a spirituous alcohol, and anhydrous gallic acid separated from the flavonoids via preparatory liquid chromatography. Background Art

[2] The definition of male climacteric disorders was suggested by Werner in 1939 year as a contrast concept for menopausal women based upon reports that men over 50 years of age complained of complex symptoms such as sensitivity, depression, memory failure, difficulty concentrating, fatigue, insomnia, facial blushing, periodic sweating, and reduced sexual appetite, which are similar to symptoms experienced by menopausal women. At present the above symptoms are variously referred to as andropause, ADAM (androgen deficiency in aging males), PADAM (partial androgen deficiency in aging males), etc.

[3] The definition of male climacteric disorders suggested at ISSAM in 2001 consisted of the following complex symptoms: first, decline of sexual desire, erectile dysfunction, especially, erection failure during sleep; second, feeling change accompanied by intellectual activity, deteriorated spatial abilities, fatigue, depression and instability; third, decline of muscle mass and strength together with loss of body mass index; fourth, loss of body hair and skin changes; fifth, bone density loss; and sixth, increased intestinal fat.

[4] Recently, testosterone administration has been suggested as a male climacteric therapy and physical, mental and sexual effects can be attained as via such therapy. Physical effects include, first, increased body fat, caused by an increase in the rate of muscle protein synthesis and an increase in skeletal muscle mass. Second, fat loss is. Third, androgen supplement therapy in normal and hypogonadism males of various ages revealed an increase in the volume of haemoglobin and erythrocyte. Fourth,

androgen supplement therapy in males suffering from congenital hypogonadism yielded increased bone mass, and in acquired hypogonadism, increased bone formation and decreased bone replacement rate and bone absorption. As to psychological effects, improvements in perception, hypochondria and general well-being were reported. The sexual effects included increased sexual desires and increased responsiveness to sexual stimulation. According to a paper by Mulligan, et al, testosterone increases sexual interest, sexual activity frequency and night erection.

[5] The administration mode of testosterone can be in the form of a skin patch preparation, an oral preparation, a pellet for subcutaneous injection or an intramuscular injection preparation. However, it is difficult to achieve the normal diurnal periodicity secretion rhythm of testosterone in the preparations except for the patch type preparation and oral preparation. In addition, side effects occur in 25% of patients using skin patch preparations, and hence interruption of the therapy in some patients was the primary problem limiting general use of the patch preparation. Further, the testosterone preparation for oral use may decrease serum concentration due to its rapid metabolic activity in the liver, and especially, methyltestosterone, an alkylated preparation, exhibits hepatotoxicity and effect on lipid metabolism in blood to increase LDL and decrease HDL which may cause cardiovascular diseases.

[6] Recently, the above mentioned methods have been suggested for treating male climacteric disorders, but they still have side effects. Therefore, there is a need for development of a therapeutical agent capable of safely being used psychologically and physically. Disclosure of Invention

Technical Problem

[7] The present inventors earnestly studied to develop a therapeutic agent for treating male climacteric disorders that has physical and psychological sexual function enhancement effects. As a result, the present inventors have discovered that flavonoids and/or anhydrous gallic acid separated and purified from the lacquer tree (Rhus verniciflua) cause no side effects and exhibit potent male climacteric disorder treatment efficacy and sexual function improvement efficacy through promotion of the secretion of testosterone. Accordingly, the present invention was completed based upon this discovery. Technical Solution

[8] Therefore, in accordance with one aspect of the present, there is provided a pharmaceutical composition for treating male climacteric disorders and improving male sexual function, comprising as active ingredients, flavonoids obtained by immersing lacquer tree (Rhus verniciflua) in a C lower alcohol or a spirituous alcohol, or by

extracting the lacquer tree with a spirituous alcohol, and/or anhydrous gallic acid separated from the flavonoids by preparatory liquid chromatography (LC). [9] In accordance with another aspect of the present, there is provided a functional health food comprising the flavonoids and/or anhydrous gallic acid. [10] In accordance with another aspect of the present, there is provided a pharmaceutical composition for improving sexual function in animals, comprising the flavonoids and/ or anhydrous gallic acid. [11] In accordance with another aspect of the present, there is provided a pharmaceutical composition for improving sexual function of animals, comprising the flavonoids and/ or anhydrous gallic acid. [12] In accordance with another aspect of the present, there is provided a drink additive prepared by mixing the flavonoids and/or the anhydrous gallic acid with an oriental herbal medicine.

Advantageous Effects

[13] The flavonoids and/or anhydrous gallic acid according to the present invention were proven to promote testosterone secretion, increase free testosterone secretion, and be safe for use by humans (confirmed by analysis on body weight and serum).

Brief Description of the Drawings [14] FIG. 1 is a graph comparing testosterone levels of male rats between a control group, a test group (RWE) administered with lacquer tree extract and a test group (F2) administered with a lacquer tree fraction;

[15] FIG. 2 is a reverse phase HPLC chromatogram of the lacquer tree extract;

[16] FIG. 3 is a reverse phase HPLC chromatogram of the lacquer tree fraction;

[17] FIG. 4 is a reverse phase HPLC chromatogram of anhydrous gallic acid;

[18] FIG. 5 is a graph comparing testosterone levels of male rats between a control group and a test group administered with lacquer tree extract; [19] FIG. 6 is a graph comparing free testosterone of male rats between a control group and a test group administered with lacquer tree extract; [20] FIG. 7 is a graph comparing the level of AST in the serum of male rats between a control group and a test group administered with anhydrous gallic acid-containing flavonoids; [21] FIG. 8 is a graph comparing the level of ALT in the serum of male rats between a control group and a test group administered with the anhydrous gallic acid-containing flavonoids; and [22] FIG. 9 is a graph comparing body weight of male rats between a control group and a test group administered with anhydrous gallic acid.

Best Mode for Carrying Out the Invention

[23] In one aspect, the present invention is directed to a pharmaceutical composition for treating male climacteric disorders and improving male sexual function comprising as active ingredients, flavonoids obtained by immersing the lacquer tree (Rhus verniciflua ) in a C lower alcohol or a spirituous alcohol, or extracting the lacquer tree with a spirituous alcohol, and/or anhydrous gallic acid separated from the flavonoids by preparatory liquid chromatography (Prep LC).

[24] The pharmaceutical composition for treating male climacteric disorders and improving male sexual function contains flavonoids extracted from the lacquer tree as an active ingredient. The flavonoids are prepared in accordance with the method as described in PCT Publication No. 2006-059861 Al (published on June 8, 2006), the disclosure of which is incorporated by reference herein in its entirety. In short, the method for preparing the flavonoids comprises: drying the lacquer tree in the shade; extracting the lacquer tree three times in a predetermined amount of water at 9O ⁰ C to 13O ⁰ C for 3 to 8 hours to obtain a hydrothermal extract; spray-drying or freeze-drying the extract; immersing the dried extract in a C lower alcohol or a spirituous alcohol, or extracting the same with a spirituous alcohol to obtain a spirituous alcohol solution; and separating the spirituous alcohol solution by reverse phase-high performance liquid chromatography (RP-HPLC).

[25] Preferably, the C lower alcohol may be methanol or ethanol in the form of spirits.

Most preferred is spirituous alcohol.

[26] In addition, the anhydrous gallic acid is separated from the flavonoids by preparatory liquid chromatography (Prep LC). Alternatively, the anhydrous gallic acid may be prepared by drying the lacquer tree, extracting the lacquer tree three times in a predetermined amount (i.e. about 10-fold the amount of dried lacquer tree) of water at 100 ⁰ C to 13O ⁰ C for 3 to 8 hours to obtain a hydrothermal extract; drying the extract by spray drying or freeze drying; immersing the dried extract in a C lower alcohol or a spirituous alcohol, or extracting the dried extract with a spirituous alcohol to obtain a spirituous alcohol solution; and separating the spirituous alcohol solution by preparatory liquid chromatography (prep LC) or reverse phase high performance liquid chromatography (RP-HPLC). Preferably, the C lower alcohol may be methanol or ethanol in the form of spirits. Most preferred is spirituous alcohol.

[27] An elution solvent useful for RP-HPLC may be a mixture of acetonitrile and tri- fluroracetic acetic acid in an appropriate ratio. The solvent A may be a mixture of 5% acetonitrile and 0.035% trifluoric acid. The solvent B may be a mixture of 50% acetonitrile and 0.025% trifluoric acid. The anhydrous gallic acid can be separated by prep LC using the elution solvent. Through the prep LC, the resulting solution is fractioned into five groups. It was confirmed from RP-HPLC that the peak 2 corresponds to gallic acid (3,4,5-trihydroxybenzoic acid, C 7H 6O 5, molecular weight: 170.12), the single

compound represented by the following formula I. Based on the confirmation of the chemical structure of the fraction 2, gallic acid is synthetically or commercially available. [28]

(D

[29] Animal tests of the gallic acid ascertained that since the gallic acid is extracted from natural plants, it is free of toxicity and thus safe for use, and furthermore, promotes the secretion of male hormones, increases sperm count and improves sperm motility, sexual appetite and sexual intercourse frequency. Therefore, the gallic acid extracted from the lacquer tree can be used in foods or medications for treating male climacteric disorders and improving sexual function.

[30] The pharmaceutical composition for improving sexual function can be used for a subject in need thereof. Those skilled in the art can readily identify those who are suspected of suffering from such diseases, conditions and disorders using standard diagnostic techniques. Examples of the diseases to which the flavonoids extracted from the lacquer tree may be applied include, but are not limited to, sexual dysfunctions such as impotence, reduced sexual appetite, premature ejaculation, sexual nerve hypersensitivity, ejaculation disorder, ejaculation contract disorder, orgasm disorder, etc.

[31] The pharmaceutical composition of the present invention can be prepared in com bination with a conventional pharmaceutically acceptable carrier. Generally, the active component is mixed with a pharmaceutically acceptable liquid or solid carrier. If necessary, additives such as solvents, dispersants, emulsifiers, buffers, stabilizers, diluents, binders, dis integrants, lubricants, etc. can be used. The composition may take the form of solid formulations such as tablets, granules, powders or capsules, or liquid formulations such as normal liquids, suspensions or oils. These formulations may also be made as dry preparations which can be used in liquid form by addition to a proper carrier before use.

[32] The pharmaceutical composition of the present invention can be administered orally or parenterally, i.e., by way of injection, drop or ointment means.

[33] The pharmaceutically acceptable carriers may be selected according to the mode of administration and formulation. In case of oral formulations, for example, the carriers include starch, lactose, refined sugar, mannite, carboxymethylcellulose, corn starch, or inorganic salts. In the preparation of oral formulations, binders, disintegrants, surfactants, lubricants, fluidity enhancers, sweetening agents, coloring agents or spices

can be additionally used.

[34] In the meantime, the parenteral formulation is prepared by dissolving or suspending the composition containing the flavonoid extracts of the present invention in a diluent, such as distilled water for injection, physiological salt solution, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol or polyethylene glycol and, if necessary, adding disinfectants, stabilizers, tonic agents or analgesics.

[35] The pharmaceutical composition of the invention can be administered via a proper route depending on the formulation. The administration mode includes, but is not specifically limited to, internal, external or injection. For injection administration, intravenous, intra-muscular, subcutaneous or intra-dermal injection is possible.

[36] The amount of the pharmaceutical composition to be administered can be properly determined according to the formulation, the administration route, the purpose of use and the patient's age, weight or symptoms. The amount of the active component in the preparation is, for example, 10 D to 2000 mg/kg (weight) a day for an adult. Of course, the dosage varies depending on the conditions and the actual dosage may be greater or less than the above range. However, those skilled in the art will appreciate that the dosage varies depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.

[37] The present invention, in an additional aspect, provides a functional healthy food for enhancing sexual function comprising the above flavonoids extracted from lacquer tree.

[38] The formulation of the functional healthy food according to the present invention can be prepared according to conventional processes in the art, for example, in the form of a powdered stock material, a capsule, or a liquid preparation.

[39] The amount of the food preparation to be administered can properly be determined according to the formulation, the administration route, the purpose of use and the patient's age, weight or symptoms. The amount of the active component in the preparation is, for example, 5 mg to 2000 mg/kg (weight) a day for an adult. Of course, the dosage varies depending on the conditions and the actual dosage may be outside the above range.

[40] The food composition according to the present invention, depending on the desired type of the composition can include one or more ingredients selected from the group consisting of conventional supplements, additives, and sweetening agents, for example, licorice, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharine, pectin, mannitol, sorbitol, xylitol, guar gum, skimmed milk, oligosaccharides, etc, in

order to increase preference or taste.

[41] It is preferable to use the above ingredient in about 0.01-90 weight % based on the weight of the total composition.

[42] It is apparent that as stated in the above the flavonoids extracted from the lacquer tree according to the present invention activates sexual function, and thus, the flavonoids can be used in the pharmaceutical in animal for treating diseases in need thereof as well as human. Therefore, the present application, in another additional aspect, provides an agent for enhancing sexual function in animal which comprises the flavonoids from the lacquer tree.

[43] The present invention, in a further aspect, provides a drink additive prepared by addition of Chinese herbal medicines to the lacquer tree extracts.

[44] The medicinal drink prepared from the drink additives containing the flavonoids of the invention can be prepared by one of the conventional methods.

[45] For example, into the lacquer extracts of the invention aged in alcohol spirits are added powdered plant extracts selected from the group consisting of Acanthopanax senticosus, Lycium chinense, licorice, dried Rehmannia glutinosa, Codonopsis lanceolata, Angelicae Gigantis Radix, undried ginseng, Liriope muscari, Eucommia ulmoides, Pleuropterus multflorus, Schisandra chinensis, Cornus officinalis, red ginseng, deer horn, garlic, Paeonia japonica, jujube, the fruit of Chinese quince, apple, grape, cherry, apricot, azalea, wild grapes, the fruit of the Actindia chinensis in an appropriate ratio, and the mixuture is soaked, aged and then filtered to give the desired product. The mixing ratio of the lacquer tree extracts and the herbal additives may be determined within the range of about 2 to 50% of the total composition.

[46] According to the present invention, the medicinal drink comprising flavonoids and/ or anhydrous gallic acid extracted from the lacquer tree has no side effects and provides advantageous effects on nutrition and tonicity of the human body. Mode for the Invention

[47] Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

[48] Examples

[49] Example 1: Separation of Flavonoids from Lacquer Tree

[50] The lacquer tree was cut into sections with a length of about 10 cm and then dried in the shade for over one month. 1OL of water was added to 1 Kg of the lacquer tree fragments and the mixture was then extracted three times at 12O ⁰ C for 4 hours. The extract was spray-dried to obtain a powder (60 g, yield: 6%).

[51] The dried crude extract was immersed in 99% spirituous alcohol (dried crude extract : spirituous alcohol = 1 : 10) for ten days and then concentrated under reduced pressure to obtain lacquer tree flavonoids. Alternatively, the alcoholic solution was collected and concentrated under reduced pressure to obtain a spirituous alcohol soluble (24g, 40% of crude extract).

[52] Example 2: Separation of Lacquer Tree Flavonoids

[53] The lacquer tree extract obtained in Example 1 was separated in powdered form using a HIDACHI JPL-7400 prep LC. 0.5g/ml of a sample was injected into an NW-50 column and detected at a flow rate of 30 ml/min at a wavelength of 274 nm. The sample was fractionalized into five groups. The fraction F2 was separated (9.5g, yield: 5%). As a result of RP-HPLC, the fraction F2 was considered to be a single substance and structure thereof was identified (Refer to FIGs. 2 and 3).

[54] Example 3: Identification of structure of Active Fraction (Fraction F2)

[55] For identification of the structure, the resulting fraction F2 was subjected to

IH-NMR and 13C-NMR. The NMR analysis ascertained that the fraction 2 was anhydrous gallic acid, chemical name 3,4,5-trihydroxybenzoic acid (C H O , molecular weight: 170.12, See FIG. 4).

[56] Example 4: Pharmaceutical preparations

[57] Formulation (tablet)

[58] Lacquer tree extract 45 mg

[59] (mixture with the same amount as in Examples 1 to 3)

[60] Lactose 80 mg

[61] Starch 17 mg

[62] Magnesium stearate 3 mg

[63] Crystalline Cellulose 10 mg

[64] In one embodiment of the present invention, provided is a tablet containing the anhydrous gallic acid as an active ingredient. The tablet can be prepared by conventional methods. Preferred formulations are conventional enteric-coated tablets (e.g. hydroxypropylmethylcellulose pthalate), sugar-coated tablets and film-coated tablets.

[65] Example 5: Preparation of Drink

[66] To 150g of the anhydrous gallic acid obtained in Example 3, there were added 16Og of high-fructose corn syrup, 2g of citric acid, 2g of sodium citrate, 0.5g of sodium benzoate, 0.5g of vitamin C, and the remaining volume of purified water, based on a total of l,000L of the drink. A trace amount of flavor was added to the mixture to give a IL drink. Then, the drink was pasteurized. Test results ascertained that the drink of the present invention was proved to be superior in terms of mouth feeling, taste and overall evaluation categories, as compared with conventional drinks.

[67] Example 6: Preparation of Drink Additive

[68] To 0.4% of the anhydrous gallic acid obtained in Example 3, there were added 85% of a deer horn extract (solid content: 0.25%), 13% of fructose, 1% of a cinnamon concentrate (35 brix or more), 0.5% of a concentrated Japanese apricot juice (60 brix or more) to prepare a drink additive. After pasteurization, the drink additive was added in an amount of 0.5% (in the range of 0.2 to 20%) by weight to general drinks (e.g. distilled spirits (also known as a "soju"), beer, Western liquors, traditional drinks, fruit drinks, etc.). Test results of the resulting drinks demonstrated that the drink additive of the present invention is excellent in terms of flavoring, taste and hangover curing.

[69] Experimental Example 1: Effect of Lacquer Tree Flavonoids on Increase in

Sexual Hormones of Rats.

[70] In this experiment, Sprague-Dawley rats weighing about 25Og were used as a control group (n=8, a no-administration group) and a test group (n=8, a group administered with the lacquer tree flavonoids of the present invention). During breeding, rats were freely taken solid feed for rats and distilled water. A solution (100 mg/kg) of the lacquer tree flavonoids obtained in Example 1 in PEG and a solution (100 mg/kg) of flavonoids of the fraction 2 obtained in Example 2 in PEG were orally administered at a predetermined time every morning for 4 weeks. After the final administration, the rats were fasted for 8 hours, anesthetized with ether, and subjected to laparotomy to collect blood from the abdominal aorta. The level of testosterone in the blood was then measured using an RIA method via an r-counter (PACKARD, U.S.). The results are shown in FIG. 1.

[71] As can be seen from the results of FIG. 1, the administration of the flavonoids of the present invention caused a 2.5-fold or more increase in testosterone.

[72] Experimental Example 2: Tests for Increase in Sexual Hormones of Rats.

[73] In this experiment, Sprague-Dawley rats weighing about 25Og were used for a control group (n=8, a no-administration group) and a test group (n=8, a group administered with the lacquer tree flavonoids in Examples 1 to 3). During breeding, rats were freely taken solid feed for rats and distilled water. A solution (100 mg/kg) of the flavonoids in PEG and a solution (100 mg/kg) of anhydrous gallic acid in PEG were orally administered at a predetermined time every morning for 4 weeks. After the final administration, the rats were fasted for 8 hours and anesthetized with ether, and then subjected to laparotomy to collect blood from the abdominal aorta. The levels of testosterone and free testosterone in the blood were then measured using an RIA method via an r-counter (PACKARD, U.S.). The results are shown in FIGS. 5 and 6.

[74] As can be seen from the results of FIG. 5, the administration of the flavonoids of the present invention caused a 1.5-fold or more increase in testosterone. As can be seen from the results of FIG. 6, the administration of the flavonoids of the present invention caused a 3-fold or more increase in free testosterone.

[75] Experimental Example 3: Sperm Function Test in Lacquer Tree Flavonoids- administered Rats [76] 200 mg/kg of the lacquer tree flavonoids was orally administered for 4 weeks in the same manner as in Experimental Example 1 and incised tail of left epididymis was then weighted in order to investigate sperm count in the tail of the epididymis and sperm motility. After incising the left spermatic duct, the tissue was placed onto a petri dish containing 3 mL of modified Tyrode's media and incubated in a 5% CO incubator at 37 ⁰ C for 10 minutes. After removing the tissue from the media, 0.1 ml of the media was mixed with a mixture consisting of 2% eosin and 3% citric acid at a mix ratio of 1 : 1 to prepare a smear sample. 100 sperm per smear sample were then observed under a microscope with 20Ox magnification. Sperm count was calculated using a Makler counting chamber. The test results are shown in Tables 1 and 2. Table 1 shows test results on sperm motility of the rats administered lacquer tree flavonoids for 8 weeks. Table 2 shows test results on sperm count of the rats administered lacquer tree flavonoids for 8 weeks.

[77] Table 1

[78] Table 2

[79] As apparent from the data shown in the above tables, the sperm motility and sperm count of the test group were significantly increased by about 49% and about 73.3%, respectively, as compared to those of the control group.

[80] Experimental Example 4: Effects of Lacquer Tree Flavonoids Administration on Increases in Sexual Appetite and Sexual Intercourse [81] Mating behavior testing was performed on male SD rats in order to verify the effects of the lacquer tree flavonoids on mating behavior of the male rats. A test material at a dose of 50 mg/kg was repeatedly administered once daily for two weeks. After the male rats were caged together with estrus-induced female rats, mating behavior was observed for five minutes. The time, in which the first mating behavior was observed, after caging together, and the frequency of mating behavior were measured on a primary date (the date on which the test material was initially ad-

ministered) and a secondary date (the date on which the test material was finally administered). The results are shown in Tables 3 and 4 below. Table 3 shows the contact- return time in which the male rats administered with lacquer tree flavonoids responded to the estrus-induced female rats. Table 4 shows an increase in the frequency at which the male rats administered with lacquer tree flavonoids mated with the estrus-induced female rats. The data were represented by ± S.E.M and the results were statistically analyzed with T-test. [82] Table 3

[83] Table 4

[84] As can be seen from the data shown in Tables 3 and 4, the contact-return time of the test group was reduced by about 5 seconds and the frequency of mating thereof was significantly increased by about 50%, as compared to the control group.

[85] Experimental Example 5: Test for Hepatotoxicity of Anhydrous gallic acid- containing flavonoids

[86] In this experiment, Sprague-Dawley rats weighing about 25Og were used for a control group (n=8, a no-administration group) and a test group (n=8, a group administered with anhydrous gallic acid-containing flavonoids in which the anhydrous gallic acid was 100 mg/kg and the flavonoids in Example 1 was 100 mg/kg). During breeding, rats were freely given solid feed for rats and distilled water. A solution (200 mg/kg) of the anhydrous gallic acid-containing flavonoids in PEG was orally administered at a predetermined time every morning for 4 weeks. After the final administration, the rats were fasted for 8 hours, anesthetized with ether, and underwent laparotomy. Blood was collected from the abdominal aorta, allowed to stand at ambient temperature and centrifuged at 3,000 rpm for 10 minutes. Only the serum in the supernatant was separated. The levels of AST and ALT in the separated serum were measured with a blood analyzer (Express PLUS (CHIRON. DIAGNOSTIC. USA)), to ascertain whether the anhydrous gallic acid was hepatotoxic. The results are shown in FIGS. 8 and 9. FIG. 7 is a graph showing the level of AST in the serum of male rats administered with anhydrous gallic acid-containing flavonoids. FIG. 8 is a graph showing the level of ALT in the serum of male rats administered with anhydrous gallic acid-containing flavonoids. It can be seen from the above results that upon oral administration, the anhydrous gallic acid-containing flavonoids were not hepatotoxic, and rather, were beneficial to the liver.

[87] Experimental Example 6: Tests for safety of anhydrous gallic acid on Beagle dogs

[88] Tests were performed to investigate the toxicity to non-rodents, to which the anhydrous gallic acid in Example 3 is orally administered once. Male and female Beagle dogs were used for a test group, to which 2,000 and 1,000 mg/kg of the test material was administered, and a control group, to which a placebo capsule containing an excipient only was administered. The number of animals in each of the groups was two and mortality, general symptoms, weight change and autopsy opinions were analyzed for two weeks.

[89] As a result, no dead animal was observed during a test period of two weeks, and temporary vomiting, brownish stool, diarrhea and anorexia (the lack of appetite) were observed. The weight change and autopsy results indicated zero toxicity. As can be seen from these test results, the fact that no dead animal was observed in the Beagle dogs with single oral administration of the anhydrous gallic acid ascertained that a minimum lethal dose (MLD) is more than 2,000 mg/kg.

[90] Experimental Example 7: Effects of Anhydrous Gallic Acid Administration on

Variation in Body Weight of Male Rats

[91] This experiment was performed to confirm whether long term administration of the anhydrous gallic acid obtained in Example 3 affects variation in body weight. Sprague- Dawley rats weighting about 25Og were divided into a control group (n=8, no- administration group) and a test group (n=8, a group administered with anhydrous gallic acid). During breeding, rats were freely taken solid feed for rats and distilled water. A solution (200 mg/kg) of the anhydrous gallic acid obtained in Example 1 in PEG was orally administered at a predetermined time every morning for 4 weeks. The weight of the rats was measured at a predetermined time in the morning once weekly. The results are shown in FIG. 9. As apparent from these results, prominent change in weight was not observed, which means that the anhydrous gallic acid has no side effects on the animals. Industrial Applicability

[92] As apparent from the foregoing, the composition according to the present invention comprising, as active ingredients, flavonoids and anhydrous gallic acid extracted from the lacquer tree were proven to promote testosterone secretion, increase free testosterone secretion, and be safe for use (confirmed by analysis on body weight and serum). Accordingly, the anhydrous gallic acid-containing flavonoids of the present invention can be widely used in foods and medications for treating male climacteric disorders and enhancing sexual function.