A PHARMACEUTICAL COMPOSITION HAVING CHLOROGENIC ACID AS ACTIVE COMPONENT FOR PREVENTING OR TREATING THE DECLINING OF MALE REPRODUCTION CAPABILITY TECHNICAL FIELD The present invention relates to a pharmaceutical composition or a health food containing chlorogenic acid as active ingredient for preventing or treating the decline of male reproduction capability, for example, decline of number of sperm or sperm motility in the mammal including human by the hormone-disrupting chemicals, and the use thereof.

BACKGROUND ART Recently, not a few of environmental protection groups have noticed the careful attention to external hormone-disrupting chemicals. External hormone-disrupting chemicals have been reported that they have similar chemical structures of estrogen, and that about 150 kinds of chemicals have been found up to now. As representative chemicals, dioxin, DDT, dieldrin, nonylphenol, bisphenol or polychlorovinylphenol have been reported. Such external hormone-disrupting chemicals have remained in the water or soil after wasting, which causes the storage of external hormone-disrupting chemicals in animal or human body by absorbing them from the contaminated nature.

Recent researches about hormone-disrupting chemicals revealed that such chemicals cause the malformation, decline of number of sperm, decline of sperm motility or the increase of female characters from male in wild animals, and that such chemicals also cause the decline of number of sperm, decline of sperm motility or the increase of reproductive organ disease in human (Toppari, J. et al., Environ. Health Perspect., 104 Supplement 4, 741-803, 1996).

Dioxin, a polyhalogenated aromatic hydrocarbon, is the most toxic chemical among external hormone-disrupting chemicals, which generates during the combustion of waste or Pb containing gasoline or the pulp manufacturing process, and contaminates the soil or water by remaining in the nature. The dioxin remained in the nature is delivered to the human body through the natural food chain, and it is stored in human body as lipophilic substance.

It has been reported that the concentration of dioxin in the air of Japanese metropolitan cities is 0. 3-1. 65 pg/m3 according to the research of Japanese Environmental Organization in 1997, and that such concentration of dioxin in European or American cities is about 0. 1 pg/m3. The fact that the dioxin concentration in the air of Japan is higher than that of other nations means the rapid elevation of dioxin concentration in the air according to the industrialization in accumulated area. It is estimated that the ordinary Japanese people absorb the dioxin from the nature in the amount of 0. 52-3. 53 pg/lKg of human weight.

In Korea, there is no specific report regarding the dioxin concentration in the nature. It is estimated that the dioxin concentration in the air is not less than that of other industrialized nation, because the rapid industrialization has been made in a short period. According to the report of animal experiment, in the case of dioxin exposure during the fetal, newborn or growing step, the male reproductive organ has been affected by dioxin. It results in various adverse effects, such as, the decline of number of sperm, decline of sperm motility or mal-growing of male reproductive organ.

Therefore, it is known that dioxin is the most toxic chemical to the reproductive organ in mammal and human among the hormone-disrupting chemicals (Roman, B. L. et al., Toxicol, Appl, Pharmacol., 150, 240-253, 1998).

Thus, it is regarded as the most urgent matter to develop the drug for preventing and treating the diseases caused by dioxin.

However, there has been little reported regarding the material to protect the human reproductive function from dioxin until now. Red ginseng extract is regarded as active material for protecting the harmful effect of dioxin in guinea pig (Kim, W. et al., BJU Inteanational, 83, 842-849, 1999). On the other hand, green tea has been reported as protective material against dioxin if drinking green tea is made for a long time (Fifth international green tea symposium, Seoul, 1999).

Further, recent research of our inventor showed that natural phenol or polyphenol derivatives from the leaves or fruits of dicotyledonous plant have an effect to prevent or treat the decline of male reproductive function against hormone-disrupting chemicals, such as, dioxin.

However, we did not find which material can prevent or treat the decline of male reproductive function against hormone-disrupting chemicals at that time.

The present invention reveals the active material for preventing or treating decline of male reproductive function, and this invention has been completed by finding chlorogenic acid as active material.

DISCLOSURE OF INVENTION The object of the present invention is to provide a natural or synthesized chlorogenic acid for preventing or treating the decline of male reproductive function caused by hormone-disrupting chemicals.

Another object of the present invention is to provide a preparation method of chlorogenic acid by purifying or isolating the extract from Rosaceae fruit, for example, apple, pear or peach ; coffee bean ; cacao bean ; or seed of grape. Especially, the preferred origin of chlorogenic acid is green bean or roasted bean of coffee.

Further object of the present invention is to provide a health food or a pharmaceutical composition containing chlorogenic acid as active ingredient for preventing or treating the decline of male reproductive function caused by hormone-disrupting chemicals.

BEST MODE FOR CARRYING OUT THE INVENTION The chlorogenic acid of the present invention is naturally present in the fruits or leaves of dicotyledonous plant. Especially, it is present in the green coffee bean in an amount of 5. 5-10 wt%. It can be extracted using mixed solution of methanol and water, and can be isolated using HPLC-UV [Bicchi et al., J. Agric. Food Chenl., 43, ppl549-1555 (1995)].

As a reference, natural phenol and polyphenol derivatives including chlorogenic acid can be obtained from the premature or mature apple fruit.

The IUPAC chemical name of chlorogenic acid is 3- [ [3- (3, 4-Dihydroxyphenyl) -1-oxo-2-propenyl] oxy]-1, 4, 5-trihydroxycyclohexanecarboxylic acid represented by following formula (C16H18O9, M. W. =354. 30).

Chlorogenic acid It can be also named as 3-caffeoylquinic acid and caffeic acid is produced at the time of hydrolysis. A small amount of isomers, such as, isochlorogenic acid or neochlorogenic acid is contained in naturally present chlorogenic acid.

Table 1 shows the composition of natural phenols and polyphenols in the premature or mature apple fruit.

Table 1 Component Amount (%) Quantitative method caffeic acid 2. 0 1. 0 HPLC method chlorogenic acid 22. 05. 0 HPLC method para-coumaric acid 2. 01. 0 HPLC method prolectin 2. 0 1. 0 HPLC method prolisin 3. 0 2. 0 HPLC method (+)-catechin 5. 0 2. 0 HPLC method (-)-epicatechin 10. 0 2. 0 HPLC method condensed tannin 45. 05. 0 Acid-hydrolysis method The animal experiment shows that chlorogenic acid, a major component of natural phenols and polyphenols, has an effect to prevent or treat the decline of male reproductive function against hormone-disrupting chemicals, such as, dioxin.

For this experiment, 4 weeks aged male rats (Sprague-Dawley) are employed and these rats are divided into a few groups for detecting the effect of preventing or treating the decline of male reproductive function by chlorogenic acid.

To detect such effect, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is injected to the experimental group of rats to induce the decline of male reproductive function. For detecting the decline or the recovery of male reproductive function, the weight of prostate gland and spermatic vesicle after post-mortem is measured. The average weight of prostate gland and spermatic vesicle of rats injected with dioxin is much less than that of control group and the weight of prostate gland and spermatic vesicle is recovered at normal level by the injection of chlorogenic acid of the present invention to the experimental group. This result proves the effect for preventing or treating the decline of male reproductive function by chlorogenic acid.

The decline of sperm motility caused by dioxin and the effect for preventing or treating the decline of male reproductive function by chlorogenic acid are observed through the optical microscope. Even though the injection of dioxin declines the sperm motility about 27% compared to control group, the injection of chlorogenic acid recovers the sperm motility at normal level. Further, the decline of number of sperm by dioxin and the effect for preventing or treating the decline of male reproductive function by chlorogenic acid are confirmed by measuring the number of sperms through the optical microscope. The number of sperms declined by the injection of dioxin can be recovered at normal number of sperms by the injection of chlorogenic acid.

According to the result of the animal experiment, the decline of number of sperm, decline of sperm motility and decline of weight portion of prostate gland and spermatic vesicle to body induced by dioxin can be recovered at normal level by the injection of chlorogenic acid. Chlorogenic acid is, therefore, proved as a material to have an effect to prevent or treat the decline of male reproductive function against hormone-disrupting chemicals, such as, dioxin. Further, chlorogenic acid is also proved as useful material to have an effect to prevent or treat the decline of male reproductive function at the least concentration compared to other natural phenols or polyphenols.

Health food containing chlorogenic acid as active ingredient can be provided in a conventional method. As an example, oral preparation, such as, tablet, capsule or soft capsule can be manufactured by mixing with diluents or any nutrients.

The pharmaceutical composition containing chlorogenic acid as active ingredient can be manufactured by mixing with additives, such as, carriers and/or diluents. Of course, any formulations including oral formulations can be prepared, for example, tablets, capsules, injections or edible solutions.

The dosage of chlorogenic acid in such pharmaceutical composition can be varied by the purpose of administration. Generally, 0. 1-100 mg of chlorogenic acid to 1 Kg body weight is available.

The present invention will be more specifically explained by the following example. However, it should be understood that the example is intended to illustrate but not in any manner to limit the scope of the present invention.

EXAMPLES (Preparation Example 1) Preparation of chlorogenic acid from apple extract The fruit of apple was washed and squeezed. After treatment of pectin-decomposing enzyme to obtained squeezed juice, the juice was filtered and purified with hot water using column. Using 65% of ethanol, the purified juice was extracted and concentrated in reduced pressure. Finally, a powder of apple extract was obtained by spray-drying. The content of polyphenol in apple extract was more than 50% and main components of polyphenol were tannin, caffeic acid, chlorogenic acid, para-coumaric acid, prolectin, prolisin, catechin and epicatechin. Chlorogenic acid (95% purity) was obtained by separating from polyphenols using HPLC.

(Preparation Example 2) Preparation of chlorogenic acid from coffee extract The green coffee bean was cut into about 5 mm size. After removing unregular coffee bean, the extraction was carried out using deionized water. Finally, a powder of coffee extract was obtained by spray-drying. The content of polyphenol in coffee extract was more than 50% and main components of polyphenol were chlorogenic acid and tannin.

Chlorogenic acid (97% purity) was obtained by separating from polyphenols using HPLC.

(Preparation Example 3) Preparation of chlorogenic acid from cacao extract Using 60% of acetone, the extraction was carried out from dried cacao bean and cacao shell during 24 hours. Obtained cacao extract was centrifuged, and supernatant was obtained by the separation. Using 60% of acetone, the residue was extracted and centrifuged to obtain the supernatant for 4 more times. Obtained supernatant was concentrated and filtered. Finally, cacao extract was obtained by removing chlorophyl and by carrying out the extraction using acetone. The content of polyphenol in cacao extract was more than 50% and main components of polyphenol were chlorogenic acid and tannin. Chlorogenic acid (94% purity) was obtained by separating from polyphenols using HPLC., (Example 1) Animal experiment protocol The experimental protocol was designed to detect the effect of chlorogenic acid against 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by measuring the weight of prostate gland and spermatic vesicle, number of sperm and sperm motility.

(1) Experimental animal 40 numbers of 4 weeks aged male rats (Sprague-Dawley) are employed, and adapted to experimental surroundings for 1 week before experiment.

(2) Experimental material 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (NIH, USA) was used, and marketed chlorogenic acid (98%, Aldrich, USA) was also used for testing material. They were all stored in a dark place.

TCDD was dissolved with acetone and diluted with cone oil. Such diluted TCDD was intraperitoneally injected only once in a concentration of 15 ug/kg. Chlorogenic acid was diluted with distilled water and diluted chlorogenic acid was intraperitoneally injected every day after 1 week from the date of TCDD injection in each concentration of 11 mg/kg, 22 mg/kg into 2 groups respectively. Each of them was fully stirred and solubilized before use.

(3) Design of experimental groups Group A, B, C and control group consist of 10 numbers of rats as shown in following scheme. 1 2 3 4 5 6 TCDD Nontreatment A group L I TCDD Injection of chlorogenic acid in 1Irng/hg every day B group TCDD Injection of chlorogenic acid in 22mg/kg every day C group Nontreatment Control group (solution) (Example 2) Measurement of change of reproductive organ and sperm of experimental animal (1) Measurement of weight of accessory sex glands The weight of prostate gland and spermatic vesicle of experimental rats was measured after removing the lipid.

(2) Sperm motility Right side of caudal epididymis is removed and put on the 35 mm petri dish containing 3 ml of HBSS (pH 7. 0) at 37°C. Using surgical knife No. 15, caudal epididymis was scratched 2 or 3 times. After closing the cover, the petri dish was shaked and 1 or 2 drop of solution was taken by pipet. 1 or 2 drop of solution was laid on slide glass and observed by 100x optical microscope to find the sperm motility of about 200 sperms.

The moving sperm portion was calculated in percent ratio.

(3) Sperm head counting Right side of testicle was weighed after removing the epithelium and put on the tube with 20 ml of distilled water. After complete homogenization, sonication for 5 minutes was carried out in sonicator. 2 ml of treated solution was distilled 5 fold by adding 8 ml of distilled water and distilled solution was transferred to hemocytometer using pipet. The number of sperm was counted with 400x optical microscope.

Caculation equation = (number of sperm head x square factor x hemocytometer factor x dilution factor)/weight of testicle = (number of sperm head x 1 x 104 x (20x5))/weight of testicle (Example 3) Result of experiment (1) Weight of accessory sex glands Accessory sex glands can be classified into prostate gland and spermatic vesicle. The weight of such accessory sex glands was divided into the weight of rats. The result was shown in Table 2.

The group injected by TCDD showed a remarkable decline of weight portion of accessory sex glands into the weight of rat, whereas the group recovered and injected by chlorogenic acid showed the recovery of weight portion of accessory sex glands into the weight of rat at normal level.

Table 2. Weight portion of accessory sex glands (w/w%) weight of prostate weight of spermatic Group gland/weight of rat vesicle/weight of rat A 0. 0220. 006 0. 254 ~ 0. 052 B 0. 033 0. 004 0. 366 ~ 0. 034 C 0. 034 0. 002 0. 354 ~ 0. 023 Control 0. 040 0. 005 1 0. 070 (2) Sperm motility Table 3 showed the percent of sperm motility among 200 sperms.

The sperm motility was declined about 27% by the injection of TCDD and the injection of chlorogenic acid showed the recovery of sperm motility at normal level Table 3. Sperm motility Group Sperm motility A 52. 15 3. 51 % B 63.60 ~ 3.09% C70. 10 5. 93% Control 79. 49 ~ 3.51% (3) Sperm head counting Table 4 showed the result of sperm head counting. The number of sperm head declined by the injection of TCDD was recovered by the injection of chlorogenic acid at normal level.

Number of sperm = (number of sperm head x 1 x 104 x (20 x 5))/weight of testicle Table 4. Sperm head counting Group Number of sperm head A (89. 7 + 8. 6) x 10 (' B (104. 8 ~ 9.7) # 106 C (102. 0 8. 4) x lOt' Control (108. 8 5. 6) x 10h TCDD has been known the strongest hormone-disrupting chemicals in dioxin derivatives. Therefore, the remarkable decline of weight portion of accessory sex glands, sperm motility and decline of number of sperms have been induced by the injection of TCDD.

However, the decline of weight portion of accessory sex glands, sperm motility and decline of number of sperms have been recovered by the injection of chlorogenic acid at normal level, which proves that chlorogenic acid has an effect for preventing or treating the decline of male reproductive function caused by hormone-disrupting chemicals.