COMPOSITION FOR PREVENTING AND TREATING MALE INFERTILITY, CONTAINING MIXED HERBAL EXTRACT AS ACTIVE INGREDIENT AND USE THEREOF

Abstract

A composition and health functional food for treating male infertility using a mixed herbal mixture. Through an in vitro sperm mobility promotion effect evaluation method experiment for investigating effects of an herbal extract of Morinda officinalis, Cuscuta seed, and/or Allium cepa of the present invention on changes in sperm count and mobility in human semen, it was verified that the composition increased sperm mobility. In addition, through an in vivo varicocele animal model evaluation method for investigating effects of the herbal extract on changes in sperm count and mobility in an in vivo varicocele animal model, it was verified that the composition samples of the present invention increased the sperm count in the semen obtained from spermiducts of a sample-administered group among varicocele-induced groups. Therefore, the herbal extract can be used in a pharmaceutical composition, health functional food, and health supplement food for treating male infertility.

Claims

1. A pharmaceutical composition comprising a combination of extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb as an active ingredient for treating male infertility.

2. The pharmaceutical composition of claim 1, wherein the combination of extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb has a range of mixed ratios based on the dried weight of each herb of 0.01-100:0.01-1:1-100 (w/w).

3. The pharmaceutical composition of claim 1, wherein the male infertility is selected from the group consisting of an adverse response of an anti-cancer agent, azoospermia, oligospermia, asthenospermia, and teratospermia.

4. A health functional food comprising a combination of extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb as an active ingredient for the improvement of male infertility.

5. The health functional food of claim 4, wherein the health food is selected from the group consisting of powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leached tea, or beverage type.

6. (canceled)

7. A method of treatment of male infertility comprising administering the pharmaceutical composition of claim 1 to a subject in need of treatment of male infertility.

8. (canceled)

9. The method of claim 7, further comprising the step of: obtaining extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb; mixing the extracts together to obtain the combination of extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb; and administering the combination of extracts to the subject in need of treatment of male infertility.

10. The method of claim 8, wherein the combination of extracts from Morinda Officinalis root, Cuscuta seed, and Allium cepa bulb has a range of mixed ratios based on the dried weight of each herb of 0.01-100:0.01-1:1-100 (w/w).

11. The method of claim 7, wherein the male infertility is selected from the group consisting of an adverse response of an anti-cancer agent, azoospermia, oligospermia, asthenospermia, and teratospermia.

12. A method of manufacturing a medicament for the treatment of male infertility in a human or mammal comprising the steps of extracting a composition from Morinda Officinalis root, extracting a composition from Cuscuta seed, and extracting a composition from Allium cepa bulb; and mixing the compositions together at mixed ratios ranging from 0.01-100:0.01-1:1-100, based on the dried weight of each composition (w/w).

Description

DESCRIPTION OF DRAWINGS

[0050] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[0051] FIG. 1 shows the improving effect of test sample on the human seminal count and human seminal motility in human semen (the preliminary test on each test samples of CINTM1 (10:1:10), CINTM1 (5:1:5), and CINTM1 (1:1:1) was performed before the test);

[0052] FIG. 2 shows the improving effect of test sample on the human seminal count and human seminal motility in human semen between extract of each herb and combined herb (CINTher-1: extract of Morinda Officinalis root; CINTher-2: extract of Cuscuta seed; CINTher-3: extract of Allium cepa bulb; CINTM1: extract of combined herb mixed with 10:1:10, respectively);

[0053] FIG. 3 shows the improving effect of test sample on the sperm count in the varicocoele-induced animal group (CTR: Control, OP: Operation);

[0054] FIG. 4 presents the improving effect of test sample on the sperm motility in the varicocoele-induced animal group (CTR: Control, OP: Operation).

BEST MODE FOR CARRYING OUT THE INVENTION

[0055] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0056] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

[0057] The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1

Preparation of Each Component

[0058] 1-1. The Extract of Morinda officinalis Root

[0059] 3,000 g of dried root of Morinda officinalis purchased from Kyung-dong market (Dukhyundang Pharmacy) located in Seoul were washed and added to 6 L of 95% ethanol to perform extraction with sonication extraction for 90 mins 3 times. The residue was filtered with filter paper to afford extract and the filtrated extract was concentrated under vaccuo. The concentrated extract was dried with freeze dryer (ScanVac, Labogene) to afford 139.4 g (Yield: 4.6%) of dried extract of Morinda officinalis root (designated as CINTher-1 hereinafter), which is used as a comparative test sample by dissolving into physiological saline solution and vigorously stirring for 2 mins to store at 4 C. prior to use in following experiment.

1-2. The Extract of Cuscuta Seed

[0060] 5.2 kg of dried seed of Cuscuta Chinensis purchased from Kyung-dong market (Backjangsaeng Pharmacy) located in Seoul were washed, pulverized, and added to 5 L of 95% ethanol to perform extraction with sonication extraction for 90 mins 2 times. The residue was filtered with filter paper to afford extract and the filtrated extract was concentrated under vaccuo. The concentrated extract was dried with freeze dryer (ScanVac, Labogene) to afford 264.3 g (Yield: 4.36%) of dried extract of Cuscuta seed (designated as CINTher-2 hereinafter), which is used as a comparative test sample by dissolving into physiological saline solution and vigorously stirring for 2 mins to store at 4 C. prior to use in following experiment.

1-3. The Extract of Allium cepa Bulb

[0061] 1,000 g of bulb of Allium cepa purchased from Changnyeong market located in Kyungsangnamdo (Korea) were washed, dried and added to 5 L of 70% ethanol to perform extraction with reflux extraction for 2 hours at 70 C. The residue was filtered with filter paper to afford extract and the filtrated extract was concentrated under vaccuo. The concentrated extract was dried with freeze dryer (ScanVac, Labogene) to afford 13.8 g (Yield: 1.38%) of dried extract of Allium cepa bulb (designated as CINTher-3 hereinafter), which is used as a comparative test sample by dissolving into physiological saline solution and vigorously stirring for 2 mins to store at 4 C. prior to use in following experiment.

Example 2

Preparation of Combined Formulation (CINTM1)

[0062] The dried extract of Morinda officinalis root, Cuscuta seed and Allium cepa bulb prepared in Example 1 was thoroughly mixed with different mixed weigh ratios (10:1:10/5:1:5/1:1:1) using by mixer (Vortex genie-2, scientific industries, USA) to obtain the various kinds of invention formulation (designated as CINTM1 hereinafter), which are used as a test samples in following experiment.

Experimental Example 1

In Vitro Sperm Quality Promoting Effect Evaluation

[0063] In order to investigate the effect of the inventive extract obtained in Examples on the sperm number and sperm motility of human spermatozoa, following in vitro experiment was performed according the procedure disclosed in the literature (Martin Blomberg Jensen, Poul J. Bjerrum, Torben E. Jessen, John E. Nielsen, Ulla N. Joensen, et al. (2011) Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Human Reproduction, Vol. 26, No. 6, pp. 1307-1317).

1-1. Procedure

[0064] After determining the number of sperm and sperm motility of human semen delivered from called patients volunteers suffering from infertility (Urology division of Chonbuk National University Hospital in South Korea, 20 to 50 years old, 85 patients), the semen was poured into incubator maintaining 37 C. The liquefaction of the semen was sufficiently completed and then 200 L of test sample was added to 20 L of semen to the extent to reach 2 mg/ml of final concentration. Three hour after the addition, the sperm motility was determined by using counting chamber (MAKLER counting chamber, self-medical instruments, Haifa, Israel) and microscope (Axiovert25, ZEISS, USA).

[0065] 3H buffer solution {Hams F-10(N6635, Sigma chemical Co/USA)+0.4% HAS (A1635, Sigma Chemical Co. USA)+12 mM HEPES (H4034, Sigma Chemical Co. USA) [3H, pH 7.4]} was used as a control group. The test samples were also dissolved in the same buffer solution as mentioned the above, adjusted to pH 7.4 and centrifuged by centrifuge apparatus (A32010(1), LABOGENE, 13,000 rpm, 2 mins, 4 C.) to afford the supernatant for determination of sperm count and motility.

[0066] The sperm motility was calculated according to following empirical formulae 1.

Percentage of motility=A (the mean number of motile sperm)/B (total number of sperms) [Empirical formulae 1]

1-2. Test Result

[0067] The sperm motility in the group treated with three kinds of inventive combinations, i.e., CINTM1(10:1:10), CINTM1(5:1:5), and CINTM1(1:1:1) was sharply increased by 70% in the group CINTM1(10:1:10) at the concentration of 0.05 mg/ml, 62% in the group CINTM1(5:1:5) at the concentration of 0.5 mg/ml and 63% in the group CINTM1(1:1:1) at the concentration of 0.5 mg/ml, respectively. Among the test sample groups, the CINTM1 (10:1:10) group showed most potent increasing effect on sperm motility comparing with the other groups with different mixed ratio and the comparative test group treated with the extract of sole herb, i.e., CINTher-1, CINTher-2 and CINTher-3 (See FIG. 1).

[0068] Specifically, the test groups treated with CINTM1(10:1:10) showed unexpectedly superior effect on the sperm motility at the same concentration (59.5%, 0.05 mg/ml) comparing with the comparative test group treated with the extract of sole herb, i.e., CINTher-1(55%), CINTher-2 (53.5%) and CINTher-3(48.0%), respectively. (See FIG. 2).

[0069] Accordingly, it has been confirmed that the combination of each herb (CINTM1) showed synergistic effect on sperm motility activity.

Experimental Example 2

In Vivo Sperm Quality Promoting Effect Evaluation

[0070] In order to investigate the effect of the inventive extract obtained in Examples on the sperm number and sperm motility of human spermatozoa, following in vivo experiment was performed according the procedure disclosed in the literature (Zhang L T, Kim H K, Choi B R, Zhao C, Lee S W, Jang K Y, et al. (2014) Analysis of testicular-internal spermatic vein variation and the recreation of varicocoele in a Sprague-Dawley rat model. Andrology. 2, 466-73).

2-1. Establishment of In Vivo Varicocoele Animal Model

[0071] In order to establish In vivo varicocoele animal model for use in following experiment, 9 weeks old Sprague-Dawley rats weighing 300 to 350 g (purchased from KOATECH Co. Ltd located at Pyeongtaek city, KOREA) were bred after acclimating to environment for 1 week. The rats divided with four rats per cage, were fed standard rat chow and freely access to purified water. The rats were maintained in the animal facility under constantly controlled environmental conditions (internal temperature: 18 C. to 23 C., relative humidity: 40 to 60%) and 12 hours light-dark cycle).

[0072] Each rat was intraperitoneally anaesthetized using by a mixture of Ketamine (50 mg/kg) and Xylazine (25 mg/kg) and the renal vein connected to venae cava was exposed after approaching left renal through a midline abdominal incision. The left renal vein was ligated with a 5.0 nylon suture with a metal probe (outer diameter: 0.85 mm) and the metal probe was withdrawn, which leads to 50% reduction in the diameter of the left renal vein and a consistent constriction of the vessel at the point of the ligation. ISV (Internal spermatic vein) branch was ligated to induce varicocoele for use as a varicocoele-induced animal group and four weeks after the ligation, the test samples dissolved in physiological saline solution were orally administrated into the experimental rats.

2-2. Evaluation of Potency in in Vivo Animal Model

[0073] 2 ml/day of test samples or physiological saline solution (PSA) had been orally administrated into two test groups for 28 days after the operation, i.e., varicocoele-induced animal group {(1) PSA: 2 ml/day, (2) CINTM1(10:1:10):100 mg/kg/2 ml/day, (3) CINTM1(10:1:10):200 mg/kg/2 ml/day} and normal control group {(1) PSA:2 ml/day, (2) CINTM1(10:1:10):100 mg/kg/2 ml/day, (3) CINTM1(10:1:10):200 mg/kg/2 ml/day} and then the weight of rats was weighed. The seminal duct of rat was delivered under general anesthesia. The sperm count and motility in the seminal duct were determined and the weight of bilateral testis and epididymis was weighed to compare.

2-3. Test Result

[0074] As shown in FIG. 3, the sperm count in the varicocoele-induced animal group treated with inventive combination showed sharply increased comparing with the group treated with PSA. The sperm count in the varicocoele-induced animal group treated with inventive combination was 20.9112.2510.sup.6/ml (100 mg/kg/2 ml/day) and 28.638.9010.sup.6/ml (200 mg/kg/2 ml/day) while that in the varicocoele-induced animal group treated with PSA was only 18.259.5310.sup.6/ml. (See FIG. 3).

[0075] As shown in FIG. 4, the sperm motility in the varicocoele-induced animal group treated with inventive combination showed sharply increased comparing with the group treated with PSA. The sperm motility in the varicocoele-induced animal group treated with inventive combination was 53.820.7% (100 mg/kg/2 ml/day) and 53.312.1% (200 mg/kg/2 ml/day) while that in the varicocoele-induced animal group treated with PSA was only 31.318.8%. (See FIG. 4).

Mode for Invention

[0076] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

1. Preparation of Powder

[0077]

TABLE-US-00001 CINTM1 20 mg Lactose 100 mg Talc 10 mg

[0078] Powder preparation was prepared by mixing above components and filling sealed package.

2. Preparation of Tablet

[0079]

TABLE-US-00002 CINTM1 10 mg Corn Starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

[0080] Tablet preparation was prepared by mixing above components and entabletting.

3. Preparation of Capsule

[0081]

TABLE-US-00003 CINTM1 10 mg Crystalline cellulose 3 mg Lactose 14.8 mg Magnesium stearate 0.2 mg

[0082] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

4. Preparation of Injection

[0083]

TABLE-US-00004 CINTM1 10 mg Mannitol 180 mg Sterilized distilled water for injection 2974 mg Na.sub.2HPO.sub.412H.sub.20 26 mg

[0084] Injection preparation was prepared by dissolving active component, and then filling all the components in 2 ml per ample by conventional injection preparation method.

5. Preparation of Liquid

[0085]

TABLE-US-00005 CINTM1 20 mg Isomerized glucose 10 g Mannitol 5 g distilled water optimum amount

[0086] Liquid preparation was prepared by dissolving active component in distilled water, adding lemon flavor, mixing together with distilled water and then filling all the components in 100 ml brown bottle and sterilizing by conventional liquid preparation method.

6. Preparation of Health Food

[0087]

TABLE-US-00006 CINTM1 1000 mg Vitamin mixture optimum amount Vitamin A acetate 70 g Vitamin E 1.0 mg Vitamin B.sub.1 0.13 mg Vitamin B.sub.2 0.15 mg Vitamin B.sub.6 0.5 mg Vitamin B.sub.12 0.2 g Vitamin C 10 mg Biotin 10 g Amide nicotinic acid 1.7 mg Folic acid 50 g Calcium pantothenic acid 0.5 mg Mineral mixture optimum amount Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Monopotassium phosphate 15 mg Dicalcium phosphate 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

[0088] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

7. Preparation of Health Beverage

[0089]

TABLE-US-00007 CINTM1 1000 mg Citric acid 1000 mg Oligosaccharide 100 g Apricot concentration 2 g Taurine 1 g Distilled water 900 ml

[0090] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 C. for 1 hour, filtered and then filling all the components in 2 L ample and sterilizing by conventional health beverage preparation method.

[0091] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0092] As described in the present invention, the combined herb composition shows potent treating effect on human infertility, which is confirmed by various experiments, for example, In vitro sperm quality promoting effect evaluation as well as in vivo varicocele animal model evaluation method for investigating effects of the herbal extract on changes in sperm count and mobility in an in vivo varicocele animal model, it was verified that the inventive combined extract showed more potent increasing effect on the sperm count and sperm motility. Therefore, the herbal extract of the present invention can be usefully used in a pharmaceutical composition, health functional food, and health supplement food for preventing and treating male infertility.