Drug combination for the treatment of infertility and its preparation methods and applications

Abstract

The present invention provides a drug combination for the treatment of infertility and its crude drug comprises the medical ingredients with their weight proportions listed as below: 10-20 amounts by weight of Rehmannia, 10-20 amounts by weight of Chinese Peony, 10-20 amounts by weight of Asiatic Dogwood, 10-20 amounts by weight of Chinese Yam, 10-20 amounts by weight of Poria, 15-25 amounts by weight of Astragalus, 15-25 amounts by weight of Codonopsis, 10-20 amounts by weight of Epimedium and 10-20 amounts by weight of Jujube. The compatibility of this drug combination invented is precise, appropriate and possess outstanding curative effects. As shown in many experiments, the drug combination invented is safe and reliable, its toxic & side-effect is low and it has obvious improvement effects on female infertility caused by androgen, fallopian tube inflammation, tubal blockage and other factors. Furthermore, it can not only significantly improve the success rate of pregnancy, but also improve immunity and enhance the body's anti-fatigue and hypoxia tolerance. In short, the invention provides a safe and reliable new choice for clinical medication.

Claims

1. A method of treating infertility in a subject suffering therefrom, comprising administrating to the subject a pharmaceutically effective dosage of a composition which is prepared by the following steps: (1) obtaining 80 g dried Nelumbo nucifera leaves and cutting said leaves into pieces and extracting with 500 ml distilled water at 4° C. for 12 hours; then (2) filtering through a Whatman paper No. 1; and (3) concentrating by evaporation; and (4) lyophilizing to yield Nelumbo nucifera leaves extract (NLE); then (5) adding NLE to a plurality of crude drugs and then add a solvent, and then extracting to produce said composition; wherein a crude drug is defined as a plant material prior to extraction; and wherein the plurality of crude drugs include: 15 kg of Rehmannia, 15 kg of Chinese Peony, 15 kg of Asiatic Dogwood, 15 kg of Chinese Yam, 15 kg of Poria, 20 kg of Astragalus, 20 kg of Codonopsis, 15 kg of Epimedium, 15 kg of Jujube, and 60 kg of Acacia extracts.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1: Preparative Method for the Drug Combination of Present Invention

(1) Presciption:

(2) 15 kg of Rehmannia, 15 kg of Chinese Peony, 15 kg of Asiatic Dogwood, 15 kg of Chinese Yam, 15 kg of Poria, 20 kg of Astragalus, 20 kg of Codonopsis, 15 kg of Epimedium and 15 kg of Jujube.

(3) Preparative Method:

(4) Respectively taking all kinds of crude drugs, adding water independently and decocting for three times. For the first time, it is required to add the water equivalent to 6 times of the crude drug weight, soak for 1 hour and then decocted for 2 hours. For the second time, it is required to add the water equivalent to 5 times of the crude drug weight, soak for 1 hour. For the third time, it is required to add the water equivalent to 4 times of the crude drug weight, decocted for 1 hour, filter out, combine the filtrate, heat and concentrate until the relative density has reached 1.09-1.12 under 60-65° C. Finally, it is required to carry out spray dehydration of above concentrated solution by use of high speed centrifugal spray dryer and prepare into dry extract powders of various crude drugs (80-120 meshes).

(5) Mixing all kinds of dry extract powders, adding appropriate amount of fillers and prepare drug granules.

Embodiment 2: Preparative Method for the Drug Combination of Present Invention

(6) Presciption:

(7) 15 kg of Rehmannia, 15 kg of Chinese Peony, 15 kg of Asiatic Dogwood, 15 kg of Chinese Yam, 15 kg of Poria, 20 kg of Astragalus, 20 kg of Codonopsis, 15 kg of Epimedium and 15 kg of Jujube, 75 kg of Paricalcitol, and 60 kg of Acacia extracts. Preparative method:

(8) Nelumbo nucifera leaves were harvested from field and the leaves were cleaned with water and air-dried. 80 g dried leaves were cut into small pieces and extracted with 500 ml distilled water in 4° C. in a period of 12 hours. The extract was filtered through a Whatman paper No. 1, and concentrated by evaporation, and lyophilized to yield Nelumbo leaf extract (NLE). Respectively taking all kinds of crude drugs, adding water and decocting for three times after the mixing. For the first time, it is required to add the NLE to 6 times of the crude drug weight, soak for 1 hour and then decocted for 2 hours. For the second time, it is required to add the NLE to 5 times of the crude drug weight and decocted for 1 hour. For the third time, it is required to add the NLE to 7 times of the crude drug weight, decocted for 1 hour, filter out, combine the filtrate, heat and concentrate until the relative density has reached 1.09-1.12 under 60-65° C. Finally, it is required to carry out spray dehydration of above concentrated solution by use of high speed centrifugal spray dryer and prepare into dry extract powders of various crude drugs (80-120 meshes). Mixing all kinds of dry extract powders, adding appropriate amount of fillers and prepare capsules.

(9) The crude drugs can consists of 15 kg of Rehmannia, 15 kg of Chinese Peony, 15 kg of Asiatic Dogwood, 15 kg of Chinese Yam, 15 kg of Poria, 20 kg of Astragalus, 20 kg of Codonopsis, 15 kg of Epimedium and 15 kg of Jujube, 75 kg of Paricalcitol, and 60 kg of Acacia extracts.

(10) The Acacia extracts of the present invention are extracted from Acacia heartwood, bark, branches, flowers, twigs, roots and leaves. Extracts with preferred xanthine oxidase inhibitory activity is extracted from heartwood, and the second preferred one is extracted from bark. The Acacia extract by organic solvent, such as ethanol, of the present invention is further separated in liquid-liquid fraction with ethyl acetate, n-butyl alcohol and water to generate ethyl acetate fraction, n-butyl alcohol fraction and water fraction. The ethyl acetate fraction with better xanthine oxidase inhibitory activity can isolate eight major compounds, including 3,7,8,3′,4′-pentahydroxyflavone (Melanoxetin), 7,8,3′,4′-tetrahydroxyflavon, 3,4,2′,3′,4′-pentahydroxy trans-chalcone (Okanin), 7,8,3′,4′-tetrahydroxy-3-methoxyflavone (Transilitin), 3,7,8,3′-tetrahydroxy-4′-methoxyflavone, 7,8,3′-trihydroxy-3,4′-dimethoxyflavone, 7,3′,4′-trihydroxyflavone and 7,3′,4′-trihydroxy-3-methoxyflavone, wherein the substances with better xanthine oxidase inhibitory activity are melanoxetin and okanin, and the best one is okanin. The xanthine oxidase inhibitory activity of melanoxetin and okanin is 17 fold and 63 fold higher than current gout treating drug allopurinol. Therefore, they are potential substitutes for allopurinol which has side effect. The Acacia of the present invention includes but is not limited to Acacia acinacea, Acacia albida, Acacia aneura, Acacia Arabica, Acacia auriculiformis, Acacia baileyana, Acacia baileyana, Acacia bealbat, Acacia binervia, Acacia brachybotrya, Acacia bussei, Acacia bynoeana, Acacia caesia, Acacia calamifolia, Acacia cardiophylla, Acacia catechu, Acacia cavenia, Acacia concinna, Acacia confusa, Acacia cornigera, Acacia cultriformis, Acacia cultriformis, Acacia cyanophylla, Acacia cyclopis, Acacia dealbara, Acacia decora, Acacia decurrens, Acacia elongate, Acacia falcate, Acacia famesiana, Acacia fimbriate, Acacia giraffae, Acacia gregii, Acacia gummifera, Acacia holosericea, Acacia homalophylla, Acacia horrida, Acacia howittii, Acacia implexa, Acacia juniperina, Acacia karroo, Acacia kettlewelliae, Acacia koa, Acacia lenticularis, Acacia leprosa, Acacia leucophloea, Acacia longifolia, Acacia mangium, Acacia mearnsii, Acacia melanoxylon, Acacia mellifera, Acacia merrillii, Acacia mollissima, Acacia nigrescens, Acacia nilotica, Acacia paniculata, Acacia paradoxa, Acacia pendula, Acacia pennata, Acacia penninervis, Acacia podalyriifolia, Acacia pravissima, Acacia prominens, Acacia pruinosa, Acacia pubescens, Acacia pycnantha, Acacia retinodes, Acacia richii, Acacia rigens, Acacia rubida, Acacia salicina, Acacia Senegal, Acacia seyal, Acacia sinuate, Acacia spectabilis, Acacia spirocarpa, Acacia suaveolens, Acacia terminalis, Acacia vestita, Acacia victoriae, Acacia woodii, wherein the preferable Acacia is Acacia melanoxylon, Acacia nigrescens or Acacia confusa, and the most preferred is Acacia confusa.

Embodiment 3: Preparative Method for the Drug Combination of Present Invention

(11) Presciption:

(12) 20 kg of Rehmannia, 10 kg of Chinese Peony, 10 kg of Asiatic Dogwood, 10 kg of Chinese Yam, 10 kg of Poria, 15 kg of Astragalus, 15 kg of Codonopsis, 10 kg of Epimedium and 10 kg of Jujube.

(13) Preparative Method:

(14) Taking various crude drugs, carrying out the ethanol soakage extraction with 55-65% v/v ethanol after mixing. After recycling the ethanol with extracting solution, it is required to concentrate, add appropriate amount of excipients and prepare tablets.

Embodiment 4: Preparative Method for the Drug Combination of Present Invention

(15) Presciption:

(16) 10 kg of Rehmannia, 20 kg of Chinese Peony, 20 kg of Asiatic Dogwood, 20 kg of Chinese Yam, 20 kg of Poria, 25 kg of Astragalus, 25 kg of Codonopsis, 20 kg of Epimedium and 20 kg of Jujube, 75 kg of Paricalcitol. Paricalcitol is a vitamin D.sub.2 derived sterol lacking the carbon-19 methylene group found in all natural vitamin D metabolites. A novel class of vitamin D-related compounds, namely the 1α-hydroxy-19-nor-vitamin D analogs, as well as a general method for their chemical synthesis are disclosed in U.S. Pat. No. 5,710,294 and U.S. Pat. No. 5,342,975. Furthermore, U.S. Pat. No. 6,359,012 provides the method for making 24(s)-hydroxyvitamin D.sub.2, which is the first sterospecific synthesis of 24(s)-hydroxyvitamin D.sub.2 by coupling of (S)-(+)-2,3-dimethyl-2-triethylsilyloxybutyraldehyde and a vitamin D phosphine oxide derivative to form a C-3 and C-24 diprotected trans-vitamin D.sub.2 which is then deprotected and irradiated to yield the 24(S)-hydroxyvitamin D.sub.2.

(17) Preparative Method:

(18) Taking various crude drugs and carrying out the ethanol reflux extraction with 80-95% v/v ethanol after mixing. After recycling the ethanol with extracting solution, it is required to leave the concentrated solution for stand-by application. Adding water to gruffs and decocting for 2 times, the amount of water addition shall be 6-10 times equivalent to the crude drug weight at each time and it is required to decocted for 1 hour, collect the water decoction, merge with the concentrated solution for condensation and drying and add appropriate amount of excipients and prepare pills.

(19) The following testing examples are used to specify the beneficial effects of present invention. The drug combination obtained from the above embodiment 1 of present invention is taken as the test drug to conduct the following experimental investigations, which can manifest the remarkable effects of this drug combination.

(20) Among all testing drugs, the dosage shall be calculated as per the crude drug weight (g/kg) equivalent to the corresponding raw material extracted.

Testing Example 1: Effects on Infertility

(21) (1) Effects of the drug combination of present invention on mice with sterility induced by androgens

(22) 1. Experimental Materials

(23) TP (testosterone propionate), the drug combination of present invention, Clomifenecitrate capsules (commercially available), Kunming mice. Gynecological endocrine ELISA kit and instrument, electronic balance, low speed autobalancing centrifuge, etc.

(24) 2. Experimental Methods and Results

(25) 2.1 Animal Grouping and Model Building

(26) 120 Kunming female mice (20 days old) were randomly divided into 6 groups according to their weights and there were 20 in each group. 20 mice in the normal control group were subcutaneously injected with saline and the remaining 100 mice were used in modeling and subcutaneously injected with TP (testosterone propionate). The modeling method is described like this, 100 mice were subcutaneously injected with 0.06 mL TP (testosterone propionate), 4d/time, continuous injection for 2 weeks and drug withdrawal for 1 weeks. Those mice after modelling were randomly divided into three groups, i.e. model control group, Clomifenecitrate capsules group and HDG(high-dose group), MDG (medium-dose group) and LDG (low-dose group) of the drug combination in the present invention.

(27) Experimental animals were fed separately in cages (2 cages in each group) and the experimental conditions include natural illumination, room temperature 17-20° C., humidity 50%-70%, creation of 12-hour illumination & 12-hour dark environment inside the space. Throughout the experiment, the mice were able to consume freely and drink water. Complete pellet feeds were used in feeding and the drinking water was replaced on daily basis.

(28) 2.2 Medication for Female Mice

(29) Blank control group: Intragastrically administrated with distilled water on daily basis at a dose of 2 ml/d;

(30) Model control group: Intragastrically administrated with distilled water on daily basis and at a dose of 2 ml/d;

(31) Clomifenecitrate capsules group: Intragastrically administrated with Clomifenecitrate capsules suspension at a dose of 18 mg/kg

(32) HDG(high-dose group) of drug combination in the present invention: Intragastrically administrated with the drug combination at a dose of 6 g/kg (calculated as per the crude drug weight);

(33) MDG(medium-dose group) of drug combination in the present invention: Intragastrically administrated with the drug combination at a dose of 4 g/kg;

(34) LDG (low-dose group) of drug combination in the present invention: Intragastrically administrated with the drug combination at a dose of 2 g/kg;

(35) After 4 weeks of administration, the drug was suspended for 48 h, 10 mice were executed in each group and the remaining 10 mice were left to carry out the conception rate test.

(36) 3. Experimental Results

(37) 3.1 Effect of the Drug Combination in the Present Invention on the Estradiol Content in the Sera of Laboratory Animals.

(38) Irrigation was stopped for 48 h, orbital blood of mice was collected, serum was separated and enzyme linked immunosorbent assay was used to detect E2. The results are recorded as shown in Table 1.

(39) Comparison of E2 content in the mice sera of each group: x±s

(40) TABLE-US-00001 Group category Number of animals E.sub.2/(ug.Math.L.sup.-1) Normal control group 10 9.95 ± 2.43  Model control group 10 5.64 ± 1.65* Clomifenecitrate capsules 10 12.76 ± 4.98## group HDG (high-dose group) of 10 10.59 ± 2.83## drug combination in the present invention MDG (medium-dose group) 10 7.80 ± 3.95# of drug combination in the present invention LDG (low-dose group) of 10 5.72 ± 2.04  drug combination in the present invention Note: comparison with the normal control group, *P < 0.05; comparison with the model control group, #P < 0.05, ##P < 0.01.

(41) Note: comparison with the normal control group, *P<0.05; comparison with the model control group, #P<0.05, ##P<0.01.

(42) As indicated in Table 1, in the HDG(high-dose group) and MDG(medium-dose group) of drug combination in the present invention, the estrogen level of modeled mice significantly increased if compared with that of the model control group, indicating that the drug combination in the present invention possesses the role of enabling it to be back to normal. Among the rest, the effects of HDG(high-dose group) was close to that of Clomifenecitrate capsules group.

(43) 3.2 Effects of the drug combination in the present invention on the quantities of follicles, oocytes, interstitial gland composition and estrogen receptor (ER) among experimental animals.

(44) Mice were executed and their ovaries and uterus were removed. Ovaries were fixed with 100 g formaldehyde and tissue slices were observed under the ordinary optical microscope after the process of HE tinction and oil red 0 tinction. Uterus were fixed with 100 g/L formaldehyde and tissue slices were imbedded with paraffin and undergone the process of 4 μm slicing and ER marking. Light microscopy was used for observation after the hematoxylin counter staining. IHC was used to detect ER and the record results are as shown in Table 2.

(45) The quantities of follicles, oocytes, interstitial gland composition and comparison of estrogen receptor (ER) expression rate x±s among mice in every group in Table 2.

(46) TABLE-US-00002 Number of Quantity of Quantity of Interstitial gland ER expression Group category animals follicles/piece oocytes/piece composition rate/% Normal control group 10 14.6 ± 3.0  14.6 ± 3.0   27.00 ± 7.90  36.64 ± 6.27  Model control group 10 10.3 ± 2.6  3.1 ± 1.2**  42.00 ± 10.02* 68.00 ± 9.98** Clomifenecitrate 10 37.6 ± 6.4## 8.7 ± 2.2#  24.96 ± 5.00  14.25 ± 4.34## capsules group HDG (high-dose 10 24.5 ± 4.1## 6.7 ± 2.5## 38.52 ± 1.49* 31.20 ± 4.79## group) of drug combination in the present invention MDG (medium-dose 10 20.9 ± 3.9## 5.2 ± 2.1## 34.68 ± 6.99* 17.21 ± 5.74## group) of drug combination in the present invention LDG (low-dose group) 10 18.2 ± 2.4#  4.7 ± 1.9## 32.76 ± 4.97* 30.48 ± 4.06## of drug combination in the present invention Note: comparison with the normal control group, *P < 0.05, **P < 0.01; comparison with the model control group, #P < 0.05, ##P < 0.01.

(47) Note: comparison with the normal control group, *P<0.05,**P<0.01; comparison with the model control group, #P<0.05, ##P<0.01.

(48) As indicated in the experimental results, HDG, MDG and LDG of drug combination in the present invention can increase the quantities of follicles and oocytes, develop interstitial glands, promote the follicular development and generate ovulation and corpus luteum. The effect of modeling on interstitial substance was not obvious and Clomifenecitrate capsules group had insignificant effect on the interstitial gland after medication. Among the experimental animals in HDG, MDG and LDG of drug combination in the present invention, the levels of interstitial glands after medication were higher than that of the normal control group, explaining indirectly that the drug combination in the present invention exert their efficacies by regulating the overall function of reproductive organs, rather than only stimulating ovarian follicles.

(49) 3.3 Conception Conditions of Mated Female Mice after the Medication.

(50) To put 10 mice from each cage into the cage where 4 normally-matured male mice. 10 d after the mating, uterus of female mice were dissected to observe the conception conditions. The record results are as shown in Table 3.

(51) TABLE-US-00003 TABLE 3 Conception conditions of mated female mice after the medication. Number of Number of Rate of Group category animals conception/mice conception % Normal control group 10 6 60.0 Model control group 10 2 20.0 Clomifenecitrate 10 6 60.0 capsules group HDG (high-dose 10 5 50.0 group) of drug combination in the present invention MDG (medium-dose 10 5 50.0 group) of drug combination in the present invention LDG (low-dose 10 3 30.0 group) of drug combination in the present invention

(52) For the HDG and LDG of drug combination of the present invention, the PR(pregnancy rate) was slightly lower than that of Clomifene citrate capsules group and normal control group, indicating that the HDG and LDG of drug combination can effectively restore the physiological functions of modelled experimental animals.

(53) (2) Effects of the Drug Combination of Present Invention on Mice with Fallopian Tube Inflammatory Obstructive Infertility

(54) 72 wistar female rats were randomly divided into 6 groups according to their weights and there were 12 rats in each group. Except for 12 rats in the blank control group, the remaining 60 rats were put into the modelling experiment aimed to induce the fallopian tube inflammatory obstructive infertility. Rats in the modelling group were fasted for 24 h (food was forbidden, drinking permitted), 20% urethane (5 ml.Math.kg.sup.−1) was used in anaesthesia, ventral hair was cut away to open the abdominal cavity after iodine and alcohol disinfection. Next, 4# scalp needle was used to inject 20% phenol paste of 0.01 ml into ovarian ducts on both sides at the uterine bifurcation and sutured the wound. After the operation, 5 groups were randomly divided and there were 12 rats in each group, i.e. model control group (equal volume of distilled water was given), Fuyankang tablets group (0.41 g.Math.kg.sup.−1), as well as HDG, MDG and LDG of the drug combination of the present invention. 30 d after respectively continuous intragastric administration, pharmacodynamic hemorheology detection was conducted by drawing blood and uterus was also taken for observation by naked eye. The record results are as shown in Table 4 and Table 5.

(55) TABLE-US-00004 TABLE 4 Comparison of fallopian tube patency in modelled rats Number Number of Number Number Patency of fallopian of of rate Group category animals tube patency blockage (%) Normal control 12 24 24 0 100 group Model control 12 24 4 20 16.67 group Fuyankang tablets 12 24 14 10 58.33.sup.## group HDG (high-dose 12 24 11 13 45.83.sup.## group) of drug combination in the present invention MDG (medium- 12 24 8 16 33.33.sup.## dose group) of drug combination in the present invention LDG (low-dose 12 24 5 19 20.83.sup.# group) of drug combination in the present invention Note: comparison with the model control group, .sup.#P < 0.05, .sup.##P < 0.01;

(56) Note: comparison with the model control group, #P<0.05, ## P<0.01;

(57) The tubal patency rate of rats in the model group was 16.67% and the number of blockage was obviously higher than that of the normal control group, indicating that the injection of 20% 0.01 mL phenol paste into the ovarian duct may lead to obstruction due to ureteritis. In HDG, MDG and LDG of the drug combination of the present invention, the tubal patency rate of rats was higher than that of Fuyankang tablets group. For this reason, the drug combination in the present invention can improve the symptoms of fallopian tube obstruction.

(58) TABLE-US-00005 TABLE 5 Effects of the drug combination in the present invention on the hemorheology in rats Number of PV (plasma Group category animals High cut Low cut viscosity) Normal control 12 2.33 ± 0.35 9.85 ± 2.25 2.37 ± 0.82 group Model control 12 3.28 ± 1.02 16.03 ± 3.67  3.56 ± 0.68 group Fuyankang 12  2.58 ± 0.69* 13.49 ± 3.62* 2.79 ± 0.75 tablets group HDG (high-dose 12  2.49 ± 0.92*  10.94 ± 2.63**  2.43 ± 0.52* group) of drug combination in the present invention MDG (medium- 12  2.53 ± 0.97* 12.23 ± 2.74* 2.72 ± 0.69 dose group) of drug combination in the present invention LDG (low-dose 12 3.57 ± 1.20 14.92 ± 3.18  3.38 ± 1.14 group) of drug combination in the present invention Note: comparison with the model control group, *P < 0.05, #**P < 0.01;

(59) Note: comparison with the model control group, *P<0.05, #**P<0.01;

(60) The high cut, low cut and PV (plasma viscosity) of model group rose if compared with the normal control group, indicating that abnormal hemorheologic indexes occurred in rats with obstruction due to ureteritis. In HDG, MDG and LDG of the drug combination of the present invention, the whole blood viscosities (low & high cut) of rats significantly decreased if compared with that of the model group and no obvious difference if compared with Fuyankang tablets group. For the HDG group, the difference was significant if compared with the model group. It is quite evident that the drug combination in the present invention has reducing effect on the whole blood viscosities (low & high cut) and PV (plasma viscosity) of 20% rats with fallopian tube inflammatory obstructive infertility caused by phenol paste.

Testing Example 2 a Study on the Effects of Anti-Fatigue and Hypoxia Tolerance Efficacy

(61) Test materials, drugs and animals were the same as that in testing example 1.

(62) 160 healthy Kunming mice (weights 22±2 g) were randomly divided into 8 groups and each of two groups was taken as an experimental group. I group was the control group and intragastric administration of equivalent normal saline was arranged. For II-IV groups, the intragastric administration of drug combination in the present invention was arranged as per three dosages, i.e. 6(HDG), 4 (MDG) and 2 (LDG)g/kg, 1 time/d and constant administration for 7 d. 30 min after the end of last administration, a group of mice were tied with load equivalent to 5% of the mice weight. Next, these mice were put into a glass jar. Physical exhaustion was considered when the head of mice was plunged into the water for 10 s and failed to come to the surface. In this case, the time from the commencement of swimming to the physical exhaustion was recorded as the load swimming time of mice. Another group of mice were put into a 500 mL ground mouth &wide-mouth bottle filled with 15 g soda lime (1 bottle for each mouse). Next, Vaseline was used to seal the bottle neck and started the timing. Stop watch was used to record the time from the hermetization of bottle neck to the death time of mice, which was recorded as the hypoxia time of mice.

(63) TABLE-US-00006 TABLE 6 Effects of oral administration of the drug combination on the mice activity Admin- Number istration Load Hypoxia of mice dosage swimming time Group category (piece) (g/kg) time (min) (min) Normal control 20 —  8.40 ± 1.25 81.25 ± 23.58 group HDG (high-dose 20 6 12.23 ± 3.40 106.30 ± 21.57  group) of drug combination in the present invention MDG (medium- 20 4 11.54 ± 1.62 97.26 ± 18.35 dose group) of drug combination in the present invention LDG (low-dose 20 2 10.02 ± 1.14 90.49 ± 15.94 group) of drug combination in the present invention

(64) In the HDG, MDG and LDG of drug combination of the present invention, the load swimming time and hypoxia time of mice were higher than that of the normal control group, indicating that the drug combination in the present invention can improve the mice activity and strengthen the physique of mice.

Testing Example 3 Effects of the Drug Combination in the Present Invention on the Immune Function of Mice

(65) Test materials, drugs and animals were the same as that in testing example 1.

(66) 140 SPF-grade Kunming male mice (body weights 18-22 g) were taken and divided into 7 groups and there were 20 mice in each group. Except that 20 mice in the blank control group were given equivalent normal saline, the remaining mice administrated with the drug combination (HDG, MDG and LDG) of the present invention. The intragastric administration was conducted on daily basis with a dose of 20 ml/kg every day, 1 time for 1 d and continue for 30 d. After these mice were executed, the following tests were conducted: (1) ConA-induced mouse spleen lymphocyte transformation test (MTT method); (2) Determination of numbers of antibody-producing cells (modified Jerne's method); (3) Determination of serum hemolysin (blood agglutination test); (4) Mouse abdominal cavity macrophage swallows the chicken red blood cell experiment (semi-in-vivo method). The record results are as shown in Table 7 and Table 8.

(67) TABLE-US-00007 TABLE 7 Effects of the drug combination in the present invention on ConA-induced mouse spleen lymphocyte transformation ability, the number of antibody-producing cells, serum hemolysin ( x ± s, n = 20) Lymphocytes Number of hemolysis Dosage/ proliferation plaque (1 × 10/10.sup.8 Group category (g/kg) assay [D(λ)] number of splenocyte) Antibody volume Blank control group — 0.2439 ± 0.0613 182.4 ± 20.3 120.3 ± 16.4 HDG (high-dose group) 6  0.3367 ± 0.0635*  212.7 ± 16.9**  149.7 ± 14.9* of drug combination in the present invention MDG (medium-dose 4 0.3148 ± 0.0597 199.2 ± 14.5 134.5 ± 13.3 group) of drug combination in the present invention LDG (low-dose group) 2 0.2871 ± 0.0840 192.8 ± 13.0 129.8 ± 13.7 of drug combination in the present invention Note: comparison with the blank control group, *P < 0.05, **P < 0.010;

(68) Note: comparison with the blank control group, *P<0.05, **P<0.010;

(69) As indicated in Table 7, if compared with the blank control group, the HDG, MDG and LDG of drug combination in the present invention increased in terms of lymphocyte proliferation ability, hemolytic plaque number and antibody volume, indicating that drug combination in the present invention can promote the lymphocyte proliferation and transformation ability in mice, enhance the antibody-producing cell proliferation in mice and increase the level of serum hemolysin in mice.

(70) TABLE-US-00008 TABLE 8 Effects of the drug combination in the present invention on mouse abdominal cavity macrophage swallows the chicken red blood cell ( x ± s, n = 20) Dosage/ Phagocytic Pyagocytic Group category (g/kg) rate/% index Blank control group — 24.3 ± 4.6 0.497 ± 0.098 HDG (high-dose 6 31.7 ± 4.9  0.763 ± 0.182* group) of drug combination in the present invention MDG (medium-dose 4 27.5 ± 4.3 0.628 ± 0.131 group) of drug combination in the present invention LDG (low-dose group) 2 25.9 ± 4.0 0.512 ± 0.125 of drug combination in the present invention Note: comparison with the blank control group, *P < 0.05.

(71) Note: comparison with the blank control group, *P<0.05.

(72) As indicated in Table 8, the phagocytic indexes of HDG, MDG and LDG in the drug combination in the present invention were significantly higher than that of blank control group, indicating that it plays a certain role of promoting the phagocytic function of mice's peritoneal macrophage.

Testing Example 4 Safety Evaluation

(73) Test materials, drugs and animals were the same as that in testing example 1.

(74) 1. Acute Toxicity Test

(75) The mice were intragastrically-administrated with 50 g (crude drug)/kg of test preparation and no animal deaths were found during the testing period. In this case, it can be determined that the maximum tolerance dose for single dosage was 50 g (crude drug)/kg, which was equivalent to 125 times as high as the daily consumption of humankind (calculated as per the weight of 60 kg/each person, dosage of capsule: 2 capsules at each time, one time every day, 12 g of crude drug is contained in each capsule, daily consumption per person is 24 g, 0.4 g/kg/day).

(76) 2. Long-Term Toxicity Test

(77) Continuous intragastric administration at a dosage of 20 g/kg were provided to rats for 10 weeks and there were no toxicity changes in terms of general clinical symptoms, weight gain, indicators of hematology and blood biochemistry examination, related organ systems and pathological tissues among animals, if compared with the distilled water in control group. 4 weeks after the suspension of drug, delayed toxicity was not found in the examination of the above same items, indicating that the oral successive medication of the drug combination in the present invention have no obvious toxic side effects.

(78) In summary, the compatibility of this drug combination invented is precise, appropriate and possess outstanding curative effects. As shown in many experiments, the drug combination invented is safe and reliable, its toxic & side-effect is low and it has obvious improvement effects on female infertility caused by androgen, fallopian tube inflammation, tubal blockage and other factors. Furthermore, it can not only significantly improve the success rate of pregnancy, but also improve immunity and enhance the body's anti-fatigue and hypoxia tolerance. In short, the invention provides a safe and reliable new choice for clinical medication.