阅读说明：本技术 一种林蛙油冻干粉补肾药理作用的研究方法 (Research method for kidney-tonifying pharmacological effect of oviductus ranae freeze-dried powder ) 是由 张凤春 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种林蛙油冻干粉补肾药理作用的研究方法,包括步研究林蛙油冻干粉对小鼠网状内皮系统炭粒廓清能力的影响、研究林蛙油冻干粉对幼年雄性小鼠的生殖附性器官精液囊、睾丸、包皮腺、免疫器官胸腺和脾脏的重量指数的影响、研究林蛙油冻干粉对肾阳虚大鼠内分泌功能的影响、研究林蛙油冻干粉对去势小鼠精液囊与包皮腺重量指数的影响、研究林蛙油冻干粉对精子数及活精子百分率的影响和依据上述研究做出整体评估,该研究方法可从肾功能影响的多方面对林蛙油冻干粉进行研究,形成系统的评价方法,对林蛙油冻干工艺的发展和冻干制品的推广具有重大意义。(The invention discloses a method for researching the kidney-tonifying pharmacological effect of oviductus ranae freeze-dried powder, which comprises the steps of researching the influence of the oviductus ranae freeze-dried powder on the carbon particle clearance capability of a reticuloendothelial system of a mouse, researching the influence of the oviductus ranae freeze-dried powder on the weight indexes of an organ seminal vesicle, a testis, a glandula preputialis, a thymus gland and a spleen of a male young mouse, researching the influence of the oviductus ranae freeze-dried powder on the endocrine function of a rat with kidney yang deficiency, researching the influence of the oviductus ranae freeze-dried powder on the weight indexes of the sperm vesicle and the glandula preputialis of a castrated mouse, researching the influence of the oviductus ranae freeze-dried powder, the research method can research the oviductus ranae freeze-dried powder from multiple aspects influenced by renal function to form a systematic evaluation method, and has great significance for the development of oviductus ranae freeze-drying process and the popularization of freeze-dried products.)

1. A research method for the kidney-tonifying pharmacological effect of oviductus ranae freeze-dried powder is characterized by comprising the following steps:

the method comprises the following steps: research on influence of oviductus ranae lyophilized powder on carbon particle clearance capability of reticuloendothelial system of mouse

Dividing experimental male KM mice into several groups, setting blank control group, blank model group, experimental group and control group, performing corresponding treatment, intravenously injecting India ink into all groups of mice with the weight of 0.1mL/10g, and measuring OD respectively by taking blood twice at a certain time interval₁And OD₂The liver and spleen are sacrificed and taken after blood collection is finished, analyzed and weighed, and the clearance index and the phagocytosis coefficient are calculated;

step two: study on the influence of lyophilized powder of oviductus Ranae on the weight index of seminal vesicle, testis, glandula preputialis, thymus and spleen of reproductive organs of young male mice

Dividing the male KM mice for experiments into a plurality of groups in equal number, setting a blank control group, a blank model group, an experimental group and a control group, carrying out corresponding treatment, killing and weighing 24 hours after the last treatment, taking thymus, spleen, seminal vesicle, testis and glandula preputiales, respectively weighing, and taking the ratio of organ weight to body weight as an index for calculation;

step three: research on influence of oviductus ranae lyophilized powder on endocrine function of rats with kidney yang deficiency

Dividing SD male rats into several groups, setting blank control group, blank model group, experimental group and control group, performing corresponding treatment, and measuring cortisol, testosterone, epinephrine and norepinephrine content in blood serum 1h after the last administration;

step four: research on influence of oviductus Ranae lyophilized powder on weight index of seminal fluid vesicle and glandula preputipes of castrated mouse

After deep anesthesia castration treatment is carried out on male KM mice for experiments, the mice are equally divided into a plurality of groups, a blank control group, an experimental group and a control group are arranged and correspondingly treated, and after continuous treatment for 10 days, seminal vesicle glands and preputial glands of the mice in each group are killed and sheared to be weighed;

step five: research on influence of oviductus Ranae lyophilized powder on sperm count and percentage of viable sperm

Dividing SD male rats into several groups, setting blank control group, experimental group and control group, performing corresponding treatment, killing, dissecting, taking epididymis, and calculating total sperm count, dead sperm percentage and live sperm percentage 24h after the last treatment;

step six: and comprehensively evaluating according to the data results measured in the first step to the fifth step.

2. The method for studying the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder according to claim 1, wherein the blank control group is a normal saline treatment group.

3. The method for researching the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder as claimed in claim 1, wherein the experimental group is an oviductus ranae lyophilized powder suspension administration group which comprises three or more administration groups with different dosages.

4. The method for researching the kidney-tonifying pharmacological effect of the oviductus ranae freeze-dried powder as claimed in claim 1, wherein the control group comprises an oviductus ranae suspension administration group and a corresponding prior art medicine administration group.

5. The method for studying the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder as claimed in claim 1, wherein the India ink in the first step is diluted 4 times with 1% gelatin solution and then injected into tail vein.

6. The method for studying the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder according to claim 1, wherein OD is measured by orbital hemospasia 2min after injection in the step one₁OD determination by orbital bleeding 10min after injection₂Value, calculated phagocytosis index K ═ (lgOD)₁-lgOD₂)/(T₂-T₁) Wherein T is time, T₁Is 2min, T₂Is at 10 min.

7. The method for studying the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder according to claim 1, wherein the phagocytic coefficient α in the first step is × K (body weight/(liver weight + spleen weight))^1/3。

8. The method for studying the kidney-tonifying pharmacological effect of the oviductus ranae lyophilized powder, as claimed in claim 1, wherein in the fifth step, the total number of sperms, the percentage of dead sperms and the percentage of live sperms are calculated by trypan blue staining method.

Technical Field

The invention relates to the technical field of health-care food, in particular to a research method for kidney-tonifying pharmacological effects of oviductus ranae freeze-dried powder.

Background

The wood frog, also known as the frog, is a famous economic frog seed integrating medicinal, nourishing and beautifying functions in China. The oviduct of female forest frog in maturation period contains bioactive factors such as 18 kinds of amino acids, 13 kinds of inorganic elements, 9 kinds of vitamins and multiple composite polypeptides, and is especially rich in three kinds of sex hormones, i.e. estradiol, testosterone and progesterone. The estradiol and the testosterone are respectively the hormones with the strongest physiological action in female hormone and male hormone, and have obvious effects of nourishing yin, strengthening kidney, stimulating immunologic function and regulating mechanism.

The wood frog oil directly obtained from female wood frog bodies is dry wood frog oil, belongs to oil substances which are not easy to digest by human bodies, and simultaneously has virus or parasitic ova and strong blood fishy smell, and can not be directly eaten without processing. The processing method of the prior art is to cook the wood frog oil at high temperature (100 ℃) to achieve the purposes of removing fishy smell and degerming. However, the long-time high-temperature cooking causes the loss of nutrients of the rana japonica oil seriously, and active substances with efficacy are damaged, such as unsaturated fatty acid, vitamin E, vitamin C, estradiol and the like, which seriously affect the efficacy of the rana japonica oil. In addition, the high-temperature cooking can only eliminate most of fishy smell, the prepared forest frog oil product still has a silky fishy smell, and meanwhile, the forest frog oil after the high-temperature cooking is not soft enough, so that the taste is influenced. Therefore, more and more technologies are directed to the research of forest frog oil freeze-drying, the freeze-drying process enables the forest frog oil to have the effect of convenient and quick use, but the research of the prior art is less aiming at whether the freeze-dried forest frog oil has functional influence or not, and the research of pharmacological action of the forest frog oil freeze-dried powder in the kidney tonifying aspect lacks a systematic research method, so that the development process and the wide popularization of the forest frog oil product are hindered.

Therefore, how to provide a systematic research method for the pharmacological effect of the oviductus ranae lyophilized powder on kidney tonifying is a problem to be solved urgently by technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a research method for the kidney tonifying pharmacological effect of oviductus ranae freeze-dried powder, which takes rats commonly used in laboratories as research objects to research the influence on renal function in multiple aspects so as to achieve the effect of systematically and comprehensively evaluating the kidney tonifying effect of oviductus ranae.

In order to achieve the purpose, the invention adopts the following technical scheme:

a research method of kidney-tonifying pharmacological effects of oviductus ranae lyophilized powder comprises the following steps:

the method comprises the following steps: research on influence of oviductus ranae lyophilized powder on carbon particle clearance capability of reticuloendothelial system of mouse

Dividing experimental male KM mice into several groups, setting blank control group, blank model group, experimental group and control group, performing corresponding treatment, intravenously injecting India ink into all groups of mice with the weight of 0.1mL/10g, and measuring OD respectively by taking blood twice at a certain time interval₁And OD₂The liver and spleen are sacrificed and taken after blood collection is finished, analyzed and weighed, and the clearance index and the phagocytosis coefficient are calculated;

step two: study on the influence of lyophilized powder of oviductus Ranae on the weight index of seminal vesicle, testis, glandula preputialis, thymus and spleen of reproductive organs of young male mice

Dividing the male KM mice for experiments into a plurality of groups in equal number, setting a blank control group, a blank model group, an experimental group and a control group, carrying out corresponding treatment, killing and weighing 24 hours after the last treatment, taking thymus, spleen, seminal vesicle, testis and glandula preputiales, respectively weighing, and taking the ratio of organ weight to body weight as an index for calculation;

step three: research on influence of oviductus ranae lyophilized powder on endocrine function of rats with kidney yang deficiency

Dividing SD male rats into several groups, setting blank control group, blank model group, experimental group and control group, performing corresponding treatment, and measuring cortisol, testosterone, epinephrine and norepinephrine content in blood serum 1h after the last administration;

step four: research on influence of oviductus Ranae lyophilized powder on weight index of seminal fluid vesicle and glandula preputipes of castrated mouse

After deep anesthesia castration treatment is carried out on male KM mice for experiments, the mice are equally divided into a plurality of groups, a blank control group, an experimental group and a control group are arranged and correspondingly treated, and after continuous treatment for 10 days, seminal vesicle glands and preputial glands of the mice in each group are killed and sheared to be weighed;

step five: research on influence of oviductus Ranae lyophilized powder on sperm count and percentage of viable sperm

Dividing SD male rats into several groups, setting blank control group, experimental group and control group, performing corresponding treatment, killing, dissecting, taking epididymis, and calculating total sperm count, dead sperm percentage and live sperm percentage 24h after the last treatment;

step six: and comprehensively evaluating according to the data results measured in the first step to the fifth step.

Further, the blank control group was a normal saline treatment group.

Furthermore, the experimental group is a rana japonica oil freeze-dried powder suspension administration group which comprises three or more administration groups with different dosages.

Further, the control group comprises a rana japonica oil suspension administration group and a corresponding prior art drug administration group.

Further, the India ink in the first step is diluted 4 times with 1% gelatin solution and then injected into tail vein.

Further, in the step one, OD is determined by taking orbital blood 2min after injection₁OD determination by orbital bleeding 10min after injection₂Value, calculated phagocytosis index K ═ (lgOD)₁-lgOD₂)/(T₂-T₁) Wherein T is time, T₁Is 2min, T₂Is at 10 min.

Further, the phagocytic coefficient α in the first step is × K (body weight/(liver weight + spleen weight))^1/3。

Further, in the fifth step, the total number of sperms, the percentage of dead sperms and the percentage of live sperms are calculated by trypan blue staining.

Through the technical scheme, compared with the prior art, the invention provides a research method for the kidney-tonifying pharmacological effect of the oviductus ranae freeze-dried powder, the oviductus ranae freeze-dried powder can be researched from multiple aspects influenced by renal function to form a systematic evaluation method, and the research method has great significance for the development and popularization of the oviductus ranae freeze-drying process.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental Material

1. Test drug

Name oviductus ranae freeze-dried powder

Provisioning of the Unit Heilongjiang Sibao Biotech Co., Ltd

Batch number 20171101

The content meets the requirement

Physical and chemical properties the product is light yellow powder.

2. Test animal

Strain SD rat, kunming strain mouse.

The source is provided by experimental animals and animal experiment centers in Qingdao city, and the qualification number is SCXK (Lu) 20140001.

The body weight is shown in each test.

Sex male.

The number of animals is shown in each experiment.

3. Major drugs and reagents

4. Main instrument

101A-1E type electrothermal blowing dry box	Shanghai laboratory instruments Ltd
		FA2004 analytical electronic balance	Shanghai Liangping instrument and meter
Inverted microscope	Olympus corporation of Japan
		Victorx3 enzyme-labeling instrument	PerkinElmer Co
TDL-4 model low-speed desk centrifuge	Shanghai Anxiang scientific instrument factory
		108mL hand tissue homogenizer	TIANJIN GLASS INSTRUMENT FACTORY
H2050R model desk type high speed freezing centrifuge	HUNAN XIANGYI LABORATORY INSTRUMENTS DEVELOPMENT Co.,Ltd.

(1) Influence of oviductus Ranae lyophilized powder on carbon particle clearance capability of reticuloendothelial system of mouse

Taking 70 male KM mice with the weight of 18-22 g, randomly dividing the mice into 7 groups, wherein each group comprises 10 mice ① normal control group (physiological saline is perfused into the stomach of 0.5mL/d), ② blank model group (cyclophosphamide is 30mg/kg), ② LWY-freeze-dried powder low-dose dry pre-treatment group (0.07g/kg oviductus ranae freeze-dried powder suspension), ④ LWY-freeze-dried powder medium-dose dry pre-treatment group (0.14g/kg oviductus ranae freeze-dried powder suspension), ⑤ LWY-freeze-dried powder high-dose dry pre-treatment group (0.28g/kg oviductus ranae freeze-dried powder suspension), ② LWY group (0.14g/kg oviductus ranae suspension), ⑦ Zhenqi strengthening particle suspension (3.90g/kg Zhenqi strengthening particle suspension), ② group, subcutaneously injecting cyclophosphamide into a ring-making mold (30mg/kg) on the 1 st day of the experiment, 3 th day and 5 days, beginning to 467 group (gastric saline is perfused into the stomach of ③ - ⑥ group) on the first day of the stomach-makingThe oviductus Ranae lyophilized powder suspension, ⑦ groups of intragastric administration glossy privet and astragalus root strengthening granule suspension, 1 time per day, and continuous administration for 14 days, 24h after the last administration, injecting diluted India ink (diluted 4 times with 1% gelatin solution) 0.1mL/10g body weight into caudal vein of all groups of mice, immediately timing, respectively taking blood from orbit at 2 nd and 10 th min, sucking 20 μ l with microsampler (soaked with heparin in advance), respectively adding into 2mL of 0.1% sodium carbonate solution, shaking, measuring OD value at 600nm wavelength, and 2min blood sample is OD₁OD 10min blood sample₂The mice after blood collection were sacrificed to take their livers and spleens, the surrounding tissues were removed, weighed on an electronic analytical balance, and the clearance index K and phagocytosis coefficient calculated α. the results are shown in table 1.

Phagocytic index K ═ l gado₁-lgOD₂)/(T₂-T₁)

Phagocytic coefficient α ═ body weight/(liver weight + spleen weight) × K^1/3

Note: t is time, T₁Is 2min, T₂Is at 10 min.

TABLE 1 influence of lyophilized powder of oviductus Ranae on the carbon particle cleaning ability of mice (± s; n ═ 10)

Note comparison with Normal group # P<0.05，^##P<0.01，^###P<0.001, compared with model group^*P<0.05，^**P<0.01，^***P<0.001, compared with LWY group^〇P<0.05，^〇〇P<0.01，^〇〇〇P<0.001。

The clearance index indicates the rate at which foreign bodies in the blood stream are cleared by macrophages, reflecting the phagocytic function of mononuclear macrophages. The mononuclear macrophage system is an important system in keeping the environment in the organism stable, and plays an important role in the occurrence, development or prognosis of various diseases because the mononuclear macrophage system can rapidly remove various pathogenic substances, such as pathogenic microorganisms and toxins thereof, aged, dead or mutant cells, blood clots, fibrinolysis products, complement fragments, antigen-antibody complexes and the like.

The results show that: compared with the normal group, the phagocytic index and the phagocytic coefficient of the model group are obviously reduced (P is less than 0.01), which indicates that the model is successfully made; LWY compared with the model group, the administration groups of lyophilized powder have higher phagocytic index and phagocytic coefficient than the model group, and have significant difference (P <0.05, P <0.01 or P <0.001), which indicates that the oviductus Ranae lyophilized powder has enhancement effect on phagocytic function of mononuclear macrophage; compared with the crude wood frog oil group, the lyophilized wood frog oil powder has the advantages that the phagocytosis index (K) of the crude wood frog oil group is more obvious (P is less than 0.05), and the phagocytosis coefficient (alpha) of the dried wood frog oil group is more obvious (P is less than 0.001).

(2) Effect of lyophilized powder of oviductus Ranae on weight index of seminal vesicle, testis, glandula preputialis, thymus and spleen of reproductive organs of young male mice

70 male KM mice are selected, and the weight of the male KM mice is 18-22 g. The samples were randomly divided into 7 groups of 10. Normal control group (physiological saline solution intragastric administration 0.5mL/d), blank model group (cyclophosphamide 30mg/kg), lyophilized powder low dose dry pre-treatment group (rana japonica oil lyophilized powder suspension of 0.07 g/kg), lyophilized powder medium dose dry pre-treatment group (rana japonica oil lyophilized powder suspension of 0.14 g/kg), lyophilized powder high dose dry pre-treatment group (rana japonica oil lyophilized powder suspension of 0.28 g/kg), and dried powder medium dose dry pre-treatment group (rana japonica oil suspension of 0.14 g/kg), and dried glossy privet fruit and astragalus strengthening particle group (glossy privet fruit and astragalus strengthening particle suspension of 3.90 g/kg). Secondly, performing subcutaneous injection of cyclophosphamide for molding (30mg/kg) on days 1, 3 and 5 from the beginning of the experiment, starting to perform intragastric administration of physiological saline (0.5mL/d) on the first day of molding, and starting to perform intragastric administration of forest frog oil lyophilized powder suspension and seventh to perform intragastric administration of glossy privet fruit and astragalus strengthening granules for 1 time every day for 14 days. 24h after the last dose, mice were sacrificed, weighed, thymus, spleen, seminal fluid vesicles, testis, sebaceous glands were excised, surrounding tissues were removed, and weighed on an electronic analytical balance, indexed by the ratio of organ weight (mg) to body weight (g). The results are shown in Table 2.

TABLE 2 Effect of oviductus Ranae lyophilized powder on mouse immune organ and reproductive organ

Note that the comparison with the normal group^#P<0.05，^##P<0.01，^###P<0.001, compared with model group^*P<0.05，^**P<0.01，^***P<0.001, compared with LWY group^〇P<0.05，^〇〇P<0.01，^〇〇〇P<0.001。

The results show that: compared with the normal group, the weights of the preputial gland, the testis and the seminal vesicle of the mouse in the model group are reduced and have significant difference (P <0.05 or P <0.001), which proves that the model building is successful. Compared with the LWY-lyophilized powder administration groups, the weight of thymus and spleen of the model group has no significant difference (P >0.05), and the oviductus ranae lyophilized powder has no obvious influence on immune organs such as thymus and spleen of mice. LWY the periderm gland, testis, seminal vesicle of each group administered with lyophilized powder are increased and have significant difference (P <0.05, P <0.01 or P <0.001) compared with the model group, which shows that the lyophilized powder of oviductus Ranae has enhancement effect on mouse genital organ. Compared with the crude wood frog oil group, the crude wood frog oil group has more obvious effect (P <0.05 or P <0.01) on the weight increase of the glandular preputiales, the testicles and the seminal vesicles of mice under the same dosage, and has no significant difference on the weight of the thymus and the spleen.

(3) Influence of oviductus Ranae lyophilized powder on endocrine function of rat with kidney yang deficiency

70 SD male rats with the weight of 160-180 g are taken. The groups were randomly divided into 7 groups of 10 individuals. Normal control group (physiological saline is filled into stomach by 1mL/d), blank model group (hydrocortisone by 15mg/kg), lyophilized powder low dose dry pre-group (rana japonica oil lyophilized powder suspension of 0.05 g/kg), lyophilized powder medium dose dry pre-group (rana japonica oil lyophilized powder suspension of 0.10 g/kg), lyophilized powder high dose dry pre-group (rana japonica oil lyophilized powder suspension of 0.20 g/kg), dried powder of phi LWY group (rana japonica oil suspension of 0.10 g/kg), and capsule group of phi guin (capsule suspension of 1.458 g/kg). And (c) intramuscular injection of hydrocortisone every day for molding for 2 weeks. Administration groups III-III start intragastric administration while molding, and groups III-III start intragastric administration with 1mL/d of physiological saline and continue until the 6 th day after molding is finished. The animals of the groups are quickly (within 15 s) blood is taken from the retroorbital venous plexus 1h after the last administration, and the content of cortisol, testosterone, adrenaline and noradrenaline in the blood serum is measured. The results are shown in Table 3.

TABLE 3 influence of oviductus Ranae lyophilized powder on endocrine function of rats with kidney yang deficiency

Note that the comparison with the normal group^#P<0.05，^##P<0.01，^###P<0.001, compared with model group^*P<0.05，^**P<0.01，^***P<0.001, compared with LWY group^〇P<0.05，^〇〇P<0.01，^〇〇〇P<0.001。

Testosterone is a steroid hormone and has the effects of maintaining muscle strength and quality, maintaining bone density and strength, refreshing and improving physical strength. Cortisol is a hormone produced by the adrenal glands in response to stress, and is a substance that maintains the body's normal physiological functions under stress. Adrenalin is a hormone secreted by the human body to provide more energy for physical activity, resulting in a faster response. Norepinephrine is secreted in the face of short-term stress, behaves somewhat like epinephrine, and as a stress hormone, its increase promotes the storage of food energy and increases the alertness of the brain.

The results show that: compared with the normal control group, the model group has significant reduction of corticosterone, testosterone, norepinephrine and epinephrine (P <0.05 or P <0.001), which indicates that the model building is successful. LWY Freeze-dried powder is administered with cortisol, testosterone, norepinephrine and epinephrine which are all higher and significantly different than those in the model group (P <0.05, P <0.01 or P < 0.001). Experimental results show that the oviductus ranae lyophilized powder has an effect of increasing the endocrine function of rats with kidney yang deficiency, and compared with oviductus ranae crude oil groups under the same dosage, the oviductus ranae lyophilized powder has a more obvious effect (P is less than 0.01) of enhancing epinephrine by the oviductus ranae crude oil groups, and has no significant difference on the contents of corticosterone, testosterone and norepinephrine.

(4) Influence of oviductus Ranae lyophilized powder on weight index of seminal fluid sac and glandula preputialis of castrated mouse

60 male KM mice are selected, and the weight of the mice is 25-30 g. All mice were deeply anesthetized with chloral hydrate, then placed on an operating table, the skin of the testis was sterilized with iodine and alcohol sequentially, the skin was cut with surgical scissors, about 0.5cm long, the subcutaneous tissue was separated with forceps, the left and right testis were pulled out, and the epididymis was removed together, the wound was sutured, and the wound was sterilized with yellow liquid medicine. The mice after the operation are placed around the electric heating fan for heat preservation until the mice are revived. Mice were given intramuscular injection of penicillin sodium injection (0.46g/kg) for 3 days to prevent post-operative infection. At 3d post-surgery, all mice were randomized into 6 groups: the composition comprises a castration group (physiological saline is used for intragastric administration for 0.5mL/d), a LWY-freeze-dried powder low-dose dry preparation group (0.07g/kg of oviductus ranae freeze-dried powder suspension), a LWY-freeze-dried powder medium-dose dry preparation group (0.14g/kg of oviductus ranae freeze-dried powder suspension), a LWY-freeze-dried powder high-dose dry preparation group (0.28g/kg of oviductus ranae freeze-dried powder suspension), a fifthly LWY group (0.14g/kg of oviductus ranae suspension), and a testosterone propionate group (testosterone propionate 20mg/kg intramuscular injection). ② to fifthly, the medicine is administrated once a day by intragastric administration, and sixthly, the testosterone propionate is injected intramuscularly at the rate of 20mg/kg once every other day for 10 days. After 10d all animals were sacrificed, the seminal vesicle glands and the glandulae preputiales of each group of mice were excised, the surrounding fat and the like were removed, and the wet weight thereof was immediately weighed on an electronic analytical balance. The results are shown in Table 4.

TABLE 4 influence of oviductus Ranae lyophilized powder on weight index of seminal fluid vesicle and glandula preputialis of castrated mouse

Group of	Dosage (g/kg)	Seminal fluid vesicle (mg/g)	Glandula preputipes (mg/g)
				Group of castration	-	3.882±0.447	3.157±0.350
LWY Freeze-dried powder group	0.070	4.239±0.261*	3.671±0.514*
				LWY Freeze-dried powder group	0.140	4.380±0.302**〇〇	3.897±0.441***
LWY Freeze-dried powder group	0.280	5.031±0.382***	4.052±1.115*
				LWY group	0.140	5.384±0.548**	4.399±0.604***
Testosterone propionate group	0.020	6.038±0.762***	3.924±0.504**

Note that the comparison with the normal group^#P<0.05，^##P<0.01，^###P<0.001, compared with model group^*P<0.05，^**P<0.01，^***P<0.001, compared with LWY group^〇P<0.05，^〇〇P<0.01，^〇〇〇P<0.001。

The results show that: LWY the seminal fluid vesicles and the glandulae preputiales of each administration group of the freeze-dried powder are increased in weight and have significant difference (P <0.05, P <0.01 or P <0.001) compared with the castrated group, which shows that the freeze-dried powder of the wood frog oil can significantly increase the weight of the seminal fluid vesicles and the glandulae preputiales of the castrated mice. Compared with the wood frog crude oil group, the wood frog crude oil group has more obvious effect (P is less than 0.01) on the increase of the seminal vesicle weight of the castrated mice under the same dosage of the wood frog oil freeze-dried powder, and has no obvious difference on the glandular preputium weight.

(5) Influence of oviductus Ranae lyophilized powder on sperm count and percentage of viable sperm

Taking 60 male adult SD rats with the weight of 200-280 g, randomly dividing the SD rats into 6 groups, namely, a blank control group (1 mL/d of physiological saline for intragastric administration), a LWY-freeze-dried powder low-dose group (0.05g/kg of oviductus ranae freeze-dried powder suspension), a LWY-freeze-dried powder medium-dose group (0.10g/kg of oviductus ranae freeze-dried powder suspension), a LWY-freeze-dried powder high-dose group (0.20g/kg of oviductus ranae freeze-dried powder suspension), a fifthly LWY-group (0.10g/kg of oviductus ranae freeze-dried powder suspension) and a xiong capsule group (1 g/kg of andrao capsule). Firstly, 1mL/d of normal saline is infused into the stomach every day, and secondly, 1 time of infusion and administration are carried out every day for 17 days. 24 hours after the last administration, the femoral artery is bled to kill the animal, the epididymis is dissected and taken out, the tail end of the epididymis is cut into a cross shape by a scissors and is placed in a thick test tube with 10mL of physiological salt, the temperature is kept at 37 ℃ for 10min, the suspension is dropped into a cell counting plate, the cell is stained by a vital dye trypan blue, the counting is carried out on the blood cell counting plate according to a white blood cell counting method, and the living condition of the cell is convenient to determine. Trypan blue is unable to penetrate the normal intact cell membrane of living cells, so living cells are not stained. The cell membrane permeability of dead cells is increased, and the dye enters the cells to stain the cells (blue). The total number of sperm, percentage of dead sperm and percentage of viable sperm were calculated. The results are shown in Table 5.

TABLE 5 Effect of oviductus Ranae lyophilized powder on sperm count and percentage of viable sperm

Note: comparison with blank control group^*P<0.05，^**P<0.01，^***P<0.001, compared with LWY group^〇P<0.05，^〇〇P<0.01，^〇〇〇P<0.001。

The results show that: LWY-lyophilized powder administration groups all had increased total sperm count, viable sperm count, and viable sperm percentage, and had significant difference (P <0.05, P <0.01) compared with blank group. Compared with the blank group, the number of dead sperms and the percentage of dead sperms are both reduced, and the freeze-dried powder of the wood frog oil has significant difference (P <0.05, P <0.01) compared with the blank group, which indicates that the wood frog oil has enhancement effect on the number and activity of the sperms of the rat, and compared with the wood frog oil group under the same dosage, the wood frog oil freeze-dried powder has more significant effect (P <0.05) on the total number of the sperms and the number of the live sperms, and has no significant difference on the number of the dead sperms.

Through the evaluation of the step system, the oviductus ranae freeze-dried powder has better effect compared with common oviductus ranae products and other medicine components with corresponding functions, so that the evaluation method makes an overall evaluation on the kidney effect of the oviductus ranae freeze-dried powder and has higher reagent reference significance.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.