CN109045014B

CN109045014B - Application of vitexin in preparing medicine for treating diabetic function and low fertility

Info

Publication number: CN109045014B
Application number: CN201810708970.5A
Authority: CN
Inventors: 余建强; 李志梅; 刘宁; 吴璟; 李玉香
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2021-02-26
Anticipated expiration: 2038-07-02
Also published as: CN109045014A

Abstract

The invention discloses an application of Vitexin in preparing a medicament for treating diabetic hypofunction and fertility. The experimental result of the invention shows that when the vitexin with the dosage of 40mg/kg is used in the safe dosage range, the body mass, the reproductive organ weight reduction, the sperm motility and the survival rate are low, the serum endocrine hormone disorder, the sexual function deficiency, the fertility deficiency and the like of the diabetic patient can be obviously improved, and the vitexin has the effects of preventing the diabetic function and the fertility deficiency.

Description

Application of vitexin in preparing medicine for treating diabetic function and low fertility

Technical Field

The invention relates to application of vitexin, in particular to application of vitexin in preparing a medicament for treating diabetic function and low fertility.

Background

Diabetes is one of the major health problems in the world and is characterized by hyperglycemia due to defects in insulin secretion and/or insulin action, which has become the ninth leading cause of death. The international diabetes association (IDF) estimates that by 2017 there are 4.51 billion (18-99 years old) diabetic patients worldwide, and by 2045 these numbers are expected to rise to 6.93 billion. Sexual dysfunction and poor fertility and/or infertility associated with diabetes are major secondary complications in diabetic animals and humans compared to non-diabetic patients. About 90% of diabetic patients develop sexual dysfunction, including decreased libido, impotence, and infertility; meanwhile, decrease in the weight of accessory organs (epididymis, prostate and seminal vesicle), decrease in sperm quality, endocrine disorders, significant decrease in leydig cells and supporting cells and spermatogenic dysfunction were observed in Streptozotocin (STZ) -induced animal models of diabetes.

Vitexin, a c-glycosylated flavonoid, is found in many traditional Chinese medicines, such as pearl millet, hawthorn, mung bean, bamboo, etc. Studies have shown that oral administration of vitexin significantly reduces postprandial blood glucose in sucrose-loaded renal glycemic mice and STZ-induced diabetic rats, suggesting a potential anti-diabetic effect of vitexin, but no studies on the effect of vitexin on the reproductive system of diabetic patients have been reported. The medicine has extremely high potential value and social significance when being developed into the medicine for treating the diabetic hypofunction and the low fertility.

Disclosure of Invention

The invention aims to provide the application of vitexin in preparing a medicament for treating diabetic function and low fertility through researching the pharmacological action of the vitexin.

The invention achieves the purpose through the following technical scheme:

the invention provides an application of vitexin in preparing a medicament for treating diabetic hypofunction and low fertility, wherein the structural formula of the vitexin is shown as a formula (1):

formula (1).

Specifically, the diabetes is developed 72 hours after 5 days of continuous intraperitoneal injection of streptozotocin (45mg/kg), and is treated for 9 weeks.

Specifically, the single application dose of vitexin is limited to a dose that does not cause central inhibition.

Specifically, the single application dose of the vitexin is 10-40mg/kg.

Preferably, the single application dose of the vitexin is 10 mg/kg.

Preferably, the single application dose of the vitexin is 20 mg/kg.

Preferably, the single application dose of the vitexin is 40mg/kg.

Specifically, the single application dosage of the vitexin is 10mg/kg-40mg/kg for mice, and the single application dosage of 70kg for standard weight people is 1.59mg/kg-6.35 mg/kg.

In particular, the medicament is formulated for administration via the gastrointestinal tract or parenterally.

Preferably, the dosage form of the medicament is a pharmaceutically allowable oral dosage form or an injection dosage form.

In particular to the application of vitexin as a unique active ingredient in preparing a medicament for treating diabetic hypofunction and fertility.

The application of vitexin in preparing the medicine for treating diabetic function and low fertility provided by the invention has the following beneficial effects:

(1) under the condition of not reducing the dosage of blood sugar, the vitexin can obviously increase the body mass, the weight of gonadal organs, the sperm motility and the survival rate of diabetic mice, and obviously improve the sexual function and the low fertility;

(2) the vitexin can obviously improve the serum sex hormone T, FSH, LH, GnRH levels of diabetic mice and improve the spermatogenic function.

The vitexin is proved to have the function of treating the diabetic function and the fertility hypofunction for the first time, and can be used for preparing the medicine for treating the diabetic function and the fertility hypofunction.

Drawings

FIG. 1 shows the effect of vitexin on blood glucose in diabetic mice.

FIG. 2 is a graph of the effect of vitexin on the cross-incubation period in diabetic mice.

FIG. 3 is a graph showing the effect of vitexin on insertion latency in diabetic mice.

FIG. 4 is a graph showing the effect of vitexin on ejaculation latency in diabetic mice.

FIG. 5 is a graph showing the effect of vitexin on post ejaculation refractory period in diabetic mice.

FIG. 6 is a graph of the effect of vitexin on the frequency of straddling diabetic mice.

FIG. 7 is a graph showing the effect of vitexin on the frequency of insertion in diabetic mice.

FIG. 8 is a graph showing the effect of vitexin on sperm motility in diabetic mice.

FIG. 9 is a graph showing the effect of vitexin on sperm motility in diabetic mice.

FIG. 10 is a graph of the effect of vitexin on serum testosterone levels in diabetic mice.

FIG. 11 is a graph showing the effect of vitexin on serum gonadotropin-releasing hormone levels in diabetic mice.

FIG. 12 is a graph showing the effect of vitexin on serum FSH levels in diabetic mice.

FIG. 13 is a graph of the effect of vitexin on serum luteinizing hormone levels in diabetic mice.

FIG. 14 shows the staining pattern (X400) of vitexin for pathological change HE of testis tissue in diabetic mice (FIG. 14A is normal group; FIG. 14B is model group; FIG. 14C is sildenafil-positive drug group; FIG. 14D is vitexin 10mg/kg group; FIG. 14E is vitexin 20mg/kg group; FIG. 14F is vitexin 40mg/kg group).

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1

The use of vitexin in preparing medicine for treating diabetic hypofunction and fertility is disclosed, wherein the structural formula of vitexin is shown as formula (1):

formula (1).

Wherein, the single application dose of the vitexin is limited to the dose which does not cause central inhibition, the single application dose of the vitexin is 10mg/kg of ICR mice, and the dosage form of the medicine is a solution dosage form.

Example 2

formula (1).

Wherein, the single application dose of the vitexin is limited to the dose which does not cause central inhibition, the single application dose of the vitexin is 20mg/kg of ICR mice, and the dosage form of the medicine is a powder injection dosage form.

Example 3

formula (1).

Wherein, the single application dose of the vitexin is limited to the dose which does not cause central inhibition, the single application dose of the vitexin is 40mg/kg of ICR mice, and the dosage form of the medicine is a powder injection dosage form.

The following animal experiments further illustrate the effects of the above examples 1 to 3:

first, experimental material

1.1 animal treatment

9 weeks old, ICR mice, 25-30g, purchased from experimental animals center of Ningxia medical university, animal production license number: NCXK 2013-. Culturing in SPF barrier system at room temperature of 22 + -2 deg.C and humidity of 40-60% for 12 hr each day in light and dark. The experiment is carried out after one week of adaptive feeding, and the food and the water are freely taken.

1.2 Experimental drugs and instruments

Vitexin (Beijing Zhongke quality test, 99.65%, M-023-

1.3 grouping and administration of Experimental animals

Adult male ICR mice (SPF grade) with the weight of 25-30g (n is 160) are bred adaptively in a barrier system for 1 week, and then are randomly divided into a diabetes model construction group (n is 140) and a normal control group (n is 20), the diabetes model construction group is continuously injected with streptozotocin (45mg/kg) in the abdominal cavity for 5 days and 0.1ml/10g, the normal group is given with the same dose of sodium citrate buffer solution, and blood is collected at the tail tip 72h after the last administration for measuring fasting blood glucose, the blood glucose of the model construction group is larger than 16.7mmmol/L and is brought into the model group, the mice after the model is divided into the model group, the sildenafil group, the model group, the insulin group, the vitexin low, medium and high dose group and the normal group according to a complete random grouping method, and the mice are continuously administered for 62 days. The body weights of the mice in each group were weighed and recorded weekly, and fasting blood glucose was measured with a glucometer at 0,10,20, 40, and 62 days after treatment. After the last administration, the patient is fasted overnight for 12 hours without water prohibition, blood is collected from the orbit, and testis, epididymis and seminal vesicle are separated for histopathology and molecular biology detection.

1.4 Diabetes Mellitus (DM) model building

The diabetes model construction group is continuously injected with streptozotocin (45mg/kg) in the abdominal cavity for 5 days, the normal group is given with the same dose of sodium citrate buffer solution, and the blood sampling is carried out 72h after the last administration to measure the fasting blood glucose, wherein the blood glucose is larger than 16.7mmmol/L and is included in the model group.

Second, the experimental procedure

(one) blood glucose determination

1.1 Experimental methods:

blood glucose levels were measured with a glucometer at the tail tip on

days

0,10,20, 40, and 62 of the experiment, respectively.

1.2 Experimental results:

as can be seen from fig. 1, compared to the normal group; blood glucose was significantly elevated and the difference was significant in the diabetes model group (p < 0.01); compared with the model group, the blood sugar values of the vitexin and sildenafil treatment groups are not different, but are kept at a stable level (p >0.05), and the vitexin is suggested to have no influence on the blood sugar of diabetic mice.

(II) body weight and testis, epididymis and seminal vesicle weight

2.1 Experimental methods

Before daily dosing, the weight was weighed and recorded weekly. After the last administration, mice of each experimental group were sacrificed under anesthesia, testis, epididymis and seminal vesicle were isolated, adipose tissues around the testis, epididymis and seminal vesicle were removed, weighed, and organ coefficients were calculated.

2.2 results of the experiment

As can be seen from table 1, the model group body mass and the weights of testis, epididymis and seminal vesicle were significantly decreased (p <0.01) compared to the normal group, and the diabetic mouse body mass was significantly increased (p <0.05) and the testis, epididymis and seminal vesicle were significantly increased (p <0.05) after administration of vitexin (20mg/40mg/kg) and sildenafil treatment compared to the model group, suggesting that vitexin could improve the weight decrease and gonadal organ decrease caused by diabetes.

TABLE 1 Effect of Vitexin on body Mass, testis, epididymis and seminal vesicle glands in diabetic mice

(comparison with normal group:^#p<0.05，^##p<0.01; comparison with model groups: p<0.05,**p<0.01)

(III) sexual behavior test

3.1 Experimental methods

The female mice were injected subcutaneously with 5mg/kg of estradiol benzoate 54 hours before the experiment and with 0.5mg/kg of progesterone 6 hours before the experiment. Female mice with good estrus were screened for the experiment before the experiment. The experiment was terminated immediately after the asexual impulse in both male and female mice. Placing a male mouse in a (32cm multiplied by 16cm) transparent cage box, performing dark red light, performing sexual behavior training 3 days before the experiment, placing the male mouse in the transparent cage box to adapt for 10 minutes before the experiment begins, then placing a female mouse with good estrus, immediately recording the riding and crossing latency of the male mouse within 40 minutes by using a camera, inserting the latency, ejaculation latency, refractory period after ejaculation, riding and inserting frequency. The experiments were carried out at 17:00-23:00 pm, and sexual behavior experiments were carried out at 0 day and 62 days, respectively.

3.2 results of the experiment

As can be seen from the results of fig. 2-7, the 0-day-old model group had significantly prolonged refractory period and ejaculation latency period after ejaculation (p <0.01, p <0.05), straddling latency period, insertion latency period, straddling frequency, and insertion frequency (p >0.05) compared with the normal group; the model group at 62 days has obviously prolonged straddling latency, insertion latency, ejaculation latency and post-ejaculation refractory period, and remarkably reduced straddling frequency and insertion frequency (p < 0.01). Compared with a model group, the treatment with vitexin (40mg/kg) can significantly reduce the straddling latency, the insertion latency and the post-ejaculation refractory period (p <0.01), and significantly increase the straddling frequency and the insertion frequency (p < 0.05); the treatment of sildenafil reduces the straddling latency and the insertion latency (p <0.01), obviously increases the straddling frequency and the insertion frequency (p <0.05), and indicates that vitexin has the effect of improving the sexual hypofunction of diabetic mice.

(IV) fertility test

4.1 Experimental methods

Female mice, male mice, 8 weeks old, which were not mated, were selected: 1:2, closing the male mice for 3 days, respectively checking whether the female mice have the vaginal embolus in the next morning and afternoon, if the vaginal embolus is checked, the female mice are considered to be 0 day of pregnancy, taking out the female mice which are not observed to have the vaginal embolus, putting the female mice into another cage, and closing each male mouse and 6 female mice 3 times. Weighing female mice with vaginal embolus every three days, separately raising in cages until parturition if weight is increased, and calculating fertility rate, mating rate, pregnancy rate and number of offspring born. The fertility rate is the number of pregnant female mice/total mating female mice, the mating rate is the number of female mice for checking vaginal embolus/total mating female mice, and the pregnancy rate is the number of pregnant female mice/female mice for checking vaginal embolus.

4.2 results of the experiment

As can be seen from Table 2, the mating rate, the fertility rate and the number of offspring born were significantly decreased (p <0.01) in the model group compared to the normal group, while the pregnancy rate was not different, and the number of offspring born was significantly increased (p <0.01) in the normal vitexin-added group. The mating rate was significantly increased after administration of vitexin treatment (40mg/kg,20mg/kg) compared to the model group. Fertility rate and number of offspring born (p <0.05, p < 0.01); the number of born fetus of filial generation is obviously increased after sildenafil treatment (p is less than 0.01), and vitexin has the effect of improving the hypofertility of diabetic mice.

TABLE 2 Effect of Vitexin on fertility in diabetic mice

(comparison with normal group:^##p<0.01; comparison with model groups: p<0.05,**p<0.01)

(V) sperm motility and survival rate

5.1 Experimental methods

At the 70 th day of the experiment, the mice were sacrificed by anesthesia, epididymis was separated, weighed, epididymis tails cut off, minced, placed in 5ml of physiological saline pre-warmed in advance at 37 ℃, incubated for 5 minutes to allow the sperm to swim out, and filtered through a 200-mesh sieve to obtain a sperm suspension. And (3) dripping 10 mu l of filtrate on a blood counting plate, observing under a 400-time lens, counting 100 sperms, counting the movable and immovable sperms, and calculating the sperm motility. 20 mul of sperm suspension was aspirated, an equal volume of 0.5% eosin was added, incubated at room temperature for 2 minutes, smeared onto a clean slide, dead sperm stained pink, live sperm not stained, 100 sperm counted, and sperm motility was calculated.

5.2 results of the experiment

As can be seen from fig. 8-9, both sperm motility and sperm motility rate were significantly reduced in the model group compared to the normal group (p < 0.01). Sperm motility and survival rate (p <0.01, p <0.05) were significantly improved after vitexin treatment (40mg/kg,20mg/kg) compared to the model group. The sildenafil group has no difference in the sperm motility and the survival rate, and the vitexin has the effect of improving the low sperm motility and the survival rate of the diabetic mice.

(VI) measurement of serum sex hormone

6.1 Experimental methods

On the 70 th day of the experiment, blood is collected from the orbit at the 8:00 earliest day, 3500r/min and centrifuged for 15 minutes to obtain serum, which is subpackaged and stored at-80 ℃ for detection. Testosterone, follicle stimulating hormone, luteinizing hormone and gonadotropin releasing hormone are all detected by an ELISA method.

6.2 results of the experiment

As can be seen from fig. 10-13, the serum testosterone, follicle stimulating hormone and luteinizing hormone levels in the model group mice were significantly reduced and gonadotropin releasing hormone was significantly increased (p <0.01) compared to the normal group. Compared with a model group, after the vitexin (40mg/kg and 20mg/kg) and sildenafil are administered, testosterone, follicle stimulating hormone, luteinizing hormone reduction and gonadotropin releasing hormone ((p <0.01 and p < 0.05)) increase can be obviously improved, and the vitexin has the effect of improving the endocrine hormone disorder of diabetic mice.

(seventhly) testis histopathology test

7.1 Experimental methods

Taking right testis, fixing with Boniu stationary liquid for 24 hr, gradient dehydrating, removing xylene, soaking in wax for 2 hr, embedding with conventional paraffin, taking 5 μm thick continuous slice, staining with eosin-Hematoxylin (HE)

7.2 results of the experiment

The seminiferous tubules of the testis tissue of the normal control group mouse are regularly and tightly arranged, spermatogonia and spermatocytes at different developmental stages are radially arranged in about 5-7 layers, densely formed sperms can be seen in the tube cavity, and the testis interstitial cells are tightly arranged (see fig. 14A). The seminiferous tubules of the mouse testis tissue in the diabetes model group are obviously atrophied, the spermatogenic cells at all levels are unclear in level, disordered in arrangement and obviously reduced in number, interstitial tissues among the seminiferous tubules are partially disappeared, epithelial cells of the seminiferous tubules are subjected to lots of necrosis, edema and vacuole-like structures, the number of the testicular interstitial cells is obviously reduced, spermatids are not seen in most of the canals or the number of sperms is obviously reduced, and the spermatogenic phenomenon is obviously inhibited (see fig. 14B). The vitexin treatment group has certain recovery effect on testis tissues of diabetic mice, wherein the vitexin (40mg/kg) group has the best effect. The complete appearance of the boundary membrane of the convoluted and fine seminiferous tubules is more regular under the mirror, the distribution and arrangement of all levels of spermatogenic cells are gradually compact and regular, a small amount of sperms can be seen in the tubules, and the structure is close to that of a normal control group (see fig. 14F); the sildenafil treatment group has poor recovery effect on the testis structure, spermatids of the testis are not regularly arranged, a large amount of vacuoles are still found in the interstitial cells of the testis, and a small amount of sperms can be seen in the lumen of the seminiferous tubule (see figure 14C), which suggests that the vitexin has the effect of improving pathological damage of the testis tissue of the diabetic mouse.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. The application of vitexin in preparing the medicine for treating diabetic function and low fertility is characterized in that: the structural formula of the vitexin is shown as a formula (1):

formula (1)

The single application dosage of the vitexin is 10mg/kg-40mg/kg for mice, and the single application dosage of 70kg for standard weight people is 1.59mg/kg-6.35 mg/kg.

2. The use of vitexin of claim 1 in the preparation of a medicament for the treatment of diabetic dysfunction and fertility disorders, wherein: the single application dose of the vitexin is 10mg/kg of mice.

3. The use of vitexin of claim 1 in the preparation of a medicament for the treatment of diabetic dysfunction and fertility disorders, wherein: the single application dose of the vitexin is 20mg/kg of mice.

4. The use of vitexin of claim 1 in the preparation of a medicament for the treatment of diabetic dysfunction and fertility disorders, wherein: the single application dose of the vitexin is 40mg/kg of mice.

5. Use of vitexin of any of claims 1-4 in the preparation of a medicament for the treatment of diabetic dysfunction and fertility decline, wherein: the medicament is formulated for administration via the gastrointestinal tract or parenterally.

6. The use of vitexin of claim 5 in the preparation of a medicament for the treatment of diabetic hypofunction and fertility, wherein: the dosage form of the medicine is a pharmaceutically allowable oral dosage form, an injection dosage form or a powder injection.