阅读说明：本技术 一种林蛙油冻干粉抗衰老药理作用的研究方法 (Research method for anti-aging 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抗衰老药理作用的研究方法,包括体重及力竭时间测试、对大鼠血清中一氧化氮、丙二醛、超氧化物歧化酶、谷胱甘肽还原酶及过氧化氢酶活力的影响和依据上述研究做出整体评估,该研究方法可从引起衰老的机理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 anti-aging pharmacological effects of oviductus ranae freeze-dried powder, which comprises the steps of testing body weight and exhaustion time, and carrying out overall evaluation on the influences of activities of nitric oxide, malondialdehyde, superoxide dismutase, glutathione reductase and catalase in rat serum according to the research.)

1. A research method for anti-aging pharmacological effects of oviductus ranae lyophilized powder is characterized by comprising the following steps:

the method comprises the following steps: body weight and exhaustion time test

Carrying out adaptive feeding on SD male rats for one week for experiments, after eliminating rats which do not adapt to movement, dividing the rest rats into a plurality of groups, setting a static control group, a movement control group, an experiment group and a control group, carrying out corresponding treatment, recording the weight every week in the treatment process, continuously treating for 30d, carrying out a load-bearing 5% swimming exhaustion experiment after last administration for 60min, leading the rats to swim to exhaustion, and recording the swimming time when exhaustion;

step two: influence on activity of nitric oxide, malondialdehyde, superoxide dismutase, glutathione reductase and catalase in rat serum

Weighing 24h after exhaustion test, taking blood from abdominal aorta after anesthesia, centrifuging at 4 deg.C and 3000r/min to obtain serum, and measuring the content of nitric oxide and malondialdehyde and the activity of superoxide dismutase, glutathione reductase and catalase;

step three: and comprehensively evaluating according to the data results measured in the first step to the third step.

2. The method for researching the anti-aging pharmacological effect of the oviductus ranae freeze-dried powder according to claim 1, wherein the static control group in the step one is treated by normal saline for intragastric administration at a rate of 1mL/d, and no exhaustion experiment is performed after the last treatment.

3. The method for researching anti-aging pharmacological effects of the oviductus ranae freeze-dried powder according to claim 1, wherein the exercise control group in the first step is treated by normal saline for intragastric administration at a rate of 1mL/d, and a exhaustion experiment is performed after the last treatment.

4. The method for researching the anti-aging pharmacological effect of the oviductus ranae lyophilized powder as claimed in claim 1, wherein the treatment of the experimental group in the first step is gastric lavage of the oviductus ranae lyophilized powder suspension, and the exhaustion experiment is performed after the last treatment.

5. The method for researching the anti-aging pharmacological effect of the oviductus ranae freeze-dried powder according to claim 4, wherein the experimental group comprises three or more administration groups with different dosages.

6. The method for researching the anti-aging pharmacological effect of the oviductus ranae freeze-dried powder as claimed in claim 1, wherein the treatment of the control group in the first step is gastric gavage of an anti-aging health-care product in the prior art, and a exhaustion experiment is performed after the last treatment.

7. The method for researching the anti-aging pharmacological effect of the oviductus ranae freeze-dried powder according to claim 1, wherein the standard of exhaustion in the exhaustion experiment is measured by that the rat does not expose out of the water surface within 10s after sinking, and the swimming time during exhaustion is recorded.

Technical Field

The invention relates to the technical field of health-care food, in particular to a research method for anti-aging 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 the hormones with the strongest physiological action in female hormone and male hormone respectively, have obvious effects of nourishing yin, strengthening kidney, stimulating immunity and regulating mechanism, and in addition, the rana japonica oil has good anti-aging effect and is one of excellent health care products in daily life.

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, and the research of the pharmacological action of the forest frog oil freeze-dried powder in the aspect of anti-aging 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 anti-aging pharmacological effect of the oviductus ranae freeze-dried powder 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 method for researching the anti-aging pharmacological effect of oviductus ranae freeze-dried powder, which takes rats commonly used in laboratories as research objects to research the mechanisms in various aspects of aging, so as to achieve the effect of systematically and comprehensively evaluating the anti-aging effect of oviductus ranae.

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

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

the method comprises the following steps: body weight and exhaustion time test

Carrying out adaptive feeding on SD male rats for one week for experiments, after eliminating rats which do not adapt to movement, dividing the rest rats into a plurality of groups, setting a static control group, a movement control group, an experiment group and a control group, carrying out corresponding treatment, recording the weight every week in the treatment process, continuously treating for 30d, carrying out a load-bearing 5% swimming exhaustion experiment after last administration for 60min, leading the rats to swim to exhaustion, and recording the swimming time when exhaustion;

step two: influence on activity of nitric oxide, malondialdehyde, superoxide dismutase, glutathione reductase and catalase in rat serum

Weighing 24h after exhaustion test, taking blood from abdominal aorta after anesthesia, centrifuging at 4 deg.C and 3000r/min to obtain serum, and measuring the content of nitric oxide and malondialdehyde and the activity of superoxide dismutase, glutathione reductase and catalase;

step three: and comprehensively evaluating according to the data results measured in the first step to the third step.

Further, the treatment of the stationary control group in the step one is performed by perfusing the stomach with physiological saline for 1mL/d, and no exhaustion experiment is performed after the last treatment.

Further, the exercise control group in the step one is treated by normal saline for intragastric administration by 1mL/d, and a exhaustion experiment is carried out after the last treatment.

Further, the experiment group in the first step is processed by perfusing oviductus ranae lyophilized powder suspension, and performing exhaustion experiment after the last treatment.

Further, the experimental group comprises three or more different dosage administration groups.

Further, the treatment of the control group in the step one is realized by gavage of the anti-aging health care product in the prior art, and a exhaustion experiment is carried out after the last treatment.

Furthermore, the standard of exhaustion in the exhaustion experiment is measured in the degree that the rat does not expose out of the water surface after sinking for 10s, and the swimming time of exhaustion is recorded.

Through the technical scheme, compared with the prior art, the invention provides a method for researching the anti-aging pharmacological effect of the oviductus ranae freeze-dried powder, the oviductus ranae freeze-dried powder can be researched from various factors causing aging to form a systematic evaluation method, and the 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 strain rats.

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

Name of medicine	Manufacturer of the product	Batch number
			Oviductus Ranae lyophilized powder	Heilongjiang Sibao Biotechnology Ltd	20171101
75% ethanol	SHANDONG LIRCON MEDICAL TECHNOLOGY INCORPORATED Co.	171129D1
			Physiological saline	HARBIN MEDISAN PHARMACEUTICAL Co.,Ltd.	171004D5
MDA kit	American R&DSystems Inc	ELA05990
			SOD kit	American R&DSystems Inc	ELA05990
CAT kit	American R&DSystems Inc	ELA05990
			Urea nitrogen kit	Nanjing institute of biological engineering	C013-2
CAT kit	Nanjing institute of biological engineering	A007-1-1
			MDA kit	Nanjing institute of biological engineering	A003-1
NO kit	Nanjing institute of biological engineering	A013-2
			SOD kit	Nanjing institute of biological engineering	A001-3
GR kit	Nanjing institute of biological engineering	A062

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
		VICTOR X3 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) Body weight and exhaustion time test

70 male SD male rats with the weight of 0.18 +/-0.02 kg are taken, the temperature in a laboratory is kept at 22 +/-2 ℃ in the whole experiment process, the relative humidity is 55-75%, and the illumination time is naturally changed. All experimental rats were routinely fed basal diet and distilled water, free diet. The experimental time was 34 days and the administration time was 30 days. After the experimental rats are bred adaptively for one week, the experimental rats are screened according to the 20min exercise amount, the rats which are not suitable for swimming training are eliminated, the remaining 56 rats are randomly grouped into 7 groups, and the rest control group (the physiological saline is perfused into the stomach for 1 mL/d); ② a sports control group (the stomach is filled with physiological saline 1 mL/d); ③ LWY-lyophilized powder low dose drying group (0.05g/kg oviductus ranae lyophilized powder suspension); LWY-lyophilized powder middle dose drying group (0.10g/kg oviductus ranae lyophilized powder suspension); LWY-lyophilized powder high dose drying group (0.20g/kg oviductus Ranae lyophilized powder suspension); LWY groups (0.10g/kg oviductus Ranae suspension); seventhly, the decoction minister and the health care group (0.30g/kg decoction minister and the health care Gongjinjiyukang capsule) eat and drink water freely every day, and the stomach is infused and administrated 1 time every day. The weight was taken weekly and the body weight of each rat was recorded. The experiment of swimming exhaustion with load of 5% is carried out 60min after the last administration, and the glass swimming grooves of 100cm × 50cm × 60cm are used as the training devices for rat swimming, and the water depth is 50 cm. The water temperature is 31 +/-2 ℃, in order to prevent the rat from floating on the water surface, a Jiabao 'AP 1500' type water pump is particularly arranged at the bottom of the swimming tank to form flowing water, so that the rat can swim to exhaustion. (the time from the beginning of swimming to exhaustion of the rat is exhaustion exercise time, the exhaustion standard is measured in the degree that the rat does not expose out of the water surface after sinking for 10s, and the swimming time during exhaustion is recorded.) t test statistical treatment, and the results are shown in Table 1.

TABLE 1 influence of lyophilized powder of oviductus Ranae on weight and exhaustion time of rat: (n＝8)

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 weight change and exhaustion time can reflect the influence of the training on the body and the adaptation condition of the body to the training.

The results show that: weight: the body weight of the swimming control group was not much different from that of the stationary control group, compared with that of the stationary control group. LWY-lyophilized powder of each administration group showed a weight increase and a high significance (P <0.001) compared with swimming control group. The lyophilized powder of oviductus Ranae can increase the weight of rats. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage.

Exhaustion time: LWY-lyophilized powder administration groups showed prolonged exhaustion time and significant effect (P <0.001) compared with swimming control group. The freeze-dried powder of the rana japonica oil can increase the exhaustion time of rats and has certain anti-aging effect. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage.

(2) Influence on NO, MDA, SOD, GR, CAT activities in rat serum

According to the above experimental method, weighing is carried out 24 hours after the exhaustion test, chloral hydrate is appropriately anesthetized, blood is taken from the abdominal aorta, centrifugation is carried out at 4 ℃ and 3000r/min, serum is prepared by separation, and the content of Nitric Oxide (NO) and Malondialdehyde (MDA), the activity of Superoxide Dismutase (SOD), Glutathione Reductase (GR) and Catalase (Catalase, CAT) are determined strictly according to the kit instructions. t-test statistical treatment, results are shown in table 2.

TABLE 2 influence of lyophilized powder of oviductus Ranae on the activities of NO, MDA, SOD, GR and CAT in rat serum ((n＝8)

Note: comparison with static control group^#P<0.05，^##P<0.01，^###P<0.001, compared with exercise 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。

Nitric Oxide (NO) carries an unpaired electron and is extremely unstable in vivo, a property that is exactly the same as that of other free radicals. So that the two are very easy to combine to react. Thereby greatly reducing the number of free radicals in vivo. Aging or other diseases are mostly related to the generation of excessive free radicals, and the reduction of the NO content can play a role in resisting oxidation. Malondialdehyde (MDA) is a stable form of lipid peroxide formed by oxygen radicals oxidizing phospholipids on cell membranes, and can reflect the degree of lipid peroxidation in the body. Its production can also exacerbate membrane damage. Superoxide Dismutase (SOD) is an active substance derived from living bodies and can eliminate harmful substances generated in the metabolism process of organisms. SOD is continuously supplemented for human body, and has special anti-aging effect. Glutathione Reductase (GR) catalyzes the reduction of oxidized Glutathione in the body to Glutathione, which can bind to oxygen radicals and scavenge oxygenated free radicals inside the body to reduce damage of free radicals to the body. Catalase (CAT) exists in the peroxide in red blood cells and certain tissues, and has the main function of catalyzing H2O2 to be decomposed into H2O and O2, so that H2O2 does not react with O2 under the action of iron chelate to generate harmful-OH, and the Catalase has a protective effect on organisms.

The results show that: NO: compared with the stationary control group, the NO content of the movement control group is increased, and the movement control group has a very significant difference (P < 0.001). LWY-the NO content was reduced and was very significantly different (P <0.001) in each group compared to the exercise control group. The oviductus ranae lyophilized powder can reduce the NO content of rats and has good antioxidant effect. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage.

MDA: compared with the stationary control group, the exercise control group has higher MDA content and has very significant difference (P < 0.001). LWY-each administration group had a very significant difference (P <0.01 or P <0.001) in terms of the decrease in MDA content as compared to the exercise control group; the oviductus ranae freeze-dried powder can reduce the MDA content of rats and has the function of oxidation resistance. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage.

SOD (superoxide dismutase): compared with the normal control group, the SOD content of the model group is reduced, and the model group has extremely significant difference (P < 0.001). LWY comparing each administration group with the model group, the SOD content is increased, and has significant difference (P <0.001), which indicates that the oviductus Ranae lyophilized powder can increase rat SOD activity, and has antioxidant effect. Compared with the wood frog crude oil group, the wood frog oil freeze-dried powder has more obvious enhancement effect (P is less than 0.05) in the intervention group under the same dosage.

GR: compared with the stationary control group, the content of the moving control group is reduced, and the moving control group has a very significant difference (P < 0.001). LWY the GR content of each administration group of the freeze-dried powder is higher than that of the exercise control group, and the GR content is very significant (P <0.001), which indicates that the oviductus ranae freeze-dried powder can improve the GR activity of rats and has the function of antioxidation. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage.

CAT: compared with the stationary control group, the CAT content of the moving control group is reduced, and the moving control group has a very significant difference (P < 0.001). LWY-lyophilized powder administration groups and exercise control groups have obviously increased CAT content and have very significant difference (P < 0.001); the freeze-dried powder of the wood frog oil can improve the CAT activity of rats and has the function of anti-aging. Compared with the crude wood frog oil group, the freeze-dried wood frog oil powder has no significant difference under the same dosage. .

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 anti-aging 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.