CN1563308A - Antianxity effect of egg of Rana Cnensinensis and preparation method - Google Patents

Abstract

The invention discloses a wood frog egg oil with anti-anxiety effect and a preparation method thereof. It can be used to prepare pharmaceutical preparations with anxiolytic effect. The Rana egg oil can be prepared by pressing, _CO2 supercritical extraction and solvent extraction, etc. Oral preparations can be made into soft capsules, tablets, capsules, nano-dry milk, oral liquids and other dosage forms, and injections can be made into injection oils , liposome injection, etc. The obtained product can be applied to the body through different routes such as oral administration and injection, and these routes allow the frog egg oil to exert an anti-anxiety effect after entering the body. The invention provides a new approach for the application of the egg oil of the wood frog.

Description

Rana egg oil with anti-anxiety effect and preparation method thereof

Technical field:

The invention relates to the technical field of medicines, in particular to a wood frog egg oil with anti-anxiety effect and a preparation method thereof.

Background technology: Rana Temporaria Chensinensis David, also known as Rana Temporaria, belongs to the Chordate Phylum, Dimorpha, Anura, Ranaidae, Ranainae, Rana, and Chinese Rana species. Rana is a traditional valuable tonic and nourishing Chinese medicinal material in my country. According to historical records, Rana and its fallopian tubes (toad oil) nourish but not dry, moisten the lungs and promote body fluid, enter the kidneys, clear the lungs and moisten the throat. The medical books of all dynasties all record it as a tonic, and its products are toad oil (fora oviduct) and the dry whole body of toad (rana).

In recent years, Chinese scholar Li Chengyi has carried out solvent extraction and analysis on Rana roe, which proves that Rana roe is rich in amino acids, more than 30 kinds of trace elements, a variety of unsaturated fatty acids, vitamins D, E, estrogen and progesterone, etc. Biologically active substances (Chinese Medicine and Natural Medicine 1994; 29: 712). The research of our research group proves that Rana roe contains multiple types of components such as fatty acid and fatty acid amide (Chinese Journal of Medicinal Chemistry, 2001, 11(4): 224 225).

The development and comprehensive utilization of Rana chinensis has been paid more and more attention by people in recent years, therefore, the patents of Rana chinensis are increasing year by year. Search domestic and foreign patents with keywords such as wood frog, husky toad, hushi leech, clam toad, and snow toad, but found no patents from other countries. There are 74 Chinese patents, including 15 invention patents involving wood frog eggs . Among these 15 patents: there is one patent authorization (ZL 97116918.7), and fourteen patent applications. Chinese patent 99119379.2 "Rana eggs and their application" protects the effects of Rana eggs on immune regulation, anti-aging, aphrodisiac, eyesight, blood fat regulation and women's menopausal syndrome prevention. Chinese patent 01106213.4 "Using CO2 Supercritical Extraction Method for Rana Egg Oil", 01106211.8 "Hypolipid-lowering Nutritional Health Soft Capsule and Its Preparation Method", 01106212.6 "A Rana Egg Oil Nutritional Health Capsule with Lowering Blood Lipid Effect and Its Preparation 01106209.6 "Anti-aging, blood fat-lowering nutrition and health soft capsules and its preparation method", 01106210.X "education, blood fat-lowering nutrition and health care soft capsules and its preparation method" are 5 items applied by the same agency on the same day Patents, all related to the supercritical extraction process of Chinese Rana eggs. 01112272.2 Xueha Shuzhi Soft Capsule discloses the lipid-lowering effect of Rana egg oil. Chinese patent 01113910.2 "Supercritical Extraction Process of Rana Egg Oil", 01106240.1 "Rana Egg Oil Soft Capsule and Its Production Technology", 01114070.4 "Rana Egg Oil Injection and Its Preparation Technology" are the three patents applied by our research group. A patent protects the extraction process and preparation process of the product, enabling the key process of the product to have exclusive intellectual property rights. These methods revealed that the Rana roe contains rich nutrients and has various regulatory effects on the body.

Our research group prepared Rana egg oil by pressing, CO2 supercritical extraction, and solvent extraction, and made the obtained products into preparations for the body in different ways. After these methods made Rana egg oil enter the body, it was found that its Has an anxiolytic effect. Therefore, in the present invention, the Rana ova oil extracted in different ways is made into various preparations, and the Rana ova oil is introduced into the body in different ways to exert its anti-anxiety effect.

Invention content:

The object of the present invention is to provide an anti-anxiety effect of Rana ovale oil and its preparation method, so that Rana ovale oil can be used to prepare pharmaceutical preparations with anxiolytic effect. Preparations can be made into oral and injection preparations. Oral preparations can be made into soft capsules, tablets, capsules, nano-dried emulsions, oral liquids and other dosage forms, and injections can be made into injection oils.

Liposome injection, etc. The method for obtaining the wood frog egg oil can be prepared by pressing, and the selected clean wood frog eggs are soaked in oil overnight, and then squeezed with a press to obtain the wood frog egg oil solution. The oil for soaking is vegetable oil such as soybean oil, salad oil, peanut oil, sesame oil, evening primrose oil, perilla oil, olive oil, and animal oil such as fish oil and squalene. The obtaining method can also be prepared by the method of CO2 supercritical extraction. The selected clean Rana eggs are crushed into coarse powder, and the CO2 supercritical extraction device is used at a pressure of 5-50MPa and a temperature of 17-75°C at a concentration of 0-50%. Extract for 30-360 minutes under the condition of ethanol as entrainer, CO2 flow rate 13.7-68 liters/hour, then separate and separate egg oil in the separation tank, filter the egg oil after standing still, and recover ethanol under reduced pressure to obtain the finished product. The obtaining method can also be prepared by solvent extraction. After extracting the cleaned wood frog eggs with ether, acetone, chloroform, petroleum ether, ethanol and other solvents, the solvent is recovered under reduced pressure to obtain the wood frog egg oil.

Compared with previous inventions, the present invention has the following improvements:

1. Using pressing, CO2 supercritical extraction, ether, acetone, chloroform, petroleum ether, ethanol and other solvent extractions, various methods are used to prepare Rana egg oil.

2. Making the extract of wood frog eggs into oral dosage forms such as soft capsules, tablets, capsules, nano-dried milk, oral liquids, and injections such as oil injections and liposome injections.

3. Rana egg oil raw materials and preparations have significant anxiolytic effects. Drugs with anti-anxiety effects can be prepared, which provides a new application path for Rana egg oil.

The inventiveness of the present invention can be embodied by the following content:

1. Extraction of Rana eggs

1.1 Equipment The HA121-50-05 supercritical extraction device was provided by Nantong Huaan Supercritical Extraction Co., Ltd. The TTC525×2 direct cooling _CO2 supercritical extraction device was provided by Tieling Tianyuan Biological Co., Ltd. Squeezers and vertical centrifuges are provided by Liaoyang Pharmaceutical Machinery Factory.

1.2 Reagents

Absolute ethanol was provided by Shenyang Reagent No. 1 Factory, batch number: 991203. Anhydrous diethyl ether was provided by Shenyang Reagent No. 1 Factory, batch number: 991219. Salad oil comes from Yingkou Bohai Oil Co., Ltd. Stability injection, product of People's Pharmaceutical Factory of Tianjin Amino Acid Company, batch number 010424. Jiajing Anding Injection, a product of Henan Tianfang Pharmaceutical Co., Ltd., batch number 010101260.

1.3 Extraction method

1.3.1 Ether extraction of Rana chinensis eggs

Take the Rana chinensis eggs after removing the oviducts, dry them naturally, pick out impurities, grind them into a 40-mesh coarse powder with a pulverizer, soak them in 1:2 (W/V) ether for 24 hours, and then pour out the supernatant Add the same amount of diethyl ether and immerse three times in the same method, collect and filter the immersion liquid, use a decompression device to recover diethyl ether under reduced pressure on a water bath, and after no diethyl ether is evaporated, pour the yellow oily liquid in the distillation bottle into a beaker Put it at room temperature for a week, and after dispersing the ether, light yellow-brown egg oil is obtained, with a yield of 20%.

1.3.2 Ethanol extraction of Rana chinensis eggs

Take and remove the Rana chinensis ovum after removing the oviduct of Rana chinensis, dry naturally, pick out impurities, pulverize into a coarse powder with a pulverizer, and cold-soak with 1: 2 (W/V) ethanol for 24 hours, then pour out the supernatant, and then Add the same amount of ethanol and immerse three times in the same method, collect and filter the immersion liquid, connect to the decompression device, and recover the ethanol under reduced pressure on the water bath. After no ethanol is evaporated, pour the yellow oily liquid in the distillation bottle into a beaker at room temperature. After standing for a week, the ethanol was evaporated to obtain a light yellow-brown egg oil with a yield of 18%.

1.3.3 Acetone extraction of Rana chinensis eggs

Take the Rana chinensis ovum after removing the oviduct of Rana chinensis and dry it naturally, pick out impurities, grind it into a coarse powder with a pulverizer, then cold-soak with 1:2 (W/V) acetone for 24 hours, then pour out the supernatant, and then Add the same amount of acetone and immerse three times in the same method, collect and filter the immersion liquid, connect to the decompression device, and recover acetone under reduced pressure on the water bath. After no acetone is evaporated, pour the yellow oily liquid in the distillation bottle into a beaker at room temperature. After standing for a week, the acetone was evaporated to obtain a light yellow-brown egg oil with a yield of 17%.

1.3.4 Chloroform extraction of Rana chinensis eggs

Take the Rana chinensis ovum after removing the oviduct of Rana chinensis and dry it naturally, pick out impurities, grind it into a coarse powder with a pulverizer, and then cold-soak it with 1:2 (W/V) chloroform for 24 hours, then pour out the supernatant, and then Add the same amount of chloroform and soak three times in the same way, collect and filter the soaking liquid, connect to the decompression device, and recover the chloroform under reduced pressure on the water bath. After no chloroform is evaporated, pour the yellow oily liquid in the distillation bottle into the beaker at room temperature After standing for a week, the chloroform was evaporated to obtain a light yellow-brown egg oil with a yield of 20%.

1.3.5 Petroleum ether extraction of Rana chinensis eggs

The Rana chinensis ovum after removing the oviduct of Rana chinensis is dried naturally, and impurities are removed, crushed into a coarse powder with a pulverizer, and then cold soaked with 1:2 (W/V) petroleum ether for 24 hours, and then the supernatant is poured out. Add the same amount of petroleum ether and dip three times in the same method, collect and filter the immersion liquid, connect to the decompression device, and recover petroleum ether under reduced pressure on the water bath. After no petroleum ether is evaporated, pour the yellow oily liquid in the distillation bottle into Put it in a beaker at room temperature for a week, evaporate the petroleum ether to obtain a light yellow-brown egg oil, and the yield is 18%.

1.3.6 Supercritical _CO2 extraction of Rana chinensis eggs

Take the Rana sinensis after removing the oviducts, let it fully dry in the shade, carefully peel off the dried eggs, collect them together, sort out the impurities, crush the eggs into coarse powder, and extract them with a CO ₂ supercritical extraction device. Rana chinensis eggs are crushed into coarse powder, and extracted at a temperature of 31-33° C., a pressure of more than 35 MPa, and a time of 180 minutes, using 5% ethanol as an entrainer to extract is the optimum condition. Yield is 12%

1.3.5 Extraction of Rana eggs by pressing

After removing the fallopian tubes of Rana chinensis, the eggs of Rana chinensis are naturally dried, and the impurities are removed, and oils are used to clean the eggs of Rana sylvatica (respectively use soybean oil, salad oil, peanut oil, sesame oil, evening primrose oil, perilla oil, olive oil, etc.) Vegetable oils such as oil, animal oils such as fish oil and squalene) are soaked overnight, and squeezed with a press to obtain the wood frog egg oil solution. The yield is 15%.

2 Preparation method of Rana egg oil

2.1 Preparation of Rana egg oil soft capsules

Get wood frog egg oil 25kg. Let the raw materials stand still, filter, mix the filtrate with vitamin E as an antioxidant, sterilize with dry heat sterilization after filling nitrogen, take the raw materials and gelatin solution and make soft capsules by compression method, each soft capsule has a capacity of about 0.4ml, capsule The heart weighs about 400mg.

2.2 Preparation of Rana egg oil injection

Rana egg oil injection uses Rana egg oil and other ingredients and preparation auxiliary materials, such as diluents, emulsifiers, co-solvents, antioxidants, etc. Other injection auxiliary materials that help the preparation to form and stabilize can also be used. The ratio of each component in the formula of Rana egg oil injection is: Rana egg oil 5-70%, soybean oil 0-80%, phospholipid 0-90%, Span 80 0-1%, Tween 80 0-1 %, glycerin 0-30%, vitamin E 0-5%, water for injection 40-70%.

2.3 Preparation of Rana egg oil capsules

Get wood frog egg oil 20kg. Filter, absorb the filtrate with 100kg of edible calcium carbonate, mix evenly, and make No. 1 capsule, each capsule weighs 300mg.

2.4 Preparation of Rana egg oil tablet

Weigh 300g of calcium carbonate and 50g of Rana egg oil and mix well. Separately take starch and add water to make a paste, add it to the mixed powder to make large rice granules, dry at 60°C for 2 hours, add appropriate amount of talcum powder to adjust fluidity, and press with a 10mm die (flat right angle) of a tablet machine. Tablet weight 550mg.

2.5 Preparation of Rana egg oil nano dry emulsion,

Take 20kg of wood frog egg oil, 20kg of salad oil, mix evenly, take another 4kg of phospholipids and dissolve them with an appropriate amount of ethanol, mix 1kg of Tween 80, 2kg of poloxamer and 400kg of distilled water, slowly change the oil phase and slowly add it to the water phase to prepare an emulsion . Thereafter, dry milk was prepared by spray drying. A mixture of 10 kg of sucrose, 10 kg of mannitol and 10 kg of sodium carboxymethyl cellulose is used as an auxiliary material and mixed with Rana egg oil spray dry powder to form dry milk.

2.6 Preparation of Rana egg oil oral liquid

Take 10kg of phospholipids and dissolve them in ethanol to make a saturated solution, add 10kg of wood frog egg oil, mix well, take another 1kg of Tween 80, 2kg of sucrose, dissolve in 40kg of distilled water, slowly pour the wood frog egg oil and phospholipid ethanol solution into water Get it right away.

3 Pharmacological study of Rana egg oil

3.1 Experimental materials

3.1.1 Animals

Kunming mice, aged 5-6 weeks, weighing 18-22g; Wistar rats, aged 6-7 weeks, weighing 120-130g, both male and female. All were provided by the animal room of Shenyang Pharmaceutical University, and the animal qualification certificate was Liao Shi Dong Zhi Zi [2000] No. 042. The animal environment facility certificate is Liao Shi Dong Huan Zi [1999] No. 062. The issuing authority is the Liaoning Provincial Laboratory Animal Management Committee. The animals were kept in a sound-proof animal room with a temperature of 24±2°C, 50-60% relative humidity, 12 hours of light (08:00--20:00 hours), 12 hours of darkness, and the animals had free access to food and water. After adapting to the above environment for one week, it was used in the experiment.

3.1.2 Instruments

The small animal spontaneous activity recorder was provided by Beijing Pharmaceutical Institute. The mice climbed the ladder, and the rats raised the cross maze device by referring to the model described in "Modern Pharmacological Experimental Methods". Elevated plus maze for rats: The plus maze is a wooden structure and painted black. It includes two opposite open arms (open arms) of 50cm×10cm ² and two opposite closed arms (close arms) of 50×10×40cm ³ , the upper part of the closed arms is open. There is an open area of 10cm×10cm ² in the center of the maze. The maze is 50cm above the ground. Staircase for mice: A closed glass box containing 5 identical stairs of 2.5 cm height. Stair side wall heights were kept constant to allow animals to have the same distribution of standing on all levels of the stairs.

3.2 Experimental methods and results

3.2.1 Effect of Rana egg oil on mice climbing ladders

100 mice, half male and half male, were randomly divided into 5 groups according to body weight and gender balance, and administered by intragastric administration. Blank control group (equal volume salad oil), positive control group (clonazepam 2mg·kg ^-1 ), wood frog egg oil group (1.4, 2.8, 5.6g·kg ^-1 ), mice were placed in the At the bottom of the box, facing away from the stairs, the mouse behavior was filmed with a digital video camera, and the experimenter left to reduce disturbance. The number of ladders climbed (according to the fact that all limbs climbed the stairs) and the number of standing of mice were recorded within 3 minutes. Experiments were carried out under the red light from 18:00 to 21:00 every day, and the experimental box was cleaned quickly after each animal experiment to eliminate the interference of olfactory cues on the next animal.

Anxiety responses, such as increased alertness, occur when mice are introduced to a new environment. The ladder climbing behavior evaluation explores the indicators of behavior or activity, and standing upright is used as a parameter of anxiety state. Anxiolytics reduce the number of standing at a dose that does not reduce the number of ladders climbed. It can be seen from Table 1 that compared with the blank control group, the number of climbed ladders decreased but there was no significant difference between the mice taking Rana egg oil group, but the number of standing was significantly reduced. It is suggested that Rana egg oil has anti-anxiety effect, and it has a certain dose-dependent relationship.

  Table 1 Effect of Rana egg oil on mice climbing ladders ( x±s)n=20

Groups Dose (g/kg) Number of stairs climbed Number of standing

Control group - 11.6±3.3 9.9±2.7

Clonazepam 0.002 10.9±2.7 5.5±1.5**

Rana egg oil 1.4 11.1±3.0 8.6±2.9

Rana egg oil 2.8 10.1±3.0 7.0±2.9*

Rana egg oil 5.6 10.4±2.4 5.9±1.9**

  Compared with the control group, **p<0.01, *p<0.05

3.2.2 Comparison of anti-anxiety activities of different solvent extracts of Rana ovale oil

Get 112 mice, divide 8 groups randomly by body weight, be respectively blank control group ip salad oil 10ml/kg, positive control group ip.clonazepam 2mg/kg, 6 groups of administration groups (ethanol extract, ether extract , acetone extract, chloroform extract, petroleum ether extract, CO ₂ supercritical extract, ip400mg/kg), determine the anxiolytic activity of the extract obtained from extracting Rana chinensis eggs with different solvents according to the climbing ladder test method,

The results showed that the number of ladders climbed and the number of standing on the ladders of mice in each group were significantly reduced, suggesting that each extract has the effect of inhibiting the activity of mice climbing ladders, indicating that each extract has anti-anxiety activity. Comparing the data of each group, there is no significant difference in the number of climbing ladders and standing on the ladders of the animals, and the inhibitory effect of the extracts of each group on the behavior of standing on the ladder of mice is generally stronger than that of climbing the ladder. This is exactly what anxiolytics do. On the basis of anti-anxiety effect, there is no significant difference between different extracts.

Table 2 Comparison of anxiolytic activity of different solvent extracts of Rana oval oil

Group Dose (mg/kg) Number of stairs climbed Number of standing

Control group - 15.9±5.3 11.6±4.0

Clonazepam 2 8.7±6.3** 3.1±3.1**

CO ₂ Supercritical 400 10.4±5.4* 5.0±3.7**

Ethanol extract 400 10.8±3.3** 5.6±3.1**

Diethyl ether extract 400 11.5±5.1* 5.7±4.2**

Acetone extract 400 11.1±5.0* 5.1±3.8**

Chloroform extract 400 11.3±4.1* 6.3±3.9**

Petroleum ether extract 400 12.1±3.9* 6.8±2.6**

Compared with the control group, **p<0.01, *p<0.05

3.2.3 Effect of Rana egg oil soft capsules on mice climbing ladders

100 mice were taken, half male and half male, randomly divided into 5 groups according to body weight, and administered by intragastric administration (ig) respectively. Blank control group (ig salad oil 10ml/kg), positive control group (ig clonazepam 2mg/kg), wood frog egg oil group (ig wood frog egg oil 5.6, 2.8, 1.4g/kg), 30min after administration The number of ladders climbed and the number of standing mice within 3 min were recorded.

The results showed that there was no significant difference in the number of climbing stairs between the mice taking the Rana egg oil soft capsule group and the blank group, but the number of standing was significantly reduced. It is suggested that the dosage of Rana egg oil soft capsule that does not reduce the number of climbing stairs can reduce the number of standing in mice, suggesting that it has anti-anxiety effect (Table 3).

          Table 3 Effects of Rana egg oil soft capsules on mice climbing ladders

Group Dose (g/kg) Number of climbs Number of standing

Control group - 11.60±3.35 10.30±3.87

Clonazepam 0.002 11.20±1.45 5.45±2.19**

Rana egg oil soft capsule 5.6 10.15±7.46 5.95±4.45**

Rana egg oil soft capsule 2.8 10.90±6.36 7.25±5.35*

Rana egg oil soft capsule 1.4 14.20±4.68 7.50±4.08*

Compared with the control group, **p<0.01, *p<0.05

3.2.4 Rana egg oil anti-anxiety time-effect experiment

Seventy mice, half male and half male, were randomly divided into 7 groups according to body weight and gender balance, and administered intraperitoneally. The dose given to the mice in the Rana egg oil group was 1.2 g·kg ^-1 . The mice were placed on the bottom of the box after administration for 0 min (equal volume salad oil), 15, 30, 60, 120, 180, and 240 min, respectively, and the number of climbing ladders and standing numbers of the mice within 3 min were recorded.

The results are shown in Table 4, there was no significant difference in the number of climbs of the test mice administered at different times. After intraperitoneal administration for 15 minutes, there was no significant difference in the number of mice standing. After administration for 30 minutes and 60 minutes, the number of mice standing was significantly reduced, and the effect was stronger at 30 minutes; after 120 minutes until 240 minutes, the number of mice standing gradually increased with the administration time. It suggested that the anti-anxiety effect gradually weakened.

Table 4 Rana egg oil anti-anxiety time-effect experiment ( x±s)n=10

Group Dose (g/kg) Determination time (minutes) Number of climbs Number of standing

Control group - 0 20.4±1.3 15.9±2.8

Rana egg oil 1.2 15 20.0±2.2 14.3±3.5

Rana egg oil 1.2 30 20.0±0.5 11.9±3.1**

Rana egg oil 1.2 60 20.0±1.9 12.6±2.0**

Rana egg oil 1.2 120 20.4±2.2 13.8±3.9

Rana egg oil 1.2 180 21.3±3.0 14.3±4.7

Rana egg oil 1.2 240 20.6±3.1 14.4±4.4

Compared with the control group, **p<0.01, *p<0.05

3.2.5 Effect of Rana egg oil on elevated plus maze anxiety model rats

60 rats, half male and half male, were randomly divided into 5 groups according to body weight: blank control group (gavage (ig) equal volume salad oil), positive control group (ig diazepam 2mg/kg), wood frog egg oil group (ig forest Frog egg oil 4.8, 2.4, 1.2g/kg), all experiments were carried out at 18:00-21:00. After 30 minutes of ig administration, the rats were placed in the center of the elevated cross maze, with the head facing the closed arms, and the observer was 1.2 meters away from the center of the maze. During the experimental period (5min), the number of times the rats entered the open arm and the closed arm and the residence time (based on whether all limbs entered or exited the arm) were recorded. Observation indicators: Latency and stay in the central departure room, continuous stay in open and closed passages, number of entry times in open and closed passages

The rat elevated plus maze experiment is to use the animal's exploration characteristics of novel environments and the fear of hanging open arms to form the animal's conflict state. It is used to evaluate the anxiety behavior of animals. If the cross maze is 50cm from the ground, it is equivalent to a person standing on a cliff, and rats will have fear and anxiety. Rats in the control group mainly stayed in the closed arm after entering the maze, and the number of times of entering the open arm and the time of staying in the open arm were short. High and medium doses of Rana ovale oil can significantly prolong the continuous stay time of rats in the open pathway, and there is no significant difference in the starting latency of the rats in each group in the central chamber of the maze; The number of times and the time of staying in the open arm were significantly increased; the time of rats in the stability group and the Rana egg oil group staying in the closed arm was significantly shorter than that of the blank control group; the time of the rats in the Rana egg oil group staying in the central chamber was shorter significantly increased in the blank group. The extended stay time of the test animals in the open arm suggests that Rana egg oil has a certain anti-anxiety effect, and diazepam can also increase the number of times rats enter the open pathway and the stay time (Table 5).

Table 5 Anxiolytic effect of EORTCD on the elevated plus maze model

Enter into open arm times

Dose Number of Animals Departure Latency During Open Arm Stays Closed Arm Stays Central Chamber Stays Number of Entry Closed Arms

group

Number of times

(g/kg) (only) (min) (min) ((min) (min) (times/5min)

/5min)

Control group - 12 1.42±1.61 2.17±4.20 288.75±19.11 3.75±4.88 0.25±0.45 2.67±2.90

Stability 0.002 12 1.92±1.98 13.58±12.83 254.08±33.89 ^* 32.33±28.47 ^** 2.00±1.55 ^** 4.42±1.30 ^*

Rana egg oil 4.8 12 1.92±1.98 25.25±27.89 ^** 238.67±44.03 ^* 36.33±28.83 ^** 1.91±1.00 ^** 4.00±1.95 ^*

Rana egg oil 2.4 12 1.92±1.83 19.83±33.02 251.00±51.83 ^* 29.50±28.93 ^** 1.58±2.54 5.00±2.52 ^*

Rana egg oil 1.2 12 1.33±0.49 16.17±21.44 267.00±34.51 16.83±12.04 ^** 1.00±1.13 ^* 3.33±1.44

Compared with the control group, ^* P<0.05, ^** P<0.01

Detailed ways:

The following examples are for better illustrating the present invention, and are not intended to limit the scope of the invention.

Example 1:

Rana egg oil soft capsule

formula

Rana egg oil 25kg

Vitamin E 5kg

According to a certain process, it is made into 75000 soft capsules of Rana egg oil

Preparation method: Take 25kg of Rana egg oil. Let the raw materials stand still, filter, mix the filtrate with antioxidant vitamin E, sterilize with dry heat sterilization after nitrogen filling, take the raw materials and gelatin solution and make soft capsules by compression method, each soft capsule has a capacity of about 0.4ml, The heart weighs about 400mg.

Example 2

Rana egg oil injection

formula

Rana egg oil 50kg

Soybean oil for injection 100kg

Phospholipids 20kg

Span 80 10kg

Tween 80 5kg

Vitamin E 20kg

Glycerin 50kg

Water for injection 500kg

600,000 sticks of Rana egg oil injection formulated according to a certain process

Preparation method: Take Rana egg oil and soybean oil for injection according to the formula and mix them to obtain the oil phase for later use; in addition, disperse phospholipids and glycerin in water for injection to obtain the water phase. Add the oil phase, Span 80, Tween 80 and vitamin E into the water phase, emulsify, homogenize with a homogenizer, filter, pack in 1ml ampoules, sterilize, inspect, print, and package.

Example 3

Rana egg oil capsules

formula

Rana egg oil 20kg

Calcium carbonate 100kg

Formulated into Rana egg oil capsules 400,000 capsules according to a certain process

Preparation method: Take 20kg of Rana egg oil. Filter, absorb the filtrate with 100kg of edible calcium carbonate, mix evenly, and make No. 1 capsule, each capsule weighs 300mg.

Example 4

Rana Egg Oil Tablets

formula

Rana egg oil 50kg

Calcium carbonate 300kg

Starch 20kg

Talc powder 3kg

Distilled water 50kg

Made into 600,000 pieces of Rana egg oil tablets according to a certain process

Preparation method: Weigh 300g of calcium carbonate, 50g of Rana egg oil and mix well. Separately take starch and add water to make a paste, add it to the mixed powder to make large rice granules, dry at 60°C for 2 hours, add appropriate amount of talcum powder to adjust fluidity, and press with a 10mm die (flat right angle) of a tablet machine. Tablet weight 550mg.

Example 5

Rana egg oil nano dry milk

formula

Rana egg oil 20kg

salad oil 20kg

Phospholipids 4kg

Tween 80 1kg

Poloxamer 2kg

Distilled water 400kg

Sucrose 10kg

Mannitol 10kg

Sodium Carboxymethyl Cellulose 10kg

Made into 600,000 pieces of Rana egg oil tablets according to a certain process

Preparation method: take 20kg of wood frog egg oil, 20kg of salad oil, mix evenly, take another 4kg of phospholipids and dissolve them with an appropriate amount of ethanol, mix 1kg of Tween 80, 2kg of poloxamer and 400kg of distilled water, slowly replace the oil phase and add to the water phase Prepare the emulsion. Thereafter, dry milk was prepared by spray drying. A mixture of 10 kg of sucrose, 10 kg of mannitol, and 10 kg of sodium carboxymethyl cellulose was used as an auxiliary material and mixed with Rana egg oil spray dry powder to form 80 kg of dry milk.

Example 5

Rana egg oil oral liquid

formula

Rana egg oil 10kg

Phospholipids 10kg

Tween 80 1kg,

Ethanol 40kg

Distilled water 40kg

Sucrose 2kg

Made into 600,000 pieces of Rana egg oil tablets according to a certain process

Preparation method: take 10kg of phospholipids and dissolve them in ethanol to make a saturated solution, add 10kg of wood frog egg oil, mix well, and take 1kg of Tween 80, 2kg of sucrose, dissolve them in 40kg of distilled water, slowly dissolve the wood frog egg oil and phospholipid ethanol solution Pour it into the water phase.

Claims (6)

1. Rana egg oil with anti-anxiety effect, characterized in that: Rana egg oil can be used as a pharmaceutical preparation for preparing anxiolytic state. 2. The Rana egg oil with anti-anxiety effect according to claim 1 is characterized in that: the preparation can be made into oral and injection preparations, and the oral preparation can be made into soft capsules, tablets, capsules, nano dry milk, Oral liquid and other dosage forms, injection can be made into injection oil, liposome injection, etc. 3. A method for preparing the anxiolytic Rana roe oil as claimed in claim 1, characterized in that: the obtaining method can be prepared by pressing, soaking the selected clean Rana ova in oil overnight, and then using Squeeze with a press to obtain the wood frog egg oil solution. 4. The method for preparing Rana egg oil with anti-anxiety effect according to claim 3, characterized in that: the oil for soaking is soybean oil, salad oil, peanut oil, sesame oil, evening primrose oil, perilla oil, Vegetable oils such as olive oil, animal oils such as fish oil and squalene. 5. A method for preparing the anxiolytic Rana ova oil as claimed in claim 1, characterized in that: the obtaining method can be prepared by CO2 supercritical extraction, and the cleaned Rana ova are crushed into coarse powder , with a CO2 supercritical extraction device, at a pressure of 5-50 MPa, a temperature of 17-75 ° C, and 0-50% ethanol as an entrainer for extraction for 30-360 minutes, a CO2 flow rate of 13.7-68 liters/hour, and then separate Separate and separate the egg oil in the tank, filter the egg oil after standing still, and recover ethanol under reduced pressure to obtain the finished product. 6. A preparation method of the anxiolytic Rana ova oil as claimed in claim 1, characterized in that: the obtaining method can be prepared by solvent extraction, and the selected clean Rana ovum is extracted with ether, acetone, chloroform, etc. , petroleum ether, ethanol and other solvents, and recover the solvent under reduced pressure to obtain Rana egg oil.