CN105193824B - A kind of ganodenic acid acid activity position and its preparation method and application - Google Patents

Abstract

The invention discloses a ganoderma lucidum triterpene acid active part and its preparation method and its application in the preparation of sleep-improving health care products and sedative and tranquilizing medicines. The method comprises: crushing the ganoderma lucidum fruiting body medicinal material to obtain dry powder, and adding compound enzymes to the dry powder The enzymolysis liquid was obtained by inactivating the enzyme, cooling, and suction filtration; the enzymolysis liquid was added to petroleum ether for extraction to remove fat, and then ethyl acetate was added for extraction to obtain the ethyl acetate extract of Ganoderma lucidum; the ethyl acetate extract of Ganoderma lucidum was first Alkaline, then wash with water until neutral to obtain an alkalized water layer, then acidify the alkalized water layer, and then extract crude ganoderma acid through ethyl acetate; separate the crude ganoderma acid through a silica gel column, and use chloroform and methanol system gradient Eluted, identified by thin-layer chromatography, filtered, and evaporated to dryness, the active part of triterpene acid of Ganoderma lucidum was obtained. The active part of the ganoderma triterpene acid of the present invention can be obtained by enzymatic hydrolysis with complex enzymes, and then through extraction, separation and other steps, and can be used to prepare sleep-improving health care products and sedative and tranquilizing drugs.

Description

A kind of Ganoderma lucidum triterpene acid active part and its preparation method and application

technical field

The invention relates to the technical field of Ganoderma lucidum extract, in particular to an active part of Ganoderma lucidum triterpene acid and its preparation method and its application in the preparation of sleep-improving health care products and tranquilizing drugs.

Background technique

Sleep is an essential physiological process for humans and higher animals, and is the basis for maintaining physical strength and health. Insomnia is a common sleep disorder that refers to the inability to fall asleep or stay asleep. Chronic insomnia probably affects about 15% of the population, and transient insomnia affects about 80% of the population each year. At present, the treatment of insomnia is mainly to take artificial synthetic drugs, but synthetic drugs have serious drug dependence, cognitive and motor impairment, drug withdrawal rebound and other adverse reactions. Therefore, there is an urgent need to find and develop safe and effective sedative and tranquillizing drugs, so as to break through the development bottleneck of sedative and tranquilizing drugs and dietary supplements.

Ganoderma lucidum is the dry fruiting body of Polyporaceae Chizhi [Ganoderma lucidum (Leyss.ex.Fr.) Karst.] or Zizhi [Ganoderma japonicum (Fr.) Lloyd]. It is flat in nature, sweet in taste and warm in taste. The Guixin, Liver, and Spleen meridians have been known as "immortal grass" and "auspicious grass" since ancient times. They have extremely high medicinal value and are precious fungi and algae medicinal materials in my country. The Chinese people have used it as medicine for more than 2,000 years. In the ancient book "Liezi" of the Zhou Dynasty, there is a saying "On the rotten soil, there are mushrooms". The "sedative" effect of Ganoderma lucidum was first recorded in "Shen Nong's Materia Medica", the 2005 edition of "Zhejiang Province Traditional Chinese Medicine Processing Standards" and the 2010 edition of "People's Republic of China Pharmacopoeia" both recorded that Ganoderma lucidum has the effect of "supplementing qi and calming the nerves". Among the various chemical components of Ganoderma lucidum, Ganoderma lucidum polysaccharides and triterpenoids are the main functional components. In my country, the content of Ganoderma lucidum polysaccharides is used to evaluate the quality of Ganoderma lucidum; in Japan and other countries, the content of Ganoderma triterpene acids is used as identification The quality standard of Ganoderma lucidum products.

Modern pharmacology reports on the sedative and tranquilizing effect of Ganoderma lucidum are rare. Wang and Tang et al. studied the hypnotic effect of Ganoderma lucidum extract on the human body (Wang Xiangli, Meng Zhaoyang, Wang Cuiping. 60 cases of insomnia treated with Ganoderma lucidum liquid[J]. China Acta Medical Journal, 2001,16(1):47-49; TANG Wen-bo, GAO Yi-huai, CHEN Guo-liang, et al. ARandomized, double-blind and placebo-controlled study of a Ganoderma Lucidum polysaccharide extract(ganopoly) in neurasthenia[J].Journal of Medicinal Food,2005,8(1):53-58), Chu and other studies have proved that the water extract of Ganoderma lucidum fruiting body can prolong the sleep time of rats in the pentobarbital sodium sleep experiment, thinking that Ganoderma lucidum potentes pentobarbital-induced sleep via a GABAergic mechanism[J].Pharmacology, Biochemistry and Behavior, 2007,86(4):693-698); Cui et al found that Ganoderma lucidum crude polysaccharide 80mg/kg can prolong the total sleep time and non-rapid eye movement sleep time of rats without affecting slow wave sleep and rapid eye movement sleep time , TNF-α levels in rat serum, hypothalamus and dorsal raphe nucleus were significantly increased (CUI Xiang-yu, CUI Su-ying, ZHANG Juan, et al.Extract of Ganoderma lucidum prolongs sleep time in rats[J].Journal of Ethnopharmacology, 2012, 139(3):796-800). CHO et al. used radioligand receptor binding experiments to prove that the ethanol extract of Ganoderma lucidum fruiting bodies has a moderate affinity for GABA _A receptors, while the water extract has a very weak affinity for GABA _A receptors (CHOSueng-mock, SHIMIZU Makoto, LEE C -justin, et al.Hypnotic effects and bindingstudies for GABAa and5-HT2c receptors of traditional medicinal plants used in Asia for insomnia[J].Journal of Pharmacology,2010,132(1):225-232); Triterpenes and Poria cocos extracts have the function of synergistically improving sleep, but Ganoderma lucidum polysaccharides do not have this pharmacological effect, and its mechanism of action is not clear (Jia Wei, Wu Min, Zhang Jinsong, etc. Preliminary study on the function of improving sleep function of Ganoderma lucidum components[J]. Edible fungi Journal, 2005, 12(3):43-47). Research by Jiang Haitao and others found that Ganoderma lucidum extract has the effect of improving sleep, but there is no obvious linear relationship between the effect of improving sleep and the dose of the test substance, and its mechanism of action and material basis are not clear (Jiang Haitao, Ren Yuanhao, Yu Weiyan etc. Functional research of Ganoderma lucidum extract in sleep improvement[J]. Shi Zhen Guoyue Guoyao, 2008,19(9):2231-2232); Wang Yuhong, Cao Sufang, etc. all conducted research on the sedative and tranquilizing effect of Ganoderma lucidum fruiting body extract However, the possible material basis was not traced in the paper (Wang Yuhong. Research on Ganoderma lucidum improving sleep function[J]. Shanxi Medical Journal, 2011, 40(9): 878-880; Cao Sufang, Wang Xinrui, Zhang Chen, etc. About Research on Ganoderma lucidum improving sleep function [J]. Zhejiang Edible Fungi, 2009,17(6):52-54).

Therefore, although Ganoderma lucidum is a precious fungus and algae medicinal material in my country, and its "replenishing qi and tranquilizing" effect has been used medicinally for thousands of years, modern pharmacology has not yet clarified the corresponding active and functional components, and most pharmacological experiments only stay on the original medicinal material. And on the basis of crude extract, there is no report that Ganoderma lucidum extract is further refined to active parts and used to prepare calming and tranquilizing health care products and medicines.

Contents of the invention

The invention provides the active part of ganoderma triterpene acid and its preparation method and its application in the preparation of sleep-improving health care products and sedative and tranquilizing drugs. It can be obtained and can be used to prepare sleep-improving health care products and sedative and tranquilizing drugs.

A preparation method for the active part of ganoderma triterpene acid, comprising the following steps:

(1) Crushing the Ganoderma lucidum fruiting body medicinal material to obtain dry powder, adding compound enzyme to enzymolyze the dry powder in water, inactivating the enzyme, cooling, and suction filtration to obtain the enzymatic hydrolyzate;

(2) Add the enzymolysis solution obtained in step (1) to petroleum ether for extraction and degreasing, then continue to add the enzymolysis solution after degreasing to ethyl acetate for extraction to obtain the extraction part of ethyl acetate, and evaporate the ethyl acetate layer Solvent, obtain Ganoderma lucidum ethyl acetate extract;

(3) Dissolve the ganoderma ganoderma ethyl acetate extract prepared in step (2) in ethyl acetate, alkalize to pH 9-10 first, then wash with water until neutral to obtain an alkalized water layer, and then acidify the alkalized water layer to pH 2-3, and then extracted with ethyl acetate to obtain crude ganoderma acid;

(4) The crude ganoderma acid prepared in step (3) is separated through a silica gel column, eluted with a gradient of chloroform and methanol, and the eluate is collected, identified by thin-layer chromatography, filtered, and evaporated to dryness to obtain the ganoderma triterpenes Acid active site.

In the present invention, through the enzymatic hydrolysis of the complex enzyme and the cooperation of each extraction step, the triterpene acid compounds in the ganoderma lucidum can be extracted more fully and comprehensively, and the triterpene acid active part of the ganoderma lucidum has better sedative and tranquilizing activity.

Following as preferred technical scheme of the present invention:

In step (1), crush the fruiting body of Ganoderma lucidum to obtain dry powder, including: firstly take the fruiting body of Ganoderma lucidum, dry at 55°C-75°C, pulverize, and sieve through 80-120 mesh to obtain dry powder; further preferably, include: firstly take the fruiting body of Ganoderma lucidum The medicinal materials are dried at 65°C, pulverized, and sieved through a 100-mesh sieve to obtain a dry powder.

The compound enzyme is composed of the following components in weight percentage:

Neutral protease 5% to 95%;

Cellulase 2.5%~48%;

Pectinase 2.5% to 48%.

Further preferably, the complex enzyme is composed of the following components in weight percentage:

Neutral protease 30% to 80%;

Cellulase 10%~35%;

Pectinase 10% to 35%.

The main components of the plant cell wall are pectin and cellulose. The pectinase in the compound enzyme can enzymatically hydrolyze the pectin, and the cellulase can enzymolyze the cellulose to release the active ingredients in the plant cells. The neutral protease can The protein and other macromolecular substances are clarified to increase the yield of active ingredients. The use of complex enzymes can shorten the extraction time of ganoderma triterpene acids and increase its yield, and can more fully and comprehensively extract triterpene acid compounds in ganoderma lucidum.

The added amount of the compound enzyme is 1.0%-5.0% of the dry powder mass.

The solid-liquid ratio of the water to the dry powder is 10mL-30mL:1g, more preferably 20mL:1g.

The enzymolysis conditions are: temperature 35-70° C., pH 3.5-7, and enzymolysis time 4.5-8 hours.

In step (3), the ethyl acetate extract of Ganoderma lucidum is first alkalized, and the triterpene acid in it is salified, dissolved in water, and then extracted with ethyl acetate to remove alkaline and neutral impurities; then the alkalized washing solution is acidified The salified triterpene acid is released, and ethyl acetate is used to continue extracting to obtain the purified triterpene acid component. The role of alkalization and acidification is to purify triterpene acids, mainly to remove neutral triterpenes.

In step (4), the volume ratio of chloroform to methanol in the chloroform and methanol system is 1-10:1. Further preferably, the volume ratio of chloroform to methanol in the chloroform and methanol system is 2 to 8:1, and the eluate whose volume ratio of chloroform to methanol is 2 to 8:1 is collected, and the above eluent contains more triterpenoid compounds.

In the present invention, in the obtained ganoderma triterpene acid active part, the weight percent content of triterpene acid is 5% to 70%, and the content of triterpene acid is determined by a spectrophotometer.

The active part of ganoderma lucidum triterpene acid obtained by the preparation method of the invention can be used to prepare sleep-improving health care products and sedative and tranquilizing drugs.

In order to detect the performance of the sedative and tranquilizing active part of the active part of the ganoderma triterpene acid, the active part of the ganoderma triterpene acid obtained in the present invention is configured into a 25-100 mg/ml medicinal liquid, which is used for prolonging the sleep time experiment of pentobarbital sodium, pentobarbital sodium, etc. In the barbital sodium subthreshold dose hypnosis experiment and the barbital sodium sleep latency experimental model, the effects of different doses of samples on the sleep behavior of experimental mice were observed respectively. The results show that the active part of ganoderma triterpene acid has significant sedative and tranquilizing activity, and can be used to prepare health care products for improving sleep or auxiliary drugs for treating insomnia, sleep disorders and other diseases.

The active part of ganoderma lucidum triterpene acid of the present invention can be a pharmaceutical composition composed of pharmaceutically acceptable auxiliary materials.

The active part of ganoderma triterpene acid can be used alone or combined with several parts, and further combined with auxiliary materials. The dosage forms include: tablets, capsules, pills, granules, suspensions, dropping pills, oral liquid preparations, etc.

The carriers and excipients of the active part of ganoderma lucidum triterpene acid in the present invention include carriers and excipients commonly used in pharmacy, such as solvents, disintegrants, flavoring agents, preservatives, coloring agents, binders and the like.

The active part of ganoderma triterpene acid has obvious sedative and tranquilizing effects, which can prolong the sleep time and shorten the sleep latency of pentobarbital sodium mice; in the subthreshold dose hypnosis experiment of pentobarbital sodium, it can significantly increase the and significantly shorten the sleep latency of mice in the barbital sodium sleep latency experiment, indicating that the active part of Ganoderma lucidum triterpene acid has a good sedative and tranquilizing effect and has the potential for development.

Compared with prior art, the present invention has following advantage:

The preparation method of the active part of the ganoderma triterpene acid of the present invention, wherein the content and purity of the ganoderma triterpene acid are greatly improved. Existing methods usually use chloroform, ethyl acetate and other reagents to directly extract Ganoderma lucidum fruiting body dry powder or ethanol extract, which is mixed with a large amount of impurities such as fatty acids and neutral triterpenes.

The preparation method of the ganoderma triterpene acid active part of the present invention optimizes the extraction process of the ganoderma triterpene acid, respectively adopts enzymatic method and chromatography to pre-preparate the ganoderma triterpene acid and its active part before and after extraction. The treatment and refining, on the one hand, can more fully and comprehensively extract triterpene acid compounds in Ganoderma lucidum, and on the other hand, can increase the yield of triterpene acids in Ganoderma lucidum. The process is simple and the yield is high; the equipment is simple and suitable for industrial production.

The active part of the ganoderma triterpene acid of the present invention has obvious sedative and tranquilizing effects, can be used to prepare sleep-improving health care products and sedative and tranquilizing drugs, is beneficial to market promotion and utilization, and has broad application prospects.

Description of drawings

Fig. 1 is the comparative figure of the impact of the ganoderma triterpene acid active site prepared in embodiment 1 on prolonging the sleep latency experiment of pentobarbital sodium;

Fig. 2 is the comparative figure of the impact of the ganoderma triterpene acid active site prepared in embodiment 1 on prolonging the sleep time experiment of pentobarbital sodium;

Fig. 3 is a comparison chart of the effect of the active part of ganoderma lucidum triterpene acid prepared in Example 1 on the sleep latency experiment of barbital sodium.

Detailed ways

Embodiment 1 (preparation of ganoderma triterpene acid active site)

(1) Take Ganoderma lucidum fruiting body medicinal material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), dry at 65°C, pulverize, and pass through a 100-mesh sieve to obtain dry powder; add distilled water to the dry powder at a material-to-liquid ratio of 1g: 20mL, and heat at a temperature of 50°C and pH5. 5, add 3.0% of the compound enzyme relative to the mass of the dry powder, the compound enzyme is composed of 50% neutral protease, 25% cellulase and 25% pectinase in percentage by weight, and the enzymolysis time is 6h. Enzyme, cooling, suction filtration, obtain enzymatic hydrolysis solution, set aside;

(2) Take the enzymolysis solution prepared in the above step (1), add petroleum ether to extract and remove fat, then continue to add ethyl acetate to the enzymolysis solution after degreasing to extract, to obtain the extraction part of ethyl acetate, and evaporate the ethyl acetate to dryness Ester layer solvent to obtain ethyl acetate extract;

(3) Dissolve the Ganoderma ganoderma ethyl acetate extract prepared in the above step (2) in ethyl acetate, alkalinize to pH 9.5, wash with water to neutrality, acidify the alkalized water layer to pH 2.5, and pass through ethyl acetate Extract the crude ganoderma acid, set aside;

(4) Take the crude ganoderma acid prepared in the above step (3), separate it through a silica gel column, and use the gradient elution of chloroform and methanol system with a volume ratio of 1 to 10:1, and collect the chloroform and methanol with a volume ratio of 2 to 8 ︰1 eluent, thin-layer chromatographic detection, filtration, and evaporation to dryness is the active part of ganoderma triterpene acid.

In the active parts of triterpene acids of Ganoderma lucidum prepared in Example 1, the weight percentage of triterpene acids in the active parts of triterpene acids of Ganoderma lucidum was measured by a spectrophotometer was 65%.

Embodiment 2 (preparation of ganoderma triterpene acid active site)

(1) Take Ganoderma lucidum fruiting body medicinal material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), dry at 65°C, pulverize, and sieve through a 100-mesh sieve to obtain dry powder; add distilled water to the dry powder at a material-to-liquid ratio of 1g︰15mL, and heat at a temperature of 40°C and pH4. Under the condition of 0, add 2.0% compound enzyme relative to the mass of the dry powder, the compound enzyme is composed of 30% neutral protease, 35% cellulase and 35% pectinase in weight percentage, the enzymolysis time is 5h, and the Enzyme, cooling, suction filtration, obtain enzymatic hydrolysis solution, set aside;

(2) Take the enzymolysis solution prepared in the above step (1), add petroleum ether to extract and remove fat, then continue to add ethyl acetate to the enzymolysis solution after degreasing to extract, to obtain the extraction part of ethyl acetate, and evaporate the ethyl acetate to dryness Ester layer solvent to obtain ethyl acetate extract;

(3) Dissolve the ganoderma ganoderma ethyl acetate extract prepared in the above step (2) in ethyl acetate, alkalinize to pH 9, wash with water until neutral, acidify the alkalized water layer to pH 2, and then extract with ethyl acetate Obtain crude ganoderma acid, set aside;

(4) Take the crude ganoderma acid prepared in the above step (3), separate it through a silica gel column, and use the gradient elution of chloroform and methanol system with a volume ratio of 1 to 10:1, and collect the chloroform and methanol with a volume ratio of 2 to 8 ︰1 eluent, thin-layer chromatographic detection, filtration, and evaporation to dryness is the active part of ganoderma triterpene acid.

In the active parts of triterpene acids of Ganoderma lucidum prepared in Example 2, the weight percentage of triterpene acids in the active parts of triterpene acids of Ganoderma lucidum was measured by a spectrophotometer was 45%.

Embodiment 3 (preparation of ganoderma triterpene acid active site)

(1) Take Ganoderma lucidum fruiting body medicinal material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), dry at 65°C, pulverize, and pass through a 100-mesh sieve to obtain dry powder; add distilled water to the dry powder at a material-to-liquid ratio of 1g: 25mL, and heat at a temperature of 60°C and pH6. Under the condition of 0, add 4.0% compound enzyme relative to the mass of the dry powder, the compound enzyme is composed of 80% neutral protease, 10% cellulase and 10% pectinase in weight percentage, the enzymolysis time is 7h, and the Enzyme, cooling, suction filtration, obtain enzymatic hydrolysis solution, set aside;

(2) Take the enzymolysis solution prepared in the above step (1), add petroleum ether to extract and remove fat, then continue to add ethyl acetate to the enzymolysis solution after degreasing to extract, to obtain the extraction part of ethyl acetate, and evaporate the ethyl acetate to dryness Ester layer solvent to obtain ethyl acetate extract;

(3) Dissolve the ganoderma ganoderma ethyl acetate extract prepared in the above step (2) in ethyl acetate, alkalinize to pH 10, wash with water to neutrality, acidify the alkalized water layer to pH 3, and then extract with ethyl acetate Obtain crude ganoderma acid, set aside;

(4) Take the crude ganoderma acid prepared in the above step (3), separate it through a silica gel column, and use the gradient elution of chloroform and methanol system with a volume ratio of 1 to 10:1, and collect the chloroform and methanol with a volume ratio of 2 to 8 ︰1 eluent, thin-layer chromatographic detection, filtration, and evaporation to dryness is the active part of ganoderma triterpene acid.

In the active parts of ganoderma triterpene acids prepared in Example 3, the content of triterpene acids in the active parts of ganoderma lucidum triterpene acids was 55% by weight as measured by a spectrophotometer.

Embodiment 4 (the effect of the active part of ganoderma triterpene acid on the sleep of experimental mice)

1. Experimental materials

1.1 Experimental drugs

Ganoderma lucidum was purchased from Zhejiang Wuyangtang Pharmaceutical Co., Ltd.; the triterpene acid active part of Ganoderma lucidum prepared in Example 1 of the present invention.

Diazepam, aladdin company; pentobarbital sodium, barbital sodium, merck company

1.2 Experimental animals: The experiment uses clean-grade healthy ICR mice, weighing 18-22g, single male, provided by the Experimental Animal Center of Zhejiang Academy of Medical Sciences, the experimental animal production license number SCXK (Zhejiang) 2014-0001; In the SPF grade animal room of the Experimental Animal Center of the Academy of Sciences, the experimental conditions were maintained at a temperature of 25°C, a relative humidity of 40%-70%, a light-dark cycle of 12h:12h, and free access to water and food.

2. Experimental method

Experimental animals were randomly divided into 5 groups according to body weight, 10 in each group: blank control group (5% tween 80, 10ml/kg·bw), positive control group (diazepam, 2.5mg/kg·bw), Ganoderma lucidum triterpenes Low-dose group (25mg/kg·bw), middle-dose group (50mg/kg·bw), high-dose group (100mg/kg·bw), where bw represents body weight. Animals in each group were gavaged once a day to observe their direct sleep effect, and the experimental treatment was carried out 30 days later. Animal experiments were carried out in a quiet environment from 10:00 to 14:00 every day.

2.1 In the present invention, the active part of Ganoderma lucidum triterpene acid is on the influence of prolonging pentobarbital sodium sleep time experiment:

On the basis of pentobarbital sodium hypnosis, observe whether each test substance prolongs the sleep time. If the sleep time is prolonged, it shows that the test substance and pentobarbital sodium have a synergistic effect. Mice in each group were administered once a day for 30 consecutive days, and were fasted for 12 hours before the last administration. 60 minutes after the last intragastric administration, the mice in each group were given 48 mg/kg·bw pentobarbital sodium (i.p.), and the injection volume was 0.1 mL/10 g·bw. The mouse's righting reflex disappears for more than 60 seconds as the criterion for falling asleep. The recovery of the righting reflex is the awakening of the animal. The time between the disappearance of the righting reflex and the recovery of the righting reflex is the sleep time of the animal. Sleep duration of sodium-injected mice.

2.2 The effect of the active part of Ganoderma lucidum triterpene acid on mice in the subthreshold dose hypnosis experiment of pentobarbital sodium:

Mice in each group were administered once a day for 30 consecutive days, and were fasted for 12 hours before the last administration. 60 minutes after the last gavage administration, the animals in each group were given i.p. 32mg/kg bw pentobarbital sodium, the injection volume was 0.1mL/10g bw, and the righting reflex of the mice disappeared for more than 60s as the criterion for falling asleep , record the number of animals falling asleep in each group within 30 minutes and calculate the incidence of sleep, and the incidence of sleep = number of sleeping animals in each group/total number of animals in each group×100%.

2.3 The effect of active parts of Ganoderma lucidum triterpene acid on mice in barbiturate sodium sleep latency experiment

On the basis of barbital sodium hypnosis, observe whether the test substance can shorten the sleep latency of animals, and if the latency is shortened, it shows that the test substance and barbital sodium have a synergistic effect. Mice in each group were administered once a day for 30 consecutive days, and were fasted for 12 hours before the last administration. 60 minutes after the last gavage administration in each dosage group, the animals in each group were given barbiturate sodium (i.p.) at 280 mg/kg bw, and the injection volume was 0.1 mL/10 g bw. Sleep index, from the injection of barbital sodium to the animal falling asleep is the sleep latency period, and it is observed whether each group of test substances can shorten the sleep latency period of barbital sodium.

The sedative and tranquilizing effect of the active part of ganoderma lucidum triterpene acid in the present invention is evaluated comprehensively by the above-mentioned indicators.

3. Experimental results

3.1 In the present invention, the active part of Ganoderma lucidum triterpene acid is on the influence of prolonging pentobarbital sodium sleep time experiment

Fig. 1 is the comparison diagram of the effect of the active part of ganoderma lucidum triterpene acid prepared in Example 1 on prolonging the sleep latency experiment of pentobarbital sodium. Fig. 2 is the comparison diagram of the effect of the active part of ganoderma lucidum triterpene acid prepared in Example 1 on prolonging the sleep time experiment of pentobarbital sodium. The results are shown in Figure 1 and Figure 2, and the data show that all dosage groups of the active part of Ganoderma lucidum triterpene acid can significantly increase the sleep time of mice and shorten the sleep latency period. The sleep duration of the high-dose group of active parts of triterpene acids of Ganoderma lucidum was stronger than that of the positive control group.

3.2 Influence of Ganoderma lucidum triterpene acid active part on subthreshold dose hypnosis experiment of pentobarbital sodium in the present invention

The results are shown in Table 1. It can be seen from the experimental results that the active part of ganoderma triterpene acid 100 mg/kg bw has a certain synergistic effect on the subthreshold hypnotic dose of pentobarbital sodium, and the effect is stronger than that of diazepam 2.5 mg/kg.

Table 1 Effect of Ganoderma lucidum triterpene acid on pentobarbital sodium subthreshold dose hypnosis test

3.3 Influence of Ganoderma lucidum triterpene acid active part on barbiturate sodium sleep latency experiment in the present invention

Figure 3 is a comparison chart of the effect of the active parts of the triterpene acids of Ganoderma lucidum on the sleep latency experiment of barbiturate sodium. Bital sodium sleep latency, its effect of promoting sleep is remarkable.

The above experimental results show that the ganoderma triterpene acid active site prepared by the present invention can significantly prolong the sleep time of mice in the pentobarbital sodium sleep experiment, shorten its sleep latency; and improve the pentobarbital sodium subthreshold dose hypnosis test At the same time, it also significantly shortened the sleep latency of mice in the barbital sodium sleep latency experiment, indicating that the active part of ganoderma triterpene acid has obvious sedative and tranquilizing effects.

Embodiment 5 (preparation of dropping pill)

Weigh 400g polyethylene glycol 4000 respectively, melt it on a water bath, then add 500g dry powder of the active part of Ganoderma lucidum triterpene acid, stir evenly, pour it into the insulation tube, adjust the constant temperature device, and make the liquid drip at 80-90°C Put it into cooled liquid paraffin (temperature ± 4°C). After dripping, pour the pills into the filter paper to absorb the paraffin oil, add a small amount of talcum powder, and mix well to obtain 1000 ganoderma triterpene acid dripping pills.

Embodiment 6 (preparation of capsule)

Ganoderma lucidum triterpene acid active part dry powder 1000g, mixed with 500g medicinal starch, dried, and made into capsules at 0.45g per capsule.

Embodiment 7 (preparation of tablet)

Ganoderma lucidum triterpene acid active part dry powder 1000g, starch 500g, mixed evenly, granulated with appropriate amount of ethanol, granulated by a granulator, compressed into tablets, 0.35g per tablet.

Embodiment 8 (preparation of granules)

Ganoderma lucidum triterpene acid active part dry powder 1500g, starch 1000g, sugar powder 400g, mix evenly, granulate with appropriate amount of ethanol, dry, granulate, and pack.

Claims (3)

1. a kind of preparation method at ganodenic acid acid activity position, which is characterized in that include the following steps：

(1) ganoderma lucidum fruitbody pulverizing medicinal materials are obtained into dry powder, complex enzyme zymohydrolysis is added in dry powder in water, through enzyme deactivation, cooling, pumping Filter, obtains enzymolysis liquid；

Ganoderma lucidum fruitbody pulverizing medicinal materials are obtained into dry powder, including：Ganoderma lucidum fruitbody medicinal material is first taken, 55 DEG C~75 DEG C dry, pulverize, It crosses 80~120 mesh and sieves to obtain dry powder；

The complex enzyme is grouped as by the group of following weight percentage：

Neutral proteinase 30%~80%；

Cellulase 10%~35%；

Pectase 10%~35%；

The addition of the complex enzyme is the 1.0%~5.0% of dry powder quality；

The solid-liquid ratio of the water and dry powder is 10mL~30mL：1g；

The condition of the enzymolysis is：35~70 DEG C of temperature, pH3.5~7,4.5~8h of enzymolysis time；

(2) petroleum ether extraction grease removal is added in the enzymolysis liquid obtained in step (1), then continuously adds the enzymolysis liquid after grease removal Ethyl acetate extracts, and obtains Ethyl acetate fraction, volatilizes ethyl acetate layer solvent, obtains ganoderma lucidum ethyl acetate extract；

(3) ganoderma lucidum ethyl acetate extract prepared by step (2) is dissolved in ethyl acetate, first alkalized to pH 9~10, then wash To neutrality, alkalization water layer is obtained, alkalization water layer is then acidified to pH 2~3, then thick ganoderic acid is extracted to obtain by ethyl acetate；

(4) the thick ganoderic acid for preparing step (3) collects elution through silica gel post separation with chloroform and methanol system gradient elution Liquid is known through thin-layer chromatography inspection, filters, is evaporated to get ganodenic acid acid activity position；

The volume ratio of chloroform and methanol is 1~10 ︰ 1 in the chloroform and methanol system.

2. ganodenic acid acid activity position prepared by preparation method according to claim 1.

3. ganodenic acid acid activity according to claim 2 position is preparing improvement sleep effect health products and tranquilizing and allaying excitement Application in drug.