CN105193824A - Tritepenoidic acid active site of ganoderma lucidum, method for preparing tritepenoidic acid active site and application thereof - Google Patents
Tritepenoidic acid active site of ganoderma lucidum, method for preparing tritepenoidic acid active site and application thereof Download PDFInfo
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Abstract
The invention discloses a tritepenoidic acid active site of ganoderma lucidum, a method for preparing the tritepenoidic acid active site and application of the tritepenoidic acid active site to preparing sleep quality improving healthcare products and mind tranquillizing medicine. The method includes smashing ganoderma lucidum medicinal materials to obtain dry powder, adding compound enzymes into the dry powder in water to carry out enzymatic hydrolysis and carrying out enzyme deactivation, cooling and pumping filtration to obtain enzymatic hydrolysis solution; adding the enzymatic hydrolysis solution into petroleum ether to extract the enzymatic hydrolysis solution, removing grease, adding ethyl acetate into the enzymatic hydrolysis solution to extract the enzymatic hydrolysis solution so as to obtain ethyl acetate extracts of the ganoderma lucidum; alkalizing the ethyl acetate extracts of the ganoderma lucidum, then washing the ethyl acetate extracts of the ganoderma lucidum until the ethyl acetate extracts of the ganoderma lucidum are neutral to obtain alkalized water layers, then acidifying the alkalized water layers and extracting the alkalized water layers by the aid of ethyl acetate to obtain crude ganoderma lucidum acid; separating the crude ganoderma lucidum acid by the aid of silica gel columns, eluting the crude ganoderma lucidum acid by the aid of chloroform and methanol systems in a gradient manner and carrying out thin-layer chromatography identification, filtering and drying by distillation to obtain the tritepenoidic acid active site of the ganoderma lucidum. The tritepenoidic acid active site, the method and the application have the advantage that the tritepenoidic acid active site of the ganoderma lucidum is subjected to enzymatic hydrolysis by the aid of the compound enzymes, can be obtained by the aid of steps of extraction, separation and the like and can be used for preparing the sleep quality improving healthcare products and the mind tranquillizing medicine.
Description
Technical field
The present invention relates to Ganoderma extract technical field, be specifically related to a kind of Ganoderma triterpenoids acid activity position and preparation method thereof and preparing the application in improving water flood health product and tranquillizing and allaying excitement medicine.
Background technology
Sleep is the necessary physiological process of the mankind and higher mammal, is to preserve one's strength and healthy basis.Insomnia is a kind of common sleep disorder, refers to can not fall asleep or maintain sleep state.Chronic insomnia probably affects the crowd of about 15%, the crowd of the annual impact about 80% of transience insomnia.The current treatment for insomnia is to take synthetic medicine, but synthetic drug exists serious drug dependence, cognition and the untoward reaction such as locomotor damage, drug withdrawal knock-on.Therefore, need searching badly and develop tranquillizing and allaying excitement medicine safely and effectively, to break through the development bottleneck of tranquillizing and allaying excitement medicine and dietary supplement.
Ganoderma is the dry sporophore of Polyporaceae Ganoderma lucidum (Leyss. Ex Fr.) Karst. [Ganodermalucidum (Leyss.ex.Fr.) Karst.] or Ganoderma [Ganodermajaponicum (Fr.) Lloyd], and its property is put down; Sweet in the mouth, temperature.GUIXIN, liver, spleen channel, just have the title of " Herba mesonae chinensis ", " Ganoderma ", medical value is high from ancient times, is the Homonemeae medical material of China's preciousness.Our people as the medicinal history having more than 2000 year, just has it " on rotten earth, having bacterium person sesame " in the ancient books Zhou Dynasty " row " book." calming the nerves " effect beginning of Ganoderma is loaded in Shennong's Herbal, and versions " Zhejiang Province's prepared slices of Chinese crude drugs concocted specification " in 2005 and the version Pharmacopoeia of the People's Republic of China in 2010 all describe Ganoderma and have " benefiting vital QI for tranquillizing " effect.In the number of chemical composition of Ganoderma, ganoderan and triterpenoid compound are its main compositions, and China evaluates the quality of Ganoderma quality with the content of ganoderan; In the standard of Japan and other countries then using the content of Ganoderma triterpenoids acid as qualification Garnoderma product quality.
Modern pharmacology is actually rare about the research report of Ganoderma tranquilizing effect, Wang and Tang etc. have studied the syngignoscism (Wang Xiangli of Ganoderma extract to human body, Meng Zhaoyang, Wang Cuiping. lucidum bacteria liquid treatment of insomnia patients 60 example [J]. Chinese Medicine journal, 2001,16 (1): 47-49, TANGWen-bo, GAOYi-huai, CHENGuo-liang, etal.ARandomized, double-blindandplacebo-controlledstudyofaGanodermaLucidu mpolysaccharideextract (ganopoly) inneurasthenia [J] .JournalofMedicinalFood, 2005, 8 (1): 53-58), the research such as Chu proves that Ganoderma sporophore water extract can extend the length of one's sleep of rat in pentobarbital sodium sleep experiments, think that ganoderan has Benzodiazepine sample effect (ChuQing-ping, WANGLi-en, CuiXiang-yu, etal.ExtractofGanodermalucidumpotentiatespentobarbital-i nducedsleepviaaGABAergicmechanism [J] .Pharmacology, BiochemistryandBehavior, 2007, 86 (4): 693-698), the research such as Cui finds that ganoderma lucidum crude polysaccharide 80mg/kg can prolong rats total sleep time and length of one's sleep no rapid eye movement phase and do not affect S sleep and fast quick-action eye length of one's sleep phase, the TNF-alpha levels of rat blood serum, hypothalamus and dorsal part rapheal nuclei significantly raises (CUIXiang-yu, CUISu-ying, ZHANGJuan, etal.ExtractofGanodermalucidumprolongssleeptimeinrats [J] .JournalofEthnopharmacology, 2012,139 (3): 796-800).The application of radiation ligand receptor Binding experiment such as CHO prove that Ganoderma sporophore ethanol extract is to GABA
areceptor has moderate strength affinity, and water extract is to GABA
aextremely weak (the CHOSueng-mock of affinity of receptor, SHIMIZUMakoto, LEEC-justin, etal.HypnoticeffectsandbindingstudiesforGABAaand5-HT2cre ceptorsoftraditionalmedicinalplantsusedinAsiaforinsomnia [J] .JournalofPharmacology, 2010,132 (1): 225-232); The research such as Jia Wei finds that Ganoderma triterpenoids and Poria extract have the function of collaborative improving water flood, ganoderan does not but have this pharmacological action, its mechanism of action indefinite (Jia Wei, Wu Min, Jinsong ZHANG etc. Ganoderma composition improving water flood function test pre-test [J]. edible fungi journal, 2005,12 (3): 43-47).The research such as Jiang Haitao finds that Ganoderma extract has the effect of improving water flood, but the effect of its improving water flood and tested material dosage do not have obvious linear relationship, and its mechanism of action and material base also indefinite (Jiang Haitao, Ren Yuanhao, Yu Weiyan etc. the functional study of Ganoderma extract in sleep improvement [J]. time precious traditional Chinese medical science traditional Chinese medicines, 2008,19 (9): 2231-2232); Wang Yuhong, Cao Sufang etc. are all studied the tranquilizing effect of Ganoderma sporophore extract, but Wen Zhongjun do not review possible material base (Wang Yuhong. the research [J] of Ganoderma improving water flood function. Shanxi Medicine magazine, 2011,40 (9): 878-880; Cao Sufang, Wang Xinrui, Zhang Chen, etc. about the research [J] of Ganoderma improving water flood function. Zhejiang edible fungi, 2009,17 (6): 52-54).
Therefore, although Ganoderma is the Homonemeae medical material of China's preciousness, its " benefiting vital QI for tranquillizing " effect medicinal history of existing thousands of years, but modern pharmacology is not yet very clear and definite to its corresponding active functional component, most pharmacological evaluation only rests on the basis of crude drug and crude extract, there is no Ganoderma extract and is refined to active site further and for the preparation of the report of tranquillizing and allaying excitement health product and medicine.
Summary of the invention
The invention provides a kind of Ganoderma triterpenoids acid activity position and preparation method thereof and preparing the application in improving water flood health product and tranquillizing and allaying excitement medicine, complex enzyme zymohydrolysis is passed through at Ganoderma triterpenoids acid activity position, can obtain through steps such as extraction, separation again, can be used for preparing improving water flood health product and tranquillizing and allaying excitement medicine.
The preparation method at Ganoderma triterpenoids acid activity position, comprises the following steps:
(1) Ganoderma sporophore pulverizing medicinal materials is obtained dry powder, dry powder adds complex enzyme zymohydrolysis in water, through enzyme denaturing, cooling, sucking filtration, obtains enzymolysis solution;
(2) enzymolysis solution obtained in step (1) is added petroleum ether extraction grease removal, then the enzymolysis solution after grease removal is continued to add extraction into ethyl acetate, obtain Ethyl acetate fraction, volatilize ethyl acetate layer solvent, obtain Ganoderma ethyl acetate extract;
(3) Ganoderma ethyl acetate extract prepared by step (2) is dissolved in ethyl acetate, first alkalize to pH9 ~ 10, then be washed to neutrality, obtain the water layer that alkalizes, then will alkalize aqueous layer acidified to pH2 ~ 3, then obtain thick Ganodenic acid by extraction into ethyl acetate;
(4) by thick Ganodenic acid prepared by step (3), be separated through silicagel column, with chloroform and methanol system gradient elution, collect eluent, through thin layer chromatography inspection knowledge, filtration, evaporate to dryness, obtain Ganoderma triterpenoids acid activity position.
In the present invention, by the enzymolysis of compound enzyme and the cooperation of each extraction step, can more fully, extract the compound of triterpene acids in Ganoderma all sidedly, it is active that Ganoderma triterpenoids acid activity position has good tranquillizing and allaying excitement.
Following as the preferred technical solution of the present invention:
In step (1), Ganoderma sporophore pulverizing medicinal materials is obtained dry powder, comprising: first get Ganoderma sporophore medical material, 55 DEG C ~ 75 DEG C dry, pulverize, and cross 80 ~ 120 mesh sieves and obtain dry powder; Further preferably, comprising: first get Ganoderma sporophore medical material, 65 DEG C dry, pulverize, and cross 100 mesh sieves and obtain dry powder.
Described compound enzyme, is made up of the component of following weight percentage:
Neutral protease 5% ~ 95%;
Cellulase 2.5% ~ 48%;
Pectase 2.5% ~ 48%.
Further preferably, described compound enzyme, is made up of the component of following weight percentage:
Neutral protease 30% ~ 80%;
Cellulase 10% ~ 35%;
Pectase 10% ~ 35%.
The main component of plant cell wall is pectin and cellulose, pectase in compound enzyme can enzymolysis pectin wherein, cellulase degradation cellulose wherein, effective ingredient in plant cell is discharged, neutral protease can carry out clarifying treatment to macromolecular substances such as protein wherein, thus improves the yield of effective ingredient.The use of compound enzyme can be shortened the extraction time of Ganoderma triterpenoids acid and improve its yield, simultaneously can more fully, extract the compound of triterpene acids in Ganoderma all sidedly, it is active that Ganoderma triterpenoids acid activity position has good tranquillizing and allaying excitement.
The addition of described compound enzyme is 1.0% ~ 5.0% of dry powder quality.
Described water and the solid-liquid ratio of dry powder are 10mL ~ 30mL:1g, more preferably 20mL:1g.
Described enzymatic hydrolysis condition is: temperature 35 ~ 70 DEG C, pH3.5 ~ 7, enzymolysis time 4.5 ~ 8h.
In step (3), Ganoderma ethyl acetate extract first alkalizes, and by triterpenic acid salify wherein, is dissolved in the water, and is then extracted with ethyl acetate and removes alkalescence, neutral impurity; Then the triterpenic acid of salify is discharged the acidify of alkalization water lotion, continue by ethyl acetate the triterpenic acid composition that extraction obtains purification.The effect of alkalization, acidify is purification triterpenic acid, the main neutral triterpene removed wherein.
In step (4), in described chloroform and methanol system, the volume ratio of chloroform and methanol is 1 ~ 10 ︰ 1.Further preferably, in described chloroform and methanol system, chloroform is 2 ~ 8 ︰ 1 with the volume ratio of methanol, and the volume ratio of collecting chloroform and methanol is the eluent of 2 ~ 8 ︰ 1, contains the compound of more triterpene acids in above-mentioned eluent.
In the present invention, in the Ganoderma triterpenoids acid activity position obtained, the weight percentage of triterpenic acid is 5% to 70%, and the content of its triterpenic acid adopts spectrophotometric determination.
The Ganoderma triterpenoids acid activity position that preparation method of the present invention obtains, can be used for preparing improving water flood effect health product and tranquillizing and allaying excitement medicine.
In order to detect the performance of the tranquillizing and allaying excitement active site at Ganoderma triterpenoids acid activity position, the Ganoderma triterpenoids acid activity position of gained of the present invention is configured to 25 ~ 100mg/ml medicinal liquid, for extending in the pentobarbital sodium experiment length of one's sleep, pentobarbital sodium sub-threshold dose hypnosis experiment, barbital sodium Sleep latency experimental model, observe sample respectively at different dosages on the impact of experiment mice sleep behavior.It is active that result shows that Ganoderma triterpenoids acid activity position has significant tranquillizing and allaying excitement, can be used for preparing the health product of improving water flood or the ancillary drug of the disease such as Cure for insomnia, sleep disorder.
The pharmaceutical composition that Ganoderma triterpenoids acid activity position of the present invention can form with pharmaceutically acceptable adjuvant.
Ganoderma triterpenoids acid activity position can independent or several part combination, and further with auxiliary material combination, dosage form comprises: tablet, capsule, pill, granule, suspensoid, drop pill, oral liquid etc.
The carrier at Ganoderma triterpenoids acid activity position of the present invention and excipient comprise carrier and excipient, such as solvent, disintegrating agent, correctives, antiseptic, coloring agent, the binding agent etc. of the application of pharmaceutics routine.
Ganoderma triterpenoids acid activity position has obvious tranquilizing effect, can extend the length of one's sleep of pentobarbital sodium mice, shortens its Sleep latency; In pentobarbital sodium sub-threshold dose hypnosis experiment, significantly can increase the quantity of sleep mice; And significantly shorten the Sleep latency of barbital sodium Sleep latency experiment small mouse, show that Ganoderma triterpenoids acid activity position has good tranquilizing effect, there are the potentiality of exploitation.
Compared with prior art, tool of the present invention has the following advantages:
The preparation method at Ganoderma triterpenoids acid activity position of the present invention, content and the purity of wherein Ganoderma triterpenoids acid significantly promote.The reagent such as the multiplex chloroform of existing method, ethyl acetate directly extract Ganoderma sporophore dry powder or ethanol extract, have wherein mixed the impurity such as a large amount of fatty acids, neutral triterpene.
The preparation method at Ganoderma triterpenoids acid activity position of the present invention, the extraction process of Ganoderma triterpenoids acid is optimized, enzyme process and chromatography is adopted to carry out pretreatment with refining to Ganoderma triterpenoids acid and its active site respectively before extraction with after extraction, on the one hand can more fully, extract the compound of triterpene acids in Ganoderma all sidedly, can improve the yield of Ganoderma triterpenoids acid on the other hand, its technique is simple, yield is high; Equipment is simple, is applicable to commercial production.
Ganoderma triterpenoids acid activity position of the present invention, has obvious tranquilizing effect, can be used for preparing improving water flood effect health product and tranquillizing and allaying excitement medicine, is conducive to market-oriented utilization and extention, possesses wide application prospect.
Accompanying drawing explanation
Fig. 1 is the embodiment 1 Ganoderma triterpenoids acid activity position of preparing affects comparison diagram to what extend the experiment of pentobarbital sodium sleep latency;
Fig. 2 is the embodiment 1 Ganoderma triterpenoids acid activity position of preparing affects comparison diagram to what extend experiment pentobarbital sodium length of one's sleep;
Fig. 3 be embodiment 1 prepare Ganoderma triterpenoids acid activity position barbital sodium Sleep latency is tested affect comparison diagram.
Detailed description of the invention
Embodiment 1 (preparation at Ganoderma triterpenoids acid activity position)
(1) get Ganoderma sporophore medical material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), 65 DEG C dry, pulverize, and cross 100 mesh sieves and obtain dry powder; Dry powder adds distilled water by solid-liquid ratio 1g ︰ 20mL, under temperature 50 C, pH5.5 condition, add the compound enzyme of the quality 3.0% relative to dry powder, compound enzyme, be made up of weight percentage 50% neutral protease, 25% cellulase and 25% pectase, enzymolysis time is 6h, enzyme denaturing, cooling, sucking filtration, obtain enzymolysis solution, for subsequent use;
(2) enzymolysis solution that above-mentioned steps (1) prepares is got, add petroleum ether extraction grease removal, then the enzymolysis solution after grease removal is continued to add extraction into ethyl acetate, obtain Ethyl acetate fraction, volatilize ethyl acetate layer solvent, obtain ethyl acetate extract;
(3) get the Ganoderma ethyl acetate extract that above-mentioned steps (2) prepares and be dissolved in ethyl acetate, alkalize to pH9.5, be washed to neutrality, alkalize aqueous layer acidified to pH2.5, then obtain thick Ganodenic acid by extraction into ethyl acetate, for subsequent use;
(4) the thick Ganodenic acid that above-mentioned steps (3) prepares is got, be separated through silicagel column, with chloroform and methanol system gradient elution that volume ratio is 1 ~ 10 ︰ 1, the volume ratio of collecting chloroform and methanol is the eluent of 2 ~ 8 ︰ 1, and thin layer chromatography inspection knowledge, filtration, evaporate to dryness are Ganoderma triterpenoids acid activity position.
In Ganoderma triterpenoids acid activity position prepared by embodiment 1, adopt spectrophotometric determination, in Ganoderma triterpenoids acid activity position, the weight percentage of triterpenic acid is 65%.
Embodiment 2 (preparation at Ganoderma triterpenoids acid activity position)
(1) get Ganoderma sporophore medical material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), 65 DEG C dry, pulverize, and cross 100 mesh sieves and obtain dry powder; Dry powder adds distilled water by solid-liquid ratio 1g ︰ 15mL, under temperature 40 DEG C, pH4.0 condition, add the compound enzyme of the quality 2.0% relative to dry powder, compound enzyme, be made up of weight percentage 30% neutral protease, 35% cellulase and 35% pectase, enzymolysis time is 5h, enzyme denaturing, cooling, sucking filtration, obtain enzymolysis solution, for subsequent use;
(2) enzymolysis solution that above-mentioned steps (1) prepares is got, add petroleum ether extraction grease removal, then the enzymolysis solution after grease removal is continued to add extraction into ethyl acetate, obtain Ethyl acetate fraction, volatilize ethyl acetate layer solvent, obtain ethyl acetate extract;
(3) get the Ganoderma ethyl acetate extract that above-mentioned steps (2) prepares and be dissolved in ethyl acetate, alkalize to pH9, be washed to neutrality, alkalize aqueous layer acidified to pH2, then obtain thick Ganodenic acid by extraction into ethyl acetate, for subsequent use;
(4) the thick Ganodenic acid that above-mentioned steps (3) prepares is got, be separated through silicagel column, with chloroform and methanol system gradient elution that volume ratio is 1 ~ 10 ︰ 1, the volume ratio of collecting chloroform and methanol is the eluent of 2 ~ 8 ︰ 1, and thin layer chromatography inspection knowledge, filtration, evaporate to dryness are Ganoderma triterpenoids acid activity position.
In Ganoderma triterpenoids acid activity position prepared by embodiment 2, adopt spectrophotometric determination, in Ganoderma triterpenoids acid activity position, the weight percentage of triterpenic acid is 45%.
Embodiment 3 (preparation at Ganoderma triterpenoids acid activity position)
(1) get Ganoderma sporophore medical material (Zhejiang Wuyangtang Pharmaceutical Co., Ltd.), 65 DEG C dry, pulverize, and cross 100 mesh sieves and obtain dry powder; Dry powder adds distilled water by solid-liquid ratio 1g ︰ 25mL, under temperature 60 C, pH6.0 condition, add the compound enzyme of the quality 4.0% relative to dry powder, compound enzyme, be made up of weight percentage 80% neutral protease, 10% cellulase and 10% pectase, enzymolysis time is 7h, enzyme denaturing, cooling, sucking filtration, obtain enzymolysis solution, for subsequent use;
(2) enzymolysis solution that above-mentioned steps (1) prepares is got, add petroleum ether extraction grease removal, then the enzymolysis solution after grease removal is continued to add extraction into ethyl acetate, obtain Ethyl acetate fraction, volatilize ethyl acetate layer solvent, obtain ethyl acetate extract;
(3) get the Ganoderma ethyl acetate extract that above-mentioned steps (2) prepares and be dissolved in ethyl acetate, alkalize to pH10, be washed to neutrality, alkalize aqueous layer acidified to pH3, then obtain thick Ganodenic acid by extraction into ethyl acetate, for subsequent use;
(4) the thick Ganodenic acid that above-mentioned steps (3) prepares is got, be separated through silicagel column, with chloroform and methanol system gradient elution that volume ratio is 1 ~ 10 ︰ 1, the volume ratio of collecting chloroform and methanol is the eluent of 2 ~ 8 ︰ 1, and thin layer chromatography inspection knowledge, filtration, evaporate to dryness are Ganoderma triterpenoids acid activity position.
In Ganoderma triterpenoids acid activity position prepared by embodiment 3, adopt spectrophotometric determination, in Ganoderma triterpenoids acid activity position, the weight percentage of triterpenic acid is 55%.
Embodiment 4 (impact slept on experiment mice in Ganoderma triterpenoids acid activity position)
1, experiment material
1.1 Experimental agents
Ganoderma is purchased from Zhejiang Wuyangtang Pharmaceutical Co., Ltd.; The Ganoderma triterpenoids acid activity position that the embodiment of the present invention 1 prepares.
Diazepam, aladdin company; Pentobarbital sodium, barbital sodium, merck company
1.2 laboratory animals: the healthy ICR mice of cleaning grade is selected in experiment, body weight 18-22g, and single male, Zhejiang Academy of Medical Sciences Experimental Animal Center provides, laboratory animal produces card credit number SCXK (Zhejiang) 2014-0001; Raise in Zhejiang Academy of Medical Sciences Experimental Animal Center SPF level Animal House, experiment condition keeps temperature to be 25 DEG C, and relative humidity is 40%-70%, and fight-darkness cycle is 12h:12h, freely drinks water and ingests.
2, experimental technique
Laboratory animal is divided into 5 groups at random according to body weight, often organize 10: blank group (5%tween80,10ml/kgbw), positive controls (diazepam, 2.5mg/kgbw), Ganoderma triterpenoids acid low dose group (25mg/kgbw), middle dosage group (50mg/kgbw), high dose group (100mg/kgbw), wherein, that bw representative is body weight (bodyweight).Each treated animal gavage every day 1 time, observes its direct sleep effect, carries out experimental station reason after 30d.Zoopery was carried out in 10:00-14:00 quiet environment every day.
In 2.1 the present invention, Ganoderma triterpenoids acid activity position is on the impact extending the pentobarbital sodium experiment length of one's sleep:
On the basis of pentobarbital sodium hypnosis, observe each tested material and whether extend the length of one's sleep, if extend the length of one's sleep, then illustrate that tested material and pentobarbital sodium have synergism.Each group of mice administration every day 1 time, continuous 30d, before last administration, water is can't help in 12h fasting.After last gastric infusion 60min, each group mice gives 48mg/kgbw pentobarbital sodium (i.p.), and injection volume is 0.1mL/10gbw.Using more than the righting reflex loss 60s of mice as sleep criterion, righting reflex recovers to be animal awakening, righting reflex loss is the animal sleep time to recovering during this period of time, and can more each tested material extend the length of one's sleep of pentobarbital sodium injection mice.
2.2 Ganoderma triterpenoids acid activity positions are on the impact of pentobarbital sodium sub-threshold dose hypnosis experiment small mouse:
Each group of mice administration every day 1 time, continuous 30d, before last administration, water is can't help in 12h fasting.After last gastric infusion 60min, each treated animal i.p. gives 32mg/kgbw pentobarbital sodium, injection volume is 0.1mL/10gbw, more than 60s is reached as sleep criterion using the righting reflex loss of mice, each group sleep number of animals in record 30min also calculates sleep incidence rate, sleep incidence rate=often organize sleeping animal number/every treated animal sum × 100%.
2.3 Ganoderma triterpenoids acid activity positions are on the impact of barbital sodium Sleep latency experiment small mouse
On the basis of barbital sodium hypnosis, observe the dropping asleep latency whether tested material can shorten animal, if shorter latencies, then illustrate that tested material and barbital sodium have synergism.Each group of mice administration every day 1 time, continuous 30d, before last administration, water is can't help in 12h fasting.Each dosage group is after last gastric infusion 60min, each treated animal gives barbital sodium (i.p.) by 280mg/kgbw, injection volume is 0.1mL/10gbw, using mice more than righting reflex loss 60s as sleep index, fall asleep from injection barbital sodium to animal and be Sleep latency, observe each group of tested material and can shorten barbital sodium Sleep latency.
Comprehensive above-mentioned indices comments the tranquillizing and allaying excitement effect at Ganoderma triterpenoids acid activity position in the present invention.
3, experimental result
In 3.1 the present invention, Ganoderma triterpenoids acid activity position is on the impact extending the pentobarbital sodium experiment length of one's sleep
Fig. 1 is the embodiment 1 Ganoderma triterpenoids acid activity position of preparing affects comparison diagram to what extend the experiment of pentobarbital sodium sleep latency.Fig. 2 is the embodiment 1 Ganoderma triterpenoids acid activity position of preparing affects comparison diagram to what extend experiment pentobarbital sodium length of one's sleep.The results are shown in Figure 1 and Fig. 2, data show, each dosage group in Ganoderma triterpenoids acid activity position all significantly can increase the length of one's sleep of mice, and shorten Sleep latency.The length of one's sleep of Ganoderma triterpenoids acid activity position high dose group is better than positive controls.
The impact that in 3.2 the present invention, Ganoderma triterpenoids acid activity position is tested the hypnosis of pentobarbital sodium sub-threshold dose
The results are shown in Table 1, from experimental result, Ganoderma triterpenoids acid activity position 100mg/kgbw has certain synergism to pentobarbital sodium sub-threshold lull dosage, and action effect is better than diazepam 2.5mg/kg.
The acid of table 1 Ganoderma triterpenoids is on the impact of pentobarbital sodium sub-threshold dose hypnosis experiment
The impact that in 3.3 the present invention, Ganoderma triterpenoids acid activity position is tested barbital sodium Sleep latency
Fig. 3 be Ganoderma triterpenoids acid activity position barbital sodium Sleep latency is tested affect comparison diagram, the results are shown in Figure 3, data show, the basic, normal, high dosage group in Ganoderma triterpenoids acid activity position all significantly can shorten mice barbital sodium Sleep latency, the Be very effective of its hypnotic.
Above experimental result shows, the Ganoderma triterpenoids acid activity position that the present invention prepares, can length of one's sleep of significant prolongation pentobarbital sodium sleep experiments small mouse, shortens its Sleep latency; And improve the sleep rate of pentobarbital sodium sub-threshold dose hypnosis experiment small mouse; Meanwhile, also significantly shorten the Sleep latency of barbital sodium Sleep latency experiment small mouse, illustrate that Ganoderma triterpenoids acid activity position has obvious tranquilizing effect.
Embodiment 5 (preparation of drop pill)
Take 400g Macrogol 4000 respectively, water-bath is melted, then adds Ganoderma triterpenoids acid activity position 500g dried powder, stir, in impouring insulating tube, regulating thermostatic device, makes medicinal liquid at 80-90 DEG C, instill (temperature ± 4 DEG C) in cooled liquid paraffin, after dripping off, paraffin oil is blotted by pill impouring filter paper, add a small amount of Pulvis Talci again, mixing, obtain Ganoderma triterpenoids acid drop pill 1000.
Embodiment 6 (preparation of capsule)
Ganoderma triterpenoids acid activity position dried powder 1000g, mixs homogeneously with medical starch 500g, dries, makes capsule by every 0.45g.
Embodiment 7 (preparation of tablet)
Ganoderma triterpenoids acid activity position dried powder 1000g, starch 500g, mix homogeneously, granulates by ethanol in proper amount, through pelletizing machine granulate, tabletting, every sheet 0.35g.
Embodiment 8 (preparation of granule)
Ganoderma triterpenoids acid activity position dried powder 1500g, starch 1000g, Icing Sugar 400g, mix homogeneously, granulates by ethanol in proper amount, drying, granulate, subpackage and get final product.
Claims (10)
1. the preparation method at Ganoderma triterpenoids acid activity position, is characterized in that, comprises the following steps:
(1) Ganoderma sporophore pulverizing medicinal materials is obtained dry powder, dry powder adds complex enzyme zymohydrolysis in water, through enzyme denaturing, cooling, sucking filtration, obtains enzymolysis solution;
(2) enzymolysis solution obtained in step (1) is added petroleum ether extraction grease removal, then the enzymolysis solution after grease removal is continued to add extraction into ethyl acetate, obtain Ethyl acetate fraction, volatilize ethyl acetate layer solvent, obtain Ganoderma ethyl acetate extract;
(3) Ganoderma ethyl acetate extract prepared by step (2) is dissolved in ethyl acetate, first alkalize to pH9 ~ 10, then be washed to neutrality, obtain the water layer that alkalizes, then will alkalize aqueous layer acidified to pH2 ~ 3, then obtain thick Ganodenic acid by extraction into ethyl acetate;
(4) by thick Ganodenic acid prepared by step (3), be separated through silicagel column, with chloroform and methanol system gradient elution, collect eluent, through thin layer chromatography inspection knowledge, filtration, evaporate to dryness, obtain Ganoderma triterpenoids acid activity position.
2. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (1), Ganoderma sporophore pulverizing medicinal materials is obtained dry powder, comprising: first get Ganoderma sporophore medical material, 55 DEG C ~ 75 DEG C dryings, pulverize, cross 80 ~ 120 mesh sieves and obtain dry powder.
3. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (1), described compound enzyme, is made up of the component of following weight percentage:
Neutral protease 5% ~ 95%;
Cellulase 2.5% ~ 48%;
Pectase 2.5% ~ 48%.
4. the preparation method at Ganoderma triterpenoids acid activity position according to claim 3, is characterized in that, in step (1), described compound enzyme, is made up of the component of following weight percentage:
Neutral protease 30% ~ 80%;
Cellulase 10% ~ 35%;
Pectase 10% ~ 35%.
5. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (1), the addition of described compound enzyme is 1.0% ~ 5.0% of dry powder quality.
6. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (1), described water and the solid-liquid ratio of dry powder are 10mL ~ 30mL:1g.
7. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (1), the condition of described enzymolysis is: temperature 35 ~ 70 DEG C, pH3.5 ~ 7, enzymolysis time 4.5 ~ 8h.
8. the preparation method at Ganoderma triterpenoids acid activity position according to claim 1, is characterized in that, in step (4), in described chloroform and methanol system, the volume ratio of chloroform and methanol is 1 ~ 10 ︰ 1.
9. Ganoderma triterpenoids acid activity position prepared by the preparation method according to any one of claim 1 ~ 8.
10. the application in improving water flood effect health product and tranquillizing and allaying excitement medicine is being prepared at Ganoderma triterpenoids acid activity position according to claim 9.
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| CN110974855A (en) * | 2019-12-30 | 2020-04-10 | 中国科学院微生物研究所 | Method for enriching ganoderic acid A into ganoderma lucidum spore oil |
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| CN104761612A (en) * | 2015-02-03 | 2015-07-08 | 安发（福建）生物科技有限公司 | Ganoderma triterpene acid extraction separation process method |
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| CN106722875A (en) * | 2016-12-07 | 2017-05-31 | 盐城工学院 | The extracting method and Ganoderma lucidum of a kind of ganodenic acid |
| CN110974855A (en) * | 2019-12-30 | 2020-04-10 | 中国科学院微生物研究所 | Method for enriching ganoderic acid A into ganoderma lucidum spore oil |
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