CN104547104B - Ramulus Ziziphi Spinosae leaf extract and its preparation method and application - Google Patents
Ramulus Ziziphi Spinosae leaf extract and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology field, it is related to Ramulus Ziziphi Spinosae leaf extract and its production and use, further relates to application of the Ramulus Ziziphi Spinosae leaf extract in tranquilizing soporific, antidepressant is prepared.The chemical composition of extract is mainly flavones ingredient and triterpenes components.Its preparation method is that wild jujube branches and leaves are extracted through water or alcoholic solvent, be concentrated under reduced pressure to obtain medicinal extract, medicinal extract is dispersed in water, after purification with macroreticular resin, merge 40% 60% and 70% 90% ethanol eluates, be concentrated under reduced pressure to give ethanol elution thing, spectrophotometry each several part general flavone content is 45.5% 60.7%, total triterpene contentses are 66.7% 73.5%, and it is 15.9% 21.1% to merge two parts to obtain in extract general flavone content, total triterpene contentses 43.3% 47.8%.Using a variety of separation means, isolated 16 compounds from Ramulus Ziziphi Spinosae leaf extract.Pharmacology test proves that Ramulus Ziziphi Spinosae leaf extract has tranquilizing soporific, antidepressant effect, in the medicine that can be applied to treatment central nervous system.
Description
Technical field
The present invention relates to Chemistry for Chinese Traditional Medicine technical field, it is related to Ramulus Ziziphi Spinosae leaf extract and its preparation method and application, specifically
It is related to Ramulus Ziziphi Spinosae leaf extract and preparation method thereof and the extract answering in terms for the treatment of tranquilizing soporific, antidepressant is prepared
With.
Background technology
Epidemiology survey shows that western countries have different degrees of insomniac to be about 35.2%.One system of ministry of Health of China
Meter data displays that current China insomniac is up to 1,200,000~1,400,000 person-times, and insomnia rate is up to 10%~20%.Estimate according to expert
Meter, to the year two thousand twenty, global insomniac will be more than 700,000,000.
Chronic insomnia can cause human immunologic function to reduce and metabolic disorder, aggravate original cardiovascular and cerebrovascular, digestive system
Etc. disease, insomnia can aggravate depression and anxiety obstacle again, cause serious psychological hygiene.Depression is a kind of common
Phrenoblabia, its current incidence of disease has leapt to the 4th of world's common disease, and depression will in scholarly forecast 20 years from now on
Rise to second largest common disease.According to statistics, about 5%~6% people suffers from depression to varying degrees in crowd.With life
Movable joint plays continuous quickening, and social pressures are increasing, and incidence of depression increases year by year, has had a strong impact on the life matter of people
Amount.
There are various treatment insomnias, antidepressant medicine in the market, although disease can be controlled, but still suffer from long-term treatment
Shortcoming in terms of the not good and adverse reaction of effect, such as somnifacient benzodiazepine (such as estazolam, Flurazepam, Quazepam and for horse
Dissolve in west) there are apparent side effect, including spirit and Behavioral change, memory disorders, certain tolerance is produced during long-term prescription, long
Dependence and habituation may occur for clothes, and knock-on and withrawal symptom (insomnia, anxiety, excitement, tremble) are likely to occur during drug withdrawal, can
Residual action on daytime etc. can occur.Medicine for treating depression is such as：Tricyclic drugs, MAOI and selection
Property 5-HT cell reabsorption inhibitors etc., though there is definite curative effect, there is a variety of side effects and contraindication, patient is not resistant to simultaneously
By, it is difficult to reach satisfactory effect, high recurrence rate.Therefore, countries in the world scholar all found from plant potent, nontoxic activity into
Divide and carry out developing new drug.The extraction mask work of traditional Chinese medicine active ingredient is just absorbed in extensively by countries in the world, from Chinese medicine
Effective position or monomer are extracted, effective means is provided for the discovery of new drug.
Wild jujube (Ziziphus jujuba Mill var.spinosa (Bunge) Hu ex H.Fu Chow) is Rhamnaceae
Jujube, mild-natured, sweet acid, with good Appetizing spleen-tonifying, promote the production of body fluid to quench thirst, help digestion only stagnant curative effect.《Sheng Nong's herbal classic》
Described in, wild jujube can " five viscera settling, macrobiosis of making light of one's life by commiting suicide ".Spina date seed is clinically usually used in treatment neurasthenia, vexed insomnia, many
Dream, night sweat, easily frightened etc. disease.Wild jujube branches and leaves of the present invention, are rhamnaceae plant wild jujube (Ziziphus jujuba Mill
Var.spinosa (Bunge) Hu ex H.Fu Chow) branch and leaf.Ramulus Ziziphi Spinosae claims " quil ", cures mainly detumescence, purulence of bursting, analgesic.
Wild Jujube Leaf is also referred to as spine leaf, cures mainly shin ulcer on the shank, with anesthetic effect, it is among the people be used for it is soaked on behalf of the tea, treatment hypertension, insomnia,
According to clinical verification, Wild Jujube Leaf also has good therapeutic effect to coronary heart disease.Wild jujube tree is wild resource, in recent years, because some areas are wild
Production-goods source is destroyed, and the wild jujube resource of some remote knobs is not developed, causes the price of spina date seed year by year
Increase, its branches and leaves is also intended only as feed use, causes the significant wastage of resource.The chemical composition and pharmacology to spina date seed are made at present
It is relatively many with studying.It is reported that containing flavonoids, alkaloids, saponins, fat oil, polysaccharide and organic acid in spina date seed
The multiple compounds such as class.And about the chemical constitution study of wild jujube branches and leaves, have scholar carried out Wild jujube leaf total flavonoids measure and
Triterpenes, saponins and flavones ingredient in HPLC-PDA-ESI-MS/MS methods identification Wild Jujube Leaf.In terms of pharmacological action, wild jujube
The research of benevolence tranquilizing soporific antidepressant effect is more deep, and wild jujube branches and leaves anti-insomnia and antidepressant report are less.
The content of the invention
Resource is utilized in order to effective, the present invention provides the preparation method of Ramulus Ziziphi Spinosae leaf extract and monomeric compound, with
And in the application of the medicines for central nervous system such as treating insomnia depression.
The purpose of the present invention is achieved by the following technical solution：
1. the preparation of Ramulus Ziziphi Spinosae leaf extract：
(1) wild jujube branches and leaves are crushed, wild jujube branches and leaves powder is that 30%-80% alcoholic solutions are extracted with water or volumetric concentration, dense
Shorten medicinal extract into；
(2) medicinal extract adds 5~10 times of distilled water dissolving；
(3) by liquid obtained by step (2) by macroporous resin column chromatography, successively with water, 10%-30% ethanol, 40%-
The solution elution of 60% ethanol and 70%-90% ethanol；
(4) water, 10%-30% ethanol eluates are discarded, merges 40%-60% and 70%-90% ethanol eluates, decompression
It is concentrated to dryness, obtains Ramulus Ziziphi Spinosae leaf extract.
In step (1), pulverizing medicinal materials mesh number is 10-40 mesh, preferably 20 mesh.
Described alcoholic solution is the aqueous solution of methanol, ethanol or n-butanol, preferred alcohol solution, the preferred 40%- of concentration
80%.
Described water or the consumption of alcoholic solution are measured for 8-20 times of wild jujube branches and leaves, preferably 15-20 times.
Described extraction method therefor is backflow, cold soaking or homogenate extraction, preferably each 1min~1h, refluxing extraction.
In described refluxing extraction, extraction time is 1~3 hour, and reflux temperature is 70~90 DEG C, preferably 1 hour.
The cold soaking time is 1~3 hour, preferably 3 hours.
The homogenate extraction time is 20 seconds~3 minutes, preferably 1 minute.
In step (2), the consumption for adding distilled water is 5~10 times of medicinal extract weight, under conditions of 60~80 DEG C or ultrasound
Dissolve medicinal extract；
In step (3), described macroreticular resin is D101, AB-8, HPD-100, HPD-400 or HPD-450, preferably
D101, HPD-100 or AB-8.
Step (3) comprises the following steps：
(a) post waterborne is used, is rinsed to phenolsulfuric acid test solution reaction negative, water consumption is 10~15 times of column volume, flow velocity
For 1~2 column volume/hour；
(b) ethanol solution for being 10%-30% with volumetric concentration is eluted, and is rinsed to hydrochloric acid magnesium powder reacting positive, ethanol is used
Measure as 10~20 times of column volume, flow velocity is 1~2 column volume/hour；It is preferred that 10%, 30%.
(c) the ethanol solution upper prop for being 40%-60% with volumetric concentration, it is post to rinse to the negative ethanol consumption of hydrochloric acid magnesium powder
10~20 times of volume, flow velocity is 1~2 column volume/hour；It is preferred that 40%, 60%.
(d) the ethanol solution upper prop for being 70-90% with volumetric concentration, rinses to Liebrmann Burchard and reacts cloudy
Property, ethanol consumption is 10~20 times of column volume, and flow velocity is 1~2 column volume/hour；It is preferred that 70%, 90%.
2. the separation of wild jujube branches and leaves monomeric compound
Utilize a variety of separation means, including silica gel, Ju Xian An ﹑ Sephadex LH-20 and ODS column chromatographys and preparation HPLC
Deng, isolated from wild jujube branches and leaves ethanol extract and obtain 16 compounds, according to physicochemical property and spectral data, it is determined that
The structure of compound, wherein compound 3,4,7,8,15 and 16 are isolated from jujube first.
Above-mentioned 16 compounds are by following method preparation：
Through polyamide column chromatography after 40%-60% ethanol eluate is concentrated, with ethanol-water system 0:100-90:100
Gradient elution, wherein 30% ethanol stream part under being eluted through polyamide column chromatography is again through polyamide column chromatography, with dichloromethane-first
Alcohol or petroleum ether-acetone (100:20-100:30) gradient elution.Obtain three stream parts.Stream part 1 is through Sephadex LH-20 gels
Column chromatography, then through preparing the isolated compound 1 of efficient liquid phase.Through ODS column chromatographys and repeatedly, Sephadex LH-20 coagulate stream part 2
Glue column chromatography, obtains compound 2 and compound 3.Stream part 3 is obtained through Sephadex LH-20 gel column chromatographies and preparation efficient liquid phase
To compound 4, compound 5, compound 6, compound 7 and compound 8.
Through silica gel column chromatography after 70%-90% ethanol eluate is concentrated, with methylene chloride-methanol or petroleum ether-the third
Ketone (100:0-100:10) gradient elution, obtains 5 stream parts (Fr.1~Fr.5).Fr.2 is through silica gel column chromatography, Sephadex
The means such as LH-20 gel column chromatographies and recrystallization obtain compound 9, compound 10, chemical combination 11, compound 12 and compound 13.
Fr.3 is through silica gel column chromatography, with methylene chloride-methanol/ethyl acetate system gradient (100:1-100:20) elute, then use
Sephadex LH-20 gel column chromatographies are purified, and the means such as preparative high-performance liquid chromatographic separation obtain compound 14.Fr.5 is through silica gel
Column chromatography, with methylene chloride-methanol-water (100:10:0-100:20:1) gradient elution, then with ODS column chromatographys and Sephadex
The means such as LH-20 gel column chromatographies obtain compound 15 and compound 16.
Present invention also offers a kind of pharmaceutical composition, Ramulus Ziziphi Spinosae leaf extract as described above is included.Described wild jujube
Branches and leaves extract can be mixed with clinically-acceptable solid pharmaceutical preparation or liquid preparation, solid with pharmaceutically acceptable carrier
Preparation includes granule, tablet, capsule etc., and liquid preparation includes, oral liquid and injecting fluid preparation.
3. Ramulus Ziziphi Spinosae leaf extract anti-insomnia, the research of antidepressant effect
(1) Ramulus Ziziphi Spinosae leaf extract causes the influence that mouse falls asleep to above threshold dose of sodium pentobarbitone
Kunming mouse is randomly divided into blank control group (physiological saline), spina date seed crude extract group, positive drug group, wild jujube
Branches and leaves 40%-60% ethanol elution things group, 70%-90% ethanol elution thing groups and Ramulus Ziziphi Spinosae leaf extract group (embodiment 1), often
Group 8, continuous gavage administration 5d, in after last dose 60min, intraperitoneal injection yellow Jackets 50mgkg-1 (above threshold dosage
Refer to the dosage that 90% mouse falls asleep), observe and record mouse generation sleep number of elements, Sleep latency and sleep time.
To inject after yellow Jackets, righting reflex loss is defined as the positive up to more than 1min persons in 15min, to inject yellow Jackets
Occurs righting reflex loss in 15min afterwards for Sleep latency；With animal righting reflex loss to reverting to sleep time.
Total data is handled using the statistical softwares of Spss 16.0.As a result show each administration group to above threshold dose of sodium pentobarbitone
The Sleep latency that mouse falls asleep is caused there are no significant influence, in addition to wild jujube branches and leaves 70%-90% eluate groups, other are each to be administered
Group can dramatically increase mouse sleep time (P<0.01).
(2) influence of the Ramulus Ziziphi Spinosae leaf extract to p-chlorophcnylalaninc (PCPA) induced insomnia rat blood serum cell factor
Research shows that the cell factor of maincenter participates in the regulation of sleep, and wherein interleukin 1 (IL-1) and tumour is bad
Necrosis factor-α (TNF-α) plays an important role in sleep regulation.IL-1 is a kind of cell factor for having and causing to sleep effect,
When sleep deprivation, the accumulation of IL-1 and other sleep-promoting substances in vivo indicates the accumulation of sleep, and these materials are to body
With sleep pressure, so that sleep " knock-on " phenomenon formed after sleep deprivation, to compensate lost sleep.IL-I includes IL-1
α, IL-1 β and IL-1Ra (IL-1 receptor antagonists).IL-1 β are the principal modes in blood circulation, and physiological function is stronger, so
At present mostly based on IL-1 β researchs.TNF-α with IL-1 there is similar sleep regulation to act on.TNF-α influences in immune system
Play a part of intermediary in sleep procedure, can be by improving intracerebral serotonin (5-HT) turnover rate, enhancing 5-HT can nerve
The function of member, extends the slow wave sleep time.This research observes Ramulus Ziziphi Spinosae leaf extract to p-chlorophcnylalaninc (PCPA) induced insomnia
The influence of rat blood serum cell factor.
From SD rats, blank group, model group, Ramulus Ziziphi Spinosae Leave extract group, spina date seed crude extract group, sun are randomly divided into
Property medicine group, wild jujube branches and leaves 40%-60% ethanol elution things group, 70%-90% ethanol elution thing groups and Ramulus Ziziphi Spinosae leaf extract group
(embodiment 1), every group 8, in addition to blank group, other each groups are in every morning 8:00~9:00 disposable celiac is injected
PCPA300mg/kg, continuous injection two days, after modeling success, blank group is filled with the physiological saline of respective volume respectively with model group
Stomach, the corresponding solution of extract of other each group rats difference gavage, positive drug group diazepam gavage, once a day, even
It is continuous 10 days.Blood 3ml is taken in last dose 24h posterior orbits, 1h, 1500r/min centrifugation 30min is stood, supernatant is taken, using enzyme-linked
The content of interleukin-1 ' beta ' (IL-1 β) and tumor necrosis factor-alpha (TNF-α) in immunoabsorption detection each group serum.Knot
Really, IL-1 β, the content of TNF-α are reduced in model group rats serum, and being compared with blank group has significant difference (P<0.01)；
Positive drug group, wild jujube branches and leaves crude extract group, the ethanol elution thing group of wild jujube branches and leaves 40%, spina date seed crude extract group and Ramulus Ziziphi Spinosae
The IL-1 β, TNF-α of the rat of leaf extract group content rise, being compared with model group has significant difference (P<0.05 or P<
0.01).Show that wild jujube branches and leaves crude extract, wild jujube branches and leaves 40%-60% ethanol elutions thing and Ramulus Ziziphi Spinosae leaf extract group have
The effect of tranquilizing soporific may be relevant with the content by changing the cell factor relevant with sleep, and wild jujube branches and leaves 40%-
60% ethanol elution thing plays a major role in tranquilizing soporific.Specific test result is shown in embodiment 7-8.
(3) mouse tail suspension and forced swim test
The principle of the antidepression model of mouse tail suspension and forced swim test is to provide one without avoidable compressing for it
Environment, makes the mouse in experiment produce the state of a kind of " behavioral despair and distortion " or " to suppressing environment adjustment failure ", similar
In the depressive state of the mankind, the classical model of antidepressant screening is widely used in.Specific process of the test is as follows：
1. tail-suspention test
Kunming mouse is randomly divided into blank group, positive drug group (amitriptyline, 20mgkg-1), Ramulus Ziziphi Spinosae Leave extract
Group, spina date seed crude extract group, wild jujube branches and leaves 40%-60% ethanol eluates group, 70%-90% ethanol eluates group and wild jujube
Branches and leaves extract group (embodiment 5), every group 8.In addition to blank group gavage gives the physiological saline of respective volume, remaining each group is equal
Gavage gives corresponding medicine respectively, 1 time a day, successive administration 10 days, and blank group gives the physiological saline of respective volume, Yu Mo
After secondary administration 30min, immediately with adhesive plaster by mouse tail away from being bonded at tail point 2cm on balance bracket, it is in projecting state, head to make it
From desktop about 15cm, both sides separate sight with plank, adapt to after 2min, determine dead time of the mouse in 4min.Experiment knot
Fruit shows：Compared with blank group, positive drug group, Ramulus Ziziphi Spinosae Leave extract group, spina date seed crude extract, 70%-90% ethanol are washed
De- liquid group and Ramulus Ziziphi Spinosae leaf extract group can substantially reduce dead time (P during mouse tail suspension<0.001), 40%-60% second
Alcohol eluate group is close to blank group level.
2. forced swim test
Experimental animal is grouped same tail-suspention test, and mouse is put into diameter 20cm, depth of water 10cm bucket, and water temperature is kept
(23 ± 1) DEG C, are adapted to after 2min, and determining dead time of the mouse in 4min, (dead time is to stop struggle in water, in drift
Floating state, duration of the only tiny limb motion to keep head to keep afloat).FST experimental results are shown：With blank
Group compares, positive drug group, wild jujube branches and leaves crude extract group, spina date seed crude extract group, the equal energy of 70%-90% ethanol elution thing groups
Substantially reduce dead time (P during mouse forced swimming test<0.001), the close sky of wild jujube branches and leaves 40%-60% ethanol elution things group
White group level.As a result show, Ramulus Ziziphi Spinosae Leave extract 70%-90% ethanol elutions thing and Ramulus Ziziphi Spinosae leaf extract group are respectively provided with anti-
Depression is acted on, and 70%-90% ethanol elution things play a major role in antidepression.Specific test result is shown in embodiment 7.
Beneficial effects of the present invention are as follows：
1. raw material used is wild jujube branches and leaves in the present invention, sour jujube resources are made to obtain sufficient development and utilization.
2. Ramulus Ziziphi Spinosae leaf extract prepared by the method for the invention, with the effect in terms of tranquilizing soporific, antidepression,
Medicine applied to exploitation sedative hypnotic has broad application prospects.
3. the extracting method of the present invention uses traditional extraction equipment, cheap, easy to be reasonable, do not used in technique
Poisonous organic solvent, it is pollution-free, be conducive to environmental protection, suitable for industrialized production, general flavone content is 15.9%-
21.1%；Total triterpene contentses are 43.3%-47.8%.
To make the present invention easier to understand, it is further elaborated with reference to specific embodiment.It is interpreted as these
Embodiment is only illustrative of the invention and is not intended to limit the scope of the invention, NM specific experiment side in the following example
Method, is generally carried out according to normal experiment method.
Embodiment：
Embodiment 1
After wild jujube branches and leaves 100g is crushed, the ethanol solution heating and refluxing extraction using 20 times of amount concentration as 80% 3 times, every time
1h, is recovered under reduced pressure solvent, is concentrated to give medicinal extract, then adds the distilled water of 10 times of medicinal extract weight, and leaching is dissolved under conditions of ultrasound
Cream, by above-mentioned solution by D101 macroreticular resin chromatographic columns, the consumption of macroreticular resin is 10 times of medicinal extract weight, is first washed with water
It is de-, rinse to phenolsulfuric acid test solution reaction negative, flow velocity is 1 column volume/hour, with volumetric concentration be successively then 10%,
30%th, 40%, 60%, 70% and 90% ethanol solution gradient elution, flow velocity is respectively 1 column volume/hour, consumption difference
For 10 times of column volume, water and 10-30% ethanol eluates are discarded, 40%-60% and 70%-90% ethanol eluates are obtained, point
It is total three in 58.2%, 70%-90% ethanol eluates that light photometry, which measures general flavone content in 40%-60% ethanol eluates,
The content of terpene is 73.5%.Eluent is merged, is concentrated under reduced pressure into dry, is obtained in Ramulus Ziziphi Spinosae leaf extract, Ramulus Ziziphi Spinosae leaf extract
General flavone content is 20.4%, and the content of total triterpene is 47.8%.
Embodiment 2
After wild jujube branches and leaves 100g is crushed, the ethanol solution homogenate extraction using 8 times of amount concentration as 30% 2 times, each 1min,
Recycling design, is concentrated to give medicinal extract, then adds the distilled water of 5 times of medicinal extract weight, medicinal extract is dissolved under conditions of ultrasound, will be above-mentioned
Solution is by D101 macroreticular resin chromatographic columns, and the consumption of macroreticular resin is 10 times of medicinal extract weight, is first eluted with water, and is rinsed to benzene
Phenol-sulfuric acid test solution reaction negative, flow velocity is 1 column volume/hour, with volumetric concentration be successively then 10%, 30%, 40%,
60%th, 70% and 80% ethanol solution gradient elution, flow velocity is respectively 1 column volume/hour, and consumption is respectively column volume
10 times, water and 10-30% ethanol eluates are discarded, 40%-60% and 70%-90% ethanol eluates, spectrophotometric are obtained respectively
It is that total triterpene contains in 50.0%, 70%-90% ethanol eluates that method, which measures general flavone content in 40%-60% ethanol eluates,
Measure as 66.7%.Eluent is merged, is concentrated under reduced pressure into dry, general flavone in Ramulus Ziziphi Spinosae leaf extract, Ramulus Ziziphi Spinosae leaf extract is obtained
Content is 16.9%, and the content of total triterpene is 44.0%.
Embodiment 3
After wild jujube branches and leaves 100g is crushed, extracted 3 times with 10 times of amount aqueous solution cold soakings, each 3h, recycling design is concentrated to give
Medicinal extract, then adds the distilled water of 8 times of medicinal extract weight, medicinal extract is dissolved under conditions of ultrasound, and above-mentioned solution is big by D101
Hole resin chromatography post, the consumption of macroreticular resin is 10 times of medicinal extract weight, is first eluted with water, and is rinsed anti-to phenolsulfuric acid test solution
Should be negative, flow velocity is 1 column volume/hour, then successively with the second that volumetric concentration is 10%, 30%, 40%, 60% and 70%
Alcoholic solution gradient elution, flow velocity is respectively 1 column volume/hour, and consumption is respectively 10 times of column volume, discards water and 10%-
30% ethanol eluate, obtains 40%-60% and 70%-90% ethanol eluates respectively, and AAS measures 40%-60% second
General flavone content is that the content of total triterpene in 45.5%, 70%-90% ethanol eluates is 66.7% in alcohol eluen.Will elution
Liquid merges, and is concentrated under reduced pressure into dry, and it is 15.9%, total three to obtain in Ramulus Ziziphi Spinosae leaf extract, Ramulus Ziziphi Spinosae leaf extract general flavone content
The content of terpene is 43.3%.
Embodiment 4
After wild jujube branches and leaves 100g is crushed, extracted 3 times with 8 times of amount n-butanol hot dippings, each 3h, recycling design is concentrated to give
Medicinal extract, then adds the distilled water of 10 times of medicinal extract weight, medicinal extract is dissolved under conditions of ultrasound, above-mentioned solution is passed through into HPD-
100 macroreticular resin chromatographic columns, the consumption of macroreticular resin is 8 times of medicinal extract weight, is first eluted with water, and is rinsed to phenolsulfuric acid examination
Liquid reaction negative, flow velocity is 1 column volume/hour, with volumetric concentration is successively then water, 30%, 40%, 60%, 70% and
90% ethanol solution gradient elution, flow velocity is respectively 1 column volume/hour, and consumption is respectively 10 times of column volume, discards water
And 30% ethanol eluate, respectively 40%-60% and 70%-90% ethanol eluates, AAS measures 40%-60%
General flavone content is that the content of total triterpene in 47.1%, 70%-90% ethanol eluates is 67.7% in ethanol eluate.It will wash
De- liquid merges, and is concentrated under reduced pressure into dry, and general flavone content is 16.4% in Ramulus Ziziphi Spinosae leaf extract, and the content of total triterpene is
44.1%.
Embodiment 5
After wild jujube branches and leaves 100g is crushed, the methanol solution heating and refluxing extraction using 15 times of amount concentration as 50% 2 times, every time
1.5h, is recovered under reduced pressure solvent, is concentrated to give medicinal extract, then adds the distilled water of 10 times of medicinal extract weight, is dissolved under conditions of ultrasound
Medicinal extract, by above-mentioned solution by AB-8 macroreticular resin chromatographic columns, the consumption of macroreticular resin is 10 times of medicinal extract weight, is first washed with water
It is de-, rinse to phenolsulfuric acid test solution reaction negative, flow velocity is 1 column volume/hour, with volumetric concentration be successively then 30%,
40%th, 60%, 70% and 80% ethanol solution gradient elution, flow velocity is respectively 1 column volume/hour, and consumption is respectively post
10 times of volume, discard water and 10%-30% ethanol eluates, 40%-60% and 70%-80% ethanol eluates are obtained respectively,
It is total in 60.7%, 70%-90% ethanol eluates that AAS, which measures general flavone content in 40%-60% ethanol eluates,
The content of triterpene is 72.4%.Eluent is merged, is concentrated under reduced pressure into dry, general flavone content is in Ramulus Ziziphi Spinosae leaf extract
21.1%, the content of total triterpene is 46.9%.
The separation identification of the monomeric compound of embodiment 6
(1) through polyamide column chromatography after 40%-60% ethanol eluate is concentrated, with 0%-90% ethanol waters ladder
Degree elution, wherein 30% ethanol stream part under being eluted through polyamide column chromatography uses methylene chloride-methanol again through polyamide column chromatography
(5:1-3:1) gradient elution.Obtain 4 stream parts.By stream part 1 through Sephadex LH-20 gel column chromatographies, then inverted preparation
Type efficient liquid phase is separated, methanol-water (53：47) elute, obtain the FNS of compound 1；By stream part 2 through ODS
Column chromatography and repeatedly Sephadex LH-20 gel column chromatographies, obtain the rutin of compound 2 and the Quercetin -3-O- β-D- of compound 3
Glucoside；Stream part 3 is separated through Sephadex LH-20 gel column chromatographies, then inverted preparative efficient liquid phase, methanol-water
(36：64) elute, obtain the Quercetin-3-O- α of compound 4-L-arabinose base (1 → 2)-α-L- rhamnoses and the mountain of compound 5
How phenol；Stream part 4 is subjected to silica gel column chromatography, with methylene chloride-methanol (100:0-100:50) gradient elution, through Sephadex
LH-20 gel column chromatographies, with petroleum ether-methylene chloride-methanol (4:1:1) elute, obtain the Quercetin of compound 6, compound 7
(+)-catechin and the-pentahydroxyflavane of 2R, 3R-3,5,7,3 ', 5 ' of compound 8.
Fr.4 is subjected to silica gel column chromatography, with methylene chloride-methanol-water (100:0:0-0:0:100) gradient elution, flow point
Sephadex LH-20 gel column chromatographies obtain compound 9lupeol and compound 10maslinic through ODS column chromatographys and repeatedly
acid。
(2) by 70%-90% ethanol eluate through silica gel column chromatography, with methylene chloride-methanol (100:0-100:10)
Gradient elution, obtains 6 stream parts (Fr.1~Fr.6).
1) Fr.2 carries out silica gel column chromatography, with petroleum ether-ethyl acetate (100:0-1:1) gradient elution, stream part is through repeatedly
The means such as the purifying of Sephadex LH-20 gel column chromatographies and recrystallization obtain the oleanolic acid of compound 11, the white birch fat of compound 12
Acid, compound 13epiceanothic acid and the alphitolic acid of compound 14.
2) Fr.3 is subjected to silica gel column chromatography, with methylene chloride-methanol (100:0-100:50) gradient elution, stream part warp
Sephadex LH-20 gel column chromatographies are purified, then carry out Reverse phase preparative efficient liquid phase separation, with methanol-water (40:60) wash
It is de-, obtain compound 15 (R)-PLA methyl esters and compound 16 (S) -3- (3- indyls) methyl lactate.
Structural Identification
It is main to utilize spectrum, including ultraviolet, mass spectrum and nuclear magnetic resoance spectrum (1HNMR、13) etc. CNMR analysis means, identify it
Structure.
The physicochemical constant and dominant spectral data of compound
The FNS of compound 1
Molecular formula C28H31O15, Yellow amorphous powder, ferric trichloride-potassium ferricyanide reacting positive, hydrochloric acid-magnesium powder,
Molish reactions are the positive.1H-NMR (300MHz, DMSO-d6) spectrum δ：7.98 (2H, d, J=8.1Hz, H-2 ', 6 '), 6.87
(2H, d, J=8.1Hz, H-3 ', 5 '), 6.32 (1H, d, J=1.8Hz, H-8), 6.16 (1H, J=1.8Hz, H-6), 5.30
(1H, d, J=7.2Hz, H-1 "), 4.38 (1H, brs, H-1 " '), 2.5-3.8 (10H, m, sugar), 0.98 (3H, d, J=
6.0Hz,CH3)。13C-NMR (75MHz, DMSO-d6)。
Quercetin-3-O- the β of compound 3-D-Glucose glycosides
Molecular formula C21H20O12, Yellow amorphous powder, ferric trichloride-potassium ferricyanide reacting positive, hydrochloric acid-magnesium powder,
Molish reactions are the positive.1H-NMR(300MHz,DMSO-d6) spectrum δ:6.18(1H,brs,H-6)、6.40(1H,brs,H-
8), 6.83 (1H, d, J=9Hz, H-5 '), 7.56 (2H, m, H-6 ', 2 '), 12.61 (1H, s, 5-OH) are Quercetin parent nucleus proton
Signal.5.45 (1H, d, J=7.2Hz) are sugared anomeric proton signal, and determine that glycosides is strong according to coupling constant and be configured as β types.13C-
NMR(125MHz,DMSO-d6)：It is shown in Table 1.
Quercetin-3-O- the α of compound 4-L-arabinose base (1 → 2)-alpha-L-rhamnoside
Molecular formula C26H28O15, yellow indefiniteness powder, ESI-MS m/z：603[M+Na]+,579[M-H]-,615[M-
Cl]-.Ferric trichloride-potassium ferricyanide reacting positive, hydrochloric acid-magnesium powder, Molish reactions are the positive.1H-NMR(600MHz,
DMSO-d6)δ：6.16 (1H, brs, H-6), 6.35 (1H, brs, H-8), 6.87 (1H, d, J=8.3Hz, H-5 '), 7.25 (1H,
Brd, J=8.3Hz, H-6 '), 7.33 (1H, brs, 2 '), 12.73 (1H, s, 5-OH) be Quercetin parent nucleus proton signal.5.30
(1H, brs, H-1 "), 4.09 (1H, d, J=6.84Hz, H-1 " ') are sugared anomeric proton signal.13C-NMR (125MHz, DMSO-
d6)：It is shown in Table 1.
Compound 7 (+)-catechin
Molecular formula C15H14O6：White amorphous powder.It is a yellow blackening under uviol lamp (254nm), volume fraction is
10% ethanol solution of sulfuric acid colour developing is in brown color.1H-NMR (300MHz, DMSO-d6) spectrum δ：4.48 (1H, d, 7.2Hz, H-2),
3.82 (1H, m, H-3), 2.35 (1H, dd, J=15.9,8.1Hz, H-4), 2.66 (1H, dd, J=15.9,5.4Hz, H-4),
6.60 (1H, dd, J=8.1,1.8Hz, H-6 '), 6.69 (1H, d, J=8.1Hz, H-5 '), 6.72 (1H, d, J=1.8Hz, H-
2 '), 5.69 (1H, d, J=2.1Hz, H-6), 5.89 (1H, d, J=2.4Hz, H-8).C-2 is inferred by the coupling constant of 2 hydrogen
With C-3 hydrogen into anti-configuration.13C-NMR (75MHz, DMSO-d6)：It is shown in Table 2.
- the pentahydroxy of 2R, 3R-3,5,7,3 ', the 5 ' flavane of compound 8
Molecular formula C15H14O6, red unformed powder, 20D=-35 ° of [α] (c1.6, MeOH).It is under uviol lamp (254nm)
One yellow blackening, volume fraction is in brown color for 10% ethanol solution of sulfuric acid colour developing.1H-NMR (300MHz, DMSO-d6) spectrum δ：
δ 4.72 (1H, s, H-2), 4.00 (1H, s, H-3), 2.67 (1H, dd, J=16.2,4.2Hz, H-4) and 2.44 (1H, d, J=
3.3Hz, H-4), 6.66 (2H, m, H-2 ', H-6 '), 6.88 (1H, s, H-4 '), 5.71 (1H, d, J=1.8Hz, H-6), 5.88
(1H, d, J=1.8Hz, H-8), (1H, s) is presented unimodal to 2 hydrogen 4.72, does not split point C-2 and C-3 hydrogen of deduction into cis structure
Type.13C-NMR (75MHz, DMSO-d6)：It is shown in Table 2.
Compound 15 (R)-PLA methyl esters
Molecular formula C10H11O3：White crystals, [α] 20D=9.2 ° of (c0.4, CHCl3), volume fraction is 10% sulfuric acid ethanol
Solution shows purple.1H-NMR (300MHz, DMSO-d6) spectrum δ：2.80 (1H, dd, J=13.8,8.1Hz), 2.93 (1H, dd, J=
13.8th, 5.1Hz) it is CH2Proton signal, 4.23 (1H, dd, J=4.8,8.1Hz) for even oxygen CH proton signals, 3.58 (3H,
S) it is OCH3Proton signal, 7.24 (5H, m) be phenyl protons signal.13C-NMR(75MHz,DMSO-d6)：It is shown in Table 3.
Compound 16 (S) -3- (3- indyls) methyl lactate
Molecular formula C12H12O3:White powder, [α] 20D=-24 ° of (c1.0, CHCl3), bismuth potassium iodide reacting positive.1H-
NMR(300Hz,CDCl3) spectrum δ：8.08 (1H, s, H-NH), 7.60 (1H, d, J=7.8Hz), 7.34 (1H, d, J=8.1Hz),
7.18 (1H, t, J=9Hz) and 7.12 (1H, t, J=9Hz) are proton signal on adjacent disubstituted benzenes ring, and 7.08 (1H s) is indoles
Ring hydrogen signal, 3.30 (1H, dd, J=14.7Hz, 4.2Hz), 3.18 (1H, dd, J=14.7Hz, 6Hz) are CH2Proton is believed
Number, 4.52 (1H, t, J=5.25Hz) are the CH proton signals of even oxygen.13C-NMR(75MHz,CDCl3)：It is shown in Table 3.
The compound 3,4 of table 113CNMR chemical displacement values
The compound 7,8 of table 213CNMR chemical displacement values
| Carbon atom | Compound 5 (δ, solvent DMSO-d6) | Compound 6 (δ, solvent DMSO-d6) |
| 1 | - | 174.7 |
| 2 | 81.1 | 78.1 |
| 3 | 66.4 | 64.9 |
| 4 | 27.9 | 28.3 |
| 5 | 156.5 | 155.8 |
| 6 | 93.9 | 95.2 |
| 7 | 156.3 | 156.6 |
| 8 | 95.2 | 94.2 |
| 9 | 155.4 | 156.3 |
| 10 | 99.2 | 98.6 |
| 1′ | 130.7 | 130.7 |
| 2′ | 114.6 | 114.9 |
| 3′ | 144.9 | 144.5 |
| 4′ | 144.9 | 118.0 |
| 5′ | 115.2 | 144.4 |
| 6′ | 118.5 | 114.8 |
The compound 15,16 of table 313C-NMR chemical displacement values
| Carbon atom | Compound 15 (δ, solvent DMSO-d6) | Compound 16 (δ, solvent C DCl3) |
| 1 | 174.0 | 174.7 |
| 2 | 71.3 | 70.8 |
| 3 | 39.6 | 30.3 |
| 1′ | 137.7 | - |
| 2′ | 129.4 | 123.2 |
| 3′ | 128.1 | 110.1 |
| 4′ | 126.3 | 118.8 |
| 5′ | 128.1 | 119.5 |
| 6′ | 129.4 | 122.1 |
| 7′ | - | 111.2 |
| 8′ | - | 136.1 |
| 9′ | - | 127.6 |
| -OCH3 | 51.4 | 52.4 |
In addition the rutin of compound 2, the Kaempferol of compound 5,6 Quercetins, 9lupeol, 10maslinic acid, 11 neat piers
Tartaric acid, 12 betulic acids, 13epiceanothic acid, 14 alphitolic acids etc. are by using three kinds of different solvents and reference substance
Thin layer method, which is identified, altogether comes.
Above threshold dose of sodium pentobarbitone causes the influence that mouse falls asleep to 7 pairs of embodiment
Kunming mouse is randomly divided into 6 groups, every group 8, blank control group (physiological saline), positive controls (Gastrodin
Piece, 50mgkg-1), spina date seed crude extract, wild jujube branches and leaves crude extract, wild jujube branches and leaves 40%-60% ethanol elutions thing it is (real
Apply example 1), wild jujube branches and leaves 70%-90% ethanol elutions thing (embodiment 1), Ramulus Ziziphi Spinosae leaf extract group (embodiment 1), it is continuous to fill
5d is administered in stomach, and in after last dose 60min, yellow Jackets 50mgkg is injected intraperitoneally-1(above threshold dosage refers to 90% mouse and entered
The dosage slept), observe and record mouse generation sleep number of elements, Sleep latency and sleep time.To inject amobarbital
Righting reflex loss is defined as the positive up to more than 1min persons in 15min after sodium, is turned over injecting after yellow Jackets in 15min
It is Sleep latency that normal reflection, which disappears,；With animal righting reflex loss to reverting to sleep time.As a result withTable
Show.Paired t-test is relatively used between group and group.One-way analysis of variance is relatively used between multigroup, total data is equal
Handled using the statistical softwares of Spss 16.0.As a result show that each administration group causes mouse to fall asleep above threshold dose of sodium pentobarbitone
Sleep latency there are no significant influence, in addition to wild jujube branches and leaves 70%-90% ethanol elution thing groups, other each equal energy of administration group
Dramatically increase mouse sleep time (P<0.01).It the results are shown in Table 4.
Above threshold dose of sodium pentobarbitone causes the influence that mouse falls asleep to 4 pairs of table
* represents the P ＜ 0.01 compared with blank group
The influence of embodiment 8 Ramulus Ziziphi Spinosae leaf extract p-chlorophcnylalaninc (PCPA) induced insomnia rat blood serum cell factor
With will healthy cleaning grade male SD rat 70, be divided into 7 groups, blank group, model group, positive drug group (medicine),
Wild jujube branches and leaves crude extract group, spina date seed crude extract, wild jujube branches and leaves crude extract group, wild jujube branches and leaves 40%-60% ethanol are washed
De- thing (embodiment 1) and wild jujube branches and leaves 70%-90% ethanol elutions thing (embodiment 1), Ramulus Ziziphi Spinosae leaf extract group (embodiment
1).In addition to Normal group, PCPA300mg/kg is disposably injected intraperitoneally in other each groups, continuous injection two days, is observed after 30h
Rat behavior, replicates rat and has a sleepless night completely model.Rat after modeling success, except blank group and model group give same volume respectively
Long-pending physiological saline, other each groups every press 1mL/100g gavages, wild jujube branches and leaves crude extract group and spina date seed crude extract group
And 40%-60% wild jujubes branches and leaves ethanol elution thing and 70%-90% wild jujube branches and leaves ethanol elution agents amount are by the crude drug amount converted
5.4g/kg is administered, and positive drug group is administered by 0.9mg/mL concentration, 1 time a day, continuous 10d.Each group is tested in last dose 24h
Posterior orbit takes blood 3mL, stands 1h, 4 DEG C are overnight, 1500r/min centrifugation 30min, take in supernatant, rat blood serum IL-1 β and
The content of TNF-α is detected using Enzyme-linked Immunosorbent Assay (ELISA) method, is operated in strict accordance with kit, be the results are shown in Table 5.
The each group rat blood serum IL-1 β of table 5, TNF-α comparision contents(pg·mL-1)
Statistical method：Variance analysis；##P ＜ 0.01, are compared with blank group；*P ＜ 0.05,**P ＜ 0.01 and model group ratio
Compared with.
Result is shown in table 5, and IL-1 β, the content of TNF-α are reduced in model group rats serum, and being compared with blank group has
Significant difference (P<0.01)；Positive drug group, spina date seed crude extract group, wild jujube branches and leaves crude extract group, wild jujube branches and leaves 40%-
The rat IL-1 β, TNF-α of 60% ethanol elution thing group and Ramulus Ziziphi Spinosae leaf extract group content rise, being compared with model group has
Significant difference (P<0.05 or P<0.01).As a result show that wild jujube branches and leaves crude extract and wild jujube branches and leaves 40%-60% ethanol are washed
The content of the de- thing cell factor relevant with sleep with Ramulus Ziziphi Spinosae leaf extract group pair improves significantly, and points out Ramulus Ziziphi Spinosae
Leaf Cu Tiquwu wild jujubes branches and leaves 40%-60% ethanol elution thing groups and Ramulus Ziziphi Spinosae leaf extract group are respectively provided with the work of tranquilizing soporific
With.
The antidepressant effect research of the Ramulus Ziziphi Spinosae leaf extract of embodiment 9
(1) tail-suspention test
Kunming mouse is randomly divided into 6 groups：Blank group, positive drug group (amitriptyline, 20mgkg-1) and wild jujube branches and leaves it is thick
Extract group, spina date seed crude extract group, wild jujube branches and leaves crude extract group, wild jujube branches and leaves 40%-60% ethanol eluates group (are implemented
Example 5), wild jujube branches and leaves 70%-90% ethanol eluates group (embodiment 5), Ramulus Ziziphi Spinosae leaf extract group (embodiment 5), every group 8
Only.In addition to blank group gives the physiological saline of respective volume, respectively gavage gives corresponding medicine to remaining each group, and dosage is by folding
The crude drug amount 8g/kg administrations of calculation.1 time a day, successive administration 10 days, blank group is incited somebody to action with adhesive plaster immediately in after last dose 30min
Mouse tail makes it be in projecting state away from being bonded at tail point 2cm on balance bracket, head from desktop about 15cm, both sides plank every
Disconnected sight, is adapted to after 2min, determines dead time of the mouse in 4min.Experimental result is shown：Compared with blank group, positive drug
Group, Ramulus Ziziphi Spinosae Leave extract group, 70%-90% ethanol eluates group and Ramulus Ziziphi Spinosae leaf extract group can substantially reduce mouse and hang
Dead time (P during tail<0.001), 40%-60% ethanol elutions thing group is close to blank group level.It the results are shown in Table 6.
(2) forced swim test
The same tail-suspention test of animal packet, mouse is put into diameter 20cm, depth of water 10cm bucket, water temperature holding (23 ±
1) DEG C, adapt to after 2min, determining dead time of the mouse in 4min, (dead time is to stop struggling in water, in floating shape
The duration of state, only tiny limb motion to keep head to keep afloat).FST experimental results are shown：With blank group ratio
Compared with positive drug group, wild jujube branches and leaves crude extract group, wild jujube branches and leaves 70%-90% ethanol elution thing groups and Ramulus Ziziphi Spinosae leaf extract
Group can substantially reduce the dead time (P ＜ 0.001) during mouse forced swimming test, the close sky of 40%-60% ethanol elution things group
White group level, it is consistent with tail-suspention test result.It the results are shown in Table 6.
The measure of dead time in the tail-suspention test of table 6 and forced swim test
In table 6 tail-suspention test and forced swim test result show except wild jujube branches and leaves 40%-60% ethanol elution thing groups without
Outside the outstanding tail dead time and forced swimming dead time for significantly shortening mouse, remaining each group can significantly shorten the motionless of mouse
Time (P ＜ 0.001), it is Wild Jujube Leaf antidepression position to point out 70%-90% ethanol elutions thing.
The granule of the leaf extract containing Ramulus Ziziphi Spinosae of embodiment 10 and its preparation
| Prescription： | Composition | % (weight) |
| Wild jujube branches and leaves crude extract | 15 | |
| Sucrose | 65 | |
| Pregelatinized starch | 10 | |
| Dextrin | 10 |
Preparation method：Ramulus Ziziphi Spinosae leaf extract, sucrose, pregelatinized starch and dextrin are well mixed, appropriate 75% ethanol is added
Softwood is made, 18 mesh sieve series wet granulars, 50 DEG C of oven drying 30min cross 18 mesh sieve whole grains after taking-up, packing produces Ramulus Ziziphi Spinosae
Leaf extract granule.
The tablet of the crude extract of branches and leaves containing wild jujube of embodiment 11 and its preparation
| Prescription： | Composition | % (weight) |
| Ramulus Ziziphi Spinosae leaf extract | 15 | |
| Microcrystalline cellulose | 77 | |
| Sodium carboxymethyl starch | 7.5 | |
| Magnesium stearate | 0.5 |
Preparation method：Ramulus Ziziphi Spinosae leaf extract, microcrystalline cellulose, sodium carboxymethyl starch are well mixed, appropriate 75% second is added
Softwood is made in alcohol, and softwood crosses the 18 wet grains of mesh sieve series, and wet grain dries 30min in 50 DEG C of baking ovens, and 18 mesh sieve whole grains are crossed after taking-up, plus
Enter magnesium stearate, mix, tabletting produces Ramulus Ziziphi Spinosae leaf extract tablet.
The capsule of the leaf extract containing Ramulus Ziziphi Spinosae of embodiment 12 and its preparation
| Prescription： | Composition | % (weight) |
| Ramulus Ziziphi Spinosae leaf extract | 15 | |
| Dextrin | 45 | |
| Starch | 40 |
Preparation method：Take the Ramulus Ziziphi Spinosae leaf extract of recipe quantity to crush 80 mesh sieves, add starch, dextrin, well mixed, loading
Capsule, produces Ramulus Ziziphi Spinosae leaf extract capsule.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention principle it
Interior made any modification, equivalent substitution and improvements etc., should be included in the scope of the protection.
Claims (10)
1. a kind of Ramulus Ziziphi Spinosae leaf extract, it is characterised in that be made by the steps：
(1) wild jujube branches and leaves are crushed, wild jujube branches and leaves powder is that 30%-80% alcoholic solutions are extracted with water or volumetric concentration, is condensed into
Medicinal extract；
(2) medicinal extract adds 5~10 times of distilled water dissolving；
(3) by liquid obtained by step (2) by macroporous resin column chromatography, successively with water, 10%-30% ethanol, 40%-60% second
The solution elution of alcohol and 70%-90% ethanol；
(4) water, 10%-30% ethanol eluates are discarded, merges 40%-60% and 70%-90% ethanol eluates, is concentrated under reduced pressure
To dry, Ramulus Ziziphi Spinosae leaf extract is obtained；
Macroreticular resin described in step (3) is D101, HPD-100 or AB-8；
Described Ramulus Ziziphi Spinosae leaf extract includes Quercetin -3-O- β-D-Glucose glycosides, Quercetin -3-O- α-L-arabinose base
(1 → 2)-alpha-L-rhamnoside, (+)-catechin,-pentahydroxyflavane of 2R, 3R-3,5,7,3 ', 5 ', (R) -3- benzene
Base methyl lactate, (S) -3- (3- indyls) methyl lactate.
2. Ramulus Ziziphi Spinosae leaf extract as claimed in claim 1, it is characterised in that described Quercetin -3-O- β-D-Glucose
Glycosides, Quercetin -3-O- α-L-arabinose base (1 → 2)-alpha-L-rhamnoside, (+)-catechin, 2R, 3R-3,5,7,3 ', 5 ' -
Pentahydroxyflavane, (R)-PLA methyl esters, (S) -3- (3- indyls) methyl lactate are obtained by the following method
Arrive：
(1), through polyamide column chromatography, it will be washed after 40%-60% ethanol eluate concentration with 0%-90% ethanol water gradients
It is de-, wherein 30% ethanol stream part under being eluted through polyamide column chromatography is again through polyamide column chromatography, with methylene chloride-methanol 5:1-
3:1 gradient elution, obtains 4 stream parts；Stream part 1 is efficient through Sephadex LH-20 gel column chromatographies, then inverted preparative
Liquid phase separation, methanol-water 53：47 elutions, obtain the FNS of compound 1；By stream part 2 through ODS column chromatographys and
Sephadex LH-20 gel column chromatographies, obtain the rutin of compound 2 and the Quercetin-3-O- β of compound 3-D-Glucose glycosides repeatedly；
Stream part 3 is separated through Sephadex LH-20 gel column chromatographies, then inverted preparative efficient liquid phase, methanol-water 36：64 wash
It is de-, obtain the Quercetin-3-O- α of compound 4-L-arabinose base (1 → 2)-α-L- rhamnoses and the Kaempferol of compound 5；Will stream
Part 4 carries out silica gel column chromatographies, with methylene chloride-methanol 100:0-100:50 gradient elutions, through Sephadex LH-20 gel columns
Chromatogram, with petroleum ether-methylene chloride-methanol 4:1:1 elution, obtains the Quercetin of compound 6, compound 7 (+)-catechin and change
- the pentahydroxyflavane of 2R, 3R-3,5,7,3 ', 5 ' of compound 8；Fr.4 is subjected to silica gel column chromatography, with dichloromethane-
Methanol-water 100:0:0-0:0:100 gradient elutions, flow point Sephadex LH-20 gel column chromatographies through ODS column chromatographys and repeatedly
Obtain compound 9lupeol and compound 10maslinic acid；
(2) by 70%-90% ethanol eluate through silica gel column chromatography, with methylene chloride-methanol 100:0-100:10 gradients are washed
It is de-, obtain 6 stream parts of Fr.1~Fr.6；Fr.2 carries out silica gel column chromatography, with petroleum ether-ethyl acetate 100:0-1:1 gradient is washed
De-, stream part obtains the oleanolic acid of compound 11, change through the means such as the purifying of Sephadex LH-20 gel column chromatographies repeatedly and recrystallization
The betulic acid of compound 12, compound 13epiceanothic acid and the alphitolic acid of compound 14；Fr.3 is subjected to silica gel column layer
Analysis, with methylene chloride-methanol 100:0-100:50 gradient elutions, stream part is purified through Sephadex LH-20 gel column chromatographies, then
Reverse phase preparative efficient liquid phase separation is carried out, with methanol-water 40:60 elutions, obtain compound 15 (R)-PLA methyl esters
With compound 16 (S) -3- (3- indyls) methyl lactate.
3. Ramulus Ziziphi Spinosae leaf extract according to claim 1, it is characterised in that：Alcoholic solution concentration in step (1) is
40%-80%.
4. Ramulus Ziziphi Spinosae leaf extract according to claim 1, it is characterised in that：Alcoholic solution described in step (1) is first
The aqueous solution of alcohol, ethanol or n-butanol, described water or the consumption of alcoholic solution are 8-20 times of wild jujube branches and leaves and measured.
5. Ramulus Ziziphi Spinosae leaf extract according to claim 1, it is characterised in that：The weight of macroreticular resin described in step (3)
Amount consumption is 10~20 times of medicinal extract weight.
6. Ramulus Ziziphi Spinosae leaf extract according to claim 1, it is characterised in that：General flavone content is 15.9%-21.1%,
Total triterpene contentses are 43.3%-47.8%.
7. a kind of pharmaceutical composition, includes the Ramulus Ziziphi Spinosae leaf extract described in claim 1-6 any one.
8. the composition described in Ramulus Ziziphi Spinosae leaf extract or claim 7 described in claim 1-6 any one is anti-in preparation
Control the application in medicine for central nervous system.
9. application according to claim 8, it is characterised in that described medicine for central nervous system be hypnotic sedative agent or
Antidepressants.
10. pharmaceutical composition described in Ramulus Ziziphi Spinosae leaf extract or claim 7 and medicine described in claim 1-6 any one
Acceptable carrier combines and solid pharmaceutical preparation or liquid preparation is made on.
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