CN106478622B - A kind of compound and preparation method thereof extracted from Tinospora sinensis - Google Patents
A kind of compound and preparation method thereof extracted from Tinospora sinensis Download PDFInfo
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- CN106478622B CN106478622B CN201610744333.4A CN201610744333A CN106478622B CN 106478622 B CN106478622 B CN 106478622B CN 201610744333 A CN201610744333 A CN 201610744333A CN 106478622 B CN106478622 B CN 106478622B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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Abstract
The present invention discloses a kind of compound, is named as Tinospora sinensis alkali.In addition, the present invention discloses a kind of preparation method extracted from Tinospora sinensis and be separated to Tinospora sinensis alkali, the preparation method utilizes acid alcohol extracting, after concentration is redissolved, and is directly chromatographed using column, eluate merges the Tinospora sinensis alkali for recrystallizing and can obtaining high-purity.In addition, separating palmatine, vacation palmatine that purity is up to 90%~95% using this preparation method.The preparation method simple process, stabilization, favorable reproducibility, and Tinospora sinensis alkali, palmatine, false palmatine pure compounds can be once obtained simultaneously, it saves the time, reduce consumption, it is suitble to industrialized production, there is positive effect to the comprehensive utilization of Tinospora sinensis herb resource and exploitation is accelerated, have a good application prospect.
Description
Technical field
The present invention relates to a kind of compound and preparation method thereof, especially a kind of compound extracted from Tinospora sinensis
Tinospora sinensis alkali and preparation method thereof.
Background technique
Tinospora sinensis Tinospora sinensis (Lour.) Merr. is Menispermaceae Tinospora plant China black ox
The drying rattan of gallbladder, alias Herba Lycopodii, Chinese tinospora stem, blue veins rattan etc., bitter is slightly cold, return liver warp, has relaxing tendons and activating collaterals, wind-dispelling
The effect of analgesic, be mainly used for rheumatic arthralgia, muscle arteries and veins contraction, joint stuffiness, traumatic injury, main product in Guangdong, Guangxi, Yunnan,
The ground such as Hunan are Guangdong and Guangxi Provinces area parts of generic medicinal plants.
Palmatine, false palmatine belong to quaternary amines alkaloid component, and the molecular structural formula of the two is as follows, palmatine
(also known as fibrauretine) and its inorganic acid salt, acylate or derivative etc., there are many pharmacological activity and clinical values, such as town
Bitterly, hypoglycemic, tune rouge, anti-hypertension, anti-inflammatory, antibacterial, antiviral etc., are widely used in field of medicaments, and demand is very big, but because existing
The equal Shortcomings of manufacture craft of capable various palmatine high sterlings, are encountered by which results in palmatine patent medicine and lack raw material, supply
The awkward situation that should not be asked；False palmatine causes its sterling low yield, so that city because lacking the research for isolating and purifying aspect
False palmatine chemical reference substance on sale is extremely rare, at high price on face.
The research not about the purifying preparation of false palmatine is reported in the prior art, and the purifying preparation side about palmatine
Method, which is predominantly extracted from plants, to be isolated and purified or is prepared using chemistry, biotechnology purifying, but each Shortcomings of two ways
Place.Such as application No. is the Chinese patent application of CN103393780A " a kind of extracting method of high-purity coptis chinensis total alkaloid ",
Total alkaloid has only been extracted from plants using the heavy method saltoutd again of the molten alkali of acid in it, but does not carry out the purifying of monomer, unknown
Impurity is more, is unfavorable for further producing；Such as application No. is the Chinese patent application of CN102247440A, " one kind is mentioned from herba fibraureae recisae
The method for taking fibrauretin coarse alkali " is impregnated using sulfuric acid, decompression is extracted, the heavy method saltoutd of alkali has to lower bar of purity
Horse spit of fland crude product, and the content of palmatine is relatively low, impurity is on the high side, can not directly use；Such as application No. is in CN104610252A
State's patent application " the extraction preparation method of palmatin hydrochloride and its application in preparation prevention and treatment colon cancer drug ",
The palmatin hydrochloride product purity for extracting preparation is also merely greater than 85%；Such as application No. is the Chinese patent applications of CN101058575A
" the Hydrolysis kinetics method and its application of palmatin hydrochloride ", although content can be obtained up to 99% palmatin hydrochloride, it must
Repeated multiple times acid processing and alkali process must be carried out, complex steps, energy consumption is big, is not suitable for large-scale production；Such as application No. is
The Chinese patent application " method of monomeric compound is isolated and purified from Chinese Drug Rhizomes of Coptis " of CN102285982, it is inverse using high speed
Flow chromatography purifies to obtain the palmatine of 98% or more purity, but used method is higher to technology, equipment requirement, is not suitable for
Industrialized production.
In addition, generally using a variety of organic solvents and synthesis using the method that chemical synthesis, biotechnology prepare palmatine
Raw material generates chemical impurity and residual in the synthesis process, and there are biggish potential risks.Such as application No. is CN103980271A
Chinese patent application " preparation method of fibrauretine and the like ", application No. is the Chinese patent applications of CN103408544A
" a kind of preparation method of fibrauretine ", application No. is a kind of Chinese patent application of CN102040604 " synthesis sides of fibrauretine
Method ", application No. is the Chinese patent application of CN101804054A " purposes, preparation and the preparation of synthetic palmatine ", application No. is
The Chinese patent application " new preparation process of palmatine " of CN102875545A, application No. is the Chinese patents of CN104561179A
Application " a kind of microbial secondary metabolite palmatin hydrochloride and its application " etc., method employed in these patents generally makes
With a variety of organic solvents and synthesis material, cost of manufacture is higher, generates chemical impurity and residual in the synthesis process, there are larger
Potential risk.
Due to complicated component in Tinospora sinensis, mentioned in extraction using different Extraction solvents and extracting mode is extracted
Take physical property matter that there is larger difference.Extraction work according to document report in the prior art, about Tinospora sinensis chemical component
Skill, such as: appoint gorgeous, Tang Qianrui, the chemical component of hardwood Tinospora sinensis can study [J] research and development of natural products,
2008,20:278-279,282, it is extracted using 95% alcohol reflux in document, then with silica gel column layer color after being extracted with ethyl acetate
Spectrum separation, but palmatine, false palmatine are not obtained therefrom；Han Dan caulis sinomenii extraction and preparation technique and the control of Herba Lycopodii quality
The Beijing technique study [D] processed: Beijing University of Chinese Medicine, 2014 are extracted using -70% alcohol reflux of hydrochloric acid and combine neutral alumina
Aluminium column chromatography carried out the enrichment of alkaloid, but and not up to monomer isolate and purify.
Palmatine, false palmatine belong to quaternary ammonium Alkaloid, should be soluble in acid flux material, and existing document report is about from medicine
It isolates and purifies to obtain the extracting method of palmatine Alkaloid in material, also mostly be extracted using acid flux material.Such as: Tian Jinfeng radix scrophulariae
Separate with the chemical component of the coptis and its Chongqing bioactivity research [D]: Southwest University, 2013 use 70% that hydrochloric acid is acidified
Alcohol dipping extracts cooperation high speed adverse current chromatogram separation, finds palmatine monomer in product；Fibrauretine extracts pure in journey shellfish herba fibraureae recisae
The change Sichuan technical study [D]: Chengdu University of Traditional Chinese Medicine, 2008 are extracted solvent using dilute sulfuric acid, are extracted with infusion process, in conjunction with
Macroreticular resin is enriched with alkaloid position, reuses adsorption chromatography and is refining to obtain fibrauretine.
Summary of the invention
The purpose of the present invention is to provide a kind of compound, the compound is to extract to be separated to from Tinospora sinensis
, it is named as Tinospora sinensis alkali, the preparation method that separation Tinospora sinensis alkali is extracted from Tinospora sinensis can be simultaneously
Extraction separation and purification palmatine, false palmatine high sterling from Tinospora sinensis, and purity is all up to 90%~95%.The system
Preparation Method reduces influence of the raw material impurity to extract property, and separation product property is more stable, extraction separation and purification stability
More preferably, while the comprehensive utilization of Tinospora sinensis herb resource is improved, there is very big practical significance, market efficiency and academic valence
Value.
To achieve the above object, the technical scheme adopted by the invention is as follows: a kind of compound, the structural formula of the compound
Are as follows:
.The compound is to extract to be separated to from Tinospora sinensis, is named as Tinospora sinensis alkali, Tinospora sinensis
Alkali is new native compound, and inventor is further purified to obtain the high sterling that its content reaches 90%-95%, and uses chromatography, light
The technologies such as spectrum and nuclear-magnetism.
As the preferred embodiment of compound of the present invention, the compound can be prepared into pharmaceutically acceptable
Salt, including hydrochloride, hydrobromate, hydriodate, hydrofluoride, sulfate, nitrate, phosphate, formates, acetate,
Propionate, oxalate, malonate, butyrate, lactate, mesylate, esilate, tosilate, maleic acid
Salt, benzoate, succinate, picrate, tartrate, citrate, fumarate；Preferably hydrochloride.
In addition, the present invention provides a kind of preparation method that separation Tinospora sinensis alkali is extracted from Tinospora sinensis, it is described
Method the following steps are included:
(1) Tinospora sinensis medicinal raw material is crushed；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, Plus acidic alcohol reflux extracts three
Secondary, the volume for the acidic ethanol being added every time is 5~15 times of amounts of Tinospora sinensis volume, is heated to reflux every time 2~5 hours；
Acidic ethanol extracting solution is filtered and merged, recycles, be concentrated into small size, NaOH solution is added and adjusts pH to neutrality, continues to be concentrated
It to paste, is dried under reduced pressure, obtains Tinospora sinensis extract；
(3) it isolates and purifies Tinospora sinensis alkali: Tinospora sinensis extract being completely dissolved using organic solvent, is then adopted
Sample method is mixed with solution dry method, Tinospora sinensis extract is uniformly admixed in sample chromatographic silica gel, it is dry, it loads on separation and uses
Chromatographic silica gel post bed top is successively mixed with low polar organic solvent, low polar organic solvent with highly polar organic solvent
Solvent, the elution of highly polar organic solvent, collecting eluent by TLC test, it is higher will to contain Tinospora sinensis alkali number
And the few fraction of impurity merges, and is concentrated, and it is dry, it carries out recrystallization processing, filtering for crystallizing and washes away residual impurity, after measured, obtain
90%~95% Tinospora sinensis alkali sterling is up to purity of the present invention.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, Tinospora sinensis is the Tinospora of Menispermaceae Tinospora plant Tinospora sinensis in the step (1)
The drying rattan of sinensis (Lour.) Merr..
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, middle Tinospora sinensis medicinal raw material to the partial size that crushes of the step (1) is 50~200 mesh.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the pH of the acidic ethanol in the step (2) are 2~4, and the mass percentage of ethyl alcohol is 40%~90%.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the acid solution in acidic ethanol in the step (2) are inorganic acid or organic acid, inorganic acid include hydrochloric acid, sulfuric acid,
Nitric acid, phosphoric acid, organic acid include formic acid, acetic acid, propionic acid, benzoic acid, salicylic acid, tartaric acid.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
The organic solvent of mode, the middle dissolution Tinospora sinensis extract of the step (3) includes ethyl acetate, methanol, dehydrated alcohol.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the production method that chromatographic silica gel post bed is used in separation in the step (3) are as follows: take have been placed in 105 DEG C of thermostatic driers plus
The column chromatography silica gel of thermal activation 0.5~1 hour and clean glass chromatography column is done, using low polar organic solvent wet process upper prop, is used in combination
Punching press obtains separation chromatographic silica gel post bed to smooth, fine and close, consolidation to low polar organic solvent repeatedly.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the partial size of column chromatography silica gel used in the step (3) are 80~200 mesh.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the low polar organic solvent in the step (3) includes petroleum ether, chloroform, methylene chloride or benzene, highly polar organic solvent
For methanol or dehydrated alcohol, mixed organic solvents are the mixed solvent of low polar organic solvent and highly polar organic solvent.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, in the step (3) when elution by the way of gradient elution, the percent by volume of gradient elution agent is that low polarity is organic
Solvent: highly polar organic solvent=100:0~0:100.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the quality of chromatographic silica gel used in the step (3) are 1~1.5 times of Tinospora sinensis extract quality, separation
With 5~15 times that chromatographic silica gel additional amount is Tinospora sinensis extract quality.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, thin layer chromatography in the step (3) are as follows: lamellae: -0.5% sodium carboxymethylcellulose of silica G, expanding body
System are as follows: ethyl acetate and methanol mixed solution, wherein the volume ratio of ethyl acetate and methanol is 4:1~8:3, point sample: dotted point
Sample is set and is saturated 15min in expansion cylinder, starts to spread out, and exhibition develops the color away from being 8-12 centimetres: prior to being inspected under ultraviolet light 365nm, after
Improvement bismuth potassium iodide test solution is given in spray, and Tinospora sinensis alkali will appear clear orange red spot under room temperature.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the solvent of solvent fraction is methanol or acetonitrile in the step (3).
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, mobile phase is the methanol aqueous solution of 5%-80% or the acetonitrile solution of 5%-80% in the step (3).
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, recrystallization method in the step (3) are as follows: take the target fraction being concentrated and dried, the mixed of methanol-water few as far as possible is added
Solution is closed, solute is heated to and is completely dissolved, opening stands thoroughly cooling, is precipitated to crystal, filters out crystal, and with above-mentioned on a small quantity
Methanol aqueous solution washs plane of crystal, after draining, takes out crystal, is placed in do and collects and dry in clean glassware or dry, claims
Amount, obtains the Tinospora sinensis alkali sterling.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the method for the middle measurement Tinospora sinensis alkali content of the step (3) are as follows: contained using HPLC method measurement Tinospora sinensis alkali
Amount, chromatographic condition are as follows: C18 column, 4.6 × 250mm, flow velocity: 0.6-1.5ml/min；Sample volume: 10-20 μ l；Area normalization method
It is quantitative；System condition: mobile phase: acetonitrile: 0.10% potassium dihydrogen phosphate 0.65% octane sulfonate sodium solution (solution proportion 24:
76~20:80)；Detection wavelength: 345nm.
Preferred implementation as the preparation method of the present invention for extracting separation Tinospora sinensis alkali from Tinospora sinensis
Mode, the HPLC method measure the C18 column bed production method in Tinospora sinensis alkali content are as follows: take and do clean glass chromatography column, make
With methanol or acetonitrile wet method dress post, and punching press reuses 5% methanol aqueous solution or 5% acetonitrile to smooth, fine and close, consolidation repeatedly
Aqueous solution rinses, and balance profit column must be separated with chromatography C18 column bed.
The preparation method of the present invention that separation Tinospora sinensis alkali is extracted from Tinospora sinensis, preparation method letter
Just, the comprehensive study for Tinospora sinensis medicinal material provides further infrastructural support, improves Tinospora sinensis herb resource
Comprehensive utilization, provide production technology for the research of native compound Tinospora sinensis alkali and the production of chemical reference substance, have
Very big practical significance, market efficiency and learning value.
Separation palmatine, false palmatine sterling are extracted simultaneously in addition, the present invention provides a kind of from Tinospora sinensis
Preparation method, which is characterized in that the described method comprises the following steps:
(1) Tinospora sinensis medicinal raw material is crushed；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, Plus acidic alcohol reflux extracts three
Secondary, the volume for the acidic ethanol being added every time is 5~15 times of amounts of Tinospora sinensis volume, is heated to reflux every time 2~5 hours；
Acidic ethanol extracting solution is filtered and merged, recycles, be concentrated into small size, NaOH solution is added and adjusts pH to neutrality, continues to be concentrated
It to paste, is dried under reduced pressure, obtains Tinospora sinensis extract；
(3) palmatine, false palmatine are isolated and purified: being completely dissolved Tinospora sinensis extract using organic solvent, so
Sample method is mixed using solution dry method afterwards, Tinospora sinensis extract is uniformly admixed in sample chromatographic silica gel, it is dry, it loads on point
It is successively mixed with low polar organic solvent, low polar organic solvent and highly polar organic solvent from chromatographic silica gel post bed top
Organic solvent, the elution of highly polar organic solvent are closed, eluent is collected, by TLC test, palmatine, false palmatine will be contained
It measures higher and few impurity fraction to merge respectively, be concentrated, it is dry, it carries out recrystallization processing respectively, filtering for crystallizing and washes away remnants
Impurity obtains palmatine, vacation palmatine sterling that purity of the present invention is up to 90%~95% after measured.
As the preparation method of the present invention for extracting separation palmatine, false palmatine sterling simultaneously from Tinospora sinensis
Preferred embodiment, thin layer chromatography in the step (3) are as follows: lamellae: -0.5% carboxymethyl cellulose of silica G
Sodium, used expansion system are as follows: 1. toluene: isopropanol: ethyl acetate: methanol: dense ammonia (6:1.5:3:6:1)；2. ethyl acetate:
Methanol (3:1)；3. ethyl acetate: methanol (2:1) after mixing by solvent pours into expansion cylinder；Point sample: spot application,
It sets and is saturated 15min in expansion cylinder, start to spread out, colour developing: exhibition prior to inspecting under ultraviolet light 365nm, is sprayed afterwards away from being 8~12 centimetres
Improvement bismuth potassium iodide test solution is given, palmatine, false palmatine will appear clear orange red spot under room temperature.
The preparation method of extraction separation and purification palmatine, false palmatine of the present invention simultaneously from Tinospora sinensis, benefit
With acid alcohol extracting, after concentration is redissolved, directly chromatographed using column, eluate, which merges recrystallization, can obtain bar horse of high-purity
Spit of fland, false palmatine, purity are up to 90%~95%, and simple process, stabilization, favorable reproducibility, and can once obtain simultaneously bar
Two kinds of Ma Ting, false palmatine pure compounds, save the time, reduce consumption, to the comprehensive benefit of quickening Tinospora sinensis herb resource
There is positive effect with exploitation, to develop using palmatine, false palmatine as the production of the new drug development of raw material or chemical reference substance
Preparation provide new industrialized manufacturing technique method, have a good application prospect.
Detailed description of the invention
Fig. 1 is the chemical structural drawing of compound of the present invention；
Fig. 2 is the carbon-13 nmr spectra of compound of the present invention；
Fig. 3 is the nuclear magnetic resonance spectroscopy of compound of the present invention.
Specific embodiment
It is right below in conjunction with the accompanying drawings and the specific embodiments to better illustrate the object, technical solutions and advantages of the present invention
The present invention is described further.
Embodiment 1
A kind of embodiment of the preparation method of the present invention that separation Tinospora sinensis alkali is extracted from Tinospora sinensis, institute
State preparation method the following steps are included:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 50 mesh；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, be added and be acidified to pH=using hydrochloric acid
2 85% ethyl alcohol about 1L, is heated to reflux 3 times, merges alcohol extract, and NaOH solution is added and adjusts pH=7 and is concentrated into paste, molten
Agent recycling；60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 19.7g；
(3) isolate and purify Tinospora sinensis alkali: appropriate ethyl acetate is added into Tinospora sinensis extract to be made to dissolve, molten
Liquid is admixed in about 23g silica gel, water-bath drying, and loading on silicagel column, (glass column internal diameter is 5cm, and the long 55cm of shaft, wet process filling is
Activated silica gel about 220g) top, use methylene chloride: methanol (100:0-0:100) gradient elution, every 150ml make a fraction
Amount, tests through thin-layer chromatography, the fraction that alkali number containing Tinospora sinensis is higher and impurity is few is merged respectively, is concentrated to dryness, and uses
Methanol few as far as possible makes to redissolve, and in C18 column, (glass silica gel column internal diameter is 3cm, and the long 55cm of shaft, wet process filling C18 is about for dropwise addition
100g), it is eluted using 5%-80% aqueous methanol gradient, and recycling design, is tested through thin-layer chromatography, Tinospora sinensis will be contained
The fraction that alkali number is higher and impurity is few merges respectively, is concentrated to dryness, and recrystallization processing obtains Tinospora sinensis alkali of the present invention,
Its purity is 91.8% after measured.
Present inventor combines pertinent literature to report with technologies such as chromatography, spectrum and nuclear-magnetisms, it was demonstrated that this has been determined
The structure of compound described in embodiment, as shown in Fig. 1.The carbon-13 nmr spectra of compound described in the present embodiment such as 2 institute of attached drawing
Show, the nuclear magnetic resonance spectroscopy of compound described in the present embodiment is as shown in Figure 3.
Embodiment 2
A kind of embodiment of the preparation method of the present invention that separation Tinospora sinensis alkali is extracted from Tinospora sinensis, institute
State preparation method the following steps are included:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 200 mesh；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, addition makes to be acidified with acetic acid to pH=
4 40% ethyl alcohol about 1L, is heated to reflux 3 times, merges alcohol extract, and NaOH solution is added and adjusts pH=7 and is concentrated into paste, molten
Agent recycling, 60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 20.4g；
(3) isolate and purify Tinospora sinensis alkali: appropriate ethyl acetate is added into Tinospora sinensis extract to be made to dissolve, molten
Liquid is admixed in about 22g silica gel, water-bath drying, and loading on silicagel column, (glass column internal diameter is 5cm, and the long 55cm of shaft, wet process filling is
Activated silica gel about 225g) top, use methylene chloride: methanol (100:0-0:100) gradient elution, every 150ml make a fraction
Amount, tests through thin-layer chromatography, the fraction that alkali number containing Tinospora sinensis is higher and impurity is few is merged respectively, is concentrated to dryness, and uses
Methanol few as far as possible makes to redissolve, and in C18 column, (glass silica gel column internal diameter is 3cm, and the long 55cm of shaft, wet process filling C18 is about for dropwise addition
100g), it is eluted using 5%-80% aqueous methanol gradient, and recycling design, is tested through thin-layer chromatography, Tinospora sinensis will be contained
The fraction that alkali number is higher and impurity is few merges respectively, is concentrated to dryness, and recrystallization processing obtains Tinospora sinensis alkali of the present invention,
Its purity is 92.1% after measured.
Present inventor combines pertinent literature to report with technologies such as chromatography, spectrum and nuclear-magnetisms, it was demonstrated that this has been determined
The structure of compound described in embodiment, as shown in Fig. 1.The carbon-13 nmr spectra of compound described in the present embodiment such as 2 institute of attached drawing
Show, the nuclear magnetic resonance spectroscopy of compound described in the present embodiment is as shown in Figure 3.
Embodiment 3
A kind of embodiment of the preparation method of the present invention that separation Tinospora sinensis alkali is extracted from Tinospora sinensis, institute
State preparation method the following steps are included:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 150 mesh；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, be added and be acidified to pH=using sulfuric acid
3 80% ethyl alcohol about 1L, is heated to reflux 3 times, merges alcohol extract, and NaOH solution is added and adjusts pH=7 and is concentrated into paste, molten
Agent recycling, 60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 20.9g；
(3) isolate and purify Tinospora sinensis alkali: appropriate ethyl acetate is added into Tinospora sinensis extract to be made to dissolve, molten
Liquid is admixed in about 24g silica gel, water-bath drying, and loading on silicagel column, (glass column internal diameter is 5cm, and the long 55cm of shaft, wet process filling is
Activated silica gel about 220g) top, use methylene chloride: methanol (100:0-0:100) gradient elution, every 150ml make a fraction
Amount, tests through thin-layer chromatography, the fraction that alkali number containing Tinospora sinensis is higher and impurity is few is merged respectively, is concentrated to dryness, and uses
Methanol few as far as possible makes to redissolve, and in C18 column, (glass silica gel column internal diameter is 3cm, and the long 55cm of shaft, wet process filling C18 is about for dropwise addition
100g), it is eluted using 5%-80% aqueous methanol gradient, and recycling design, is tested through thin-layer chromatography, Tinospora sinensis will be contained
The fraction that alkali number is higher and impurity is few merges respectively, is concentrated to dryness, and recycling design, and recrystallization processing obtains in of the present invention
Magnificent tinosporine, its purity is 92.5% after measured.
Present inventor combines pertinent literature to report with technologies such as chromatography, spectrum and nuclear-magnetisms, it was demonstrated that this has been determined
The structure of compound described in embodiment, as shown in Fig. 1.The carbon-13 nmr spectra of compound described in the present embodiment such as 2 institute of attached drawing
Show, the nuclear magnetic resonance spectroscopy of compound described in the present embodiment is as shown in Figure 3.
Embodiment 4
A kind of reality of the preparation method of the present invention for extracting separation palmatine, false palmatine simultaneously from Tinospora sinensis
Example is applied, the preparation method comprises the following steps:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 50 mesh；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, be added and be acidified to pH=using formic acid
3 85% ethyl alcohol about 1L, is heated to reflux 3 times, merges alcohol extract, and NaOH solution is added and adjusts PH=7 and is concentrated into paste, molten
Agent recycling；60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 19.5g；
(3) palmatine, false palmatine are isolated and purified: be added into Tinospora sinensis extract appropriate ethyl acetate make it is molten
Solution, solution admixes in about 22g silica gel, water-bath drying, load on silicagel column (glass silica gel column internal diameter is 5cm, the long 55cm of shaft,
Wet process fills activated silica gel about 220g) top, use methylene chloride: methanol (100:0~0:100) gradient elution, every 150ml
Make a fraction amount, tested through thin-layer chromatography, will be merged respectively containing palmatine, the fraction that false palmatine amount is higher and impurity is few,
It is concentrated into dry doubling recycling design, recrystallization processing obtains palmatine of the present invention, false palmatine, and palmatine purity is after measured
94.5%, false palmatine purity is 93.8%.
Embodiment 5
A kind of reality of the preparation method of the present invention for extracting separation palmatine, false palmatine simultaneously from Tinospora sinensis
Example is applied, the preparation method comprises the following steps:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 50 mesh；
(2) it prepares Tinospora sinensis extract: the Tinospora sinensis crushed being taken to be added using phosphoric acid to pH=3
85% ethyl alcohol about 1L, be heated to reflux 3 times, merge alcohol extract, be added NaOH solution adjust pH=7 simultaneously be concentrated into paste, it is molten
Agent recycling；60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 20.2g；
(3) palmatine, false palmatine are isolated and purified: be added into Tinospora sinensis extract appropriate ethyl acetate make it is molten
Solution, solution admixes in about 23g silica gel, water-bath drying, load on silicagel column (glass silica gel column internal diameter is 5cm, the long 55cm of shaft,
Wet process fills activated silica gel about 230g) top, use methylene chloride: methanol (100:0~0:100) gradient elution, every 150ml
Make a fraction amount, tested through thin-layer chromatography, will be merged respectively containing palmatine, the fraction that false palmatine amount is higher and impurity is few,
It is concentrated into dry doubling recycling design, recrystallization processing obtains palmatine of the present invention, false palmatine, and palmatine purity is after measured
94.6%, false palmatine purity is 93.8%.
Embodiment 6
A kind of reality of the preparation method of the present invention for extracting separation palmatine, false palmatine simultaneously from Tinospora sinensis
Example is applied, the preparation method comprises the following steps:
(1) it crushes Tinospora sinensis medicinal raw material: taking Tinospora sinensis medicinal material 100g, being crushed to partial size is 50 mesh；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, be added and be acidified to pH=using nitric acid
3 85% ethyl alcohol about 1L, is heated to reflux 3 times, merges alcohol extract, and NaOH solution is added and adjusts pH=7 and is concentrated into paste, molten
Agent recycling；60 DEG C are dried under reduced pressure to after doing, and obtain Tinospora sinensis extract about 20.4g；
(3) palmatine, false palmatine are isolated and purified: be added into Tinospora sinensis extract appropriate ethyl acetate make it is molten
Solution, solution admixes in about 23g silica gel, water-bath drying, load on silicagel column (glass silica gel column internal diameter is 5cm, the long 55cm of shaft,
Wet process fills activated silica gel about 220g) top, use petroleum ether: methanol (100:0~0:100) gradient elution, every 150ml make
One fraction amount, is tested through thin-layer chromatography, will be merged respectively containing palmatine, the fraction that false palmatine amount is higher and impurity is few, dense
It is reduced to dry doubling recycling design, recrystallization processing obtains palmatine of the present invention, false palmatine, and palmatine purity is after measured
94.7%, false palmatine purity is 94.0%.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (9)
1. a kind of preparation method of compound, which is characterized in that the described method comprises the following steps:
(1) Tinospora sinensis medicinal raw material is crushed；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, Plus acidic alcohol reflux extracts three times, often
The volume of the acidic ethanol of secondary addition is 5~15 times of amounts of Tinospora sinensis volume, is heated to reflux every time 2~5 hours；Filtering is simultaneously
Merge acidic ethanol extracting solution, recycle, be concentrated into small size, NaOH solution is added and adjusts pH to neutrality, continues to be concentrated into thick paste
Shape is dried under reduced pressure, and obtains Tinospora sinensis extract；
(3) it isolates and purifies: being completely dissolved Tinospora sinensis extract using organic solvent, sample is then mixed using solution dry method
Method uniformly admixes Tinospora sinensis extract in sample chromatographic silica gel, dry, loads on separation chromatographic silica gel post bed top
End, successively with low polar organic solvent, low polar organic solvent and highly polar organic solvent mixed organic solvents, highly polar have
Solvent elution, collects eluent, and by TLC test, the fraction that alkali number containing Tinospora sinensis is higher and impurity is few is closed
And be concentrated, it is dry, it carries out recrystallization processing, filtering for crystallizing and washes away residual impurity, after measured, obtain the purity and be up to
90%~95% compound；
The structural formula of gained compound are as follows:
The pH of acidic ethanol in the step (2) is 2~4, and the mass percentage of ethyl alcohol is 40%~90%；
Low polar organic solvent in the step (3) is selected from petroleum ether, chloroform, methylene chloride or benzene, highly polar organic solvent
For methanol or dehydrated alcohol, mixed organic solvents are the mixed solvent of low polar organic solvent and highly polar organic solvent；
The production method that chromatographic silica gel post bed is used in separation in the step (3) are as follows: take to have been placed in 105 DEG C of thermostatic driers and heat
The column chromatography silica gel of activation 0.5~1 hour and clean glass chromatography column is done, using low polar organic solvent wet process upper prop, and with low
Punching press obtains separation chromatographic silica gel post bed to smooth, fine and close, consolidation to polar organic solvent repeatedly.
2. as described in claim 1 from the preparation method of compound, which is characterized in that Tinospora sinensis is in the step (1)
The drying rattan of Tinospora sinensis；
It is 50~200 mesh that Tinospora sinensis medicinal raw material to partial size is crushed in the step (1)；
The acid solution in acidic ethanol in the step (2) is inorganic acid or organic acid, inorganic acid be selected from hydrochloric acid, sulfuric acid,
Nitric acid, phosphoric acid, organic acid are selected from formic acid, acetic acid, propionic acid, benzoic acid, salicylic acid, tartaric acid；
The organic solvent of dissolution Tinospora sinensis extract is selected from ethyl acetate, methanol, dehydrated alcohol in the step (3).
3. the preparation method of compound as described in claim 1, which is characterized in that column layer used in the step (3)
The partial size for analysing silica gel is 80~200 mesh.
4. the preparation method of compound as claimed in claim 1 or 3, which is characterized in that using ladder when elution in the step (3)
The mode of elution is spent, the percent by volume of gradient elution agent is low polar organic solvent: highly polar organic solvent=100:0~0:
100。
5. the preparation method of compound as described in claim 1, which is characterized in that chromatography silicon used in the step (3)
The quality of glue is 1~1.5 times of Tinospora sinensis extract quality, and separation is that Tinospora sinensis extracts with chromatographic silica gel additional amount
5~15 times of amount of substance.
6. the preparation method of compound as described in claim 1, which is characterized in that thin-layer chromatographic analysis side in the step (3)
Method are as follows: lamellae: system is unfolded in -0.5% sodium carboxymethylcellulose of silica G are as follows: ethyl acetate and methanol mixed solution, wherein
The volume ratio of ethyl acetate and methanol is 4:1~8:3, and point sample: spot application is set and is saturated 15min in expansion cylinder, starts to spread out,
Exhibition is away from being 8-12 centimetres, and colour developing: prior to inspecting under ultraviolet light 365nm, improvement bismuth potassium iodide test solution is given in rear spray, and China is green under room temperature
Taurine will appear clear orange red spot.
7. the preparation method of compound as claimed in claim 6, which is characterized in that recrystallization method in the step (3) are as follows:
The target fraction being concentrated and dried is taken, the mixed liquor of methanol-water few as far as possible is added, is heated to solute and is completely dissolved, opening is stood
It is thoroughly cooling, it is precipitated to crystal, filters out crystal, and wash plane of crystal with a small amount of above-mentioned methanol aqueous solution, after draining, take out
Crystal is placed in do and collects and dry in clean glassware or dry, and weighs, obtains the Tinospora sinensis alkali sterling.
8. a kind of preparation method for extracting separation palmatine, false palmatine sterling simultaneously from Tinospora sinensis, which is characterized in that
It the described method comprises the following steps:
(1) Tinospora sinensis medicinal raw material is crushed；
(2) it prepares Tinospora sinensis extract: taking the Tinospora sinensis crushed, Plus acidic alcohol reflux extracts three times, often
The volume of the acidic ethanol of secondary addition is 5~15 times of amounts of Tinospora sinensis volume, is heated to reflux every time 2~5 hours；Filtering is simultaneously
Merge acidic ethanol extracting solution, recycle, be concentrated into small size, NaOH solution is added and adjusts pH to neutrality, continues to be concentrated into thick paste
Shape is dried under reduced pressure, and obtains Tinospora sinensis extract；
(3) palmatine, false palmatine are isolated and purified: Tinospora sinensis extract being completely dissolved using organic solvent, is then adopted
Sample method is mixed with solution dry method, Tinospora sinensis extract is uniformly admixed in sample chromatographic silica gel, it is dry, it loads on separation and uses
Chromatographic silica gel post bed top is successively mixed with low polar organic solvent, low polar organic solvent with highly polar organic solvent
Solvent, the elution of highly polar organic solvent, collect eluent, by TLC test, will contain palmatine, false palmatine amount compared with
High and few impurity fraction merges respectively, is concentrated, dry, carries out recrystallization processing respectively, filtering for crystallizing and washes away residual impurity,
After measured, palmatine, vacation palmatine sterling that the purity is up to 90%~95% are respectively obtained；
The pH of acidic ethanol in the step (2) is 2~4, and the mass percentage of ethyl alcohol is 40%~90%；
Low polar organic solvent in the step (3) is selected from petroleum ether, chloroform, methylene chloride or benzene, highly polar organic solvent
For methanol or dehydrated alcohol, mixed organic solvents are the mixed solvent of low polar organic solvent and highly polar organic solvent；
The production method that chromatographic silica gel post bed is used in separation in the step (3) are as follows: take to have been placed in 105 DEG C of thermostatic driers and heat
The column chromatography silica gel of activation 0.5~1 hour and clean glass chromatography column is done, using low polar organic solvent wet process upper prop, and with low
Punching press obtains separation chromatographic silica gel post bed to smooth, fine and close, consolidation to polar organic solvent repeatedly.
9. extract the preparation method of separation palmatine, false palmatine sterling simultaneously from Tinospora sinensis as claimed in claim 8,
It is characterized in that, thin layer chromatography in the step (3) are as follows: lamellae: -0.5% sodium carboxymethylcellulose of silica G,
Used expansion system are as follows: 1. toluene: isopropanol: ethyl acetate: methanol: dense ammonia=6:1.5:3:6:1；2. ethyl acetate: first
Alcohol=3:1；3. ethyl acetate: methanol=2:1 after mixing by solvent pours into expansion cylinder；Point sample: spot application is set
It is saturated 15min in expansion cylinder, starts to spread out, exhibition is away from being 8~12 centimetres, and colour developing: prior to inspecting under ultraviolet light 365nm, rear spray is given
Bismuth potassium iodide test solution is improved, palmatine, false palmatine will appear clear orange red spot under room temperature.
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| CN104561179A (en) * | 2014-11-13 | 2015-04-29 | 云南农业大学 | Microbial secondary metabolite palmatine hydrochloride and application thereof |
| CN104610252A (en) * | 2015-01-07 | 2015-05-13 | 中国药科大学 | Extraction preparation method of palmatine chloride and application thereof to preparation of medicine for preventing and treating colorectal cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104561179A (en) * | 2014-11-13 | 2015-04-29 | 云南农业大学 | Microbial secondary metabolite palmatine hydrochloride and application thereof |
| CN104610252A (en) * | 2015-01-07 | 2015-05-13 | 中国药科大学 | Extraction preparation method of palmatine chloride and application thereof to preparation of medicine for preventing and treating colorectal cancer |
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