CN103301177B - The preparation of Mahonia dolichostylis total alkaloids and method of quality control thereof - Google Patents
The preparation of Mahonia dolichostylis total alkaloids and method of quality control thereof Download PDFInfo
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Abstract
The invention discloses a kind of from plant Mahonia dolichostylis extract total alkaloids and method of quality control thereof, it is that Mahonia dolichostylis is dried the coarse powder of stem is size-reduced, alcohol hydrochloric acid reflux, extract, concentrate, add water suspendible, macroporous adsorbent resin column chromatography eluting (ethanol water system), concentrate, vacuum drying, obtain Mahonia dolichostylis total alkaloids, preparation method is reasonable, stable, with short production cycle, it is suitable for industrialized production, present invention thin layer chromatography qualitative identification total alkaloids, use high effective liquid chromatography for measuring Mahonia dolichostylis total alkaloid content, method is accurate, stable, simple to operate.The toxicity of the Mahonia dolichostylis total alkaloids extract that the present invention prepares is little, has the pharmacological actions such as antiinflammatory, analgesia, antidiarrheal and anti-icterohepatitis.
Description
Technical field
The present invention relates to total alkaloids extract preparation field, the preparation of a kind of Mahonia dolichostylis total alkaloids
And method of quality control.
Background technology
Mahonia dolichostylis (Mahonia duclouxiana Gagnep.) is the big merit of Berberidaceae Berberidaceae ten
Labor belongs to a kind of Mahonia plant, and resource is the abundantest, and its nature and flavor bitter cold enters liver, stomach, large intestine channel, has heat-clearing and toxic substances removing
Reduce phlegm the effectiveness of dampness removing, cure mainly pulmonary tuberculosis, hectic fever, cough, spit blood, weakness of the waist and knees, dizziness and tinnitus, insomnia；Intestinal class diarrhoea, jaundice
Type hepatitis, conjunctival congestion and swelling pain.Modern scientific research finds, rich in protoberberine active component, He Kai in Mahonia fortunei (Lindl.) Fedde platymiscium
Families etc. are separated from Mahonia fortunei (Lindl.) Fedde obtains 5 compounds, is identified respectively as Jatrorrhizine chloride palmatine hydrochloride hydrochloric acid Radix Berberidis Amurensis
Alkali isotetrandrine and cupreol ([autograph] Mahonia dolichostylis chemical constitution study [author] He Kaijia, Liu Buming, Lu
Wen Jie, Guangxi traditional Chinese medicine academy [periodical name] West China pharmaceutical journal, 2008,23 (2): 72-174), and differentiate and thin layer through chemistry
Chromatographic identification, it was demonstrated that its alkaloid component mainly has Jatrorrhizine chloride palmatine hydrochloride berberine hydrochloride ([autograph] long column ten
Discriminating [author] Liu Xiang of big contribution, He Kaijia, Cai Quanling, etc., Guangxi traditional Chinese medicine academy [periodical name] Colleges Of Traditional Chinese Medicine Of Guangxi
Journal, 2008,11 (1): 48-49).
Mahonia dolichostylis at Guangxi aboundresources, but, due to for a long time about Mahonia dolichostylis research relatively
Few, the exploitation to Mahonia dolichostylis at present relatively lag behind, and mainly direct crude drug is prepared decoction and taken, this biography
System administrated method takes the shortcomings such as inconvenience, curative effect is unstable, quality is uncontrollable.Along with the development of medicine science and technology, refined
The efficient characteristic of natural plant extracts low toxicity more and more favored by people, modern scientific research also finds long column ten
Big contribution extract has the pharmacological actions such as antiinflammatory, analgesia, antidiarrheal, protection icterohepatitis, and therefore a kind of long column ten of research and development is big
The preparation of contribution total alkaloids extract and method of quality control thereof are a need for, and are beneficial to the medicine that exploitation Mahonia dolichostylis is new
By approach, and provide the foundation of science for making full use of and improving its using value of herb resource.
Summary of the invention
It is an object of the invention to, it is provided that one has the pharmacological actions such as antiinflammatory, analgesia, antidiarrheal, protection icterohepatitis
The preparation method of Mahonia dolichostylis total alkaloids, and the method for quality control of Mahonia dolichostylis total alkaloids.
The present invention is achieved in that
The preparation method of Mahonia dolichostylis total alkaloids of the present invention, comprises the following steps:
1, ethanol-hydrogen chloride heating and refluxing extraction:
1. take Mahonia dolichostylis and be dried the coarse powder of stem, add 5-15 times amount 50%-90% volumetric concentration by weight ratio
Ethanol-hydrogen chloride (100:0.5)；
2. reflux, extract, 1-4 time at temperature is 60-90 DEG C, each 0.5-2.5 hour, filters, merging filtrate；
3. filtrate recycling ethanol, and it is concentrated into without ethanol taste, it is 0.1-0.2 g crude drug amount/mL that dilute obtains concentration
Mahonia dolichostylis extracting solution, treat that upper prop is used.
2, Flavonoids by Macroporous Adsorption Resin is isolated and purified:
1. AB-8 or the D101 macroporous adsorbent resin of low pole is selected；
2. absorption: upper prop Mahonia dolichostylis extract concentration is 0.1-0.2 g crude drug amount/mL, and upper prop long column ten is big
Contribution extracting liquid volume is fill macroporous adsorbent resin volume 5-10 times, loading flow velocity 1-3 BV/h[note: in BV/h represents post
Unit interval (h) flows through the average liquid measure of unit volume resin]；
3. eluting: be eluant with 10-50% ethanol；Eluting agent is 5-10 BV；Elution flow rate is 1-3 BV/h;
Collect 5-10 times of eluent, decompression recycling ethanol, be concentrated to dryness, 50-80 DEG C of vacuum drying, i.e. can get Mahonia dolichostylis
Total alkaloids extract.
The method of quality control of Mahonia dolichostylis total alkaloids of the present invention, comprises the following steps:
1, the indentification by TLC of total alkaloids:
Take Mahonia dolichostylis extract powder 10 mg, put in 10 ml volumetric flasks, add methanol to scale, shake up, as
Need testing solution.Take Mahonia dolichostylis medicinal powder 1 g, add methanol 15 ml, supersound process (power 160W, frequency 40kHz)
15 minutes, filter, as control medicinal material solution.Separately take berberine hydrochloride reference substance, Jatrorrhizine chloride reference substance, palmatine hydrochloride
Reference substance, adds methanol and makes every 1 ml mixed solution containing 0.5 mg, as reference substance solution.Test according to thin layer chromatography, inhale
Take each 2 μ l of above two solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-methanol-isopropanol-dense
Ammonia solution (volume ratio: 6:3:1.5:1.5:0.5) is developing solvent, puts in the vapour-saturated expansion cylinder of ammonia, launches, and takes out, dries,
Put and inspect under uviol lamp (365 nm).In test sample chromatograph, with control medicinal material, the position that reference substance is identical, aobvious same color
Speckle.
2, the assay (algoscopy is commented in a survey more) of total alkaloids
1. chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With acetonitrile-
0.05 mol/L potassium phosphate buffer (volume ratio: 3:7) (phosphoric acid tune pH value to 3.0), for flowing phase；Detection wavelength is
265 nm。
2. the preparation of need testing solution: take Mahonia dolichostylis extract about 40 mg, accurately weighed, put tool plug taper
In Ping, accurate addition water 50 ml, weighed weight, supersound process (power 160W, frequency 40kHz) 5 minutes, let cool to room temperature, then
Weighed weight, supplies the weight of less loss with water, filters, accurate absorption subsequent filtrate 2 ml, puts in 10 ml volumetric flasks, adds water to carve
Degree, shakes up, and filters, takes subsequent filtrate, to obtain final product.
3. the preparation of reference substance solution: accurately weighed berberine hydrochloride reference substance is appropriate, add acetonitrile-water (volume ratio: 3:
7) solution makes every 1 ml solution containing 20 μ g, to obtain final product.
4. algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid,
Measure, with the peak area of berberine hydrochloride reference substance for comparison, calculate berberine hydrochloride, Jatrorrhizine chloride and hydrochloric acid bar horse respectively
The content in spit of fland, to obtain final product.Mahonia dolichostylis total alkaloid content in terms of berberine hydrochloride, hydrochloric berberine (C20H19ClNO4),
Jatrorrhizine chloride (C20H20ClNO4) and palmatine hydrochloride (C21H22ClNO4) total amount must not be less than 50.0%.
5. result: use preparation and the method for quality control of the present invention, empirical tests is tested, the Mahonia dolichostylis of preparation
Total alkaloid content in terms of berberine hydrochloride, the total amount of hydrochloric berberine, Jatrorrhizine chloride and palmatine hydrochloride up to 50% with
On.
Described Mahonia dolichostylis total alkaloids antiinflammatory, analgesia, antidiarrheal, protection icterohepatitis medicine in.
Compared with prior art, the substantive distinguishing features that the present invention highlights with significantly progress is:
1, the present invention prepares the Mahonia dolichostylis total alkaloids of content more than 50% first from Mahonia dolichostylis and carries
Taking thing, wherein the stable content of three kinds of main alkaloid is in more excellent level, and process conditions reasonably stability is feasible.
2, this extraction process is simple, consumes energy low, and in technical process, main employing ethanol-hydrogen chloride extracts, through the biggest
Macroporous adsorbent resin column separating purification i.e. can get the Mahonia dolichostylis total alkaloids extract that content is higher, makes in raw material harmful
The less residue of solvent, meets country to food and the requirement of medicine.
3, the present invention use thin layer chromatography differentiate total alkaloids, content assaying method use high performance liquid chromatography, one
Survey the formula of discussing and select model workers more and 3 kinds of alkaloids are carried out synchronism detection, Jatrorrhizine chloride, the comparison of palmatine hydrochloride assay can not be provided
Product, the reference substance that only need to differentiate and position.This method is simple, accurate, feasible, it is possible to resolve in actual application, reference substance lacks
Accuse, with multi objective, the difficulty that cost is high, preferably apply to Chinese medicine multicomponent quality control.
4, by Mahonia dolichostylis total alkaloids is studied, a kind of Mahonia dolichostylis total alkaloids is established
Preparation and the further exploitation of method of quality control, beneficially Mahonia dolichostylis Related product, and wide for exploitation
Western special medicinal material, exploitation has high-tech, a high value-added product, improves the market competitiveness, it will produce potential and can not
The social benefit estimated and economic benefit.
Accompanying drawing explanation
Fig. 1 is the preparation technology flow chart of Mahonia dolichostylis total alkaloids.
Fig. 2 is mixing reference substance solution high-efficient liquid phase chromatogram.
Fig. 3 is Mahonia dolichostylis total alkaloids high-efficient liquid phase chromatogram.
As can be seen from Figure 1, the preparation method of described Mahonia dolichostylis total alkaloids comprises the following steps:
Mahonia dolichostylis is dried stem-pulverizing-ethanol-hydrogen chloride heating and refluxing extraction-concentration-recovery ethanol-crude extract
Add water suspendible-macroporous adsorbent resin column chromatography-alcohol-water eluting-concentration-vacuum drying, obtains Mahonia dolichostylis total alkaloids
Extract.
Find out from Fig. 2 and Fig. 3, the peak value of various compositions in high-efficient liquid phase chromatogram: 1-Jatrorrhizine chloride, 2-hydrochloric acid bar horse
Spit of fland, 3-berberine hydrochloride.
Detailed description of the invention
Embodiment one:
Take Mahonia dolichostylis and be ground into coarse powder 500 g, extract with the ethanol-hydrogen chloride (100:0.5) of 10 times amount 50%, in
80 DEG C of heating in water bath 3 times, each 2.5 hours, filter, and filtrate reduced in volume, without alcohol taste, adds water and is settled to 5000 ml(0.1 g
Medical material/ml), four layers of filtered through gauze, to clear liquor, filter, and cross AB-8 macroporous adsorptive resins (sample solution is 5 times of resin volumes),
Upper column flow rate is 1 BV/h.With 2 times of water washings, water lotion discards, and with the ethanol elution of 5 times 20%, elution flow rate is 1 BV/h.
Collecting eluent, eluent is evaporated to extractum, 60 DEG C of vacuum drying, i.e. obtains carrying of Mahonia dolichostylis total alkaloids
Taking thing, measurement result, the content of three kinds of main alkaloid of Mahonia dolichostylis is 59.40%.
Embodiment two:
Take Mahonia dolichostylis and be ground into coarse powder 1 kg, extract with the ethanol-hydrogen chloride (100:0.5) of 15 times amount 70%, in 80
DEG C heating in water bath 3 times, each 1 hour, filters, filtrate reduced in volume without alcohol taste, add water be settled to 6250 ml(0.16g medical materials/
Ml), four layers of filtered through gauze, to clear liquor, filter, and cross AB-8 macroporous adsorptive resins (sample solution is 6 times of resin volumes), upper prop
Flow velocity is 2 BV/h.With 2 times of water washings, water lotion discards, and with the ethanol elution of 10 times 10%, collects eluent, and elution flow rate is
2 BV/h.Eluent is evaporated to extractum, 60 DEG C of vacuum drying, i.e. obtains the extract of Mahonia dolichostylis total alkaloids,
Measurement result, the content of three kinds of main alkaloid of Mahonia dolichostylis is 57.17%.
Embodiment three:
Take Mahonia dolichostylis and be ground into coarse powder 1 kg, extract with the ethanol-hydrogen chloride (100:0.5) of 5 times amount 90%, in 80
DEG C heating in water bath 3 times, each 0.5 hour, filters, filtrate reduced in volume without alcohol taste, add water be settled to 5000 ml(0.2g medical materials/
Ml), upper column flow rate is 3 BV/h.Four layers of filtered through gauze, to clear liquor, filter, and (sample solution is to cross D101 macroporous adsorptive resins
10 times of resin volumes), with 2 times of water washings, water lotion discards, and with the ethanol elution of 5 times 30%, elution flow rate is 3 BV/h.Collect
Eluent, eluent is evaporated to extractum, 60 DEG C of vacuum drying, i.e. obtains the extract of Mahonia dolichostylis total alkaloids,
Measurement result, the content of three kinds of main alkaloid of Mahonia dolichostylis is 55.35%.
The method of quality control of Mahonia dolichostylis total alkaloids:
The indentification by TLC of total alkaloids:
Take Mahonia dolichostylis medicinal powder 1 g, add methanol 15 ml, ultrasonic 15 min, filter, molten as control medicinal material
Liquid.Take Mahonia dolichostylis extract powder 10 mg, put in 10 ml volumetric flasks, add methanol to scale, shake up, as test sample
Solution.Separately take berberine hydrochloride reference substance, Jatrorrhizine chloride reference substance, palmatine hydrochloride reference substance, add methanol and make every 1 ml
Containing the mixed solution of 0.5 mg, as reference substance solution.Test according to thin layer chromatography (annex VI B), draw above two solution
Each 2 μ l, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-methanol-isopropanol-strong ammonia solution (volume ratio:
6:3:1.5:1.5:0.5) it is developing solvent, puts in the vapour-saturated expansion cylinder of ammonia, launch, take out, dry, put uviol lamp (365
Nm) inspect under.In test sample chromatograph, with control medicinal material, the position that reference substance is identical, the speckle of aobvious same color.
The assay (algoscopy is commented in a survey more) of total alkaloids
1, chromatographic condition and system suitability are with octadecylsilane chemically bonded silica as filler；With acetonitrile-0.05
Mol/L potassium phosphate buffer (volume ratio: 3:7) (phosphoric acid tune pH value to 3.0) is flowing phase；Detection wavelength is 265 nm.
2, the preparation of need testing solution takes Mahonia dolichostylis extract about 40 mg, accurately weighed, puts tool plug conical flask
In, accurate addition water 50 ml, weighed weight, supersound process (power 160W, frequency 40kHz) 5 minutes, let cool to room temperature, then claim
Determine weight, supply the weight of less loss with water, filter, accurate absorption subsequent filtrate 2 ml, put in 10 ml volumetric flasks, add water to scale,
Shake up, filter, take subsequent filtrate, to obtain final product.
3, reference substance solution to prepare accurately weighed berberine hydrochloride reference substance appropriate, add acetonitrile-water (volume ratio: 3:7)
Solution makes every 1 ml solution containing 20 μ g, to obtain final product.
4, algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, surveys
Fixed, with the peak area of berberine hydrochloride reference substance for comparison, calculate berberine hydrochloride, Jatrorrhizine chloride and palmatine hydrochloride respectively
Content, to obtain final product.
Mahonia dolichostylis total alkaloid content in terms of berberine hydrochloride, hydrochloric berberine (C20H19ClNO4), hydrochloric acid medicine
Root alkali (C20H20ClNO4) and palmatine hydrochloride (C21H22ClNO4) total amount must not be less than 50.0%.
Content assaying method test is as follows:
1, one survey comment more ultimate principle composition (in the range of linearity) in certain scope amount (quality or concentration) and
Detector response is directly proportional, it may be assumed that W=f A.(W indicated concentration, A represents response value).When multi objective quality evaluation, with medical material
In a certain typical composition (having reference substance supplier) be internal reference thing, set up the relative correction factor between this composition and other compositions,
Then the content (s is internal reference thing, and k is other components) of other components is calculated by correction factor.
fsk=fs/fk=Ws×Ak/(Wk×As) (1)
Wk=Ws×Ak/( fsk×As) (2)
Jateorhizine, palmatine and berberine are all benzylisoquinoline alkaloid.Three's parent nucleus is identical, chemical constitution phase
Seemingly, a suitable survey comments method to carry out content detection more.
2, the range of linearity prepares the mixing reference substance solution of series mass concentration I-VI, and accurate absorption 20 μ l sample introductions, to enter
Sample amount (μ g) carries out regression treatment to integrating peak areas value, obtains Jatrorrhizine chloride, palmatine hydrochloride, berberine hydrochloride regression equation
It is respectively as follows: Y=5929410C+52037 (r=0.9997)；Y=6004427C+113862 (r=0.9997)；Y=5481101C+
197679 (r=0.9997).Three is respectively at 0.0697-1.3944 μ g, 0.2070-4.1400 μ g, 0.1086-2.172 μ
G range internal linear relation is good.
3, correction factor calculates with berberine hydrochloride for internal reference thing, surveys according to one and comments formula (1) under ultimate principle item more,
Calculating berberine hydrochloride is to Jatrorrhizine chloride, the correction factor of palmatine hydrochloride respectively, the results are shown in Table 1.
4, precision test precision draws same need testing solution, continuous sample introduction 5 times, hydrochloric acid under above-mentioned chromatographic condition
The RSD% of jateorhizine palmatine hydrochloride berberine hydrochloride is respectively 1.13% 0.92% 1.39%.
5, stability test takes same need testing solution, at interval of certain time sample introduction in 24h, measures.Hydrochloric acid medicine root
The RSD% of alkali palmatine hydrochloride berberine hydrochloride is respectively 0.93% 0.64% 1.12%.Show that need testing solution was at 24 hours
Interior stable.
6, replica test takes same batch sample and prepares need testing solution 6 parts, measures by drafting method, result hydrochloric acid medicine
Root alkali, palmatine hydrochloride peak, the average content of berberine hydrochloride are respectively 14.90%, 32.96%, 10.21%, and RSD is respectively
0.85%、1.14%、0.91%。
7, recovery test takes the Mahonia dolichostylis extract of known content, accurate addition a certain amount of reference substance mixing
Solution, prepares sample by need testing solution processing method, measure, calculate average recovery, Jatrorrhizine chloride, palmatine hydrochloride,
Berberine hydrochloride average recovery rate is respectively 103.0%, 102.8%, 100.7%, and RSD is respectively 1.21%, 0.91%, 1.35%.
Methodology result of the test shows that this method is simple, accurate, feasible, it can be ensured that the quality of this product.
With the inventive method extract Mahonia dolichostylis total alkaloids extract toxicity little, have simultaneously antiinflammatory, analgesia,
The pharmacological action such as antidiarrheal, anti-icterohepatitis, through toxicology, pharmacodynamics test, result is as follows:
Experimental animal Kunming mouse, 18-22 g, male and female dual-purpose, cleaning grade, by Guangxi Medical University's laboratory animal
The heart provides, credit number: SCXK osmanthus 2003-0003.
1, acute toxicity test
Take mice 50, be randomly divided into 5 groups, often group 10.After water 12 h is can't help in fasting, mice is filled by 20 ml/kg body weight
Stomach gives Mahonia dolichostylis extract 1 time.Continuous Observation 14 days after administration, record mice crawler behavior, defecation and diet
Situation.Result: having no that animal has the situation such as dystropy, death, body weight increases normal.
2, antiinflammatory action test
2.1 impacts that Oleum Tiglii caused mice auricle swelling
Taking male mice 40, be randomly divided into blank group, aspirin (0.2 g/kg) group, Mahonia dolichostylis carries
Take thing high and low dose (5.0,2.5g crude drug/kg) group, often group 10.In addition to Normal group gives equal-volume distilled water, remaining
Each group gastric infusion every day 1 time, continuous 7d.1h after last is administered, takes 0.05 ml 2% Oleum Tiglii with microsyringe and is applied to
Mouse right ear, by drawing cervical vertebra to put to death mice after 4h.Immediately with the card punch that bore is 8 mm, identical along mice left and right auricle
Round auricle is laid at position, weighs the most respectively, using the difference of two auricle weight (mg) as swelling, and calculates inhibitory rate of intumesce
(%).Inhibitory rate of intumesce=(matched group ear swelling degree-administration group ear swelling degree)/matched group ear swelling degree × 100%.The results are shown in Table
2.Result shows, compares with blank group, and mice auricle swelling is had significantly by Mahonia dolichostylis extract each dosage group
Inhibitory action, prompting has antiinflammatory action.
2.2 impacts on mouse writhing reaction
Taking mice 40, male and female half and half, be randomly divided into blank group, aspirin (0.2 g/kg) group, long column ten is big
Contribution extract high and low dose (5.0,2.5g crude drug/kg) group, often group 10.Except Normal group gives equal-volume distilled water
Outward, remaining respectively organizes gastric infusion every day 1 time, continuous 7d.1h after last is administered, every mice is by 0.1 ml/10g body weight abdominal cavity
Inject 0.6% acetum, each writhing number of times organizing mice in observing immediately and record 20 minutes.The results are shown in Table 3.Result shows,
Compare with blank group, Mahonia dolichostylis extract each dosage group substantially reduce acetic acid induced mice writhing number of times (* * P <
0.01), prompting has analgesic activity.
2.3 impacts on Folium Sennae induced mice diarrhoea
Take mice 40, male and female half and half, be randomly divided into blank group, abdomen can amine (1.4g/kg) group, Mahonia dolichostylis
Extract high and low dose (5.0,2.5g crude drug/kg) group, often group 10.In addition to Normal group gives equal-volume distilled water, its
Remaining each group gastric infusion every day 1 time, continuous 3 d.After last is administered 1 h, press 0.5 ml/ with 8% Folium Sennae water decoction for each group
20g body weight gavage, point cage observation, Mus one cage, laid absorbent paper in cage, change absorbent paper per hour, record diarrhea of mouse is hidden
Muck sum in phase and 8 h.The results are shown in Table 4.Result shows, compares with blank group, Mahonia dolichostylis extract
Each dosage group and Mahonia bealei (Fort.) Carr. extract high dose group have obvious antagonism, table to the diarrhea of mouse caused by Folium Sennae
Extend now the accumulation diarrhoea number of times suffering from diarrhoea incubation period and reduce within the identical time, and Mahonia dolichostylis anti-diarrhea effect has
A certain amount effect relationship, action effect is slightly stronger than Mahonia bealei (Fort.) Carr..
2.4 jaundice eliminating experiments
Take mice 50, male and female half and half, be randomly divided into blank group, model control group, bifendate (0.2g/kg)
Group, Mahonia dolichostylis extract high and low dose (5.0,2.5g crude drug/kg) group, often group 10.Except Normal group and model
Group gives outside equal-volume distilled water, each group gastric infusion every day 1 time, continuous 7 d.Semen arachidis hypogaeae is given except Normal group ig in 7d
Outside oil, remaining respectively organizes mice ig 0.6% α-naphthyl isothiocyanate peanut oil solution 90 mg/kg, is further continued for gastric infusion 2 d.Mice
After giving α-naphthyl isothiocyanate 48h, pluck eyeball take blood, with kit measurement serum total bilirubin (T-BIL) content and Gu Bingzhuan
Ammonia enzyme (ALT) activity.The results are shown in Table 5.Result shows, compares with model group, and Mahonia dolichostylis extract high dose group can drop
Low jaundice mouse model serum T-BIL content and ALT activity, prompting has jaundice eliminating fall enzyme anti-liver injury effect.
In sum, Mahonia dolichostylis total alkaloids preparation method of the present invention is reasonable, stable, with short production cycle, is suitable for
Industrialized production.The present invention uses thin layer chromatography qualitative identification total alkaloids, uses high effective liquid chromatography for measuring long column ten
Big contribution total alkaloid content, method is accurate, stable, simple to operate, adapts to industrial needs, can effectively control long
The quality of post Mahonia fortunei (Lindl.) Fedde total alkaloids extract.Mahonia dolichostylis total alkaloids extract toxicity is little, has antiinflammatory, town
Bitterly, the pharmacological action such as antidiarrheal, anti-icterohepatitis, there is good DEVELOPMENT PROSPECT.
Claims (3)
1. the preparation method of a Mahonia dolichostylis total alkaloids, it is characterised in that its step prepared is as follows:
(1) ethanol-hydrogen chloride heating and refluxing extraction:
1. Mahonia dolichostylis is dried stem, pulverizes, adds the ethanol-salt of 5-15 times amount 50-90% volumetric concentration by weight
Acid, wherein ethanol: hydrochloric acid is 100:0.5；
2. heating and refluxing extraction 3 times at temperature is 60-90 DEG C, each 0.5-2.5 h, filter, merging filtrate；
3. filtrate recycling ethanol, and be concentrated into without ethanol taste, it is diluted with water to the long column that concentration is 0.1-0.2g crude drug amount/mL
Mahonia fortunei (Lindl.) Fedde extracting solution, treats that upper prop is used；
(2) macroporous adsorbent resin is isolated and purified:
1. the macroporous adsorbent resin of low pole is selected；
2. absorption: upper prop Mahonia dolichostylis extract concentration is 0.1-0.2 g crude drug amount/mL, and upper prop Mahonia dolichostylis carries
Taking liquid and amass 5-10 times into filling macroporous adsorbent resin volume, upper column flow rate is 1-3 BV/h；
3. eluting: eluant is 10-70% ethanol, eluting agent is fill macroporous adsorbent resin volume 5-10 times, eluting
Flow velocity is 1-3 BV/h, collects 5-10 times of eluent, decompression recycling ethanol, is concentrated to dryness, and 50-80 DEG C of vacuum drying obtains length
Post Mahonia fortunei (Lindl.) Fedde total alkaloids extract；
The macroporous adsorbent resin of described low pole is AB-8 or D101 macroporous adsorbent resin.
The preparation method of Mahonia dolichostylis total alkaloids the most according to claim 1, it is characterised in that its detection method
For: use the thin layer chromatography of total alkaloids to carry out qualitative identification: to take Mahonia dolichostylis extract powder 10 mg, put 10 ml and hold
In measuring bottle, add methanol to scale, shake up, as need testing solution；Take Mahonia dolichostylis medicinal powder 1g, add methanol 15 ml,
Supersound process 15 minutes, filters, as control medicinal material solution；Separately take berberine hydrochloride reference substance, Jatrorrhizine chloride reference substance, salt
Acid palmatine reference substance, adds methanol and makes every 1 ml mixed solution containing 0.5 mg, as reference substance solution；According to thin layer chromatography
Test, draws each 2 μ l of above two solution, puts respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-methanol-different
Propanol-strong ammonia solution is developing solvent, puts in the vapour-saturated expansion cylinder of ammonia, launches, and takes out, dries, put under uviol lamp 365 nm
Inspect；In test sample chromatograph, with control medicinal material, the position that reference substance is identical, the speckle of aobvious same color；
Described toluene-ethyl acetate-methanol-isopropanol-strong ammonia solution volume ratio 6:3:1.5:1.5:0.5；
The power 160W of described supersound process, frequency 40kHz；
The assay step of described total alkaloids is as follows:
(1) chromatographic condition and system suitability: with octadecylsilane chemically bonded silica as filler；With acetonitrile-0.05
Mol/L potassium phosphate buffer is flowing phase；Detection wavelength is 265 nm；
In described buffer, acetonitrile: volume ratio 3:7 of potassium dihydrogen phosphate, adjust pH value to 3.0 with phosphoric acid；
(2) preparation of need testing solution: take Mahonia dolichostylis extract 40 mg, accurately weighed, put in tool plug conical flask, essence
Close addition water 50 ml, weighed weight, power 160W, the supersound process of frequency 40kHz 5 minutes, let cool to room temperature, more weighed heavy
Amount, supplies the weight of less loss with water, filters, accurate absorption subsequent filtrate 2 ml, puts in 10 ml volumetric flasks, adds water to scale, shake
Even, filter, take subsequent filtrate, to obtain final product；
(3) preparation of reference substance solution: accurately weighed berberine hydrochloride reference substance is appropriate, adds acetonitrile-aqueous solution and makes every 1 mL
Containing the solution of 20 μ g, to obtain final product；Wherein, acetonitrile and volume ratio 3:7 of water；
(4) algoscopy: precision draws reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures,
With the peak area of berberine hydrochloride reference substance for comparison, calculate berberine hydrochloride, Jatrorrhizine chloride and palmatine hydrochloride respectively
Content, to obtain final product；Mahonia dolichostylis total alkaloid content in terms of berberine hydrochloride, hydrochloric berberine, Jatrorrhizine chloride and salt
The total amount of acid palmatine must not be less than 50.0%.
3. the total alkaloids obtained by the Mahonia dolichostylis total alkaloids preparation method described in claim 1 prepare antiinflammatory,
Application in analgesia and antidiarrheal.
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| 功劳木总生物碱的提取与分离纯化;田在福;《万方数据企业知识服务平台-学位，网址：d.wangfangdata.com.cn.Thesis_Y1737949.aspx》;20110531;第I页第1-2段，第II页第1段，第1页第1段，第8页第2-3段，第12页第1段，第29页倒数第4段 * |
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Application publication date: 20130918 Assignee: Guangxi Houde Pharmaceutical Co.,Ltd. Assignor: GUANGXI INSTITUTE OF CHINESE MEDICINE & PHARMACEUTICAL SCIENCE Contract record no.: X2022450000405 Denomination of invention: Preparation and quality control of total alkaloids from Mahonia longissima Granted publication date: 20160810 License type: Common License Record date: 20221227 |