CN106237310B - Preparation method of traditional Chinese medicine extract - Google Patents
Preparation method of traditional Chinese medicine extract Download PDFInfo
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- CN106237310B CN106237310B CN201610887675.1A CN201610887675A CN106237310B CN 106237310 B CN106237310 B CN 106237310B CN 201610887675 A CN201610887675 A CN 201610887675A CN 106237310 B CN106237310 B CN 106237310B
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- crude drug
- macroporous resin
- chinese medicine
- traditional chinese
- water
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Classifications
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/42—Cucurbitaceae (Cucumber family)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- A—HUMAN NECESSITIES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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Abstract
The invention discloses a method which comprises the following steps: (1) extracting crude drug with water to obtain water extractive solution; the crude drug comprises fructus Trichosanthis, ramulus Cinnamomi, radix Paeoniae alba, rhizoma Zingiberis recens, fructus Jujubae and Glycyrrhrizae radix; (2) taking the water extract, centrifuging and filtering to obtain a sample solution; (3) and (4) taking the sample solution, and purifying by HPD-100 macroporous resin to obtain the traditional Chinese medicine extract. The preparation method of the traditional Chinese medicine extract effectively reserves various active ingredients in the traditional Chinese medicine, and the dripping pill prepared by taking the obtained traditional Chinese medicine extract as the raw material reduces the dosage of patients and improves the curative effect.
Description
Technical Field
The invention relates to a preparation method of a traditional Chinese medicine extract and the traditional Chinese medicine extract prepared by the method.
Background
The trichosanthes kirilowii and cassia twig decoction is from the golden lack essence of Zhang Zhongjing of eastern Han, mainly comprises trichosanthes kirilowii, cassia twig, white paeony root, ginger, Chinese date, liquorice and the like, and has the efficacies of muscle relieving, nutrient regulating, nutrient nourishing, body fluid production promoting, and tendon and vessel softening [1 ].
Studies have reported that the trichosanthes kirilowii and cassia twig decoction and the trichosanthes kirilowii and cassia twig granules have good brain protection effect [2-7 ]. However, the dosage of the medicine is relatively large, and the adaptability is not high. In order to reduce the dosage of the patients and improve the curative effect, further improvement is needed.
[1] Tianwenxi, Guizhi Tang type prescription in jin Kui Yao L ü e (gold Kui Yao L ü e) has the effect of regulating yin and yang [ J ]. Chinese basic medicine journal of traditional Chinese medicine 2010,08: 646-.
[2] Zhangyuqin, Lihuang, xu wen, xu wei, Huang pian, zhu ke dan, Chen Li Dian. the granules of Gua sui Zhi Gui have blood brain barrier permeability and neuroprotective effect on rats with cerebral ischemia reperfusion injury [ J ]. Chinese medicine J2015, 05: 1410-.
[3] Dawn, li zu fang, maojing, huhaixia, linrue, chen li dian, trichosanthes and cassia twig decoction has protective effect on PC12 cell damage caused by glutamic acid [ J ]. Fujian traditional Chinese medicine 2015,02:35-36.
[4] Lin Yu, Xuwei, Zhangyuqin, Li Huang, xu, the study of apoptosis of rat neurons and primary hippocampal neurons of Trichosanthes angustifolia Cassia twig particles in resisting ischemic cerebral apoplexy [ J ] rehabilitation proceedings 2015,01:38-43.
[5]Huang J,Tao J,Xue X,Yang S,Han P,Lin Z,Xu W,Lin J,Peng J,ChenL.Gua Lou Gui Zhi decoction exerts neuroprotective effects on post-strokespasticity via the modulation of glutamate levels and AMPA receptorexpression.Int J Mol Med.2013 Apr；31(4):841-8.
[6]Hu H,Li Z,Zhu X,Lin R,Lin J,Peng J,Tao J,Chen L.Gua Lou Gui Zhidecoction suppresses LPS-induced activation of the TLR4/NF-κB pathway in BV-2murine microglial cells.Int J Mol Med.2013 Jun；31(6):1327-32.
[7]Hu H,Li Z,Zhu X,Lin R,Peng J,Tao J,Chen L.GuaLou GuiZhi decoctioninhibits LPS-induced microglial cell motility through the MAPK signalingpathway.Int J Mol Med.2013 Dec；32(6):1281-6.11.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of a traditional Chinese medicine extract and the traditional Chinese medicine extract prepared by the method.
The invention provides a preparation method of a traditional Chinese medicine extract, which comprises the following steps:
(1) extracting crude drug with water to obtain water extractive solution; the crude drug comprises fructus Trichosanthis, ramulus Cinnamomi, radix Paeoniae alba, rhizoma Zingiberis recens, fructus Jujubae and Glycyrrhrizae radix;
(2) taking the water extract, centrifuging and filtering to obtain a sample solution;
(3) and (4) taking the sample solution, and purifying by HPD-100 macroporous resin to obtain the traditional Chinese medicine extract.
Further, in the step (1), the weight ratio of the trichosanthes kirilowii maxim, the cassia twig, the white paeony root, the ginger, the Chinese date and the liquorice in the crude drugs is 30: 9: 9: 9: 9: 6.
further, in the step (1), the water extraction is to add 10 times of water and decoct for 2 times, each time for 1.5 hours.
Further, the crude drug concentration of the upper sample solution in the step (2) is 0.2-0.4 g crude drug/mL; the preferred crude drug concentration is 0.3g crude drug/mL.
Further, in the macroporous resin purification of the step (3), the adsorption flow rate is 1-3 BV.h-1Preferably 2 BV.h-1。
Further, in the macroporous resin purification in the step (3), an eluent is 70-90% of ethanol.
Further, in the macroporous resin purification of the step (3), the dosage of the eluent is 6 BV.
Further, in the macroporous resin purification of the step (3), the elution flow rate is 1-3 BV.h-1Preferably 2 BV.h-1。
The invention also provides a traditional Chinese medicine extract which is prepared by the method.
Further, the extract contains the following components per 100 mL: 49.68 + -0.03 mg of paeoniflorin, 14.15 + -0.05 mg of albiflorin, 0.88 + -0.02 mg of cinnamic acid, 1.07 + -0.02 mg of gingerol, 10.55 + -0.01 mg of liquiritin, 8.12 + -0.03 mg of glycyrrhizic acid and 0.38 + -0.01 mg of liquiritigenin.
The preparation method of the traditional Chinese medicine extract effectively reserves various active ingredients in the traditional Chinese medicine, and the dripping pill prepared by taking the obtained traditional Chinese medicine extract as the raw material reduces the dosage of patients and improves the curative effect.
BV: resin bed volume.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the top view of HPLC of mixed control, and the bottom view of HPLC of sample solution, 1: albiflorin; 2, paeoniflorin; 3, liquiritin; 4, liquiritigenin; 5, cinnamic acid; 6, glycyrrhizic acid; 7: 6-gingerol.
Fig. 2 shows the results of the adsorption leakage curve examination.
Detailed Description
Example 1 preparation of the extract of the present invention
Prescription: 30g of trichosanthes kirilowii maxim, 9g of cassia twig, 9g of white paeony root, 9g of ginger, 9g of Chinese date and 6g of liquorice.
Taking the raw materials in the formula proportion, adding 10 times (720mL) of water, decocting for 2 times, each time for 1.5h, mixing decoctions, filtering, concentrating to obtain solution with concentration of 0.3g crude drug/mL, centrifuging for 30min at 3000 r.min-1Filtering to obtain a sample solution; separating and purifying the sample solution by an HPD-100 type macroporous resin column, wherein the purification process comprises the following steps: the maximum sample loading amount is 2.5g crude drug g-1The adsorption flow rate is 2 BV.h-1Eluent is 70% ethanol, and the elution flow rate is 3 BV.h-1The dosage of the eluent is 6 BV; mixing, and collecting 70% ethanol eluate.
Example 2 dropping pills prepared from the extract of the present invention as a raw material
Concentrating the traditional Chinese medicine extract prepared in the embodiment 1, drying under reduced pressure, and crushing to obtain the trichosanthes kirilowii maxim and cassia twig decoction extract powder.
Mixing polyethylene glycol 4000, polyethylene glycol 6000 and the extract powder of the trichosanthes kirilowii and cassia twig decoction at a ratio of 3:2:1, heating and melting at 90 ℃, and dripping into pills according to the following conditions: the dripping speed is 20 drops/min-1The dropping distance is 2cm, and the condensation temperature is 10 ℃.
Example 3 Process screening for the preparation method of the invention
1 instruments and drugs
Model U-3000 high performance liquid chromatograph (Daian, USA); an XS105 model electronic balance (Mettler-Torledo instruments, Inc.), a TGL-16G model bench centrifuge (Shanghai Tingn scientific Instrument plant); ELX-800 type multifunctional microplate reader (diken (shanghai) trade ltd); inverted microscope (OLYMPUS, japan).
Trichosanthis radix (batch No. 20151213), radix Paeoniae alba (batch No. 20151211), purchased from Dechang medicinal decoction pieces Limited, Anhui; ramulus Cinnamomi (batch No. 160403), available from Yonggang decoction pieces works, Inc., of Bozhou; licorice (batch No. 15060901) obtained from Fuzhou Huichun decoction pieces of Chinese medicinal materials, Co., Ltd; ginger and Chinese date are sold in the market; paeoniflorin (batch No. 110736-201539), albiflorin (batch No. 16021410), cinnamic acid (batch No. 110786-200503), liquiritin (batch No. 111610-201106), purchased from the institute of pharmaceutical and biological products of China; 6-gingerol (batch: PZ9M6R1), glycyrrhizic acid (batch: Z30A6B1), glycyrrhizin (batch: ZMO314BD14), available from Shanghai-derived leaf Biotech Co., Ltd; HPD-100 type macroporous resins available from Zhengzhou Qinshi scientific and technological Limited; d101 and NKA-9 type macroporous resin is purchased from Cangzhou Baoyen adsorption material science and technology limited; chromatographic grade acetonitrile was purchased from merck, germany; the water is ultrapure water.
PC12 cells were purchased from cz's biotechnology limited; phosphate buffer solution, DMEM high-sugar medium, penicillin-streptomycin double antibody and fetal bovine serum, all purchased from Saimer Feishale Biochemical (Beijing) Co., Ltd; trypsin was purchased from Life Technologies, usa; glutamic acid, thiazole blue were purchased from SIGMA, usa; dimethyl sulfoxide was purchased from national pharmaceutical group chemical agents, ltd.
2 methods and results
2.17 measurement of contents of index Components
2.2.1 preparation of Mixed reference solution A proper amount of paeoniflorin, albiflorin, cinnamic acid, 6-gingerol, liquiritin, glycyrrhizic acid, and liquiritigenin reference are precisely weighed, dissolved and diluted with methanol, and shaken well to obtain each reference stock solution; precisely absorbing appropriate amount of each control stock solution, diluting with methanol to desired volume, and shaking.
2.2.2 chromatographic conditions DIKMA C18Chromatography column (250 mm. times.4.6 mm, 5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid, and the gradient elution condition is shown in table 1; flow rate 0.8 mL/min-1(ii) a The column temperature is 30 ℃;the sample injection volume is 10 mu L, and the detection wavelength is 236 nm; the chromatogram is shown in FIG. 1.
TABLE 1 HPLC gradient elution conditions
2.2 preparation of the sample solution the medicinal materials are weighed according to the prescription proportion, added with 10 times of water to be decocted and extracted for 2 times, each time lasts for 1.5h, the extract is filtered, merged and concentrated, distilled water is added to a certain volume, and the mixture is centrifuged for 30min (3000 r.min)-1) And carrying out suction filtration to obtain a sample liquid.
2.3 pretreatment of macroporous resin is taken, 95% ethanol is added into the macroporous resin for soaking for 24h, the macroporous resin is filled into a column by a wet method after full swelling, the column is eluted by the 95% ethanol until the effluent liquid is not white and turbid by adding 5 times of water, and the effluent liquid is washed by water until no alcohol smell exists for standby.
2.4 macroporous resin model screening
2.4.1 dynamic adsorption of macroporous resin the sample liquid is passed through 3 types of treated resin columns (column volume 20mL) for dynamic adsorption, after complete adsorption, the effluent is washed with water until the effluent is nearly colorless, and then eluted with 100mL ethanol. Respectively collecting the residual liquid, the water washing liquid and the ethanol eluent. The contents of the 7 index components were measured, and the optimum resins were selected using the specific adsorption amounts and the resolution as evaluation indexes, and the results are shown in tables 2 and 3.
TABLE 2 screening results of specific adsorption capacity of macroporous resins
Specific adsorption capacity ═ MSample loading liquid-MResidual liquid-MWater washing liquid)/MMacroporous resin weight
TABLE 3 macroporous resin resolution screening results
Resolution is MEthanol washing liquid/MAmount of adsorption×100％
The results show that the specific adsorption capacity of the D101 macroporous resin is relatively large, and the resolution ratio of the HPD-100 macroporous resin is relatively high, so that the HPD-100 and D101 macroporous resins are further examined.
2.4.2MTT Activity Screen logarithmic phase PC12 cells were seeded into 96-well plates at 200. mu.L/well containing 2X 104Individual cells, cultured overnight for experimental use. The medium was discarded and washed with PBS gently, and the mixture was randomly divided into 10 groups, a normal group and a model group (glutamic acid-damaged group, concentration 30 mmol. multidot.L)-1) HPD-100 group (100, 200, 300 and 400. mu.g.mL)-1) And D101 groups (100, 200, 300 and 400. mu.g.mL), the normal group, the model group added with 1640 medium of 1% FBS, the HPD-100 group and the D101 group added with 1640 medium of 1% FBS of different sample concentrations, each well was 100. mu.L, after 24 hours of culture, the normal group added with 100. mu.L of 1640 medium of 1% FBS, the model group, the HPD-100 group and the D101 group added with 100. mu.L of 1640 medium containing 30 mmol.L-11640 medium of 1% FBS for glutamic acid (final concentration of glutamic acid 15 mmol. multidot.L)-1) And continuously culturing for 6h, and detecting the activity of each group of cells by an MTT method. The experiment was repeated 3 times, each set was set to 6 duplicate wells, and the results are shown in Table 4
TABLE 4 MTT method Activity screening results
P < 0.01 compared to normal group; p < 0.05 compared to model group; compared with the corresponding concentration of the HPD-100 group,#P＜0.05。
the results show that the survival rate of the model group is obviously reduced (P is less than 0.01), and the survival rate of the HPD-100 group is 100 mu g/mL and 400 mu g/mL-1After concentration administration, the survival rate is obviously higher than the corresponding concentration of the D101 group (P is less than 0.05), and 200 mu g/mL of the HPD-100 group-1Survival after concentration administrationThe rate is obviously higher than that of a model group (P is less than 0.05); comprehensively considering, HPD-100 type macroporous resin is selected as the best resin for purifying the trichosanthes kirilowii maxim and cassia twig decoction.
2.5 optimization of purification Process parameters
2.5.1 investigation of sample concentration 20mL 5 parts of HPD-100 wet resin was precisely measured and subjected to wet column packing to prepare 0.1, 0.2, 0.3, 0.4, 0.5g crude drug/mL-1The 5 kinds of sample solutions with different concentrations are respectively passed through HPD-100 resin columns for dynamic adsorption, after complete adsorption, the effluent is washed by water until the effluent is nearly colorless, then the ethanol is used for elution, the ethanol eluate is collected, and the contents of 7 index components in the eluate are measured, which is shown in Table 5.
TABLE 5 examination of the concentration of the sample liquid
The results showed that the concentration of the sample solution reached 0.3g crude drug/mL-1When the content of the 7 index components reaches the maximum value and the concentration continues to increase, the content decreases, so 0.3g crude drug/mL is selected-1Is the loading concentration.
2.5.2 examination of adsorption flow Rate 20mL 3 parts of HPD-100 wet resin were precisely measured, and wet-packed into a column, 0.3g crude drug/mL-1The sample loading liquid is respectively 1 BV.h, 2 BV.h and 3 BV.h-1Dynamic adsorption is carried out at the flow rate, after complete adsorption, the effluent is washed by water until the effluent is nearly colorless, then the ethanol is used for elution, the ethanol eluate is collected, and the content of 7 index components in the eluate is measured, which is shown in Table 6.
TABLE 6 examination of adsorption flow Rate
The results show that the content of the 7 index components is along with the adsorption flow velocityIs increased when the adsorption flow rate is 1, 2 BV.h-1The content difference is not large, and the flow rate is selected to be 2 BV.h in consideration of the actual production process and the shortening of the production period-1Is the adsorption flow rate.
2.5.3 examination of the Drain Curve, 20mL of the treated HPD-100 wet resin was precisely measured, and the column was packed by wet method, 0.3g crude drug/mL-1At a flow rate of 2 BV.h-1The effluent was loaded and collected in fractions, one for each column volume (20mL), and the content of 7 marker components was determined and a leakage curve was plotted, see FIG. 2.
The results show that paeoniflorin, albiflorin, liquiritin and glycyrrhizic acid are obviously leaked at the 6 th part, cinnamic acid, 6-gingerol and liquiritigenin are not leaked yet, and in order to reduce the loss of effective components as much as possible, the maximum sample loading amount of the medicine is determined to be 2.5g crude drug/g-1。
2.5.4 examination of eluent 20mL 5 parts of HPD-100 wet resin, wet packing, 0.3g crude drug/mL-1The flow rate of the sample solution is 2 BV.h for 100mL-1Loading, dynamic adsorption, washing with water until the effluent is nearly colorless after complete adsorption, eluting with 10%, 30%, 50%, 70% and 90% ethanol respectively, collecting the ethanol eluates, and determining the content of 7 index components in the eluates, as shown in Table 7.
TABLE 7 examination of eluents
The results show that 70% and 90% ethanol have better elution capability.
2.5.5 examination of eluent dosage the well-treated HPD-100 wet resin 20mL was measured precisely and packed into column by wet method, 0.3g crude drug/mL was taken-1The flow rate of the sample solution is 2 BV.h for 100mL-1Loading, dynamic adsorbing, washing with water until the effluent is nearly colorless after complete adsorption, eluting with 70% ethanol, and collecting the effluent by stages, each column volume (20mL)A portion was collected and the content of 7 index components was determined, as shown in Table 8.
Table 8 results of investigation of eluent amount
The results show that the amount of eluent is preferably 6 BV.
2.5.6 examination of elution flow Rate A well-treated HPD-100 wet resin 20mL was precisely measured, wet-packed into a column, and 0.3g crude drug in mL was taken-1The flow rate of the sample solution is 2 BV.h for 100mL-1Loading, dynamic adsorption, washing with water until the effluent is nearly colorless, eluting with 6BV 70% ethanol at 1, 2 and 3 BV.h respectively-1The ethanol eluate was collected and the contents of 7 index components were measured, as shown in Table 9.
TABLE 9 examination of elution flow Rate
Comprehensively considering, selecting 2 BV.h-1The elution flow rate.
2.5.7 validation test was carried out according to the optimum process conditions, 3 batches were carried out, conditions: the sample concentration was 0.3g crude drug/mL-1The maximum sample loading is 2.5 g.g-1The adsorption flow rate is 2 BV.h-1Eluent is 70% ethanol, and the elution flow rate is 2 BV.h-1Eluting with 6BV eluent, collecting eluent, measuring the contents of 7 index components, and determining the component retention rate as evaluation index, as shown in Table 10.
Table 10 verifies the results of the experiment
Component retention rate of MAfter purification/MBefore purification×100％
The average mass of the purified sample obtained by the experiment is 0.329mg, and under the optimal process, the HPD-100 type macroporous resin has obvious effect of reducing the extractum rate of the trichosanthes kirilowii maxim and cassia twig decoction and has higher component retention rate.
The beneficial effects of the dropping pills of example 2 of the present invention are demonstrated by the following test examples.
Test examples
In the specification of the preparation of the trichosanthes kirilowii maxim and cassia twig granules (the batch is 2013S0001) in hospital of the second people of Hospital of Fujian province, 12 g/bag is marked, 3 bags are taken every day, and the dosage required for taking the granules is 36 g.d-1(ii) a After the dripping pill is changed into the dripping pill, the daily prescription amount of the medicinal materials is purified by macroporous resin to obtain 0.79g of extract, and because the optimal process matrix dosage of the formed dripping pill is determined to be 5 times of the extract dosage, namely 3.95g, the dosage of the final dripping pill is 4.74 g.d-1The average pill weight of each pill is 35mg, thus effectively reducing the dosage of patients.
In conclusion, the preparation method of the traditional Chinese medicine extract effectively reserves various active ingredients in the traditional Chinese medicine, and the dripping pill prepared by using the obtained traditional Chinese medicine extract as the raw material reduces the dosage of patients and improves the curative effect.
Claims (6)
1. A preparation method of a traditional Chinese medicine extract is characterized by comprising the following steps: it comprises the following steps:
(1) extracting crude drug with water to obtain water extractive solution; the crude drug comprises fructus Trichosanthis, ramulus Cinnamomi, radix Paeoniae alba, rhizoma Zingiberis recens, fructus Jujubae and Glycyrrhrizae radix;
(2) taking the water extract, centrifuging and filtering to obtain a sample solution;
(3) purifying the supernatant with HPD-100 macroporous resin to obtain Chinese medicinal extract;
in the step (1), the weight ratio of the trichosanthes kirilowii maxim, the cassia twig, the white paeony root, the ginger, the Chinese date and the liquorice in the crude drugs is 30: 9: 9: 9: 9: 6;
in the step (1), 10 times of water is added into the water for decoction for 2 times, and each time lasts for 1.5 hours;
the crude drug concentration of the upper sample solution in the step (2) is 0.2-0.4 g crude drug/mL;
in the macroporous resin purification of the step (3), the adsorption flow rate is 1-3 BV.h-1；
In the macroporous resin purification of the step (3), an eluent is 70-90% of ethanol;
in the macroporous resin purification of the step (3), the dosage of the eluent is 6 BV;
in the macroporous resin purification of the step (3), the elution flow rate is 1-3 BV.h-1。
2. The method of claim 1, wherein: in the step (2), the crude drug concentration of the upper sample solution is 0.3g crude drug/mL.
3. The method of claim 1, wherein: in the macroporous resin purification of the step (3), the adsorption flow rate is 2 BV.h-1。
4. The method of claim 1, wherein: in the macroporous resin purification of the step (3), the elution flow rate is 2 BV.h-1。
5. A traditional Chinese medicine extract is characterized in that: it is prepared by the method of any one of claims 1 to 4.
6. The herbal extract as claimed in claim 5, wherein: the extract contains the following components in each 100 mL: 49.68 + -0.03 mg of paeoniflorin, 14.15 + -0.05 mg of albiflorin, 0.88 + -0.02 mg of cinnamic acid, 1.07 + -0.02 mg of gingerol, 10.55 + -0.01 mg of liquiritin, 8.12 + -0.03 mg of glycyrrhizic acid and 0.38 + -0.01 mg of liquiritigenin.
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