CN101975829A - Screening method and application of alpha-glucosidase inhibitor - Google Patents
Screening method and application of alpha-glucosidase inhibitor Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicines. The screening method of an alpha-glucosidase inhibitor comprises the following steps of: (1) establishing in vitro screening models; establishing in vitro screening models of alpha-glucosidase and a substrate PNPG (Para Nitrobenzene-Beta-D-Galactose Pyranoside); (2) establishing a component library: selecting traditional Chinese medicines, systematically extracting each traditional Chinese medicine with various (more than two kinds) solvents according to a similar compatible principle according to the polarities from low to high, wherein multiple extracting parts can be obtained for each traditional Chinese medicine by adopting the method; and (3) screening: blending all the extracting parts of the traditional Chinese medicines into a solution with a certain concentration, adding the solution to a reaction model, and calculating the inhibition rate. The screening method is simple, reasonable and easy to operate and can be used for finally screening out traditional Chinese medicines and traditional Chinese medicine components having obvious inhibition effects on the alpha-glucosidase. In addition, the screened traditional Chinese medicines and the traditional Chinese medicine components can be used for developing products aiming to inhibit the alpha-glucosidase and particularly used for developing drugs for reducing the blood sugar and has industrialization significance.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to the screening technique that suppresses the alpha-glucosidase activity component in the Chinese medicine, also relate to the application of Chinese medical extract in addition.
Background technology
Along with the raising of people's living standard, the change of dietary structure, the diabetic increases sharply and is the trend of becoming younger, and (Diabetes mellitus DM) has become one of the main chronic disease of serious threat human health to diabetes.Two important indicators that diabetes detect are fasting blood glucose level and level of postprandial blood sugar, epidemiological study shows, the symptom that occurs at first in the onset diabetes process is postprandial hyperglycemia often, and postprandial hyperglycemia is than the various complication of the easier initiation of fasting blood-glucose, also be that glucose tolerates the key factor that impaired patient develops into type II diabetes, therefore controlling postprandial hyperglycemia is the important measures of prevention and treatment diabetes.Alpha-glucosidase restrainer (α-glucosidase inhibitors, α-GI) by suppressing the activity of various alpha-glucosidases on the intestinal brush border, the hydrolysis of control compound sugar and disaccharide, reduce postprandial blood sugar concentration, reduce the stimulation of hyperglycaemia to pancreas, improve insulin receptor susceptibility, and do not cause hypoglycemia, so alpha-glucosidase restrainer repeatedly is recommended as a line medicine that reduces postprandial blood sugar by Asian-Pacific area diabetes medicament guide; In addition, alpha-glucosidase restrainer can also be treated multiple diseases such as virus infections, tumour, lysosomal storage disease, obesity, high triglyceride blood fat disease, hepatitis.
At present, being applied to clinical alpha-glucosidase restrainer all is the biosynthesizing or the patent medicine that narrows, and kind is few, price is expensive, spinoff is big.There are abundant natural resources of Chinese medicinal materials and huge herbal species and the prescription of quantity in China, as: the treatment of putting down in writing in the Compendium of Material Medica of Li Shizhen (1518-1593 A.D.) quench one's thirst Chinese medicine commonly used have 140 surplus kind, the anti-diabetic Chinese patent drug that registration is declared by China has nearly 40 kinds.Yet guide's thing of the inhibition alpha-glucosidase activity that pharmacology is clear and definite or medicine are but very limited.Therefore, the screening alpha-glucosidase restrainer has important researching value from traditional Chinese medicine.
(High Throughout Screening HTS) is the important step that new drug is found to high flux screening.By screening, can find to rise in the Chinese medicine active component of therapeutic action, the action target spot of clear and definite Chinese medicine is the development traditional medicine, realize the modernization of Chinese medicine, promotes the effective way of new drug research.At present, right-nitrobenzophenone-α-D-glucopyranoside (PNPG) method that the alpha-glucosidase restrainer in-vitro screening method mainly contains, glucose oxidase method and immobilized enzyme method, but adopt spectrophotometric method mostly as detection means, reagent consumption is big, poor reproducibility, especially for complex sample or the screening of pigment disturbed specimen is arranged, the false positive height, have in addition do not have activity in vivo fully.
At present disclosed patent about the alpha-glucosidase restrainer screening, the widespread usage spectrophotometer carries out the mensuration that enzyme is lived; General single solvent extract part in the foundation of component pool at certain flavor Chinese medicine.For example, Chen Qun etc. (application number 200810162970.6) are by microwave heating method, garden burnet is further taken off albumen, centrifugal back extract from supernatant and obtained having alpha-glucosidase and suppress active extract after water extracts; Huang Xinli etc. (application number 02125869.4) have obtained alpha-glucosidase restrainer with water, alcohol or the water alcohol mixed liquor extract of mulberry leaf by anion-cation exchange resin or macroreticular resin; Wu Hui equality (200710131472.0) is extracted selfheal water and liquid extract has carried out the active screening of alpha-glucosidase inhibition.
Summary of the invention
The invention provides a kind of screening technique of alpha-glucosidase restrainer, the method advantages of simple, easy operating has filtered out the active component with remarkable inhibition effect.Another object of the present invention provides the application of alpha-glucosidase restrainer, with single active site or a plurality of active site applied in any combination in being the research and development of the medicine or the health products of purpose to suppress alpha-glucosidase.
The technical scheme that the present invention adopts for achieving the above object is: the screening of alpha-glucosidase restrainer, specifically divided for three steps:
1) foundation of in-vitro screening model
Set up the vitro reactions model of alpha-glucosidase and substrate PNPG, use HPLC and detect hydrolysate PNP,, calculate the inhibiting rate of each extract part alpha-glucosidase by the concentration change of PNP;
(2) foundation of component pool
That chooses some has the Chinese medicine of remarkable result to treating diabetes, according to " similar compatibility " principle, ascending according to polarity, with multiple (〉=2) solvent every flavor Chinese medicine being carried out system extracts, adopt this method, every flavor Chinese medicine obtains a plurality of extract parts, and the preferred 5 kinds of solvents of the present invention are respectively normal hexane, methylene chloride, ethyl acetate, methyl alcohol, water;
(3) screening
Each extract part of Chinese medicine is made into certain density solution, joins reaction and calculating inhibiting rate in the reaction model; Because the inhibition activity of different traditional Chinese medicinal components differs bigger, for suppressing active higher component, carry out multiple sieve according to identical method behind the concentration dilution, suppress active higher active site up to filtering out;
The application of alpha-glucosidase restrainer of the present invention, there is Chinese medicine or the Application in Food that suppresses effect at selected traditional Chinese medicine extraction position in preparation to alpha-glucosidase; Particularly reduce the medicine or the Application in Food of postprandial blood sugar, treatment or prevention diabetes and diabetic complication in preparation.
The application of described alpha-glucosidase restrainer, selected traditional Chinese medicine extraction position is meant: what screening obtained has the traditional Chinese medicine extraction position of remarkable inhibition effect to be meant the water extract part of garden burnet, Chinese cassia tree, reticulate millettia, cloves, Chinese torreyanut, cymose buckwheat rhizome, FRUCTUS TERMINALIAE IMMATURUS, granatum, the radix paeoniae rubrathe, cynomorium songaricum to alpha-glucosidase; The methanol extraction position of Chinese medicine Chinese torreyanut, cymose buckwheat rhizome, Chinese gall, garden burnet, FRUCTUS TERMINALIAE IMMATURUS, granatum, reticulate millettia, Chinese cassia tree, rheum officinale, Ramulus euonymi; The ethyl acetate extraction position of Chinese medicine Chinese gall, Chinese cassia tree, rheum officinale, the wind-weed, reticulate millettia, ramulus mori, the radix paeoniae rubrathe, HERBA DENDROBII, Chinese yam, cynomorium songaricum; The methylene chloride extract part of Chinese herb rhubarb, Chinese gall, Radix Glycyrrhizae, Poria cocos, the root of bidentate achyranthes, the wind-weed, reticulate millettia, the root bark of Chinese wolf-berry, sealwort, cairo morningglory root or leaf; The normal hexane extract part of Chinese medicine Chinese gall, the root of bidentate achyranthes, great burdock achene, Poria cocos, Radix Glycyrrhizae, rheum officinale, sealwort, XIAKUQIU, cymose buckwheat rhizome, radix polygonati officinalis.
Described preparation has the medicine of remarkable inhibition effect to alpha-glucosidase, formulations such as tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol can be adopted, the known controlled release of modern medicine circle or slow release formulation or nanometer formulation can also be adopted.
Screening technique advantages of simple of the present invention, easy operating, can carry out the screening of alpha-glucosidase restrainer to Chinese medicine, use the model that HPLC has set up the in-vitro screening alpha-glucosidase restrainer, the modelling principle: alpha-glucosidase is a key selectivity enzyme, PNPG can be hydrolyzed to glucose and PNP, as shown in Figure 4, by HPLC to product P NP quantitative test, if having alpha-glucosidase, the Chinese medicine sample suppresses active, the corresponding minimizing of the amount of product P NP, by the concentration change of PNP, the calculation sample inhibiting rate.Finishing screen is selected Chinese medicine and the traditional Chinese medicinal components that alpha-glucosidase is had remarkable inhibition effect.The Chinese medicine that filters out of the present invention and traditional Chinese medicinal components can be used for to be the research and development of the product of purpose, especially to can be used for the exploitation of hypoglycemic medicine to have the industrialization meaning to suppress alpha-glucosidase in addition.
Description of drawings
Fig. 1 is that the present invention sets up traditional Chinese medicinal components storehouse system extraction process flow diagram;
Fig. 2 is the HPLC figure of PNPG of the present invention and PNP.
Fig. 3 is the canonical plotting of PNP of the present invention.
Embodiment
The present invention is elaborated by following concrete enforcement, but is not limited only to following examples.
1, the foundation of alpha-glucosidase restrainer in-vitro screening model
(1) alpha-glucosidase suppresses active mensuration: experiment is carried out in 1.5 mL centrifuge tubes, and the reaction cumulative volume is 160 μ L:10 μ L, 0.067 molL
-1, the phosphate buffered solution of pH 6.8,30 μ L concentration, 0.2 UmL
-1Alpha-glucosidase, the concussion mixing is hatched 20 min for 37 ℃, adds 40 μ L concentration, 8.0 mM PNPG and opens reaction, and the concussion mixing behind 37 ℃ of reaction 30 min, adds 80 μ L concentration, 0.2 M Na
2CO
3Cessation reaction adds ultrapure water and is diluted to 500 μ L, and the vibration mixing behind 0.45 μ m membrane filtration, detects with HPLC, by the concentration change of product P NP, calculates the activity of alpha-glucosidase.The principle of alpha-glucosidase hydrolysis substrate PNPG:
Invention replaces phosphate buffered solution with certain density extraction sample, suppresses active mensuration; Acarbose with same concentrations replaces the positive contrast of phosphate buffered solution, finds the easy spontaneous hydrolysis of substrate PNPG in the experimentation, and therefore replacing extract and enzyme liquid with isopyknic buffer solution is blank.Being calculated as follows of inhibiting rate:
In the formula,
AThe concentration of PNP (is deducted corresponding blank, mmoL/L) when not adding testing sample;
BFor the concentration of PNP behind the adding testing sample (is deducted corresponding blank, mmoL/L).
(2) the HPLC testing conditions of PNP
Chromatographic column: Sino Chrom ODS-BP(5 μ m, 4.6 mm * 250 mm); Moving phase: A is an acetonitrile, and B is the aqueous solution that contains 0.1% formic acid, and condition of gradient elution is: 0～8 min, 20%～30% A; 8～13 min, 30%～80% A; 13～18 min, 80%～20% A; 18～28 min, 20% A; Flow velocity: 1.0mLmin
-1Sample size: 20 μ L; Column temperature: normal temperature; Detect wavelength: 314 nm.Under above-mentioned testing conditions, by the external standard method contrast, the retention time of determining PNP is 14.467 min, as shown in Figure 2.
The making of typical curve: accurately take by weighing 0.020 9 g PNP standard items, use the phosphate buffered solution ultrasonic dissolution, constant volume is in 25 mL volumetric flasks, be configured to the PNP mother liquor of 6 mmol/L, mother liquor is diluted to 0.002 5,0.005,0.01,0.05,0.1,0.2,0.4,0.8 mmol/L.Measuring according to above-mentioned chromatographic condition, is horizontal ordinate with concentration, and peak area is an ordinate, and the drawing standard working curve as shown in Figure 3, is tried to achieve regression equation and is
y=197.4
x+ 0.819 3, R
2=0.999 8, illustrate that PNP is good in 0.002 5 ~ 0.8 mmol/L scope internal linear relation.
2, the foundation of component pool
At first select 50 flavors treatment of diabetes to be had the Chinese medicine of remarkable result, see Table 1, selecting normal hexane, methylene chloride, ethyl acetate, 5 kinds of solvents of first alcohol and water that every flavor Chinese medicine is carried out system successively extracts: take by weighing Chinese medicine material, oven dry is pulverized, accurately raw material 200 g are pulverized in weighing, according to Chinese medicine: solvent (m:v)=1:4 ultrasonic Extraction 1 h, filter paper filtering, medicinal extract concentrates to such an extent that Chinese medicine must be got the position with Rotary Evaporators, soak slag and continue to extract with next solvent, idiographic flow as shown in Figure 1;
By above extraction, every kind of Chinese medicine is cut into 5 parts according to the polarity size, be respectively normal hexane extract part, methylene chloride extract part, ethyl acetate extraction position, methanol extraction position and water extract part, set up the component pool that contains 250 traditional Chinese medicine extraction positions thus.
3, the screening of water extract part
The water extract part is made into 64 mgmL
-1Aqueous solution joins in the reaction model reaction and calculates inhibiting rate, is 100% component to inhibiting rate, dilute 10 times of multiple sieves, up to the inhibiting rate size that compares each component.According to this screening scheme, invention is at sample concentration 0.64 mgmL
-1Filtered out down and suppressed active 10 stronger components: garden burnet Chinese cassia tree reticulate millettia cloves Chinese torreyanut cymose buckwheat rhizome FRUCTUS TERMINALIAE IMMATURUS granatum the radix paeoniae rubrathe cynomorium songaricum acarbose.Concrete inhibition is active as shown in table 2
Table 2 water extract part is to the inhibition activity of alpha-glucosidase
| Water extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) | Water extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) |
| Garden burnet | 0.64 | 100.00 | FRUCTUS TERMINALIAE IMMATURUS | 0.64 | 95.26 |
| Chinese cassia tree | 0.64 | 99.72 | Granatum | 0.64 | 95.19 |
| Reticulate millettia | 0.64 | 99.60 | The radix paeoniae rubrathe | 0.64 | 75.88 |
| Cloves | 0.64 | 99.05 | Cynomorium songaricum | 0.64 | 74.52 |
| Chinese torreyanut | 0.64 | 96.84 | Cymose buckwheat rhizome | 0.64 | 95.36 |
| Acarbose | 0.64 | 8.73 | ? | ? | ? |
4, the screening at methanol extraction position
Consider the influence of organic solvent to alpha-glucosidase activity, the methanol extraction position is made into methanol solution, concentration is than water extract part (128 mgmL that double
1), get 5 μ L and join in the reaction model; Replace the negative contrast of phosphate buffered solution of equal volume simultaneously with 5 μ L methyl alcohol, all the other operation stepss are with the screening of water extract part.Invention is at sample concentration 0.64 mgmL
-1Filtered out down and suppressed active 10 stronger components: Chinese torreyanut cymose buckwheat rhizome Chinese gall garden burnet FRUCTUS TERMINALIAE IMMATURUS granatum reticulate millettia Chinese cassia tree rheum officinale Ramulus euonymi acarbose.Concrete inhibition is active as shown in table 3
Table 3 methanol extraction position is to the inhibition activity of alpha-glucosidase
| The methanol extraction part | Inhibition concentration (mgMl -1） | Inhibiting rate (%) | The methanol extraction part | Inhibition concentration (mgMl -1） | Inhibiting rate (%) |
| Chinese torreyanut | 0.64 | 99.69 | Reticulate millettia | 0.64 | 92.45 |
| Cymose buckwheat rhizome | 0.64 | 99.59 | Chinese cassia tree | 0.64 | 90.73 |
| Chinese gall | 0.64 | 93.39 | Rheum officinale | 0.64 | 88.37 |
| Garden burnet | 0.64 | 93.23 | Ramulus euonymi | 0.64 | 81.28 |
| FRUCTUS TERMINALIAE IMMATURUS | 0.64 | 92.66 | Granatum | 0.64 | 92.46 |
| Acarbose | 0.64 | 8.73 | ? | ? | ? |
5, the screening at ethyl acetate extraction position
Acetone and dimethyl sulfoxide (DMSO) (DMSO) all have dissolubility preferably to extract, but there is obvious suppression in acetone to enzymatic activity, and it is solvent that DMSO is selected in invention, the negative control of DMSO is set simultaneously, the same methanolic extract of concrete operations step.Invention is at sample concentration 0.64 mgmL
-1Filtered out down and suppressed active 10 stronger components: Chinese gall Chinese cassia tree rheum officinale the wind-weed reticulate millettia acarbose ramulus mori radix paeoniae rubrathe HERBA DENDROBII Chinese yam cynomorium songaricum.Concrete inhibition is active as shown in table 4
Table 4 ethyl acetate extraction position is to the inhibition activity of alpha-glucosidase
| The ethyl acetate extraction part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) | The ethyl acetate extraction part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) |
| Chinese gall | 0.64 | 92.94 | The radix paeoniae rubrathe | 0.64 | 19.51 |
| Chinese cassia tree | 0.64 | 74.88 | HERBA DENDROBII | 0.64 | 17.73 |
| Rheum officinale | 0.64 | 72.61 | Chinese yam | 0.64 | 16.55 |
| The wind-weed | 0.64 | 63.82 | Cynomorium songaricum | 0.64 | 14.64 |
| Reticulate millettia | 0.64 | 38.68 | Ramulus mori | 0.64 | 25.59 |
| Acarbose | 0.64 | 8.73 | ? | ? | ? |
6, the screening of methylene chloride extract part
Screening technique is with the screening at ethyl acetate extraction position.Invention is at sample concentration 6.4 mgmL
-1Filtered out down and suppressed active 10 stronger components: rheum officinale Chinese gall Radix Glycyrrhizae Poria cocos the root of bidentate achyranthes wind-weed reticulate millettia root bark of Chinese wolf-berry sealwort cairo morningglory root or leaf acarbose.Concrete inhibition is active as shown in table 5
Table 5 methylene chloride extract part is to the inhibition activity of alpha-glucosidase
| Methylene chloride extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) | Methylene chloride extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) |
| Rheum officinale | 6.4 | 100.00 | Reticulate millettia | 6.4 | 90.81 |
| Chinese gall | 6.4 | 100.00 | The root bark of Chinese wolf-berry | 6.4 | 90.76 |
| Radix Glycyrrhizae | 6.4 | 99.17 | Sealwort | 6.4 | 85.51 |
| Poria cocos | 6.4 | 96.83 | Cairo morningglory root or leaf | 6.4 | 84.94 |
| The root of bidentate achyranthes | 6.4 | 94.30 | The wind-weed | 6.4 | 93.27 |
| Acarbose | 6.4 | 21.29 | ? | ? | ? |
7, the screening of normal hexane extract part
Screening technique is with the screening of ethyl acetate extract.Invention is at sample concentration 6.4 mgmL
-1Filtered out down and suppressed active 10 stronger components: Chinese gall the root of bidentate achyranthes great burdock achene Poria cocos Radix Glycyrrhizae rheum officinale sealwort XIAKUQIU cymose buckwheat rhizome radix polygonati officinalis acarbose.Concrete inhibition is active as shown in table 6.
Table 6 normal hexane extract part is to the inhibition activity of alpha-glucosidase
| Normal hexane extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) | Normal hexane extracts part | Inhibition concentration (mgmL -1） | Inhibiting rate (%) |
| Chinese gall | 6.4 | 99.61 | Sealwort | 6.4 | 80.31 |
| The root of bidentate achyranthes | 6.4 | 99.53 | XIAKUQIU | 6.4 | 79.13 |
| Great burdock achene | 6.4 | 98.23 | Cymose buckwheat rhizome | 6.4 | 78.62 |
| Poria cocos | 6.4 | 93.57 | Radix polygonati officinalis | 6.4 | 77.78 |
| Radix Glycyrrhizae | 6.4 | 92.42 | Rheum officinale | 6.4 | 91.96 |
| Acarbose | 6.4 | 21.29 | ? | ? | ? |
8, use
The present invention's screening has obtained suppressing the active component of alpha-glucosidase, independent or the process appropriate combination of these components, can mix with pharmaceutically acceptable auxiliary interpolation composition, according to corresponding conventional medicine formulation method, preparation alpha-glucosidase restrainer class medicine, this medicine or pharmaceutical composition can adopt formulations such as tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol, can also adopt the known controlled release of modern medicine circle or slow release formulation or nanometer formulation; Described component also can be added in the various food, as the health food of control of blood sugar separately or through appropriate combination simultaneously.
Claims (6)
1. the screening technique of alpha-glucosidase restrainer is characterized in that: screening is concrete to divide for three steps:
(1) foundation of in-vitro screening model
Set up the vitro reactions model of alpha-glucosidase and substrate PNPG, use HPLC and detect hydrolysate PNP,, calculate the inhibiting rate of each extract part alpha-glucosidase by the concentration change of PNP;
(2) foundation of component pool
That chooses some has the Chinese medicine of remarkable result to treating diabetes, according to " similar compatibility " principle, ascending according to polarity, with multiple (〉=2 kinds) solvent every flavor Chinese medicine is carried out system and extract, adopt this method, every flavor Chinese medicine obtains a plurality of extract parts;
(3) screening
Each extract part of Chinese medicine is made into certain density solution, joins reaction and calculating inhibiting rate in the reaction model; Because the inhibition activity of different traditional Chinese medicinal components differs bigger, for suppressing active higher component, carry out multiple sieve according to identical method behind the concentration dilution, suppress active higher active site up to filtering out.
2. according to the screening technique of the described alpha-glucosidase restrainer of claim 1, it is characterized in that: the foundation of (2) component pool: with multiple 5 kinds of solvent solvents every flavor Chinese medicine being carried out system and extract, is respectively normal hexane, methylene chloride, ethyl acetate, methyl alcohol, water.
3. the application of alpha-glucosidase restrainer is characterized in that: the extract part of selected Chinese medicine is the Chinese medicine or the Application in Food of purpose in preparation to suppress alpha-glucosidase activity.
4. the application of alpha-glucosidase restrainer is characterized in that: selected traditional Chinese medicine extraction position reduces the medicine or the Application in Food of postprandial blood sugar, treatment or prevention diabetes and diabetic complication in preparation.
5. according to the application of claim 3 or 4 described alpha-glucosidase restrainers, it is characterized in that: preparation has the medicine that suppresses effect to alpha-glucosidase, adopt formulations such as tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol, or controlled release or slow release formulation or nanometer formulation.
6. according to the application of claim 3 or 4 described alpha-glucosidase restrainers, it is characterized in that: what screening obtained has the traditional Chinese medicine extraction position of remarkable inhibition effect to be meant the water extract part of garden burnet, Chinese cassia tree, reticulate millettia, cloves, Chinese torreyanut, cymose buckwheat rhizome, FRUCTUS TERMINALIAE IMMATURUS, granatum, the radix paeoniae rubrathe, cynomorium songaricum to alpha-glucosidase; The methanol extraction position of Chinese medicine Chinese torreyanut, cymose buckwheat rhizome, Chinese gall, garden burnet, FRUCTUS TERMINALIAE IMMATURUS, granatum, reticulate millettia, Chinese cassia tree, rheum officinale, Ramulus euonymi; The ethyl acetate extraction position of Chinese medicine Chinese gall, Chinese cassia tree, rheum officinale, the wind-weed, reticulate millettia, ramulus mori, the radix paeoniae rubrathe, HERBA DENDROBII, Chinese yam, cynomorium songaricum; The methylene chloride extract part of Chinese herb rhubarb, Chinese gall, Radix Glycyrrhizae, Poria cocos, the root of bidentate achyranthes, the wind-weed, reticulate millettia, the root bark of Chinese wolf-berry, sealwort, cairo morningglory root or leaf; The normal hexane extract part of Chinese medicine Chinese gall, the root of bidentate achyranthes, great burdock achene, Poria cocos, Radix Glycyrrhizae, rheum officinale, sealwort, XIAKUQIU, cymose buckwheat rhizome, radix polygonati officinalis.
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