阅读说明：本技术 一种柑橘属中药的在线活性筛选方法 (On-line activity screening method of citrus traditional Chinese medicine ) 是由 曹君 闫天赐 乐紫璇 顾郁欣 于 2021-07-29 设计创作，主要内容包括：本发明涉及酶抑制剂筛选领域,针对传统的酶抑制剂筛选方法效率低的问题,提供一种柑橘属中药的在线活性筛选方法,采用电泳介导的微量分析法EMMA,在毛细管中用脂肪酶催化水解底物的一步反应,然后在紫外400nm处直接测定产物峰面积实现脂肪酶抑制剂的在线筛选,最后利用分子对接技术对脂肪酶抑制剂的筛选结果进行验证。本发明将酶抑制剂的筛选与毛细管电泳相结合,不仅实现了脂肪酶抑制剂的在线筛选,也极大地提高了酶抑制剂筛选的效率,与传统的酶抑制剂筛选方法相比,本方法具有更短的分析时间、更高的分离效率、更少的试剂消耗。(The invention relates to the field of enzyme inhibitor screening, and provides an on-line activity screening method of citrus traditional Chinese medicines aiming at the problem of low efficiency of the traditional enzyme inhibitor screening method. The method combines the screening of the enzyme inhibitor with capillary electrophoresis, not only realizes the on-line screening of the lipase inhibitor, but also greatly improves the screening efficiency of the enzyme inhibitor.)

1. An on-line activity screening method of citrus traditional Chinese medicine is characterized in that an electrophoresis-mediated microanalysis method EMMA is adopted, a one-step reaction of a hydrolysis substrate is catalyzed by lipase in a capillary, then the peak area of a product is directly measured at the ultraviolet 400nm to realize the on-line screening of the lipase inhibitor, and finally the screening result of the lipase inhibitor is verified by utilizing a molecular docking technology;

the electrophoresis-mediated microanalysis method comprises the following steps:

(1) pretreatment: injecting background electrolyte solution BGE into a capillary in advance;

(2) and (3) injection: firstly, injecting a lipase solution into a capillary, and then injecting a substrate solution containing or not containing an inhibitor;

(3) mixing: applying a voltage of +1.0kV to fully mix the lipase and the substrate solution;

(4) hatching: the capillary tube is left to stand without external pressure and voltage;

(5) separation: the product was separated from unreacted substrate at constant voltage.

2. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the BGE of step (1) is selected from the group consisting of: borax, disodium hydrogen phosphate, sodium acetate and Tris-HCl buffer solution.

3. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the concentration of BGE in step (1) is 10-40 mM.

4. The on-line activity screening method for citrus traditional Chinese medicine according to claim 1, 2 or 3, wherein the BGE of step (1) has a pH of 6.4-9.0.

5. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the preparation method of the substrate solution in the step (2) comprises: dissolving 0.1% -0.2% (w/v) 4-nitrophenyl laurate in 5-10mM sodium acetate containing 1% -2% Triton X-100, heating in boiling water to aid dissolution, and cooling the solution to room temperature to obtain a substrate solution.

6. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1 or 5, wherein in the step (2), after each sample injection of the capillary tube, the sample injection end is immersed in deionized water for cleaning.

7. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the mixing conditions in step (3) are as follows: the +1.0kV voltage is applied for 5-35 s.

8. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the standing time in step (4) is 1-5 min.

9. The on-line activity screening method of citrus traditional Chinese medicine according to claim 1, wherein the constant voltage in step (5) is 20-25 kV.

Technical Field

The invention relates to the field of enzyme inhibitor screening, in particular to an on-line activity screening method for citrus traditional Chinese medicines.

Background

Obesity, which has been considered by the World Health Organization (WHO) as the largest epidemic in humans, is an important risk factor for a range of pathological conditions, such as hypertension, kidney disease, diabetes, hyperlipidemia, coronary heart disease, psychiatric disorders and certain cancers. Studies have shown that obesity is caused by excessive or abnormal accumulation of fat. Triglycerides are the most abundant fat types in the body and are stored mainly in adipose tissue. Lipases, also known as triacylglycerol acylhydrolases, are a class of esterases that play a key role in the complete digestion of triglycerides, being able to hydrolyze from 50% to 70% of dietary fat to glycerol and fatty acids. Therefore, inhibiting the activity of lipase can effectively inhibit the hydrolysis and absorption of fat, thereby achieving the effect of losing weight. Inhibition of lipase activity is a hot topic against obesity and its related diseases. Orlistat is the only lipase inhibitor approved for long-term weight management. However, it often shows strong gastrointestinal side effects such as flatulence, nausea, diarrhea and vomiting. Therefore, it is very urgent and essential to find safe, effective, novel lipase inhibitors for the treatment of obesity.

Natural products are of great interest because of their abundance in biologically active compounds. Citrus fruits are the main edible fruits in all countries in the world and are also traditional medicinal plants in China. It is one of the most important dietary sources of flavonoids. Citrus flavones contain several phenolic hydroxyl groups, such as glycosides like hesperidin and naringin. Related studies have shown that citrus flavonoids possess many biological activities, such as antioxidant, antiviral, anti-inflammatory, anticancer, and anti-atherosclerotic properties. Research shows that the citrus flavone is used for regulating antioxidant indexes through directly or indirectly mediating AMPK, PPAR and NF-kB signal channels so as to treat obesity, but the influence of the citrus flavone on the activity of lipase is rarely reported. Therefore, the screening of lipase inhibitors from citrus flavonoids was chosen in this experiment.

The traditional enzyme assay method is to incubate the enzyme and substrate off-line and then measure the result with a spectrophotometer or enzyme reader. However, this method is low in automation, long in time, large in sample consumption, and not suitable for high-throughput screening of enzyme inhibitors. In recent years, several new enzymatic analytical methods, such as ultrafiltration, hollow fiber immobilization, and magnetic nanoparticle immobilization, have been used for enzyme inhibitor screening experiments. However, these methods can only be used for off-line analysis. Capillary Electrophoresis (CE) has attracted considerable attention today as an on-line enzymatic analytical tool. Research shows that CE is widely applied to screening of enzyme inhibitors and determination of enzyme kinetic parameters due to the advantages of short analysis time, easy automation, high separation efficiency, low reagent consumption and the like. Electrophoresis-mediated microanalysis (EMMA) is a widely used modality in CE. In EMMA, a capillary is used as a microreactor in which reactants can be injected sequentially into the same capillary for enzymatic reactions, separations, and detection. The method of EMMA was used by Lilin Tang et al to screen tyrosinase inhibitors in traditional Chinese medicine. Jing Han et al screened a Chinese medicine with cathepsin B inhibitory activity from 12 Chinese medicine extracts by EMMA method. Haiong Wang et al developed an EMMA method for screening aminopeptidase N inhibitors (Rong Hailong. 2015. study of the on-line screening method for aminopeptidase N inhibitors by capillary electrophoresis. Doctorl Disservation, university of Shandong.). However, because lipase substrates are difficult to dissolve and have poor stability after dissolution, screening of lipase inhibitors by the EMMA method has been recently reported.

Disclosure of Invention

The invention aims to overcome the problem of low efficiency of the traditional enzyme inhibitor screening method, and provides the on-line activity screening method of the citrus traditional Chinese medicine, which combines the screening of the enzyme inhibitor with capillary electrophoresis, thereby not only realizing the on-line screening of the lipase inhibitor, but also greatly improving the screening efficiency of the enzyme inhibitor. Compared with the traditional enzyme inhibitor screening method, the method has the advantages of shorter analysis time, higher separation efficiency and less reagent consumption.

In order to achieve the purpose, the invention adopts the following technical scheme:

an on-line activity screening method of citrus traditional Chinese medicine adopts an electrophoresis-mediated microanalysis method EMMA, one-step reaction of a substrate is catalyzed and hydrolyzed by lipase in a capillary, then the peak area of a product is directly measured at the ultraviolet 400nm to realize on-line screening of the lipase inhibitor, and finally, the molecular docking technology is utilized to verify the screening result of the lipase inhibitor;

the electrophoresis-mediated microanalysis method comprises the following steps:

(1) pretreatment: injecting background electrolyte solution BGE into a capillary in advance;

(2) and (3) injection: firstly, injecting a lipase solution into a capillary, and then injecting a substrate solution containing or not containing an inhibitor;

(3) mixing: applying a voltage of +1.0kV to fully mix the lipase and the substrate solution;

(4) hatching: the capillary tube is left to stand without external pressure and voltage;

(5) separation: the product was separated from unreacted substrate at constant voltage.

The invention develops a method (EMMA method) for screening lipase inhibitors on line mediated by capillary electrophoresis, and evaluates the inhibition activity of citrus traditional Chinese medicinal materials by combining a molecular docking technology. By measuring the Michaelis constant (K) of lipase_m) To evaluate the effectiveness of the EMMA method and to measure the half maximal Inhibitory Concentration (IC) of orlistat, a commercially available lipase inhibitor₅₀) And inhibition constant (K)_i). Meanwhile, the inhibition effect of the citrus traditional Chinese medicine extract and several flavonoid compounds on lipase is evaluated by using the method, and the compounds with lipase inhibition potential are screened out. In addition, the type and content of flavonoid compounds in the extract were analyzed by ultra performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Finally, the inhibition effect of the flavonoid compound on the lipase is theoretically verified by utilizing molecular docking research.

Preferably, the BGE of step (1) is selected from: borax, disodium hydrogen phosphate, sodium acetate and Tris-HCl buffer solution. Preferably, the BGE in the step (1) is borax

Preferably, the BGE in step (1) is at a concentration of 10-40 mM. Further preferably, the BGE in step (1) is at a concentration of 20 mM.

Preferably, the BGE of step (1) has a pH of 6.4-9.0. Further preferably, the BGE of step (1) has a pH of 8.8.

Preferably, the preparation method of the substrate solution in the step (2) comprises the following steps: dissolving 0.1% -0.2% (w/v) 4-nitrophenyl laurate in 5-10mM sodium acetate containing 1% -2% Triton X-100, heating in boiling water to aid dissolution, and cooling the solution to room temperature to obtain a substrate solution.

Preferably, in the step (2), after each sample injection of the capillary, the sample injection end is immersed in deionized water for cleaning. To prevent mutual contamination.

Preferably, the mixing conditions in step (3) are as follows: the +1.0kV voltage is applied for 5-35 s. More preferably, the mixing time in step (3) is 25 s.

Preferably, the standing time in the step (4) is 1-5 min. Further preferably, the standing time in the step (4) is 3 min.

Preferably, the constant voltage in step (5) is 20-25 kV.

Therefore, the beneficial effects of the invention are as follows: (1) an on-line screening method of lipase inhibitors based on capillary electrophoresis is established; (2) the screening result of the lipase inhibitor is verified by utilizing a molecular docking technology; (3) the method is rapid, efficient, low in cost and suitable for high-throughput screening of enzyme inhibitors.

Drawings

FIG. 1 is an electrophoretogram of different types of background buffers of examples 1-4 after in-line reaction;

FIG. 2 is a graph of apparent mobility and peak area for the products of examples 5-8 at different borax concentrations;

FIG. 3 is a graph of apparent mobility and peak area for the products of examples 9-13 borax at different pH values;

FIG. 4 is a graph of peak areas of the products of examples 14-18 at various mixing times;

FIG. 5 is a graph of the peak areas of the products of examples 19-23 at different incubation times;

FIG. 6 is a graph (a) and a double reciprocal plot (b) of the Michaelis equation for lipase enzymatic reactions at different substrate concentrations (0.2 to 4 mM);

fig. 7 is a graph of the inhibition of orlistat;

FIG. 8 is a molecular docking diagram of lipase with hesperetin (A), nobiletin (B), hesperetin (C) and naringenin (D).

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.

First, reagent preparation

1. Background electrolyte solution (BGE): borax was dissolved in ultrapure water to a final concentration of 20mM and the pH was adjusted with 1.0M boric acid and 1.0M NaOH solution.

2. Enzyme solution: the lipase was dissolved in 20mM borax solution to a final concentration of 50mg/mL, and the enzyme solution was placed in a 1.5mL centrifuge tube and stored in a refrigerator at 4 ℃.

3. Substrate solution: 0.1% (w/v) of 4-nitrophenyl laurate was dissolved in 5mM sodium acetate containing 1% Triton X-100 and heated in boiling water for 2min to aid dissolution, and the solution was then cooled to room temperature for further use.

4. Target analyte solution: eight flavonoids were dissolved in methanol and diluted with 50% methanol (methanol: DMSO ═ 1:1) to appropriate concentrations. All solutions were filtered through a 0.22 μm nylon membrane before use.

5. Preparing a medicinal material extracting solution: pulverizing dried Citrus medicinal materials with high speed pulverizer, and sieving with 40 mesh sieve. 500mg of fruit powder and 50mL of methanol were added to the capped Erlenmeyer flask, and the mixture was then sonicated at 40kHz for 40 minutes. After ultrasonic extraction, the mixture was filtered. The filtrate was evaporated to dryness in a water bath at 75 ℃ and the residue was dissolved in 4mL of 50% DMSO (methanol: DMSO ═ 1: 1). After complete dissolution, the solution was centrifuged at 13,000rpm for 5 minutes and the supernatant collected in a centrifuge tube for use.

Second, use the instrument

1. The EMMA method was performed on an Agilent 7100HP3D CE system equipped with a diode array detector and Agilent chemical workstation software. Bare fused silica capillary tubes (50 μm inner diameter; 48.5cm full length; 40cm effective length) for CE separation were purchased from perpetual ruifeng chromatography equipment ltd (hebei, china). The capillary was thermostated at 25 ℃ and the detector set at UV wavelength 400 nm. The pH of the solution was measured and adjusted using a PHS-3C pH meter purchased from shanghai instruments and electrosciences instruments ltd (shanghai, china). The new capillary tubes were rinsed with 1M NaOH and 0.1M NaOH for 10 minutes, respectively, and then with deionized water for 5 minutes before use. Before the start of the experiment, the capillary was rinsed with running buffer for 3 to 5 minutes to stabilize the capillary. Between runs, the capillary was sequentially rinsed with 0.1M NaOH for 2 minutes, water for 2 minutes, and BGE solution for 3 minutes. At the end of each day experiment, the capillary was rinsed with deionized water for 3 minutes to ensure its interior surface was clean.

2. The UHPLC-Q-TOF/MS system for qualitative analysis was equipped with an autosampler, column oven, diode array detector, binary pump, online degasser and Agilent 6560QTOF MS system equipped with ESI source. Chromatographic separation of target analytes in Agilent SB-C₁₈On a column (50 mm. times.2.1 mm. times.1.8 μm). The mobile phases were 0.1% formic acid (phase a) and acetonitrile (phase B). The step of elution gradient was as follows: 0-2 min, 5-20% of phase B; 20-30% of phase B in 2-4 min; 4-6 min, 30% of phase B; 30-100% of phase B in 6-8 min; at 8-9 minutes, 100% of phase B; 100% -5% of phase B in 9-10 min. The flow rate was 0.4mL/min and the amount of sample was 2. mu.L. The negative ion mode was used for QTOF-MS analysis, with mass to charge ratio ranges recorded as 100 to 1000 Da. The optimal ionization parameters are: capillary voltage, 3.5 kV; atomizer, 35 psig; fragmentation voltage, 175V; the temperature of the drying gas is 300 ℃; the temperature of the sheath gas is 350 ℃; the flow rates of the drying gas and the sheath gas were 10L/min and 11L/min, respectively.

Thirdly, establishment of screening method

1. Examples 1-4BGE species selection

Examples 1-4 the effect of four common running buffers on the peak area, peak symmetry and migration time of the product was investigated, 20mM sodium borate, disodium hydrogen phosphate, sodium acetate and Tris-HCl buffers, respectively. The other experimental conditions are the same and are as follows: sampling for 6s under the pressure of 50 mbar; mixing the samples, 1.0kV multiplied by 25 s; incubation time, 3 minutes; separation voltage: 25 kV; the lipase concentration: 50 mg/mL; substrate concentration: 1 mg/mL; detection wavelength: 400 nm; the column temperature was 25 ℃.

As shown in FIG. 1, the migration times of the products in these four buffer solutions are not very different. And the peak area of the product was larger in the sodium acetate buffer and borax buffer compared to the other two buffers, probably because the buffers affected the enzyme-substrate interaction. In addition, there was a peak of impurities in the sodium acetate buffer, which could be a by-product during the reaction. And, in borax buffer, the product peak symmetry is best. Combining all of the above factors, borax buffer solution was found to be the best of all four buffer solutions.

2. Examples 5-8 optimization of BGE concentration

Examples 5-8 the peak areas of the products at four concentrations of borax solution were investigated, 10mM, 20mM, 30mM and 40mM respectively. The other experimental conditions are the same and are as follows: sampling for 6s under the pressure of 50 mbar; mixing the samples, 1.0kV multiplied by 25 s; incubation time, 3 minutes; separation voltage: 25 kV; the lipase concentration: 50 mg/mL; substrate concentration: 1 mg/mL; detection wavelength: 400 nm; the column temperature was 25 ℃.

As shown in FIG. 2, the peak area of the product increased and then decreased with increasing borax concentration, and reached a maximum at a concentration of 20 mM. The reason may be that a low concentration of borax (10mM) is insufficient to allow effective ionization of the reaction product, while a high concentration of sodium borate (40mM) inhibits lipase activity. Furthermore, the results show that the apparent mobility of the product decreases with increasing borax concentration. This is because high concentrations of buffer can generate excessive Joule heat and increase its viscosity to lower the EOF, thereby extending migration time. Apparent mobility (. mu.)_a) Can be calculated by formula (2)

Wherein U is a voltage, t_pAs migration time of the product, L_aFor the full length of the capillary, L_eIs the effective capillary length. Taken together, sodium borate buffer solution at a concentration of 20mM was best suited for separation and had the greatest target analyte response and lower Joule heat generation.

3. Examples 9-13 optimization of BGE solution pH

Examples 9-13 the effect of BGE solution pH on sample separation, which plays a crucial role in sample separation by affecting the activity of EOF and enzymes, was investigated, respectively for borax buffer solutions with the same concentration (20mM) but pH values of 6.4, 7.0, 8.0, 8.8 and 9.0, respectively. The other experimental conditions are the same and are as follows: sampling for 6s under the pressure of 50 mbar; mixing the samples, 1.0kV multiplied by 25 s; incubation time, 3 minutes; separation voltage: 25 kV; the lipase concentration: 50 mg/mL; substrate concentration: 1 mg/mL; detection wavelength: 400 nm; the column temperature was 25 ℃.

As can be seen from the results in FIG. 3, the peak area of the product was maximal at pH 8.8, indicating that the lipase activity was maximal at a buffer pH of 8.8. At the same time, the apparent flowability of the product did not change significantly, indicating that the pH had little effect on the EOF. In summary, a buffer with a pH of 8.8 was selected for subsequent experiments.

4. Examples 14-18 Effect of reaction time on reaction product yield

The reaction time has a great influence on the on-line enzymatic reaction. In the EMMA procedure, it is important to mix the enzyme and substrate thoroughly. Thus, examples 14-18 investigated the effect of mixing time (5, 10, 15, 20, 25, 30, 35s) on the yield of the reaction product 4-nitrophenol (pNP) by applying a mixing voltage of +1.0 kV. The other experimental conditions are the same and are as follows: sampling for 6s under the pressure of 50 mbar; separation voltage: 25 kV; the lipase concentration: 50 mg/mL; substrate concentration: 1 mg/mL; detection wavelength: 400 nm; the column temperature was 25 ℃.

FIG. 4 shows the relationship between mixing time and product peak area. It can be seen that the peak area of the product increases with increasing mixing time, with peak area reaching a maximum at 25s mixing time. When the mixing time exceeded 25s, the peak area of the product decreased. Therefore, a mixing time of 25s was chosen for subsequent experiments.

5. Examples 19-23 Effect of incubation time on reaction product yield

Examples 19-23 the effect of incubation times (1, 2, 3, 4, 5 minutes) was investigated on the basis of the mixing time of 25s selected in examples 14-18. As shown in fig. 5, the formation of the product was almost linear within 3 minutes, while a decrease in the product formation rate was observed after 3 minutes, probably due to insufficient amount of substrate. Therefore, an incubation time of 3 minutes was chosen as the optimal time.

Fourth, result verification and application in on-line activity screening of citrus traditional Chinese medicine

1. Determination of kinetic parameters of lipases

K_mDepending on the reaction conditions and the type of enzyme, it may roughly indicate the affinity between the enzyme and the substrate. As shown in FIGS. 6a and 6b, a Lineweaver-Burk nonlinear curve and a double reciprocal plot were constructed by measuring the peak area of the product at 6 different substrate concentrations (0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4 mM). According to equation (3):

the resulting linear regression equation with reciprocal y of 0.02830x +0.02103, R²When the lipase K is calculated as 0.9900_mThe value was 1.345 mM. Substantially consistent with the values reported in the literature as determined by the EMMA method (1.28mM-3.44 mM).

To validate this method, an inhibition profile was established using the known inhibitor orlistat by varying its concentration in the range of 0.001 to 2000 μ g/mL (fig. 7). IC of orlistat₅₀0.01512 μ g/mL, which is lower than the off-line method, indicating that the EMMA method developed in this study is suitable for screening lipase inhibitors. On the other hand, K calculated by the formula (4)_iThe value was 0.0087. mu.g/mL.

2. Screening of Lipase inhibitors from Citrus flavones

Eight common flavonoids (concentration is 150 mug/mL) in citrus are selected for screening lipase inhibitors. The inhibition was measured in comparison with a blank control without inhibitor added and is shown in table 1. The results show that the eight flavonoid compounds all show inhibition potential, the inhibition rate range is 30-46%, and the results are consistent with the results of previous researches. In addition, the inhibition rates of the four flavonoid aglycones are all over 40 percent, and the inhibition rates of the four flavonoid glycosides are all lower than 40 percent. The reason may be that the presence of sugar groups may reduce the inhibitory activity of the compound. In addition, the inhibitory activities of the four flavonoid aglycones were compared, and the inhibitory activity was found to be highest when the hydroxyl group was at the 7' -position. In addition, by comparing nobiletin and hesperetin, it was found that the more methoxy groups on the benzene ring, the poorer the inhibitory activity.

TABLE 1 inhibition of flavonoids

Compound (I)	Inhibition rate	Compound (I)	Inhibition rate
				Hesperidin	30.53％	Hesperetin	43.42％
Rutachinin	38.66％	Naringenin	45.66％
				Neohesperidin	35.01％	Nobiletin	41.17％
Naringin	33.05％	Orange peel essence	43.69％

3. Molecular docking study

In order to further verify the results of inhibition experiments, molecular docking research is adopted to simulate the combination mode of the crystal structures of hesperetin, nobiletin, hesperetin and naringenin and lipase. The best binding conformation is seen in fig. 8, and the amino acid residues for which lipases hydrogen bond with the four compounds are listed in table 2. Docking results show that the binding energy of naringin is lowest, which indicates that the inhibitory activity is highest and is consistent with the inhibition experiment results. In addition, naringin forms hydrogen bonds with TRY-115, ASP-80, HIS-152 and TRP-86, indicating that TRY-115, ASP-80, HIS-152 and TRP-86 are closely related to the catalytic activity of lipase. Catalytic activity of lipase. The formation of hydrogen bonds between the inhibitor and the amino acid residues of the lipase affects the interaction of the enzyme with the substrate. Furthermore, the results indicate that the more hydroxyl groups on the phenyl ring, the more hydrogen bonds are formed, which indicates that the binding ability of the inhibitor to lipase is related to the type and number of substituents on the phenyl ring.

TABLE 2 docking results of hesperetin, cinnamyl element, hesperetin, naringenin, and lipase

4. Screening lipase inhibitor from citrus Chinese medicinal material

The inhibitor screening method established by the experiment is adopted to research the inhibition activity of the six citrus extracts, and the inhibition rate results are shown in table 3. In addition, eight flavonoid compound mixed standard substances are used as a reference, and UHPLC-Q-TOF/MS is adopted to analyze the types and the contents of the flavonoid compounds in the extract, and the results are shown in a table 4. Among the citrus extracts measured, the inhibitory activity of the tangerine pith extract was the highest, and the inhibitory rate was 40.67%.

TABLE 3 inhibition of methanol extract of citrus drug

Medicinal materials	Inhibition rate	Medicinal materials	Inhibition rate
				Fructus Aurantii	34.29％	Finger citron	22.94％
Dried orange peel	31.50％	Immature bitter orange	21.10％
				Tangerine pith	40.67％	Exocarpium Citri Grandis	30.89％

TABLE 4 content of target analytes in Citrus crude drugs

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.