Smilax glabra quality evaluation method based on xanthine oxidase inhibitory activity

Abstract

The invention relates to a rhizoma smilacis glabrae quality evaluation method based on xanthine oxidase inhibitory activity. According to the method, the quantity of uric acid generated by xanthine under the catalytic action of xanthine oxidase is measured, and the semi-inhibitory concentration (IC50) of the xanthine oxidase is used as an evaluation index, so that the quality of rhizoma smilacis glabrae is evaluated. Specifically, when the IC50 value of the xanthine oxidase inhibitory activity of the rhizoma smilacis glabrae is lower than the standard, the rhizoma smilacis glabrae is effective. Compared with a traditional quality control method depending on chemical component analysis, the method has the advantages that the quality evaluation of the rhizoma smilacis glabrae is directly associated with the efficacy of the rhizoma smilacis glabrae, the detection cost is reduced, and the operation process is simplified. Through simple and convenient uric acid generation amount detection, the method can effectively judge the quality of the rhizoma smilacis glabrae medicinal material, and has popularization and application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of quality evaluation of traditional Chinese medicines, and particularly relates to a method for evaluating the quality of smilax glabra based on xanthine oxidase inhibition activity. Background Art

[0002] Smilax glabra is the dried rhizome of Smilax glabra Roxb., a plant of the Liliaceae family. It is a variety included in the previous editions of the Chinese Pharmacopoeia. Since 2005, the Chinese Pharmacopoeia has formulated a method for determining the content of Smilax glabra using astilbin as the index component, and stipulates a minimum limit of 0.45%. Chinese herbal medicine production and business units in many provinces and regions in China have continuously reported that the limit of astilbin content stipulated in the first part of the Chinese Pharmacopoeia is too high, and it is difficult for domestic Smilax glabra to meet this limit requirement. The Zhejiang Provincial Institute of Food and Drug Inspection has collected and purchased a total of 17 batches of samples from Hunan, Guangdong, Zhejiang, Jiangxi and other provinces and cities for determination. As a result, 13 batches of genuine Smilax glabra did not meet the minimum limit requirements, of which one batch of genuine samples did not detect astilbin, and one batch of genuine samples contained only 0.07% astilbin, indicating that the content determination method stipulated in the Chinese Pharmacopoeia is difficult to accurately control the quality of Smilax glabra.

[0003] Astilbin, neoastilbin, neoisoastilbin, isoastilbin, flavonoids, isoflavones, 5-O-caffeoylshikimic acid are the main components of Smilax glabra, but whether the above components are the effective components of Smilax glabra needs to be studied and clarified, and the content determination method with the above components as indicators is difficult to reasonably evaluate the medicinal material quality of Smilax glabra, and the quality evaluation method of Smilax glabra needs to be improved. In the field of traditional Chinese medicine, Smilax glabra is widely used in clinical practice because of its remarkable effect in treating hyperuricemia and gout, and becomes the core Chinese medicine for treating gout. Xanthine oxidase is a key enzyme in the uric acid metabolic pathway, and the inhibition of its activity can effectively reduce the generation of uric acid, thereby playing a key role in the treatment of hyperuricemia and gout. Based on this, the present invention proposes a Smilax glabra quality evaluation method based on xanthine oxidase inhibitory activity, which is not only directly related to the drug effect of Smilax glabra, but also through biological activity evaluation, a kind of Smilax glabra quality evaluation means closely connected with clinical efficacy is provided. This method can evaluate the quality of Smilax glabra medicinal materials more objectively and reasonably, and provides a new technical path for the modernization and standardization of traditional Chinese medicine. Summary of the invention

[0004] The present invention aims to solve the deficiencies of existing Smilax glabra medicinal material quality evaluation methods, and proposes a new quality evaluation method, which is directly related to drug efficacy, based on the determination of xanthine oxidase inhibitory activity, so as to objectively and reasonably evaluate the quality of Smilax glabra medicinal materials. The method of the present invention has the following characteristics: detection using conventional laboratory instruments and equipment, easy operation, and low cost; by the determination of xanthine oxidase inhibitory activity, the drug efficacy of Smilax glabra is directly reflected, and the accuracy of evaluation is improved; the method is highly applicable, suitable for laboratories and production enterprises of different sizes, and helps to unify the quality standard of Smilax glabra medicinal materials; the method of the present invention not only improves the efficiency of Smilax glabra medicinal material quality evaluation, but also helps to ensure the efficacy and safety of medicinal materials, which is of great significance for improving the modernization level of traditional Chinese medicine.

[0005] In order to achieve the purpose of the above invention, the present invention provides the following technical solutions:

[0006] A method for evaluating the quality of Smilax glabra based on xanthine oxidase inhibitory activity is developed by measuring the amount of uric acid generated by xanthine under the catalysis of xanthine oxidase, using the half inhibition concentration (IC 50 ) was used as the evaluation index to assess the quality of Smilax glabra.

[0007] In the above technical solution, further, the method comprises the following steps:

[0008] Step 1, preparing the tested Rhizoma Smilacis Glabrae into powder, and treating it to obtain Rhizoma Smilacis Glabrae extract; taking the Rhizoma Smilacis Glabrae extract, and adding phosphate buffer to prepare Rhizoma Smilacis Glabrae extract solutions of different concentrations;

[0009] Step 2, take a uric acid reference substance, add sodium hydroxide to dissolve, add water to dilute to prepare a reference solution, store at 4°C for standby use; the sodium hydroxide concentration is usually 0.1 mol / L;

[0010] Step 3, take xanthine, add phosphate buffer to prepare xanthine solution, store at 4°C for later use;

[0011] Step 4, taking xanthine oxidase, adding phosphate buffer to prepare xanthine oxidase solution, and storing at 4°C for later use;

[0012] Step 5, take the xanthine oxidase solution, add phosphate buffer, incubate at 37°C for 30 minutes, add xanthine solution, incubate at 37°C for 120 minutes, and place in a boiling water bath for 5 minutes to obtain a blank test solution;

[0013] Step 6: Take the same volume of xanthine oxidase solution as in step 5, add the same volume of Smilax glabra extract solution as in step 5, incubate at 37° C. for 30 min, add the same volume of xanthine solution as in step 5, incubate at 37° C. for 120 min, and place in a boiling water bath for 5 min to obtain a sample test solution;

[0014] Step 7, using a chromatographic column, with 0.1% glacial acetic acid-methanol (90:10) as the mobile phase, the detection wavelength is 291nm, the column temperature is 30°C, and the injection volume is 5μL for determination;

[0015] Step 8: Using the peak area as a variable, calculate the inhibition rate of xanthine oxidase at each concentration of Smilax glabra extract solution according to the following formula: Where A is the peak area of ​​uric acid in the blank solution, and B is the peak area of ​​uric acid in the sample solution. The half inhibition concentration (IC 50 ); comparing the half inhibition concentration with the standard. If the half inhibition concentration does not exceed the standard, the tested Smilax glabra sample is an effective Smilax glabra.

[0016] Furthermore, the treatment in step 1 to obtain the Smilax glabra extract includes: placing the Smilax glabra powder in a container, adding 70% ethanol by volume, ultrasonic extraction, supplementing the missing weight with 70% ethanol again, shaking well, taking the supernatant and then steaming it, adding water to dissolve it, and freeze-drying it to obtain the Smilax glabra extract.

[0017] Furthermore, the pH of the phosphate buffer is 7.2-7.4.

[0018] Furthermore, the concentration of the Smilax glabra extract solution of different concentrations is selected from 20 to 1.25 mg / mL.

[0019] Furthermore, the uric acid concentration in the reference solution in step 2 is 50 μg / mL, the concentration of the xanthine solution in step 3 is 2 mmol / L, the concentration of the xanthine oxidase solution in step 4 is 0.5 U / mL, the xanthine solution and the xanthine oxidase solution in step 5 are 50 μL, and the volume of the phosphate buffer is 100 μL.

[0020] Further, the standard described in step 8 is 20.0 mg / mL.

[0021] The method of the present invention also carried out a methodological investigation, including linear range, precision, repeatability, stability and other aspects. The results showed that the method was stable and feasible. Using the above-established quality evaluation method for Smilax glabra, 19 batches of Smilax glabra samples were measured, and the results all met the requirements. Through the determination, it was found that among all the Smilax glabra samples confirmed as authentic, those that did not contain astilbin or had a low content of astilbin had an IC value of xanthine oxidase inhibition of 1.34. 50 The value was lower than that of some samples with higher astilbin content, and all genuine Smilax glabra samples showed an inhibitory effect on xanthine oxidase. This result shows that the method of the present invention can more effectively evaluate the quality of Smilax glabra.

[0022] The beneficial effects of the present invention are:

[0023] 1. Improve evaluation accuracy: Through the determination of xanthine oxidase inhibitory activity, the present invention provides a quality evaluation method directly related to the medicinal effect of Smilax glabra. Compared with the traditional chemical component analysis method, it can more accurately reflect the actual therapeutic effect of the medicinal material.

[0024] 2. Reduce testing costs: The testing conditions and instruments used in the present invention are all conventional laboratory configurations, and no expensive special equipment is required. Compared with traditional chemical composition analysis methods, there is no need to purchase expensive reference materials, thereby significantly reducing the cost of quality evaluation.

[0025] 3. Promote the modernization of traditional Chinese medicine: The method of the present invention provides a new technical means for the modernization and standardization of traditional Chinese medicine, which helps to promote the development of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the chromatogram of uric acid reference substance;

[0027] Figure 2 It is the sample test liquid chromatogram;

[0028] Figure 3 is the blank assay liquid chromatogram;

[0029] Figure 4 It is the chromatogram of the assay liquid of domestic samples at 20, 15, 10, 5, 2.5, and 1.25 mg / mL;

[0030] Figure 5 This is the chromatogram of the test liquid of the sample produced in Vietnam at 20, 15, 10, 5, 2.5, and 1.25 mg / mL;

[0031] Figure 6 It is domestically produced Smilax glabra IC 50 Measurement results;

[0032] Figure 7 It is Vietnamese-produced Smilax glabra IC50 Measurement results. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The experimental methods for which specific conditions are not specified in the embodiments are generally conventional methods in the art. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0034] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0035] Example 1

[0036] The method for evaluating the quality of Smilax glabra based on xanthine oxidase inhibition activity comprises the following steps:

[0037] Step 1, take 5.0g of the powder of the Chinese smilax glabra sample (passed through a No. 2 sieve), accurately weigh it, put it in a stoppered conical flask, add 200mL of 70% ethanol, weigh it, ultrasonically extract it for 30min, weigh it again, make up the missing weight with 70% ethanol, shake it well, centrifuge it at 3000rmp / min for 10min, take the supernatant and evaporate it at 45°C, add 10mL of water to dissolve it, and freeze-dry it to obtain the Chinese smilax glabra extract; take the Chinese smilax glabra extract, accurately weigh it, add phosphate buffer (pH7.2-7.4) to prepare Chinese smilax glabra extract solutions with concentrations of 20, 15, 10, 5, 2.5, and 1.25mg / mL respectively;

[0038] Step 2: Take a uric acid reference substance, weigh it accurately, add 1.0 mL of 0.1 mol / L sodium hydroxide to dissolve it, dilute it with water to make a reference substance solution with a concentration of 50 μg / mL, and store it at 4°C for later use;

[0039] Step 3, take xanthine, weigh accurately, add phosphate buffer (pH7.2-7.4) to prepare a xanthine solution with a concentration of 2mmol / L, store at 4°C for later use;

[0040] Step 4, take xanthine oxidase, weigh accurately, add phosphate buffer (pH 7.2-7.4) to make a xanthine oxidase solution with a concentration of 0.5 U / mL, store at 4°C for later use;

[0041] Step 5: Use a Shimadzu WondaCract ODS-2 (4.6 mm × 250 mm, 5 μm) chromatographic column with a mobile phase of 0.1% acetic acid-methanol and isocratic elution (10% methanol); detection wavelength 291 nm, column temperature 30°C, injection volume 5 μL.

[0042] Step 6, for 19 batches of Smilax glabra samples from different sources, respectively, domestic (label G1-G11), Vietnam (label Y1-Y8), specific information see table. The collected Smilax glabra samples, a part of the samples are collected, and the collected samples are identified as Liliaceae plant Smilax glabra Roxb. by Chen Hao, the head of the Chinese medicine institute of Zhejiang Food and Drug Inspection Institute. A part of the samples are purchased from the decoction company, and are identified as the dry rhizome of Smilax glabra Roxb. according to the Chinese Pharmacopoeia. That is, they are all genuine. The collected Smilax glabra samples are divided into two categories according to the origin of origin, and those produced in China are called domestic Smilax glabra, and those produced in Vietnam are called Vietnamese Smilax glabra. All samples confirmed as genuine Smilax glabra were tested for astilbin content according to the method specified in the "Chinese Pharmacopoeia" under the "Astellin content determination" item of Smilax glabra varieties. Among them, the astilbin content of domestic samples numbered G3 and G6 and all Vietnamese Smilax glabra samples was higher than 0.45%, which met the standard requirements of the "Chinese Pharmacopoeia". The astilbin content of domestic samples numbered G1, G2, G4, G5, G7, G8, G9, G10, and G11 was lower than 0.45%, which did not meet the standard requirements of the "Chinese Pharmacopoeia".

[0043] Table 1 Source information of Smilax glabra samples

[0044]

[0045] Step 7, xanthine oxidase inhibitory activity determination: take 50 μL of xanthine oxidase solution, add 100 μL of phosphate buffer (pH 7.2-7.4), incubate at 37° C. for 30 min, add 50 μL of xanthine solution, incubate at 37° C. for 120 min, and place in a boiling water bath for 5 min to obtain a blank assay solution; take 50 μL of xanthine oxidase solution, add 100 μL of Smilax glabra extract solution, incubate at 37° C. for 30 min, add 50 μL of xanthine solution, incubate at 37° C. for 120 min, and place in a boiling water bath for 5 min to obtain a sample assay solution; using peak area as a variable, calculate the inhibition rate of Smilax glabra on xanthine oxidase at each concentration according to the following formula: X%, X%=(AB) / A×100%, where A is the uric acid peak area in the blank assay solution, and B is the uric acid peak area in the sample assay solution; using GraphPadPrism 7.0 software analysis, the half inhibition concentration (IC 50 ).

[0046] Table 2 Results of xanthine oxidase inhibition activity determination of Smilax glabra (n=3, i.e., parallel times)

[0047]

[0048] It can be seen from this example that all the tested Smilax glabra extracts can significantly inhibit the activity of xanthine oxidase, and their half inhibition concentration (IC 50 ) were all lower than 20 mg / mL, so it was set as the threshold. The setting of this threshold provided a clear quantitative indicator for the efficacy of Smilax glabra.

[0049] In summary, the present invention aims at the limitations of the existing quality evaluation methods of Smilax glabra medicinal materials and proposes a novel quality evaluation method, which is directly related to the efficacy of Smilax glabra. The core of this method is to quantitatively analyze the inhibitory activity of Smilax glabra extract on xanthine oxidase by measuring and calculating the half inhibitory concentration (IC50) of xanthine oxidase. 50 ) is used to evaluate the quality of Smilax glabra medicinal materials. Specifically, using the method of the embodiment of the present invention, the concentration of uric acid in the reference solution is 50 μg / mL, the concentration of the xanthine solution is 2 mmol / L, the concentration of the xanthine oxidase solution is 0.5 U / mL, the volume of the xanthine solution and the xanthine oxidase solution is 50 μL, and the volume of the phosphate buffer is 100 μL, the IC value of the Smilax glabra extract is 50 When the value is lower than 20 mg / mL, it can be identified as effective Rhizoma Smilacis Glabrae. Compared with the existing methods, the method described in the present invention provides a more objective and accurate quality evaluation standard.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made to it on the basis of the present invention, such as continuing to expand the number of Smilax glabra samples, and further optimizing the evaluation criteria of the present method by collecting and analyzing the half-inhibitory concentration values ​​of xanthine oxidase inhibitory activity of more reference samples. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for evaluating the quality of Smilax glabra based on xanthine oxidase inhibitory activity, characterized in that: The amount of uric acid produced by xanthine under the catalysis of xanthine oxidase was measured, and the half-inhibitory concentration (IC 50 ) was used as the evaluation index to assess the quality of Smilax glabra.

2. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 1, wherein: The method comprises the following steps: Step 1, preparing the tested Rhizoma Smilacis Glabrae into powder, and treating it to obtain Rhizoma Smilacis Glabrae extract; taking the Rhizoma Smilacis Glabrae extract, and adding phosphate buffer to prepare Rhizoma Smilacis Glabrae extract solutions of different concentrations; Step 2, take a uric acid reference substance, add sodium hydroxide to dissolve it, add water to dilute it to make a reference substance solution, store it at 4°C for later use; Step 3, take xanthine, add phosphate buffer to prepare xanthine solution, store at 4°C for later use; Step 4, taking xanthine oxidase, adding phosphate buffer to prepare xanthine oxidase solution, and storing at 4°C for later use; Step 5, take the xanthine oxidase solution, add phosphate buffer, incubate at 37°C for 30 minutes, add xanthine solution, incubate at 37°C for 120 minutes, and place in a boiling water bath for 5 minutes to obtain a blank test solution; Step 6: Take the same volume of xanthine oxidase solution as in step 5, add the same volume of Smilax glabra extract solution as in step 5, incubate at 37° C. for 30 min, add the same volume of xanthine solution as in step 5, incubate at 37° C. for 120 min, and place in a boiling water bath for 5 min to obtain a sample test solution; Step 7, using a chromatographic column, with 0.1% glacial acetic acid-methanol (90:10) as the mobile phase, the detection wavelength is 291nm, the column temperature is 30°C, and the injection volume is 5μL for determination; Step 8: Using the peak area as a variable, calculate the inhibition rate of xanthine oxidase at each concentration of Smilax glabra extract solution according to the following formula: Where A is the peak area of ​​uric acid in the blank solution, and B is the peak area of ​​uric acid in the sample solution. The half inhibition concentration (IC 50 ); comparing the half inhibition concentration with the standard. If the half inhibition concentration does not exceed the standard, the tested Smilax glabra sample is an effective Smilax glabra.

3. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 2, wherein: The treatment in step 1 to obtain the Smilax glabra extract includes: placing the Smilax glabra powder in a container, adding 70% ethanol, ultrasonic extraction, supplementing the missing weight with 70% ethanol again, shaking well, taking the supernatant and then steaming it, adding water to dissolve it, and freeze-drying it to obtain the Smilax glabra extract.

4. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 2, wherein: The pH of the phosphate buffer is 7.2-7.

4.

5. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 2, wherein: The concentration of the Smilax glabra extract solution of different concentrations is selected from 20 to 1.25 mg / mL.

6. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 2, characterized in that: The uric acid concentration in the reference solution in step 2 is 50 μg / mL, the concentration of the xanthine solution in step 3 is 2 mmol / L, the concentration of the xanthine oxidase solution in step 4 is 0.5 U / mL, the xanthine solution and xanthine oxidase solution in step 5 are 50 μL, and the volume of the phosphate buffer is 100 μL.

7. The method for evaluating the quality of Rhizoma Smilacis Glabrae according to claim 6, characterized in that: The standard described in step 8 is 20.0 mg / mL.

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