CN108037234B

CN108037234B - Quality detection method of abrus herb hepatitis granules

Info

Publication number: CN108037234B
Application number: CN201711269360.1A
Authority: CN
Inventors: 韦智灵; 王永江; 郑文雅
Original assignee: Guangdong Shaxi Pharmaceutical Co Ltd
Current assignee: Guangdong Shaxi Pharmaceutical Co Ltd
Priority date: 2017-12-05
Filing date: 2017-12-05
Publication date: 2020-02-07
Anticipated expiration: 2037-12-05
Also published as: CN108037234A

Abstract

The invention discloses a quality detection method of abrus herb hepatitis granules, which comprises qualitative identification by thin-layer chromatography and content detection of hesperidin. The invention establishes the thin-layer chromatography qualitative identification of the herba hyperici japonici in the herba abri hepatitis granules for the first time, and performs content determination on hesperidin.

Description

Quality detection method of abrus herb hepatitis granules

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a quality detection method of abrus herb hepatitis granules.

Background

The abrus herb hepatitis granule consists of 6 traditional Chinese medicines of abrus herb, oriental wormwood, hypericum japonicum, myrtle root, blumea balsamifera and aralia armata, and is collected on page 94 of the third volume of the standard traditional Chinese medicine prescription preparation of the Ministry of health of the people's republic of China, with the standard number: WS3-B-0561-91 has good curative effect on acute hepatitis jaundice type or anicteric hepatitis jaundice type, fast jaundice treatment, virus removal, liver protection and effective reduction of sequelae of hepatitis patients. The quality standard of the abrus herb hepatitis granules only comprises character, granule inspection items and microorganism limit inspection items, but thin-layer identification items and content measurement items do not exist, the controllability of the quality standard is too low, the internal quality of the product is difficult to monitor, and the quality control method needs to be improved.

①, the method adopts a 366nm ultraviolet lamp for inspection, 366nm wavelength is not commonly used in the thin layer identification method, visible light, 254nm and 365nm ultraviolet light sources are arranged in the inspection device of 0502 thin layer chromatography in the four parts of the version 2015 of Chinese pharmacopoeia, a plurality of laboratories do not have the 366nm ultraviolet lamp, which indicates that the method is not easy to popularize and apply ②, in addition, the thin layer diagram has large main spot Rf value, the main spot has run to the front edge of the solvent, and does not accord with the Rf requirement of 2 thin layer chromatography in the four parts of the version 2015 of Chinese 050pharmacopoeia (preferably between 0.2 and 0.8). ③, the identification method has not carried out durability test, and can not ensure the reliability of the established method for daily inspection and method.

The contents of chlorogenic acid, quercetin, protocatechuic aldehyde, protocatechuic acid and gallic acid in the abrus herb hepatitis particles are simultaneously measured by adopting HPLC (high performance liquid chromatography) in the literature. However, it is known that quercetin, protocatechualdehyde, protocatechuic acid, gallic acid and the like are widely present in medicinal plants, and thus lack specificity as an index component.

In addition, the content of chlorogenic acid is measured in thin layer identification of abrus cantoniensis hance hepatitis particles and measurement of chlorogenic acid and quercetin reported by Chengjing and other documents, and the chlorogenic acid is an active ingredient of oriental wormwood in the prescription. It should be noted that according to the different harvesting seasons, the chinese pharmacopoeia 2015 specifies that the harvesting in spring is called "capillary artemisia" and the harvesting in autumn is called "floral capillary artemisia". Chlorogenic acid is used as index for determining herba Artemisiae Scopariae content, and should not be less than 0.50%; the scoparone lactone is an index for measuring the content of herba Artemisiae Scopariae and should not be less than 0.20%. However, the prescription of the abrus herb hepatitis granule does not indicate the season in which the oriental wormwood is collected. Scholars report that the content of chlorogenic acid in capillary wormwood is 0.81%, the content of chlorogenic acid in oriental wormwood is 0.46%, and the content of chlorogenic acid in capillary wormwood is higher than that in oriental wormwood. This shows that the use of chlorogenic acid as the content index component of abrus herb hepatitis particles is unreasonable, and the transfer rate of the content and the content of chlorogenic acid in abrus herb hepatitis particles can not be determined to evaluate the controllability of the product quality.

In the process of preparing a test solution from high performance liquid chromatography for simultaneously measuring the content of protocatechuic acid, protocatechuic aldehyde and gallic acid in abrus herb hepatitis particles reported in the documents of Hou Xiaotao and the like, a sample needs to be dried at low temperature to constant weight and then extracted by ultrasound, and the defects of complex operation and long time consumption exist.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the quality detection method of the abrus herb hepatitis particles, which has the advantages of good specificity, strong durability and good stability, and can effectively ensure the stability and controllability of the product quality.

The invention is realized by the following technical scheme:

a quality detection method of abrus herb hepatitis granules is characterized by comprising the following steps of qualitative identification by thin-layer chromatography and content detection of hesperidin:

(1) qualitative identification of herba Hyperici Japonici by thin layer chromatography:

taking 3g of the product, grinding, adding 25ml of water, carrying out ultrasonic treatment for 20-40 minutes, filtering, adding dilute hydrochloric acid to adjust the pH value to 1-2, extracting with ethyl acetate for 1-3 times, 20ml each time, combining ethyl acetate extract, evaporating to dryness, and adding 1ml of ethanol into residues to dissolve the residues to obtain a test solution; taking 1g of herba Hyperici Japonici as a reference medicinal material, adding 25ml of water, heating and refluxing for 20-40 min, filtering, and preparing herba Hyperici Japonici reference medicinal material solution by the same method from "adjusting pH to 1-2 with dilute hydrochloric acid" for use; and (3) performing thin-layer chromatography test, sucking 5-10 mu l of each of the two solutions, respectively dropping the two solutions on the same silica gel G thin-layer plate, and mixing the two solutions in a volume ratio of dichloromethane: ethyl acetate: methanol: formic acid: developing with water at ratio of 12:10:2:1:1 as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, heating at 105 deg.C for 5-10 min, and viewing under 365nm ultraviolet lamp; in the chromatogram of the test solution, spots with the same color are arranged at the positions corresponding to those of the chromatogram of the reference solution;

(2) measuring the content of hesperidin:

the determination is carried out by adopting a high performance liquid chromatography, and the specific method and the steps are as follows:

octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; acetonitrile-water in a volume ratio of 17:83 is a mobile phase; the detection wavelength is 283 nm; the number of theoretical plates is not less than 5000 calculated according to hesperidin peak;

preparing a reference substance solution by precisely weighing an appropriate amount of hesperidin reference substance, and adding methanol to obtain a solution containing 15 μ g of hesperidin per 1 ml;

preparing a test solution, grinding the test solution, sieving the ground test solution by a No. five sieve, precisely weighing 2.0g of the test solution, placing the test solution into a conical flask with a plug, precisely adding 50ml of 10-50% methanol, weighing the test solution, carrying out ultrasonic treatment for 30-60 minutes, taking out the test solution, cooling the test solution, weighing the test solution again, complementing the loss weight by 10-50% methanol, shaking the test solution uniformly, filtering the test solution, and taking a subsequent filtrate to obtain the test solution;

the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

Preferably, in the step (3), the sample solution is prepared by: taking the product, grinding, sieving by a fifth sieve, taking 2.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 10% methanol, weighing, ultrasonically treating for 45 minutes, taking out, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.

The invention carries out thin-layer qualitative identification on the hypericum japonicum in the abrus herb hepatitis particles, can be inspected under a 365nm ultraviolet lamp, has strong specificity, moderate Rf value of about 0.2-0.5, has good separation degree (R is more than 1.5) of two main spots, has durability, ensures the reliability of the method, and is suitable for popularization and application.

The invention selects hesperidin as an index component for content determination, wherein the hesperidin is an index component and an active component of the traditional Chinese medicine of the Yingpo, and the Yingpo is a medicinal material with the largest dosage in the prescription. The abrus cantoniensis hance is a monarch drug in the prescription, and has no content determination item according to the quality standard of the first part of the 2015 edition of Chinese pharmacopoeia. Further referring to the literature, Abrus cantoniensis Hance is collected in the sixth stage of hong Kong Chinese medicinal materials, and the content limit of abrine in Abrus cantoniensis Hance is not less than 0.025% calculated on the basis of dry product. Because the abrine is slightly soluble in water, and the process of the abrus herb hepatitis particles adopts water decoction of medicinal materials, the extraction rate of abrine is low directly. Therefore, HPLC is adopted to detect the content of abrin, and the test result shows that the content is extremely low, so that the content is not suitable to be measured. Therefore, the method for determining the content of the hesperidin in the abrus herb hepatitis particles is reasonable, the hesperidin has certain specificity and stable physical and chemical properties, the introduction of system errors is reduced, the accuracy and the reproducibility of the determination result are improved, and the stability and the quality controllability of the abrus herb hepatitis particles are ensured.

The invention adopts a direct ultrasonic extraction method to prepare the test solution, and saves the operation of ultrasonic extraction and measurement after drying at low temperature to constant weight. The method is simple, convenient, time-saving and environment-friendly to operate, avoids unnecessary loss of index components caused by complicated operation process, and reflects the real content of the hesperidin index components to the maximum extent.

Compared with the prior art, the invention has the following beneficial effects:

the invention establishes the thin-layer chromatography qualitative identification of the herba hyperici japonici in the herba abri hepatitis granules for the first time, and performs content determination on hesperidin.

Drawings

FIG. 1 is a chromatogram obtained by thin layer chromatography of herba Hyperici Japonici, wherein ① is a negative sample of herba Hyperici Japonici, ② - ④ is a sample of herba abri hepatitis granule, ⑤ is a control drug of herba Hyperici Japonici;

FIG. 2 is a thin layer chromatogram for identification of Hypericum japonicum from 10 test samples, wherein ① is a negative sample of Hypericum japonicum,

is 10 batches of abrus herb hepatitis particle samples,

is herba Hyperici Japonici reference material;

FIG. 3 is a graph of the standard curve of a hesperidin control;

FIG. 4 is an HPLC chart of a hesperidin control;

FIG. 5 is an HPLC chart of negative control solution without eagle;

FIG. 6 is an HPLC chart of a sample of Abrus cantoniensis hepatitis particles.

Detailed Description

The present invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.

Example 1:

and (3) verification of methodology:

1. specialization inspection

Two spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution of herba Hyperici Japonici. And in the chromatography of the negative sample of the agrimonia pilosa, no interference exists at the corresponding position. The method is specially used for identifying the hypericum japonicum medicinal material. The results are shown in FIG. 1.

2. Durability examination

2.1 study of thin-layer plates from different manufacturers

The influence of three thin-layer chromatography plates, namely a hand-laid plate (100X 200mm, thickness 0.25mm), a prefabricated plate (Qingdao spectral separation materials Co., Ltd., 20161022, 100X 200mm, thickness 0.20-0.25 mm) and a Merck plate (thickness 0.25mm), on the chromatographic behavior was examined respectively. The Rf value of the hand-paved main spot is about 0.3-0.4, which is larger than that of a precast slab and a Merck slab, and the three types of thin-layer plates can obtain good separation effects, which shows that the method has durability to the change of the types of the thin-layer plates.

2.2 investigating the influence of the unfolding temperature on the chromatographic behavior

And (3) inspecting the influence of different temperatures of 8 ℃, 21 ℃, 26 ℃ and 30 ℃ on the separation effect of the main spots in the spectrum. The results show that the main spot Rf values of the maps obtained at the four temperatures are basically consistent and have no significant influence along with the temperature change, which indicates that the method has durability to the variation of the development temperature. Therefore, no particular specification is made in the text for the development temperature.

2.3 investigating the influence of relative humidity on the chromatographic behavior

Mu.l of each sample solution is spotted on a silica gel G thin layer plate, pre-balanced for 30min under the conditions of relative humidity of 18%, 42%, 65% and 88%, and developed. The results show that different relative humidities have little influence on the main spot of the chromatogram of the test solution, and good separation effect can be obtained, which indicates that the method has durability to the variation of the relative humidity of the sample solution. Therefore, no particular specification is made in the text for the relative humidity of the spread.

2.4 investigating the influence of Pre-saturation time on chromatographic behavior

And comparing whether different pre-saturation times of 0min, 15min and 30min have obvious influence on the thin-layer chromatography. The result shows that the separation condition of the main spots of the chromatogram of each test solution is good, and the method has durability to different pre-saturation times. Therefore, the pre-saturation time is not specified in the text.

2.5 investigating the influence of different sample sizes on the chromatographic behavior

Whether the different spot numbers 3. mu.l, 5. mu.l, 8. mu.l and 10. mu.l had a significant effect on thin layer chromatography was examined. In the test chromatogram with the above 4 sample application amounts, the main spots can be effectively separated. When the amount of the 3. mu.l spot was used, the main spot was slightly blurred due to the small amount of the spot, but could be recognized. When the sample amount is 5 mul, 8 mul or 10 mul, the thin layer chromatogram has better effect, and the main spot is clear and easy to distinguish. And considering the content difference among different batches of products, determining the sample amount to be 5-10 mu l in the text.

2.6 investigating the influence of different spreading agents on the chromatographic behavior

And (3) inspecting an expansion system of petroleum ether-ethyl acetate-methanol-formic acid with a volume ratio boiling range of 60-90 ℃, wherein the Rf value of the compound spot to be detected is about 0.2, adjacent spots interfere the compound spot to be detected, and the separation degree cannot meet the requirement. And (3) inspecting an expansion system with the volume ratio of ethyl acetate-acetone-formic acid-water being 5:3:0.5:05, wherein the Rf value of the compound spot to be detected is about 0.9, adjacent spots interfere with the compound spot to be detected, and the separation degree cannot meet the requirement. And (3) observing an expansion system with the volume ratio of ethyl acetate-methanol-water-formic acid being 100:1:1:2, wherein the Rf value of the spot of the compound to be detected is about 0.5, and adjacent spots do not have interference on the spot of the compound to be detected but have trailing spots. And (3) inspecting an expansion system with the volume ratio of dichloromethane-ethyl acetate-methanol-formic acid-water of 12:10:2:1:1, wherein the Rf value of the spot of the compound to be detected is about 0.5, spots with the same color are displayed on the positions, corresponding to the chromatogram of the control medicinal material of the hypericum japonicum in the chromatogram of the test solution, the main spot in the thin-layer diagram is clear and easy to distinguish, the separation degree is good, and adjacent spots are not interfered. Therefore, a developing system with a volume ratio of dichloromethane-ethyl acetate-methanol-formic acid-water of 12:10:2:1:1 is selected.

2.710 examination of test articles

Respectively taking 10 batches of herba abri hepatitis granules with different production periods, preparing test solution, and inspecting, wherein the result is shown in figure 2. In 10 test sample chromatograms, spots of the same color appear at the positions corresponding to those of the reference material chromatograms.

In conclusion, negative control in the thin-layer chromatography qualitative identification of the hypericum japonicum is free of interference at a corresponding position, which indicates that the method is strong in specificity; the Rf value is moderate and is about 0.2-0.5, and the two main spots have good separation degree (R is more than 1.5); meanwhile, six factors of thin-layer plates of different manufacturers, different temperatures, different relative humidities, different pre-saturation times, different sample amounts and different developing agents are examined, so that the chromatographic behavior of the herba hyperici japonici control medicinal material is not significantly influenced, and the method is durable, ensures the reliability and is suitable for popularization and application.

(2) Measuring the content of hesperidin:

preparing a test solution, grinding the test solution, sieving the ground test solution by a No. five sieve, precisely weighing 2.0g of the test solution, placing the test solution into a conical flask with a plug, precisely adding 50ml of 10% methanol, weighing the test solution, ultrasonically treating the test solution for 45 minutes, taking out the test solution, cooling the test solution, weighing the test solution again, complementing the weight loss by 10% methanol, shaking the test solution uniformly, filtering the test solution, and taking a subsequent filtrate to obtain the test solution;

The product contains hesperidin (C) in each bag₂₈H₃₄O₁₅) Calculated, the content of the active ingredient should not be less than 2.0 mg.

And (3) verification of methodology:

2 optimization of extraction Process

2.1 examination of different extraction modes

Weighing 2.0g of herba abri hepatitis granule, weighing two parts, adding methanol 25ml, weighing, ultrasonically extracting one part for 30min, heating and refluxing the other part for 30min, cooling, supplementing weight, filtering with 0.45 μm microporous membrane, and sampling to obtain the final product shown in Table 1. The result shows that the content of the heating reflux extraction is slightly higher than that of the ultrasonic extraction, the SPSS single-factor analysis shows that no significant difference exists, and the ultrasonic extraction is selected in consideration of the advantages of convenience and easiness in operation, environmental protection and the like of the ultrasonic extraction.

TABLE 1 comparison of the contents of different extraction methods

2.2 investigation of different extraction solvents

Weighing herba abri hepatitis granule 2.0g and two parts, adding anhydrous ethanol 25ml into one part, weighing, adding methanol 25ml into the other part, ultrasonic extracting for 30min, cooling, supplementing weight, filtering with 0.45 μm microporous membrane, and sampling. The results show that methanol has better extraction efficiency than absolute ethanol. Further examining the extraction effect of 50% methanol and 10% methanol, the result shows that the extraction content of 10% methanol is the highest, and the result is shown in Table 2.

TABLE 2 Effect of different extraction media on the content

2.3 orthogonal test

Selecting methanol concentration, solvent times and extraction time as investigation factors according to single factor investigation result, respectively setting 3 levels according to L (3)⁴) (one level is blank) the orthogonal tables were tested as shown in Table 3, and the results of the orthogonal tests are shown in tables 4 and 5.

TABLE 3 orthogonal test factor horizon

TABLE 4 results of orthogonal experiments

TABLE 5 ANOVA TABLE

As can be seen from the above table, the influence of each factor on the content measurement result is in the order of methanol concentration (a) > solvent amount (B) > extraction time (C), and none of the three factors has significant difference. The best protocol for the orthogonal test was A1B3C2, considering that the addition of 100ml of solvent to the Erlenmeyer flask results in over-balance range. While the mean values for the two factors, 50ml and 100ml, differed 4.566, analysis of variance indicated no significant difference in solvent usage groups. Therefore, the best experimental protocol obtained by combination is: A1B2C2, 50ml 10% methanol, ultrasonic extracting for 45 min.

3 System suitability test

3.1 specificity test

And (3) respectively carrying out sample injection analysis on the eagle-lacking berth-reducing negative solution, the abrus herb hepatitis particle test solution and the hesperidin reference solution, wherein as shown in fig. 4-6, the hesperidin peak is completely separated from other similar component peaks (the separation degree is more than 1.5), and the eagle-lacking berth-reducing negative sample solution has no interference at the same peak emergence time of the hesperidin reference solution.

3.2 Linear relationship investigation

Taking the hesperidin reference substance solution and 1ml hesperidin reference substance solution, respectively placing in 2ml, 5ml, 10ml, 25ml, 50ml and 100ml volumetric flasks, adding methanol to scale, shaking up, measuring according to the above chromatographic conditions, and performing linear regression on the mass concentration (X) by using the peak area (Y). The standard curve equation of hesperidin is that Y is 21838.6976X +3169.8100.01 (R)²1.0000) at a concentration of 1.2904-129.0362 μ g/ml, as shown in fig. 3.

3.3 precision test

The same control solution (hesperidin concentration 12.9036 μ g/ml) was precisely pipetted 10 μ l and injected into a high performance liquid chromatograph, and the results obtained by the method are shown in Table 6, the peak area RSD was 1.11%, and the precision of the instrument was found to be good.

TABLE 6 results of precision test

3.4 repeatability test

6 parts of the same batch of samples (QL002) are precisely weighed, a sample solution to be tested is prepared, peak areas are recorded, the content is calculated, and the average content of hesperidin is 132.2 mu g/g and the RSD is 0.53 percent, which shows that the method has good repeatability.

Table 7 repeatability test results (n ═ 6)

3.5 sample application recovery test

A sample (lot No. QL002) of 1.0g in known content was weighed precisely, 6 parts in total were put in a conical flask with a stopper, 50ml of a control solution (7.9186. mu.g/ml) was added precisely, and the recovery rate was measured by the procedure under "3.3" and calculated, the results are shown in Table 8.

Table 8 sample recovery test results (n ═ 6)

3.6 durability test

3.6.1 stability test

2.0g of the same test solution (QL002) is taken, the test solution is prepared according to the preparation method of the test solution under the content determination item, the determination is carried out at different times respectively, the result is shown in the table 9, and the test solution is stable within 5 h.

TABLE 9 stability test results

3.6.2 column temperature investigation

2.0g of the same two test sample solutions (QL002) were taken, and the test sample solutions were prepared according to the methods for preparing the test sample solutions under the item of content measurement, and the results are shown in Table 10 for each column temperature. The test result shows that the content measurement result is not significantly influenced within 25-40 ℃, and the preferable temperature is 30 ℃.

TABLE 10 influence of different column temperatures on the assay

3.6.3 investigation of flow Rate

2.0g of the same sample solution (QL002) was used to prepare a sample solution according to the method for preparing a sample solution under the assay, and the three flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min were measured, respectively, and the results are shown in Table 11. Test results show that the flow rate of 0.8-1.2 ml/min has no significant influence on the content measurement result, and the flow rate is preferably 1.0 ml/min.

TABLE 11 influence of different flow rates on the results of the assay

3.6.4 investigation of different wavelengths

2.0g of the same sample solution (QL002) was used to prepare a sample solution according to the method for preparing a sample solution under the item of content measurement, and the measurement was carried out at five wavelengths of 278nm, 281nm, 283nm, 285nm and 287nm, respectively, and the results are shown in Table 12. The test result shows that the content measurement result is not significantly influenced within 283 +/-5 nm, and is preferably 283 nm.

TABLE 12 Effect of different wavelengths on the results of the assay

3.6.5 test for investigating the composition of mobile phase

And (3) inspecting the methanol-water mobile phase system, and displaying the result that compared with the acetonitrile-water mobile phase system, the peak shape of the compound to be detected is wide, and the theoretical plate number is slightly low, so that the acetonitrile-water mobile phase system is selected.

When the mobile phase proportion is acetonitrile-water (18:82), the result shows that the adjacent peaks in the test solution cause interference to the peak to be measured, the separation degree is less than 1.5, and the content measurement requirement cannot be met. When the mobile phase ratio is acetonitrile-water (16:84), the separation from adjacent peaks is good, and the analysis time is greatly increased and the efficiency is reduced after the peak production time of the hesperidin reference product is delayed. On the premise of ensuring that the hesperidin reference substance meets the adaptability of a liquid phase system, the method has proper analysis time, so that the mobile phase is acetonitrile-water (17: 83).

3.710 batch kidney nourishing and tranquilizing pill sample content determination

Taking 10 batches of abrus herb hepatitis particles to be tested, preparing a test solution according to a preparation method of the test solution under the content measurement item, recording peak areas and calculating the content, wherein the results are shown in a table 13.

TABLE 1310 measurement of hepatitis granule content of batch abrus herb

Claims

1. A quality detection method of abrus herb hepatitis granules is characterized by comprising the following steps of qualitative identification by thin-layer chromatography and content detection of hesperidin:

(2) measuring the content of hesperidin:

octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; acetonitrile-water in a volume ratio of 17:83 is a mobile phase; the detection wavelength is 283nm, and the flow rate is 1.0 ml/min; the number of theoretical plates is not less than 5000 calculated according to hesperidin peak;

2. The quality detection method of abrus herb hepatitis particles as claimed in claim 1, wherein in the step (2), the preparation of the test solution is as follows: taking the product, grinding, sieving by a fifth sieve, taking 2.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 10% methanol, weighing, ultrasonically treating for 45 minutes, taking out, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.