Study on Quality Standard of Tibetan Medicine Corydalis dasyptera Maxim.

Yi SHI, Yunfeng HAN, Xi TANG, Jie GAO, Lin PAN, Yi ZHANG, Xianrong LAI*, Duojie LONGZHU

1.School of Ethnic Medicine, Chengdu University of Chinese Medicine, Chengdu 611137, China; 2.College of Life Sciences, Qinghai Normal University, Xining 810008, China

Abstract [Objectives]To establish the quality standard of Tibetan medicine Corydalis dasyptera Maxim.[Methods]According to the research method of drug quality standard in the appendix of 2020 edition of Chinese Pharmacopoeia, 8 batches of C. dasyptera Maxim.from different habitats were studied by character identification, microscopic identification and TLC identification.The content of water, total ash, acid-insoluble ash and alcohol-soluble extract was determined, and the content of protopine in medicinal materials was determined by high performance liquid chromatography(HPLC).[Results]The properties and microscopic characteristics of C. dasyptera Maxim.were determined.The TLC characteristic spots of the medicinal materials were clear, the degree of separation was good, and the specificity was strong.Both the test sample and the control sample showed the same yellow-green spots in the corresponding position.It was tentatively determined that the water content of C. dasyptera Maxim.should not exceed 14.0%, the total ash content should not exceed 14.0%, the acid-insoluble ash content should not exceed 3.0%, and the alcohol-soluble extract content should not be less than 18.0%.There was a good linear relationship between the concentration of protopine and the peak area in the range of 16.64-166.40 μg·10-3(r=0.999 6).The average recovery rate was 98.47% and the RSD was 1.21%(n=6).The content of protopine in 8 batches of C. dasyptera Maxim.was 0.023%-0.093%.[Conclusions]The established quality research method is simple, stable and reliable, and can be used for the quality control of C. dasyptera Maxim.

Key words Corydalis dasyptera Maxim., Microscopic identification, TLC identification, Content determination

1 Introduction

CorydalisdasypteraMaxim.is a perennial herb in the Papaveraceae family[1].Its Tibetan medicine name is Gezhai Siwa[2].After uprooting it in summer and autumn, it was washed, dried in shade or dried in the sun.It tastes bitter and cold, and has the effects of clearing away heat and detoxification, soothing the liver and gallbladder, reducing swelling and relieving pain.It is mainly used in the treatment of febrile diseases, icteric hepatitis, meat poisoning, traumatic bleeding, sore and ulcer, carbuncle and jaundice[3-4].It is mainly distributed in Sichuan, Qinghai, Gansu, Tibet and other places, and grows in alpine grasslands, rock crevices, thickets or stone beaches at an altitude of 3 400-4 800 meters[5].

The main chemical components ofC.dasypteraMaxim.are alkaloids[6-7].In 1997, Wang Hengshanetal.[8]studied the chemical constituents ofC.dasypteraMaxim., and successfully isolated five alkaloids—tetrahydrocorysamine, tetrahydrocoptisine, corytuberine, corysamine and coptisine from the roots of the medicinal materials.In 2017, Ye Juetal.[9]qualitatively identified the chemical components ofC.dasypteraMaxim.by systematic pre-test, and confirmed that it may contain alkaloids, flavonoids, sugars, tannins, phenols, anthraquinones, saponins and volatile oils, and the content of total alkaloids was determined by acid dye colorimetry.As a traditional medicine commonly used in Tibetan areas, there are no records and literature reports on the quality control ofC.dasypteraMaxim., and there is a lack of reasonable means of quality control.As a result, racemosa,C.hendersoknii,C.adunca,C.conspersaMaxim.and other commercial medicinal materials are mistaken forC.dasypteraMaxim., which is not conducive to the safety and effectiveness of clinical use.Therefore, it is necessary to conduct an in-depth study and establish the quality standard of medicinal materials, so as to provide a scientific basis for the deep research and development ofC.dasypteraMaxim.In this study,C.dasypteraMaxim.was identified by character, microscopic observation and TLC, and its water, ash and alcohol-soluble extracts were investigated according to the method ofChinesePharmacopoeia2020(IV).At the same time, the content ofC.dasypteraMaxim.was determined by using protopine as the index.

2 Instruments and testing medicinal materials

2.1 InstrumentsAgilent 1260 high performance liquid chromatograph(Agilent); BH200 microscope(Shunyu Optical Technology Co., Ltd.); FW-100 high speed universal crusher(Beijing Zhongxing Weiye Instrument Co., Ltd.); FA1004 electronic balance(Shanghai Liangping Instrument Co., Ltd., accuracy of 1/10 000); CPA225D electronic balance(Sartorius Scientific Instruments Co., Ltd., accuracy of 1/100 000); DHG-8145 electrothermal thermostatic blast drying oven(Shanghai Yuejin Medical Equipment Co., Ltd.); ZF-20D black box ultraviolet analyzer(Shanghai Gucun Electro-Optic Instrument Factory); SB-8200DTS ultrasonic cleaning instrument(Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); DZKW-S-4 electric thermostat water bath(Beijing Yongguangming Medical Instrument Factory).

2.2 Testing medicinal materials8 batches of medicinal materials(serial number: DLHJ1-8)were identified as dried whole herb ofC.dasypteraMaxim.in the family Papaveraceae by Dr.Li Qien of Qinghai University and Dr.Duojie Longzhu of Qinghai Normal University.The sources of medicinal materials are shown in Table 1.Protopine reference substance(Desite, batch number of DST191226-113, purity of 98.0%); silica gel G thin layer plate(Qingdao Ocean Chemical Co., Ltd.); acetonitrile is chromatographically pure, water is ultra-pure water, and other reagents are analytically pure.

Table 1 Sample sources of Corydalis dasyptera Maxim.

3 Plant morphology and characters of medicinal materials

3.1 Plant morphologyIt is a perennial herb with a height of about 8-15 cm.Its tubers are bunched, cylindrical or rod-shaped, or slightly inflated in the middle and lower parts.Its rhizome is shortened and has a dark-brown residual leaf sheath.Stems are longitudinally angular, with upper part foliate, and lower part bare.There are a few basal leaves, long stipitate, and the leaf blade is long ovate, pinnately parted.Lobule basal margin has scabrous and white edges, the abaxial surface is densely covered with scabrous white protuberant cistolith, and the surface is slightly inconspicuous; 2 cauline leaves are nearly opposite at the upper part of the stem.The leaves are oblong, 4-8 cm long and have short stalks.Other features are the same as those of basal leaves.Racemes are densely flowered; bracts rhombic, lower part cuneate, upper part flabellate, margin with cartilaginous rough hair.The flowers are yellowish; 2 sepals; 4 petals, 2 whorls, outer petals 1.5-2.5 cm long, apex with cristate protuberance, spur cylindric, 2-row seeds(Fig.1).

Fig.1 Plant morphological diagram

3.2 Characters of medicinal materialsThe appearance, color, smell and other shape characteristics of 8 batches ofC.dasypteraMaxim.were basically the same, but there were only some slight differences in the size ofC.dasypteraMaxim.The root is long cylindrical, twisted by several fine roots, the surface and fine roots are brown, coated or mixed with grayish brown or dark brown suberized tissue.It is crisp and easy to break.Flowers are yellow to dark yellow, racemes capitate.It is bitter in taste(Fig.2).

Fig.2 Picture of Corydalis dasyptera Maxim.

4 Microscopic identification

The vessels, leaf epidermis cells, stomata and corolla epidermis cells were observed by conventional microscopic identification methods and photographed.

The surface of leaf epidermis cells is polygonal, covered with small sand crystals and the anticlinal wall is straight.The stomata are indeterminate, and there were 4-5 accessory cells.Corolla epidermis cell is quasi-polygonal, anticlinal wall is slightly curved, and the surface of some cells is covered with small sand crystals.Leaves and bracts have aristate cuspate fragments, yellowish green.The vessel is spiral, areolae and scalariform, with a diameter of 5-44 μm.Occasionally, unicellular non-glandular hairs with blunt head and small sand crystals are seen(Fig.3).

Fig.3 Characteristic diagram of Corydalis dasyptera Maxim.powder

5 TLC identification

2 g powder of 8 batches ofC.dasypteraMaxim.(serial number: DLHJ1-8)was mixed with 30 mL of trichloromethane-methanol-concentrated ammonia test solution(5∶1∶0.1)and treated with ultrasound for 30 min.After cooling, it was filtered, the filtrate was evaporated, and the residue was dissolved by adding 1 mL methanol, which was used as the test solution.In addition, the protopine reference substance was added with methanol to prepare a solution(1 mg/mL), which was used as the control solution.According to the thin layer chromatography(four general rules 0502 ofChinesePharmacopoeia2020 edition), 5-7 μL of test solution and 4 μL of reference solution were pipetted and placed on the same silica gel G thin layer plate respectively.Cyclohexane-ethyl acetate-diethylamine(16∶3∶1)was used as the developer, placed in the developing chamber pre-saturated with ammonia vapor for 20 min, developed, removed, dried and sprayed with dilute bismuth potassium iodide test solution.In the chromatogram of the sample, the spots of the same color were shown in the corresponding position of the chromatogram of the reference substance.The results are shown in Fig.4.

Note: 1: Protopine reference solution; 2-9: different batches of C. dasyptera Maxim.(corresponding to the serial number DLHJ1-8 in Table 1).

6 Content determination

6.1 Chromatographic conditions and system applicability testOctadecyl silane bonded silica gel was used as filler; acetonitrile-triethylamine acetic acid solution(triethylamine 8 mL, glacial acetic acid 30 mL, diluted to 1 000 mL by adding water)(18∶82)was used as mobile phase; the detection wavelength was 289 nm; the column temperature was 30 ℃, and the number of theoretical plates should not be less than 6 000 according to the protopine peak.

6.2 Solution preparation

6.2.1Preparation of reference solution[10].A proper amount of protopine reference substance was taken, weighed accurately, and methanol was added to make a solution(8.0 μg/mL)to obtain the reference solution.

6.2.2Preparation of test solution[10].About 1 g of the powder ofC.dasypteraMaxim.was taken, weighed accurately, placed in a conical flask with a stopper, and 50 mL of 60% methanol was added accurately, and was weighed.After soaking for 15 min, ultrasonic treatment(power 250 W, frequency 33 kHz)was carried out for 30 min before it was cooled and weighed.60% methanol was used to make up for the lost weight, and after being shaken well, filtered, subsequent filtrate was taken and the test solution was obtained.

6.3 Methodological investigation

6.3.1Investigation of linear relationship.2, 4, 6, 8, 10, 12, 14, 16, 18, 20 μL of reference solution under in Section6.2.1was precisely pipetted and injected into the liquid chromatograph, it was determined according to the chromatographic condition of Section6.1, and the chromatogram was recorded.The linear regression equation was obtained by linear regression with the peak area as the ordinate and the protopine amount(μg·10-3)as the abscissa.The regression equation wasy=1.163 1x+2.48(R2=0.999 6).The results showed that there was a good linear relationship for the protopine amount in the range of 16.64-166.40 μg·10-3.

6.3.2Precision test.10 μL of the same sample solution was precisely pipetted, and the sample was injected continuously for 6 times in the same day according to the chromatographic condition of Section6.1, and the peak area in the chromatogram was recorded.The results showed that theRSDof precision peak area value of protopine was 2.90%, indicating that the precision of the instrument was good.

6.3.3Stability test.According to the chromatographic conditions of Section6.1, 10 μL of the same sample solution was precisely pipetted and injected into high performance liquid chromatograph at 0, 1, 2, 4, 8, 12 and 24 h, respectively, and the chromatographic peak area of protopine was recorded.The results showed that theRSDof protopine content was 2.34%, indicating that the sample solution was stable within 24 h.

6.3.4Replication test.Six samples of the same batch were precisely weighed, and the sample solution was prepared according to the method of Section6.2.2.According to the chromatographic conditions of Section6.1, 10 μL of each sample solution was precisely pipetted and injected into liquid chromatograph to determine the peak area value and calculate the content of protopine.The results showed that theRSDwas less than 3%, indicating that the method had good repeatability.

6.3.5Sample recovery test.8 mL of reference solution(52.0 μg/mL)was precisely pipetted and placed in a conical flask with a stopper, and methanol was evaporated in a water bath.1.0 g of medicinal powder with known content was taken again(a total of 6 portions), precisely weighed and placed in the above conical flask.50 mL of 60% methanol was precisely weighed, and the sample solution was prepared according to the above method.Under the above chromatographic conditions, the sample injected was determined, the content was calculated, and the recovery rate of this method was investigated.The recovery rate was 98.47% and theRSDwas 1.21%, which indicated that the method was reliable and met the requirements.The results are shown in Table 2.

Table 2 Sample recovery rate

6.4 Determination of the content of samples10 μL of reference solution and 10 μL of 8 batches ofC.dasypteraMaxim.were precisely pipetted and injected into high performance liquid chromatograph(HPLC).The determination results are shown in Table 3.

Table 3 Determination results of protopine in Corydalis dasyptera Maxim.from different sources(n=3)

7 Determination of water, total ash, acid-insoluble ash and extracts

7.1 Water8 batches of powder ofC.dasypteraMaxim.(DLHJ1-8)were weighed and determined according to the water determination method(the second method of 0832 of the four general rules ofChinesePharmacopoeia2020 edition).The results are shown in Table 4.The water content of 8 batches ofC.dasypteraMaxim.varied from 10.96% to 13.78%, with an average of 12.94%.According to the determination results, it is tentatively determined that the water content of this product should not exceed 14.0%.

7.2 Total ash6 batches of powder ofC.dasypteraMaxim.(DLHJ1, 4-8)were taken and weighed precisely, and the total ash content was determined according to 2302 of the four general rules ofChinesePharmacopoeia2020.The results are shown in Table 4.The total ash content of 6 batches ofC.dasypteraMaxim.varied from 8.79% to 12.61%, with an average of 10.27%.According to the determination results, the total ash content should not exceed 14.0%.

7.3 Acid-insoluble ash6 batches of powder ofC.dasypteraMaxim.(DLHJ1, 4-8)were weighed precisely and measured according to the acid-insoluble ash method under 2302 of the four general rules ofChinesePharmacopoeia2020.The results are shown in Table 4.The total ash content of 6 batches ofC.dasypteraMaxim.varied from 1.17% to 4.62%, with an average of 2.32%.According to the determination results, the acid-insoluble ash content should not exceed 3.0%.

7.4 Alcohol-soluble extract6 batches of powder ofC.dasypteraMaxim.(DLHJ1, 4-8)were weighed precisely and measured with 75% ethanol as solvent according to the method for the determination of alcohol-soluble extracts(2201 of the four general rules ofChinesePharmacopoeia2020).The results are shown in Table 4.The results of 6 batches of alcohol-soluble extracts ofC.dasypteraMaxim.ranged from 18.35% to 25.51%, with an average of 21.96%.According to the determination results, it is tentatively determined that the alcohol-soluble extract of this product should not be less than 18.0%.

Table 4 Determination results of water, total ash, acid-insoluble ash and extract of Corydalis dasyptera Maxim.in 8 batches (n=3, %)

8 Discussion

In this study, with reference to the TLC identification system ofC.conspersaMaxim.andC.impatiens(Pall.)Fisch[10], cyclohexane-ethyl acetate-diethylamine(16∶3∶1)(pre-saturated in ammonia vapor for 20 min)and petroleum ether-ether-acetone(pre-saturated in ammonia vapor for 15 min)were investigated.Results showed that cyclohexane-ethyl acetate-diethylamine(16∶3∶1)(pre-saturated in ammonia vapor for 20 min)was used as the best developing agent, and the protopine spots inC.dasypteraMaxim.could be well separated.

The extract ofC.dasypteraMaxim.was determined according to the method of determination of alcohol-soluble extract(2201 of the four general rules ofChinesePharmacopoeia2020).50% ethanol, 75% ethanol and 95% ethanol were used as solvents, respectively, and the extracts were determined by hot dipping method and cold dipping method.The determination results were as follows: hot dipping method > cold dipping method, 75% ethanol > 50% ethanol > 95% ethanol.Therefore, according to the determination results, the hot dipping method with 75% ethanol as solvent was used for determination.

At present, there are still many problems in Tibetan medicine, such as confusion of source, low level of quality standards and even lack of quality standards, so the variety identification, quality control and evaluation of Tibetan medicine still have a long way to go, which is an important part of the modernization of Tibetan medicine[11-12].In this study, a TLC method for the identification of protopine and a HPLC method for the determination of protopine inC.dasypteraMaxim.were established for the first time.Its repeatability was good, stable and reliable.At the same time, the water, total ash, acid-insoluble ash and alcohol-soluble extract in the medicinal materials were determined and the preliminary limit requirements were made.This provides a basis for the formulation of the quality standard of this medicinal material, and is of great significance to the development and utilization of its resources.