Quality,Standard,of,Zijinbiao
Jianlong LAN, Yuebu HAILAI, Cijia DIJIU, Hongna SU, Li LI, Zhengming YANG, Yuan LIU
1. College of Pharmacy, Southwest Minzu University, Chengdu 610041, China; 2. Sichuan Provincial Qiang-Yi Medicinal Resources Protection and Utilization Technology Engineering Laboratory, Chengdu 610225, China; 3. Tibetan Plateau Ethnic Medicinal Resources Protection and Utilization Key Laboratory of National Ethnic Affairs Commission of the People’s Republic of China, Chengdu 610225, China; 4. Institute of Qinghai-Tibetan Plateau Research, Southwest Minzu University, Chengdu 610041, China
Abstract [Objectives] To establish the quality standard for Zijinbiao. [Methods] Microscopic identification, physical and chemical identification, and thin-layer chromatography (TLC) were used to qualitatively identify Zijinbiao. The moisture, total ash, acid-insoluble ash, and alcohol-soluble extract content were determined. The content of Plumbagin was determined by high performance liquid chromatography (HPLC). [Results] Microscopic identification, physical and chemical identification and thin layer identification features were remarkable. The moisture, total ash, acid-insoluble ash, and extract content of the 15 batches of samples were 7.49%-11.84%, 2.43%-5.81%, 0.59%-3.18% and 13.80%-20.45%, respectively. The linear equation of Plumbagin was Y=38 094X, R2=0.999 6. Plumbagin had a good linear relationship in the range of 0.01-0.53 mg/mL. [Conclusions] This method is specific and reproducible, and can be used for quality control of Zijinbiao.
Key words Zijinbiao, Plumbagin, Microscopic identification, Thin-layer chromatography (TLC), High performance liquid chromatography (HPLC)
Zijinbiao, also named Zijinlian, Xiaolanxue, Qixingjian,etc., is a commonly used medicinal material among multi-ethnic folks and is widely used in the medicine of Yi, Bai, Tibetan, Jingpo, Qiang and other ethnic minorities. It has been recorded in classics such asDictionaryofTraditionalChineseMedicine,ChineseHerbalMedicinesofYunnanProvince,SelectedChineseHerbalMedicinesofYunnanProvince,ChineseHerbalMedicinesofGuizhouProvince,etc.Dictionary of Traditional Chinese Medicine recorded that Zijinbiao is the dry root of Ceratostigma minus Stapf ex Prain[1].AnnalsofTraditionalChineseMedicineinYunnan[2]and Botanical Medicines of Yi Medicine[3]recorded that Zijinbiao is the dry root ofCeratostigmawillmottianumStapf. At present, the research ofC.minusStapf ex Prain mainly focuses on plant geography research, biochemical research, tissue culture and rapid propagation[4-6], while the research ofC.willmottianumStapf is mainly concentrated on chemical components and pharmacological effects[14-15], quality standard research[16], cultivation science[7-10],etc., and as the original plant of Zijinbiao, it was included in theQualityStandardofChineseMedicinalMaterialsforEthnicMedicinalMaterialsinGuizhouProvince(2003 edition), with only provisions of traits and physical and chemical identification[11].
According to our previous survey and research on the clinical use of Yi medicine, the original plant base of Zijinbiao was identified as the dry root ofC.minusStapf ex Prain andC.willmottianumStapf. Through the research on the quality standard of the multi-base variety Zijinbiao, this study is intended to provide a theoretical basis for the improvement of the standard of Zijinbiao medicinal materials, and provide data support for the establishment of theSichuanProvincialStandardsforEthnicMedicinalMaterials(2022 edition).
2.1 InstrumentAglent1260 high performance liquid chromatography instrument; JC-2F-7 dark box type three-purpose UV analyzer (Qingdao Juchuang Environmental Protection Group Co., Ltd.); KQ-300DE digital controlled ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); ME new classic electronic balance (Mettler Toledo Instruments (Shanghai) Co., Ltd.).
2.2 Reagents and drugsPlumbagin (purity 99.76%, Chengdu Refmedic Technology Co., Ltd.), silica gel GF254 for thin-layer chromatography (Qingdao Haiyang Chemical Co., Ltd.). Methanol was of chromatographic grade; ethanol and other reagents were of analytical grade; water was ultrapure water.
2.3 DrugsFifteen batches of Zijinbiao samples were collected and were identified by Professor Liu Yuan from Southwest Minzu University as dry roots ofCeratostigmaminus Stapf ex Prain andCeratostigmawillmottianumStapf. Among them, ZJB-1-ZJB-10 wasC.minusStapf ex Prain and ZJB-11-ZJB-15 wasC.willmottianumStapf.
3.1 Microscopic identificationTook a small amount of Zijinbiao powder (yellow-brown), put it on a glass slide, prepared slide with dilute glycerol and chloral hydrate, and observed under a microscope. The ducts were reticulated ducts, very common, 15-50 μm in diameter. Starch granules were many, the single granules were spherical, the umbilical point was star-shaped, and the complex granules were composed of 2-3 or more granules. The fibers were scattered singly or in bundles. Stone cells were round, square or rectangular, with thick walls and obvious hole patterns, as shown in Fig.1.
Note: 1. reticulated ducts; 2-3. starch granules; 4-5. fiber; 6. stone cells.
3.2 TLC identificationTook 0.5 g of the sample powder, added 20 mL of ethanol, ultrasonically treated it for 30 min, filtered, evaporated the filtrate to dry state, added 1 mL of ethanol to the residue to dissolve it, and used it as the test solution. Took another Plumbagin reference substance, added ethanol to prepare 1 mL of solution containing 0.5 mg, used it as the reference substance solution.
Pipetted 5 μL of each of the above two solutions using the TLC method in General Rules 0502 ofChinesePharmacopoeia(2002 Edition) Volume IV, separately dotted the solutions on the same silica gel GF254thin layer plate respectively, used petroleum ether (60-90 ℃)-ethyl acetate-glacial acetic acid (7∶1∶0.5) as the developing solvent, developed, took out, and air dried. Viewed under UV light (254 nm). In the chromatogram of the test substance, there were spots of the same color at the positions corresponding to the chromatograms of the reference medicinal materials, as shown in Fig.2.
Note: 1. Plumbagin reference substance; 2-16. Zijinbiao samples.
3.3 Physical and chemical identificationTook 2 g of Zijinbiao powder, added 20 mL of water, soaked for 15 min, heated and boiled for 2 min, filtered, added dilute hydrochloric acid to the filtrate to make it acidic, and added 10 mL of ether, shook till the ether layer turned yellow, added sodium hydroxide test solution to make it strongly alkaline, shook till the water layer turned purple red.
3.4 Determination of moisture and ash contentsThe moisture content and total ash and acid insoluble ash content were determined using the method in General Rules 0832 and General Rules 2302 ofChinesePharmacopoeia(2020 Edition) Volume IV. The results were shown in Table 2.
Table 1 Information of Zijinbiao samples
Table 2 Determination results of moisture, total ash, acid insoluble ash and extract of Zijinbiao %
3.5 Determination of extractUsing 30% ethanol as a solvent, the extract was determined in accordance with hot dip method for alcohol-soluble extracts in General Rules 2201 and General Rules 2302 ofChinesePharmacopoeia(2020 Edition) Volume IV. The results were shown in Table 3.
Table 3 Determination results of extract of Zijinbiao %
3.6 Determination of contents
3.6.1Chromatographic conditions. Chromatographic column: Diamonsil C18(4.6 mm×250 mm, 5 μm); mobile phase acetonitrile-pure water was 70∶30, isocratic elution; the detection wavelength was 265 nm; column temperature was 30 ℃; volume flow rate was 1.0 mL/min; the injection volume was 10 μL. Under these chromatographic conditions, the theoretical plate number should not be less than 5 000 according to the Plumbagin peak. The chromatogram is shown in Fig.3.
Note: A. Ceratostigma minus Stapf ex Prain; B. Ceratostigma willmottianum Stapf; C. Plumbagin.
3.6.2Preparation of test solution. Took about 0.5 g of Zijinbiao powder, precisely weighed it, put it in a conical flask with a stopper, precisely added 25 mL of dilute ethanol, closed the stopper, weighed it, and ultrasonically treated it (at power 500 W and frequency 40 kHz) for 30 min, cooled down, weighed again, made up for the lost weight with dilute ethanol, shook up and filtered. Took the filtrate to obtain the test solution.
3.6.3Linear relationship test. Precisely weighed 10.66 mg of Plumbagin reference substance, added methanol to prepare a solution containing 1.066 mg of Plumbagin per 1 mL, and obtained the reference substance solution. Precisely pipetted 0.05, 0.10, 0.25, 0.50, 1.00, 2.50 mL of the reference solution, put them in a 5 mL volumetric flask, added methanol to the desired scale, and shook up. Injected into the liquid chromatograph, measured the peak area in accordance with chromatographic conditions in Section3.6.1, plotted the standard curve with the reference substance as the abscissa and the peak area as the ordinate. The regression equation wasY= 38 094X(R2=0.999 6). The results show that Plumbagin has good linearity in the range of 0.01-0.53 mg/mL.
3.6.4Precision test. Precisely pipetted 10 μL of Plumbagin reference solution containing 0.053 3 mg/mL, and injected and measured in accordance with chromatographic conditions in Section3.6.1. TheRSDof measured Plumbagin peak area was 0.30%, indicating that the instrument has high precision.
3.6.5Stability test. Precisely weighed about 0.5 g of the sample (ZJB-3), and prepared the test solution using the method in Section3.6.2. At 0, 4, 8, 12, and 24 h, injected the samples under the chromatographic conditions in Section3.6.1, and theRSDof the measured Plumbagin peak area was 1.89%, indicating that the test sample is stable within 24 h.
3.6.6Reproducibility test. Precisely weighed 6 parts of sample (ZJB-6), each about 0.5 g, prepared the sample solution in accordance with the method in Section3.6.2, injected 10 μL under the chromatographic conditions in Section3.6.1, theRSDof the measured Plumbagin peak area was 2.01%, indicating the method has good reproducibility.
3.6.7Sample recovery test. Precisely weighed 6 parts of the sample (ZJB-6), each 0.25 g, put in a stoppered conical flask, separately added 1 mL of Plumbagin reference solution, prepared the sample solution in accordance with the method in Section3.6.2, injected samples under the chromatographic conditions in Section3.6.1, measured the content of Plumbagin, and calculated the recovery rate. The results were shown in Table 4.
Table 4 Results of sample recovery test
3.6.8Determination of sample content. Took 10 batches of medicinal materials, prepared the test solution using the method in Section3.6.2, injected and measured under the chromatographic conditions in Section3.6.1, and calculated the content. The Plumbagin contents of ZJB-1, ZJB-2, ZJB-3, ZJB-4, ZJB-5, ZJB-6, ZJB-7, ZJB-8, ZJB-9, ZJB-10, ZJB-11, ZJB-12, ZJB-13, ZJB-14 and ZJB-15 were 0.17%, 0.40%, 0.50%, 0.72%, 0.50%, 0.41%, 0.40%, 0.39%, 0.89%, 0.48%, 1.02%, 2.01%, 0.88%, 1.75%, and 1.33%, respectively; the mean was 0.79%.
4.1 TLC identificationThrough investigating different developing systems in this experiment, the final developing conditions were determined as petroleum ether-ethyl acetate-glacial acetic acid (7∶1∶0.5). The petroleum ether is volatile, when the temperature increases, the polarity of the expansion system becomes larger, resulting in a largerRfvalue. Therefore, the ambient temperature should be controlled in the experiment.
4.2 Limit of moisture, ash and extract contentsCombined with the relevant provisions of General Rules 0212 ofChinesePharmacopoeia(2020 Edition) Volume IV and General Rules for the Verification of Medicinal Materials and Decoction Pieces, it is tentatively decided that the moisture content should not exceed 13%, the total ash content should not exceed 7.0%, and the acid-insoluble ash content should not exceed 4%, and the extract should not be less than 11%.
4.3 Determination and limit of Plumbagin contentIn this experiment, the extraction solvents (30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, dilute ethanol, 70% ethanol, and ethanol) and extraction methods (ultrasonic extraction, reflux extraction) were investigated. The results showed that the use of dilute ethanol as the extraction solvent to extract Plumbagin by ultrasonic has a significant effect. The results of Plumbagin content in 15 batches of Zijinbiao were in the range of 0.17%-2.01%, with an average value of 0.79%, showing a significant difference. It is tentatively decided that the content should not be lower than 0.12% based on the dried product of Zijinbiao.
This experiment established the physical and chemical, microscopic and TLC identification method for Zijiniao medicinal materials. The method is simple and reliable. Besides, it determined the moisture, ash, acid-insoluble ash, alcohol-soluble extract and Plumbagin content of 15 batches of Zijinbiao medicinal materials from different origins. It established the quality standard for Zijinbiao medicinal materials, which is of great significance to effectively control the quality of Zijinbiao.