Establishment of Fingerprint of Cryptolepis buchananii Roem. et Schult. by HPLC

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1. Hechi Food and Drug Inspection Institute, Hechi 547000, China; 2. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] This study aimed to establish the fingerprint of Cryptolepis buchananii Roem. et Schult. by HPLC. [Methods] Taking the peak of chlorogenic acid as the references, the 10 batches of C. buchananii Roem. et Schult. materials were detected by HPLC under the conditions of column of Thermo ODS-2 Hypersil (5 μm, 4.6 mm×250 mm), detection wavelength of 323 nm, column temperature of 25 ℃, mobile phase of methanol-0.1% phosphoric acid solution, gradient elution and injection volume of 20 μL. The common peak identification and similarity evaluation were carried out according to Similarity Evaluation System for Chromatographic Fingerprint of Chinese Traditional Medicine (Version 2004 A). [Results] Among the HPLC fingerprints of the 10 batches of C. buchananii Roem. et Schult. materials, there are 13 common peaks, and their similarity is all greater than 0.9, indicating good similarity. [Conclusions] This study can provide a scientific basis for the quality evaluation of C. buchananii Roem. et Schult.

Key words Cryptolepis buchananii Roem. et Schult., High-performance liquid chromatography, Fingerprint, Common peak, Similarity

1 Introduction

CryptolepisbuchananiiRoem. et Schult. is generally used as medicine with its stems and leaves[1], also known as Daye Baiyeteng, Niujiaoteng, Duanchangcao[2], Banjianiu and Daanxiao[3]. It is widely distributed in Guangdong, Guangxi, Yunnan and Guizhou, and is commonly used in Guangxi.C.buchananiiRoem. et Schult. is bitter in taste, cold in nature, and poisonous, with effects of relaxing tendons, activating collaterals, relieving swelling and pain and detoxifying. In Guangxi folk, it is mainly used for the treatment of low back pain, abdominal pain, traumatic injury, sputum, fracture, carbuncle, eczema[4], snake bite[5], breast cancer, edema[6]. At present, the research on the chemical constituents ofC.buchananiiRoem. et Schult. focuses on cardiac glycosides, alkaloids and phenylpropanoids[7]. Modern pharmacological studies have shown that it has analgesic, anti-inflammatory[8-10], blood sugar-regulating, blood lipid-regulating[11-13], liver-protecting[14-15], cardiotonic, antibacterial[16], antioxidant[17-18], immunity-regulating[19]and other pharmacological effects. There is little research on the quality control ofC.buchananiiRoem. et Schult., a folk medicine. In this paper, the HPLC fingerprint ofC.buchananiiRoem. et Schult. from different producing areas in Guangxi was established by HPLC, in order to provide a scientific basis for the effective evaluation and quality control ofC.buchananiiRoem. et Schult. and the development of its medicinal resources.

2 Materials

2.1 InstrumentsAgilent 1260 high-performance liquid chromatograph with high-pressure quaternary pump (G-1311C), standard autosampler (G-1329B), diode detector (G-1314B) DAD, Agilent 1260 series chromatography data working station, intelligent column oven (G-1316A) (Agilent, USA); Thermo ODS-2 Hypersil (5 μm, 4.6 mm×250 mm) column; KQ-5200 ultrasonic cleaning instrument (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); Sartorius BP211D electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Simplicity 185 ultrapure water system (MILLIPORE, USA); HH-S6 digital constant-temperature water bath (Jintan Medical Instrument Factory); TGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory).

2.2 Reagents and plant materialA total of 10 batches ofC.buchananiiRoem. et Schult. samples were collected from different producing areas of Guangxi (Table 1). They were identified by Lu Hailin, senior experimental teacher of Guangxi University of Chinese Medicine. The used reagents and drugs included chlorogenic acid standard (batch No. 110753-201415, China Food and Drug Administration Institute, for content determination), methanol (chromatographically pure, Fisher, USA), phosphoric acid (chromatographically pure, Tianjin Kemiou Chemical Reagent Co., Ltd.) and ultrapure water (homemade). The other reagents used were of analytical grade.

3 Methods and results

3.1 Chromatographic conditionsThe chromatographic conditions were as follows: column, Thermo ODS-2 Hypersil (5 μm,

Table 1 Sources ofCryptolepisbuchananiiRoem. et Schult. materials

Sample codeSourceCollection timeSample codeSourceCollection timeS1Gaofeng, NanningNovember, 2015S6Yilingyan, NanningJanuary, 2016S2Jinxiu County, LaibinJanuary, 2016S7Mingxiu District, NanningSeptember, 2015S3Wuming County, NanningNovember, 2015S8Bama, GuangxiOctober, 2015S4Chongzuo, GuangxiSeptember, 2016S9Tianlin County, BaiseNovember, 2015S5Jingxi County, BaiseDecember, 2015S10Tongling Large BarrancaNovember, 2015

4.6 mm×250 mm); detection wavelength, 323 nm; flow rate, 0.8 mL/min; column temperature, 25 ℃; injection volume, 20 μL; mobile phase, methanol-0.1% phosphoric acid solution (volume fraction); gradient elution (Table 2).

Table 2 Gradient elution program of mobile phase

Time/minVolume fraction ofmethanol∥%Volume fraction of 0.1%phosphoric acid solution∥%01090202773242773282971464456524753566634

3.2 Preparation of test solutionAn accurate amount (1.000 g) of powder ofC.buchananiiRoem. et Schult. was placed in a conical flask with cover, added with 20 mL of water, weighed, and boiled for 20 min in success. The lost weight was supplemented with water. Then, the solution was filtered, and the filtrate was collected and centrifuged at 13 000 r/min for 10 min. The supernatant was collected and passed through 0.45-μm microporous membrane to obtain a test solution.

3.3 Preparation of reference solutionAn accurate amount (3.9 mg) of the chlorogenic acid standard was placed in a brown volumetric flask, dissolved with an appropriate volume of 50% methanol, and diluted to 100 mL. Thus, the chlorogenic acid reference solution with concentration of 0.39 mg/mL was obtained, and it was filtered through 0.45-μm microporous membrane for use.

3.4 Methodological investigation

3.4.1Precision test. The powder ofC.buchananiiRoem. et Schult. material from certain producing area was prepared into test solution according to the description in Section3.2. Under the chromatographic conditions described in Section3.1, the prepared test solution was detected six times repeatedly to examine the precision of the instrument. The results show that theRSDvalues of relative retention time of the common peaks were in the range of 0%-0.13%, and theRSDvalues of their relative areas were in the range of 0%-1.5%, all less than 3.0%, meeting the requirements of the fingerprinting.

3.4.2Stability test. The powder ofC.buchananiiRoem. et Schult. material from certain producing area was prepared into test solution according to the description in Section3.2. After standing for 0, 4, 8, 12, 16, 20 and 24 h, the test solution was detected under the chromatographic conditions described in Section3.1, respectively. The results show that theRSDvalues of relative retention time of the common peaks were in the range of 0%-0.12%, and theRSDvalues of their relative areas were in the range of 0%-2.6%, all less than 3.0%, indicating that the test solution prepared is stable within 24 h.

3.4.3Reproducibility test. A total of six portions of powder ofC.buchananiiRoem. et Schult. material from certain producing area were weighed and prepared into test solutions according to Section3.2, respectively. The test solutions were subjected to chromatographic analysis under the conditions described in Section3.1. The results show that theRSDvalues of relative retention time of the common peaks ranged from 0%-0.14%, and theRSDvalues of their relative areas ranged from 0% to 2.5%, all less than 3.0%, indicating good reproducibility.

4 HPLC fingerprint establishment and technical parameters

4.1 Collection of fingerprints ofCryptolepisbuchananiiRoem. et Schult.The 10 batches ofC.buchananiiRoem. et Schult. materials were prepared into test solutions according to the description in Section3.2. According toTechnicalRequirementsforStudyingFingerprintofTraditionalChineseMedicineInjection(Provisional), the HPLC fingerprint ofC.buchananiiRoem. et Schult. was established. The chromatogram was recorded for 10 min, and it was found that no peak appeared after 60 min, so the collection time of the chromatogram was determined to be 60 min. The test solutions were analyzed chromatographically in sequence, and their HPLC chromatograms were recorded. The superimposed HPLC chromatograms of the 10 batches ofC.buchananiiRoem. et Schult. materials is shown in Fig.1.

Fig.1 Superimposed HPLC chromatogram of the 10 batches ofCryptolepisbuchananiiRoem. et Schult. samples

4.2 Determination of reference peakAccording to Technical Requirements for Studying Fingerprint of Traditional Chinese Medicine Injection (Provisional), a suitable control is selected as a reference for fingerprinting based on the nature of the chemical constituents in medicinal materials. Chlorogenic acid is one of the main chemical constituents ofC.buchananiiRoem. et Schult. It is stable in the HPLC fingerprint ofC.buchananiiRoem. et Schult. Moreover, the area of the peak of chlorogenic acid accounts for an appropriate proportion in the total peak area. Therefore, the peak No.11 of chlorogenic acid was selected as the reference peak of this experiment and labeled as S. Under the chromatographic conditions under Section3.1, the HPLC fingerprints of the chlorogenic acid reference solution and theC.buchananiiRoem. et Schult. test solution were determined, as shown in Fig.2 and Fig.3.

Fig.2 HPLC fingerprint of chlorogenic acid

Fig.3 HPLC fingerprint ofCryptolepisbuchananiiRoem. et Schult.

4.3 Marking of common peaksAccording toSimilarityEvaluationSystemforChromatographicFingerprintofChineseTraditionalMedicine(Version 2004 A), the similarity between the fingerprints of the 10 batches ofC.buchananiiRoem. et Schult. materials was analyzed. The time width was set to 0.4 min, and the common pattern of the HPLC chromatograms ofC.buchananiiRoem. et Schult. samples was extracted using the median method to establish a reference fingerprint forC.buchananiiRoem. et Schult (Fig.4). A total of 13 common chromatographic peaks were marked. Among them, the peak No.11 was chlorogenic acid, and it was also the reference peak (S). The statistical results of relative retention time and relative areas of the common chromatographic peaks are shown in Table 3 and Table 4. It is shown that theRSDvalues of relative retention time of the common peaks were all less than 3.0%, and theRSDvalues of relative areas of the common peaks were greater. The similarities between the fingerprints of the 10 batches ofC.buchananiiRoem. et Schult. samples were all greater than 0.9, and they had a good similarity with the reference fingerprint. The chromatographic behaviors of the fingerprints of the 10 batches ofC.buchananiiRoem. et Schult. samples were basically the same (Table 5).

Fig.4 Common pattern of the 10 batches ofCryptolepisbuchananiiRoem. et Schult. in HPLC fingerprints

Table 3 Relative retention time of common peaks of the 10 batches ofCryptolepisbuchananiiRoem. et Schult. samples in HPLC fingerprints

Peak No.Sample codeS1S2S3S4S5S6S7S8S9S10RSD∥%10.337 00.338 20.338 50.337 10.338 40.336 90.336 60.338 10.338 60.336 50.25 20.472 70.473 70.472 90.472 20.473 40.472 30.472 20.472 30.473 30.472 10.12 30.552 10.554 00.554 00.552 40.553 70.551 70.551 20.553 00.554 50.551 40.22 40.601 30.603 00.600 90.600 70.600 90.600 70.600 20.600 00.601 30.600 20.14 50.645 30.645 80.644 80.644 30.645 10.646 90.644 40.643 80.644 80.644 20.14 60.703 90.705 10.705 00.704 00.704 80.703 70.703 30.704 00.705 30.703 30.10 70.741 50.742 80.742 30.741 50.742 20.741 10.740 30.741 20.742 20.740 50.11 80.864 00.863 80.863 60.863 50.864 10.863 10.863 20.862 50.863 30.862 80.06 90.924 50.924 60.924 60.924 50.925 40.924 10.923 60.923 80.924 30.923 50.06 100.946 40.946 10.946 00.945 70.946 50.945 50.945 40.945 20.945 40.945 20.05 111.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 00 121.026 51.026 01.026 21.026 21.026 81.026 51.026 31.026 21.026 01.026 20.02 131.051 51.050 31.051 01.053 51.051 71.050 91.051 11.050 51.050 41.050 30.09

Table 4 Relative area of common peaks of the 10 batches ofCryptolepisbuchananiiRoem. et Schult. samples in HPLC fingerprints

Peak No.Sample codeS1S2S3S4S5S6S7S8S9S10RSD∥%10.098 00.268 70.118 50.135 60.060 10.130 60.077 00.199 00.169 60.196 043.8 20.174 10.637 00.290 10.353 40.144 10.304 70.175 70.502 90.408 10.447 546.3 30.320 40.832 40.373 90.405 50.189 20.416 20.236 00.608 10.514 80.600 343.1 40.814 00.755 00.637 20.453 20.643 00.786 01.087 51.243 01.560 11.278 737.8 50.069 90.216 70.106 80.168 90.070 50.097 10.062 00.249 80.430 10.107 573.0 60.331 20.933 80.408 30.464 30.216 80.468 20.271 40.688 90.540 60.712 544.0 70.124 40.354 00.177 20.271 80.121 50.167 30.106 50.362 60.698 00.177 770.6 80.048 40.130 80.069 80.110 60.041 80.066 00.050 60.171 20.186 70.104 552.9 90.164 90.476 90.243 70.356 30.160 60.232 10.158 60.550 50.904 40.265 667.3 100.025 20.119 40.050 40.087 80.035 30.048 80.035 40.139 50.263 50.075 582.1 111.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 01.000 00 120.669 50.646 50.544 80.431 60.575 90.676 50.919 61.007 41.092 01.085 931.4 130.051 40.154 10.072 70.208 00.049 30.076 10.056 20.174 40.186 20.112 153.8

Table 5 Similarity of HPLC fingerprints of the 10 batches ofCryptolepisbuchananiiRoem. et Schult. samples

Sample codeS1S2S3S4S5S6S7S8S9S10ReferencefingerprintS11.0000.8950.9870.9330.9900.9930.9820.9540.9050.9690.996S20.8951.0000.9410.9580.8480.9410.8250.9510.8870.9280.926S30.9870.9411.0000.9770.9760.9950.9410.9570.9040.9540.991S40.9330.9580.9771.0000.9250.9590.860.9310.8830.9010.951S50.9900.8480.9760.9251.0000.9730.9710.9150.8770.9260.979S60.9930.9410.9950.9590.9731.0000.9620.9730.9190.9790.998S70.9820.8250.9410.860.9710.9621.0000.9380.9080.9620.974S80.9540.9510.9570.9310.9150.9730.9381.0000.9740.9850.976S90.9050.8870.9040.8830.8770.9190.9080.9741.0000.9330.933S100.9690.9280.9540.9010.9260.9790.9620.9850.9331.0000.980Reference fingerprint0.9960.9260.9910.9510.9790.9980.9740.9760.9330.9801.000

5 Discussion and conclusions

5.1 Selection of mobile phaseIt has been found that the single mobile phase has poor separation, so in this experiment, gradient elution was used. The results show that each chromatographic peak was well separated. Multiple elution procedures were examined during the experiment. The elution procedure of the acetonitrile-water system had poor elution ability to the samples, and the appearance time of the peaks was also relatively lagging. In the methanol-water system, the peaks showed a phenomenon of tailing, prolonging and poor symmetry. In contrast, the methanol-0.1% phosphoric acid (volume fraction) system showed good resolution, more peaks, and more stable baseline, so methanol-0.1% phosphoric acid (volume fraction) was selected as the mobile phase.

5.2 Selection of columnIn this experiment, Elite ODS Hypersil (4.6 mm×250 mm, 5 μm) column, Welch Matenals XB-C18(4.6 mm×250 mm, 5 μm) column and Thermo ODS-2 Hypersil (4.6 mm×250 mm, 5 μm) were investigated. The results show that Thermo ODS-2 Hypersil (4.6 mm×250 mm, 5 μm) column had more peaks and good resolution, so it was selected as the HPLC column in this experiment.

5.3 Selection of detection wavelengthIn order to obtain the best absorption wavelength for the medicinal herb ofC.buchananiiRoem. et Schult., in this experiment, the DAD full-wavelength detection was performed at a wavelength of 200-400 nm using a diode detector. After eliminating a band with a severe baseline drift at a low wavelength in the 3D spectrum, an effective part from 240 to 400 nm was obtained. Focus was placed on the chromatograms obtained at 270, 280, 300, 310, 323 and 340 nm. It was observed that the chromatogram obtained at the wavelength of 323 nm had good UV absorption, more evenly distributed peaks, more stable baseline and good symmetry, so 323 nm was selected as the detection wavelength for the fingerprinting ofC.buchananiiRoem. et Schult.

5.4 Selection of column temperature and flow rateIn this experiment, the column temperatures of 20, 25, 30 and 35 ℃ and the flow rates of 0.8, 1.0 and 1.2 mL/min were examined, respectively. It was found that the chromatogram obtained under the conditions of column temperature of 25 ℃and flow rate of 0.8 mL/min was better.

5.5 Blank and extended elution testIn order to investigate the interference of the mobile phase to the sample analysis, 20 μL of the mobile phase was injected into the high-performance liquid chromatograph and analyzed under the conditions described in Section3.1. The obtained chromatogram shows that there was no impurity in the mobile phase to interfere with sample analysis.

In this paper, the fingerprints ofC.buchananiiRoem. et Schult. from different habitats were established by HPLC. The results show that the fingerprints ofC.buchananiiRoem. et Schult. had stable common peaks and good separation. The similarities between the fingerprints of the 10 batches ofC.buchananiiRoem. et Schult. samples and the reference fingerprint were greater than 0.9, indicating that the chemical composition of the 10 batches ofC.buchananiiRoem. et Schult. samples was basically the same. However, the area of each of the common peaks varied, indicating that there is a difference in the content of the same component of the medicinal herb from different habitats. This might be caused by the differences in external (e.g. climate, season and environment) and internal factors. The method established in this experiment can objectively evaluate the overall quality ofC.buchananiiRoem. et Schult., with strong specificity, good precision, reproducibility and stability. It is of great significance for the establishment of a scientific and rational quality evaluation method forC.buchananiiRoem. et Schult. and the improvement of its quality control standards.