Establishment of Specific Chromatogram of Spica Prunellae Formula Granules Based

Xinwei NONG, Mei YANG, Xiongmei HUANG, Huilian DENG, Chan WANG, Jian ZHOU

PuraPharm (Nanning) Pharmaceutical Co., Ltd., Nanning 530007, China

Abstract [Objectives] This study aimed to establish HPLC chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae. [Methods] The chromatographic conditions were as follows: column, SHISEIDO CAPCELL PAK C18 MGII column (4.6 mm×250 mm, 5 μm); mobile phase, acetonitrile-0.1% phosphoric acid; gradient elution; detection wavelength, 280 nm; flow rate, 1.0 mL/min; and column temperature, 30 ℃. The correlation between the decoction pieces, standard decoction and formula granules of Spica Prunellae was analyzed by specific chromatograms. [Results] The fingerprint chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae showed five common peaks, with good correlation. Among the five common peaks, four of them were tanshinol, protocatechuic acid, caffeic acid and rosmarinic acid. [Conclusions] The main chemical constituents of decoction pieces, standard decoction and formula granules of Spica Prunellae are basically the same. The HPLC specific chromatogram established can be used for the quality control of Spica Prunellae formula granules.

Key words Spica Prunellae, Standard decoction, Formula granule, HPLC, Fingerprint chromatogram

1 Introduction

Formula granules of traditional Chinese medicine are made through water extraction, concentration, dryness and granulation of corresponding decoction pieces. With the publication of theTechnicalRequirementsforQualityControlandStandardFormulationofChineseMedicineFormulaGranules, standards for research on formula granules have been clearly defined. Standard decoction is used as a standard reference for measuring formula granules. The active ingredients of formula granules should be basically consistent with those of the decoction of the traditional Chinese medicine. Therefore, comparison with standard decoction has become an important prerequisite for the standard formulation of Chinese medicine formula granules. Spica Prunellae is the dried ears ofPrunellavulgarisL. It has the effects of clearing away the liver-fire, improving eyesight, removing stasis and diminishing swelling[1]. At present, the reports of Spica Prunellae mainly focus on the separate study of the medicinal material and its formula granules[2-7], but the correlation between its decoction pieces, standard decoction and formula granules has not been reported. In this study, the HPLC specific chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae were established, and the correlation between them was evaluated to provide reference for the establishment of the quality standards of Spica Prunellae.

2 Instruments and reagents

2.1 InstrumentsThe apparatus and instruments used mainly included Agilent 1260 high-performance liquid chromatograph (Agilent, USA), XPE205DR electronic analytical balance (METTLER TOLEDO, Switzerland) and KQ-500DE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

2.2 ReagentsRosmarinic acid (batch No. 111871-201102, purity 99.8%), caffeic acid (batch No.110885-200102, purity 100%) and protocatechuic acid (batch No.110810-200205, purity 100%) were purchased from National Institutes for Food and Drug Control, China. Tanshinol (batch No. MUST-18060920, purity 98.38%) was purchased from Chengdu Mansite Biotechnology Co., Ltd. The acetonitrile was chromatographically pure, water used was ultrapure water and the other reagents were of analytical grade. Fifteen batches of Spica Prunellae decoction pieces and four batches of Spica Prunellae formula granules were provided by PuraPharm (Nanning) Pharmaceutical Co., Ltd. Fifteen batches of Spica Prunellae standard decoctions were prepared according to the relevant requirements ofManagementSpecificationforDecoctingRoominMedicinalInstitution. The coding and specific sources of the test materials are shown in Table 1.

3 Methods and results

3.1 Chromatographic conditionsColumn: SHISEIDO CAPCELL PAK C18MGII (250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile (A) -0.1% phosphoric acid (B); gradient elution (0-10 min, 10% A; 10-22 min, 10%→22% A; 22-37 min, 22% A; 37-62 min, 22%→38% A; 62-65 min, 38%→90% A; 65-70 min, 90% A); flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 280 nm; and injection volume: 10 μL.

3.2 Preparation of solutions

3.2.1Mixed reference solution. Accurate amounts of rosmarinic acid, caffeic acid, protocatechuic aldehyde and tanshinol refer-

Table 1 Information of the samples

SourceCode ofdecoctionpiecesCode ofstandarddecoctionCode offormulagranulesShigunhe Town, Queshan County, Henan ProvinceY1B1K1Wagang Town, Queshan County, Henan ProvinceY2B2K2Xinandian Town, Queshan County, Henan ProvinceY3B3Zhugou Town, Queshan County, Henan ProvinceY4B4Liudian Town, Queshan County, Henan ProvinceY5B5Anpeng Town, Tongbai County, Henan ProvinceY6B6Erpu Town, Miyang County, Henan ProvinceY7B7K4Shibali Town, Bozhou City, Anhui ProvinceY8B8K3Shijiuli Town, Bozhou City, Anhui ProvinceY9B9Wuma Town, Bozhou City, Anhui ProvinceY10B10Dasi Town, Bozhou City, Anhui ProvinceY11B11Qiaocheng District, Bozhou City, Anhui ProvinceY12B12Gaochun County, Nanjing City, Jiangsu ProvinceY13B13Dushan County, Guizhou ProvinceY14B14Dushan County, Guizhou ProvinceY15B15

ence substances were dissolved in a certain volume of 50% ethanol to prepare into a mixed reference solution containing 87.36 μg/mL of rosmarinic acid, 11.55 μg/mL of caffeic acid and 17.78 μg/mL of protocatechuic aldehyde and 79.30 μg/mL of tanshinol.

3.2.2Test solution of decoction pieces. An accurate amount (around 1.0 g) of the powder of Spica Prunellae decoction pieces (passed through No.2 sieve) was placed in a conical flask with stopper, added with 25 mL of water, weighed, refluxed for 30 min, cooled, and weighed again in success. The lost weight was supplemented with water. Then, the solution was filtered, and the filtrate obtained was used for analysis.

3.2.3Test solution of standard decoction. A certain amount (100 g) of Spica Prunellae decoction pieces was added with 18 times the amount of water, soaked for 30 min, decocted for 20 min after boiled with strong fire, and filtered. The filter residue was added with 16 times the amount of water, decocted for 25 min, and filtered. The water decoction of the two times was mixed together, concentrated under reduced pressure at 50 ℃, and free-dried under vacuum to obtain the paste. A certain amount of the paste was ground. Then, an accurate amount (around 0.2 g) of the powder was placed in a conical flask with stopper, added with 25 mL of homeopathic alcohol, weighed, sonicated for 30 min, cooled, and weighed again. The lost weight was supplemented with homeopathic alcohol. The solution was filtered, and the subsequent filtrate was collected.

3.2.4Test solution of formula granules. A certain amount of Spica Prunellae formula granules was ground. An accurate amount (around 0.2 g) of the powder was placed in a conical flask with stopper, added with 25 mL of 50% ethanol, weighed, sonicated for 30 min, cooled, and weighed again. The lost weight was supplemented with homeopathic alcohol. Then, the solution was filtered, and the subsequent filtrate was collected.

3.2.5Preparation of negative control solution of formula granules. The Spica Prunellae-free negative control sample, prepared according to the proportion and process of the prescription, was prepared into negative control solution according to the description in Section3.2.4.

3.3 Methodological investigation

3.3.1System applicability and specificity tests. A certain volume (10 μL) of the mixed reference solution, the test solution and the negative control solution was injected into high-performance liquid chromatogram for analysis, respectively. The obtained HPLC chromatograms are shown in Fig.1. The results show that the peaks in the samples are well separated and symmetrical, and there is no interference from the negative control, indicating that this method is specific.

Note: A.negative control; B.mixed reference; C.test solution; 1.tanshinol; 2.protocatechuic acid; 3.caffeic acid; 4.rosmarinic acid.

Fig.1 HPLC specific chromatogram of Spica Prunellae formula granules

3.3.2Precision test. The test solution of Spica Prunellae formula granules was detected chromatographically six times repeatedly, 10 μL for each time. Taking the peak 4 as the peak S, the relative retention time of the specific peaks 1-5 against the peak S was calculated. The results show that theRSDvalues of relative retention time of the specific peaks ranged from 0.20% to 0.25%, indicating that the instrument has good precision.

3.3.3Stability test. The test solution of Spica Prunellae formula granules was detected by HPLC 0, 4, 8, 12 and 24 h after its preparation, respectively. Taking peak 4 as the peak S, the relative retention time of the specific peaks 1-5 against the peak S was calculated. The results show that theRSDvalues of relative retention time of the specific peaks were in the range of 0.10%-0.23%, indicating that the test solution is stable within 24 h.

3.3.4Reproducibility test. Six portions of the powder of Spica Prunellae formula granules were weighed, prepared into test solutions, respectively according to the description in Section3.2.4, and detected according to the proposed method. Taking the peak 4 as the peak S, the relative retention time of the specific peaks 1-5 against the peak S was calculated. The results show that theRSDvalues of relative retention time of the specific peaks ranged from 0.11% to 0.30%, indicating that this method is reproducible.

3.3.5Durability test. Based on the original chromatographic conditions, the test solution of Spica Prunellae formula granules was detected at different column temperatures [(30±5)℃] and flow rates [(1.0±0.1) mL/min]to investigate the durability of the chromatographic conditions. Taking peak 4 as the peak S, the relative retention time of the specific peaks 1-5 against the peak S was calculated. The results show that at different column temperatures and flow rates, theRSDvalues of relative retention time of the specific peaks ranged from 0.19%-2.71% and 0.61%-2.74%, indicating that the durability of column temperature and flow rate in the method is better.

3.4 Establishment of specific chromatogramThe 15 batches of Spica Prunellae decoction pieces, the 15 batches of Spica Prunellae standard decoctions and the 4 batches of Spica Prunellae formula granules were detected according to the proposed method, respectively, and their HPLC specific chromatograms were established (Fig.2-Fig.5). Peak matching was performed using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine. A total of five reproducible peaks were selected as the common specific peaks. By comparison with the reference chromatogram (Fig.1), it can be seen that peak 1 is tanshinol, peak 2 is protocatechuic aldehyde, peak 3 is caffeic acid, and peak 4 is rosmarinic acid. Taking peak 4 as peak S, the relative retention time and relative area of the specific peaks 1-5 against the peak S were calculated, and the results are shown in Table 2.

Fig.2 HPLC specific chromatograms of the 15 batches of Spica Prunellae decoction pieces and reference fingerprint

Fig.3 HPLC specific chromatograms of the 15 batches of Spica Prunellae standard decoction and reference fingerprint

Fig.4 HPLC specific chromatograms of the 4 batches of Spica Prunellae formula granules and reference fingerprint

Fig.5 Comparison of HPLC specific chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae

Table 2 Relative retention time and relative area of the common peaks in the reference chromatograms

ItemSamplePeak No.1234 (S)5RelativeDecoction pieces0.1820.3810.5381.0001.609retention timeStandard decoction0.1830.380.5401.0001.611Formula granules0.1830.3820.5411.0001.614RelativeDecoction pieces0.2760.1060.5501.0000.182peak areaStandard decoction0.3180.1750.6931.0000.250Formula granules0.2970.1830.4651.0000.210

3.5 Correlation between decoction pieces, standard decoction and formula granulesAs shown in Fig.2-Fig.5 and Table 2, the chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae have five common specific peaks. The relative retention time of each of the specific peaks is stable, and the relative area of each of the specific peaks is relatively close. The similarity between the chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae was in the range of 0.987-0.996. It indicates that decoction pieces, standard decoction and formula granules of Spica Prunellae have a good correlation. Formula granules and standard decoction of Spica Prunellae have basically the same chemical constituents. This study will provide an important basis for the feasibility of traditional Chinese medicine formula granules to replace traditional decoction.

4 Discussion

4.1 Selection of experimental conditionsThe test sample was subjected to 190-400-nm full-wavelength scanning. The results show that at the wavelength of 280 nm, the number of peaks was large with abundant information, and the shape and resolution of the peaks were good, so 280 nm was chosen as the detection wavelength. The formula granules of Spica Prunellae were extracted sonically with water, 30% ethanol, 50% ethanol, 70% ethanol and ethanol, respectively. The results showed that when 50% ethanol was used as the extraction solvent, the shape of the peaks was better, and the response value was greater, so 50% ethanol as selected as the extraction solvent.

4.2 Identification of specific peaksThe peak 5 was further analyzed by mass spectrometry. Combined with literature review, it was identified as schizotenuin A. Due to the failure to purchase the reference substance, it was not positioned on the chromatograms.

4.3 Correlation analysisThe HPLC fingerprint of Spica Prunellae formula granules was established, which was simple and fast. The specific chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae were highly similar and basically consistent in chemical composition, with traceability. It can be used to conduct quality control for all links of the production process of Spica Prunellae formula granules, providing an effective means and basis for its quality control.