GUANG Li-wen, ZHANG Shuang, GAO Xin-lei, YANG Tian-ming

1. Clinical Pharmacy Department of Hainan Cancer Hospital, Haikou 570100, China

2. Hainan Cancer Prevention and Treatment Center, Haikou 570100, China

3. Quality Management Department of Haikou Pharmaceutical Factory, Haikou 570100, China

4. College of Traditional Chinese Medicine, Guangdong Vocational College of Food and Medicine, Guangzhou 510520, China

Keywords:HPLC QAMS Dianxiankang Capsules fk/s Quantitative determination

ABSTRACT

1. Introduction

Epilepsy is a common chronic brain disease characterized by abnormal discharge of brain nerves in neurology, which may lead to permanent brain injury and even death. It is composed of 15 Chinese herbal medicines such as Gastrodia, Radix Polygalae,Radix Ophiopogonis, Rhizoma Acori Tatarinowii, Bombyx Batryticatus, and Radix Salviae Miltiorrhizae. It has the effect of calming convulsions and extinguishing wind, resolving phlegm and opening orifices. It is mainly used for epilepsy patients with wind-phlegm blocking, phlegm-fire disturbing heart, fainting convulsions, and salivation. Modern studies have shown that Dianxiankang Capsule combined with sodium valproate sustainedrelease tablets can reduce the levels of tumor necrosis factor-α,interleukin-6 and serum factors in the brain tissue of patients with epilepsy, thereby improving the clinical symptoms of patients and reducing the levels of inflammatory factors [1]. Epilepsy Kang Capsule can also improve the quality of disease control in the later stage and improve the mental state of patients [2,3] ; its joint levetiracetam epilepsy patients can reduce serum S-100β protein,high-sensitivity C-reactive protein and malondialdehyde levels,improve their living ability and intelligence level, but also can increase α, β-band EEG relative power, reduce δ, θ-band EEG relative power, effectively improve their neurological function[4-7]. The current quality standard of Dianxiankang Capsule[8] and its related literature [9,10] only determine the content of one component, which cannot objectively evaluate the internal quality of Dianxiankang Capsule, and cannot ensure its clinical effectiveness. The HPLC-QAMS method uses the intrinsic function and proportional relationship between the components contained in traditional Chinese medicine to achieve simultaneous detection of multiple index components by measuring a certain component with stable quality and low cost. It has been widely used in the quality control of Chinese patent medicine compound preparations. In this experiment, gastrodin, balison glycoside E and balison in Epilepsy Capsule were used Glycoside B, Palisenoside, Polygala tenuifolia saponin, Polygala tenuifolia saponin B, Ilex japonicus Yflavanone A, methylophiopogon dihydrohomoisoflavone B β-Asarone and α-Asarone is a quantitative control indicator, which covers 4 kinds of traditional Chinese medicines in the prescription There are 10 representative ingredients in the wood, and Polygala tenuifolia saponin is the internal reference Substance, calculate its relationship with gastrodin, balison glycoside E, balison glycoside B, balison glycoside Senoside, polyglycoside B, ophiopogon methyl flavanone A, methylophiopogon Hydrogen homoisoflavone B β-Asarone and α-Relative correction factor of asarone Sub (fk/s), calculate the content of each component by fk/s, and compare with the measured value by external standard method Compare and investigate the differences between the two methods. This method reduces the inspection cost This method shortens the inspection cycle and is conducive to the popularization of the method Face control of the internal overall quality of Epilepsy Capsule provides data supp.

2. Materials and Methods

2.1 Materials

Epilepsy Kang Capsule (0.3 g per capsule) from Guangshengyuan Traditional Chinese Medicine Co., Ltd. (Batch number 2006052,2007072, 2007083, 2010091, 2010102, 2101013, 2101022,2103012, 2104030, 2105032, 2105034 and 2109043, number:S1～S12) ; the reference substances of gastrodin, tenuifolin and β-asarone (Batch No.110807-202010,111849-202106 and 112018-201802, purity 95.5%, 97.3% and 99.3%) were from China Institutes for Food and Drug Control. The reference substances Ophiopogon japonicus methyl flavanone A, parishin E, parishin B, methyl ophiopogon dihydrohomoisoflavone B, parishin, tenuigenin B and α-asarone (batch number PRF9102924, PRF9010943, PRF9061142,PRF9102925, PRF9061141, PRF8112741 and PRF8051843, purity 98.9%, 98.4%, 97.2%, 98.9%, 97.6%, 98.7% and 99.2%) were derived from Chengdu Purifa Technology Development Co., Ltd.; chromatographic grade acetonitrile, the rest of the reagents were analytically pure.Agilent high performance liquid chromatograph(Agilent, Model: 1260), Shimadzu high performance liquid chromatograph (Shimadzu Instrument Co., Ltd., Model: LC-20A) ;electronic balance (Mettler Toledo, Switzerland, model: AB265-S) ;ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Model:KQ-300VDY) ; waters Symmetry C18, Phenomenex Nucleosil C18and Dima Spursil C18columns, 250 mm×4.6 mm, 5 μm.

2.2 Methods

2.2.1 Preparation of reference solution

Precision weighing The reference substance gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B, Ophiopogon japonicus methyl flavanone A, methyl Ophiopogon japonicus dihydrohomoisoflavone B, β-asarone and α-asarone were dissolved and diluted with 50% methanol to prepare a mixed reference solution containing 0.978, 1.130, 0.356, 2.492, 3.810, 4.594, 0.292, 0.118,5.874 and 1.856 mg/mL. The stock solution was diluted 20 times with 50 % methanol to obtain a mixed reference solution with 10 components at mass concentrations of 48.9, 56.5, 17.8, 124.6, 190.5,229.7, 14.6, 5.9, 293.7 and 92.8 μg/mL, respectively.

2.2.2 Preparation of test solution

2 g of the content of Dianxiankang capsule was accurately weighed,20 mL of 50% methanol was added, and the KQ-300 VDY ultrasonic cleaner was used to extract the content of Dianxiankang capsule for 45 min. After cooling, the content of Dianxiankang capsule was fixed to 25 mL with the same solvent, and filtered (0.45 μm). The negative test samples lacking Gastrodiae Rhizoma, Polygalae Radix,Ophiopogonis Radix and Acori Tatarinowii Rhizoma prepared according to the prescription process were taken to prepare the negative test solution according to the above method.

2.2.3 Chromatographic conditions

Acetonitrile (A)-0.05% phosphoric acid as mobile phase, gradient elution (0-10 min, 17.0% A; 10-24 min, 17.0%→26.0% A ; 24-37 min, 26.0%→58.0% A; 37-48 min, 58.0% → 72.0% A; 48-70 min, 72.0%→17.0% A) ; agilent 1 260 high performance liquid chromatograph; waters Symmetry C18column, column temperature 30℃ ; detection wavelength: 220 nm (0-37 min, gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B) [11-16], 296 nm(37-48 min, methylophiopogonanone A, methylophiopogonan dihydrohomoflavone B) [17,18] and 275 nm (48-70 min, β-asarone,α-asarone) [19-22]; the flow rate was 1.0 mL/min and the injection volume was 10 μL. The number of theoretical plates should not be less than 5 500 according to the time of tenuifolin.

3. Results

3.1 Specificity test

Figure 1 HPLC chromatograms

Take an appropriate amount of reference solution and test solution according to the sample detection, record the chromatographic outflow curve (Figure 1). The results showed that No. 1～10 chromatographic peaks and adjacent impurity peaks in the test solution of Dianxiankang Capsules could be effectively separated,and the tailing factor was in accordance with the current Chinese Pharmacopoeia. The negative samples did not interfere with the quantitative analysis of gastrodin, parishin E, parishin B, parishin,tenuifolin, tenuifolin B, ophiopogonin A, methylophiopogonin B,β-asarone and α-asarone in Dianxiankang capsules.

3.2 Investigation of linear range

0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of the mixed reference stock solution was accurately pipetted and diluted with 50 % methanol to 20 mL to prepare six series of linear solutions (Ⅰ～Ⅵ). Under the above chromatographic conditions, the mass concentration and peak area of gastrodin, parishin E, parishin B, parishin, tenuifolin,tenuifolin B, ophiopogon japonicus methylflavanone A, methyl ophiopogon japonicus dihydrohomoflavone B, β-asarone and α-asarone reference substances were linearly regressed, as shown in Figure 1. It can be seen that each component has a good linear relationship within their respective ranges.

3.3 Precision, repeatability and stability experiment

Take epilepsy Kang capsule (No.: S1), according to the law to prepare a sample solution, under the above chromatographic conditions repeated injection 6 times, record the outflow chromatogram. The RSD values of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B,methylophiopogonanone A,methylophiopogonanone B, β-asarone and α-asarone were 1.25%,1.10%, 1.37%, 0.82%, 0.76%, 0.62%, 1.47%, 1.59%, 0.53% and 1.01%, respectively.

Table 1 Regression equations and linear ranges of ten reference substances

Epilepsy Kang Capsule (No.: S1) was taken, and 6 portions of the test solution were prepared according to the method. The samples were injected under the above chromatographic conditions, and the effluent chromatogram was recorded. The average contents of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B,ophiopogonanone A, methylophiopogonanone B, β-asarone and α-asarone were calculated by external standard method to be 0.816,0.943, 0.281, 1.860, 2.716, 3.531, 0.221, 0.098, 4.395 and 1.404 mg/g, respectively. The RSD values of the 10 components were 1.57%,1.51%, 1.79%, 1.13%, 1.06%, 0.99%, 1.80%, 1.88%, 0.87% and 1.36 %, respectively.

Epilepsy Kang capsule (No. S1) was taken and one portion of the test solution was prepared according to the law. The test solution was detected under the above chromatographic conditions at 0, 3,6, 10, 16 and 24 h after preparation, and the effluent chromatogram was recorded. The results showed that the test solution ofEpilepsy Kang capsule was stable for 24 h. The RSD values of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B,methylophiopogonanone A, methylophiopogonanone B, β-asarone and α-asarone were 1.31%, 1.14%, 1.35%, 0.85%, 0.78%, 0.59%,1.50%, 1.62%, 0.54% and 1.03%, respectively.

Table 2 Recoveries of ten constituents in Dianxiankang Capsules (n=9)

images/BZ_20_236_226_2312_2210.png

3.4 Sample recovery experiment

1 g content of Dianxiankang Capsules (No.: S1) was precisely weighed, and 9 portions were taken and divided into 3 groups. The mixed reference solution (the mass concentrations of 10 reference substances were 0.816, 0.941, 0.282, 1.849, 2.706, 3.524, 0.229,0.097, 4.412 and 1.395 mg/mL, respectively) of 0.8, 1.0 and 1.2 mL was precisely added to prepare the sample test solution according to the method. Under the above chromatographic conditions, the average recovery and RSD values of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B, methylophiopogonanone A,methylophiopogonanone B, β-asarone and α-asarone were obtained.99.01% (0.92%), 98.89% (1.01%), 97.03% (0.76%), 99.29% (1.14%), 98.56% (0.87 %), 100.10% (0.62 %), 96.96% (1.50 %), 97.94%(1.55 %), 100.06% (0.73 %) and 99.82% (0.51 %) respectively. The results are shown in Table 2.

3.5 Determination of Relative Correction Factor (fk/s) and Investigation of Durability

3.5.1 fk/sDetermination

Table 3fk/s of each component for Dianxiankang Capsules

Table 4Effects of instruments and columns on fk/s

3.5.2 Effects of instrument and chromatographic column on fk/s

The effects of Agilent 1260 and Shimadzu LC-20A HPLC on fk/swere investigated by Waters Symmetry C18column (5 μm, 250 mm×4.6 mm), Phenomenex Nucleosil C18column (5 μm, 250 mm×4.6 mm) and Dima Spursil C18column (5 μm, 250 mm×4.6 mm),respectively. The mixed reference solution under 1.2.1 was injected,and the effluent chromatogram was recorded. The fk/sof the other 9 components were calculated with tenuifolin (peak 5) as the internal reference, which were 0.824 5, 0.725 8, 1.018 1, 1.184 5, 0.724 6,1.308 3, 0.926 5, 0.655 1 and 1.055 0, respectively. The results are shown in Table 4, indicating that the relative correction factor has good applicability to the instrument and chromatography.

3.5.3 Effect of flow velocity on fk/s.At the flow rate of 0.8,1.0 and 1.2 mL/min, the mixed

Reference solution under 1.2.1 was injected into the sample,and the outflow chromatographic curve was recorded. Taking tenuifolin (peak 5) as the internal reference substance, the fk/sof each component was calculated, which was 0.8255, 0.722 0, 1.024 0,1.191 6, 0.725 9, 1.317 6, 0.934 4, 0.656 5 and 1.059 5, respectively,RSD＜2.0%. The results are shown in Table 5, indicating that the relative correction factor has good applicability to the flow rate.

3.5.4 Effect of column temperature on fk/s

At column temperature: 25,30,35℃, the mixed reference solution under 1.2.1 was injected into the sample, and the outflow chromatogram was recorded. The fk/sof each component was calculated by using tenuifolin (peak 5) as the internal reference substance, which was 0.822 8, 0.723 8, 1.023 4, 1.187 3, 0.725 8,1.308 3, 0.925 7, 0.650 9 and 1.0558, respectively, RSD＜2.0%. The results are shown in table 6, indicating that the relative correction factor has good applicability to column temperature.

3.5.5 Chromatographic peak positioning

In this experiment, Waters Symmetry C18column (5 μm, 250 mm×4.6 mm), Phenomenex Nucleosil C18column (5 μm, 250 mm×4.6 mm) and Dima Spursil C18column (5 μm, 250 mm×4.6 mm) were used. Under the above chromatographic conditions,Agilent 1260 and Shimadzu LC-20 A high performance liquid chromatograph were used to detect the mixed reference solution under 1.2.1. The chromatographic outflow curve and the corresponding retention time of each component were recorded.With tenuifolin (peak 5) as the reference peak, the chromatographic peaks of the components to be measured were located by the relative retention time method, and the relative retention time of the other 9 components was calculated (ts/k＝ts/tk, k is the internal reference, s is the component to be measured). The ts/kof each component was calculated to be 0.384 9, 0.576 6, 0.652 7, 0.717 4, 1.117 5, 1.327 6, 1.476 2, 1.737 7 and 1.976 3, respectively, and RSD＜2.0%. The results are shown in Table 7, indicating that the relative retention time method can be used for chromatographic peak positioning.

Table 5 Effects of flow velocity on fk/s

Table 6 Effects of column temperatures on fk/s

Table 7 Effects of instruments and chromatographic columns on ts/k

Table 8 Determination results of gastrodin, parishin E, parishin B, parishin, tenuifolin, onjisaponin B, methylophiopogonanone A,methylophiopogonanone B, β asarone and α asarone in Dianxiankang Capsules(mg/g,n=3)

3.6 Content

Determination 12 batches of Dianxiankang capsules (No. S1～S12)were taken to prepare the test solution of Dianxiankang capsules according to the method. The samples were injected under the above chromatographic conditions, and the chromatogram outflow curve was recorded. The contents of each component were calculated by ESM and HPLC-QAMS (P＞0.5), indicating that there was no significant difference between the two methods (Table 8).

4. Discussion

4.1 Selection of target components

Epilepsy Kang Capsule is composed of Gastrodia elata, Polygala tenuifolia, Ophiopogon japonicus, Acorus tatarinowii Schott,Ginseng, Salvia miltiorrhiza, Bombyx batryticatus, Arisaema bombycis, Scorpio, Borneol, Calculus bovis, Amber, Fritillaria cirrhosa, Lophatherum gracile and Ginger. In the prescription,Gastrodia elata and Bombyx batryticatus are used to stop spasm,dispel wind and dredge collaterals; acorus tatarinowii Schott phlegm resuscitation, intelligence dampness ; bile south star clearing heat and resolving phlegm, dispelling wind and calming shock, four drugs are monarch drug ; salvia miltiorrhiza for promoting blood circulation and cooling blood, clearing heart and tranquilizing mind; ginseng tonify vitality, tranquilize mind ; artificial bezoar, borneol resuscitation, clearing heat and eliminating phlegm, extinguish wind antispasmodic; yuanzhi Anshen Yizhi, Kaiqiao Ningxin ;amber Zhenjing Anshen; scorpion wind antispasmodic, Tongluo Sanjie, seven herbs as minister medicine ; the other four flavors are adjuvants. In this experiment, the active ingredients contained in the monarch drug Gastrodia elata and Acorus tatarinowii Schott were mainly taken into account, taking into account the main components contained in the ministerial drug Polygala tenuifolia and the adjuvant drug Ophiopogon japonicus. Ten active ingredients including gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B,ophiopogonanone A, methylophiopogonanone B, β-asarone and α-asarone were selected for quantitative analysis. At the same time,considering that the peak time of tenuifolin was in the middle and the quality of the reference substance was stable, it was selected as the internal reference substance.

4.2 Determination of mobile phase

The separation effects of gastrodin, parishin E, parishin B,parishin, tenuifolin, tenuifolin B, methylophiopogonanone A,methylophiopogonanone B, β-asarone and α-asarone in Dianxiankang Capsules were taken as the main indexes. At the same time, the baseline stability was taken into account, and the comparative preexperiment was carried out under the conditions of acetonitrilewater and methanol-water mobile phases. The results showed that the acetonitrile-water system was superior to methanol-water, but the separation of peak 2 and peak 3 was incomplete and could not be integrated normally. After that, acetonitrile-0.05% phosphoric acid solution [14,17] and acetonitrile-0.1% formic acid solution [18]were used to compare the pre-experiment. Finally, acetonitrile-0.05% phosphoric acid solution was selected as the mobile phase.HPLC-QAMS was used to simultaneously detect the contents of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B,ophiopogonin A, methylophiopogonin B, β-asarone and α-asarone in Dianxiankang Capsules.

4.3 Determination of sample extraction method

The comprehensive extraction effects of gastrodin,parishin E, parishin B, parishin, tenuifolin, tenuifolin B,methylophiopogonanone A, methylophiopogonanone B, β-asarone and α-asarone were used as the indexes. The extraction solvents (30%methanol, 50% methanol, 70% methanol, 100% methanol, 70%ethanol [13]) and extraction methods (ultrasonic extraction, heating reflux) were compared and investigated. The best preparation method of the test sample was selected. The results showed that the comprehensive extraction effect of 10 components was the best when 50% methanol was ultrasonically extracted. The extraction time was optimized. The preparation method of the test sample under 1.2.2 was finally determined.

In this experiment, HPLC-QAMS method was applied to the multiindex quality evaluation model of gastrodin, parishin E, parishin B, parishin, tenuifolin, tenuifolin B, ophiopogonin A, methyl ophiopogonin B, β-asarone and α-asarone in Dianxiankang capsule for the first time, which avoided the instability and high price of some reference substances. Tenuifolin, which was stable, inexpensive and moderate in content in Dianxiankang capsule, was selected as the internal reference substance. At the same time, the difference of content determination results between external standard method and HPLC-QAMS method was compared. The established method is accurate and simple, which lays a foundation for the multi-index comprehensive quality control of Dianxiankang Capsules.

Conflict of Interest:

This experiment was designed by Guan Liwen. Gao Xinlei and Yang Tianming were responsible for data collection and analysis.Guan Liwen wrote the thesis, and Zhang Shuang was responsible for the article review. All authors declare no conflict of interest.