Tang Xianglin 1, Yi Jianfeng 1, 2, Liang Qiande 1, Ma Zengchun 1, Wang Yuguang 1, Gao Yue 1* 1 Institute of Radiology and radiation, Academy of Military Medical Sciences, Beijing 100850 2 Yichun University, Yichun 336000   Chinese herbal medicine, 2013,44
Abstract: Objective To study the material basis of Radix Ginseng and Radix Aconiti by ultra performance liquid chromatography time of flight mass spectrometry (UPLC-TOF-MS), and to analyze the effect of compatibility on the dissolution of ginsenosides. Methods acquity HSS T3 (100 mm × 2.1 mm, 1.8 μ m) chromatographic column was used, acetonitrile water system (containing 0.1% formic acid) gradient elution was used, mass spectrometer was used for detection, and ginsenoside was analyzed by comparison with the reference substance. The dissolution amount of ginsenoside was expressed by the peak area of HPLC. Results after the compatibility of Radix Aconiti, the dissolution of most ginsenosides decreased linearly with the decrease of ginseng proportion; The dissolution of triol ginsenoside Re and oleanolic acid ginsenoside ro increased compared with the theoretical value; The dissolution of diol ginsenosides Rb2, RB3 and RD and their malonic acid formyl derivatives decreased compared with the theoretical value. Conclusion ginseng-AconiteAfter compatibility, variousGinsenosideThe dissolution phenomenon is different; The dissolution of these ginsenosides may be related to the efficacy of the compatibility of ginseng and aconite.
Key words: Ginseng; Aconite; Compatibility ratio; Ginsenoside; Ultra high performance liquid chromatography time of flight mass spectrometry
CLC No.: r283.21; 286.02 document code: a Article No.: 0253 -2670 (2013) 01 - 0000 - 00

        Shenfu Decoction is a famous prescription commonly used in clinic. It is often used for the deficiency of true Yang, deficiency of Yang and Qi, and sudden loss of yin and Yang Qi and blood. As early as the Song Dynasty, there were records of Shenfu Decoction in the general record of Shengji. Later, there were records of Shenfu Decoction in medical books in various periods of song, yuan, Ming and Qing Dynasties. This prescription is composed of ginseng and aconite. With the different ratio of ginseng and aconite, its usage and indications are also different, suggesting that there may be differences in its material basis. Ginsenoside is the main pharmacodynamic component of ginseng, and also the main pharmacodynamic component of Shenfu Decoction [1]. At present, there are few studies on the influence of the compatibility ratio of ginseng and Aconite on the dissolution of ginsenoside [2-4]. Generally, the method to study the compatibility ratio of two drugs is to fix the dosage of one drug and change the dosage of the other drug, but the proportion of this method is generally small [5-6]. In this study, after fixing the total amount of Radix Aconiti and changing the dosage of Radix Aconiti, the proportion of Radix Aconiti can reach 0 ~ 100%, so as to obtain the effect of human participation in the interaction of Radix Aconiti on the dissolution of various ginsenosides.
1. Instrument and material acquity UPLC synapt MS ultra high performance liquid chromatography mass spectrometry (waters), including mass lynx v4.1 mass spectrometry workstation (waters), acquity HSS T3 chromatographic column (100 mm × 2.1 mm, 1.8 μ m, waters); Refrigerated centrifuge (Heraeus labofuge 400R) and millipore simplicity. Both ginseng and aconite were purchased from the hebei'an traditional Chinese medicine market and identified as Panax ginseng C. A. mey., a perennial herb of Acanthopanax senticosus by Professor mabaiping of Institute of radiation medicine, Academy of Military Medical Sciences The dried roots and rhizomes of Aconitum carmichaelii debx. of Ranunculaceae, and the processed sub roots of Aconitum carmichaelii debx. are deposited in room 2, Institute 2, Academy of Military Medical Sciences. Ginsenosides Rg1 (batch No. 201122), re (batch No. 201027), RF (batch No. 201223), RB1 (batch No. 201021), RC (batch No. 200305), Rb2 (batch No. 200507), RD (batch No. 201017), RO (batch No. 201015) and Rh1 (batch No. 201108) were purchased from China Institute for food and drug control. Formic acid (chromatographically pure, CNW technologies), acetonitrile (chromatographically pure, Fisher Scientific) and ultrapure water were prepared by millipore pure water instrument.
2. methods and results
2.1 preparation of ginseng and aconite sample solution after crushing ginseng and aconite, fix the total mass of ginseng and aconite to 10 g, weigh and mix according to the six ratios of ginseng and aconite (10:0, 8:2, 6:4, 4:6, 2:8, 0:10), add 100 ml of pure water, soak for 30 min, decoct for 60 min in a slightly boiling state, and filter and extract the decoction through three layers of gauze; Add 50ml of water to the remaining residue, decoct for 30 min, filter and extract the decoction through three layers of gauze, and combine the two filtrates to obtain the desired ginseng Aconite Decoction in different proportions; Let the prepared Decoction stand for 12h, take 1ml of the standing supernatant, centrifuge for 10min at 10000 r/min, take the supernatant, filter it with 0.22 μ M aqueous phase microporous filter membrane, and then it is the sample solution, which is stored at 4 ℃ for testing.
2.2 UPLC chromatographic conditions: acquire HSS T3 (100 mm × 2.1 mm, 1.8 μ m) chromatographic column is used, the column temperature is 30 ℃, the sample room temperature is 4 ℃, the volume flow is 0.5 ml/min, the injection volume is 5 μ L, the mobile phases are 0.1% formic acid aqueous solution (a) and 0.1% formic acid acetonitrile solution (b), gradient elution conditions: 0 ~ 1 min, 2% B; 1～2 min，2%～ 5% B； 2～3 min，5%～20% B； 3～6 min，20%～ 30% B； 6～10 min，30%～33% B； 10～13 min， 33%～36%； 13～17 min，36%～40% B； 17～18 min， 40%～100% B； 18～19 min，100%～2% B；19～20 min，2% B。
2.3 mass spectrometric conditions waters synapt mass spectrometer, electrospray ion source (ESI), and time of flight (TOF) V mode is used for quality detection. The sample from the ultra-high performance liquid phase flows directly into the mass spectrum. The negative ion scanning range is m/z 100 ～ 1500, the capillary voltage is 3.0 kV, the taper hole voltage is 40 kV, the ion source temperature is 100 ℃, the desolvent temperature is 450 ℃, the volume flow of desolvent gas is 900 l/h, the volume flow of taper hole gas is 50 l/h, and the mass corrected mass nucleus ratio is m/z 556.2771 (leucine enkephalin).
2.4 the data obtained by mass spectrometry data analysis and liquid chromatography-mass spectrometry are processed by mass lynx 4.1 software (waters). After eliminating the background noise, make use of maker lynx XS to analyze the chromatogram to obtain the retention time, relative molecular weight, peak area and other information of the substance. Using principal component analysis, we can see the overall differences between the main groups. Origin 8.0 software is used for drawing and statistical processing of data.
2.5 separation and identification of ginsenosides using ultra-high performance liquid chromatography time of flight mass spectrometry (uplctof-ms) combined technology, under the negative ion detection mode, various ginsenosides in Ginseng Decoction were separated by ultra-high performance liquid chromatography to obtain the UPLC-TOF-MS chromatogram (Fig. 1). The relative molecular weight information was obtained from the first-order electrospray ionization spectrum. The main aglycone type, the type and quantity of linked glycosyl were determined by multi-stage tandem mass spectrometry. Finally, the structure of saponin was determined. Ginsenosides can be inferred from this method and the reference substance or literature data F3、 Rg1、Rb1、Rh1、Rd、Rb2、Rf、Ro、Re、Rc、Rg2、 Rb3、 20 glucosyl ginsenoside RF and Notoginsenoside R1 [7-10]. Among them, Rg1, Rh1, re, RF, 20 glucosyl ginsenoside RF, Rg2 and Notoginsenoside R1 are triol ginsenosides, which have the characteristic fragment m/z 475 ion of triol ginsenoside; Rb1, RC, Rb2, RB3 and RD are diol ginsenosides, which have the characteristic fragment m/z 459 ions of diol ginsenosides; Ro is oleanolic acid type ginsenoside, and ginseng contains only one kind of this type of ginsenoside. For peaks 11, 14, 17, 18 and 20, it can be observed that there is no ion peak with formic acid added in the mass spectrum data, but there is an ion peak with a mass charge ratio difference of 44, which should be the removal of a CO2 ion peak. By comparing the mass spectrum characteristics and retention time of these peaks with the literature data, they can be attributed to formyl ginsenoside malonate Rb1, formyl ginsenoside malonate RC, formyl ginsenoside malonate Rb2, formyl ginsenoside malonate RB3 and formyl ginsenoside malonate Rd. Table 1 lists the retention time and relevant ion peak information of each identified component.
2.6 see Fig. 2 for the difference analysis between the samples of ginsenoside dissolution change. The UPLC-TOF-MS chromatogram of ginseng in each of the six proportions is shown in Fig. 1. The UPLC-TOF-MS chromatogram of ginseng radix was repeated three times, and a total of 18 samples were obtained. The overall difference analysis was carried out. After the Pareto arrangement, taking the average value as the center, it can be seen that the difference between the samples with the same proportion is very small, but the difference between the samples with different proportions is relatively obvious, which indicates that the reproducibility of the experiment is relatively good.
        By fixing the total mass of ginseng and aconite, changing the proportion of people participating in aconite. After decocting together, we can get the equal volume extract containing 100%, 80%, 60%, 40%, 20% and 0% of ginseng, and the corresponding proportion of aconite is 0%, 20%, 40%, 60%, 80% and 100%. Carry out UPLC-TOF-MS analysis on the extracted solution, and the negative ion liquid chromatogram is shown in Figure 3. It can be seen from the figure that pure ginseng solution (100% ginseng) has obvious signal in the negative ion detection mode, which indicates that most substances in ginseng ionize to produce negative ions, because ginsenoside, the main component of ginseng, contains many hydroxyl groups, which is easy to lose a hydrogen ion to form negative ions. Therefore, the change of ginsenoside is reflected by the negative ion detection mode. With the decrease of ginseng proportion, the peak intensity and peak area of ginsenoside also decreased. In order to obtain direct data, the retention time, mass charge ratio and peak area in all samples were extracted by using marker lynx XS software. The same retention time and mass charge ratio are determined as the same substance, and the corresponding peak area represents its dissolution. The average value and standard deviation of peak area were obtained by using origin 8.0 for the same substance between repeated samples. With the proportion of ginseng as the variable, the dissolution amount of ginsenoside should decrease in an equal proportion straight line with the decrease of the proportion of ginseng without the addition of aconite. When aconite is present, the dissolution amount of ginsenoside changes. See Figure 4 for the changes of some main Ginsenosides with the compatibility ratio.
        According to the dissolution amount of Ginsenoside in Ginseng Decoction and the proportion of compatible drugs to Chinese ginseng, the theoretical dissolution amount of Ginsenoside in compatible decoction can be calculated. As shown in Figure 4-A, the dissolution amount of most of the triol ginsenosides decreased linearly with the decrease of ginseng ratio, which is consistent with the expected changes, such as ginsenosides Rg1, RF, Rg2, F3, 20 glucose ginsenoside RF and Notoginsenoside R1, indicating that the increase of aconite does not affect the dissolution of the above ginsenosides. However, one obvious exception is ginsenoside Re, whose dissolution amount does not decrease linearly, but is always higher than the theoretical dissolution amount when the proportion of aconite increases, and there is an obvious inflection point when the proportion of ginseng is 40%. According to the formula {rr% = [(ax/as) / MX] / [(ax /as) /mx] × 100%} for the relative dissolution rate of ginsenoside Re, the relative dissolution rates of each ratio are a 100%, B 109%, C 132%, D 177% and E 170% respectively. This shows that the increase of aconite is beneficial to the dissolution of ginsenoside Re, and when the ratio of ginseng to aconite is 4:6, it is most beneficial to the dissolution of ginsenoside Re.
        For diol ginsenoside, observe the effect of Aconite on the change of its dissolution, and the results are shown in figure 4-b. The experimental results showed that the dissolution of ginsenosides Rb2, RB3 and RD decreased lower than the theoretical value except that the dissolution of ginsenosides RB1 and RC decreased in an equal proportion. This indicates that the increase of the amount of aconite inhibits the dissolution of these ginsenosides. The turning point when the ratio of ginseng to aconite is 60% indicates that when the ratio of ginseng to aconite is 6:4, the dissolution of ginsenosides Rb2, RB3 and RD is most affected. The change curve of dissolution amount of formyl ginsenoside malonate is similar to that of diol ginsenoside. As shown in Figure 4-C, formyl ginsenoside malonate RB1 and RC decrease in an equal proportion, indicating that aconite has little effect on its dissolution. However, formyl ginsenosides Rb2, RB3 and RD showed similar changes to ginsenosides Rb2, RB3 and Rd. when the ratio of ginseng to aconite was 6:4, aconite inhibited the dissolution of these ginsenosides most significantly. Ginseng contains only one oleanolic acid type ginsenoside, namely ginsenoside ro. The influence of Aconite on its dissolution is shown in figure 4-b. On the whole, the dissolution amount of ginsenoside ro is higher than the theoretical value. When the ratio of ginseng to aconite was 6:4, there was an inflection point in the dissolution of ginsenoside RO, which showed that the dissolution efficiency of ginsenoside ro was the highest when the ratio of ginseng to aconite was 60%. The peak area of total ginsenosides is obtained by adding the peak areas of all ginsenosides. The change of total ginsenosides is shown in figure 4-D.
        In general, there is little difference between the change of total ginsenosides dissolution and the theoretical value, but there are some differences in details. When the proportion of ginseng is 100% ~ 50%, the dissolution of total ginsenosides is lower than the theoretical value; However, when the proportion of ginseng decreased to 50%, the dissolution of total ginsenosides was higher than the theoretical value. These results showed that the dissolution of total ginsenosides was inhibited by aconite when the proportion of aconite was less than 50%; When the proportion of aconite was higher than 50%, the increase of aconite stimulated the dissolution of total ginsenosides.
        3 discussion UPLC-TOF-MS is a detection method that has been widely used in the study of traditional Chinese medicine in recent years [11-13]. UPLC can improve the reproducibility of retention time, chromatographic resolution, sensitivity and speed up the operation process at the same time [14]. The compounds contained in traditional Chinese medicine have the characteristics of large quantity and similar structure. By using UPLC-TOF-MS, the complex system of traditional Chinese medicine can be separated with high resolution in a short time and the information of compounds of interest can be obtained. It is one of the most powerful tools for analyzing the complex system of traditional Chinese medicine [15-16]. By fixing the total mass of ginseng and aconite, adjusting the ratio of ginseng and aconite, the changes of ginsenoside dissolution under different ratios were studied by uplctof-ms. About 20 main ginsenosides were identified, and their respective dissolution amounts were analyzed in detail with the change of the ratio. Because the total amount of ginseng and aconite remains unchanged, changing the amount of ginseng can make the proportion of ginseng reach 0 ~ 100%. Without the influence of aconite, the dissolution amount of various ginsenosides should decrease linearly with the decrease of the proportion of ginseng. The dissolution amount of most ginsenosides decreased linearly with the decrease of the proportion of ginseng, and was not affected by the addition of aconite. However, the dissolution amount of ginsenoside Re was higher than the theoretical value, and the maximum increase occurred when the proportion of ginseng was 40%. The change of ginsenoside ro is similar to that of ginsenoside Re, but the maximum change occurs when the proportion of ginseng is 60%. The dissolution of diol ginsenosides Rb2, RB3, RD and their malonic acid formyl derivatives decreased slightly after the addition of aconite, and the change was the largest when the ratio of ginseng to aconite was 6:4, indicating that malonic acid formyl had no effect on the dissolution of ginsenosides in the compatibility of ginseng and aconite. In general, the dissolution of triol type and Oleanolic Acid Type Ginsenosides increased, and the dissolution of diol Type Ginsenosides was inhibited, which may be related to the presence of glycosides at position 6 of ginsenosides. Ginsenoside Re has pharmacological effects such as anti fatigue and inhibition of myocardial apoptosis [17], ginsenoside ro can promote splenocyte proliferation and regulate immune function [18], and the increase of ginsenoside Re and RO may be the pharmacodynamic basis of ginseng aconite compatibility. The next work will focus on the molecular mechanism of aconite to change the dissolution of several ginsenosides.
References (omitted)