Study On The Absorption And Desorption Capability And Kinetics Of Multiple Ingredients Of Shuxiong Tablets On Macroporous Resins

Shuxiong tablets is the preparation which is recorded by Chinese Pharmacopoeia 2005 Volume I, composed by Radix Notoginseng、Rhizoma Chuanxiong and Flos Carthami. Radix Notoginseng contains saponins (ginsenoside Rg1、ginsenoside Rb1、notoginsenoside R1), Rhizoma Chuanxiong contains alkaloids (chuanxiongzine) and organic acid (ferulic acid), Flos Carthami contains flavonoids (hydroxysafflor yellow A)。This paper aims to make a system research of the absorption and desorption capability and Kinetics of six active ingredients in mixed extractive solution of Shuxiong tablets on macroporous resins.1. AimBy exploring the regularity of absorption and desorption of different type of active ingredients in Chinese traditional compound medicine (Shuxiong tablets) on macroporous resins, it can provide experimental foundation for getting a good means of the purification of multiple ingredients in Chinese traditional compound medicine.2. Methods(1) Research of the determination methods of 6 ingredients in mixed extractive solution of Shuxiong tablets:HPLC was selected. ginsenoside Rg1、ginsenoside Rb1、and notoginsenoside R1:acetonitrile-water was used as a mobile phase, gradient eluting, and the detection wavelength was set at 203nm. chuanxiongzine:methanol-water(50:50) was used as a mobile phase, and the detection wavelength was set at 280nm. ferulic acid:methanol-1% acetic acid (35:65) was used as a mobile phase, and the detection wavelength was set at 320nm. hydroxysafflor yellow A:methanol-1%phosphagen acid (38:62) was used as a mobile phase, and the detection wavelength was set at 403nm. And the determination methods of 6 ingredients was inspected respectively.(2) Research of preparation method of mixed extractive solution of Shuxiong tablets:ethanol concentration、soaking time、ethanol volume and extraction time are chosen as factors and the dry extract weight and their content of total saponins are chosen as evaluation index to optimize ethanol extraction process of Radix Notoginseng by uniform design, the dry extract weight and their content of chuanxiongzine、ferulic acid、hydroxysafflor yellow A are chosen as evaluation index to detect the influence on the lixiviating efficiency of Rhizoma Chuanxiong and Flos Carthami due to different water volume; The content of chuanxiongzine、ferulic acid、hydroxysafflor yellow A are chosen as evaluation index to detect the feasibility of ethanol-precipitating method for pretreating the solution of Rhizoma Chuanxiong and Flos Carthami.(3) Research of the static absorption-desorption capability and the kinetics:①Absorption ratio and relative absorption ratio are chosen as the indexes to detect the absorption capability of the six ingredients in the mixed solution of Shuxiong tables on five resins (LSA-7、LSA-10、A-30、D101A、D-100).②Desorption quantum and elution ratio are chosen as indexes to detect desorption capability of the six ingredients on the resins (LSA-7、LSA-10、A-30、D101A、D-100),50% and 70% ethanol being alternative eluting solvent.③Absorption ratio of the six ingredients on four resins (LSA-7、A-30、D101A、D-100) is chosen as the index to detect the effect of pH (4.0-6.0).④Absorption ratio of the six ingredients on two resins (A-30、D-100) is chosen as the index to detect the effect of sample concentration.⑤On two resins (A-30、D-100), absorption ratio and relative absorption ratio of hydroxysafflor yellow A in the extractive solution of Flos Carthami and the mixed solution are chosen as the index to detect the absorption capability of them.⑥Detect the static absorption dynamic peculiarity of the six ingredients on two resins (A-30、D-100) by measuring absorption ratio of different time.(4) Research of the capability of dynamic absorption-desorption and the kinetics:①On the condition of dynamic absorption (pH5.0, flow velocity 2 BV·h-1), absorption ratio is chosen as the index to detect the effect of sample concentration on the absorption capability of six ingredients on two resins (A-30、D-100).②on the condition of dynamic absorption (flow velocity 2 BV·h-1), absorption ratio is chosen as the index to detect the effect of sample pH on the absorption capability of six ingredients on two resins (A-30、D-100)③Elution quantum and elution ratio are chosen as the indexes to detect desorption capability of six ingredients on two resins(A-30、D-100) by step eluting with ethanol of different concentration.④Comparing absorption capability of each active ingredient in individual solution of Radix Notoginseng、Rhizoma Chuanxiong and Flos Carthami on resin A-30 with that in the mixed solution by chosing the absorption ratio as the index⑤On the condition of pH5.0 and 2 BV·h-1 flow velocity, the sample solution was past through resin A-30, then the absorption dynamic capability was detected by measuring the content of six ingredients in the leaking solution or leakage ratio;⑥Detecting the desorption dynamic capability of ingredients by measuring the content(or elution ratio) of six ingredients in the solution which was eluted by 70%ethanol.(5) Primary research on step absorption isolation for purifying the mixed extractive solution of Shuxiong tablets:①According to the fact that hydroxysafflor yellow A is rarely absorbed by the first resin (A-30, pH5), absorption and elution ratio are chosen as the indexes to detect the absorption- desorption capability of hydroxysafflor yellow A on three resins(A-30、D101A、and D-100) when the value of pH is 2;②On the condition of pH2.0 and 2 BV·h-1 flow velocity, the leaking solution of first resin was past through the second resin column (D-100), then the dynamic absorption capability of hydroxysafflor yellow A on the secong column was detected by measuring the content of hydroxysafflor yellow A in the leaking solution or leakage ratio.③70% ethanol is used as eluting solvent to detect the desorption dynamic capability of hydroxysafflor yellow A on the second resin column by measuring the content of hydroxysafflor yellow A in each part of leaking solution or leakage ratio.④Original and purified dry-extract weight, and the content of six ingredients in both extract are chosen as indexes to detect the purification result of step absorption isolation3. Results(1) Researches of the content determination of six ingredients in the extractive solution of Shuxiong tablets show:In the selected chromatographic conditions, the peaks of ginsenoside Rg1、ginsenoside Rb1、notoginsenoside R1、chuanxiongzine、ferulic acid、and hydroxysafflor yellow A in the mixed extractive solution of Shuxiong tablets can be well separated from each other, the resolutions are above 1.5,the average recovery is 98%-102%with RSD of 2.5%, and the negative samples show no interference.(2) Researches on the preparation of Shuxiong tablets’extractive solution show:Radix Notoginseng powder was extracted twice with 50% ethanol after being soaked for 2.5 hours, extract time 2.5 and 2.0 hours,and solvent volumn 8 times and 6 times of crude drug, respectively. the optimal water volumn of Rhizoma Chuanxiong and Flos Carthami is 10、8 and 8 times of crude drug. After extractive solution is pretreated by ethanol precipitating (the content of crude drug is lg·mL-1, content of ethanol in the solution is 70%), the reservation ratio of chuanxiongzine、ferulic acid、hydroxysafflor yellow A are over 90%, and it would decrease the pollution of resins.(3) Researches on the static absorption-desorption capability and kinetics show:①the absorption capability of six ingredients on five resins (LSA-7、LSA-10、A-30、D101A、and D-100) line from strong to weak:ginsenoside Rb1、notoginsenoside R1、ginsenoside Rg1、chuanxiongzine、ferulic acid、hydroxysafflor yellow A, and the absorption capability of hydroxysafflor yellow A is very weak, it is hard to collect it with the five resins. Moreover, the comprehensive capability indicate no apparent difference among the five resins (P>0.05)②Resin A-30 and D-100 among the five resins were better Desorption capability on 6 ingredients. Moreover,70%ethanol can enhance the elution ratio of ingredients on resins, compared with 50% ethanol.③As to the same resin, ferulic acid、hydroxysafflor yellow A are greatly affected by the value of pH,and lower pH value, bgger absorption ratio. As to the same ingredient, different resins show different influence, among them, polar resin LSA-7 is much different from the other three resins.④When the solution concentration is low,generally the absorption ratio show the rising trend with the rising of the concentration, while the concentration is large, the absorption ratio show no apparent increase, sometimes show the lowering trend with the rising of concentration. In addition, all absorption isotherms of six ingredients can adapt to model of Langmuir or Freundlich equation (P＜0.05 or P＜0.01)⑤Absorption ratio and relative absorption ratio of hydroxysafflor yellow A in the single extractive solution of Flos Carthami on resin A-30、D-100 are bigger than that in the mixed solution, which indicates different ingredients in mixed solution may exist the competitive absorption.⑥The static absorption dynamic curve of six ingredients on resin A-30 shows:at the beginning(from 10 to 30minute) can reach the largest absorption ratio, later, absorption ratio generally rise slowly till 24 hours, which indicates single absorption principle during this absorption process. The static absorption dynamic curve of six ingredients on resin D-100 shows:except for hydroxysafflor yellow A, the other five ingredients show an apparent platform region, which indicates that there are two absorption principles during the absorption process.(4) Research of the absorption-desorption capability and kinetics show:①when concentration of crude drug in the sample solution is 0.3-0.6 g·mL-1,the dynamic absorption ratio of 6 ingredients on resin A-30 generally is larger than that on resin D-100, and when the concentration of crude drug is 0.4 g·mL-1,each ingredient has the fairly good absorption ratio.②Different ingredients show different change trend with the change of value of pH on the same resin. As to resin A-30, exept for ferulic acid,the other five ingredients almost have the same change trend, when the value of pH is five,the absorption ratio of the five ingredients shows the largest, and the ratio of ferulic acid is also optimal. While as to resin D-100, the change trend of absorption of 6 ingredients show much difference, but there is the optimal absorption ratio of 6 ingredients when the value of pH is 4. The influence on absorption capability of similar structural ingredient on resins may be different with the change of pH, for example, the absorption ratio of three saponins in Radix Notoginseng on resin A-30 and D-100 indicates different change trend. As to organic acid (e.g. ferulic acid)、flavonoids (e.g. hydroxysafflor yellow A), it can deduce the absorption capability of non-polar resin with the rising of the value of pH; as to alkaloids, such as chuanxiongzine, the absorption capability of non-polar resin can improve a little with the rising of the value of pH..③As to the same resin, although the desorption speed of six different structural ingredients are not completely same,they can almost be eluted by 70% ethanol. As to different resins, the desorption speeds of 6 different structural ingredients are distinguished, the speed of resin D-100 is faster than resin A-30, the ingredients except for ginsenoside Rb1 can almost be eluted by 50% ethanol, but in the light of integrated elution ratio from 10% to 95% ethanol, resin A-30 was prior to D-100.④The absorption ratio of active ingredients except for ginsenoside Rb1, in the extractive solution of Radix Notoginseng、Rhizoma Chuanxiong and Flos Carthami, is larger than that in the mixed solution on resin A-30, it further indicate that there is a competitive absorption among different ingredients.⑤The apparent leakage order of six ingredients on A-30 is:hydroxysafflor yellow A→three saponins→ferulic acid→chuanxiongzine. Among them, the leakage speed of hydroxysafflor yellow A is very fast, the leakage time of three saponins are close,and the leakage volume of ferulic acid is increasing slowly, chuanxiongzine begin to leak almost when the other ingredients completely leak. In addition, the leakage curve of the beginning part of six ingredients can adapt to first order (or zero order) velocity equation (P<0.01), according to this equation, we can work out the leakage curve parameters (velocity constant, leakage terminal point、AUC(?)、leakage amount、absorption amount).⑥The shapes of elution cure of 6 ingredients are similar, except for hydroxysafflor yellow A, the other five ingredients reach peak at the same time,and with 7BV of 70% ethanol,6 ingredients can capitally eluted. In addition, the end of elution curve of 6 ingredients all adapt to first order equation (P<0.01), according to this equation, we can work out the elution curve parameters(velocity constant、AUC0→∞、elution amount、elution terminal point).(5) primary researches of step absorption isolation for the purification of mixed extractive solution of Shuxiong tablets show:①When the value of pH is 2, the absorption ratio of hydroxysafflor yellow A in the extractive solution of Flos Carthami on resin A-30 is the largest among three resins (A-30、D101A、and D-100),then resin D-100. Compared with pH4, the absorption ratio of the two resins have been apparently enhanced. But in the aspect of desorption,resin D101A、D-100 have the similar capability,both prior to resin A-30. Resin D-100 can be used at the second step absorption by the absorption and desorption comprehensive capability.②On the dynamic curve(leakage curve) of the second step absorption,t he beginning part can adapt to the first order equation (P<0.01),and according to the equation, we can get that the terminal point of leakage of hydroxysafflor yellow A is 5.8 BV.③On the dynamic curve(elution curve) of the second step desorption, the terminal part can adapt to the first order equation (P＜0.01),and according to the equation,we can get that the terminal point of elution of hydroxysafflor yellow A is 3.9 BV.④Two step absorption isolation is used to purify the extractive solution of Shuxiong tablets,compared with unpurified dry extract, the purified dry extract ratio decrease 5.5 times, but the content of six ingredients increase 3-5 times, the transition ratio of active ingredients is 58%-87%.4. Conclusion(1) The HPLC method to determining the six ingredients in mixed extractive solution of Shuxiong tablets is reliable、accuracy and highly specific. It can be used for determination of the content of each ingredients during the research of absorption and desorption capability and kinetics of macroporous resms.(2) The results of ethanol extraction、water extraction and ethanol precipitating can provide the experimental basis of the preparation process of mixed extractive solution of Shuxiong talets.(3) The results of static absorption-desorption and kinetic can provide a quick way to select the optimal resin for Chinese compound medicin,moreover,it provide enlightenment on the absorption principle of multiple ingredients on resins.(4) The results of the researches of dynamic absorption-desorption capability and kinetics can provide the optimal absorption process conditions (including drug solution concentration, pH, column volume ratio, absorption or leakage terminal point) and desorption process conditions (including eluting solvent, elution terminal point and so on). Moreover, it also visually shows how to judge if the compound ingredients can be purified by one resin at the same time, and can provide scientific basis by designing step absorption isolation to purify Chinese traditional compound medicne.(5) The step absorption isolation to purify Chinese traditional compound medicine is feasible. It is a new way of joining multiple resins or multiple process conditions together, it can purify the ingredients of different peculiarity, then avoid the loss of ingredients by single process. This method will enrich the connotation of the macroporous resin absorption isolation, and it is expected to solve the key problem in purification of Chinese traditional compound medicine, but still to be tested.