Traditional Chinese processing techniques (PaoZhi) is one of the commonly used methods for changing drug property, reducing toxicity and improving effects of TCM. It was reported that VBRB had a stronger effect than Radix Bupleuri on bile secreting and hepatoprotective effects [
11], and previous study also showed the pharmacological change may be related with the change of components in VBRB [
17,
18]. However, the pharmacological study and the component study usually did separately, and temperature, pressure and soaking time were also affecting factors during vinegar processing technology. Therefore, the results couldn’t fully explain the link between components and pharmacology. This paper exclusively explored the influence of vinegar amount on hepatoprotective effect of VBRB and its corresponding change of components after controlling for other factors for the first time.
1:5, 2:5 and 3:5 were the ratios of vinegar to Radix Bupleuri which usually used in the literature. In our previous technology experiments, Vinegar could not be absorbed sufficiently by Radix Bupleuri when the ratio up to 4:5 and the content of saikosaponin b2 changed a little over the ratio of 3:5. Therefore we chose these three ratios for this study.
VBRB usually used in clinic curing liver related disease such as hypochondriac pain, hepatic adipose infiltration, hepatitis etc., which indicated its effect may mainly in liver protection. Alcohol, high lipids diet and abnormal emotion are the main cause of uninfected liver injury, and abnormal emotion especially anger induced model had the benefit of stable and at a shorter time to establish [
19,
20]. Therefore we chose this model by angering rats through clamping tails. ALT and AST, are the indexes of evaluating the severity of liver damage. When liver cells are necrotic, the index of ALT and AST increased, and the extent of increased level are accordance with the severity of liver damage. TBIL, DBIL and TBA are the indexes reflecting the secretion, excretion of the bile and the ability to detoxify of liver. Among them, TBA reflects a more sensitive liver function and its level positively correlated with the degree of liver damage. Compared the index of liver function before and after model establishment, the level of ALT, AST and TBA changed obviously which was proved that rats angered by painful stimulus, and anger lead to liver dysfunction. VBRB could improve the liver injury by decreasing the index levels of liver especially the AST and TBA levels on model induced by anger. Among them, the most effective group was group VBRB (3:5).
The efficacy of medicine is closely related to their material basis, so we analyzed the change of constituents of VBRB after processed by different amount of vinegar. The extracting rates of total extract increased with the increasing amount of vinegar in the processing, and the trend was the same in extraction parts of BC1, BC2 and BC3. The result showed that the best amount of vinegar in the processing was 3:5, and this is accordance with that of the results of pharmacology experiment. BC1, mainly containing polysaccharides, had the effect of regulate and enhance immunity [
21]. BC3 was mainly composed of low molecular weight water soluble components [
22], no reports were found on its pharmacological effects and needed further study. BC2 was mainly composed of saponins which were also the main and bioactive components of VBRB [
22‐
24], so we investigated the saponins to seeking the main active compounds of VBRB in follow-up experiment. The results showed that the native saponins (saikosaponin a, c, d) decreased, and the secondary saponin (saikosaponin b
2) increased gradually with the increasing proportion of vinegar. Modern research considered that native saponins (saikosaponin a, c, d) were the main effective compounds of Radix Bupleuri, so these saponins usually used as the index components of Radix Bupleuri [
25‐
27]. However, similar with our results, other study also showed that secondary saponins (saikosaponin b
1, b
2) were increased, while the saikosaponin a, saikosaponin c, and saikosaponin d were decreased after the vinegar-baking process [
28,
29], and a higher saikosaponin b content also follow a better hepatoprotective effect [
17], in spite of that the model they used is CCl
4 induced liver injury. It was also reported that the saikosaponin b
2 had strong pharmacological effects which could be an efficient inhibitor of early hepatitis C virus entry, including neutralization of virus particles, preventing viral attachment, and inhibiting viral entry/fusion [
30]. In this paper, high content of saikosaponin b
2 in water extract illustrated that only using the native saponins as the index component of Radix Bupleuri were not complete and realistic. With the increasing amount of vinegar, the secondary saponins even should be more important index compounds. Combined with pharmacological study, we speculated that saikosaponin b
2 may be the main active components of VBRB.