| Radix bupleuri is a commonly used traditional Chinese medicine, which was recorded in many famous Chinese medicine classics such as "Shen Nong’s Herbal Classic", "Shang Han Lun ", "Qian Jin Yi Fang", "Materia Medica truth" and so on. Radix bupleuri has a lot of efficacies, including anti-inflammatory, antipyretic, analgesic, hepatoprotective, anti-viral, anti-tumor, endocrine regulation and immune systems regulation. It has been primarily used to treat cold fever, chill and fever in turn, the feeling of oppression and illness in the chest and hypochondria, reconciling exterior and interior and dispersing stagnated liver qi for promoting bile flow and so on. Saikosaponins were regarded as the main pharmaceutical effective constituents and literature research revealed that dozens of saikosaponins had been isolated from the radix bupleuri. The decoction is the main use form of Chinese medicine in the clinic. Our early research found that saiosaponin a and b2 were the main components, which indicated that the two saikosaponins will be the real effective substance of radix bupleuri. The detail research on saikosaponin a and b2 could shed more light on the mechanism how the radix bupleuri possessed so many medicinal effects which now could not reasonably explained. Considering that drug absorption is related to the infiltration and physicochemical properties, the emphasis in this paper were put on the chariacteristics of the dissolution, aggregation and internal absorption of ssa and ssb2, meanwhile, determination method of acyl-saikosaponin and structural transformation of ssa were deeply studied. The related research work laid the foundation for deeply understanding the radix bupleuri and provided clues for the comprehensive study of the pharmacokinetics of saikosaponins.Objective:To investigate intestinal absorption and aggregation characteristics of saikosaponins, and to establish the determination method of acyl-saikosaponin and other related issues.Methods:1. The contents of saikosaponin a and b2 were determined after the different amounts of saikosaponin a and b2 were added into the equal volume boiled water.2. Different aggregates of ssa and ssb2 in different solution systems were classified by filtering through a sintered glass funnel(from G1 to G5). The contents of different samples were determined using HPLC and UV spectroscopy.3. The intestinal absorption rules in the duodenum, jejunum, ileum, and colon of ssb2, saiko decoction and Radix-TGP decoction were studied using the in situ Single Pass Perfusion Model. Macroporous resin was used to enrich the saikosaponin in the samples, and the content determination was carried out by HPLC. The spss software was used for data processing and the effective permeability(Peff) was used as the main absorption parameter for the absorbion evaluation in the rat intestine.4. Four factors and three levels orthogonal experiment was employed to optimize parameters in the acyl-saikosaponin alkaline hydrolysis process. The optimized items included p H value, ultrasonic power and ultrasonic time. The content of acylsaikosaponin in radix bupleuri purchased from different pharmacies was determined by HPLC.5. The structure transformed components of saikosaponin a in the water were separated by various chromatographic techniques. The structures were identificated by various spectroscopic techniques.Results:1. With the increasing of the added concentration of saikosaponin in water, the dissolution concentration of saikosaponin firstly increased and then decreased. And the maximum dissolution concentration of saokosaponin b2 appeared at the concentration of 0.428-0.801 mg·m L-l, however that of saikosaponin a was at 0.240 mg·m L-l.2. Particle sizes of ssa and ssb2 in the decoction of Radix bupleuri were both less than 0.45 μm, which counted almost 60% of the total. But the particle sizes of the single ssa mostly lied between 5-40 μm. The particle sizes of the single ssb2 lied in 5-40 μm and less than 0.45 μm with the almost equal amount.3. Only ssa had the strong aggregation behavior. It could form gels in the solvents such as water, methanol-water, ethanol-water and acetone-water. The SEM showed that the ssa formed gels in different solvents possessed differed appearances.4. The absorption process of ssa and ssb2 in the duodenum, jejunum, ileum, colon was passive transport and intestinal position-depended. Compared with the duodenum, the Peff of ileum and colon were significantly lower. The absorption coefficient of ssb2 was intermediate at three different concentrations. The absorption of saikosaponin a and b2 in radix bupleuri was better than Radix-TGP decoction in each bowel.5. The optimal conditions of acyl-saikosaponin alkaline hydrolysis parameter were 40 min ultrasonic time, p H 10, ultrasonic power 400 W. The contents of acylated saikosaponin in radix Bupleuri varied with its origins.6. The ssa could convert into ssb2 under weak acid condition, and then the structure transformed to ssg.Conclusion:This paper laied the foundation for the study on radix bupleuri based on the saikosaponin aggregates. |
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