Study On Chemical And Efficacy Differences Between Radix Paeoniae Alba And Radix Paeoniae Rubra

Radix Paeoniae Alba (RPA) and Radix Paeoniae Rubra (RPR) are commonly used as traditional Chinese herbal medicines. It is specified in China Pharmacopoeia (2010version) that RPR originates from wild Paeonia lactiflora Pall. and Paeonia veitchii Lynch., and RPA originates from cultivar of Paeonia lactiflora Pall. After being decorticated and boiled, the roots are usually dried in sunlight. Due to deriving from the same genus, the chemical components of RPA and RPR are similar, however, the ratios among their constituents are different, and thus their pharmacological effects show certain commoness and difference. In traditional Chinese medicine, RPA and RPR are clinically used for different diseases. According to China Pharmacopoeia, paeoniflorin, the common major compound of RPA and RPR, is the only marker for quantitative analysis to evaluate their quality. The method is unable to differentiate between the two herbs because it is short of specificity. Obviously, the quality assessment method is not reasonable. This study finds out the differences of chemical component between RPA and RPR and explain its different pharmaceutical effect via in-vitro experiment. Those experimental can provide more scientific data for establishment of new quality control method of RPA and RPR in the future.Objective1. To extract, separate, isolate and identify the chemical compound from RPA and RPR.2. To establish quality evaluation method and determine6chemical compounds from RPA and RPR by using high-performance liquid chromatography (HPLC).3. To study the influence of proliferation of vascular smooth muscle by using RPA and RPR.Methods1. The powder of RPA and RPR was extracted by reflux with80%ethanol3 times for2h each, the solid-to-solvent ratios were1:8,1:8and1:8respectively. The extract solution was filtered and concentrated. The residue was dissolved into water and extracted by petroleum ether, ethyl acetate, water-saturated n-butanol sequentially. The fraction was concentrated and separated by column chromatography and Agilent1200HPLC. The target compound was further purified and crystallized.2. The condition of HPLC for RPA and RPR analysis:Column [Waters symmetry C18(4.6×250mm,5μm)], Mobile phase [A:0.1%phosphoric acid (pH2.7), B: Acetonitrile (v/v)], Flow rate [0.8mL/min], Detection wavelength [230and270nm], Column temperature [30℃].3. Cell line A7R5, vascular smooth muscle of rats, was used to study the proliferation effect under different contraction of RPA and RPR extract. Positive control was established by1μg/mL LPS and10ng/mL PDGF and analyzed cell proliferation by using MTT method.Results1.7pure marker compounds, Gallic Acid, Paeoniflorin Sulfonate, Albiflorin, Paeoniflorin, Pentagalloyglucose, Benzoylpaeoniflorin and Benzoylalbiflorin, were prepared.2. The composition of all marker compounds in RPA and RPR were analyzed under improved HPLC condition.15batches of RPR and17batches of RPA were measured and compared with all marker compounds based on its retention time and peak area. The result showed that Albiforin, which is Monoterpene glycosides, in RPA is6times more than RPR. In China pharmacopeia, Paeoniflorin as a quality control marker of RPA and RPR can not be less than1.6%and1.8%respectively.23samples which included12RPA and11RPR samples were contained1.8%above of Paeoniflorin. It doubts that quality control marker in CP is not appropriate to differentiate between RPA and RPR. Furthermore, a special chemical compound, Paeoniflorin Sulfonate was found in HPLC fingerprint of processed RPA.3. Cell line A7R5proliferation influence was measured by MTT method and Real-Time Cell Analyzer DP. The result showed that water extraction of RPR inhibits the cell proliferation, but water extraction of RPA stimulates the cell proliferation. ConclusionThe research on quality evaluation of RPA and RPA have established a feasible and effective method that transcends the outdated quality control mode used in China Pharmacopoeia. Based on the theory and methodologies of traditional Chinese medicine, in-vitro cell line study provides more scientific evidence that different pharmacological effect between the common origin, RPA and RPR.