Study On Quality Control And In Vitro Anti-inflammatory Of Rubiae Radixet Rhizoma And Its Formula Granule

ObjectiveRubiae Radix et Rhizoma is the dried root and rhizomes of Rubia cordifolia L.. It has the functions of cooling blood, circulating blood, removing blood stasis and activating meridians, which is one of the most important traditional Chinese medicine to cure all kinds of blood diseases. Formula granule of Rubiae Radix et Rhizoma is a new product which is made through the modern manufacturing process of extracting, concentrating, drying and granulating from Rubiae Radix et Rhizoma. As a lot of ingredients were lost during the manufacturing process, there are some differences of compositions and effects between the granule and the medicine. This brings a lot of difficulties for standardization and quality control of the drugs. On one hand, it is difficult to guarantee the quality of Rubiae Radix et Rhizoma and the method for determination in Chinese Pharmacopoeia is complicated and not so operational. On the other hand, there is a lack of complete quality control system for formula granule which is a new model. So the objective of this research was to established the quality evaluation system of Rubiae Radix et Rhizoma and its formula granule by TLC identification, determination of the main componet and finger-printing technique, Which helped us to examine the quality of the medicine from different markets and discuss the rationality of the quality evaluation system. At the same time, the experiment of in vitro anti-inflammatory would be carried out, which would provide a basis for the quarlity control for Rubiae Radix et Rhizoma and its formula granule.Methods1. Quarlity analysis of Rubiae Radix et RhizomaQuarlity analysis of Rubiae Radix et Rhizoma was carried out refers to Chinese Pharmacopoeia2010. Rubimaillin and standard medicine of Rubiae Radix et Rhizoma was used as control to identify the medicine by TLC. The method to determine the content of rubimaillin and purpurin was optimized by HPLC. The UHPLC-DAD fingerprint was established, which was used to explore its effective quality evaluation methods.2. Quarlity analysis of Rubiae Radix et Rhizoma formula granuleRubimaillin and standard medicine of Rubiae Radix et Rhizoma was used as control to identify the12batches of formula granule provided by the manufacturer by TLC. The HPLC method was used to determine the content of rubimaillin and purpurin. The formula granule HPLC fingerprints of its aqueous solution and ethyl acetate extraction were established to compare with the Rubiae Radix et Rhizoma.3. Studies of in vitro anti-inflammatory effectsThe in vitro anti-inflammatory effects of Rubiae Radix et Rhizoma and its formula granule were investigated in lipopolysaccharide(LPS)-stimulated RAW264.7cells. In order to determine the effective concentration range of drugs, MTT assay was used to detect the cell activity with the impact of different drug concentrations. The cells were pretreated before LPS stimulated with Rubimaillin(2、10、50μg/mL), Purpurin(2、10、40μg/mL) and the formula granule (40、200、800μg/mL). Then ELISA and RT-PCR methods were used to detect the secretions and gene expression of TNF-a, IL-1β, IL-6and IL-10.Results1. Quarlity analysis of Rubiae Radix et RhizomaThe TLC spots showed the same colors to the reference substances in the corresponding position. The spots were clear and separabel.The method of determination for rubimaillin and purpurin was optimized. The rubimaillin had a good linearity relationship between19.34-161.20μg·mL-1(r2=0.9999), the purpurin showed a good linearity relationship whithin the range of12.16-101.30μg·mL-1(r2=0.9997), and the recovery rates of rubimaillin and purpurin were97.27%(RSD=1.91%) and100.00%(RSD=1.87%). The content of rubimaillin is0.05%-1.63%and the content of pururin is0.35%-0.91%of22batches of Rubiae Radix et Rhizoma. And there are12batches among them did not reach the limit of Chinese Pharmacopoeia.The Rubiae Radix et Rhizoma fingerprint was established. Nine common peaks were calibrated and two peaks were identified among the nine. The similarities of the ten qualified to the fingerprint was0.911-0.987, and the12unqualified was0.791-0.942.2. Quarlity analysis of Rubiae Radix et Rhizoma formula granuleThe method of TLC was established to identify the formula granule. The spots of the samples showed the same colors to the reference substances in the corresponding position. The contents of rubimaillin and purpurin in12batches formaula granules was low that the content of rubimaillin was0.0000%-0.0118%and pururin was0.0050%-0.0121%. The common fingerprint of its aqueous solution and ethyl acetate extraction was built up according to the formula granule except the one which was not similar.11common peaks of the aqueous solution and9common peaks of the ethyl acetate extraction were calibrated. The similarities of the11aqueous solutions to the fingerprint was0.886-0.995, and the11acetate extractions was0.952-0.995.The similarity of the decoction and the granul is low.3. Studies of in vitro anti-inflammatory effectsInflammatory cell model could be built with the stimulation of LPS by increaseing the amount of TNF-a, IL-1β, IL-6and IL-10. The results of ELISA shows that Rubiae Radix et Rhizoma and its formula granle could mainly inhibit the secretion of TNF-a and IL-6. The results of RT-PCR showed that all groups can express the gene of TNF-a, and inhibit the expression of the gene of IL-6.ConclusionThe research focus on the quality control and in vitro anti-inflammatory effects of Rubiae Radix et Rhizoma and its formula granule. The determination of rubimaillin and pururin was optimized and the new detect method was more operatable, stable and reliable. Method of TLC identification was established to identify the formula granule and the content of rubimailian and pururin was determined, but the indicators were not suitable for the granuls.The fingerprints of Rubiae Radix et Rhizoma and the aqueous solution and ethyl acetate extraction of its formula granule were established. The quality analysis of Rubiae Radix et Rhizoma and its formula granule showed that the quality of the medicine was not very good. At the same time,the quality evaluation system of the herbs does not apply to the formula granule. Furthermore, preliminary study on the in vitro anti-inflammatory effects of different components, parts and preparations was carried out. The study confirmed that the medicine can inhibitate of inflammatory, which provided a basis for optimization of the efficacy quality standards.