Druggability Research Of Effective Fraction From Fruits Of Rhododendron Molle G.Don In Treatment Of Rheumatoid Arthritis

Rheumatoid arthritisis a chronic inflammation and joint destructive disease.It is characterized by aggressive and symmetrical joint synovitis and multiple systemic involvement.It is mainly characterized by non-specific inflammation of the peripheral joints,the patient’s diseased joints and surrounding areas.Progressive destruction of the tissue causes dysfunction of the damaged joint.Clinical symptoms include pain, stiffness,deformity,swelling,subcutaneous nodules,and the like.Rhododendron Molle G.Don is a Rhododendron plant,first recorded in the"Shen Nong’s Herbal Classic".R.Molle is commonly used in the treatment of RA among the people.Huazhong University of Science and Technology Tongji Medical College has applied R.Molle for the treatment of RA for more than 20 years.It has good therapeutic effects and rich clinical experience.Modern pharmacological studies have shown that R.Molle has analgesic effect,inhibits adjuvant arthritis,inhibits immune response,etc.,but it has greater toxicity,large differences in the content of diterpenoids in different batches and different medicinal parts,which has become a big problem limiting its clinical application and new drug development.In order to better solve the above problems,in this experiment,taking the medicinal part of fruits with high content of diterpenoid as the study subject,Rhodojaponin Ⅲ（Rj-Ⅲ）and Rhodojaponin Ⅵ（Rj-Ⅵ）were used as index components,exploring the druggability of diterpenoid fraction from fruits of R.molle（DFRM）:firstly,establishing high performance liquid Chromatography-evaporative light-scattering Detector（HPLC-ELSD）method for determination of contentsof Rj-Ⅲ and Rj-Ⅵ in the fruits of R.molle and DFRM;using modern chromatographic techniques,systematically inspecting various parameters of the process,obtaining diterpenoid fraction from fruits of R.molle with clear composition,quality control and content of 50-60%from the R.molle;and then completing its quality standard research,acute toxicity safety evaluation and preliminary pharmacodynamics;study its mechanism of action;explore the process feasibility and possibility of becoming a anti-RA medicine of DFRM through the above studies.1 Establishment of a method for determination of DFRMThe HPLC-ELSD of Rj-Ⅲ and Ⅵ is first established in this experiment.The content was determined by using acetonitrile（A）-0.3%formic acid water（B）mobile phase system.The elution gradient was as follows:0-5 min,1%A;6-80 min,5%A;81-100min,100%A;110min,1%A,flow rate is 1.0 m L·min^-1,injection volume is 50μL,choosing evaporative light-scattering detector,temperature is 55℃,gas flow rate is 1.6L/min,gain value is 2,studying all important influencing factors.The results of methodological investigation showed that the method was accurate,reliable and repeatable.2 Study on preparation process of DFRMIn the assay,we first establishing a preparation process of DFRM by the macroporous resin-phase silica gel column chromatographic separation,and the contents of Rj-Ⅲ and Rj-Ⅵ were 50-60%.The optimum process parameters were as follows:fruits of R.molle with 8times of 70%ethanol,extracting 3 times by reflux,2 h for each time,the extract was concentrated and centrifuged,the centrifugal force was 1000×g and the centrifugal time was 8 min,then supernatant was added to the LSA-12S macroporous resin column that the ratio of diameter to height is 1:7 at a flow rate of 2 BV·h^-1,5 BV of purified water was used to remove impurities,and then 7 BV 30%ethanol was used as eluent at a flow rate of 4 BV·h^-1,the eluent of Rj-Ⅲ and Rj-Ⅵ was concentrated and stirred with phase silica gel at a weight ratio of 1:2,then added to the phase silica gel column that the ratio of diameter to height is 1:10 at a weight ratio of 1:30,12 BV dichloromethane:methanol=20:1 and 12 BV dichloromethane:methanol=10:1 were used as eluent,the eluent of Rj-Ⅲ and Rj-Ⅵ was concentrated and dried to give the diterpenoid fraction product with purity 50-60%.This process requires simple equipments and low production costs,it has high efficiency,large sample processing and protects environment capacity,with great industrialization prospects.3 Study on quality standards of DFRMStability study and quality standard for DFRM:stability study including influence factors tests,accelerated tests and long-term tests.The results showed that high temperature is the main influencing factor for its stability and component content change.The other influence factors had no obvious influence on the content of index components.Through research,a draft of the quality standard of DFRM including preparation process,traits,identification,inspection,fingerprint,content determination and storage had been drafted in order to for better control.4 Study on acute toxicity of DFRMThe acute toxicity of DFRM was evaluated by using Bliss method.The LD₅₀of DFRM to Kunming mice was 4.0 mg/kg,which was less toxic than that of Rj-Ⅲ(LD₅₀=2.8mg/kg).The dead mice and normal mice were dissected and found no significant changes in various tissues and organs.5 Study on the anti-RA activity of DFRMIn this paper,we selected the CIA rat model to evaluate the anti-RA activity of DFRM:the results showed that that high dose（0.6 mg/kg·d）of DFRM had the effect of relieving foot swelling significantly（P<0.01）,decreasing arthritis index（P<0.001）and decreasing the levels of pro-inflammatory cytokines TNF-α（P<0.001）,IL-1β（P<0.01）,IL-6（P<0.001）,inhibiting inflammatory cell infiltration,and protecting synovial cells,and the effect were equivalent to those of the Leigongtengduogan Pian（50.0 mg/kg·d）;At the same time,the therapeutic window of the DFRM was larger(the LD₅₀of the DFRM was 4.0 mg/kg,and the LD₅₀of Leigongtengduogan Pian was 100.0 mg/kg)than Leigongtengduogan Pian.6 Study on anti-inflammatory and analgesic activity of DFRMIn this experiment,the anti-inflammatory and analgesic activities of DFRM were studied by using the classical xylene-induced inflammation of the ear-swelling inflammation,acetic acid writhing analgesia test,and hot plate analgesia test.High dose（1.2 mg/kg）of DFRM behaved effect on xylene-induced ear swelling in mice,reducing the number of writhing caused by acetic acid,and increasing the threshold of mouse hot plate method,whose effect is close to the effect of each positive drug.7 Study on the mechanism of action of DFRMThe medium and high concentrations（0.3 mg/m L,1.0 mg/m L）of DFRM had obvious inhibition effects on the abnormal proliferation of T and B lymphocytes（P<0.01）.Meanwhile,in the study of anti-RA,the high dose（0.6 mg/kg·d）of DFRM significantly reduced the levels of pro-inflammatory cytokines（IL-6,IL-1β,TNF-α）.The above results indicated that the anti-RA effect of DFRM may be related to its immune function and inhibition of proinflammatory cytokine production.