Investigation Of Pharmacological Activity Of Gardenia Jasminoides Ellis And Aconitum On Arthritis Rats And Preparation Of Crocetin Liposomes

Objective:The current study aimed to investigate the mitigating effect and mechanism of Gardenia jasminoides Ellis and two Aconite species on adjuvant arthritis in rats and the preparation of crocetin liposomes.Method:1.Investigation of the mitigating effect of total iridoid glycosides?TIG?and total crocins?TC?from Gardenia jasminoides Ellis against adjuvant arthritis?AA?ratsTIG and TC in Gardenia jasminoides Ellis were fractionated by HPD₁₀₀ macroporous adsorption resin.UPLC-DAD-QTOF-MS was employed to determine the chemical contents of TIG and TC.In addition,the contents of iridoid glycosides and crocins in TIG and TC were measured by UV-Vis spectrophotometry.The efficacy and mechanism of Gardenia jasminoides Ellis TIG and TC were comprehensively investigated on Adjuvant arthritis?AA?rats model.Thirty male sprague-dawley rats were random Ly divided into 5groups,including control,model?AA?,AA+TIG?160 mg/kg/d?,AA+TC?160 mg/kg/d?and AA+DS?5 mg/kg/d?.The rats were injected with 0.1 m L of freund's complete adjuvant?FCA?at the bottom of right toes.All rats underwent administration of drugs to for 14consecutive days.The degrees of swelling of the feet were assessed at the end of administration.The ankle diameters were measured by digital vernier caliper.The ankle joint was examined by X-ray.Moreover,the pathological states of the ankle joint,spleen and thymus,stained by HE,were analyzed by microscopy.The expressions of tumor necrosis factor?TNF-??and transforming growth factor-?1?TGF-?1?in synovium of ankle were measured by immunohistochemistry.2.Preparation of crocetin liposomes and single factor investigationCrocetin was prepared by hydrolizing crocins,then determined by UHPLC-DAD-QTOF-MS.The crocetin liposomes were prepared by thin film hydration combined with ultrasound method.The particle size,particle size distribution and zeta potential were measured by laser particle sizer analyzer.The encapsulation efficiency?EE%?and drug loading rate?DL%?were determined by ultraviolet-visible spectroscopy?UV-Vis?.The preparation conditions of crocetin liposomes were optimized by single factor design.The effects of ultrasonic time,hydration time,hydration temperature,the ratio of soybean lecithin to cholesterol and the ratio of soybean lecithin to crocetin on the average particle size,EE%and DL%of crocetin liposomes were comprehensively investigated.In addition,the effects of amount of octadecylamine on the surface potentials,EE%and DL%of crocetin liposomes were investigated.3.Investigation of the mitigating effect of Aconitum stapfianum Hand.-Mazz.and Aconitum vilmorinianum Kom.against AA ratsThe effective fraction of Aconitum stapfianum Hand.-Mazz.was prepared by acid-base extraction,and the effective fraction of Aconitum vilmorinianum Kom.,methanol ultrasonic,respectively.The median lethal doses(LD₅₀)of total alkaloids of Aconitum stapfianum Hand-Mazz.?TAA?and methanol extract of Aconitum vilmorinianum Kom.?MEA?were measured by oral acute toxicity test.Then,the effective doses of TAA and MEA for therapy were calculated by the general pharmacological method.The anti-RA efficacy and mechanism of TAA and MEA were investigated on Adjuvant arthritis?AA?rats model.Thirty-six male sprague-dawley rats were random Ly divided into 6 groups,including control,model?AA?,low dose of TAA?1.5 mg/kg/d?,high dose of TAA?3.0mg/kg/d?,low dose of MEA?5 mg/kg/d?and high dose of MEV?10 mg/kg/d?.The rats were injected with 0.1 mL of freund's complete adjuvant?FCA?at the bottom of right toes.All rats underwent administration of drugs for 14 consecutive days.The ankle diameters were measured by digital vernier caliper.The degree of swelling and bone destruction of the foot was observed by X-ray examination.Moreover,the pathological states of the ankle joint,spleen and thymus,stained by HE,were analyzed by microscopy.Furthermore,the expressions of TNF-?and TGF-?1 in synovium of ankle were measured by immunohistochemistry.Results:1.Investigation of the mitigating effect of total iridoid glycosides?TIG?and total crocins?TC?from Gardenia jasminoides Ellis against adjuvant arthritis?AA?ratsThe data showed that the content of geniposide in TIG of gardenia was 53.6%,the contents of crocin-1,crocin-2 and crocin-3 in TC of gardenia were 37.2%,3.3%and 4.6%,respectively.In UV-Vis determination,contents of total iridoid glicosides in TIG and total crocins in TC were 58.2%and 54.7%,respectively.Animal pharmacological experiments showed that TIG?160 mg/kg/d?significantly reduced ankle swelling and ankle diameter of rats compared with AA group?P<0.05?.Histopathological analysis showed that TIG?160mg/kg/d?can ameliorate the infiltration of inflammatory cells in ankle joints of rats,hyperplasia of synovial lining,capillary dilatation and congestion and erosion of the bone?P<0.01?,relieving the congestion and swelling of the spleen and thymus.Furthermore,TIG?160 mg/kg/d?could reduce the expression of TNF-?and TGF-?1 in synovium of rats?P<0.01?.The administration of TC?160 mg/kg/d?also alleviated these pathological conditions.Except for the ankle joint X-ray examination score and TGF-?1 protein expression without statistical difference?P>0.05?,the other parameters were statistically different when compared with model group?P<0.05?.2.Preparation of crocetin liposomes and single factor investigationThe content of crocetin was 73.67%.The average size of crocetin liposomes was240±8.6 nm,the PDI value was 0.216±0.03,the zeta potential was+24.1±3.3 mv,the EE%was 95.5±3.2%and the DL%was 26.1±1.2%.The optimal preparation conditions for crocetin electropositive liposomes were as follows,the dosage of octadecylamine was 40mg/100 m L,ultrasonic time 40 min,hydration time 2 h,hydration temperature 40°C,the ratio of soybean lecithin to cholesterol 6:1 and the ratio of soybean lecithin to crocetin 6:1.3.Investigation of the mitigating effect of Aconitum stapfianum Hand.-Mazz.and Aconitum vilmorinianum Kom.against AA ratsThe acute toxicity test indicated that LD₅₀ of TAA and MEA were 43.002 mg/kg and150.960 mg/kg,respectively.According to calculations,the dose of TAA for therapy was1.5 mg/kg/d and 3.0 mg/kg/d and the dose of MEA was determined to be 5 mg/kg/d and 10mg/kg/d,respectivley.Animal pharmacological experiments showed that MEA?5,10mg/kg/d?administration can significantly reduced ankle swelling and ankle diameter of rats compared with AA group?P<0.05?.Ankle X-ray images and scores showed that MEA?5,10 mg/kg/d?significantly relieved joint soft tissue swelling and osteoporosis in rats?P<0.01?.HE staining results and pathological scores of the ankle showed that MEA?5,10mg/kg/d?can relieve various pathological states of ankle joints?P<0.01?.HE staining of spleen and thymus results showed that MEA?5,10 mg/kg/d?can significantly relieve the congestion and swelling of the spleen and thymus.In addition,immunohistochemistry results showed that MEA?5,10 mg/kg/d?can reduce synovial TNF-?and expression of TGF-?1?P<0.01?.TAA?1.5 mg/kg/d?alleviated the above-mentioned pathological conditions,but there was no significant difference between model and treated group?P>0.05?.However,TAA?3.0 mg/kg/d?significantly reduced the ankle diameter in rats?P<0.05?,and alleviated the above-mentioned pathological conditions?P<0.01?.Conclusion:1.The study clarified that TIG and TC in Gardenia jasminoides Ellis both possess protective effects on AA arthritis rats for the first time,but the efficacy of TIG seems more remarkable.Considering that the content of iridoid glycosides?geniposide?in gardenia was higher,the content of geniposide can be used as an assessment index of anti-inflammatory efficacy of gardenia.2.The crocetin liposomes were prepared for the first time by thin film hydration combined with ultrasound method.The crocetin liposomes possesses the advantages of small average particle size,low PDI value,high EE%and DL%.Then,single factor was designed to to optimize the preparation conditions of crocetin liposome,and the amount of octadecylamine,the ultrasound time,the hydration time,the hydration temperature,the ratio of soybean lecithin to cholesterol and the ratio of soybean lecithin to crocetin were all determined.3.The study explored for the first time the toxicity of Aconitum vilmorinianum Kom.and Aconitum stapfianum Hand-Mazz.,and determined the oral LD₅₀ of these two species of Aconitum.Generally,this maiden attempt demonstrated that both Aconitum vilmorinianum Kom.and Aconitum stapfianum Hand-Mazz.have mitigating effects on AA arthritis rats.