| Celastrus aculeatus Merr., a traditional Chinese medicine known as "guo-shan-feng", first edited in "xin hua ben cao gang yao". Its medicinal parts are roots and canes. Celastrus aculeatus Merr. is bitter, acrid, cool in literature. Its effects are clearing heat and detoxication, activating blood to relieve pain, expelling wind and dampness. It was mainly used to treat rheumatoid arthritis, gout, nephritis, cholecystitis, hypertension etc. It distributes in provinces like Zhejiang, Fujian, Jiangxi, Guangdong, Guangxi, Yunnan etc. in China and grows at an altitude of100-1000m in woodland hillside street or under shrubs. There is currently no artificial introduction and cultivation of this plant. Celastrus aculeatus Merr. can be used as decoction or topical to treat rheumatism in folk. Its decoction and tincture have achieved good effect in the clinical treatment of rheumatoid arthritis for many years. Experimental study of modern pharmacology indicated that Celastrus aculeatus Merr. had the effects of anti-inflammatory analgesic, immunomodulatory, anti-oxidation etc.Rheumatoid arthritis (RA) is one kind of chronic systemic autoimmune disorder diseases with variable clinical expressions, including the potential for destructive arthritis and significant extra-articular manifestations. If failed to timely diagnosis and properly treated,70%RA patients would suffer the destruction of bone and joint deformities after2years, and then develop into irreversible joint mutilation. It seriously affects the quality of patients’ life and shortens the average life expectancy of patients about10-15years.Celastrus aculeatus Merr. was selected as raw materials., using orthogonal design to choose better extraction process of Celastrus aculeatus Merr. And the chloroform part of ethanol extract was further purified by macroporous resin. The petroleum ether and ethyl acetate parts which extracted from ethanol extract were systematically isolated and purified by repeated silica gel, high-speed countercurrent chromatography (HSCCC) and recrystallizationt to obtain9compounds. Their structures were identified on the basis of various spectroscopic methods. Also, the biological activity of5obtained compounds was studied according to the cytotoxicity test. This paper is to lay part of the material foundation to further explore the mechanism of anti-rheumatoid arthritis effect of Celastrus aculeatus Merr., and also provide a scientific basis for developing Celastrus aculeatus Merr. to be a drug which has the effect of anti-rheumatoid arthritis.1. Study on the optimization of the extraction process of ethanol extract from Celastrus aculeatus Merr. by orthogonal designUsing the content of total triterpenoids as evaluation index and pristimerin as reference sample, choosing ethanol concentration, ultrasonic time, solid-liquid ratio, ultrasonic power as the observation factors, influencing factors of the extraction process of ethanol extract from Celastrus aculeatus Merr. were studied by L9(34) orthogonal design using UV-vis. The influence degree of observation factors was ethanol concentration>ultrasonic time>solid-liquid ratio>ultrasonic power. According to the results of orthogonal design, the preferred extraction process of ethanol extract from Celastrus aculeatus Merr. was70%ethanol solution, ultrasonic time25minutes, solid-liquid ratio1:20, ultrasonic power720W.2. Using macroporous resin to purify ethanol extract from Celastrus aculeatus Merr.According to the pharmacological experimental studies on the different parts of Celastrus aculeatus Merr. from Chen PH, chloroform part from the ethanol extract showed significant anti-inflammatory activity, and the main active ingredients were triterpenoids. So macroporous resin was used to purify the chloroform part from ethanol extract to improve the content of triterpenoids. Specific experimental procedure:Using the prefered ultrasonic extraction process to get ethanol extract. The ethanol etract was concentrated, then suspended in H2O and extracted with petroleum ether and chloroform, respectively. Chloroform part from ethanol extract was dissolved in50%ethanol and insoluble material was filtered to obtain a clear solution. The maximum sample volume, preferred sample concentration, eluent concentration, eluent consumption and model of macroporous resin were studied using UV-vis. The results were HPD100macroporous resin, sample concentration5.75mg/mL, first with5BV distilled water, then3BV30%ethanol to remove impurities, continue to use9BV55%ethanol, flow rate3BV/h. After purification, total triterpene content of chloroform part from ethanol extract of Celastrus aculeatus Merr. increased from27.29%to53.95%.3. Preliminary study of the chemical composition of Celastrus aculeatus Merr.The air dried roots and stems of Celastrus aculeatus Merr. were powdered and extracted with95%EtOH under reflux conditions at80℃for two hours to give a crude extract. This was suspended in H2O and insolubles were filtered off. The supernatant was extracted with petroleum ether (PE), and ethyl acetate (AcOEt), successively, to yield a PE-soluble fraction, and an AcOEt-soluble fraction. Separately,5mL of EtOH crude extract was evaporated in vacuo to yield a residue that was dissolved in20mL MeOH, producing the white needle-like crystals. It was identified to be dulcitol.The PE-soluble fraction was subjected to CC repeatedly (silica gel, gradient of PE-AcOEt,20:1-1:5), producing white powder, white wax and yellow powder. They were identified to be β-sitosterol, n-tritriacontane and nimbidiol, respectively.The AcOEt-soluble fraction was subjected to CC (silica gel, gradient of CHCl3-MeOH,100:0-100:1) to afford two fractions (Ⅰ and Ⅱ). Fraction II (100:1) was used for the subsequent HSCCC isolation and purification. Experimental conditions were as follows:the two phases solvent system, composed of hexane/ethyl acetate/methanol/water (1:1:1:1, v/v); revolution speed:850rpm; stationary phase: upper phase; mobile phase:lower phase; flow rate:1.5ml/min; retention of the stationary phase:74%; detection wavelength:280nm. Fractions i-ii were corresponded to pristimerin and pristimerol (new compound).The30%ethanol soluble AcOEt fraction was purified by D101column chromatography. At first, ditilled water was used to elute the resin until the elution was nearly colorless, then increasing concentrations of ethanol were used and two main fractions were obtained:fr. A (50%ethanol elution), B (80%ethanol elution). The fr. A was subjected to CC (silica gel, gradient of CHCl3-MeOH,100:0-100:1) repeatedly, producing3kinds of white crystals or powder. They were identified to be p-hydroxybenzoic acid, vanillic acid,3,5-dimethoxy-4-hydroxybenzoic acid, respectively.4. Preliminary study on the biological activity of chemical composition from Celastrus aculeatus Merr.Compounds nimbidiol, pristimerol, p-hydroxybenzoic acid, vanillic acid and3,5-dimethoxy-4-hydroxybenzoic acid were evaluated for their effects as the cytotoxicity toward the macrophage RAW264.7cells. The results showed that nimbidiol, pristimerol, vanillic acid and3,5-dimethoxy-4-hydroxybenzoic acid all had different levels of cytotoxicity, especially nimbidiol and pristimerol. Macrophages are important cells which secrete inflammatory cytokines. They are closely related to inflammatory cell function. So this experiment suggested that these4compounds may be potential anti-inflammatory active ingredients, but also the material basis of Celastrus aculeatus Merr. with anti-inflammatory activity.This paper studied using orthogonal design to choose better extraction process of Celastrus aculeatus Merr., and further studied the purification of chloroform part from the ethanol extract using macroporous resin At the same time the petroleum ether and ethyl acetate parts were systematically isolated and purified to get9compounds. These compounds were identified according to MS, NMR, IR etc. The cytotoxicity of5compounds were investigated by MTT. This paper is to lay part of the material foundation to further explore the mechanism of anti-rheumatoid arthritis effect from Celastrus aculeatus Merr., and also provide a scientific basis for Celastrus aculeatus Merr. developing to be a drug which has the effect of anti-rheumatoid arthritis. |
PDF Full Text Request