Study On The Chemical Constituents And Its Anti-inflammation Activities Of Terminalia Chebula

Terminalia chebula is the dried immature fruit of plant Terminalia chebula Retz, which can clear away the heat-evil and expel superficial evils, used to treat Yin deficiency diphtheria clinically. Up to now, Terminalia chebula Retz, the ripe fruit of Terminalia chebula Retz, has been thorough studied, while no systematic study on components and biological effects of the immature Fructus chebulae has so far been reported. Previous studies indicate that Terminalia chebula Retz contains abundant phenolic acids, which have antioxidant, antibacterial and anti-inflammatory activities. Furthermore, ethanol extracts of Terminalia chebula Retz inhibits inflammation by reducing expression of inflammatory cytokines derived from macrophage. The In vitro data also show that chebulagic acid, another main component of Terminalia chebula Retz, attenuates inflammation by inhibiting 5-LOX and COX-2.According to Chinese Pharmacopoeia, the amount of water-soluble extracts form Terminalia chebula is 50% higher than Terminalia chebula Retz, indicating that components of Terminalia chebula may also has anti-inflammatory activity. So, we extracted some components from Terminalia chebula and gained a main component, chebulanin, which was further studied on anti-inflammatory effect in vitro and in vivo. All these studies will support further research on the biological and pharmacological activities of Terminalia chebula.Methods and Results:Part 1. The extraction and chemical structure identification of anti-inflammatory components from Terminalia chebulaMethods 1. The extraction of total phenolic acids in Terminalia chebula The powder of Terminalia chebula was extracted three times with 70% acetone. The acetone soluble extract was then evaporated to concentrate and extracted respectively threetimes with benzene and ethyl acetate in succession. All ethyl acetate solution is collected and concentrated to get brownish solid. 2. HPLC detection of total phenolic acids in Terminalia chebula The total phenolic acids extracted from Terminalia chebula were loaded into chromatographed(Ultimate LP-C18(250mm×4.6mm, 5μm) and successively eluted with methanol and 0.6% phosphoric acid at 1.2 m L/min on 40℃. Data was collected at the wavelength of 278 nm. 3. Separation of total phenolic acids Total phenolic acids were completely dissolved in distilled water. Then all supernatant was collected, loaded to macroporous absorbent column and then successively eluted with 0% to 90% methanol. Eluent was collected and tested with 1% acidic Fe Cl3 to detect phenolic acids, and further analyzed with HPLC. Eluents with similar composition were pooled together, re-dissolved with methanol and natural crystallized. All crystals were collected for further use. 4. Identification of compounds Identification of compounds was conducted on the basis of results of melting point determination, UV, IR, MS and NMR.Results 1. Analysis of chromatogram Based on the HPLC chromatograms, 5 ingredients were identified in the total phenolic acids, including gallic acid(tR7.45min), Corilagin(tR20.59min), Chebulanin(tR21.53min), Chebutagic acid(tR29.76min) and Chebulinicacid(tR37.25min). 2. Macroporous absorbent column chromatography separation Four compounds were isolated by means of macroporous absorbent column chromatography from total phenolic acids, which were white acicular crystal(compound â… ), white powdered solid(compoundâ…¡), white amorphous particles(compound â…¢) and white flaky crystal(compound â…£), respectively. 3. Identification of compounds Above four compounds were identified based on results of HPLC, UV, IR, MS, NMR and published papers, which are Gallic acid, Chebulanin, Chebutagic acid and Chebulinicacid, respectively.Part 2. Study of effect of chebulanin in Terminalia chebula on NF-κB pathway and cytokines expressionMethods 1. Effect of chebulanin on proliferation of RAW264.7 RAW264.7 cells were seeded into plates and incubated overnight. A series of gradient concentration of Chebulanin and Dexamethasone were prepared and added to cells. The plates were incubated for 24 hrs at 37℃. Cell number was analyzed with CCK8 following manufacturer’s manual. The inhibition rate of proliferation was calculated as following: Inhibition rate(%) =(1-T/C) x 100%(T: OD of tested sample C: OD of control group) 2. Effect of chebulanin on m RNA expression of inflammatory factors in RAW264.7 500,000 RAW264.7 cells were seeded into 12-well plate and incubated overnight. Chebulanin was added to each well to obtain final concentrations of 100μM, 50μM and 10μM. The plate was incubated for 2 hrs at 37℃. Afterwards, LPS was added to each well at the final concentration of 100 ng/m L and the plate was incubated for another 20 hrs at 37℃. m RNA was extracted from cells and reverted to c DNA. RT-PCR was performed to detect expression of TNF-α and COX-2 with 3 duplications. 3. Effect of chebulanin on production of inflammatory factors in RAW264.7 50,000 RAW264.7 cells were seeded to 24-well plate and incubated overnight. Chebulanin was added to each well to obtain final concentrations of 100μM, 50μM and 10μM. Dexamethasone and PBS were also separately added to corresponding wells as positive and negative control. The plate was incubated for 2 hrs at 37℃. Afterwards, LPS was added to each well at the final concentration of 100 ng/m L and the plate was incubated for another 24 hrs at 37℃. All supernatant was collected and ELISA was conducted to detect TNF-α and IL-6 expression with 3 duplications. 4. Effect of chebulanin on phosphorylation of NF-κB p65 in RAW264.7 20,000 RAW264.7 cells were seeded to 6-well plate and incubated overnight. Chebulanin was added to each well to obtain final concentrations of 100μM, 50μM and 10μM. The plate was incubated for 1 hrs at 37℃. Afterwards, LPS was added to each well at the final concentration of 100 ng/m L and the plate was incubated for another 3 hrs at 37℃. Cells were lysed with SDS buffer and analyzed with western blotting to detectphosphorylation of p65.Results 1. Effect of chebulanin on proliferation of RAW264.7 Compared with the blank control group, the proliferation of RAW264.7 was not significantly inhibited(p<0.05) when the concentrations of chebulanin and dexamethasone were below 100μM and 0.5μM, respectively. Then 100μM and 0.5μM were set as the maximum dosage for the next cell experiments of chebulanin and dexamethasone. 2. Effect of chebulanin on m RNA expression of inflammatory factors in RAW264.7 100μM, 50μM and 10μM chebulanin significantly inhibit m RNA expression of TNF-α and COX-2 compared with the control group in a dose dependent manner(P<0.01). 3. Effect of chebulanin on production of inflammatory factors in RAW264.7 Chebulanin significantly reduces TNF-α production in all dosage(p<0.01) and significantly inhibits IL-6 production only on 100μM and 50μM(p<0.01). 4. Effect of chebulanin on phosphorylation of p65 in RAW264.7 Compared to LPS-treated group, phosphorylation of p65 is decreased in cells pre-treated with chebulanin in a dose dependent manner.Part 3. Study on efficacy and preliminary mechanism of chebulanin treating collagen induced arthritis in miceMethods 1. Preparation of collagen induced arthritis(CIA) in mice Collagen induced arthritis in DBA/1 mice was induced according to Brand et al description. Briefly, all mice except the normal group were administered intradermally at the base of the tail with the dose of bovine type II collagen(100 μg) emulsified with complete Freund’s adjuvant. Animals received a booster injection of bovine type II collagen(100 μg) emulsified with incomplete Freund’s adjuvant 21 days after the initial immunization. 2. Chebulanin treatment for collagen induced arthritis in mice A total of 30 male DBA/1 mice were randomly divided into five groups as follows: control group, CIA model group, low dosage group(chebulanin, 40mg/kg), middle dosage group(chebulanin 80mg/kg) and high dosage group(Chebulanin 160mg/kg). Control groupand CIA model group were only treated with1% CMC-Na by gavages. While low, middle and high dosage group were treated with 40mg/kg/day, 80mg/kg/day and 160mg/kg/day, respectively from day 39(after full development of CIA) for a duration of 28 days by gavages. Mice were observed for two more weeks after administration and sacrificed at the end of experimentation on day 80. Both ankle joints in the paw were removed and fixed in 10% neutral buffered formalin for the following Micro-CT scan, pathological examination and immunohistochemical examination. 3. Observation and assessment of arthritis severity The diet, movement and hair changes were observed everyday and the arthritis severity scores were recorded twice a week. 4. Micro-CT evaluation Right hind paws and joints were scanned and then reconstructed into a 3D-structure using Viva CT 40 Micro-CT instrument. Bone was segmented from soft tissue using the Scanco Image Processing Language software and three relative parameters(bone volume(BV), bone surface area/ BV(BS/BV) and trabecular thickness(Tb.Th)) were analyzed. 5. Joint histopathological evaluation Paws and joints were removed and fixed, after decalcification, dehydration, embedded in paraffin blocks and stained with hematoxylin and eosin(H&E) to study the degree of synovitis, cellular infiltration and bone erosion. 6. Immunohistochemical examination Tissue samples were counterstained with specific antibodies for murine TNF-α, IL-6, COX-2 and MMP-3, followed by incubation with the appropriate secondary antibodies. Staining for the specific expression marker was scored by using microscopy.Results 1. Clinical manifestations of CIA mice No collagen induced arthritis was observed during the initial immunization; but one day after the booster immunization, collagen induced arthritis was developed and then gradually lead to a 100% incidence of CIA at day 35(a total of 24 DBA/1 mice). 2. Chebulanin ameliorated the severity of arthritis in CIA mice at middle dosage and high dosage The results indicated that during 28 days treatment, chebulanin significantly decreasedthe severity of arthritis at doses of 80 and 160 mg/kg(p<0.05); Treatment with chebulanin at a dose of 40 mg/kg also ameliorated the severity of arthritis but the improvement observed was not statistically significant. The mean severity scores were maintained 5 to 6 in middle and high dosage group while the mean severity score was 11 in untreated group which showed significant difference(p<0.05) two weeks after treatment. However, the mean severity scores in low dosage group were 9 to 10 which indicated a slight trend of decreasing but without statistical significance. 3. Micro-CT evaluation The severity of joint destruction assessed by using 3D micro-CT showed that chebulanin-treated CIA mice showed markedly less joint destruction, especially at the midand high-treatment doses of 80 mg/kg and 160 mg/kg. Quantitative analysis of the BV, BS/BV and Tb.Th parameters showed that BV and TB.Th were present at a higher volume in the chebulanin-treated mice than in the controls, and this effect was dose-dependent. Similarly, the BS/BV in chebulanin-treated CIA mice was significantly lower than in untreated mice, indicating that chebulanin has a protective effect against bone damage if applied in the early stage of CIA disease. 4. Histopathological assessment Results from histopathological assessment showed that chebulanin-treated mice had significantly decreased levels of cellular infiltration, hyperplasia and bone destruction in middle dosage and high dosage. 5. The influence of chebulanin to the expression level of TNF-α å'Œ IL-6 Expression levels of TNF-α å'Œ IL-6 were significantly reduced following chebulanin treatment at middle and high dosage(p<0.05). 6. The influence of chebulanin to the expression level of COX-2 and MMP-3 Expression levels of COX-2 and MMP-3 were significantly reduced following chebulanin treatment at middle and high dosage(p<0.05).Conclusions: 1. The total phenolic acid was extracted from Terminalia chebula. And 4 compounds, including gallic acid, chebulanin, chebulagic acid and chebulinic acid, were further isolated from total phenolic acid. 2. Results based on RAW264.7 showed that chebulanin can significantly inhibitm RNA expression of TNF-α and COX-2, and reduce protein level of TNF-α and IL-6. Chebulanin also can significantly inhibit phosphorylation of NF-κB p65 induced by LPS. It indicated that chebulanin may inhibit LPS-induced inflammation throμgh regulation of NF-κB pathway. 3. Effects of chebulanin on heumatoid arthritis and expression of inflammatory factors in joint were investigated on the basis of CIA mouse model. The results showed that chebulanin attenuated RA symptom and reduced expression of inflammatory factors, such as TNF-α, IL-6, COX-2 and MMP-3.