Study on the Pharmacological Activity and Mechanism of Girardinia Diversifolia (Link) Friis
|Course||Biochemistry and Molecular Biology|
|Keywords||Girardinia diversifolia (Link) Friis Toxicity Anti-inflammatory Antibacterial Vanillic acid Activity Mechanism|
G. diversifolia (Link) Friis ("Da Xie Zi Cao" in Chinese), a perennial herb, belongs to the family Urtica Girardinia and is widely located in Shanxi, Gansu, Hubei, Hunan, Henan, Chongqing, Sichuan, Guizhou and Yunnan, et al. It has been frequently used as folk herbal medicine and widely used in many nationalities, such as Han, Miao, Uygur, and Yi. G. diversifolia (Link) Friis has a long medicinal history and been recorded in many literatures. Recently, it has been reported that the plant of Urtica L belonging to the same family as Girardinia has hypoglycemic and anti-oxidative activity, anti-rheumatoid arthritis, analgesic, and lower blood pressure, et al. G. suborbiculata C. J. Chen and G. diversifolia (Link) Friis also exhibited anti-inflammatory and analgesic. Moreover, antibacterial compounds had been isolated from G. suborbiculata C. J. Chen. At present, there is no research report of G. diversifolia (Link) Friis about hypoglycemic activity, antioxidant activity, and the effects on traumatic infection and psoriasis, and antibacterial substance and its mechanism were not reported.Based on the literature review above, we have studied the effects of G. diversifolia (Link) Friis on hypoglycemic activity, antioxidant activity, experimental trauma, experimental diarrhea, psoriasis. Moreover, we have isolated and purified polysaccharide and antibacterial compounds, and identified the chemical structures of these antibacterial compounds, and studied the acute toxicity of G. diversifolia (Link) Friis as well.The main results are summarized from six aspects as following:1. Studies on the toxicity of G diversifolia (Link) Friis in mice The experiment of acute toxicity indicated that the LD50of ethanol extract from G. diversifolia (Link) Friis in mice by oral administration was10.52g/kg and its95%confidence limit was8.55～12.93g/kg; The MTD of aqueous extract in mice by oral administration was40g/kg. The toxicity of ethanol extract was greater toxicity, however, aqueous extract was non-toxic.The results of chronic toxicity of G. diversifolia (Link) Friis in mice showed that the histopathological changes of the heart, liver, spleen, lung, kidney and testicle were observed. On day90post-beginning of oral administration, the pathological examinations showed that ethanol extract of G. diversifolia (Link) Friis had damage on the heart, liver, spleen, lung, kidney and testicle. The general clinical manifestations and histopathological observations of the heart, liver, spleen, lung, kidney and testicle showed dose-related degenerative damage in mice, but microscopic examination of the heart, liver, spleen, lung, kidney and testicle of control group all appeared normal.Compared to the control group oral administration of for90days, the weight of the low group mice by oral administration the ethanol extract from G. diversifolia (Link) Friis showed slightly decline, and the other group mice did not showed obvious difference. The ethanol extract from G. diversifolia (Link) Friis has no marked change of primal organ coefficient of mice, and the ethanol extract from G. diversifolia (Link) Friis has less impact on the body weight of mice of the indicators of the mouse hematology part, but a greater impact on blood biochemical indexes. There would be some effects in liver and renal function.2. Studies on the hypoglycemic effect of polysaccharide and its mechanismThe purity of polysaccharide was used as an index to guide the extraction, impurity removing and purification of polysaccharide from G. diversifolia (Link) Friis. The results showed that the best extraction condition of the raw polysaccharide was as follows:ratio of liquid to material was20:1; the extraction temperature was80℃; extract was concentrated to m/v1:1; the concentrated extract was precipitated by80%ethanol by adding95%ethanol and then deposited at4℃for12h; then, the precipitate was sequentially washed with petroleum ether, acetone and alcohol until the effluent became colorless; finally it was dried under vacuum. The raw polysaccharide was re-dissolved in water.10%trichloroacetic acid was chosen to remove proteins and1.5%activated charcoal was used to remove pigment. Then, it was dialyzed for about48hours and freeze-dried to obtain the refined polysaccharide from G. diversifolia (Link) Friis.We have investigated the total antioxidant activity and the superoxide anion radical (02-·) and hydroxyl radical (·OH) scavenging capacities of polysaccharides from G. diversifolia (Link) Friis by using pyrogallic acid self-oxidation method and Fenton method. The results were analyzed by nonlinear regression method and the IC50was calculated. The result showed that polysaccharides from G. diversifolia (Link) Friis exhibited the scavenging capacities of superoxide anion radical (O2-·) and hydroxyl radical (·OH). The IC50of hydroxyl radical (·OH) and superoxide anion radical(O2-·) scavenging capacities were higher than2mg/mL and5mg/mL, respectively, while both IC50for Vc were lower than1mg/mL. The scavenging capacity of·OH was stronger than that of O2-·. Polysaccharides from G. diversifolia (Link) Friis had certain antioxidant activity and it was positively correlated with the concentration. However, this activity was less than that of vitamin C.Hyperglycemic mice model with "three increasing and one reducing" symptoms, was established by intraperitoneal injection of Alloxa. The purified polysaccharide from G. diversifolia (Link) Friis was administrated by gastric perfusion. The results demonstrated that the modeling mice have higher blood glucose concentration, amount of food intake and drinking water, and serum MDA content, lower serum SOD level and thymus and spleen index compared to the normal mice. The high dose polysaccharide (400mg/kg) from G. diversifolia (Link) Friis effectively decreased the blood glucose concentration and serum MDA content, enhanced the serum SOD level and the thymus and spleen index of diabetic mice caused by Alloxan. However, metformin hydrochloride as the positive control showed better effects than the high dose polysaccharide. There is not significant effect in the low dose polysaccharide group.3. Studies on the effect of G diversifolia (Link) Friis on psoriaticThe effect of ethanol and aqueous extracts from G. diversifolia (Link) Friis on psoriatic modeling mice was investigated by observing the mitotic index of vaginal epithelium and tail scale granular of the mice. The result showed that both ethanol and aqueous extracts could promote the formation of mouse tail scale granular and inhibit the mitosis of vaginal epithelial cells. These facts indicate that ethanol and aqueous extract from G. diversifolia (Link) Friis had no effect on psoriasis, but it is a relatively weak effect compared with Methotrexate.4. Antimicrobial efficacy test of G. diversifolia (Link) Friis on infected wound The appropriate prescription of ointment for ethanol extract from G. diversifolia (Link) Friis was screened by Staphylococcus aureu susceptibility testing. Then, we further determined the efficacy of the ointment on the mouse wounds infected with Staphylococcus aureu. The results demonstrated that the ethanol extract in O/W type ointment had a better releasing rate and a significant inhibitory effect.5%ethanol extract in this type of ointment exhibited the best inhibitory effect. This ointment had an obvious therapeutic effect on the mouse wounds infected by Staphylococcus aureu.5. Extraction and isolation of Antibacterial compounds from G diversifolia (Link) FriisThe results of systematical chemical identification showed that G. diversifolia (Link) Friis may contain sugar, polysaccharide, glycosides, phenolic compound, tannins, saponin, organic acids, flavonoids, alkaloids, steroidal, steroidal and triterpenoids. However, it may not contain volatile oil, grease, cardiac glycosides, amino acids, polypeptide and protein, et al.The antibacterial activity on Staphylococcus aureu was used as the guidance for the active compounds purification procedure. The crude extract from G. diversifolia (Link) Friis was firstly extracted by ethanol and then sequentially by organic solvents with different polarity. After that, it was further purified by column chromatography and high performance liquid chromatography. Four compounds with antibacterial activity were obtained through this procedure. These compounds were determined by spectral analysis (’H-NMR,13C-NMR, MS) as follows:compound Ⅰ:diisobutyl phthalate; compound Ⅱ:6-Hydroxy-7-methoxycoumarin; compound Ⅲ:vanillic acid; compound Ⅳ:(2Z,3Z)-3-amidinoacrylic acid-5-ribofuranoside, and it was not reported previously.6. Vanillic acid Anti-inflammatory, bacteriostatic, and antibacterial mechanismCompared with the aspirin group, vanillic acid cannot degrade on the ear edema and paw oedema induced by xylene and carrageenan in mice, respectively. Vanillic acid cannot degrade contents of inflammatory mediator as MDA in blood serum, NO in paw of mice swell, and it also can not increase contents of SOD for anti-inflammatory. furthermore, it can not effectively reduce systolic and diastolic blood pressure of spontaneously hypertensive rat (SHR). So, vanillic acid has no anti-inflammatory activity. The aim was to determine the antimicrobial spectrum and antimicrobial activity of vanillic acid, the results showed that the vanillic acid had antimicrobial activity against Escherichia coli, Staphylococcus aureu, Bacillus subtilis, Salmonella, Pseudomonas aeruginosa, and Brevibacterium. The MIC of vanillic acid on these bacteria was6mg/mLThe result of inhibition growth curves against Staphylococcus aureu demonstrated that vanillic acid could inhibit the growth and reproduction of Staphylococcus aureu, but vanillic acid did not kill Staphylococcus aureu. The results showed that vanillic acid could inhibit the reproduction of DNA by combining with the genomic DNA.The membrane permeability experiment was applied for studying the antibacterial mechanism of vanillic acid on Staphylococcus aureu. The result showed the vanillic acid also could damage biofilm that would result in the impaired adhesion, colonization, growth and reproduction of bacteria and enhanced vanillic acid ability against Staphylococcus aureu. The results also showed that it could not kill them by combining with the genomic DNA or plasmic DNA.