摘要
掌叶蝎子草(Girardinia diversifolia (L ink) Friis),为荨麻科(Urtica)蝎子草属(Girardinia),多年生草本植物,又名：大蝎子草,红活麻,主要分布于我国的甘肃、陕西、湖北、湖南、河南、贵州、四川、重庆和云南等地区。掌叶蝎子草在我国民间为常用植物药,在汉族、苗族、维吾尔族、彝族等多个民族中广泛使用,药用历史悠久,许多文献有关于掌叶蝎子草民间应用的记载。近年来,研究发现与蝎子草属植物同属荨麻科的荨麻属植物具有降血糖、抗氧化活性、抗风湿性关节炎、镇痛、降血压的作用。研究发现蝎子草属的蝎子草(G. suborbiculata C. J. Chen)具有抗炎、镇痛活性,并从该种植物中分离到抗菌化合物：目前,对掌叶蝎子草的抗炎、镇痛活性进行了研究,但目前未见关于掌叶蝎子草毒性、降血糖、治疗银屑病、抗菌机制等的研究报道。
基于以上调研,我们对掌叶蝎子草毒性、抗炎、抑菌及其活性成分的作用等进行研究,主要研究内容如下：
(1)掌叶蝎子草毒性研究
掌叶蝎子草醇提浸膏对小鼠的LD50值为10.52g/kg,LD50的95%可信限为8.55-12.93g/kg；掌叶蝎子草水提浸膏对小鼠的MTD为40g/kg,掌叶蝎子草醇提浸膏毒性较大,而水提浸膏属于无毒。
小鼠经口慢性毒性研究表明：给药90d后,掌叶蝎子草醇提浸膏对小鼠心脏、肝脏、脾脏、肺、肾脏和睾丸有一定毒性,且毒性随掌叶蝎子草醇提浸膏剂量增加增大,其引起小鼠脏器组织的病理变化也越明显；而空白对照组小鼠各脏器组织显示的显微结构均正常,未出现明显病理变化。掌叶蝎子草醇提浸膏对小鼠体重影响较小,对各脏器系数无明显影响；对小鼠血液学部分指标有影响,但对血液生化指标有较大影响,可能对肝脏代谢有一定的影响。
(2)掌叶蝎子草多糖降血糖活性及作用机制研究
掌叶蝎子草多糖提取、去杂、纯化的主要方法：选取液料比20：1,80℃左右热水浸提3次,每次1h,合并提取液,浓缩至m/v(m原料：v浓缩液)的比值为1：1,加入95%的无水乙醇至醇浓度约为80%进行4℃过夜醇沉,沉淀依次用石油醚、丙酮、无水乙醇洗涤至洗脱液无色后,真空干燥得掌叶蝎子草粗多糖。将掌叶蝎子草粗多糖复溶于水,优选10%三氯乙酸脱蛋白,1.5%活性炭脱色除杂,经脱蛋白脱色处理后的多糖溶液经过透析48h除去小分子杂质,然后冷冻干燥即得掌叶蝎子草精制多糖。
测定掌叶蝎子草粗多糖的还原力、清除·OH和O2-·的活性等三个方面研究其抗氧化能力。结果显示,掌叶蝎子草粗多糖具有一定的还原能力,且随浓度的增大还原能力也逐渐增强,但相同起始浓度下其还原能力不及维生素C(Vc)。掌叶蝎子草粗多糖对·OH和O2-·也有一定的清除能力,对-OH的清除效果强于对O2-·的清除效果,其清除·OH的IC50值大于2mg/mL,清除O2-·的IC50值大于5mg/mL,但掌叶蝎子草粗多糖清除自由基的能力均不及Vc,因Vc的IC50值均小于1mg/mL。
用四氧嘧啶致高血糖小鼠模型,造模成功的高血糖小鼠完全符合糖尿病“三高一低”症状,摄食量、饮水量明显升高,血糖值明显升高,血清SOD水平明显下降,血清MDA含量明显升高,胸腺指数和脾脏指数也明显下降。给予低、高剂量掌叶蝎子草多糖治疗2W后,掌叶蝎子草多糖高剂量组(400mg/kg)小鼠血糖值明显下降,血清SOD水平也明显上升,还一定程度降低了血清MDA水平,提高了小鼠胸腺指数和脾脏指数,但治疗效果仍不及阳性对照药物盐酸二甲双胍片显著。掌叶蝎子草多糖低剂量组(200mg/kg)也能在一定程度上降低糖尿病小鼠的血糖值和血清MDA水平,提高血清SOD水平、胸腺指数和脾脏指数,但效果都不明显。
(3)掌叶蝎子草对银屑病的作用研究
掌叶蝎子草水提浸膏和醇提浸膏能促进小鼠尾部上皮细胞颗粒层的形成,从小鼠阴道上皮细胞有丝分裂的影响研究结果也进一步证实掌叶蝎子草水提浸膏和醇提浸膏能部分抑制上皮细胞的有丝分裂,而阳性药物甲氨喋吟能明显促进小鼠尾部上皮细胞颗粒层的形成和抑制小鼠阴道上皮细胞有丝分裂,说明掌叶蝎子草水提浸膏和醇提浸膏均对银屑病有一定的治疗作用,但治疗效果明显弱于药性药物甲氨喋吟。
(4)掌叶蝎子草对感染化脓伤口治疗效果研究
将金黄色葡萄球菌用涂擦的方法感染到小鼠伤口上,能使小鼠伤口感染化脓,用制备的掌叶蝎子草软膏涂沫小鼠感染金黄色葡萄球菌的伤口,明显抑制伤口的化脓,涂抹治疗5天时,伤口已经完全结痂、愈合,表明掌叶蝎子草醇提浸膏软膏对已感染化脓的伤口有明显的治疗效果。
(5)掌叶蝎子草抑菌化合物的分离纯化
经过系统的化学鉴别分析,判断掌叶蝎子草中主要化学成分为糖、多糖、苷类、酚性成分、鞣质、有机酸、黄酮类、生物碱类、甾体或三萜类,不含挥发油、油脂、强心苷等。
以对金黄色葡萄球菌的抑菌作用为依据,通过醇提、不同溶剂萃取、柱层析、高效液相色谱分离等方法分离纯化抗菌活性物质,通过波谱学方法(1H-NMR、13C-NMR、MS)对化合物进行结构鉴定。得到4个对金黄色葡萄球具有抑制作用的单体化合物,分别是：邻苯二甲酸二异丁酯,6-羟基-7-甲氧基香豆素、香草酸(3-甲氧基-4-羟基苯甲酸)和(2Z,3Z)-4-氨基丙烯酸-5-呋喃核糖苷。其中,化合物(2z,3Z)-4-氨基丙烯酸-5-呋喃核糖苷目前未见相关文献报道。
(6)掌叶蝎子草中香草酸药理活性及作用机制研究
与空白对照组比较,香草酸对小鼠耳肿胀作用无抑制作用,只有高剂量香草酸能降低小鼠足肿胀度和抑制MDA的释放,香草酸其余各剂量即不能明显降低小鼠耳胀度和足肿胀度,也不能升高小鼠血清SOD活性和抑制血清MDA的释放,降低炎足中NO含量；而阳性对照药物阿司匹林对小鼠的耳肿胀度和足肿胀度有显著抑制作用,明显升高血清SOD含量,降低血清MDA水平和炎足中NO含量。因此,结果表明香草酸对致炎引起的小鼠炎症无明显的抗炎活性。
测定香草酸抗菌谱,结果表明：香草酸对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、沙门氏菌、绿脓杆菌和短杆菌等均具有抑制作用,其最低抑菌浓度为6mg/mL。
香草酸对金黄色葡萄球菌生长曲线的影响结果表明：香草酸能够抑制金黄色葡萄球菌的生长分裂,但不能杀灭金黄色葡萄球菌。
香草酸对金黄色葡萄球菌质粒DNA和基因组DNA结合结果表明,香草酸与金黄色葡萄球菌的质粒DNA没有结合；香草酸可能与金黄色葡萄球菌的基因组DNA有结合,影响细菌DNA的复制,从而抑制金黄色葡萄球菌的生长繁殖。
香草酸能抑制金黄色葡萄球菌生物被膜的形成,并能部分破坏已形成的生物被膜,影响金黄色葡萄球菌的粘附定植,抑制生物被膜形成活性与香草酸浓度成正比,增强对金黄色葡萄球菌的抗菌能力。
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 mechanism
The 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 psoriatic
The 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) Friis
The 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 mechanism
Compared 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/mL
The 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.
引文
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