长白山区复序橐吾和狭苞橐吾生物活性成分研究
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摘要
橐吾属(Ligularia)为菊科(Compositae)千里光族(Senecioneae)多年生草本植物,全球约有150种,绝大多数种类产于亚洲,仅2种分布于欧洲,中国为橐吾属植物种类最为丰富的地区,约有110种,3变种,大部分分布于西南部至东北部。
自2008年以来,笔者曾多次随课题组对位于吉林省长白山区的延边、白山2个地区8个县市区域的橐吾属植物开展野外调查、标本采集、试验研究等工作。调查发现,长白山区分布着8种橐吾属植物(复序橐吾、狭苞橐吾、蹄叶橐吾、三角叶橐吾、单花橐吾、全缘橐吾、合苞橐吾、白毛橐吾),其中复序橐吾主要生长于海拔400-1050m之间的林缘、林下、林间及沼泽草地,在当地分布广,资源蕴藏量大；狭苞橐吾主要分布在海拔680-1450m的亚高山地带,可以认为是岳桦林下优势种。
本研究以长白山区复序橐吾、狭苞橐吾的叶和花为试验材料,利用现代生物学实验方法及分析测试手段,对2种橐吾叶和花的营养成分、脂肪酸、挥发油等脂溶性成分进行了分析；探讨了复序橐吾多糖的提取、分离纯化过程及复序橐吾多糖的理化性质、单糖组成、分子量分布及结构特征；通过体外抗氧化、抗肿瘤、免疫活性试验对复序橐吾、狭苞橐吾叶的乙醇提取物、复序橐吾多糖的生物活性进行了研究,主要内容如下：
1.利用氨基酸自动分析仪对复序橐吾、狭苞橐吾叶和花的氨基酸含量进行分析。2种橐吾均含有16种蛋白质水解氨基酸,氨基酸总量为复序橐吾叶7.64%、花8.1%,狭苞橐吾叶12.60%、花9.90%;其中含有7种人体必需氨基酸,氨基酸的配比与FAO/WHO提出的蛋白质参考模式相比较,除蛋氨酸低于模式谱外,其它各种必需氨基酸均高于模式谱,配比合理。
2.采用电感耦合等离子体发射光谱法对复序橐吾、狭苞橐吾叶和花的Al、Cu、Fe、B、 Ba、Be、Ca、Co、Cr、K、Mg、Mn、Mo、Zn、Sr、Ni、Ti、P18种矿质元素含量进行了测定。2种橐吾叶和花除富含K、Ca、Mg等常量元素外,还含有Fe、Cu、Zn、Co、Mn、Cr、Mo、Sr、 B、Ni等多种人体必需微量元素。
3.采用GC-MS联用技术对复序橐吾、狭苞橐吾花的脂肪酸和挥发油成分进行分析。复序橐吾和狭苞橐吾花中含有的主要脂肪酸有：(Z,Z)-9,12-十八碳二烯酸(24.59%,6.29%)、软脂酸(19.12%,22.32%)、9-十八碳烯酸(7.05%,4.46%)、肉豆蔻酸(6.89%,8.30%)、(Z,Z,Z)-9,12,15-十八碳三烯酸(6.87%,2.50%)、硬脂酸(5.09%,6.77%),其中人体必需脂肪酸亚油酸、饱和脂肪酸软脂酸的含量较高。
从复序橐吾花挥发油的总离子流图中分离得到49个色谱峰,共鉴定出39个化合物,主要化学成分为4-蒈烯(26.53%)、D-柠檬烯(13.33%)；从狭苞橐吾花挥发油的总离子流图中分离得到75个色谱峰,鉴定出71种化合物,含量最高的成分为1,2,3,6-四甲基-二环[2.2.2]辛烷(46.87%)。2种橐吾花挥发油都含有丰富的萜类化合物,由于萜类化合具有广泛的生物活性,所以实验结果可以为探讨2种橐吾可能具有的抗菌、抗病毒、抗肿瘤等活性研究提供化学依据。
4.以石油醚和95%乙醇为溶剂,通过超声提取得到复序橐吾、狭苞橐吾叶的石油醚、乙醇提取液,按照GC-MS条件,将提取液直接进样,测定复序橐吾和狭苞橐吾叶的脂溶性成分。结果,从复序橐吾和狭苞橐吾叶的石油醚提取液中分别鉴定出59个和28个化合物,分别占脂溶性总成分的100%、96.33%；从乙醇提取液中分别鉴定出38个和35个化合物,占脂溶性总成分的84.50%、96.35%。鉴定的成分涵盖了萜烯类、烷烃、酮、酚、酸、酯类等化合物。
5.以复序橐吾叶为材料,在用乙醇浸提的基础上,分别以石油醚、氯仿、乙酸乙酯、正丁醇不同极性溶剂进行分级萃取,综合定性分析和总黄酮含量测定推测,复序橐吾乙醇总提物及各萃取部位均含有黄酮类成分,其中以乙酸乙酯部位总黄酮含量最高；通过清除DPPH有机自由基试验,对各萃取部位的抗氧活性进行测定,各萃取部位对DPPH清除能力的强弱顺序为乙酸乙酯(fr-Acetidin)>总提物(fr EtOHex)>正丁醇(fr-BuOH)>氯仿(fr-CHCl3)>石油醚(fr-PE),基本与总黄酮含量的多少一致；同时利用高效液相色谱法对乙醇总提物和各萃取物进行HPLC指纹图谱分析,分析这些片段中化合物的异同,为寻找复序橐吾抗氧化活性成分奠定实验基础。
6.首次以复序橐吾叶为材料,采用热水煮提、乙醇沉淀、常规干燥得到复序橐吾水溶性粗多糖,并对复序橐吾多糖进行了理化性质研究,结果显示,复序橐吾多糖为含有蛋白质但不含淀粉的酸性杂多糖；通过对粗多糖进行冻融分级、除蛋白、透析处理后,进一步通过DEAE纤维素离子交换层析和Sepharose CL-6B凝胶柱层析对多糖进行分级纯化,得到了6个多糖级分；采用HPLC、HPGPC、IR方法研究了各多糖级分的单糖组成、分子量分布和结构特征；利用MTT法对复序橐吾多糖的免疫学活性测定显示,复序橐吾粗多糖及LJPA3、LJPN、对小鼠脾脏T淋巴细胞的增殖有一定的促进作用,以LJPN1的作用较强。迄今为止对复序橐吾多糖的研究尚未见报道,因此本实验对探索复序橐吾多糖的构效关系和其潜在的药用价值有重要意义。
7.采用总还原力的测定、清除羟自由基、超氧阴离子自由基试验,对复序橐吾和狭苞橐吾叶的乙醇提取物体外抗氧化活性进行了评价,三种试验结果均显示复序橐吾叶的乙醇提取物比狭苞橐吾更具有良好的抗氧化活性；同时,利用MTT法测试了复序橐吾和狭苞橐吾叶的乙醇提取物对2种人肿瘤细胞(Hela、A549)的抑制活性,体外实验结果表明,复序橐吾和狭苞橐吾叶的乙醇提取物具有一定的抗肿瘤活性,狭苞橐吾乙醇提取物在一定浓度范围内,对Hela的生长抑制作用强于复序橐吾,但2种橐吾乙醇提取物对A549的抑制作用差异不大
以上研究表明,复序橐吾、狭苞橐吾是集营养、保健、药用功能于一体,极具开发利用价值的野生药食两用植物资源,其化学成分有待开发成为天然抗氧化剂、抗肿瘤及免疫调节剂等,未来应用前景广阔。
Ligularia Cass is a genus of perennial herb plants of Compositae Senecioneae. There are about150types of Ligularia Cass, and most of them distribute in Asia, only2kinds in Europe. China is rich in Ligularia and there are about110types and3variations. Most of them distribute in southwest and north east of China.
Since2008, a large number of investigations, collections and researches have been conducted in8counties of Yanbian, Baishan Regions Jinlin province. There are eight types of Ligularia Cass (Ligularia jaluensis、Ligularia intermedia、Ligularia fischeri、Ligularia deltoiclea、Ligularia jamesii、Ligularia mongolia、Ligularia schmidtii、Ligularia villosum) in Changbai Mountain, in which Ligularia jaluensis species has rich resources and wide distribution, growing at the altitute of400-1050meters in forest edge, woodland and wet meadow; Ligularia intermedia mainly distributes at the altitude of680-1450meters in subalpine zone.We can certainly confirm that Ligularia intermedia is a dominant species in Yuehualin area in China.
In this study, the leaves and flowers of Ligularia jaluensis and Ligularia intermedia were collected from Changbai Mountain as experimental materials. By means of modern biological experimental methods and analytical tests, the nutritional ingredients, fatty acid, and volatile oil were analized from2kinds of leaves and flowers of Ligularia; and the liposoluble composition were analyzed.The study also focuses on the process of extraction. separation, purification as well as its physicochenical property, monosaccaride composition, molecular weight distribution and formation feature in Ligularia jaluensipolysaccharide. By testing the effects of external antioxidant, antineoplstic, immunocompetence to study the bilological actvity of the ethanol extraction from both Ligularia jaluensis and Ligularia intermedia leaves and the Ligularia jaluensis polysaccharide, the main conclusion is as follows:
1. By means of automatic amino acid analyzer, the amino acid contents in leaves and flowers of Ligularia jaluensis were analyzed;16kinds of proteolysis amino acid were identified from these two type of Ligularia, in which the total amount of amino acid is7.64%in leaves,8.1%in flowers,12.60%in narrow bracts,9.90%in flowers and in them there are7kinds of amino acid identified which are essential to human. Compared the amino acid ratio with refernce mode regulated by FAO/WHO, all kinds of essential amino acid content are highter than the reference mode list except for methionine. The proportion is reasonable.
2.By means of inductive coupling plasma emission specroscopy, the content of18(Al、Cu、 Fe、B、Ba、Be、Ca、Co、Cr, K、Mg、Mn、Mo、Zn、Sr、Ni、Ti、P)kinds of mineral elements in the leaves and flowers were tested. The microelements essential to human such as Fe、Cu、Zn、Co、Mn、Cr、Mo、Sr、B、Ni were also identified besides the rich Macroelements such as K、Ca、Mg in the two kinds of leaves and flowers.
3. The ingredients of fatty acid and volatile oil from the flowers of Ligularia jaluensis and Ligularia intermedia were analized by GC-MS coupling technique. The main fatty acid contents in the flowers of Ligularia jaluensis Kom and Ligularia intermedia are (Z,Z)-9,12-octadecadienoic acid (24.59%, plamitice acid6.29%), palmitic acid (19.12%,22.32%),9-octadecenoic acid (7.05%,4.46%),myristic acid (6.89%,8.30%),(Z,Z,Z)-9,12,15-octadecane triolefinic acid (6.87%,2.50%)、stearic acid(5.09%,6.77%).The essential fatty acid, linoleic acid, staturated fatty acid, palmitit acid are higher in the contents.
After separating the volatile oil from Ligularia jaluensis flowers,49chromatographic peaks were obtained, in which39chemical compounds were identified and the chemical components are4-carene (26.53%)、D-limonene (13.33%);75chromatographic peaks were obtained from the total ion flow graph after separating the volatile oil from Ligularia intermedia flowers, in which71chemical compounds were identified and Bicyclo[2.2.2]octane,1,2,3,6-tetramethyl-(46.87%) are the highest contents. The volatile oil in both flowers contents is full of terpenoid. Since terpenoids possess comprehensive biological activity, there is no doubt that these two Ligularia sibiricas have antibiosis and antiviral functions.Therefore, the results of these experiments provide sound chemical basis for the activity study of antibiosis, antivirus and anticancer.
4. Mixed with petroleum ether and95%ethanol, under the condition of GC-MS. petroleum ether and and ethanol extracting solution from leaves by ultrasonic extraction treatment were injected directly to test the inposoluble constituent in leacves of Ligularia jaluensis and Ligularia intermedia. The result shows that59chemical compounds and28chemical compounds were identified from the petroleum ethier extracting solution of Ligularia jaluensis and Ligularia intermedia leaves respectively, taking up100%、96.33%of the total fatty soluble content;38chemical compounds and35chemical compounds were identified from the ethanol extracting solution of Ligularia jaluensis and Ligularia intermedia leaves respectively, taking up84.50%、96.35%of the total fatty soluble content. The conpounds content analysis of terpene, alkane, ketone, phenol, acid, esters was covered in the test.
5. Based on the materials of the leaves of L.jaluensis and ethanol, comprhensive qualitative analysis and the total flavonoid content test show that petroleum ethe、chloroform、acetic ether、 butyl alcohol and water also contain flavonoids compounds, in which highest level of total flavonoid content exsits in ethyl acetate part. By eliminating organic free radicals DPPH test, research focused on the study of the antioxidant activity in every part of the extract; the strong-to-weak sequence of DPPH scavenging capacity is ethyl acetate (fr-Acetidin)> total standardized extracts (fr-EtOHex>n-butyl alcohol (fr-BuOH>chloroform(fr-CHCl3)>petroleum ether (fr-PE), basically consistent with total flavonoid content. Meanwhile by means of HPLC chromatography, the finger-print spectrum of all ethanol standardized extracts and the single extract were analyzed to the determine the differences in the chemical compounds fragments, establishing a sound experimental fundition for searching antioxidant activity elements in Ligularia jaluensis.
6. For the first time Ligularia jaluensis leaves were used as testing materials. By boiling extracting, ethanol precipitation and conventional drying, water-soluble, crude polysaccharide were obtained; and then to study the physicochemical property of polysaccharide. The results show that the Ligularia jaluensis polysaccharide is a kind of acide heteropolysaccharide with starchfree protain. The crude polysaccharide was treated by freeze thawing grading, deproteinizing and dialyzing. By further DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel column chromatography to fractionate and purify the Ligularia jaluensis polysaccharide,6polysaccharide fractions were obtained. HPGPC、IR were applied in studying monosaccharide composition, molecular weight distribution and the structrural features. MTT was useed to determine the activity of Ligularia jaluensis polysaccaride. The result shows that crude polysaccaride as well as LJPA3.LJPN1can accelerate the multiplication the T-lymphocytic in the mice spleen. LJPN1is superior to others. There has been no any reports on the study of the Ligularia jaluensis polysaccharide so far. So this study has great significance in developing the potential medical values in Ligularia jaluensis.
7. Total reducing power determination, eliminating hydroxy free radical and Superoxide anion free radical test were adopted to assess the external antioxidant activiy of ethanol extract in both plants leaves. The results of above three tests indicate that Ligularia jaluensis leaves possess better antioxidant activiy than that of Ligularia intermedia leaves. Meamwhile, MTT was used to test the inhibitory activity of the ethanol extract from the leaves of Ligularia jaluensis and Ligularia intermedia against two kinds of human tumor cells (Hela、A549). The results of the experiments in vitro show that the ethanol extract from both leaves of Ligularia jaluensis and Ligularia intermedia proves to have antineoplastic activity. The ethanol extract from the leaves of Ligularia intermedia has better inbibitional effect on controlling the growth of Hela in certain concentration range. But there is no great difference in inbibitional effect on A549between the ethanol extract from both leaves.
The above study shows that Ligularia jaluensis and Ligularia intermedia have multi-function on nutrition, health care, and officinal and it is worth exploiting such potential edible and medical plants. The chemical compounds in the plants remain to be developed into natural antioxidant, antineoplastic as well as immunomodulator, and so forth. In the near future we can expect extensive application prospect.
自2008年以来,笔者曾多次随课题组对位于吉林省长白山区的延边、白山2个地区8个县市区域的橐吾属植物开展野外调查、标本采集、试验研究等工作。调查发现,长白山区分布着8种橐吾属植物(复序橐吾、狭苞橐吾、蹄叶橐吾、三角叶橐吾、单花橐吾、全缘橐吾、合苞橐吾、白毛橐吾),其中复序橐吾主要生长于海拔400-1050m之间的林缘、林下、林间及沼泽草地,在当地分布广,资源蕴藏量大；狭苞橐吾主要分布在海拔680-1450m的亚高山地带,可以认为是岳桦林下优势种。
本研究以长白山区复序橐吾、狭苞橐吾的叶和花为试验材料,利用现代生物学实验方法及分析测试手段,对2种橐吾叶和花的营养成分、脂肪酸、挥发油等脂溶性成分进行了分析；探讨了复序橐吾多糖的提取、分离纯化过程及复序橐吾多糖的理化性质、单糖组成、分子量分布及结构特征；通过体外抗氧化、抗肿瘤、免疫活性试验对复序橐吾、狭苞橐吾叶的乙醇提取物、复序橐吾多糖的生物活性进行了研究,主要内容如下：
1.利用氨基酸自动分析仪对复序橐吾、狭苞橐吾叶和花的氨基酸含量进行分析。2种橐吾均含有16种蛋白质水解氨基酸,氨基酸总量为复序橐吾叶7.64%、花8.1%,狭苞橐吾叶12.60%、花9.90%;其中含有7种人体必需氨基酸,氨基酸的配比与FAO/WHO提出的蛋白质参考模式相比较,除蛋氨酸低于模式谱外,其它各种必需氨基酸均高于模式谱,配比合理。
2.采用电感耦合等离子体发射光谱法对复序橐吾、狭苞橐吾叶和花的Al、Cu、Fe、B、 Ba、Be、Ca、Co、Cr、K、Mg、Mn、Mo、Zn、Sr、Ni、Ti、P18种矿质元素含量进行了测定。2种橐吾叶和花除富含K、Ca、Mg等常量元素外,还含有Fe、Cu、Zn、Co、Mn、Cr、Mo、Sr、 B、Ni等多种人体必需微量元素。
3.采用GC-MS联用技术对复序橐吾、狭苞橐吾花的脂肪酸和挥发油成分进行分析。复序橐吾和狭苞橐吾花中含有的主要脂肪酸有：(Z,Z)-9,12-十八碳二烯酸(24.59%,6.29%)、软脂酸(19.12%,22.32%)、9-十八碳烯酸(7.05%,4.46%)、肉豆蔻酸(6.89%,8.30%)、(Z,Z,Z)-9,12,15-十八碳三烯酸(6.87%,2.50%)、硬脂酸(5.09%,6.77%),其中人体必需脂肪酸亚油酸、饱和脂肪酸软脂酸的含量较高。
从复序橐吾花挥发油的总离子流图中分离得到49个色谱峰,共鉴定出39个化合物,主要化学成分为4-蒈烯(26.53%)、D-柠檬烯(13.33%)；从狭苞橐吾花挥发油的总离子流图中分离得到75个色谱峰,鉴定出71种化合物,含量最高的成分为1,2,3,6-四甲基-二环[2.2.2]辛烷(46.87%)。2种橐吾花挥发油都含有丰富的萜类化合物,由于萜类化合具有广泛的生物活性,所以实验结果可以为探讨2种橐吾可能具有的抗菌、抗病毒、抗肿瘤等活性研究提供化学依据。
4.以石油醚和95%乙醇为溶剂,通过超声提取得到复序橐吾、狭苞橐吾叶的石油醚、乙醇提取液,按照GC-MS条件,将提取液直接进样,测定复序橐吾和狭苞橐吾叶的脂溶性成分。结果,从复序橐吾和狭苞橐吾叶的石油醚提取液中分别鉴定出59个和28个化合物,分别占脂溶性总成分的100%、96.33%；从乙醇提取液中分别鉴定出38个和35个化合物,占脂溶性总成分的84.50%、96.35%。鉴定的成分涵盖了萜烯类、烷烃、酮、酚、酸、酯类等化合物。
5.以复序橐吾叶为材料,在用乙醇浸提的基础上,分别以石油醚、氯仿、乙酸乙酯、正丁醇不同极性溶剂进行分级萃取,综合定性分析和总黄酮含量测定推测,复序橐吾乙醇总提物及各萃取部位均含有黄酮类成分,其中以乙酸乙酯部位总黄酮含量最高；通过清除DPPH有机自由基试验,对各萃取部位的抗氧活性进行测定,各萃取部位对DPPH清除能力的强弱顺序为乙酸乙酯(fr-Acetidin)>总提物(fr EtOHex)>正丁醇(fr-BuOH)>氯仿(fr-CHCl3)>石油醚(fr-PE),基本与总黄酮含量的多少一致；同时利用高效液相色谱法对乙醇总提物和各萃取物进行HPLC指纹图谱分析,分析这些片段中化合物的异同,为寻找复序橐吾抗氧化活性成分奠定实验基础。
6.首次以复序橐吾叶为材料,采用热水煮提、乙醇沉淀、常规干燥得到复序橐吾水溶性粗多糖,并对复序橐吾多糖进行了理化性质研究,结果显示,复序橐吾多糖为含有蛋白质但不含淀粉的酸性杂多糖；通过对粗多糖进行冻融分级、除蛋白、透析处理后,进一步通过DEAE纤维素离子交换层析和Sepharose CL-6B凝胶柱层析对多糖进行分级纯化,得到了6个多糖级分；采用HPLC、HPGPC、IR方法研究了各多糖级分的单糖组成、分子量分布和结构特征；利用MTT法对复序橐吾多糖的免疫学活性测定显示,复序橐吾粗多糖及LJPA3、LJPN、对小鼠脾脏T淋巴细胞的增殖有一定的促进作用,以LJPN1的作用较强。迄今为止对复序橐吾多糖的研究尚未见报道,因此本实验对探索复序橐吾多糖的构效关系和其潜在的药用价值有重要意义。
7.采用总还原力的测定、清除羟自由基、超氧阴离子自由基试验,对复序橐吾和狭苞橐吾叶的乙醇提取物体外抗氧化活性进行了评价,三种试验结果均显示复序橐吾叶的乙醇提取物比狭苞橐吾更具有良好的抗氧化活性；同时,利用MTT法测试了复序橐吾和狭苞橐吾叶的乙醇提取物对2种人肿瘤细胞(Hela、A549)的抑制活性,体外实验结果表明,复序橐吾和狭苞橐吾叶的乙醇提取物具有一定的抗肿瘤活性,狭苞橐吾乙醇提取物在一定浓度范围内,对Hela的生长抑制作用强于复序橐吾,但2种橐吾乙醇提取物对A549的抑制作用差异不大
以上研究表明,复序橐吾、狭苞橐吾是集营养、保健、药用功能于一体,极具开发利用价值的野生药食两用植物资源,其化学成分有待开发成为天然抗氧化剂、抗肿瘤及免疫调节剂等,未来应用前景广阔。
Ligularia Cass is a genus of perennial herb plants of Compositae Senecioneae. There are about150types of Ligularia Cass, and most of them distribute in Asia, only2kinds in Europe. China is rich in Ligularia and there are about110types and3variations. Most of them distribute in southwest and north east of China.
Since2008, a large number of investigations, collections and researches have been conducted in8counties of Yanbian, Baishan Regions Jinlin province. There are eight types of Ligularia Cass (Ligularia jaluensis、Ligularia intermedia、Ligularia fischeri、Ligularia deltoiclea、Ligularia jamesii、Ligularia mongolia、Ligularia schmidtii、Ligularia villosum) in Changbai Mountain, in which Ligularia jaluensis species has rich resources and wide distribution, growing at the altitute of400-1050meters in forest edge, woodland and wet meadow; Ligularia intermedia mainly distributes at the altitude of680-1450meters in subalpine zone.We can certainly confirm that Ligularia intermedia is a dominant species in Yuehualin area in China.
In this study, the leaves and flowers of Ligularia jaluensis and Ligularia intermedia were collected from Changbai Mountain as experimental materials. By means of modern biological experimental methods and analytical tests, the nutritional ingredients, fatty acid, and volatile oil were analized from2kinds of leaves and flowers of Ligularia; and the liposoluble composition were analyzed.The study also focuses on the process of extraction. separation, purification as well as its physicochenical property, monosaccaride composition, molecular weight distribution and formation feature in Ligularia jaluensipolysaccharide. By testing the effects of external antioxidant, antineoplstic, immunocompetence to study the bilological actvity of the ethanol extraction from both Ligularia jaluensis and Ligularia intermedia leaves and the Ligularia jaluensis polysaccharide, the main conclusion is as follows:
1. By means of automatic amino acid analyzer, the amino acid contents in leaves and flowers of Ligularia jaluensis were analyzed;16kinds of proteolysis amino acid were identified from these two type of Ligularia, in which the total amount of amino acid is7.64%in leaves,8.1%in flowers,12.60%in narrow bracts,9.90%in flowers and in them there are7kinds of amino acid identified which are essential to human. Compared the amino acid ratio with refernce mode regulated by FAO/WHO, all kinds of essential amino acid content are highter than the reference mode list except for methionine. The proportion is reasonable.
2.By means of inductive coupling plasma emission specroscopy, the content of18(Al、Cu、 Fe、B、Ba、Be、Ca、Co、Cr, K、Mg、Mn、Mo、Zn、Sr、Ni、Ti、P)kinds of mineral elements in the leaves and flowers were tested. The microelements essential to human such as Fe、Cu、Zn、Co、Mn、Cr、Mo、Sr、B、Ni were also identified besides the rich Macroelements such as K、Ca、Mg in the two kinds of leaves and flowers.
3. The ingredients of fatty acid and volatile oil from the flowers of Ligularia jaluensis and Ligularia intermedia were analized by GC-MS coupling technique. The main fatty acid contents in the flowers of Ligularia jaluensis Kom and Ligularia intermedia are (Z,Z)-9,12-octadecadienoic acid (24.59%, plamitice acid6.29%), palmitic acid (19.12%,22.32%),9-octadecenoic acid (7.05%,4.46%),myristic acid (6.89%,8.30%),(Z,Z,Z)-9,12,15-octadecane triolefinic acid (6.87%,2.50%)、stearic acid(5.09%,6.77%).The essential fatty acid, linoleic acid, staturated fatty acid, palmitit acid are higher in the contents.
After separating the volatile oil from Ligularia jaluensis flowers,49chromatographic peaks were obtained, in which39chemical compounds were identified and the chemical components are4-carene (26.53%)、D-limonene (13.33%);75chromatographic peaks were obtained from the total ion flow graph after separating the volatile oil from Ligularia intermedia flowers, in which71chemical compounds were identified and Bicyclo[2.2.2]octane,1,2,3,6-tetramethyl-(46.87%) are the highest contents. The volatile oil in both flowers contents is full of terpenoid. Since terpenoids possess comprehensive biological activity, there is no doubt that these two Ligularia sibiricas have antibiosis and antiviral functions.Therefore, the results of these experiments provide sound chemical basis for the activity study of antibiosis, antivirus and anticancer.
4. Mixed with petroleum ether and95%ethanol, under the condition of GC-MS. petroleum ether and and ethanol extracting solution from leaves by ultrasonic extraction treatment were injected directly to test the inposoluble constituent in leacves of Ligularia jaluensis and Ligularia intermedia. The result shows that59chemical compounds and28chemical compounds were identified from the petroleum ethier extracting solution of Ligularia jaluensis and Ligularia intermedia leaves respectively, taking up100%、96.33%of the total fatty soluble content;38chemical compounds and35chemical compounds were identified from the ethanol extracting solution of Ligularia jaluensis and Ligularia intermedia leaves respectively, taking up84.50%、96.35%of the total fatty soluble content. The conpounds content analysis of terpene, alkane, ketone, phenol, acid, esters was covered in the test.
5. Based on the materials of the leaves of L.jaluensis and ethanol, comprhensive qualitative analysis and the total flavonoid content test show that petroleum ethe、chloroform、acetic ether、 butyl alcohol and water also contain flavonoids compounds, in which highest level of total flavonoid content exsits in ethyl acetate part. By eliminating organic free radicals DPPH test, research focused on the study of the antioxidant activity in every part of the extract; the strong-to-weak sequence of DPPH scavenging capacity is ethyl acetate (fr-Acetidin)> total standardized extracts (fr-EtOHex>n-butyl alcohol (fr-BuOH>chloroform(fr-CHCl3)>petroleum ether (fr-PE), basically consistent with total flavonoid content. Meanwhile by means of HPLC chromatography, the finger-print spectrum of all ethanol standardized extracts and the single extract were analyzed to the determine the differences in the chemical compounds fragments, establishing a sound experimental fundition for searching antioxidant activity elements in Ligularia jaluensis.
6. For the first time Ligularia jaluensis leaves were used as testing materials. By boiling extracting, ethanol precipitation and conventional drying, water-soluble, crude polysaccharide were obtained; and then to study the physicochemical property of polysaccharide. The results show that the Ligularia jaluensis polysaccharide is a kind of acide heteropolysaccharide with starchfree protain. The crude polysaccharide was treated by freeze thawing grading, deproteinizing and dialyzing. By further DEAE-cellulose ion exchange chromatography and Sepharose CL-6B gel column chromatography to fractionate and purify the Ligularia jaluensis polysaccharide,6polysaccharide fractions were obtained. HPGPC、IR were applied in studying monosaccharide composition, molecular weight distribution and the structrural features. MTT was useed to determine the activity of Ligularia jaluensis polysaccaride. The result shows that crude polysaccaride as well as LJPA3.LJPN1can accelerate the multiplication the T-lymphocytic in the mice spleen. LJPN1is superior to others. There has been no any reports on the study of the Ligularia jaluensis polysaccharide so far. So this study has great significance in developing the potential medical values in Ligularia jaluensis.
7. Total reducing power determination, eliminating hydroxy free radical and Superoxide anion free radical test were adopted to assess the external antioxidant activiy of ethanol extract in both plants leaves. The results of above three tests indicate that Ligularia jaluensis leaves possess better antioxidant activiy than that of Ligularia intermedia leaves. Meamwhile, MTT was used to test the inhibitory activity of the ethanol extract from the leaves of Ligularia jaluensis and Ligularia intermedia against two kinds of human tumor cells (Hela、A549). The results of the experiments in vitro show that the ethanol extract from both leaves of Ligularia jaluensis and Ligularia intermedia proves to have antineoplastic activity. The ethanol extract from the leaves of Ligularia intermedia has better inbibitional effect on controlling the growth of Hela in certain concentration range. But there is no great difference in inbibitional effect on A549between the ethanol extract from both leaves.
The above study shows that Ligularia jaluensis and Ligularia intermedia have multi-function on nutrition, health care, and officinal and it is worth exploiting such potential edible and medical plants. The chemical compounds in the plants remain to be developed into natural antioxidant, antineoplastic as well as immunomodulator, and so forth. In the near future we can expect extensive application prospect.
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