| 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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