Genetic Diversity and Quality Assessment of Glycyrrhiza Uralensis and Antioxidant Constituents of Viscum Coloratum
|Keywords||Glycyrrhiza uralensis Fisch. population ISSR genetic diversity quality assessment germplasm Viscum coloratum (Komar) Nakai flavanone glycosides antioxidative activity|
This thesis is composed of two parts: （1） Intraspecific genetic variations and quality assessment of Glycyrrhiza uralensis Fisch. from North China; and （2） Antioxidative components from branches and leaves of Viscum coloratum （Komar） Nakai （Loranthaceae）.In the first part, we carried out investigations on intraspecific genetic variations and their correlations to the drug quality of G uralensis from North China. This species belongs to the genus Glycyrrhiza of the family Leguminosae. It is a perennial herb and mainly distributed in the northern China. Its root, named licorice, has been used as a traditional drug for at least 2000 years in China. In addition, it contains glycyrrhizin, a non-sugar sweetener, and thus is widely used in the manufacture of food, tobacco, cosmetic, etc. For these reasons, the market demands for licorice production at home and abroad were expanded enormously in recent years. Thus, overcollection has resulted in a rapid decline of the wild resources of G uralensis and caused a severe ecological disaster in North China. Therefore, our research in this thesis is of great scientific and applied values.At the first step of the investigation, we carried out a wide range of field survey in 8 provinces of the north and north-east of China, i.e., Inner Mongolia, Ningxia, Gansu, Shanxi, Shaanxi, Liaoning, Jilin and Heilongjiang. More than 300 individual samples of G uralensis were collected from 30 different producing areas. Each individual sample separated to （included） tender leaves and root, which were used to study genetic diversity and quality evaluation respectively. Field survey revealed that wild population of G uralensis are fragmentized as the result of overcollection, and it is almost impossible to obtain main root of G uralensis in producing areas mentioned above. Ecological environment, resource reserves and quality of G uralensis were under severe situation that should be paid great attention.Genetic diversity levels were assessed based on 232 individuals of 12 natural populations from Inner Mongolia, Ningxia, Gansu and Shaanxi provinces using ISSR molecular markers. Fourteen selected primers produced a total of 287 discernible bands, of which 232 （98.61%） were polymorphism. The percentage of polymorphic bands at the population level was 57.2% on average, varied from 32.06% to 80.49%. The Nei’s gene diversity （h） and Shannon’s information index （I） were 0.304 and 0.463 respectively at the species level, and 0.181 and 0.276 respectively at thepopulation level. At the regional level, both the h and the / value, 0.285 and 0.436, of populations from Inner Mongolia were the highest among four regions, while these values, 0.213 and 0.327 respectively, of populations from Gansu province showed the lowest. The coefficient of gene differentiation （Gst） and gene flow （Nm） were 0.166 and 1.236 respectively at the population level. At the regional level, the Gst of Shaanxi populations showed the highest by 0.518 but Nm was the lowest by 0.464 among the four regions. AMOVA analysis indicated that genetic variation of G uralensis is much higher among populations （60.39%） than within populations （39.61%）. A cluster analysis （UPGMA） was used to generate dendrogram based on Nei’s genetic distances among 12 populations. The results showed that four populations from Inner Mongolia （SH, EJ, BL and BY） were phylogenetically diversified from population DK from the same province, and were clustered together with three populations （ME, GS and DM） from Ningxia province. Two populations from Gansu province （HC and LC） differentiate genetically from each other. Similarly, two populations from Shaanxi province （DB and WQ） were also diversified genetically from each other.Intraspecific genetic variations and phylogenetic relationships of 139 G uralensis samples from 30 different places of eight provinces were also revealed using ISSR molecular markers. An UPGMA dendrogram based on Nei’s genetic distance indicated that 5 individuals from population DK of Inner Mongolia formed 4 branches which had 60% supporting index and were diversified far from other populations. This result indicated that population DK was genetically unique and special. It is the only population distributed at the west bank of Yellow River in our present study. Thus, the genetic differentiation of this population may be contributed to the geographical isolation and some special ecological environment. Notably, licorice produced from this place was termed as "Wangye licorice". Other populations were clustered into two phylogenetically parallel groups which had 46% supporting index: group B （population BY, SH, EJ and BL from Inner Mongolia, population GS, ME and DM from Ningxia province, and population DB from Shaanxi province） and group A （other populations of the study）. Interestingly, populations in group B were all distributed in an area around Maowusu Desert and Kubuqi Desert in Hangjinqi of Inner Mongolia. Traditionally, licorice produced in this area was called "Liangwai licorice" and famous for its high quality. Whereas, populations in group A were distributed in a wide range of areas from the east part of Inner Mongolia to Liaoning,Jilin and Heilongjiang provinces. These populations were clustered together probably due to they share some common environment conditions, i.e., mountainous region, flourish vegetation and higher humidity climate. The licorice produced from this area was traditionally called "Eastern licorice". The research results mentioned above demonstrated that the populations of G uralensis from north and northeast of China were genetically diversified to form three distinct groups each with a clear geographical distribution area, and the genetic variations may be resulted from the adaptive co-evolution of different populations to different ecological environments. Furthermore, our findings revealed for the first time that the traditional concepts and classification such as "Liangwai licorice", "Eastern licorice" and "Wangye licorice" were not only representing a commercial type from a special producing area, but also representing a unique genotype with a distinct geographic boundary. This is the first report on the intraspecific genetic variations of G. uralensis, and the results provided solid scientific evidence for the classification of commercial productions of Radix Glycyrrhiza from China.Contents of glycyrrhizic acid, liquiritin and isoliquiritin in 116 samples from 30 different places of eight provinces were determined by high performance liquid chromatograph （HPLC） method. The results showed that, referring to 2005 Chinese Pharmacopoeia, only 15.5% of the samples reached the standard of glycyrrhizic acid （≥2%）, 11.2% of the samples reached the standard of liquiritin （≥1%）, and only 9.5% of the samples conformed to the standards of both glycyrrhizic acid and liquiritin. Our findings demonstrated how a severe situation is existed in the resource quality of wild licorice in China!Based on the results of genetic diversity and quality assessment, it could be concluded that samples with content of liquiritin larger than 1% are all distributed in group A, and those with content of glycyrrhizic acid larger than 2% are also distributed in group A. The average contents of glycyrrhizic acid and liquiritin in samples group A are 1.53% and 0.61%, respectively. While in those from group B, the average contents of glycyrrhizic acid and liquiritin are only 1.13% and 0.24%. These results indicated that the quality differentiations of licorice were probably determined by their diversifications of genetic backgrounds.In the second part, we discussed the isolation, structure elucidation and their antioxidative activity of compounds from branches and leaves of Viscum coloratum（Komar） Nakai （Loranthaceae）. It is a semi-parasitic plant distributed in southwest provinces of China. The branches and leaves of this plant are traditional Chinese medicines practiced mainly for the treatment of hypertension, atherosclerosis, rheumatism, and neuralgia. It was reported that flavonoids are the major secondary metabolites of this species and the extract from V. coloratum exhibited significant antioxidant and anti-aged effect. But the antioxidative compounds are still unknown.Phytochemical research on 40% ethanol proportion of V. coloratum extracts led to the isolation of three new compounds, （2S）-homoeriodictyol 7,4’-di-O-β-D-glucopyranoside （IV）, （2R）-eriodictyol 7,4’-di-O-β-D-glucopyranoside （V） and 1,7-di-（p-hydroxyphenyl）-5-hydroxyl-cis-2,3-epoxy-1-one （VI）, along with three known ones, homoeriodictyol 7-O-β-D-glucopyranoside （I）, eriodictyol 7-O-β-D-glucopyranoside （II） and naringenin 7-O-β-D-glucopyranoside （III）. Their structures were elucidated by UV, IR, NMR, MS and CD methods. Apart from compound I, five other compounds were first reported from this plant.The colorimetric methods and Electron Paramagnetic Resonance （EPR） method were applied subsequently for further investigation on the radical scavenging activities of these compounds. The results of colorimetric assays showed that these compounds effectively scavenged hydroxyl radical and superoxide anion radical. In the hydroxyl radical scavenging assay, compound IV exhibited strongest radical scavenging effect in all tested compounds with IC50 values of 0.17mM. In the superoxide anion radical scavenging assay, compound III showed lowest radical scavenging effect in all tested compounds with IC50 values of 0.49mM. The hydroxyl radical and superoxide anion radical scavenging activities of these compounds are stronger than those of the control, epigallocatechin gallate （EGCG）, whose IC50 value are 0.58 mM and 0.53 mM, respectively. Meanwhile, the results of EPR tests also revealed these compounds had antioxidant activities in different degrees. In the hydroxyl radical scavenging assay of EPR, compound IV showed strongest radical scavenging effect in all tested compounds with IC50 values of 0.23mM. While in the superoxide anion radical scavenging assay of EPR, compound VI exhibited lowest radical scavenging effect with IC50 values of 0.27mM. The IC50 values of EGCG were 0.54 mM and 0.12 mM, respectively in hydroxyl radical and superoxide anion radical scavenging assay of EPR. The results revealed that the differences of the antioxidant activities among these compounds were probably related to water-solubility, the hydroxyl and methoxyl number, and the position of these substitutes on benzoic rings.The finding of 3 novel compounds and the results of our present research are of great value for the elucidation of bioactive substances and quality evaluation of the crude drug, Herba Visci.The important findings and creative points of my thesis were summarized as follows:1. Genetic diversity patterns of wild Glycyrrhiza uralensis populations from North China were systematically studied using ISSR molecular markers for the first time. The results revealed that wild populations of this species remain relatively higher levels of genetic diversity. Our findings suggest that overcollection of the wild licorice has not resulted in a severe decrease of the genetic diversity of this plant, therefore, the quality and output of wild resources of G uralensis could be recovered after 4 to 5 years if effective protection measures were taken. Our research results provide solid evidence for the protections of wild resource of G uralensis, and ecological environment as well.2. Intraspecific genetic variations and phylogenetic relationships of G uralensis from 8 provinces of the north and northeast of China were also studied using ISSR for the first time. The results demonstrated that three distinct groups could be divided genetically among populations of G uralensis from this area. Each group has a distinct geographical distribution area and occupies a special environmental condition, indicating the intraspecific genetic differentiations of G uralensis were probably the results of environmental adaptive co-evolution. Another important finding was the three groups revealed by ISSR were exactly consistent with the traditional commercial classifications of licorice in China, i.e., "East licorice", "Liangwai licorice" and "Wangye licorice". The results presented here are significant to the quality control and germplasm selection of this medicinal plant.3. Based on a wide range of field survey and content assessments of liquirintin, isoliquiritin and glycyrrhizic acid in 116 samples of Radix Glycyrrhiza from 8 provinces of north and northeast of China, we found astonishingly that nearly 90% of licorice collected from the traditional producing areas of Chinese licorice were not up to the standards determined by 2005 Chinese Pharmacopoeia! It is the latest report on the severe situations of the qualities of wild licorice resource in China.4. Three novel compounds: （2S）-homoeriodictyol 7,4’-di-O-β-D- glucopyranoside （IV）, （2R）-eriodictyol 7,4’-di-O-β-D-glucopyranoside （V） and l,7-di-（p-hydroxyphenyl）-5-hydroxyl-cis-2,3- epoxy-1-one （VI）, were isolated from branches and leaves of Viscum coloratum, along with three known ones: homoeriodictyol 7-O-β-D-glucopyranoside （I）, eriodictyol 7-O-β-D-glucopyranoside （II） and naringenin 7-O-β-D-glucopyranoside （III）. The structures of these compounds were elucidated by UV, IR, MS, 1HNMR, 13CNMR and CD, and their antioxidative activity were evaluated by colorimetric method and electron paramagnetic resonance （EPR）methods, respectively. The results showed these compounds exhibited effective scavenging activity on both hydroxyl radical and superoxide anion radical. The finding of 3 novel compounds and the results of our present research are of great value for the elucidation of bioactive substances and quality evaluation of the crude drug, Herba Visci.