| Licorice is one of the most frequently used medicinal materials in China,was originally listed in Shen Nong Ben Cao Jing,and have been widely applied for the effects of nourishing qi,eliminating phlegm,tonifying spleen,alleviating pain,relieving coughing,and honoured as the reconciler in traditional Chinese fomulas for thousands of years.Three original plants,Glycyrrhiza glabra L.,Glycyrrhiza uralensis Fisch.,and Glycyrrhiza inflata Bat.,are defined as licorice in the 2015 edition of the Chinese Pharmacopoeia.Studies have shown that there are significant differences in active ingredient content and pharmacological acticities among three origins of licorice.Traditional morphological identification has always been the main identification method for the three licorice original plants.However,the medicinal parts of licorice are roots and rhizomes,which are very similar in shape.It is very difficult to identify the origin of the licorice roots using morphological and microscopic identification method.Therefore,the establishment of molecular identification method of licorice will be useful for the efficient and accurate identification and reasonable use of licorice.In recent years,as a powerful supplement to traditional methods,molecular identification has been widely used in the identification of medicinal plants and animals.In 2011,4 candidate DNA fragments(rbcL,matK,psbA-trnH and ITS)of 6286 seed plant samples,collected from 75 families,141 genera and 1757 species were analyzed by China Plant Consortium for the Barcode Of Life Group.As a result,ITS sequences and ITS2 sequences were found a higher evolutionary rate than that of rbcL +matK,and the psbA-trnH sequence was proved to be the best chloroplast gene sequence.Therefore,ITS sequence and psbA-trnH sequence were selected to be the candidate DNA barcode to identify origin plants of licorice.As stipulated by Chinese Pharmacopoeia,18β-glycyrrhizin and liquiritin were marker compounds for evaluating the quality of licorice.With the technology increasingly developed,many active compounds have been separated frome licorice root,and proved to exert the anti-tumor,anti-virus,anti-inflammatory,hepatic and many other pharmacological activites.Among them.18a-glycyrrhizin,the epimer of 18β-glycyrrhizin,has been proved a better active compound for higher targeting,better lipotropy,and fewer adverse reactions.While so far,the method to distinguish these two compounds have not been established.Meanwhile,flavonoids,as another class of active compounds,whose contents were also of great importance.While,the content differences of four main flavonoids,including liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin among three origins of licorice have not been clarified so far.In this paper,we intended to distinguish and compare the three origins of licorice based on DNA sequence and chemical content differences.Firstly,large number of different origins of licorice with different morphological characteristics were used as plant materials for DNA identification for the amplification of ITS and psbA-trnH sequences.Afterwards,with the molecular identification method established,origin-clarified biennial licorice,including G.uralensis G.glabra,and G.inflata cultured under the same condition,were used as plant materials to explore the contents differences of two triterpenoids,glycyrrhizin and isoglycyrrhizin,and four flavonoids,liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin.The results of this paper are intended to facilitate licorice breeding research,assist clinical and pharmacological studies,and evaluate the potentials for molecular identification and certification of licorice roots and their products.The results of this paper are as follows:(1)We collected 238 licorice plants from 21 populations of 7 provinces,and amplified their ITS and psbA-trnH sequences.ITS sequences with a full length of 616 bp and psbA-trnH sequences with a full length of 389 bp were obtained separately.Using DNAMAN to analyze these sequences,4 variable sites were found in ITS sequences and 2 ITS haplotypes were determined,and 3 variable sites were found in psbA-trnH sequences and 4 psbA-trnH haplotypes were determined.With the combination analysis of ITS and psbA-trnH sequences,the molecular original identification method of licorice was established.(2)We established an HPLC method for simultaneously assaying the contents of glycyrrhizin and isoglycyrrhizin,which is a pair of epimer,and compared the different contents of these two triterpenoids in licorice stipulated in Chinese pharmacopoeia from the three origins.The results showed that under the selected chromatographic conditions,isoglycyrrhizin and glycyrrhizin could be separated.The linearity ranges were 0.010 70-0.214 0 μg for isoglycyrrhizin and 0.216 4-4.328 jig for glycyrrhizin.The LOD and LOQ for isoglycyrrhizin were 3.210 ng and 11.26 ng,and the LOD and LOQ for glycyrrhizin were 4.330 ng and 12.65 ng.The average recoveries(n=3)of isoglycyrrhizin and glycyrrhizin were 99.2%-100.1%and 99.8%-99.9%,respectively.It was demonstrated that the contents(n=25)of isoglycyrrhizin and glycyrrhizin in Glycyrrhiza glabra L.were the highest,(2.650±0.064 21)mg·g-1 and(37.18±0.844 3)mg-g-1 respectively;the contents(n=25)of isoglycyrrhizin and glycyrrhizin in Glycyrrhiza uralensis Fisch.were the second,(1.975±0.057 12)mg·g-1 and(29.41±0.741 2)mg·g-1 respectively;and the contents(n=25)of isoglycyrrhizin and glycyrrhizin in Glycyrrhiza inflata Bat.were the lowest,(1.604±0.043 72)mg·g-1 and(17.81±0.502 1)mg·g-1,respectively.Furthermore,the content of isoglycyrrhizin was strikingly correlated to the content of glycyrrhizin in all three species of licorice.Conclusion:The developed method is proved by methodology validation that it can be used for determination of isoglycyrrhizic acid and glycyrrhizin in licorice from the three origins,and it must be an attractive method for the quality control of licorice and will provide the instructions in directed breeding aiming at isoglycyrrhizin.(3)We established an HPLC method for simultaneously assaying the contents of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin.The results showed that under the selected chromatographic conditions,liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin could be separated.the calibration curves for liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin were Y=2×106X-2×10-1(r=1.000),Y=4×106X+3×10-1(r=1.000),Y=3×106X-1×10-1(r=0.998),and Y=5×106X+2×10-1(r=1.000)separately,and reflect a good linearity in the range of 1.15×10-2-0.230 μg,4.45×10-3-8.90×10-2μg,1.15×10-3-2.30×10-2 μg and 2.27×10-3-4.54×10-2 μg.The LOD and LOQ of liquiritin were 1.13 ng and 3.42 ng,of isoliquiritin were 0.896 ng and 2.70 ng,of liquiritigenin were 0.463 ng and 1.39 ng,and of isoliquiritigenin were 0.454 ng and 1.38 ng.The sensitivity,accuracy,precision,repeatability,durability and the average recoveries all met the requirements.It was demonstrated that the contents of liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin in three origins of licorice varies a lot(P<0.01).The contents of 4 compounds were highest in Glycyrrhiza uralensis Fisch.,shown as(20.75±0.524)mg·g-1,(4.453±0.057)mg·g-1,(0.610±0.019)mg·g-1 and(0.272±0.008)mg·g-1 separately,and intermediate in Glycyrrhiza glabra L.,shown as(6.623±0.405)mg·g-1,(1.562±0.053)mg·g-1,(0.325±0.036)mg·g-1 and(0.180±0.012)mg·g-3 separately,and lowest in Glycyrrhiza inflata Bat.,which were(2.700±0.232)mg·g-1,(0.821±0.042)mg·g-1,(0.153±0.006)mg·g-1 and(0.115±0.005)mg·g-1 separately.Furthermore,the content of liquiritin was strikingly correlated to the content of isoliquiritin,and the content of liquiritigenin was strikingly correlated to the content of isoliquiritigenin in all three origins of licorice(P<0.01).Conclusion:The developed method is proved by methodology validation that it is suitable enough for determining liquiritin,isoliquiritin,liquiritigenin and isoliquiritigenin in the three original plants of licorice,and it will be an attractive method for the quality control of licorice and provide the instructions in directed breeding aiming at flavonoids.(4)Using the molecular established in(1),40 samples of licorice slices collected from 4 main herbal material markets in China were identified successfully.Furthermore,using the HPLC method established in(2)and(3),the contents of 2 triterpenes,isoglycyrrhizin and glycyrrhizin,and 4 flavonoids,liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin in these licorice pieces were assayed by HPLC and the results were analyzed using SPSS 21.0,to evaluate the quality of licorice slices in the market. |
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