Morphology,ITS2 Molecular And HPLC Chemical Fingerprint Analysis And Identification Of Cymbopogon Distans From Its Adulterants

In recent years,interests and applications of herbal medicinal products have been noticeably augmented throughout the world due to their large potential for providing curative and/ or healing a number of complicated ailments and maintaining of health,and high value aromatic oils for industries as well.However,adulteration and use of spurious products as substitutes have become a key concern for users and industries for reasons of safety and efficacy.Therefore,accurate and reliable species identification is a prerequisite for ensuring the efficacy and safety of medicinal plants.Cymbopogon,commonly known as lemongrass,in English,is one of the most important medicinal plants and has been recognized for producing high value aromatic oils for industries which find large use in perfumery,cosmetics,and in the pharmaceutical industries.However,morphological resemblance among C.distans species and their adulterants which are procured from different markets in the form of dried or fresh plant tissues represents a serious problem for quality and safety of medicinal plants,as it supports frauds for substitution.In order to assure the quality control of C.distans species;DNA barcode,microscopic identification and HPLC fingerprint were used to discriminate C.distans and their related species.The experimental studies came to the following results:(1)Morphological characteristics of all the collected species were done by naked eyes and different parameters were evaluated.The result demonstrates that the length and the width of C.distans ranged from 35 to 50 cm/1.7-4 mm;and the leaf size of C.citratus and C.flexuous ranged from 62-70 cm /2-5 mm and 82-120 cm/0.5-1.9 cm,respectively.For leaf shape,species C.distans and C.citratus possess narrow linear and C.flexuosus displayed linear and lanceolate,based on the measurement given in Flora of China.Furthermore,the leaf shape,size and the colors could also identify the species of C.distans from its adulterants.This finding indicates that the upper epidermis in leaf of C.citratus was composed of one layer of wide elongated cells called Bulliform cells whereas in C.distans the upper epidermis consist of one layer cell,thus these feactures were very important for the anatomy identification.(2)The ITS2 sequences of 32 samples from C.distans,C.flexuosus and C.citratus were amplified and sequenced successfully,with 100% of genera being accurately distinguished;the result indicates thatthe maximum intra-specific distance of 0.000 among C.distans,which was lower than the minimum inter-specific distance between C.distans and its adulterants(0.002-0.016).In addition,no mutation were found among C.distans and C.flexuosus ITS2 sequences,whereas two mutations A-C and A-C were identified in C.citratus ITS2 sequences at the position 208 and 160,respectively.Various samples of C.distans were gathered together and could be differentiated from its adulterants by Phylogenetic tree of the family Poaceae constructed with the ITS2 sequences by using the NJ method tree.It was concluded that ITS2 could be used as DNA barcode to accurately authenticate C.distans and detect its adulterants,and can provide not only useful information for quality assessment,but also resource protection and sustainable utilization of C.distans material in markets.(3)High performance liquid chromatography(HPLC)method was developedand we found that 30 peaks of all the 6 batches of C.distans were assigned as “common peaks”,27 peaks were common among the 7 batches of C.citratus and 21 common peaks were found among 6 batches of C.flexuosus.The similarities were all more than 0.93 except 2 species of C.citratus and one species of C.distans with the value < 093.Additionally,the chromatogram fingerprint of batches of C.distans,C.flexousus and C.citratus showed that the content of C.distans was quite different from these two species and 14 common peaks were found between them and the similarities were all less than 0.93,suggesting that there was significance difference between C.distans and its adulterants.Based on the above results,we finally conclude that the DNA barcode,HPLC fingerprint and microscopic methods could effectively authenticate the quality of C.distans from their adulterants and can provide accurate and reliable information to tackle the complex quality issue of C.distans in markets.