Study On Quality Assessment Of Three Kinds Of Gentianae Macorphyllae Radix In Songpan County,Sichuan Province

Objective:To assess the quality of three kinds of Gentianae Macorphyllae Radix in SongpanCounty, Sichuan Province and to establish the relevant qualitative and quantitativemethods as the reference for further study in this region.Methods:1.To identify three kinds of Gentianae Macorphyllae Radix based on plant morphology,herbal and macroscopic characteristics, B-Z oscillation fingerprint and DNA barcoding.2.To determine the contents of Loganic acid and Gentiopicrin in three kinds ofGentianae Macorphyllae Radix in Songpan County, Sichuan Province by Ultraperformance liquid chromatography (UPLC) and to study the effects of altitude on thecontents. To determine the heavy metal Hydrargyrum and Organochlorine pesticides ofvarious Gentianae Macorphyllae Radix herbs in various regions by Atomic AbsorptionSpectroscopy and Gas Chromatography for quantitative study.Results:1.The three differs in their plant morphology, herbal and macroscopic characteristics,etc.. Gentiana macrophylla Pall.’s corolla is the eaves blue or purple blue.The partbelow the middle of Gentiana macrophylla Pall.’s root is more like “chicken leg” or“turnip” and sclerenchymatous reticulated cell and fiber can not be found under themicroscope. G.crassicaulis Duthie.’s corolla is the eaves blue or deep purple and thepart below the middle of its root most unbranched, less twisted around each other,whilethe top of G.crassicaulis Duthie.’s root has a relatively big caudex with a big slot in thecenter and spiral. Under the microscope sclerenchymatous reticulated cell and fiber cannot be found and reticulate vessels’ wall and Spiral vessels’wall were thicked.G.straminea Maxim’s corolla is chartreuse and the part below the middle of its root is separates from each other and then twist like “braids”.The sclerenchymatous reticulatedcells and fibers can be seen under the microscope. Established the B-Z oscillationfingerprint and DNA splicing barcoding methods to identity Gentianae MacorphyllaeRadix in the first time.And the B-Z oscillating fingerprints of the three distinguish fromeach other. G.crassicaulis Duthie has the longest induction period. Gentianamacrophylla Pall comes next and then is G.straminea Maxim.The oscillating timefollows from the long to short: G.straminea Maxim> Gentiana macrophylla Pall.>G.crassicaulis Duthie. The success rates of34samples for DNA extraction, PCR anddouble chains measurement are ITS(72%), psbA-trnH(84%), rbcL(92%).The sequencelength of ITS, psbA-trnH, rbcL of Gentiana macrophylla Pall., G.crassicaulis Duthie.,and G.straminea Maxim are624~628bp,315~398bp,702~703bp. The DNA and NJphylogenetic tree combined from the psbA-trnH and rbcL sequence of chloroplast andpsbA-trnH and rbcL sequence can directly reflect the genetic relationship amongQinjiao botanical origin plants. The genetic distance between the plant species ofpsbA-trnH+rbcL(0.004～0.025) is longer than the one among the species(0～0.001,0,0), while the same is true for ITS+psbA-trnH+rbcL,(0.006～0.02) and (0～0.001,0.002,0). The above two methods can be used to identify the molecule of GentianaeMacorphyllae Radix.2.The quality assessment system based on the quantity of Loganic acid and Gentiopicrinis established. The contents of Loganic acid and Gentiopicrin were simultaneouslydetermined by UPLC. Chromatographic conditions: BEH C18(2.1mm×50mm，1.7μm)column,the mobile phase consisted of acetonitrile and0.1%acetic acid with flow of0.35ml/min,the wave length was set at254nm, eventually, a better peak shape has beengot, the baseline is stable, theoretical plate number, separation and tailing facter arefound to be. The calibration curves showed that the concentration had good linearrelation with the peak area(r＞0.9998) for Loganic acid within the range of9.9to157.8μg/ml, and for gentiopicrin within the range of15.8to252.5μg/ml. The averagerecovery rates for Loganic acid and Gentiopicrin were101.23%(RSD=1.69%) and 99.78%(RSD=1.94%), respectively. The lead content in Gentianae Macorphyllae Radixand the earth conforms to the rule, so is copper and arsenic. Cadmium and mercury infew herb samples and earth have been in the excess. The contents test ofOrganochlorine pesticides in Gentianae Macorphyllae Radix herbs have been with noresult within the conditions.Conclusions:1.Plant morphology, herbal and macroscopic characteristics, B-Z oscillation fingerprint,DNA barcoding can be used for qualitative identification of Gentianae MacorphyllaeRadix among which B-Z oscillation fingerprint can repeat very well under strictexperimental conditions and operation. Various Gentianae Macorphyllae Radix differ intheir B-Z oscillation fingerprints for their different chemical elements and contents.This method, compared with the traditional one, requires less herbs, preparation, costand experimental steps. Moreover, it is more direct and sensitive, which is moresuitablefor the identification and assessment of herbs with complex contents. The root classmedicine’s DNA’s difficulty to be extracted might result from the difficulty of splittingand the distraction of the contents like polysac-charide, polyphenols and somesecondary metabolites. The improved method can extract DNA more efficiently. TheDNA and NJ phylogenetic tree combined from the psbA-trnH and rbcL sequence ofchloroplast and psbA-trnH and rbcL sequence can directly reflect the geneticrelationship among Qinjiao botanical origin plants. To calculate the genetic distance canquantify these characteristics. Therefore, based on further development of technics andmethods,“Portable scanner TCM identify-cation analysis” is possible to be designedand produced with DNA barcoding method. Those without advanced professionalknowledge and abilities might accomplish the identification task with the problem ofnot identifying the species by the herbs.2.To assess the quality of three kinds of Gentianae Macorphyllae Radix in SongpanCounty quantitatively: The contents of Loganic acid and Gentiopicrin in this region by Ultra performance liquid chromatography conform to the quality standards in ThePharmacopeia of The People’s Republic of China, furthermore, their contents are farabove those specified by Chinese Pharmacopeia. The contents of Gentiana macrophyllaPall and G.crassicaulis Duthie. are closely correlated with the altitude. UPLC can beused for the quality control of Gentianae Macorphyllae Radix because of its quickness,accuracy, sensitivity, and good repeatability. Gentianae Macorphyllae Radix in thisregion has good quality and it is quite safe on the whole, however, the ever-increasingproblem of heavy metals in excess of standards has been unavoidable. It is urgentlyrequired to protect the ecology and environment there for further development.