| Atractylodis Rhizoma is derived from the dried rhizomes of Atractylodes lancea（Thunb.）DC.or Atractylodes chinensis（DC.）Koidz.,a commonly traditional Chinese herbal medicine,which can dry dampness tofortify the spleen,dispell wind and cold and improve eyesight.In recent years,as the market demand for Atractylodis Rhizoma is increasing year by year,wild resources of Atractylodis Rhizoma are increasingly depleted,and the supply of authentic Atractylodis Rhizoma is difficult to meet the market demand.Those have caused chaos in the market of Atractylodis Rhizoma for provenance,adulterants and inferior,and difficulty in guaranteeing the quality of medicinal materials,which has seriously affected the clinical practice of Atractylodis Rhizoma.Therefore,it is of great significance to accurately identify the source of the Atractylodis Rhizoma and to effectively evaluate the quality of the herbal medicine.Objective:The macroscopical identification and DNA barcoding technology were used for authenticating the original plants,seeds and medicinal parts of five medicinal plants of the genus Atractylodes（Atractylodes chinensis（Bunge）Koidz.,Atractylodes lancea（Thunb.）DC.,Atractylodes japonica Koidz.,Atractylodes koreana（Nakai）Kitam.and Atractylodes macrocephala Koidz.）,to determine the diagnostic characters of five medicinal plants form the genus Atractylodes;at the same time,the quality grading index and standard for the seeds of A.chinensis and the detection method for various active components of Atractylodis Rhizoma were established to ensure the accuracy and quality of the provenance from the root,providing a reference for the accurate identification of Atractylodis Rhizoma and its adulterants,and the quality evaluation of the medicinal materials,and to ensure the safety and effectiveness of the clinical medication of Atractylodis Rhizoma.Methods:1.The morphological characteristics of five medicinal plants in Atractylodes genus were observed for identification studies,including their roots,stems,leaves,flowers and fruits.2.The seeds of five medicinal plants of Atractylodes genus were identified by observing and measuring their morphological characteristics,such as shape,size,color and surface ornamentation.3.A total of 39 samples of Atractylodes seeds were collected,including12 seed samples from original plants and 27 commercially available Atractylodes seed samples,and their ITS2 and psbA-trnH sequences were obtained after DNA extraction,polymerase chain reaction（PCR）and bi-directional sequencing.A total of 38 ITS/ITS2 sequences of Atractylodes genus were downloaded from Gen Bank database.The identification of Atractylodes seeds was carried out by constructing neighbor-joining（NJ）phylogenetic tree,to verify the feasibility of DNA barcoding technology for identification of Atractylodes seeds.4.The clarity,germination rate,water content,viability,1000-grain weight and other indexes were measured according to"Rules for agricultural seed testing",and SPSS 22.0 software were used for correlation,principal component and clustering analysis to establish quality grading index and standard for the seeds,combining with actual production condition of A.chinensis seeds.5.The morphological characteristics of crude drugs derived from five medicinal plants of Atractylodes genus,including shape,size,color,surface ornamentation,section traits,texture,smell and other indicators,were observed and compared using eye observation,hand touch,nose smell,mouth taste,and other methods for identification studies.6.A total of 53 medicinal materials of Atractylodes genus were collected through base collection and field excavation,and 38 ITS/ITS2 sequences of Atractylodes species were download from the Genbank database to construct the reference DNA barcode database for Atractylodes.Moreover,54 samples of the commercially available medicinal materials of Atractylodes genus were collected from herbal markets,and the species identification of these commercially available samples was achieved by constructing the neighbor-joining（NJ）phylogenetic tree.7.High performance liquid chromatography was used to establish a detection method for the content of（4E,6E,12E）-tetradecatriene-8,10-diyne-1,3-diacetate（TDDA）,atractylodin and atractylon in the Atractylodis Rhizoma,and the methodological investigation was carried out.At the same time,the contents of bioactive components of the Atractylodis Rhizoma from different origins were compared.Results:1.The morphological characteristics of original plants of five Atractylodes medicinal plants are similar,except that there are difference in the feature of rhizomes,leaf shape and degree of division,and the color of corolla,and the other four are white.Among them,the rhizomes of A.macrocephala are irregular masses,middle cauline leaves petiolate,leaf blade divided to base into 3～5 segments,lateral segments entire,corolla purplish red;the leaf shape of A.japonica is similar to A.macrocephala,corolla white,and the rhizomes are mostly nodular;the leaves of A.koreana are undivided,the base of lower and middle cauline leaves is rounded and semiamplexicaul;the leaves of A.chinensis have relatively large split changes,the upper leaves are not split,pinnately divided or lobed,or irregularly divided;and there are only a few leaves at the base of 3～5 lobed for A.lancea.In addition,29 different types of leaf and 6 typical original plants were found in different populations of A.chinensis;2.The appearance and morphological characteristics of seeds of five Atractylodes medicinal plants are similar,all of which are achenes,obovate,densely covered with white velutinous on the surface,with collar and crested hairs on the top,and the base of seeds were ringed.There are differences in color,pappus length,size and 1000-grain weight.The seeds of A.chinensis,A.lancea,A.japonica and A.koreana are mostly brown,while the seeds of A.macrocephala are usually yellowish-brown.Pappus length of achenes of A.macrocephala are longer,the other 4 achenes are shorter.The size and1000-grain weight of seeds with the order from large to small are A.macrocephala>A.koreana>A.chinensis>A.japonica>A.lancea.3.Since the psbA-trnH sequence lack sufficient identification efficiency in the Atractylodes species,the ITS2 sequence was used for the subsequent analysis.A total of 12 ITS2 sequences were obtained from the identified seed samples of Atractylodes genus.The NJ phylogenetic tree was constructed based on ITS2 sequences obtained from identified seed samples and Gen Bank database,to effectively distinguish A.lancea,A.japonica,and A.macrocephala seeds except for A.chinensis and A.koreana.The commercially available seeds samples of Atractylodes were identified based on ITS2 sequences,and the results showed that among the 14 commercially available seeds of A.chinensis,9 of the original species were A.macrocephala,2 of the original species were A.japonica,and 3 of the original species were A.chinensis or A.koreana;10 seeds of A.lancea with original species were all A.macrocephala;3 seeds of A.macrocephala were authentic.4.After the correlation,principal component,systematic clustering and K-means clustering analysis clustering analysis on the quality indexs of A.chinensis seeds,combined with actual production situation,the quality grading index and standard for the seeds was established:GradeⅠ（excellent）,the clarity of A.chinensis seeds is no less than 95%,the germination rate of A.chinensis seeds is no less than 88%,the 1000-grain weight of A.chinensis seeds is no less than 10 g,and the water content of A.chinensis seeds is not higher than 10%;GradeⅡ（good）,the clarity of A.chinensis seeds is 88%～95%,the germination rate of A.chinensis seeds is 60%～88%,and the1000-grain weight of A.chinensis seeds is no less than 10 g,the water content of A.chinensis seeds is not higher than 10%;GradeⅢ（unqualified）,the clarity of A.chinensis seeds is not higher than 88%,the germination rate of A.chinensis seeds is not higher than 60%,and the1000-grain weight of A.chinensis seeds is lower than 10 g,the water content of A.chinensis seeds is higher than 10%.5.The main differences of Atractylodes genus medicinal materials are as follows:the texture of dried rhizome of A.macrocephala is hard,and its smell and color are obviously different from the other four medicinal materials of Atractylodes genus;the dried rhizome of A.chinensis is mostly lump-shaped,with gray-black surface,yellowish cross-section with"cinnabar spots";the surface of dried rhizome of A.lancea is soil-grey,yellowish white cross-sections with"cinnabar spots",and white flocculentcry stals are precipitated after long-term storage;the surface of dried rhizome of A.japonica is black,light in weight,fibrous,and the section is whitish;the surface of dried rhizome of A.koreana is black,with many gaps in the section,light in weight,and it is a bead-shaped cylinder with uniform thickness.6.A total of 53 ITS2 sequences were obtained from the authenticated crude drugs of Atractylodes genus,and NJ phylogenetic tree was constructed based on ITS2 sequences obtained from authenticated crude drugs and Gen Bank database.The phylogenetic tree showed that A.lancea,A.japonica,and A.macrocephala can independently formed a branch separately,except for A.chinensis and A.koreana.However,the obtained psbA-trnH sequence lacks sufficient mutation sites and cannot meet species identification requirements,and the follow-up analysis adopts the ITS2 sequence.The identification results of the commercially available Atractylodes medicinal materials showed that 47 commercially available Atractylodis Rhizoma（including the commercially available medicinal materials of A.chinensis,A.lancea,and Atractylodis Rhizoma）,the original species of 38 medicinal materials was A.chinensis or A.koreana,4 medicinal materials were A.lancea,and 5 medicinal materials were A.japonica;among the 3commercially available medicinal materials of A.japonica,one of the original species was A.chinensis or A.koreana;among the 4 medicinal materials of A.macrocephala obtained from the market,one of the original species was A.chinensis or A.koreana.7.A method based on HPLC was established for the simultaneous determination the contents of TDDA,atractylodin and atractylon in the crude drugs purchased from herbal markets and drug stores.The Discovery（?）C18column（4.6 mm×250 mm,5μm）was used,and the chromatographic conditions are as follows:the mobile phase consisted of 0.2%phosphoric acid water（A）and acetonitrile（B）with gradient elution,and the detection wavelength is at 220 nm（0～65 min,atractylon）,340 nm（0～65 min,TDDA and atractylodin）,the flow rate is 1.0 m L·min-1,the column temperature is 25℃,and the injection volume is 8μL.The linear range of TDDA is0.0065～0.262μg（R2=0.9999）,the average recovery rate is 100.0%,and the RSD is 3.1%（n=9）;the linear range of atractylodin is 0.014～0.56μg（R2=0.9999）,the average recovery rate is 100.04%,and the RSD is 2.8%（n=9）;the linear range of atractylon is 0.023～0.46μg（R2=0.9975）,the average recovery rate is 98.3%,and the RSD is 4.1%（n=9）.Finally,the crude drugs produced in Chengde,Hebei have a higher content of TDDA and atractylodin,while the crude drugs produced in northeast China have a higher content of atractylon.Conclusion:1.The original plant identification characteristics of five medicinal plants of the genus Atractylodes are as follows:flower color is the first identification basis,A.macrocephala flower color is purple-red,and the other four are white;whether the leaves split is the second basis,and the leaves of A.koreana do not split,the leaves of other three kinds of original plants are split;whether the leaves with petioles is the third basis,A.japonica has white flowers and the lower leaves are three or five-lobed,with petioles,and the other two have no petioles;the degree of leaf division,A.lancea only a few leaves at the base shallow lobed,the leaves of A.chinensis split and change greatly.2.The color of the achenes of five medicinal plants of the genus Atractylodes was as the identification indicator,it can clearly distinguish the seeds of A.macrocephala from the seeds of A.chinensis,A.lancea,A.japonica,and A.koreana;the NJ phylogenetic tree constructed based on ITS2 sequences showed that A.lancea,A.japonica,and A.macrocephala can be clearly distinguished,except for A.chinensis and A.koreana;the identification results showed that there are fake products in the market of Atractylodes seeds;3.The seeds quality of A.chinensis from different regions is uneven;the clarity and germination rate of the seeds were used as the main indicators for grading the quality of A.chinensis seeds,and the 1000-grain weight and water content were used as reference indicators.The seeds quality of A.chinensis was divided into three grade,providing a reference basis for the quality control of A.chinensis seeds;4.The identification characteristics of the medicinal parts of the genus Atractylodes:the hard texture is A.macrocephala;the dried rhizomes of A.lancea and A.chinensis have"cinnabar spots"on the section,and white crystals are often precipitated on the section of A.lancea;A.chinensis,A.koreana,A.lancea,A.japonica,and A.macrocephala can be clearly distinguished combining with DNA barcode technology based on ITS2;the identification results showed that there are fakes of the crude drugs in the market;5.A method established based on HPLC to simultaneously determine the content of TDDA,atractylodin and atractylon in the Atractylodis Rhizoma,which is simple,fast,and highly accurate,and can be used for the quality evaluation of Atractylodis Rhizoma.The test results showed that there were significant differences in the chemical composition of Atractylodis Rhizoma from different sources,and the crude drugs of Atractylodes from Chengde area of Hebei contained higher TDDA and atractyloidin,while the Atractylodis Rhizoma from the northeastern area was dominated by atractylon. |
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