| Amomum villosum is the dry and mature fruit of Amomum villosum Lour..It is mainly used in damp and turbid obstruction in the middle,poor appetite,deficiency-cold in spleen and stomach,vomiting and diarrhea,pernicious vomiting,fetal irritability,and other diseases[1].Amomum villosum is mainly produced in Guangdong,Guangxi,and Yunnan.Yangchun area of Guangdong is noted as the genuine producing area of Amomum villosum.In recent years,the market source of Amomum villosum has become more and more confusing due to the unordered introduction.In addition,there is no reliable technique to distinguish Amomum villosum from different producing areas,so it is necessary to find a way to effectively distinguish Amomum villosum from different producing areas.Studying the geographical origin of Amomum villosum and resolving the identification difficulties among Amomum villosum from different sources help to protect the genuine nature of Amomum villosum and the interests of pharmaceutical farmers from the genuine producing area of Amomum villosum,which can also protect the high-quality medical resources and ensure medication safety in the clinic.Objective:42 samples of Amomum villosum were collected from 10 different producing areas in three provinces.To determine the stable isotope ratio,the content of inorganic elements,and volatile components of Amomum villosum,analyze the feasibility of different indicators to distinguish Amomum villosum from different producing area,screen out the characteristic indicators that can effectively distinguish Amomum villosum producing area,and establish the discrimination model of Amomum villosum producing area based on the characteristic indicators.Method:1.Determination of stable isotope ratio:each sample was pulverized into a homogeneous powder(200 mesh),cracked and released in high-temperature cracking furnace or oxidation furnace,transformed into gas through oxidation-reduction,and entered into stable isotope mass spectrometer(IR-MS)through carrier gas.The results were calculated by single-point correction method.2.Determination of inorganic element content:microwave digestion was used to pretreat the sample.To optimize the digestion conditions,the influence of solid-liquid ratio,mixed acid ratio,and digestion time on the determination of element content were studied.The content of inorganic elements in each sample was determined by inductively coupled plasma mass spectrometry(ICP-MS)with Rh as the internal standard and one blank solution as three parallel controls.3.Determination of volatile component content:the sample was extracted by anhydrous ethanol ultrasonic,and the continuous filtrate was taken.The Determination was performed on a gas chromatograph(Shimadzu,GC-2010plus).The temperatures were set as follows:230℃ for the sample inlet,250℃ for the flame ionization detector(FID).The split ratio was 10:1,and the sample volume was 1 μL.The program used to raise temperature was set as follows:75 to 100℃(4℃-min-1),keep for 5min,100 to 250℃(40℃·min-1),and the collection time is 15.25min.4.Linear discriminant analysis was carried out by SPSS26.0 to screen out indicators with significant difference,the irrelevant variables were eliminated,and a more accurate geographical origin discriminant model was established.Result:1.Three indicators(the ratio of δ13C,δ15N,and δD)were used to distinguish Amomum villosum from different producing areas.The discrimination rate between three provinces is 74.4%.The discrimination rate between four producing areas(Chongzuo city,Yangchun city,Wenshan state and Xishuangbanna state)is 86.2%.2.To achieve optimal digestion conditions,an orthogonal test was adopted:sample(0.2g)added with 7ml of concentrated nitric acid was chosen as the optimal digestion conditions.The digestion procedure was chosen set as follows:0-10min,150℃;10-30min,180℃.This method can not only save time but also make the sample react with the digestion solvent fully.After digestion,the sample solution is colorless without precipitation,and the acid concentration is about 5%,which meets the requirements of determination.Twenty-seven inorganic elements of Amomum villosum can be detected by ICP-MS,and the discrimination model of producing area can be established with these 27 elements,the discrimination rate between three provinces and four producing areas are both 100.0%.However,the discriminant rate of cross-validation is only 53.8%and 34.5%,respectively.3.Using only three volatile components as indicators cannot completely distinguish Amomum villosum:the correct rate of discrimination between three provinces and four producing areas are only 66.7%,58.6%,respectively.The result also shows that the content of volatile components of Amomum villosum from Guangxi and Guangdong is higher than that from Yunnan.4.All the indicators were combined to establish the geographical origin discrimination model,the discrimination rate between three provinces and four producing areas are both 100.0%.Differential indicators were screened out by stepwise discriminant analysis to establish the discriminant model of producing area.The result shows that the accuracy of the provincial model(established with indicators:δD,Cr,Zn,Cu,Ni)is 94.9%,and its cross-validation accuracy is 92.3%.The accuracy of the regional model(established with indicators:δD,δ13C,Cu,Sn Bornyl acetate)is 93.1%,and its cross-validation accuracy is 89.7%.Conclusion:1.The content of stable isotopes,volatile components and inorganic elements of Amomum villosum from different provinces are significantly different,which can be used to distinguishe Amomum villosum from different producing areas.The accuracy rate of model established with inorganic elements is higher than models established with stable isotopes or volatile components.2.Using stepwise discriminant analysis,five specific indicators were screened out from the 33 indicators tested.Discriminant model of Amomum villosum from three provinces was established based on these five indicators,and its discriminant rate is 94.9%,which is practical in practice.The accuracy of regional model from four sampling areas is low,but it has certain feasibility to discriminate Amomum villosum from its genuine producing area.3.Only Borneol acetate was screened out into the stepwise discriminant model,which indicated that the volatile components of Amomum villosum contributed little to the discriminant model,and the difference between the contents had little effect on discriminating Amomum villosum from different areas.However,the volatile components can still be used as one of the indicators to evaluate the quality of Amomum villosum.The result shows that the quality of Amomum villosum from Guangxi and Guangdong is better than those from Yunnan. |
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