| The lotus rhizome is a large underground stem of Nelumbo nucifera Gaertn(Nelumbo nucifera Gaertn).There are two species in the lotus genus,one is the lotus(Nelumbo nucifera)distributed in Asia,and the other is yellow lotus(Nelumbo lutea)distributed in the North America.Lotus rhizome has been used as a traditional Chinese medicine for thousands of years.In Chinese medicine,lotus rhizome is believed to be used to cool blood,promote fluid,and disperse blood stasis.It can be used to treat fever,polydipsia,vomiting,blood loss,etc.,and cooked utensils are beneficial for blood,invigorating the spleen,stopping diarrhea,and appetizing.Lotus rhizome has high nutritional value.The research on lotus rhizome has always focused on the basic nutrient composition,swelling gene mining,and quality research during storage and processing.The analysis of the difference in metabolites between different varieties is still lacking.Plant metabolomics,as a discipline to study plant small molecule metabolites and dynamic changes,has great potential in many aspects such as gene mining,plant agricultural traits,quality division,Chinese medicine efficacy evaluation,and crop breeding.Simple Sample processing methods,combined with a variety of analytical methods,discover the relationship between qualitative and quantitative data,and explain its biological significance.The ground part of lotus is rich in metabolites such as lotus plumule,lotus leaf,lotus flower,etc.However,the metabolites in lotus rhizome are relatively small,and the research on the metabolites of lotus rhizome is very limited.The phenotypic variation of organisms is related to their inheritance and secondary metabolites.Therefore,in-depth exploration of the metabolites in lotus rhizome is of vital significance to explain the phenotypic changes of lotus rhizome.The lotus rhizome swelling phenomenon of different varieties is very different,and the lotus rhizome has obvious browning phenomenon after storage and processing.The difference between the varieties of browning degree is different,which seriously affects the quality of the lotus rhizome.Based on the 212 lotus rhizome samples we collected,we explored the differences in the basic substances of lotus rhizome samples with different swelling degrees,and conducted in-depth exploration of the metabolites related to browning in order to explain the relationship between the changes in the shape of the lotus rhizome and its metabolites.Record the lotus rhizome expansion related indicators(such as the circumference,length,weight,etc.)of the lotus rhizome during the maturity period,and the color indicators of the lotus rhizome after drying and flouring,namely the brightness(L)and the a value of the two color channels b value.Different processing methods are used to analyze the types and contents of starch and secondary metabolites.The analysis and measurement of starch content and amylose ratio in lotus rhizome flour are measured by Megazyme kit.The secondary metabolites are measured by two methods,namely HPLC analyzes the absolute quantification of the compounds in lotus rhizome powder and the relative quantification of LC-MS,and imports the final data into multiple analysis software for analysis.Use MassHunter for qualitative analysis of compounds,Profinder for quantitative analysis of ion current diagrams,Chemdraw to estimate the cleavage law of quantifiable compounds,and drawing of corresponding graphs;Excel and Sigmaplot for data sorting and preliminary data observation,and drawing of correlation scatter plots;SPSS conducts overall normality test,correlation analysis and unsupervised PCA analysis on the data;Simca conducts supervised PLS_DA analysis to screen differential metabolites;R conducts cluster analysis and correlation heat map of the overall sample Drawing;At the same time,there are some auxiliary processing graphics software applications,such as Adobe Illustrator,Photoshop,Origin,etc.The content distribution of small molecular metabolites,starch,amylose/amylose and other metabolites in lotus rhizome of 212 samples is shown below.LC-MS analysis was performed on the samples extracted from the membrane,and a total of 56 compounds were identified.HPLC and LC-MS were used for absolute quantification and relative quantification respectively.8 compounds were absolutely quantified by HPLC,among which the mass fraction of L-tryptophan was 0.058～1.882 mg·g-1;the mass fraction of gallocatechin was 0～2.237 mg·g-1:the mass fraction of p-hydroxybenzaldehyde was 0.005～0.0257 mg·g-1;the mass fraction of chlorogenic acid is 0～1.156 mg·g-1;the mass fraction of Liriodendron Liriodendron is 0～0.092 mg·g-1;the mass fraction of scopoltin is 0～0.103mg·g-1;the mass fraction of N-trans-p-coumarin is 0～0.131mg·g-1;N-feruloyl tyramine is 0～0.526 mg·g-1,where L-Tryptophan and p-hydroxybenzaldehyde can be detected in all samples,the content of gallocatechin and L-tryptophan is relatively high,and the content of p-hydroxybenzaldehyde is low.The data was qualitatively analyzed by LC-MS,and a total of 56 compounds were obtained,including 14 amino acids,16 alkaloids,5 nucleotides,2 flavanols,3 phenylpropanoids,8 There are 1 organic acids and 8 other compounds.Among them,39 compounds were analyzed from lotus rhizome for the first time,especially the alkaloids in it,such as apophine,monobenzyl isoquinoline and amide alkaloids.And predict the cleavage law of some compounds.In the positive ion mode,the relative quantification was performed by extracting the precursor ion flow diagram of the compound,and a total of 41 chemical components were quantified.The starch content and the amylose/amylose ratio in lotus rhizome are very different.The total starch content is 19.62%(A154)～79.12%(B061),and the amylose ratio is 12.64%(A021)～32.17%(A151).The browning index L,a and b values of lotus rhizome flour are 27～75,4.18～11.70,11.13～24.28,respectively.The L value varies greatly,and the L value of most samples varies from 50 to 70.The samples accounted for 74.53%of the total number of samples.Correlation analysis was made between the above measured color indicators and HPLC absolute quantitative data and LC-MS relative quantitative data.The results of correlation with HPLC absolute quantitative data showed that the L value was significantly negatively correlated with the 7 compounds(P<0.01),and was correlated with L-tryptophan,isosamine,N-trans-p-coumarol tyramide,N-Trans-ferulyl tyramide has a moderately negative correlation,that is,the higher the content of such compounds,the darker the color of lotus rhizome flour;the a value has a significant positive correlation with 7 compounds,and it has a significant positive correlation with compounds 1,6,7,and 8.Moderately positive correlation.The b value has a significant correlation with 7 compounds.The results of correlation with LC-MS relative quantitative data show that there are 32 compounds that are significantly related to the L value(P<0.01),of which 23 compounds are significantly negatively correlated with the L value,mainly amino acids and alkaloids Compounds(such as papaverine,N-feruly1-3-methoxytyramine,indigoidine,and dehydrocetomanine),there are 9 compounds that are significantly positively correlated with the L value,mainly nucleotides And flavanol compounds(such as adenosine,catechin);33 compounds have a significant relationship with a value(P<0.05),25 compounds are significantly positively correlated with a value,and 8 compounds are significantly negative Correlation;b value was significantly correlated with 29 compounds(P<0.05),was significantly positively correlated with 22 compounds,and was significantly negatively correlated with 7 compounds.The 212 lotus rhizome flour samples were divided into 3 groups according to their L values,and the population structure of lotus rhizomes was analyzed based on absolute and relative quantitative data.It was found that the relative quantitative compounds of LC-MS were more representative for the samples classified by L value.The OPLS_DA model indicates that the two groups of samples with high and low L scores are clearly divided into two categories.The model’s interpretation rate of Y R2Y and the model’s predictive ability Q2 are 0.814 and 0.757,respectively.By analyzing the load diagram of the sample,potential biomarkers of different degrees of browning are obtained.In the group with lower L value,the compound tryptophan,(2Z)-N-[2-(3,4-dihydroxyphenyl)-2-hydroxyethyl]-3-(4-methoxy(Phenyl)-2-acrylamide,Aegeline,papaverine and apricotine are relatively low in content.Such samples are mainly alkaloids;in the group with a relatively high L value,the compounds guanosine,The content of nucleotides and flavanols such as adenosine,(epio)catechin,(epio)gallocatechin,etc.is relatively high,while the content of other components is relatively low.The perimeter of the expansion index,the perimeter of the lotus rhizome,the longitudinal length,and the perimeter of the lotus rhizome are respectively 4.95～37.23 cm,3.45～10.55 cm,5.30～36.00 cm,11.40～338.65 g,respectively,for the metabolites of 212 varieties Spearman correlation analysis with related indexes of swelling(lotus rhizome joint circumference,lotus joint circumference,quality,length)shows that the total starch content is significant with lotus rhizome circumference,intemode circumference,lotus rhizome quality,and amylopectin content.It is positively correlated and negatively correlated with amylose content.The perimeter of lotus rhizome is significantly positively correlated with the perimeter and quality of lotus rhizome nodes,and the correlation is very strong.The longitudinal length of lotus rhizome is only significantly positively correlated with quality.The absolute quantitative correlation coefficient with HPLC showed that the perimeter of lotus rhizome was significantly negatively correlated with tryptophan,gallocatechin,p-hydroxybenzaldehyde,and chlorogenic acid,while the perimeter of the lotus rhizome was positively correlated with the perimeter of the lotus rhizome.The content of the sample is higher,and the other swelling-related phenotypes,such as longitudinal length,mass,etc.These indicators are not strongly related to the components in the sample.The correlation coefficients of 41 compounds quantified by LC-MS showed that 18 compounds were significantly correlated with lotus rhizome circumference,6 compounds were significantly positively correlated,and 12 compounds were significantly negatively correlated with enlarged circumference.The 12 compounds contain 6 quinoline alkaloids.Based on the quantitative data of LC-MS,clustering,PCA and PLS_DA were performed on the collected 212 lotus rhizome samples.The cluster heat map shows that the samples are divided into 4 categories.Most tropical lotus samples are clustered in one category.The content of L-phenylalanine is relatively high,but the content of amide alkaloids is extremely low.The sample score map obtained by PCA analysis clustered tropical lotus and non-tropical lotus in two quadrants,and further PLS_DA analysis was performed to obtain the differential metabolites of this aggregation classification.It was found that the characteristic metabolites of non-tropical lotus samples were isocoumarin and citrus aurantium.Alkali,acetyl-phenylalanine,glutamine,N-p-ferulyl tyramine.The characteristic variables of the tropical lotus samples are methyl isoasine,epigallocatechin,L-tyrosine,formyl-tetrahydrofolate,L-arginine and so on. |
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