Preliminary Study On "Five Flavors" And Material Basis Of Moringa Oeifera Leaves

Moringa Oleifera Lam was native to India and it was introduced in Taiwan province in the 1910s originally.It had been widely planted in tropical and subtropical regions such as Yunnan,Guangdong,Guangxi provinces in China.Moringa oeifera leaves(MOL)were conventionally considered as food or drug for the treatment of splenometrical,jaundice,constipation,skin tumor,diabetes,hyperlipidemia and other diseases,and it had achieved better efficacy.At present,the research reports of MOL were mainly focused on chemical and pharmacological researchs,but there were few studies on its medicinal taste.MOL was an exterior herbal medicine,which was introduced into my country as the new resource of traditional Chinese medicine.In order to achieve its "Chinese medicine",it was imperative to interpret it with traditional theory of traditional Chinese medicines.The flavor theory was an important part of property theory of Chinese medicine.In this study,MOL was used as an example to analyze medicinal belongings and material foundation.Under the guidance of the flavor theory,the study combined modern technologies with the large number of scientific,objective and visual data,in order to explain the scientific connotation of the flavor of MOL and to promote the construction of high quality integration of traditional Chinese medicine into the construction of "belt and road".The research contents were as follows:1.Qualitatively analyze non-volatile and volatile chemical constituents of MOL by UPLC-Q-TOF-MS and GC-MS technologies.Quantitatively analyze the components of glucosinolates and flavonoids in MOL by UPLC technology.The 70%methanol extracts of 4 common MOL(YD,PKM1,PKM2,HH)were analyzed by UPLC-Q-TOF-MS.The 20 common non-volatile components were identified,including 5 organic acids and their esters,1 alkaloid,4 glucosinolates,10 flavonoids and their derivatives.The volatile oil of 4 kinds of MOL was analyzed by GC-MS.The 19 common volatile components were identified,including 7 aldehydes,4 ketones,3 alkanes,2 organic acids,1 terpenoid,1 nitrile,1 alkaloid.According to the difficulty of preparation and market sale solutions of glucosinolate and flavonoid reference standards,the contents of 4 glucosinolates(4-0(α-L-rhamnopyranyloxy)-benzyl glucosinolate and 4-O-(α-Lacetylrhamnopyranyloxy)-glucosinolate Ⅰ,Ⅱ,Ⅲ)and 4 flavonoids(vicenin-2,vitexin,isoquercitrin,astragalin)in 4 kinds of MOL were determined by internal standard method and external standard method using UPLC technique,respectively.The results showed that the total content of 4 glucosinolates in PKM1 variety(3.434%)was the highest,followed by PKM2(3.083%)and YD(1.867%)varieties,and the content of YD(1.861%)variety was the lowest.The total content of 4 flavonoids in YD variety(0.832%)was the highest,followed by HH(0.687%)and PKM2(0.634%)varieties,and the content of PKM1 variety(0.528%)was the lowest.2.Study the taste and odor belongings of MOL using the electronic tongue and electronic nose and pattern recognition technologies.In the taste researchs,the 4 kinds of MOL and 37 kinds of medicinal materials with typical tastes of sour,bitter,sweet,spicy and salty were determined by the electronic tongue technique.In order to eliminate the diurnal difference of the test data and increase the differentiation of the models,the electronic tongue response value was investigated in turn by the comparison of preprocessing methods(original data,normal state standardization,polar difference standardization,decentralization,normalization),the extractions of the test time(91th～120th s or 101th～120th s or 111th～120th s)and the number of tests(3th～7th times or 4th～7th times or 5th～7th times),the screening of sensors.The final data processing methods:the test data of electronic tongue was normalized by MOL(PKM1).After normalization,the mean value of 111th～120th s and 5th～7th times was taken,and the acid or salty medicinal materials and acid or salt sensors were removed at the same time.In the odor researchs,the 4 kinds of MOL and 15 kinds of medicinal materials with typical tastes of spicy and sweet,spicy and bitter,spicy were determined by the electronic nose technique.Referring to the data processing results of the electronic tongue,the test data of electronic nose was normalized with the response value of MOL(PKM1),and then the mean value of the latter three times data was taken.For the pretreated electronic nose and electronic tongue data,4 models of principal component analysis(PCA),partial least squaresdiscriminant analysis(PLS-DA),support vector machine(SVM)and artificial neural network(ANN)were analyzed to predict the taste and odor of MOL.The results of the PCA and PLS-DA showed that MOL was predicted as sweet and bitter taste with the odor of spicy.The correct recognition rate of ANN model was 83.3%higher than the SVM model(70.8%),which was predicted that PKM1 variety was bitter,PKM2 variety was sweet,YD variety was sweet,HH variety was sweet.The correct recognition rates of the ANN and SVM models were 100.0%,which was predicted that PKM1.variety was spicy,PKM2 variety was spicy and sweet,YD variety was spicy,HH variety was spicy.In summary,the taste and odor belongings of MOL were bitter,sweet and spicy.3.Study the binding situations between chemical ligands and olfactory or taste receptor proteins by molecular docking technology,in order to clarify the material foundation of the taste and odor of MOL.The above results speculated that the MOL was sweet,bitter,and spicy.According to the literature researchs,3 kinds of bitterness receptors(T2R38,T2R10 and T2R14 proteins),sweet receptors(T1R2 and T1R3 proteins)and spicy receptors(TRPV1 and OR7D4 proteins)were selected.Then molecular docking situations of the chemical components of MOL(binding site,binding energy,binding mode)were examined.The molecular docking results of bitter receptors showed that T2R10 protein could combine adenosine,4-0-(α-L-acetylrhamnopyranyloxy)-glucosinolate Ⅱ,quercetin acetylglucoside with hydrogen and hydrophobic bonds.The T2R14 protein could combine malic acid,adenosine,4-O-(α-L-acetylrhamnopyranyloxy)-glucosinolate Ⅲ,vitexin,isoquercitrin with hydrogen and hydrophobic bonds.The T2R18 protein could combine 4-O-(α-L-acetylrhamnopyranyloxy)-glucosinolate III,vicenin-2,isoquercitrin with hydrogen and hydrophobic bonds.In summary,glucosinolates and flavonoids might be material basis of bitterness.The molecular docking results of sweet receptors showed that the binding energy between all compounds and sweet proteins was<-5.0 kcal/mol.The binding modes between T1R2 and ligands were mainly hydrogen and hydrophobic bonds,and the binding modes of between T1R3 and ligands were mainly hydrogen,hydrophobic and electrostatic bonds.In summary,glucosinolates,flavonoids,organic acids and alkaloids might be material basis of sweetness.The molecular docking results of spicy receptors showed that the binding energy between TRPV1 protein and 4-O-(α-L-acetylrhamnopyranyloxy)-glucosinolate II was strong,and the binding modes were mainly hydrogen and hydrophobic bonds.The binding energy between OR7D4 protein and safranal,geranyl acetone,damascenone,linalool oxide was strong,and the binding modes were mainly hydrogen and hydrophobic bonds.In summary,glucosinolates,aldehydes,ketones,terpenoids,nitriles,acids,alkanes and alkaloids might be material basis of spicy ordor.4.Select the entry point of purgative efficacy to screen the key targets and components by network pharmacology technology.The purgative effect of MOL was selected based on the literature.On this basis,the study analyzed mechanisms of the treatment on constipation of MOL by network pharmacology.The 674 chemical composition targets were predicted through the SEA and Swiss Target Prediction databases,and the 1086 constipation targets were predicted through the Genecards database.After the two were trapped,88 common targets were obtained.Using the cytoscape software to construct the "component-target-pathway"network,the results showed that the key components might be adenosine,astragalin,fragrant acetone,2-methyl-3-octanone,palmitic acid and oleamide;and the key targets might be PTGS2,TNF,MAPK1,ADRA2A,IL-6.Among them,PTGS2 could promote bowel movements by causing columns to shrink;ADRA2A could promote secretion of gastrointestinal motion and intestinal fluid;MAPK1 could improve constipation by affecting the MAPK pathway;IL-6 and TNF belonged to promote inflammatory factors,and excessive expression would increase the stomach intestinal inflammatory state.5.Study the binding situations between chemical ligands and receptor proteins by molecular docking technology,in order to clarify the material foundation of the effect taste of MOL.Molecular docking was performed by key components(adenosine,astragalin,fragrant acetone,2-methyl-3-octanone,palmitic acid and oleamide)and target proteins(PTGS2,TNF,MAPK1,ADRA2A,IL-6)on the treatment of constipation by network pharmacology.The results showed that the binding energy between IL-6 protein and andadenosine,astragalin,fragrant acetone,oleamide was strong with hydrogen and hydrophobic bonds.The binding energy between MAPK1 protein and adenosine,astragalin,fragrant acetone was strong with hydrogen and hydrophobic bonds.The binding energy between PTGS2 protein and adenosine,astragalin,fragrant acetone,palmitic acid,oleamide was strong with hydrogen and hydrophobic bonds.The binding energy between ADRA2A protein and adenosine,astragalin,fragrant acetone,2methyl-3-octanone was strong with hydrogen,hydrophobic and electrostatic bonds.The binding energy between TNF protein and adenosine,astragalin was strong with hydrogen and hydrophobic bonds.Similarly,alkaloids,flavonoids,ketones and organic acids might be material basis of MOL in treating constipation.This study combined bionic technologies(electronic tongue and electronic nose)with pattern discriminating technologies to establish the medicinal taste identification models,then the taste and odor of MOL were predicted.The results showed that MOL were characterized by bitter,sweet and spicy.Molecular docking results showed that glucosinolates and flavonoids might be material basis of bitterness;glucosinolates,flavonoids,organic acids and alkaloids might be material basis of sweetness;glucosinolates,aldehydes,ketones,terpenoids,nitriles,acids,alkanes and alkaloids might be material basis of spicy ordor.The effect of constipation was thinked as bitterness in traditional Chinese medicine theory,and alkaloids,flavonoids,ketones,organic acids might be material basis of MOL in treating constipation.