| Objective:According to the dynamic changes of volatile and non-volatile components in the bleaching process of A tractylodis macrocephala with the water of washing rice,the traditional bleaching process of A macrocephala was optimized.Pharmacodynamic experiments were used to compare the invigorating spleen effects of raw A.macrocephala and A macrocephala with the water of washing rice,and the mechanism of invigorating spleen was discussed by metabonomics.Method:①According to the method recorded in "Processing Standards of TCM Decoction Pieces in Jiangxi Province",The raw products of A.macrocephala and bleached productsof 5 different bleaching stages were prepared(in the first and second stages,raw products were bleached with 9-fold volumn of the water of washing rice for 12 h and 24 h,respectively;in the third,fourth and fifth stages,the raw products were firstly bleached with 9-fold volumn of the water of washing rice for 24 h,and then bleached with 9-fold volumn of clean water for 12,24 and 48 h,respectively);the bleaching temperature was set at 26℃.The volatile components of raw products of A.macrocephala and its bleached products of 5 different bleaching stages were qualitatively analyzed by using headspace gas chromatography-mass spectrometry.The relative percentage of each component was calculated by peak area normalization method.②According to the method recorded in "Processing Standards of TCM Decoction Pieces in Jiangxi Province",The raw products of A.macrocephala and bleached productsof 5 different bleaching stages were prepared(in the first and second stages,raw products were bleached with 9-fold volumn of the water of washing rice for 12 h and 24 h,respectively;in the third,fourth and fifth stages,the raw products were firstly bleached with 9-fold volumn of the water of washing rice for 24 h,and then bleached with 9-fold volumn of clean water for 12,24 and 48 h,respectively,the bleaching temperature was set at 26℃.)were used as the research object.The non-volatile were detected by using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS).The 0.1%formic acid aqueous solution(A)-acetonitrile(B)was used as the mobile phase for gradient elution(0～5 min,5%～30%B;5～14 min,30%～60%B;14～23 min,60%～70%B;23～31 min,70%～95%B;31～32 min,95%～5%B;32～35 min,5%B),column temperature 40℃,injection volume 2 μL,flow rate 0.3 mL·min-1;Electrospray ionization(ESI)was selected in positive ion with a scan range of m/z 50-1 250;The data were analyzed by using PeakView 1.2 software,and the Non-volatile components of raw products of A.macrocephala and bleached products of 5 different bleaching stages were identified by reference materials,chemicalbook database and related literature;After normalization by MarkerView 1.2 software,multivariate statistical analysis was applied to screen the differential compounds,the changes of the relative contents of the differential compounds with different bleaching time were analyzed.③32 KM mice were divided into normal group,model group,raw A.macrocephala 1.092g/ml group and A.macrocephala with the water of washing rice(bleaching according to the conclusion of ②)1.092g/ml group with 8 mice in each group.The normal group was fed normally,and the other groups established mice models of spleen deficiency.After successful modeling,each group was perfused with normal saline or corresponding drugs.One week after administration,the levels of serum vasoactive intestinal peptide(VIP),tumor necrosis factor α(TNF-α)and colon TNFα and interleukin-6(IL-6)were detected.④Serum derivatization treatment.The serum samples were detected by using UPLC-Q-TOF-MS technology,and the total ion flow charts of all serum samples were obtained.Data preprocessing is carried out by Markerview 1.2.1 software,including denoising,peak extraction,peak alignment,peak identification and normalization.The pretreated data were imported into SIMCA 14.1 software,and unsupervised principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were performed.After 200 substitutions,it was confirmed that there was no over-fitting in the model,and the differential metabolites were screened according to the variable projection importance(VIP)value>1.5 and t test(P<0.05).Then the screened differential metabolites were compared with the mass spectrum data of HMDB according to their precise m/z,and finally the names of the identified differential metabolites were input into the online database Metabo Analyst(https:/:/www.metaboanaalyst.c)for related metabolic pathway analysis.Result:①The results of the volatile component study showed that a total of 49 volatile components were identified from raw products of A.macrocephala and its bleached products of 5 different bleaching stages,including 20 common volatile components such as terpinolene,cyperene and atractylon,etc.Among them,33,31,28,30,28 and 29 volatile components were identified from the raw products of A.macrocephala and the bleached products of the first to fifth stages,the relative percentages of which were 66.218%,64.711%,79.410%,65.419%,67.101%,66.818%,respectively;among them,the relative percentage of atractylon in bleached products was the highest in the fourth stage(41.206%),but was the lowest in the third stage(35.926%).Compared with the raw product,16 volatile components such as pethylbrene and β-vetivenen were added in the bleaching process,while 8 volatile components such as ethyl palmitate and β-maaliene were not detected.However,5 volatile components including 11-rotundene and(-)-valeranone in the bleaching process showed a trend of disappearance-emergence and disappearance-emergence-disappearance.②The results of the non-volatile components study showed that showed that a total of 55 chemical components were identified from raw products of A.macrocephala and its bleached products of 5 different bleaching stages,including 40 common chemical components such as atractylenolide Ⅰ,Ⅱ,Ⅲ,etc.Among them,53、47、49、49、44 and 46 chemical components were identified from the raw products of A.macrocephala and the bleached products of the firstto fifth stages.Compared with the raw product,vitexin and dihydrosyrindine were added in the bleaching process,while 9 components such aspoly-l-histidine hydrochloride mw and uridine were not detected.However,4 components including 9-hydroxy-7-oxo-7H-furo[3,2-g]chromen-4-yl β-D-glucopyranoside、4-octylbenzoic acid in the bleaching process showed a trend of disappearance-emergence.By multivariate statistical analysis,18 different compounds were screened out,among which the relative contents of atractylenolide Ⅰ,Ⅱ and Ⅲ increased first and then decreased with with the extension of bleaching time,and all of them were the lowest in raw products,and the highest in the third stage of bleaching products.③ Pharmacodynamic studies have shown that compared with the normal group,the levels of serum VIP,TNF-α and colon TNF-α and IL-6 in the model group were significantly increased(P<0.05).Compared with the model group,the levels of serum VIP,TNF-α and colon TNF-α,IL-6 in A.macrocephala with the water of washing rice group were significantly decreased(P<0.05),but not in the raw A.macrocephala group(P<0.05).④The results of metabonomics showed that compared with the normal group,the contents of 17 different metabolites in the serum of the model group were significantly increased(P<0.05).After giving 1.092g/ml of raw A.macrocephala and 1.092g/ml of A.macrocephala with the water of washing rice,the serum levels of 17 kinds of differential metabolites in mice were significantly decreased compared with model group,and the decreasing trend of differential metabolites in the A.macrocephala with the water of washing rice group was more significant(P<0.05).The metabolic pathways involved are mainly glycerophospholipid metabolism and Pentose and glucuronate interconversions.Conclusion:①In the third stage,the total relative percentage of each volatile component and the relative percentage of representative dry component as atractylone are the lowest in bleached products of A.macrocephala,i.e.the bleaching technology of relieving the dry property of A.macrocephala with the water of washing rice is bleaching with 9-fold volumn of the water of washing rice for 24 h,and then bleaching with 9-fold volumn of clean water for 12 h.②The relative contents of atractylenolide Ⅰ,Ⅱ and Ⅲ,which are index components and pharmacodynamic components,are the highest in the third stage of A.macrocephala bleaching products.Therefore,the best bleaching process to increase the content of Atractylodes(atractylenolide Ⅰ,Ⅱ,Ⅲ)is that the raw products were firstly bleached with 9-fold volumn of the water of washing rice for 24 h,and then bleached with 9-fold volumn of clean water for 12 h.③The results of pharmacodynamics and metabolomics showed that the effect of A.macrocephala with the water of washing rice on invigorating spleen is better than that of raw A.macrocephala,so the conclusion of ②is reliable.The mechanism may be related to the regulation of glycerophosphotipid metabolism and Pentose and glucuronate interconversions. |
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