The Study Of The Oligosaccharides Change Laws And Quality Standards For Processed Products Of Morinda Officinalis How Based On Quality Marker(Q-marker)

ObjectiveMorindae Officinalis Radix is the dried root of Morinda officinalis How(MO),belonging to Rubiaceae family.It is one of the commonly used traditional Chinese medicines for tonifying kidney yang in the Lingnan area of China.Clinically,MO needs to be processed into medicine,such as salt-steaming MO(SSMO),steaming MO(SMO),etc.The processing or not plays an important role in the curative effect and quality of the medicinal materials.The previous research of our research group found that the oligosaccharides composition of MO changed greatly after processing,especially the"essential" changes in SSMO and SMO.However,the current standards have some some deficiencies,such as ambiguous processing parameters and control the quality of processed products by the control methods of medicinal materials,which couldn’t effectively control and comprehensively evaluate the quality of SSMO and SMO.Based on the previous research of our group,this paper focuses on the specificity,validity,traceability and measurability of Q-markers,taking SSMO and SMO as the research objects,the Q-markers of MO processed products were systematic studied by modern scientific analysis techniques and methods.Moreover,the draft quality standards for SSMO and SMO were formulated.This paper could provide scientific basis and theoretical support for the establishment of quality standards for scientific and standardized processing of MO and MO processed products.Methods1.Identification of oligosaccharides in MO processed productsFirstly,the soil and fibrous roots of fresh MO were removed,cut into sections and dried to obtain raw MO(RMO).Then,SSMO and SMO were obtained according to the processing methods of previous research,respectively.Fingerprintings of RMO,SSMO and SMO from different origins were established by HPLC-ELSD method.And the pattern recognition methods,such as HCA,PCA and OPLS-DA,were used to elucidate the difference on chemical constituents of raw and processed products.SSMO products were used as the separation material,and were extracted by refluxing with 50%ethanol.Extract solution was concentrated to obtain the extract sample,SSMO oligosaccharides were purified,acetylated,deacetylated and purified by modern column chromatography methods,such as wk40L weakly acidic cation exchange resin,wa30 weakly basic anion exchange resin and sephadex LH-20 gel column in turn.Then,"unknown component" X was separated by preparative column and high performance analytical liquid chromatography.The structures of "unknown component" X were identified by physicochemical properties and various spectral techniques,such as ESI-MS,1H-NMR and 13C-NMR analysis methods.Finally,the amounts of the "unknown component" X were prepared by the high-efficiency preparative liquid phase method.The impact factors of "unknown components" X to processed oligosaccharides were studied by HPLC-ELSD method.2.Anti-reproductive oxidative stress activity and mechanism of MO processed oligosaccharidesFirst,the abilities of 50%ethanol extracts of SSMO and SMO to scavenge DPPH,ABTS,PITO and OH free radicals as well as reduce Fe3+ were determined.The antioxidant effects of SSMO and SMO were preliminarily evaluated,respectively.Secondly,CTX-induced reproductive injury model in male mice was used to investigate the effects of raw and processed MO oligosaccharides who on testicular tissue recovery ability,total sperm count,sperm survival rate and sperm deformity rate in mice with oxidative stress.The contents of GSH-Px,CAT and MDA in male mouse serum were detected by kit methods.The efficacy difference of raw and processed MO oligosaccharides was preliminary investigated by CTX-induced reproductive oxidative damage model.Subsequently,the anti-reproductive oxidative stress effects of raw and processed MO oligosaccharides were compared by H2O2,-induced oxidative stress injury on TM4 cells model.Finally,the effects of raw and processed MO oligosaccharides to the target proteins of Nrf2-Keap1/ARE pathway were revealed by western blot technology.3.Discovery and verification of the Q-markers for MO processed products based on spectrum-effect relationshipGRA and PLS methods were used to establish the spectrum-effect mathematical model between the fingerprintings of MO processed products and the effects of antioxidant free radicals.Entropy weight TOPSIS and GAR methods were used to establish the spectrum-effect mathematical model between the fingerprintings of MO processed products and the effects of anti-oxidative reproductive damage.These models could preliminary screening of potential Q-markers for MO processed products.Molecular docking technology was used to simulate the binding active sites between the Q-markers and Nrf2,Keap1,HO-1 as well as SOD.The effects of Q-markers on the survival rate of TM4 cells were detected by CCK-8 kits.The effects of Q-markers to GSH-Px,SOD,CAT and MDA contents in TM4 cells were detected by kit methods.The regulatory effect of oligosaccharides compounds on the expression of Nrf2,Keap1,HO-1 and SOD was detected by western blot method.4.Study on the oligosaccharides change laws and characteristic quality standards for MO processed productsThe changes of MO oligosaccharides before and after processing were investigated by HPLC-ELSD method.The contents of moisture,total ash and extractive in RMO,SSMO and SMO were determined according to the methods under the Chinese Pharmacopoeia 2020 edition of Morindae Officinalis Radix.Fingerprintings of RMO,SSMO and SMO were established by HPLC-ELSD method based on Q-markers.And the contents of Q-markers in MO processed products were simultaneously determined by HPLC-ELSD method.The above researches could provide a scientific basis for establishing the characteristic quality standards of MO processed products.Results1.Identification of oligosaccharides in MO processed productsThe similarity evaluation of HPLC-ELSD fingerprintings showed that 15 characteristic peaks in RMO,and 23 characteristic peaks in SSMO and SMO.The similarity of each batch of samples was higher than 0.9.HCA and PCA results demonstrated that raw and processed MO samples were clearly divided into two categories,namely RMO was classified as class I,SSMO and SMO were aggregated into class Ⅱ,which could effectively distinguish RMO from SSMO and SMO.PCA and OPLS-DA results revealed that the 19 differential components between raw and processed MO.Among them,"unknown components" X1-X8 were the potential differential components,and they could be caused the quality difference of raw and processed products.The structures of "unknown component" X were isolated and identificated.The results revealed that compound X2 is β-D-fructopyranose-(1→2)-β-D-fructofuranose-(1→2)-β-D-fructanose,compound X3 is β-D-fructopyranose-(1→2)-β-Dfructofuranose-(1→2)-β-D-fructofuranose-(1→2)-β-D-fructofuranose,compound X4 isβ-D-fructopyranose-(1→2)-β-D-fructofuranose-(1→2)-β-D-fructofuranose-(1→2)-β-Dfructofuranose-(1→2)-β-D-fructofuranose,compound X5 is β-D-fructopyranose-(1→2)-β-D-fructofuranose-(1→2)-β-D-fructofuranose-(1→2)-β-D-fructanose-(1→2)-β-D-fruct ofuranose-(1→2)-β-D-fructofuranose.The "unknown components" X2-X5 structures belong to inulo-oligosaccharides,which consistent with literature reports.The amounts of X2,X3 and X4 were enriched by semi-preparative liquid phase is 20 mg and the purity is more than 96.0%,respectively.When the temperature was higher than 80℃,the total RMO oligosaccharides(inulin-oligosaccharides)could generate inulo-oligosaccharides.Different concentrations of pH and NaCl solution had different effects on the generation of inulo-oligosaccharides.Temperature and pH were the main impact factor of inulo-oligosaccharides generation by comprehensive evaluation of entropy weight TOPSIS method.2.Anti-reproductive oxidative stress activity and mechanism of MO processed oligosaccharidesBoth SSMO and SMO could scavenge DPPH,ABTS,OH,PTIO free radicals and reduce Fe3+,and the effects of the processed products scavenged DPPH,ABTS and PTIO free radicals were greater than raw products.The antioxidant activity in vitro was the best when SSMO was processed for 5 h and SMO was processed for 3 h,respectively.SSMO and SMO oligosaccharides were able to inhibit CTX-induced reproductive injury in male mice,improve sperm quality and increase the level of antioxidant enzymes.Moreover,the anti-reproductive oxidative stress effect of SSMO and SMO oligosaccharides was better than RMO oligosaccharides,which was verified in H2O2-induced oxidative stress injury on TM4 cells model.Namely,25 μg/mL of SSMO and SMO oligosaccharides could inhibit oxidative stress injury of TM4 cells,and the effects of SSMO and SMO oligosaccharides were significantly better than RMO oligosaccharides,which may be related to inhibit Keapl and increase the expression levels of Nrf2,SOD-1 and HO-1.3.Discovery and verification of the Q-markers for MO processed products based on spectrum-effect relationshipThe scavenging of DPPH,ABTS,PTIO oxidative radicals and the reduction of Fe3+by SSMO were the synergistic effect of inulin-oligosaccharides and inulooligosaccharides,and the scavenging of OH radicals was related to inulin-oligosaccharides.The scavenging of DPPH,ABTS,PTIO free radicals and the reduction of Fe3+by SMO was mainly related to inulin-oligosaccharides,while the contribution of inulo-oligosaccharides was lower.In summary,1-kestose,nystose,X2,X3 and X4 could be used as candidate Q-marker components of SSMO;sucrose,nystose and X4 could be used as candidate Q-marker components of SMO.The anti-oxidative effects of SSMO and SMO oligosaccharides were superior to RMO oligosaccharides on reproduction,which may be related to nystose,1F-fructofuranosylnystose,X2,X3 and X4.The contribution degree of these compounds to the efficacy was greater than 0.98,respectively.The correlation and structure of these compounds were strong and clear,so they could be used as potential Q-markers for the quality control of SSMO and SMO.The anti-oxidative activities of the potential Q-marker components were verified,respectively.The results revealed that inulin-oligosaccharides compounds(1-kestose,nystose,1F-fructofuranosylnystose)and inulo-oligosaccharides compounds(X2,X3,X4)could interact with the target protein(Nrf2,Keapl,HO-1,SOD-1)generate binding activity,and could interact with surrounding amino acid residues.Among them,inulo-oligosaccharides compounds had stronger affinity with each target protein than inulin-oligosaccharides compounds.Therefore,nystose and X3 were screened to compare anti-oxidative activities difference between inulin-oligosaccharides and inulooligosaccharides compounds.The results manifested that both nystose and X3 could inhabit H2O2-induced TM4 cells oxidative damage by increasing the activities of antioxidant enzymes and reducing the content of MDA.Moreover,the anti-oxidative stress activity of X3 was significantly better than nystose,the reason of which may be related to activate the expression of Nrf2,Keapl,HO-1 and SOD-1 proteins in the Keap 1-Nrf2/ARE signaling pathway.Based on these findings,1-kestose,nystose,1F-fructofuranosylnystose,X2,X3 and X4 were finally identified as the common Q-marker components of SSMO and SMO.4.Study on the oligosaccharides change laws and characteristic quality standards for MO processed productsUsing Q-marker components as indicators,the change laws of oligosaccharides components in SSMO and SMO samples during processing were explored.The results revealed that inulo-oligosaccharides appeared in SSMO at about 0.5 h,and the content reached the highest point at 5～6 h;inulo-oligosaccharides appeared in SMO at about 1 h,and the content reached the highest point at 7～8 h.The contents of monosaccharides were increased,inulin-oligosaccharides were decreased,and inulo-oligosaccharides were first increased and then decreased in SSMO and SMO.Moreover,there were intersection points in a certain period of time.However,the chemical reaction time of SSMO was about 2 h earlier than that of SMO.The best processing time was screened by combing with the results of content and anti-oxidative free radical efficacy of 6 Q-marker components.Finally,it was suggested that the optimal processing time of SSMO and SMO was 4-5 h.According to the determination of the Q-marker components and the optimal processing time,the HPLC-ELSD fingerprintings of SSMO and SMO were established,respectively.The results demonstrated that 20 common characteristic peaks in SSMO and 24 common characteristic peaks in SMO were marked,and 9 compounds in the two processed products were identified.Finally,the contents of 6 Q-marker components in SSMO and SMO were simultaneously determined,including 1-kestose,nystose,1F-fructofuranosylnystose,X2,X3 and X4.It was tentatively determined that the extract of SSMO should not be less than 57.0%,and the ratio of inulin/inulo-type oligosaccharides in SSMO not be greater than 2.5.And the extract of SMO should not be less than 54.0%,and the ratio of inulin/inulo-type oligosaccharides in SMO not be greater than 4.5.The contents of Q-marker components in 15 batches of commercially SSMO were determined,which showed that 1-kestose,nystose,1F-fructofuranosylnystose of SSMO were not detected in 4 batches;X2,X3 and X4 of SSMO were not detected in 6 batches.The similarities of 9 batches of SSMO were evaluated,which showed that the similarities of 7 batches were greater than 0.95,the similarity of 1 batch was lower than 0.8,and the similarity of 1 batch was between 0.8 and 0.9.The systematic clustering results showed that the 9 batches of SSMO were clustered into two categories,and the ratios of inulin/inulo-type oligosaccharides of category I meet to the regulations of this study,while another category not meet the regulations.ConclusionIn this paper,SSMO and SMO oligosaccharides were used as the research objects.Using modern chemical separation technology,the newly formed component structure and influencing factors of MO processed oligosaccharides were separated,identified and explored for the first time,which could provid a research idea for clarifying the processing mechanism.From the whole,animal,cell and computational simulation levels,the effect of raw and processed MO oligosaccharides components on the pharmacodynamics were systematically compared.Based on the mathematical model of"oligosaccharides-spectrum-effect" relationship,the Q-markers of MO processed products were screened and verified,as well as the similarities and differences in the change laws of SSMO and SMO were revealed.These studies could lay the foundation for scientifically standardizing the processing technology of MO.Based on Q-markers,a characteristic quality evaluation system for MO processed products were established,and a draft quality standard for MO processed products were formulated.It could provide scientific reference and theoretical support for the establishment of the exclusive quality standard of MO processed products.