| Hibiscus manihot L.belongs to the Abelmoschus genus of Malvaceae family,and is an annual berb,which is a rare plant on the verge of extinction during the census of agricultural resources in the late 1980s.It possesses the efficacy of qingli dampness and heat,anti-inflammatory and analgesic acitivities,internal use of the main five drench,edema,external use of decoction scald and other pharmacological activities.Under the guidance of anti-inflammatory activity,the optimal anti-inflammatory effect of H manihot L.flower extract was determined.Then the chemical components of H manihot L.flower were systematically isolated and purified and their structures were identified for the first time.An efficient and rapid HPLC method for quality control analysis of active compounds in H.manihot L.was developed by using modern analytical method;The contents of active constituents and their antioxidant and anti-inflammatory activities at different harvest dates in northeast region of China were investigated,and the best harvest time was comfirmed;The extraction process conditions of new green extraction solvent-tea saponin solution combined with negative pressure cavitation extraction were optimized;Preparative separation and purification of target compounds from H.manihot L.flower extracts by high-speed countercurrent chromatography combined with macroporous adsorption resin were developed.The present research provided scientific basis and application technical foundation for the full exploitation and utilization of H.manihot L.resources.The results were as follows:1.Nineteen compounds were obtained from the anti-inflammatory active fraction of H.manihot L.flower extracts by using a variety of chromatographic techniques such as silica gel column chromatography,Sephadex LH-20 gel column choromatography,ODS-C18 column chromatography,high speed counter-current chromatography,et al.On the basis of chemical and spectroscopic evidences including IR,MS,1H-NMR and 13C-NMR,seventeen compounds were identified as follows:?-sitosterol(1),daucosterol(2),palmitic acid(3),heptadecanoic acid ethyl ester(4),linoleic acid methyl ester(5),hexacosendioic acid(6),quercetin(8),quercetin-3'-O-?—D-glucoside(9),myricetin(10)?hibifolin(11),hyperin(12),isoquercetin(13),rutin(14),chlorogenic acid(16),neochlorogenic acid(17),caffeic acid(18),quercetin-3-O-robibioside(19).Among them,compounds 1,2,4,5,8,9,11,14,16,17,18,19 were isolated from H.manihot L.flower for the first time.The structures of other two compounds 7 and 15 are identifying.2.A RP-HPLC method for quality control analysis was developed to simultaneously determine active ingredients in H.manihot L.,the chromatographic conditions as follows:Chromatographic column:HIQ sil C18W column(250 mm×4.6 mm i.d.,5 ?m);Mobile phase consisted of 0.5%phosphoric acid aqueous solution(A)and acetonitrile(B);Gradient elution:0-40 min,90-83%(A);40-59 min,83-67%(A);59-62 min,67-90%(A);Detection wavelength:330 nm and 254 nm;Flow rate:1 mL/min;Column temperature:30?;Injection volume:5 ?L.Under the conditions above,ten active ingredients in H.manihot L.were separated and determined for the first time.The developed method was high sensitivity,high precision and good repeatability,and was suitable for the quality control analysis of active ingredients in H.manihot L.,which provided necessary basis and means of detection for the research,development and efficient utilization of H.manihot L.resources.3.The contents of active ingredients and their antioxidant and anti-inflammatory activities in H.manihot L.from different harvest dates in northeast region of China were investigated.The variation of the active ingresients and antioxidant activities of H.manihot L.samples during different harvest dates were determined.The total phenolic and total flavonoid contents in the samples harvested at the beginning of August was higher,and the samples harvested at the beginning of August possessed excellent DPPH and ABTS radical scavenging capacity,reducing power and FRAP activity.The samples harvested at the beginning of August showed obviously higher ABTS radical scavenging capacity,reducing power and FRAP activity,which was also higher than the positive control.Hyperin,isoquercetin,hibifolin and quercetin-3'-O-?-D-glucoside were the main active compounds of H.manihot L.flower analyzed by HPLC.The contens of these four active compounds were higher at the beginning of August samples.Principal component analysis(PCA)was applied to evaluate the quality of samples with different harvest dates.The results showed that the samples harvested at the beginning of August had higher active ingredients and biological activities,which was the best time period for H.manihot L.flower quality.4.The process of surfactant-based negative pressure cavitation extraction of active compounds from H.manihot L.flower were optimized.The effects of the types of surfactant,concentration of surfactant,negative pressure,concentration of ethanol,liquid/solid ratio,extraction time and extraction temperature on the extraction yields were studied.The optimum parameters were as follows:Concentration of surfactant:0.5w/v%tea saponin(TS)Concentration of ethanol:60%Liquid/solid ratio:53:1 mL/gExtraction time:16 minExtraction temperature:61?Under the optimal parameters,the average extraction yields of rutin(RU),hyperin(HY),isoquercetin(ISQ),hibifolin(HI),myricetin(MY),quercetin-3'-O-glucoside(QOG)and quercetin(QU)were 0.297,6.188 4.847,7.334,0.174,0.627 and 0.336 mg/g,respectively.The results showed that surfactant-based negative pressure cavitation extraction(S-NPCE)was a green,effective,low energy consumption and high extraction yield extraction technique.Meanwhile,the surfactant selected possessed the advantages of environmental protection and low cost,which had great application potential and was suitable for efficient extraction of natural active compounds from plant materials.Moreover,molecular docking prediction and FT-IR validation experiments also proved that tea saponin solution could be used as an extraction solvent coupled with NPCE or other extraction methods to synergistically extract quercetin and other target flavonoids from H.manihot L.flower by forming intermolecular hydrogen bonds between the target molecules and tea saponin.In hence,the experimental results also provided important scientific basis for the rational development and comprehensive utilization of H.manihot L.resources.5.Separation and purification of chemical constituents from H.manihot L.flower extracts were achieved by macroporous adsorption resin and high-speed countercurrent chromatography.The operating parameters as follows:(1)The enrichment and separation conditions of macroporous adsorption resin were optimized:Trade name:AB-8Sample volume:3 BVDesorption solution and volume:3 BV 40%ethanol and 8 BV 70%ethanolUnder the optimal parameters,the contents of seven target compounds in the enrichment products of H.manihot L.reached 0.28%,5.45%,4.17%,6.70%,0.15%,0.59%and 0.32%with recovery yields of 84.23%,79.57%,77.84%,82.3%,78.19%,85.41%and 87.03%,respectively.(2)The separation and purification conditions of AB-8 resin enrichment products by high-speed countercurrent chromatography were determined:Solvent system:Ethyl acetate:Methanol:Water=100:3:100,v/v/vElution flow rate:2 mL/minElution speed:900 rpm/minDetection wavelength:254 nmUnder the above optimized conditions,after one run treatment by macroporous adsorption resin,high-speed countcurrent chromatography and recrystallization,chlorogenic acid,caffeic acid,quercetin-3-O-robibioside,rutin and hibifolin with purity of more than 95%were obtained,respectively.And the recovery yields were 94.37%,96.58%,95.21%,97.69%and 96.44%,respectively.Solvent system:n-Hexane:Ethyl acetate:Methanol:Water=1:5:1:5,v/v/v/vElution flow rate:2 mL/minElution speed:850 rpm/minDetection wavelength:254 nmUnder the above optimized conditions,after one run treatment by macroporous adsorption resin,high-speed countcurrent chromatography and recrystallization,hyperin,isoquercetin,hibifolin and quercetin-3'O-?-D-glucoside with purity of more than 95%were obtained,respectively.And the recovery yields were 93.91%,98.02%,95.78%and 94.81%,respectively.The developed technique was characterized by reusable solvent,high purity and easy scale-up.High-speed countcurrent chromatography was more suitable for the rapid separation and purification of natural chemical constituents in H.manihot L.compared with the conventional separation medium silica gel as the stationary phase.In addition,HPLC and ESI-MS/MS were used to verify the chemical structure of the purified compounds.6.The scale-up experiment of extraction and enrichment of chemical comstituents in H.manihot L.flowerThe extraction yields of seven target compounds in H.manihot L.flower were 0.267 mg/g,5.445 mg/g,4.411 mg/g,6.161 mg/g,0.160 mg/g,0.545 mg/g and 0.294 mg/g,respectively,which were similar to those obtained in the laboratory experiment.The results showed the vacuum cavitation suspension solid-liquid extraction separation device could achieve a better extraction efficiency on the target compounds in H.manihot L.flower.In the scale-up experiment of enrichment,the contents of seven target compounds in the enrichment products achieved 0.25%,5.13%,3.58%,6.29%,0.13%,0.51%and 0.27%with the recovery yields of 83.98%,80.07%,78.21%,81.49%,77.68%,86.27%and 87.45%,which were consistent with the results of laboratory experiments.All the results indicated that the extraction and enrichment process of chemical constituents in H.manihot L.flower was stable and reliable,which could provide the data support for the large-scale industrialization and the scientific basis for the efficient utilization and development of resources. |
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