| Prunellae spica,Xiakucao,is the dried spica of Prunella vulgaris L(PV)belonging to Labiaceae and it is widely used not only in clinical practice but also in the field of health products.With the rapid development of health industry and the increasing demand for PV resources,large-scale planting has been carried out in the places such as Queshan in Henan province,Qichun in Hubei province and Xinyu in Jiangxi province.As a Chinese herbal medicine cultivated on a large scale,the harvesting cost and time of Prunellae spica has become an important problem restricting its industrial development.In addition,the resource of stem and leaf is much larger than that of spica on the base of the biomass of different organs(stem,leaf and spica)of PV.Large amount of stem and leaf resources are discarded in the harvesting process of spica,resulting in a serious waste of resources.Therefore,it is of great significance for comprehensive utilization of resources to clarify the difference of chemical composition between stem,leaf and spica of PV and explore the utilization value of stem and leaf.The seeds of PV are easy to fall off in the process of post-harvest treatment and storage,resulting in a large number of seeds discarded as scrap waste.It can provide reference for comprehensive utilization of the abandoned resources of PV by digging into the utilization value of the PV seeds and localizing its research and development direction.Therefore,on the basis of extensive literature research at home and abroad,32 batches of samples of spica,stem,leaf and seeds and whole plant samples with different maturity were collected from the two main producing areas of PV in Queshan,Henan province and Xinyu,Jiangxi province.On the basis of qualitative and quantitative analysis and antioxidant activity study of four parts,the anti-inflammatory activity of water extracts from spica,stem and leaf and their active fractions were evaluated using nitric oxide(NO)release in the mouse macrophage(RAW264.7)induced by lipopolysaccharide(LPS)to clarify the anti-inflammatory effect and active components of stem and leaf.In order to clarify the utilization value of PV seeds,the differences between PV seeds and chia seeds and basil seeds were systematically compared concerning the appearance,turgidity and chemical compositions(phenolic acids,fatty acids and volatile oil)to explore the feasibility of using PV seeds in the development of functional food.The main results and conclusions are as follows:1.Study on chemical compositions and antioxidant activity of different parts of PVUltra high performance liquid chromatography tandem quadrupoles time of flight mass spectrometry(UPLC-QTOF-MS/MS)was used to detect the chemical constituents of spica,stem,leaf and seeds.A total of 117 compounds were detected,84 of which were detected for the first time,and 87 compounds were identified,including 32 phenolic acids and 45 triterpenoid saponins,among which there were 4 to 6 potential new triterpenoid saponins linked to monosaccharides.The main target triterpenoid saponins were isolated and identified by various column chromatography and spectral analysis methods,and two new compounds were obtained,which verified the results of mass spectrometry identification.Multivariate statistical analysis of the quality spectra of 32 batches of samples showed that the stem,leaf,spica and seeds samples clustered into one class,respectively.However,the stem,leaf and spica were closer to each other in general,and were clearly differentiated from seeds.The main different component was the high content of salviaflaside in seeds.Considering the weight proportion of seeds in spica(24.6%),it is speculated that the chemical composition of spica is the comprehensive embodiment of stem,leaf and seeds.In order to quantitatively compare the differences of phenolic acids in different parts of PV,a method was established by ultra performance liquid chromatography(UPLC)for simultaneous determination of six phenolic acids including danshensu,protocatechuic acid,protocatechuic aldehyde,caffeic acid,salviaflaside and rosmarinic acid.The results showed that danshensu,protocatechuic acid,protocatechuic aldehyde,caffeic acid,salviaflaside and rosmarinic acid were linear in the concentration range of 0.255-258,0.147-37.4,0.0320-32.6,0.143-145,0.249-254 and 0.510-520μg·mL-1.The precision,repeatability and stability met the requirements of quantitative analysis.The recoveries were 97.9%,89.8%,94.7%,95.9%,94.2%and 96.2%,respectively.The contents of danshensu,protocatechuic acid,protocatechuic aldehyde,caffeic acid,salviaflaside and rosmarinic acid in stem,leaf,spica and seeds were 0.00716%-0.550%,0.00249%-0.0255%,0.00114%-0.00831%,0.00182%-0.159%,0.00340%-0.339%and 0.203%-1.51%,respectively.Statistical analysis showed that the content of phenolic acids in seeds was lower than that in spica except for salviaflaside.The content of salviaflaside in spica was higher than that in stem and leaf,but there was no significant difference of other phenolic acids between stem and spica,and the content of procatechuic aldehyde and caffeic acid were lower than that in leaf.DPPH radical scavenging method was used to detect the antioxidant activities of 32 batches of samples from 4 parts,and the results showed that the antioxidant activities of stem and leaf were not significantly different from those of spica,but were significantly higher than those of seeds.The correlation between antioxidant activity and the content of phenolic acid in each sample was analyzed.The results showed that danshensu,procatechuic aldehyde,caffeic acid and rosmarinic acid were positively correlated with antioxidant activities,while salviaflaside was negatively correlated,suggesting that danshensu,protocatechuic aldehyde,caffeic acid and protocatechuic aldehyde were the important substances for the antioxidant activities of PV.2.Preparation and anti-inflammatory activity of extracts from PV spica,stem and leafPrunellae spica has anti-inflammatory activity and is often used to treat thyroiditis and mastitis.In order to determine the utilization value of stem and leaf,the anti-inflammatory effect of spica,stem and leaf in vitro was compared,and the active fractions were screened.The spica,stem and leaf were extracted with water,concentrated and dried to obtain S-ZTW and JY-ZTW.The water extract was divided into S-WXF,S-S,S-30%,S-50%,S-70%,S-anhydrous,JY-WXF,JY-S,JY-30%,JY-50%,JY-70%and JY-anhydrous by macroporous adsorption resin.UPLC-QTOF-MS/MS was used to characterize the components of each part,and the content of phenolic acids and total saponins in each part was determined by UPLC and perchloric acid colorimetry.The results showed that S-30%and JY-30%were mainly phenolic acids with rosmarinic acid,dedihydro-salvianolic acid B or their isomers,and the content of rosmarinic acid was 0.113%and 0.0574%,respectively.The eluting parts of 50%ethanol,70%ethanol and anhydrous ethanol are mainly triterpenoids.The contents of S-50%,S-70%and S-anhydrous total saponins were 30.0%,35.2%and 37.1%,respectively,and the contents of JY-50%,JY-70%and JY-anhydrous total saponins were 32.2%,50.8%and 40.3%,respectively.The NO release model of RAW264.7 cells induced by LPS was used to evaluate the anti-inflammatory activity of water extract and its parts.It was found that S-ZTW had significant inhibitory effect on NO release at 100μg·mL-1,and JY-ZTW had similar effect at 200μg·mL-1.However,there was no significant difference in anti-inflammatory activity between stem,leaf and spica at 200μg·mL-1.Further activity comparison showed that 30%ethanol,50%ethanol,70%ethanol and anhydrous ethanol fractions of spica,stem and leaf had significant inhibitory effect on NO release.The 50%of maximal effect(EC50)of S-30%,S-50%,S-70%and S-anhydrous were 82.7,85.4,66.6 and 46.0μg·mL-1,and EC50 of JY-30%,JY-50%,JY-70%and JY-anhydrous were 135,93.8,141 and 101μg·mL-1,respectively.The extracts with the best activity of spica,stem and leaf(S-anhydrous and JY-50%)and triterpenoid saponin 1 were selected respectively.The levels of interleukin-1β(IL-lβ)and interleukin-6(IL-6)in RAW264.7 cells induced by LPS were determined by enzyme-linked immunosorbent assay(Elisa).The results showed that at the concentration of 93.8μg·mL-1,JY-50%could effectively inhibit IL-6 production.The S-anhydrous and JY-50%at the concentration of 92.0μg·mL-1 and 187μg·mL-1 could effectively inhibit the inflammatory factor IL-1β.Compound 1 inhibited IL-1β and IL-6 at 94.0μg·mL-1.In conclusion,there was no significant difference in anti-inflammatory activity between stem and leaf extracts and spica at the same concentration of administration.After further enrichment,the anti-inflammatory effect of each part of stem and leaf was slightly lower than that of spica,and phenolic acid and triterpenoid saponins contributed to the overall anti-inflammatory effect.3.Study on seeds exploitation and utilization of PVThe seeds of PV are rich in salviaflaside and rosmarinic acid.Rosmarinic acid extract,as a natural antioxidant,has been widely used in food,medicine and cosmetics,and the extracts prepared from rosemary are also used as food additives.In order to fully explore the utilization value of seeds of PV and realize the comprehensive utilization of waste resources,the phenolic acid extract of PV seeds was prepared by enriching salviaflaside and rosmarinic acid from seeds of PV with macroporous adsorption resin.The results showed that the contents of salviaflaside and rosmarinic acid in 30%ethanol fraction were 25.8%and 6.94%,respectively,and the content of rosmarinic acid in 50%ethanol eluted fraction was 15.3%.In order to explore the feasibility of using phenolic acid extract of PV seeds as a food additive,it can be considered to directly elute with 50%ethanol after removing impurities with water,and both phenolic acids can be taken into account.The PV seeds swell with water,which are similar to those of commercially available meal replacements chia seeds and basil seeds.In order to explore the feasibility of using seeds as functional food,the characteristics,turgidity and chemical composition(phenolic acids,fatty acids and volatile oil)of seeds of PV were studied and compared with those of chia seeds and basil seeds.The results showed that the seeds of PV were smaller than those of chia seeds and basil seeds,and the seeds coat color was obviously different.The turgidity of PV seeds was 17.4,which was slightly lower than that of chia seeds(the turgidity is 25.2)and basil seeds(the turgidity is 35.6).The main phenolic acid components in the seeds of PV were salviaflaside and rosmarinic acid,with the contents of 0.579%and 0.392%,respectively,which were higher than those in chia seeds and basil seeds.The yield of n-hexane was 18.3%,palmitic acid,oleic acid,stearic acid,linoleic acid and α-linolenic acid,and the content of volatile oil in seeds was very low.In order to accurately determine the contents of 5 main fatty acids in PV seeds,a gas chromatography-mass spectrometry(GC-MS)method with n-pentadecane as internal standard was established.The contents of PV seeds,chia seed and basil seed,Sophora Flavescens seeds oil and 8 commercially available vegetable oils(linseed oil,walnut oil,sesame oil,camellia oil,grape seeds oil,perilla seeds oil,olive oil and eucommia seeds oil)were determined simultaneously.The results showed that the linear ranges of palmitic acid,oleic acid,stearic acid,linoleic acid and α-linolenic acid were 0.0250-0.800,0.128-4.05,0.0127-0.402,0.144-4.57 and 0.217-6.90g·L-1.Methodological verification met requirements.The PV seeds oil was rich in unsaturated fatty acids,in which the α-linolenic acid content was 50.0%,which was slightly lower than that of chia seeds(57.2%),similar to that of basil seeds(50.0%),and much higher than that of Sophora Flavescens seeds oil,eucommia seeds oil and linseed oil.In conclusion,the PV seeds have high expansion,rich phenolic acids and unsaturated fatty acids,especially high content of α-linolenic acid,which can be used for functional food development,so as to realize comprehensive utilization of the waste resources of PV. |
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