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OBJECTIVE: To investigate and apply the bioaccessibility of Cd in traditional Chinese medicines (TCMs) to human health risk assessment, and provide reference for the formulation of limit standards. METHODS: The contents of Cd in Coptis chinensis Franch., Ligusticum chuanxiong Hort., Lindera aggregate (Sims) Kosterm., and Curcuma kwangsiensis S.G. Lee et C.F. Liang were determined by inductively coupled plasma mass spectrometry (ICP-MS). The bioaccessibility of Cd in TCMs was investigated by in vitro physiologically based extraction test(PBET) digestion method. And the target hazard quotient (THQ) was applied to assess the health risks of Cd contents. RESULTS: The bioaccessibility of Cd for Coptis chinensis Franch., Ligusticum chuanxiong Hort., Lindera aggregate (Sims) Kosterm., and Curcuma kwangsiensis S.G. Lee et C.F. Liang was 49.0%, 40.0%, 44.9% and 65.7%, respectively. The results of risk assessment showed that before extraction, THQ values of Cd for Coptis chinensis Franch., Ligusticum chuanxiong Hort., Lindera aggregate (Sims) Kosterm., and Curcuma kwangsiensis S.G. Lee et C.F. Liang were 1.895, 1.045, 1.446 and 1.653, respectively; after extraction, THQ values of Cd for the four types of TCMs were 0.929, 0.418, 0.649 and 1.077, respectively. CONCLUSION: Cd in the four types of TCMs cannot be completely absorbed in the artificial gastrointestinal phase, and the risks of Cd are reduced after extraction. Except THQ for Curcuma kwangsiensis S.G. Lee et C.F. Liang, the THQ values are below the value of 1, indicating very low health risks for the other three types of TCMs after extraction. In this study, the bioaccessibility of heavy metals in TCMs is applied to the health risk assessment model in order to provide a scientific basis for objectively evaluating the health risks of heavy metals in TCMs and formulating a scientific and reasonable heavy metal limit standards.
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Objective To reveal fungal community composition and diversity in health and root rot of Coptis chinensis in rhizosphere soil. Methods High-throughput sequencing technology was used to characterize the fungal community composition, richness, and diversity of health and root rot Coptis chinensis in rhizosphere soil in Shizhu County of Chongqing Province. Spearman analysis was used to evaluate the correlation between soil physicochemical parameters and the first 35 most abundant fungal genera. Results More than 106 267 effective tags were obtained, and the community was composed of six phyla (Ascomycota, Zygomycota, Basidiomycota, Glomeromycota, Chytridiomycota, and Neocallimastigomycota). The fungal community diversity showed no significant difference in healthy and root rot samples. The relative abundance of Ascomycota, Basidiomycota and Chytridiomycota in root rot soil was significantly higher than that in healthy soil. Moreover, the relative abundance of Zygomycota, Glomeromycota, and Neocallimastigomycota in root rot soil was significantly lower than in healthy soil. The relative abundance of Fusarium was significantly higher in root rot samples than in healthy samples. Spearman analysis showed that the relative abundance of Fusarium was significantly positively correlated with pH and available phosphorus, but negatively correlated with alkali-hydrolyzable nitrogen. Conclusion Changes in soil physicochemical characteristics were related to the changes in soil fungal diversity.
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Objective To explore the effect of Coptis chinensis on the intestinal microflora diversity of rats with type 2 diabetes mellitus and further elucidate the mechanism of C. chinensis in treatment of type 2 diabetes mellitus. Methods SD rats were randomly divided into control group (CON), model group (M), C. chinensis group (HL), and positive control group (POS), 10 in each group respectively. Type 2 diabetes mellitus model was caused by special diet fed and ip injection of STZ together. CON with M, HL, and POS was respectively given gavage volume 0.5% CMC-Na, 4.36 g/kg C. chinensis water extract and 0.097 g/kg of metformin. The body weight and blood glucose were monitored once a week separately. After four weeks of administration, the oral glucose tolerance and insulin level were measured. After four weeks of administration, the contents of rectum were collected and sequenced by a 16 S rRNA gene sequencing. Results A total of 951OTU, 15 Phylum, 25 Class, 43 Order, 69 Family, 182 Genus and 357 Species were obtained. At the level of Phylum, the content of Actinobacteria in M was significantly higher than that in CON and HL (P < 0.05), while Deferribacteres was only detected in M. In M, Firmicutes, Spirochaetae, Tenericutes, and Elusimicrobia were higher than CON. Interestingly, they were all reduced after giving C. chinensis, meanwhile the abundance of Proteobacteria, Fusobacteria and Verrucomicrobia in HL were slightly higher than that in CON and M. Conclusion Actinobacteria and Deferribacteres were probably potential markers in type 2 diabetes mellitus. After administrating C. chinensis, the change of intestinal microflora diversity revealed C. chinensis could improve the intestinal microflora disorder of type 2 diabetes mellitus.
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Objective:To study the anti-inflammatory effect of the ethanol extract of Coptis chinensis Franch.in vitro.Methods: An inflammatory cell model was established by stimulating the mouse peritoneal macrophages in vitro with lipopolysaccharide(LPS).LPS stimulated of RAW264.7 cells for a long time after administration of intervention.Effect of ethanol extract of Coptis chinensis Franch.on RAW264.7 cell growth activity was analyzed by MTT assay.The production of tumor necrosis factor-α(TNF-α),IL-1β,IL-6,NO,prostaglandin E2(PGE2) was determined by ELISA.The expression of mRNA of TNF-α,induced nitric oxide synthase(iNos) and HO-1 was detected by real-time fluorescent quantitative PCR(RT-PCR).Results: The ethanol extract of Coptis chinensis had no inhibition effect on the scope of RAW 264.7 cells the scope of 5-80 mg/L.Each treatment group concentration of interleukin-6 (IL-6),interleukin-1β (IL-1β),TNF-α,NO,prostaglandin E2 (PGE2) content of LPS stimulation model group were significantly (P<0.01),and content was not related to concentration.Real-time quantitative (RT-PCR) showed,the concentration of each treatment group were significantly lower iNos,HO-1 and TNF-α mRNA expression (P<0.05,P<0.05,P<0.01),and content was not related to concentration.Conclusion: Coptis chinensis Franch.ethanol extract has anti-inflammatory effects in vitro,the mechanism may be related to inhibition of TNF-α,NO and other inflammatory factors and the impact of the activation of arachidonic acid (AA) metabolism.
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Objective: Using exogenous carbon monoxide (CO) donor Hematin (H) to study the changes of photosynthesis parameters and chlorophyll fluorescence characteristics in Coptis chinensis seedlings under NaCl stress and get the way for promoting the resistance ability of C. chinensis seedlings under NaCl stress. Methods: Under 100 mmol/L NaCl stress, the several physiological indexes of C. chinensis seedlings treated by H at the different concentration, such as the contents of photosynthetic pigment and the parameters of chlorophyll fluorescence and photosynthesis, were measured. Results: Under NaCl stress the photosynthetic physiology of C. chinensis seedlings was inhibited, but the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids could be obviously increased after the treatment of H at different concentration. The levels of the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) in C. chinensis seedlings were all decreased, which would gradually increase with the time and concentration increase of NaCl stress, while the concentration of CO2 (Ci) increased with the opposite tendency. These results indicated that the main influencing factor of decreasing Pn of C. chinensis was non-stomatal factors. After being treated with different exogenous substances, the Pn, Gs, and Tr were all increased in different degrees, but the level of Ci was decreased. And the maximum fluorescence (Fm), photochemical efficiency of photosystem II (Fv/Fm), potential photochemical efficiency (Fv/Fo), photochemical efficiency (Fv'/Fm'), PSII actual photochemical efficiency (ΦPSII), photochemical quenching coefficient (qP), electronic transfer rate (ETR), and photochemistry rate (PCR) were obviously increased, while the levels of minimal fluorescence (Fo), non-photochemical quenching coefficient (NPQ), and heat dissipation rate (HDR) were effectively decreased. Conclusion: Exogenous CO H with the appropriate concentration of 2.0 mmol/L could reduce the excess excitation dissipation, improve photochemical electron transport efficiency, and efficiently protect C. chinensis leaves from PSII damage by significantly alleviating the damages of salt stress to C. chinensis so that H could obviously promote the salt resistance to C. chinensis under salt stress.
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Objective: To find get the method of alleviating salinity damage to Coptis chinensis under NaCl stress by studying the seed generation and seedling physiological nature. Methods: Several physiological indexes such as germination vigor, germination rate, germination index, and vigor parameters of C. chinensis were measured after treated by 5-aminolevulinic acid (ALA) under salt stress (NaCl with the concentration of 100 mmol∙L-1 NaCl). And other parameters such as memberane permeability, H2O2 content, production rate of superoxide anion, contents of soluble surge, soluble protein, free proline, and malondialdehyde (MDA), and activities of superoxide (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were also measured. Results: The seed germination indexes of C. chinensis under NaCl stress have an obvious inhibition. But after the treatment of ALA, germination indexes were all increased. The results also showed that the treatment of exogenous ALA obviously increased the contents of soluble sugars, free proline, and soluble protein, decreased the contents of MDA, H2O2, and production rate of superoxide anion. Meanwhile, the results also indicated that ALA could improve the activities of SOD, POD, CAT, and APX. Conclusion: Exogenous ALA with appropriate concentration could significantly alleviate the damages to the seeds and seedlings of C. chinensis under NaCl stress and promote the salt resistance of the seeds and seedlings through improving the germination indexes and activities of anti-oxidase, decreasing the memberane permeability, and so on.
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Objective: To establish the system of tissue culture regeneration and gene transformation for Coptis chinensis. Methods: The cotyledon and hypocotyl of C. chinensis were used as explants, and the effects of different basic media with different plant growth regulators on in vitro tissue culture regeneration were compared; The embryogenic calli of C. chinensis were used as recipients for genetic transformation by particle bombardment method. Results: The induction rates of cotyledon and hypocotyl were 86.31% and 54.34%, respectively; The 6, 7-V medium was more suitable for callus induction and proliferation rate of C. chinensis than the MS medium. The optimal hormone combination for callus induction was 0.5 mg/L 2, 4-D + 0.5 mg/L KT in MS medium; Either the appropriate concentration of cytokinin alone or the combination of both cytokinin and auxin could induce the continuous proliferation of calli; However, only the hormone combination 0.5 mg/L KT + 0.5 mg/L IAA and 1.0 mg/L 6-BA + 0.5 mg/L NAA could generate the somatic embryos. 6, 7-V medium in absence of hormone could maintain the continuous growth of the embryogenic calli. The somatic embryos could germinate and grow up to plantlets on 6, 7-V medium containing 1 mg/L GA3 + 0.5 mg/L IBA. The embryogenic calli were transformed by particle bombardment method and screened under 3 mg/L Basta, then the activity of β-glucuronidase was detected. PCR analysis showed that the bar gene was successfully transferred to the regenerated plants. Conclusion: The tissue culture regeneration and genetic transformation system of C. chinensis is established, which lays the foundation for its genetic improvement.
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Objective: To study the effect of nitrogen (N), phosphorus (P), and potassium (K) fertilizers on the sclerotial formation and germination of southern blight of Coptis chinensis so as to provide scientific basis for its integrated pest management (IPM). Methods: Preparation of different concentrations of N, P and K was added into soil and the mycelium and sclerotia were put on the soil, and then the effect of the fertilizers on the sclerotial formation and germination was observed. Results: The treatment of CO(NH 2)2 and NH4HCO3 had significant inhibition on sclerotial formation and germination. The treatment of NaH 2PO4, KCl, and KNO3 showed the facilitation in various degrees to promote the sclerotial formation and germination. The treatment of KH2PO4 had no significant effect on sclerotial germination, but it had significant inhibitory activity to sclerotial formation. Conclusion: In the production, the partial N, while less K and P fertilizers should be put to control the sclerotial formation and germination of southern blight of C. chinensis.
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Objective: To research the in vitro biotransformation characteristics of cinnamaldehyde in rat intestine and the influence of Coptis chinensis on its metabolism. Methods: RP-HPLC Method was applied for analyzing the content of cinnamaldehyde as well as its metabolite cinnamic acid. The stability of cinnamaldehyde in extracts of rat small intestinal mucosa side, serosal side, and homogenate was investigated, respectively. Also the impact of C. chinensis on this process was estimated. Results: The results showed that cinnamaldehyde in rat intestine was unstable and could rapidly be transformed into cinnamic acid. C. chinensis had inhibitation on cinnamaldehyde metabolism. Conclusion: Cinnamaldehyde could be metabolized in rat different intestinal extracts immediately and the main metabolite is cinnamic acid. C. chinensis shows inhibiting effect on the cinnamaldehyde metabolism.
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Objective To establish a determining method for the content of berberine hydrochloride in Coptis chinensis Franch.and Xianglian pills.Method The sample was extracted and developed,the berberine hydrochloride was determined by thin layer chromatograph-fluorescence of spectrophotometry with Benzen-ethylaceate-isopropanol-methanol-ammonia water(6:3:1.5:1.5:0.5) as the developing system and Ex=365 nm,Em=409 nm.Result The calibration curve was linear in the range of 0.473～ 3.784 ?g,r=0.999 3.The recovery rate in Coptis chinensis Franch.was 99.03%,RSD=1.86%,and in Xianglian pills was 97.10%,RSD=1.09%.Conclusion The method is simple and accurate.
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Object To establish the fingerprint spectrum of Coptis chinensis Franch. by HPCE. Methods The correlation relativity of retention time and peak area of each chromatographic peak in different batches of medicinal materials were explored respectively, and beberine was chosen as the reference to calculate the relative retention time and the ratio of peak area for each chromatographic peak. Results The correlation coefficients of relative retention time and the ratio of each chromatographic peak were all higher than 0.999 0 in different batches of medicinal materials. Conclusion The established fingerprint spectrum is stable and credible, and could be used as characteristic fingerprint spectrum for C. chinensis.
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Object To investigate the berberine in Coptis chinensis Franch. processed with various quantity of Evodia rutaecarpa (Juss.) Benth. and the components absorbed from E. rutaecarpa. Methods Taking berberine, evodiamine and rutaecarpine as targets, HPLC method was used to determine the components of C. chinensis, E. rutaecarpa, C. chinensis processed with 10%, 20%, 30%, 40%, 50% E. rutaecarpa. Results The content of berberine in C. chinensis processed with E. rutaecarpa decreased, and that of C. chinensis processed with 20% and 30% E. rutaecarpa was higher than the rest. C. chinensis processed with E. rutaecarpa absorbed the components of E. rutaecarpa really. Conclusion The suitable quantity of E. rutaecarpa is 20% when processing for C. chinensis.
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Object To study the regularity of changes on quality and yield of Coptis chinensis Franch. with its different age and month at different altitude. Methods Weighting by stoving and colorimetric analysis were used to determine the content of berberine and the weight of C. chinensis in different age and month at different altitude. Results The yield and quality of C. chinensis grown in low altitude region are better than that in high altitude region; the wight of C. chinensis in five years old is the biggest. The content of berberine of C. chinensis in six years old is the highest. And the weight and berberine content of C. chinensis in July is the highest in one year. Conclusion Results of the study may provide references for C. chinensis production and the selection of the best harvest period.
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Objective To discuss the genetic diversity of Coptis chinensis.Methods The genetic diversity of 78 individuals from seven populations was analyzed by inter-simple sequence repeat(ISSR).Results Twelve primers were selected to produce highly reproducible ISSR bands.Among 106 amplified bands,72 showed polymorphism,the percentage of polymorphic bands reached to 67.92%.Nei's gene diversity index(H)was 0.180 3,Shannon information index(I)was 0.283 2,Gst was 0.681 5.The genetic distance coefficient and genetic similarity were 0.089 4—0.184 6 and 0.832 1—0.912 7,respectively.ConclusionC.chinensis holds high genetic diversity and the majority of genetic variation occurs among the populations.By cluster analysis,the geographical distribution is very obvious.The ISSR marker could be used for the analysis of the genetic diversity and genetic variation of C.chinensis.
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Objective To reveal the genetic relationship among populations of cultivated Coptis chinensis.Methods Twenty four populations of cultivated C.chinensis from different habitats were employed to be analyzed by the approach of sequence-related amplified polymorphism(SRAP).Systematic relationship was constructed based on the UPGMA method by Treeconw software.Results A total of 276 bands were scored,among which 120 were polymorphic bands.The average percentage of polymorphic bands was 43.48%,indicating that the materials in the test have low genetic diversity.Genetic similarity coefficients were changed from 0.877 0 to 0.951 9.By cluster analysis,the geographical distribution was not very obvious,but it was also showed some of the cultivated C.chinensis from the same region were in the same group.Conclusion Different germplasms diversity of cultivated C.chinensis population is lower and genetic background is more single.
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Objective To clone and sequence the cDNA encoding(S)-N-methylcoclaurine-3'-hydroxylase from Coptis chinensis.Methods The cDNA,encoding(S)-N-methylcoclaurine-3'-hydroxylase,was amplified by RT-PCR with cDNA library of tender leaf as the template.Results The full-length cDNA of(S)-N-methylcoclaurine-3'-hydroxylase(named as CYP80B3) had 1 680 bp with an open reading frame encoding 488 amino acids of protein.The CYP80B3 had 95%,82%,70%,and 68% amino acid sequence homology to the sequence of(S)-N-methylcoclaurine-3'-hydroxylase from C.japonica,Thalictrum flavum,Eschscholzia californica and Papaver somniferum,respectively.The sequence was reported to the GenBank and coded as EF492879.Comparison of sequence with(S)-N-methylcoclaurine-3'-hydroxylase from C.japonica showed CYP80B3 possessed the same functional regions involved with 3'-hydroxylation of(S)-N-methylcoclaurine.Conclusion The cDNA encoding CYP80B3 from C.chinensis was cloned and reported.This work underlays the first step for exploring the pathway of benzylisoquinoline alkaloid biosynthesis and for improving the content of benzylisoquinoline alkaloid in C.chinensis.
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0.05), but the yields of four-year-old and five-year-old Dahuaye and Wuguangye samples were significantly higher than that of Xiaohuaye sample (P0.05). Conclusion The varieties of Dahuaye and Wuguangye are excellent in higher yield and better quality, and both of them can be used as origin seeds in the GAP plantation of C. chinensis.