[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

[Quality evaluation of Huocao based on UPLC fingerprint and multi-component content determination].

Huocao(a traditional Chinese herbal medicine) moxibustion is a characteristic technology in Yi medicine suitable for cold-dampness diseases. Huocao, as the moxibustion material, is confusedly used in clinical practice and little is known about its quality control. In this study, UPLC method was used to establish the chemical fingerprint of non-volatile components in Huocao, and the contents of eight phenolic acids such as chlorogenic acid were determined. Multivariate statistical analysis was performed to obtain the indicator components of Huocao for quality evaluation, and thus a comprehensive evaluation system for the quality of Huocao was built. The UPLC fingerprints of 49 batches of Huocao were established, and there were 20 common peaks, of which eight phenolic acids including neochlorogenic acid and chlorogenic acid were identified. Except for three batches of Huocao, the similarity of the other 46 batches was higher than 0.89, suggesting that the established fingerprint method could be used for quality control of the medicinal herb. The correlation coefficient between entropy weight score of the eight phenolic acids and comprehensive fingerprint score in Huocao was 0.875(P<0.01), which indicated that the eight phenolic acids could be used as indicator components for the quality evaluation of Huocao. Furthermore, in multivariate statistical analysis on the common peaks of fingerprint and the contents of the eight phenolic acids, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C were screened to be the indicator components. The results revealed that the proposed method achieved a simple and accurate quality control of Huocao based on UPLC fingerprint and multi-component content determination, which provided useful data for establishing the quality standard of Huocao.

[Main components from cultivated and wild Nardostachyos Radix et Rhizoma by LC-MS and GC-MS].

In this study, ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry(UPLC-Q-TOF-MS) and gas chromatography-mass spectrometry(GC-MS) were combined with non-targeted metabonomic analysis based on multivariate statistics analysis, and the content of five indicative components in nardosinone was determined and compared by UPLC. The main chemical components of Nardostachyos Radix et Rhizoma with imitative wild cultivation and wild Nardostachyos Radix et Rhizoma were comprehensively analyzed. The results of multivariate statistical analysis based on liquid chromatography-mass spectrometry(LC-MS) and GC-MS were consistent. G1 and G2 of the imitative wild cultivation group and G8-G19 of the wild group were clustered into category 1, while G7 of the wild group and G3-G6 of the imitative wild cultivation group were clustered into category 2. After removing the outlier data of G1, G2, and G7, G3-G6 of the imitative wild cultivation group were clustered into one category, and G8-G19 of the wild group were clustered into the other category. Twenty-six chemical components were identified according to the positive and negative ion modes detected by LC-MS. The content of five indicative components(VIP>1.5) was determined using UPLC, revealing that chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, linarin, nardosinone, and total content in the imitative wild cultivation group were 1.85, 1.52, 1.26, 0.90, 2.93, and 2.56 times those in the wild group, respectively. OPLS-DA based on GC-MS obtained 10 diffe-rential peaks. Among them, the relative content of α-humulene and aristolene in the imitative wild cultivation group were extremely significantly(P<0.01) and significantly(P<0.05) higher than that in the wild group, while the relative content of 7 components such as 5,6-epoxy-3-hydroxy-7-megastigmen-9-one, γ-eudesmol, and juniper camphor and 12-isopropyl-1,5,9-trimethyl-4,8,13-cyclotetrade-catriene-1,3-diol was extremely significantly(P<0.01) and significantly(P<0.05) lower than that in the wild group, respectively. Therefore, the main chemical components of the imitative wild cultivation group and wild group were basically the same. However, the content of non-volatile components in the imitative wild cultivation group was higher than that in the wild group, and the content of some volatile components was opposite. This study provides scientific data for the comprehensive evaluation of the quality of Nardostachyos Radix et Rhizoma with imitative wild cultivation and wild Nardostachyos Radix et Rhizoma.

[Chemical constituents and anti-liver fibrosis mechanism of Meconopsis quintuplinervia based on UPLC-Q-Exactive-MS/MS and network pharmacology].

In this study, UPLC-Q-Exactive-MS/MS was used to rapidly analyze the chemical constituents of Meconopsis quintupli-nervia, and the anti-liver fibrosis mechanism of M. quintuplinervia was preliminarily analyzed by network pharmacology, molecular docking, and cell experiments. The chemical constituents of M. quintuplinervia were identified according to the information of MS~1 and MS~2, as well as the data in the literature and databases. SwissTargetPrediction and TargetNet were used to predict the potential targets. The targets related to liver fibrosis were collected from GeneCards and OMIM. The protein-protein interaction(PPI) network was constructed by STRING. Cytoscape 3.6.1 was used to construct and analyze the "constituent-target-disease" network to obtain key targets and their corresponding constituents in the network. DAVID 6.8 was used for GO analysis and KEGG signaling pathway enrichment analysis. Finally, the preliminary verification was carried out by molecular docking and cell experiments. As a result, 106 chemical constituents were identified from M. quintuplinervia, including 66 flavonoids, 16 alkaloids, 18 phenolic acids, 1 anthocyanin, and 5 other constituents. Among them, 3 constituents were identified as potential new compounds, and 59 constituents were reported in M. quintuplinervia for the first time. Network pharmacology analysis showed that M. quintuplinervia presumably acted on AKT1, SRC, JUN, EGFR, STAT3, HSP90 AA1, MAPK3, and other core targets through luteolin, isorhamnetin, quercetin, apigenin, kaempferide, amurine, 2-methylflavinantine, allocryptopine, the multi and other active compounds, thereby regulating the PI3 K/AKT signaling pathway, pathways in cancer, proteoglycans in cancer, FoxO signaling pathway, and other pathways to exert anti-liver fibrosis effects. M. quintuplinervia extract(MQE) could significantly down-regulate PI3 K and AKT protein levels in the HSC-T6 cell model induced by TGF-ß1, suggesting that MQE may have the ability to regulate the PI3 K/AKT signaling pathway. The findings of this study indicated that the anti-liver fibrosis effect of M. quintuplinervia had multi-constituent, multi-target, and multi-pathway characteristics, which may provide a scientific basis for the research on the pharmacodynamic materials, action mechanism, and quality markers of M. quintupli-nervia.

[UPLC fingerprint and indicator components of Boenninghausenia albiflora var. albiflora].

The fingerprint of Boenninghausenia albiflora var. albiflora was established by ultra performance liquid chromatography(UPLC), and the content of 12 active components including chlorogenic acid was determined. Multivariate statistical analysis was used to explore the indicator components of B. albiflora var. albiflora and a comprehensive evaluation system was created for the quality of B. albiflora var. albiflora. In this study, 33 batches of B. albiflora var. albiflora with different sources were collected and studied, and the UPLC fingerprint of B. albiflora var. albiflora was developed. There were 37 common peaks, of which 12 components were identified, and the content of these 12 components was measured. In combination of the common peaks and the content of chemical components, multivariate statistical analysis was performed, and the results showed that 6 components [daphnoretin, isoimperatorin, astragalin, imperatorin, neochlorogenic acid, and isoquercitrin(weight coefficient>0.1)] were selected as chemical markers for the quality of B. albiflora var. albiflora. Technique for order of preference by similarity to ideal solution(TOPSIS) analysis and chemometrics revealed that the quality of S32, S28 and S29 were superior, while that of S12, S7 and S16 were inferior. The quality evaluation method of B. albiflora var. albiflora constructed in this study was accurate and reliable, with simpleness and easiness to operate. It is suggested that the 6 above-mentioned active components could be used as indicator components for quality control of B. albiflora var. al-biflora. The samples were harvested during the flowering and fruiting period, which is from the beginning of July to the end of August.

[Randomized controlled trial on cervical spondylotic radiculopathy of wind-cold-damp type treated with acupuncture and thunder-fire moxibustion].

OBJECTIVE: To explore the therapeutic effect of acupuncture combined with thunder-fire moxibustion on cervical spondylotic radiculopathy of wind-cold-damp type and its impacts on the conduction velocity of F wave of median nerve and ulnar nerve, as well as hypersensitive C-responsive protein (hs-CRP), interleukin 6 (IL-6), IL-1ß and tumor necrosis factor α (TNF-α) in serum. METHODS: A total of 94 patients with cervical spondylotic radiculopathy of wind-cold-damp type were randomly divided into a control group and a combined therapy group, 47 cases in each group. In the control group, acupuncture was applied to Fengchi (GV20), Tianzhu (BL10), Neck-Jiaji (EX-B5), Dazhui (GV14) and others, 30 min each time, once daily, 5 times a week, totally for 4 weeks. In the combined therapy group, on the base of the treatment as the control group, the thunder-fire moxibustion was adopted over GV20, EX-B5, GV14, Jianjing (ST21) and Tianzong (SI11), 20 min each time, once every other day, for 4 weeks in total. After the treatment, the curative effect was compared in the patients between two groups. The short-form McGill pain questionnaire (SF-MPQ), the neck specificity scale and the MOS 36-item short form health survey (SF-36) were scored. The conduction velocity of F wave in median nerve and ulnar nerve was detected by electromyography. The expression of hs-CRP was measured by immunoturbidimetry. IL-6, IL-1ß and TNF-α in serum were determined by enzyme linked immunosorbent assay. RESULTS: The total effective rate in the combined therapy group was 95.7% (45/47), which was higher than 80.9% (38/47) in the control group (P<0.05). Compared with their own pretreatment, the scores of SF-MPQ, neck specificity scale and SF-36 were all obviously improved after treatment in the patients of either group (P<0.05), while the conduction velocity of F wave in median nerve and ulnar nerve was accelerated and hs-CRP, IL-6, IL-1ß and TNF-α in serum were decreased (P<0.05). Compared with the control group, the improvements in the scores of SF-MPQ, neck specificity scale and SF-36 were more obvious (P<0.05), the increase of the conduction velocity of F wave in median nerve and ulnar nerve and the decrease of hs-CRP, IL-6, IL-1ß and TNF-α in serum were more remarkable (P<0.05) in the combined therapy group. CONCLUSION: Acupuncture combined with thunder-fire moxibustion relieves the clinical symptoms of cervical spondylotic radiculopathy of wind-cold-damp type.

[Diarrhea-predominant irritable bowel syndrome of spleen deficiency and damp excess treated with fire needling therapy with filiform needle and acupoint application therapy: a randomized controlled trial].

OBJECTIVE: To compare the clinical therapeutic effect on diarrhea-predominant irritable bowel syndrome (IBS-D) of spleen deficiency and damp excess among fire needling therapy with filiform needle combined with acupoint application therapy, simple acupoint application therapy and pinaverium bromide tablets, and explore the mechanism on the improvements in IBS-D. METHODS: A total of 150 patients with IBS-D of spleen deficiency and damp excess were randomized into a combined treatment group (50 cases, 14 cases dropped off), an acupoint application group (50 cases, 16 cases dropped off ) and a western medication group (50 cases, 13 cases dropped off ). In the western medication group, pinaverium bromide tablets were prescribed for oral administration, 50 mg per time, three times a day. In the acupoint application group, the herbal plaster of shenling baizhu powder mixed with fresh ginger juice was placed on Shenque (CV 8), once every two days. In the combined treatment group, on the base of the treatment as the acupoint application group, the fire needling therapy with filiform needle was exerted at Tianshu (ST 25), Shangjuxu (ST 37), Yinlingquan (SP 9) and Dachangshu (BL 25), once every two days. The duration of treatment in each group was 4 weeks. Separately, before treatment and after 7 and 28 days of treatment, the score of clinical symptoms and the score of quality of life scale for IBS (IBS-QOL) were observed in each group. Before treatment and after 28 days of treatment, the levels of , and T lymphocytes, and the ratio of / in peripheral blood of patients were detected successively; as well as the expressions of 5-HT3R mRNA and 5-HT4R mRNA in colon mucosal tissues in each group separately. RESULTS: Except the score for abdominal pain in the acupoint application group and the scores for abdominal pain and abdominal distention in the western medication group after 7 days of treatment, the scores for abdominal pain, abdominal distention, defecation frequency, stool form and incomplete bowel movement after 7 and 28 days of treatment were all reduced as compared with those before treatment in each group separately (P<0.05). After 7 days of treatment, the scores for abdominal distention, defecation frequency and stool form in the combined treatment group were all lower than those in the western medication group (P<0.05). After 28 days of treatment, the score of each clinical symptom in the combined treatment group was lower than either of the rest two groups respectively (P<0.05); and the scores for abdominal pain, abdominal distention and incomplete bowel movement in the acupoint application group were lower than those in the western medication group separately (P<0.05). After 7 and 28 days of treatment, the score of each item in IBS-QOL was increased as compared with that before treatment in each group (P<0.05). After 7 days of treatment, except the scores for heterosexual relationship and human relations, scores of the other items, other scores of IBS-QOL in the combined treatment group were higher than those in the western medication group separately (P<0.05); while the scores of anxiety and behavior disorder in the acupoint application group were higher than those in the western medication group (P<0.05). After 28 days of treatment, the each score of IBS-QOL in the combined treatment group was higher than the other two groups respectively (P<0.05), while the scores of anxiety, behavior disorder, dietary control, social reaction and human relations in the acupoint application group were all higher than the western medication group (P<0.05). After treatment, the levels of and T lymphocytes and the ratio of /in peripheral blood, as well as the expressions of 5-HT4R mRNA in colon tissue were all increased in each group (P<0.05), but the levels of T lymphocyte and 5-HT3R mRNA expressions in colon tissue were reduced in each group (P<0.05). After treatment, regarding the levels of and T lymphocytes and the ratio of /in peripheral blood, as well as the expressions of 5-HT4R mRNA in colon tissue, the values in the combined treatment group were all higher than either of the rest two groups respectively (P<0.05), while the values in the acupoint application group were higher than the western medication group (P<0.05). Concerning to the level of T lymphocytes and 5-HT3R mRNA expression, the values in the combined treatment group were lower than the rest two groups respectively (P<0.05), and the values in the acupoint application group were lower than the western medication group (P<0.05). CONCLUSION: The combined treatment with the fire needling therapy with filiform needle and the acupoint application therapy effectively relieves the clinical symptoms, improves the quality of life and strengthens the immunity in the patients with IBS-D. The therapeutic effect of this combined regimen is better than either simple acupoint application therapy or the oral medication of pinaverium bromide. The mechanism may be related to the regulation of the levels of T lymphocyte subsets and the modulation of the expressions of 5-HT3R and 5-HT4R mRNA in colon tissue.

[Contrasted study on pharmacokinetics of Tibetan medicine Renqing Mangjue compatible with Zuota].

To provide insights into the mechanism for the attenuate-synergistic effect of Zuota to Tibetan medicine Renqing Mangjue, a contrasted study was carried out on the pharmacokinetics of brucine and strychnine in mice plasm, which are active and toxicant ingredient in the Tibetan medicine Renqing Mangjue. LC-MS/MS was used to detect simultaneously the concentrations of brucine and strychnine in mice plasm at-different time intervals after administration parallelly and randomly, and the pharmacokinetic software Kinetica 5. 0 was selected to non-compartmental analysis (NCA) for data, and statistical analysis software SPSS 19. 0 was used for significance test on the pharmacokinetic parameters. A reliable LC-MS/MS method was established for the determination of brucine and strychnine in blood plasma, which are consistent with the requirements of the preclinical pharmacokinetic study confirmed by the methodology. The linear concentration ranges of brucine and strychnine were 0.301-104.4 µg · L(-1) (r = 0.999 5) and 0.305-106 µg · L(-1) (r = 0.999 7), respectively; The intra-day and inter-day variable coefficients were both less than 10.0% with good precision; The average extraction recoveries of brucine and strychnine were 116.23% and 112.82%, and RSD were 3.2% and 2.3% separately;The average matrix effects of brucine and strychnine were 122.48% and 116.36%, and RSD were 7.7% and 4.4%, respectively. The pharmacokinetic results showed that AUCtot of brucine and strychnine in Zuota group were both increased remarkably (P < 0.05), and the Cmax of brucine in Zuota group was about 5.25-fold higher than that of brucine in non-Zuota group (P < 0.05). The Tmax of brucine and strychnine reduced to one-eighth and one-quarter respectively compared with those in Non-Zuota group. In addition, the eliminations of brucine and strychnine in vivo were accelerated after the compatibility of Zuota. A significant difference (P < 0.05) occurred at the MRT0-t, of brucine, while the MRT0-∞ and Lz of strychnine were statistically significant upon the inspection level α = 0.1. It was found that the absorption degree of brucine and strychnine in Zuota group increased in the range of the safe dose (or concentration), while their elimination rates were accelerated, which may be one of the mechanisms for attenuate-synergistic effect of Zuota to Tibetan medicine Renqing Mangjue.

[Determination of quercetin and kaempferol in Fagopyrum spp. of different species and different parts by HPLC].

OBJECTIVE: An HPLC method was established for determination of quercetin and kaempferol in Fagopyrum spp. of differrent species and different plant parts. METHODS: The chromatographic separation was carried out on a DIKMA diamonsil (250 mm x 4.6 mm, 5 µm) chromatographic column. The mobile phase was acetonitrile (A) and 0.1% phosphoric acid solution (B) at the flow rate of 1.0 mL/min. The detection wavelength was set at 260 nm and the column temperature was 25 degrees C. RESULTS: The descending order of quercetin content in the samples of different parts of Fagopyrum tataricum was seed (6.12 mg/g) > leaf (1.47 mg/g) > stem (0.34 mg/g); quercetin was not found in root. Only a few kaempferol was found in seed (0.42 mg/g) and leaf of Xiqiao 1 (0.09 mg/g) of Fagopyrum tataricum. There were a few quercetin was determined in the whole plant of Fagopyrum esculentum, and kaempferol was not been found. The total and mean value concentration of quercetin and kaempferol of Fagopyrum tataricum were much higher than that of Fagopyrum esculentum. In different species of Fagopyrum tataricum planted in the same area, the content of quercetin and kaempferol was similar, they were not been found in Fagopyrum esculentum. CONCLUSION: This method is simple, rapid, accurate and reliable for quality evaluation of Fagopyrum spp. and its commercial products.