Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis / 中国中药杂志

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.

Protective effects of aqueous extracts from Sedum sarmentosum growing under different light intensity on CCl_4-induced damage in HepG2 cells / 中国中药杂志

To explore the effect of light intensity in cultivating environment on the hepetoprotective activity of Sedum sarmentosum, S. sarmentosum were planted under five water treatments for 60 days, namely 100% full sunlight(G1), 77% full sunlight(G2), 60% full sunlight(G3), 38% full sunlight(G4), and 16% full sunlight(G5) and CCl_4 drug-induced liver injury model in vitro was used. Cell viability, cell cycle, and cell apoptosis were individually detected by MTT, PI single staining, and Annexin-V FITC/PI double staining assays. Additionally, ALT, AST and antioxidant index in supernatant were determined by colorimetry. And the relationship among the protective effects, chemical composition and antioxidant activity were also analyzed. The results showed that S. sarmentosum aqueous extract could significantly improve the HepG2 cell viability. Among the five S. sarmentosum groups, the cell viability of G1(100% full sunlight) treatment was the highest, and the cell apoptosis was the least. Meanwhile, the level of ALT, AST, and MDA in G1 was the lowest, but it achieved the highest level of SOD and GSH. Moderate light shading(60% full light) also improved the effect of protecting liver and reducing the enzyme. It was found that cell viability was positively correlated with ferricion reducing capacity. ALT activity was positively correlated with isorhamnetin content. Taken together, different light intensity had great influence on hepatoprotective effect of S. sarmentosum, which may be related to its antioxidant capacity. From the perspective of hepetoprotective activity, S. sarmentosum should be planted under full light.

Identification of Chrysanthemum indicum and its adulterants based on ITS2 barcode / 中国中药杂志

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.

Effects of light intensity on growth, quality and antioxidant activities of Sedum sarmentosum / 中国中药杂志

The present study was conducted to explore the effect of light intensity on growth, bioactivity compounds accumulation and anti-oxidative activity of Sedum sarmentosum. The growth, yield, contents of total flavonoids, total phenolic, quercetin, kaempferol and isorhamnetin, and antioxidant activities were assessed in S. sarmentosum under five light intensities, namely 100% full sunlight (G1), 77% full sunlight (G2), 60% full sunlight (G3), 38% full sunlight (G4), and 16% full sunlight (G5). The results showed that light intensity significantly affected the growth and the chemical compounds accumulation. With the decrease of light intensity, the maximum branch length and the average internode distance increased. G2 treatment greatly promoted the numbers of leaf layers and branches, and G3 treatment remarkably improved the yield. The highest total flavonoids and phenolic contents were obtained in G3 treatment. Meanwhile, the highest quercetin and isorhamnetin contents were obtained in G1 treatment. The difference of kaempferol content was not significant. In addition, based on DPPH, FTC and FRAP methods, the antioxidant activities of the aqueous extracts under G1 treatment were superior to the others. The results indicated that more than 60% full sunlight was the optimum light intensity condition to achieve high yield and quality of S. sarmentosum.

Protective effects of Sedum sarmentosum under different soil moisture in CCl₄-induced damage in HepG2 cells / 中国中药杂志

At present, there were few studies about the effects of cultivation measures on the quality and pharmacological activity of medicinal plants. To explore the hepetoprotective activity of Sedum sarmentosum aqueous extracts after different water treatments, S. sarmentosum were planted under five water treatments for 60 days, namely 15%-20% FC (field capacity, S1), 35%-40% FC (S2), 55%-60% FC (S3), 75%-80% FC(S4), and 95%-100% FC (S5) and CCl₄ drug-induced liver injury model in vitro was used. Cell viability, cell cycle, and cell apoptosis were individually detected by MTT, PI single staining, and Annexin-V FITC/PI double staining assays. Additionally, ALT, AST and antioxidant index in supernatant were determined by colorimetry. The results showed that, compared with the model group, S. sarmentosum aqueous extract could significantly improve the HepG2 cell viability. Among the five S. sarmentosum groups, the cell viability of S4 (75%-80% FC) treatment was the highest, and the cell apoptosis was the least. Meanwhile, the level of ALT, AST, and MDA in S4 was the lowest, but it achieved the highest level of SOD and GSH. Taken together, different water treatments had great influence on hepatoprotective effect of S. sarmentosum, and the soil moisture of the 75%-80% FC is beneficial to the hepetoprotective activity of S. sarmentosum.

Quality classification criteria of Paeonia suffruticosa seeds / 中国中药杂志

In order to establish the quality classification criteria of Paeonia suffruticosa seeds, thirty-one batches of P. suffruticosa seeds from different provenances were selected. The seed rooting rate, seed germination rate, seed purity, seed viability, 1,000-seed weight and moisture content were determined and analyzed through SPSS 20.0 software. Seed rooting rate, seed germination rate and seed purity were selected as the main index for classification, while 1,000-seed weight, seed viability and moisture content could be used as important references. The seed quality grading of P. suffruticosa was set as three grades. The seed quality of each grade should meet following requirements: For the first grade seeds, seed rooting rate ≥ 80%, seed germination rate ≥ 80%, seed purity ≥ 90%, seed viability ≥ 80%, 1,000-seed weight ≥ 250 g, moisture content, ≤ 10. For the second grade seeds, seed rooting rate ≥ 50%, seed germination rate ≥ 60%, seed purity ≥ 70%, seed viability ≥ 75%, 1,000-seed weight ≥ 225 g, moisture content ≤ 10. For the third grade seeds, seed rooting rate ≥ 20%, seed germination rate ≥ 45%, seed purity ≥ 60%, seed viability ≥ 45%, 1,000-seed weight ≥ 205 g, moisture content ≤ 10. The quality classification criteria of P. suffruticosa seeds have been initially established.

Effects of flower bud removal and artificial pollination on growth and yield of Tulipa edulis / 中国中药杂志

The study was conducted to explore the response of growth and yield of Tulipa edulis to flower bud removal and artificial pollination. And flower bud removal and artificial pollination were carried out in the squaring period and bloom stage respectively. The morphological index and biomass indicators were determined and the yield was counted in harvest time. Result showed that flower bud removal was beneficial to the growth of T. edulis, resulting in increasing growth index, biomass as well as the yield of bulb. The diameter and dry weight of T. edulis fruit by artificial pollination were increased significantly compared with the control. Seed setting percentage increased to 100%, and the number of seed as well as the single grain weight increased by 69.03% and 16.48%, respectively, which did not significantly affect the bulb production. In conclusion, Flower bud removal treatment accelerates bulb biomass increase, so as to improve its yield. Artificial pollination raised significantly seed setting percentage, seed number as well as the single grain weight.