Objective: To select suitable references genes of Bupleurum scorzonerifolium for tissue expression analyses, and study the tissue expression characteristics of the key enzyme genes of saikosaponins biosynthesis. Methods: Five candidate reference genes including Actin, α-tubulin, β-tubulin, Cyclophilin, and EF-1α were chosen. The stability of these candidate reference genes was investigated by using four softwares (Delta CT, BestKeeper, NormFinder, and GeNorm). The stability of these candidate reference genes was tested and verified by real-time quantitative PCR. Used the stable reference gene, the tissue expression characteristics of the saikosaponins biosynthesis key enzyme genes (HMGR, IPPI, FPS, SS, and β-AS) was analyzed by qRT-PCR. Results: The average expression stability of the five candidate reference genes from high to low was β-tubulin > Cyclophilin > Actin > EF-1α > α-tubulin. Β-tubulin was the most suitable reference gene for tissue expression analysis in B. scorzonerifolium. HMGR expression level was roots > stems and fruits > leaves, IPPI expression level was roots > stems > fruits and leaves, FPS expression level was leaves > roots > stems and fruits, SS expression level was leaves > fruits > roots > stems, β-AS expression level was leaves > roots > fruits > stems. HMGR was significant positive correlated with IPPI, and FPS was significant positive correlated with β-AS (P < 0.05). Conclusion: β-tubulin gene was confirmed as the most suitable reference gene in different tissues of B. scorzonerifolium. It provided a methodological basis for the tissue expression analysis on the functional genes of B. scorzonerifolium. The expression pattern of five key enzyme genes of saikosaponins biosynthesis in different tissues had obvious differentiation, which might be involved in regulating the flow of saikosaponins synthesis and accumulation in various tissues of B. scorzonerifolium.

Objective To explore the effect of ecological factors and the expression of key enzyme genes on the synthesis and accumulation of ginsenosides. Methods Cultivated four-year-old ginseng were used as test materials, the expression of key enzyme genes (HMGR, FPS, SS, SE, DS, β-AS, CYP82D47, CYP716A47) in the biosynthesis of ginsenosides of roots in different growth periods was determined by real-time quantitative PCR, determination of the content of eight ginsenosides (ginsenoside Rg1, Re, Rf, Rb1, Rb2, Rb3, Rc, Rd) in roots by HPLC, the meteorological data were collected by a small weather station, correlation analysis was performed with SPSS. Results The expression of key enzyme genes in the period of flowering to fruit ripening was higher than the root growing after fruit period and the withering period, the expression of key enzyme genes involved in the synthesis of ginsenosides was influenced by each other, and the expression of key enzyme genes in ginseng roots showed a positive correlation with the accumulation of ginsenosides; The contents of ginsenoside Rg1, Rb1, Re, and Rc were higher in the roots of ginseng, eight kinds of monomer ginsenosides content dynamic changes trend is different; Temperature, photosynthetically active radiation, soil water potential are important ecological factors for ginsenosides synthesis in roots, temperature was significantly negatively correlated with ginsenoside Rb1 and Rd (P < 0.05), PAR can significantly promote the formation of ginsenoside Rg1 (P < 0.05), soil water potential was significantly negatively correlated with ginsenoside Rb1 (P < 0.05); Grey correlation analysis results showed that the major ecological factors that influenced ginsenosides content in ginseng roots were temperature, PAR and relative humidity, the grey correlation between the expression of the key enzyme genes with the content of ginsenosides is less than ecological factors with the content of ginsenosides, under the guidance of ecological factors, the expression of the key enzyme genes regulate the synthesis and accumulation of ginsenosides. Conclusion The dynamic changes of the expression of key enzyme genes and the content of ginsenosides in ginseng were determined, it provides a theoretical basis for elucidating the physiological and ecological mechanism of ginsenoside synthesis and the quality control of Radix Ginseng.

Objective To establish an HPLC method for simultaneous determination of loganin, chiratin, secoxyloganin, rosmarinic acid and calceolarioside B in Shuangxiangpaishi granules. Method HPLC colunmn was Alltima C18 column (4.6 mm×250 mm, 5 μm); mobile phase consisted of acetonitrile-methanol (1: 3) (A) -0.1% phosphate acid solution (B) with gradient elution; the column temperature was 35°C; the flow rate was 1.0 ml/min; all the injection volume was 20 μl; loganin, chiratin and secoxyloganin were detected at 240 nm, rosmarinic acid and calceolarioside B were detected at 330nm. Results The above mentioned five main ingredients had linearity in the given concentration range at 5.91-118.20 μg/ml (r=0.9995), 4.10-82.00 μg/ml (r=0.9991), 8.13-162.60 μg/ml (r=0.9993), 12.57-251.40 μg/ml (r=0.9998), 4.95-99.00μg/ml (r=0.9997), respectively. The average recoveries (n=6) and RSD were 96.88 (1.31), 99.09 (1.47%), 98.29 (1.59), 98.82 (1.42) and 97.51 (0.86), respectively. Conclusion This dual-wavelength HPLC method could simultaneously determine the contents of the five ingredients in Shuangxiangpaishi granules and can be used to evaluate their quality. The method is simple, accurate, sensitive and repeatable. It could be used as an efficient strategy for systematic quality evaluation of Shuangxiangpaishi granules.

Target identification of bioactive compounds is one of the key issues in chemical biology and drug discovery. With the development of science technology, a variety of methods and technologies for target identification have been reported. They can be fundamentally categorized into two approaches: the direct method based on affinity chromatography which is mainly to detect the combination of drug and targets, and the indirect one which is mainly to predict the drug target and action mechanism by physiological reaction and biochemical marker. This review aims to describe the two approaches for target identification.

Objective: The research indicated that the nature of Chinese medicine is mainly related to body's substance and energy metabolism. The purpose of the study is to elucidate the substance basis for warm nature of Poria cocos (called Fuling (FL) in Chinese). Methods: In terms of the effects of its separated fractions on the substance and energy metabolism in rat models of cold-deficiency with Aconiti Lateralis Radix Praeparata (called Fuzi (FZ) in Chinese), with hot nature, as reference drug. Biochemical indexes in the material metabolism, energy metabolism, endocrine system, nervous system and nucleotide system were determined, then analyzed by additive, cluster and principal component analysis (PCA). Results: The medicinal natures of oligosaccharides and amino acids fractions were attributable to plain and crude polysaccharides, volatile oils and triterpenoids fractions were attributable to mild warm. Conclusion: The nature of FL was regarded as mild warm based on the old records of Chinese medicine and fractions of crude polysaccharides, volatile oils and triterpenoids might be the main substance basis for the warm nature of FL. It is the first time that substance basis of FL was elucidated from view point of medicinal nature.

Cistanches Herba, known as "Ginseng of the desert", is authenticated from the dried succulent stems of Cistanche deserticola and Cistanche tubulosa. As a famous remedy in China for tonic the kidney, it is used to treat "kidney-deficiency syndrome"-induced diseases such as infertility, forgetfulness, hearing lost, chronic constipation, etc.. As various biological activities, including anti-aging, antioxidant, estrogenic, anti-osteoporotic, and anti-inflammation effects, have been discovered, here we reviewed Cistanches Herba in biological characteristics, chemical constituents, and pharmacological activities.

Objective To investigate the relationship between the content of flavonoids and the key enzyme genes expression in different tissues of Bupleurum chinense and B. scorzonerifolium. Methods The roots, stems, leaves, and fruits of B. chinense and B. scorzonerifolium were used as test materials, determination of flavonoids (rutin, quercetin, kaempferol, and isorhamnetin) in different tissues by HPLC, determination of total flavonoids by UV spectrophotometry, the tissues expression of key enzyme genes (IFS, F3H, and DFR) in flavonoids synthesis was determined by real-time quantitative PCR, correlation analysis was performed with SPSS. Results The content of flavonoids in the aerial parts (stems, leaves, and fruits) of B. chinense and B. scorzonerifolium was significantly higher than that in roots, the content of flavonoids was mainly rutin, and the content of rutin in the leaves of B. chinense leaves was up to 106.961 mg/g; The distribution of total flavonoids in B. chinense and B. scorzonerifolium was obviously different, the content was from high to low: leaves ≥ fruit > stem > root; The expression of B. chinense IFS, F3H, and DFR gene in the aerial parts was much higher than that in roots, IFS gene was significantly positive correlated with rutin (P < 0.05), F3H gene was significantly positive correlated with DFR gene (P < 0.05), but the expression of IFS, F3H, and DFR gene in each tissues of B. scorzonerifolium was at lower level. Conclusion The content of flavonoids in different parts of B. chinense and B. scorzonerifolium was consistent with the expression of flavonoids synthesis key enzyme genes, the differential expression of key enzyme genes regulates the synthesis and accumulation of flavonoids in different tissues.

Aim To observe the regulatory effects of astragalus polysaccharide (APS) on AKT/PI3K signa-ling pathway in lung cancer A549 cells. Methods The autophagy model was established by xanthine oxidase (XOD). Then the intervention was carried out with 100, 200, 400 mg • L"1 of APS and 3-methylade-nine (3-MA). Cell Counting Kit-8(CCK-8) method, transmission electron microscope and Western blot were used to detect the effect of APS on the proliferation, autophagy and autophagy marker molecules respectively in lung cancer A549 autophagy model. Results Compared with blank group, cell growth of the model group was accelerated; the number of autophagosomes increased , and the expressions of microtubule-associated protein 1 light chain-3B (LC3B) and PI3K significantly increased, while p62 and AKT markedly decreased (P < 0. 05) . Compared with model group, cell growth slowed down, and the number of autophagosomes decreased; the expression of LC3B and PI3K significantly decreased. However, p62 AND AKT expression significantly increased (P <0. 05) in 200 or 400 mg • L"'of APS-treatedgroups. Compared with 3-MA-treated group,the expression of LC3B significantly decreased in 200 mg • L"1 of APS-treated group(P <0. 05), and PI3K significantly decreased in 400 mg • L"1 of APS-treated group (P < 0. 05). Conclusion APS may exert anti-tumor effect by down-regulating PI3K/AKT signaling pathway of human lung cancer A549 cells.

OBJECTIVE: To establish the fingerprint of Aconitum Flarum Hand. Mazz Busch. METHODS: UPLC analysis was performed on ACQUITY UPLC BEH C18 column(2.1 mm×50 mm, 1.7 μm). Gradient elution was conducted. Mobile A was 0.2% formic acid and 0.005% TFA in water; mobile B was methanol; and mobile C was acetonitrile. The flow rate was 0.3 mL·min-1. The detection was carried out at 275 nm. The column temperature was maitained at 35℃. The injection volume was 1.0 μL. RESULTS: The UPLC fingerprint of Aconitum Flarum Hand. Mazz was established, and 14 common peaks were found including the peaks of two major constituents. The similarity factors ranged from 0.850 to 0.980. In addition, there was significant difference between wild and cultivated samples. CONCLUSION: This UPLC method has satisfactory precision, stability, repeatability and specificity. It provides a scientific method for identifying wild and cultivated Aconitum Flarum Hand. Mazz using fingerprint.

OBJECTIVE: To establish the UPLC fingerprint of Gardeniae Fructus for providing reference for effective quality control. METHODS: The samples were analyzed on a Waters ACQUITY UPLC BEH C18(2.1 mm × 50 mm, 1.7 μm) column maintained at 30℃ and eluted with methanol and water containing 0.02% phosphoric acid as mobile phases in a linear gradient mode. The flow rate was set at 0.4 mL·min-1 and the injection volume was 0.2 μL. The detection wavelengths were set at 238 nm (0-5.4 min) and 440 nm (5.4-10 min). RESULTS: The fingerprints of 16 batches of samples were analyzed with similarity evaluation, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The common mode of the fingerprint was established with 24 common peaks, and four of them were identified, which were genipin 1-gentiobioside (4), gardenoside (6), crocin I (15), and crocin II (17). The similarity degrees of the 16 batches of samples were between 0.986 0 and 0.995 0.The samples were divided into two groups, ZheJiang (S1-S6) and other areas (S7-S16), analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Eleven significantly different components were found, including genipin1-gentiobioside, gardenoside, crocin I, crocin II and some others. CONCLUSION: The establishment of UPLC fingerprint of Gardeniae Fructus from different producing areas and the application of chemical pattern recognition can provide more comprehensive references for the quality control and evaluation of genuine Gardeniae Fructus from Zhejiang Province.