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: 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.

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.

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 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 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.

BACKGROUND: Gypenosides have antioxidant properties, with beneficial effects such as reducing blood pressure, anti-aging and anti-tumor, but the specific protective mechanism is not clear. It is also unknown whether gypenosides have effect on the proliferation and differentiation of oxidative stress-damaged osteoblasts. OBJECTIVE: To investigate the mechanism by which gypenosides alleviate oxidative stress injury in rat osteoblasts and the effect on the proliferation and differentiation of oxidatively damaged osteoblasts. METHODS: Monolayer cell culture method was used to separate neonatal rat skull cells for the culture of osteoblasts. In this experiment, there were three groups, with normal culture medium as blank group, normal culture medium+oxidative damage as control group, and normal culture medium containing gypenosides and oxidative damage as experimental group. Osteoblasts in the experimental and control groups were cultured in the culture medium containing 150 μmol/L H2O2. After 3 and 5 days of intervention, cell counting kit-8 method was used to detect the effects of gypenosides on oxidative damage of osteoblasts. Alkaline phosphatase staining was used to detect alkaline phosphatase activity on day 7 after induction. Alizarin red staining was used on day 21 of induction to observe osteoblast mineralization. Western blot was used to detect the expression of NOX4, bone morphogenetic protein 2 and Smad4. The experimental protocol was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. RESULTS AND CONCLUSION: Gypenosides could promote the proliferation of oxidatively damaged osteoblasts. The results of alkaline phosphatase staining and alizarin red staining showed that gypenosides could promote the differentiation of oxidatively damaged osteoblasts. Compared with the control group, gypenosides could downregulate the expression of NOX4 protein and upregulate the expression of bone morphogenetic protein 2 and Smad4 protein in the experimental group, with statistically significant results (P < 0.05). All these findings indicate that gypenosides have a protective effect on H2O2-induced oxidative stress injury in osteoblasts, and promote the proliferation and differentiation of damaged osteoblasts. The mechanism may be related to the inhibition of Nox4 protein expression and the activation of bone morphogenetic protein/Smad pathway.

Objective To investigate the chemical constituents from Disporum cantoniense. Methods Compounds were isolated and purified by macroporus resin, ods, Sephadex LH-20, MCI-gel CHP20 resin column chromatography and preparative HPLC. The structures were identified by spectral analysis. Results Twenty compounds were isolated and identified as 2-hydroxybenzyl alcohol (1), p-hydroxybenzaldehyde (2), 4-hydroxyacetophenone (3), 3,5-dimethoxy-4-hydroxy-benzaldehyde (4), 4-(4-hydroxyphenyl)-2- butanone (5), neoliquiritin (6), 2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucoside (7), (E)-1-(4’-hydroxyphenyl)-but-1-en-3-one (8), isoquercitrin (9), 3-hydroxy-1-(4-hydroxy-3,5-dimethoxy-phenyl)-1-propanone (10), icariol A2 (11), ecdysterone (12), glansreginic acid (13), hesperidin (14), ononin (15), quercetin (16), isorhamnetin-3-O-β-D-glucoside (17), (E)-4-(4-hydroxy-3-methoxyphenyl) but-3-en-2-one (18), (-)-secoisolariciresinol (19), and luteolin (20). Conclusion Compounds 1, 3-8, 10-15, and 17-19 are isolated from the genus of Disporum for the first time.

Objective To study the chemical components from the roots of Smilacina henryi. Methods Silica gel, Sephadex LH-20, and preparation of thin layer chromatography techniques were used for isolation, and 1D, 2D-NMR, IR, and HR-ESI-MS data were used for structure identification. Results Seven steroidal components were obtained and its chemical structures were elucidated as (25S)-5α-spirostan-9(11)-en-3β,17α-diol 3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside (1), (25S)-5α-spirostan-9(11)-en- 3β,17α-diol (2), (25S)-5α-spirostan-9(11)-en-3β,17α-diol 3-O-β-D-glucopyranoside (3), diosgenin (4), aspidistrin (5), henryioside A (6), and henryioside B (7). Conclusion Compound 1 is a new steroidal saponin named henryioside D, and compounds 2-5 are isolated from the plant for the first time.