Screening and validation of RT-qPCR reference genes of Viola yedoensis under different light intensities / 中国中药杂志

Screening the reference genes that were stably expressed under different light intensities for Viola yedoensis could provide reference for the related molecular research. In this study, 11 candidate reference genes were detected by RT-qPCR for tissues of V. yedoensis treated with different light intensities. Ge Norm, Norm Finder, Best Keeper, and Ref Finder website were used to comprehensively evaluate the reference genes, and verify the stability of the reference gene based on CAT1. Finally, the ideal reference gene was determined. The results showed that CYP, Actin, and SAMDC had small Ct value ranges and stable expression. Ge Norm demonstrated that CYP, SAMDC, and Actin were suitable reference genes. Norm Finder showed that the expression of α-TUB was the most stable. Best Keeper recommended CYP, Actin, and SAMDC as reference genes. Ref Finder assessed that SAMDC, CYP, α-TUB, and Actin had better stability, while GAPDH had the worst stability. The expression trend of CAT1 gene was consistent when calibrated with SAMDC, CYP, and Actin, while it was quite different from that calibrated with GAPDH. In summary, SAMDC, CYP, and Actin can be used as ideal reference genes for the gene expression profiling of V. yedoensis under different light intensities.

Effects of light intensity on material and energy metabolism of Viola yedoensis / 中国中药杂志

Light energy is an important factor affecting plant growth. The hypothesis of "light-cold and heat property" holds that the original plants of traditional Chinese medicines(TCM) with cold property can obtain more energy to maintain growth in high light intensity environment, whereas the original plants of TCM with heat property prefer weak light environment. The current experiment investigated the effects of different light intensities on primary metabolites levels, energy levels, cell apoptosis, and leaves ultrastructure of Viola yedoensis, the original plants of TCM Violae Herba with cold property. There were five treatment groups of V. yedoensis, which was planted under Li1(8 500 lx),Li2(7 250 lx),Li3(6 000 lx),Li4(4 750 lx),Li5(3 500 lx)LEDs light intensity conditions, respectively. After harvest, primary metabolites levels, contents of ATP, ADP, AMP, activities of ATP synthesis and hydrolysis related enzyme, as well as cell apoptosis activation degree were measured, and transmission electron microscopy technique was used to observe leaves ultrastructure. The results showed that the total sugar, total protein, contents of ATP, ADP and AMP, activities of NADH dehydrogenase, cytochrome C reductase, ATP synthase and ATP hydrolase were positively correlated with light intensities(P<0.05). The crude fat content, activities of SDH and CCO enzyme showed a trend of increasing first and then decreasing, the highest value were found in Li2 group and Li3 group respectively(P<0.05). The vitality of caspase-3 and caspase-9 was negatively correlated with light intensities(P<0.05). The structure of chloroplast and mitochondria were normal and intact in Li1-Li3 groups, and the damage degree of Li4 and Li5 groups increased with the decrease of light intensities. The analysis of results indicated that the structure of chloroplast and mitochondria of V. yedoensis is normal under the light intensity of 6 000-8 500 lx, which can obtain more energy to maintain its growth and metabolism. When the light intensity is lower than 4 750 lx, the chloroplast morphology and mitochondrial membrane are damaged, affecting the metabolism of material and energy. There was no significant difference in energy charge of V. yedoensis in the light intensity range of 3 500～8 500 lx. The effect of light intensity on energy metabolism of V. yedoensis accords with the hypothesis of "light-cold and heat property".

Effects of light intensity on cleaning away heat property of Viola yedoensis / 中国中药杂志

The current study was conducted to explore the effects of light intensity in cultivating environment on the cleaning away heat property of Viola yedoensis. In the present study, we established the acute inflammation model of ICR mice by injecting carrageenan. We compared the effects of V. yedoensis grown under different light intensities(100%, 80%, 50%, 35% and 5% of full sunlight) on mice body temperature, thermal radiation and the swelling degree of foot tissue before and after modeling observing by thermal infrared imaging technique and weighing method. The changes of energy metabolism related enzymes in liver were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the effects of V. yedoensis grown under different light intensities on human lung cancer cell A549 proliferation were explored with MTT method. The results showed that the body temperature of all groups of mice in V. yedoensis group were significantly lower than that of the blank group, except 5% full sunlight group, and the body temperature declined in positive proportion to light intensity. V. yedoensis group could alleviate foot swelling, reduce SDH activity in liver tissue(especially 100% full sunlight group and 80% full sunlight group were significantly lower than model group), and the degree of alleviating and reducing was positively correlated with light intensity. There was no significant difference in the activity of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase in liver tissue among treatments. The contents of IL-1β, IL-6, TNF-α, PGE_2 in foot tissue of mice in V. yedoensis groups were significantly lower than those in model group. Among them, the lowest levels of IL-1β, IL-6, TNF-α, PGE_2 were found in 80% full sunlight group, and there was no significant difference in TNF-α among different groups. The effects of V. yedoensis aqueous extract on A549 cell line proliferation inhibition rate increased with the light intensities of V. yedoensis cultivating environment. And the effects of V. yedoensis grown under 100% of full sunlight showed significantly higher A549 cell line proliferation inhibition rate compared with other groups(P<0.05). In summary, the light intensity of V. yedoensis cultivating environment is positively correlated with the cleaning away heat property of V. yedoensis, which conforms to the "light-cold and heat property" hypothesis,The V. yedoensis should be planted under full light according.

Comparative Analysis of Volatile Organic Compounds in Viola yedoensis by Needle Trap,Static Headspace and Hydrodistillation Methods Coupled with GC-MS / 中国实验方剂学杂志

Objective: To systemically investigate the volatile organic compounds (VOCs) of Viola yedoensis, and to compare the VOCs differences of V. yedoensis obtained by the needle trap, static headspace and hydrostillation methods. Method: The needle trap, static headspace and hydrostillation methods coupled with gas chromatography-mass spectrometer (GC-MS) were developed for isolation and identification of the VOCs in V. yedoensis. The relative content of each component was obtained by peak area normalization with a triple-bed needle packed with Tenax, Carbopack X and Carboxen 1000 sorbents. Result: The 112 compounds were trapped by using needle trap, mainly moderate volatile components, including aldehydes, ketones, alcohols, monoterpenes, sesquiterpenoids and aromatic compounds. The static headspace and hydrodistillation methods were allowed to obtain 37 (mainly the high-volatile components) and 78 compounds (mainly low-volatile components), respectively. Only 13 common volatile components were detected in all these three methods. Conclusion: The results clearly demonstrated that the needle trap method is an alternative method for sampling VOCs of herbs, characterized by fast analysis, simple operation, good enrichment effect and high sensitivity.These three methods for VOCs analysis are complementary for each other.

Effects of light intensities on growth,physiological characteristic and chemical composition of Viola yedoensis / 中国中药杂志

The study is aimed to investigate the effects of light intensities on growth,photosynthetic physiology,antioxidant systems and chemical composition of Viola yedoensis and provide cultivation references for V.yedoensis.Five groups of V.yedoensis were planted under five light intensities conditions,namely 100%,80%,50%,35%,5%of full sunlight,and then morphological index,growth,chlorophyll fluorescence parameters,photosynthetic parameters and antioxidant enzyme system indexes were measured during harvest.The results showed that there was no significant difference in the biomass of V.yedoensis among 35% -100%full sunlight,but the biomass of those were significantly higher than that in the 5%full sunlight treatment(P<0.05).The net photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO_2 concentration and water use efficiency increased firstly and then decreased with the decrease of light intensity;F_m,F_v/F_mand Yield in 5% full sunlight treatment were significantly lower than those in the other four groups(P<0.05).The structure of chloroplast was normal under light intensity ranged from 50%to 100% full sunlight.The lamellar concentration of chloroplast matrix decreased and the starch granules decreased in 35% full sunlight treatment,and the margin of lamellar layer of chloroplast and substrate were blurred,and the starch granules were small and the number of starch granules decreased significantly under 5% full sunlight.MDA content in 5%full sunlight treatment was significantly higher than those in the other four groups(P<0.05).The total coumarin content and total flavonoid content decreased with the decrease of light intensity.In summary,the light in-tensity range suitable for the growth of V.yedoensis is wide(ranging from 35% to 100% full sunlight).The content of flavonoids and coumarins is positively correlated with light intensity.

Simultaneous Determination of Chlorogenic Acid, Aesculetin, Rutin, Acacetin-7-O-β-D-glucoside, Querce-tin and Luteolin in the Extract of Viola Yedoensis Makino by HPLC / 中国药师

Objective:To establish an HPLC method for the simultaneous determination of chlorogenic acid , aesculetin, rutin, acacetin-7-O-β-D-glucoside, quercetin and luteolin in the extract of Viola yedoensis Makino.Methods:The HPLC analysis was carried out on a Hypersil ODS C18 column (250 mm ×4.6 mm, 5μm) with 0.1％phosphoric acid (A)-methanol (B) as the mobile phase with gradient elution at the flow rate of 1.0 ml· min-1 .The detection wavelength was 345 nm and the column temperature was 35℃. Results:Good linear relationship was found within the range of 4.3175-172.7000 mg· L-1 for chlorogenic acid, 2.7350-109.4000 mg· L1 for aesculetin, 6.9800-279.2000 mg · L-1 for rutin, 3.7200-148.8000 mg · L-1 for acacetin-7-O-β-D-glucoside, 4.1350-165.4000 mg· L-1 for quercetin, and 3.3950-135.8000 mg · L-1 for luteolin.The average recovery was 99.06％, 98.84％, 98.77％, 99.40％, 98.53％and 98.71％, respectively.The content of chlorogenic acid , aesculetin, rutin, acacetin-7-O-β-D-glucoside, quercetin and luteolin in the extract of Viola yedoensis Makino was 1.6290, 1.1910, 4.5850, 2.2810, 3.1790 and 1.9710 mg· g-1, respectively.Conclusion:The method is accurate and reliable with good reproducibility , which can be used for the quality control of the extract of Viola yedoensis Makino.

ldentification of Viola yedoensis Makino and Viola inconspicua by FT-l R / 中国药科大学学报

The different extracts and of Viola yedoensis Makino and Viola inconspicua were analyzed and identi-fied by Fourier transform infrared spectroscopy (FT-IR). One-dimensional infrared spectrum showed that the extracts of Viola yedoensis Makino and Viola inconspicua contained the aromatics,volatile substances and glyco-sides,with not significant differences from each other. However,different extraction sites of the two medicinal materials in second derivation spectrum were obviously different,especially the number of automatic peaks and peak intensity in the range of 970 800 cm-1. Viola yedoensis Makino displayed 5 automatic peaks,6 automatic peaks and 6 automatic peaks,while Viola inconspicua displayed 7 automatic peaks,4 peaks,4 peaks in the second derivation spectrum of petroleum ether extraction site,chloroform extraction site and the ethyl acetate extraction site. In addition,the peak position of the strongest peak in the second derivative of the ethyl acetate extraction site was 1 467 cm-1,while the strongest peak of the Viola inconspicua was at 1 384 cm-1,so the two medicinal mate-rials can be distinguished by the strongest peak position of ethyl acetate extraction site in second derivation spec-trum. Studies demonstrated that one-dimensional infrared spectroscopy combined with the second-order derivative analysis could achieve the accurate identification between Viola yedoensis Makino and Viola inconspicua. This research provides new ideas and new methods for the identification of Viola and other adulterants.

Chemical constituents of Viola yedoensis / 中草药

Objective To investigate the chemical constituents of the whole plants of Viola yedoensis. Methods The chemical constituents were isolated and purified by column chromatography over silica gel and Sephadex LH-20, as well as on the semi-preparative HPLC. The structures of the isolates were identified by the NMR spectroscopic method. Results Twenty-three compounds were isolated and their structures were identified as pubinernoid A (1), (2R,6R,9R)-2,9-dihydroxy-4-megastigmen-3-one (2), 3S,5R-dihydroxy-6R,7-megastigmadien-9-one (3), dehydrovomifoliol (4), blumenol A (5), blumenol B (6), oleanolic acid (7), 2α,3α-dihydroxyurs-12-ene-28-oic acid (8), 1α,2α,3β-trihydroxyolean-12-ene-28-oic acid (9), 2α,19α-dihydroxyursolic acid (10), 3α-hydroxyfriedel-2-one (11), 7-oxopetrosterol (12), 7-oxositosterol (13), syringaresinol (14), lariciresinol (15), daphneticin (16), umbelliferone (17), trans-p-hydroxycinnamic acid methyl ester (18), p-hydroxyphenylpropionic acid (19), p-hydrobenzaldehyde (20), p-methoxybenzaldehyde (21), p-methoxybenzoic acid (22), and 5-hydroxymethyl-2-furfural (23). Conclusion Compounds 1-16 and 18-23 are isolated from the genus Viol for the first time, and compound 17 is isolated from the plant for the first time.

Anti-complement alkaloids from whole plants of Viola yedoensis / 中国中药杂志

Fifteen alkaloids were isolated from the 95% ethanol extract of the whole plants of Viola yedoensis by various column chromatographic techniques such as silica gel and Sephadex LH-20. Their structures were identified as neoechinulin A（1）,N-benzoyl-L-p-hydroxy-phenylalaninol（2）,aurantiamide acetate（3）,aurantiamide（4）,anabellamide（5）,trichosanatine（6）,indole-3-carboxylic acid methyl ester（7）,3-carboxyindole（8）,N-trans-feruloyl-tyramine（9）,paprazine（10）,7'-（3', 4'-dihydroxyphenyl）-N-[(4-methoxyphenyl)ethyl]propenamide（11）,cannabisin F（12）,N-(4-hydroxyphenethyl)octacosanamide（13）,N-(4-hydroxyphenethyl)hexacosanamide（14）and N-benzoyl-L-phenylalaninol（15）. All the compounds except 3 and 4 were isolated from this plant for the first time. These alkaloids exhibited anti-complement activity against the classical pathway(CP)and the alternative pathway(AP)with the CH50 and AP50 values ranging from 0.12 to 0.33 g•L⁻¹ and 0.22 to 0.50 g•L⁻¹, respectively. Preliminary mechanism study using complement-depleted sera showed that these alkaloids acted on different complement components in the complement activation cascade.

Adsorption Properties for Separation of Apigenin from Viola yedoensis on LSA-10 Resin / 中草药·英文版

Objective: To explore the adsorption properties for the separation of apigenin from Viola yedoensis on LSA-10 resin. Methods: After different types of macroporous resins were optimized, the effects of initial concentration, temperature, pH value, and other factors on resin adsorption were studied, and the kinetics and thermodynamics in the process of the static adsorption of LSA-10 resin for the apigenin separation from V. yedoensis were also investigated. Results: The initial concentration of 4.0 mg/mL, temperature of 50 °C, and pH 5 were suitable for the resin adsorption, the experimental data of adsorption isotherms of LSA-10 resin were validated to fit the Freunclich and Langmuir equation, the adsorption process of apigenin was fitted to the first order adsorption kinetics equation, and the adsorption rate was mainly affected by film diffusion. The thermodynamic parameters such as adsorption enthalpy change (δ. H > 0), adsorption free energy change (δ. G 0) were investigated. Conclusion: The adsorption for the separation of apigenin on LSA-10 resin was an entropy-driven spontaneous process of decalescence and entropy increase, which belongs to physical adsorption. LSA-10 resin is suitable for the industrial separation of apigenin from V. yedoensis. © 2014 Tianjin Press of Chinese Herbal Medicines.

Antipyretic Effects of Liposoluble Fractions of Viola yedoensis / 中草药·英文版

Objective: To clarify the antipyretic effect of the Chinese materia medica, Violae Herba (Viola yedoensis), and its active fractions by examining the effects of V. yedoensis extracts with differing polarities on body temperature, total white blood cell (WBC) count, WBC differential count, and total serum complement of rabbits with lipopolysaccharide (LPS)-induced fever. Methods: The rabbits were treated with water and ethanolic extracts of V. yedoensis, as well as petroleum ether, ethyl acetate, and n-butanol fractions of the ethanolic extract at low-, mid- and high- doses. The LPS was injected via the ear vein of rabbits in model and treatment groups 30 min post-gavage. Their body temperature was measured at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 h after the LPS challenge to calculate the temperature changes and thermal response index. After the last temperature measurement, blood samples were collected to determine the blood cell counts and total serum complement (CH50) level. Results: Compared with the model group, body temperature was significantly lower in the low-dose ethanolic extract group, low- and mid-dose petroleum ether fraction groups, and all three ethyl acetate fraction groups. Serum CH50 levels were lower in all treatment groups, except the ethanolic extract groups, than that in the model group, with no significant difference. V. yedoensis had no significant effect on the blood cells of febrile rabbits challenged with LPS for 6 h. Conclusions: The antipyretic effects of V. yedoensis are strong, and its active fractions are the petroleum ether and ethyl acetate fractions of ethanolic extract.

Chemical constituents of volatile oil of Viola yedoensis Makino from two habitats / 中成药

AIM:To analyse the chemical constituents of volatile components from Viola yedoensis Makino. METHODS: The essential oils from Viola yedoensis Makino(collected from Jinhua city and Changchun city) were extracted by steam distillation.The chemical compounds were separated and identified by GC-MS.The relative content of each component was determined by area normalization. RESULTS: Twenty-five and twenty-three constituents were separated from Viola yedoensis Makino collected from Changchun city and Jinhua city;twenty-one and twenty-two compounds were identified respectively.The ratios of identification of total compounds were 88.00% and 91.30% respectively. CONCLUSION: There are significant difference between two varieties of Viola yedoensis Makino,the main coustituents in each plant are n-hexadecanoic acid(16.77%)and phytol(26.97%).