β-ecdysone content and antioxidant capacity in different organs of Brazilian ginseng / Teor de β-ecdisona e capacidade antioxidante em diferentes órgãos de ginseng brasileiro

ABSTRACT: Plants that contain antioxidant compounds have attracted increasing interest for their vital role in the attenuation of oxidative damage caused by free radicals and in the treatment of various diseases. The present study investigated the β-ecdysone content and the antioxidant activity of Brazilian ginseng (Pfaffia glomerata) extracts obtained from inflorescences, stems, and roots. The P. glomerata extracts were tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, β-carotene bleaching test, and phosphomolybdenum method. The β-ecdysone content of P. glomerata extracts was measured by high-performance liquid chromatography (HPLC). The P. glomerata inflorescences showed the strongest DPPH radical scavenging activity and the strongest antioxidant activity in the β-carotene bleaching assay and phosphomolybdenum test. The roots showed the lowest antioxidant capacity in all of the assays. The concentration of β-ecdysone in the plant organs followed the following decreasing order: inflorescences > stems > roots. The present study showed that P. glomerata inflorescence extract had high antioxidant capacity that could be attributed to the presence of β-ecdysone.

RESUMO: Plantas que contêm compostos antioxidantes têm atraído interesse crescente por seu papel fundamental na atenuação de danos oxidativos causados pelos radicais livres e no tratamento de várias doenças. O presente estudo investigou o conteúdo de β-ecdysone e a atividade antioxidante de extratos de ginseng brasileiro (Pfaffia glomerata) obtidos a partir das inflorescências, caules e raízes. Os extratos de Pfaffia glomerata foram testados para atividade antioxidante usando o método sequestrante do radical 2,2-difenil-1-picrilhidrazil (DPPH), sistema modelo β-caroteno-linoleato e método de fosfomolibdênio. O conteúdo de β-ecdisona dos extratos de P. glomerata foi medido por cromatografia líquida de alta eficência (CLAE). As inflorescências de P. glomerata mostraram a maior atividade sequestrante de radical DPPH e a maior atividade antioxidante no ensaio β-caroteno-linoleato e no teste de fosfomolibdênio. As raízes mostraram a menor capacidade antioxidante em todos os ensaios. A concentração de β-ecdisona nos órgãos da planta seguiu a seguinte ordem decrescente: inflorescências > caules > raízes. Os resultados indicaram uma correlação positiva entre conteúdo de β-ecdisona e atividade sequestrante de radical DPPH. O presente estudo mostrou que o extrato das inflorescências de P. glomerata teve alta atividade antioxidante que poderia ser atribuída à presença de β-ecdisona.

Identification and HPLC Quantification of a Phytoecdysone and Three Phenolic Glycosides in Lamium takesimense Nakai

The herbs of Lamium takesimense Nakai (Lamiaceae) is used to treat spasmodic and inflammatory disease. The four polar compounds, ecdysterone, isoacteoside, rutin and lamiuside C, were isolated and identified from the BuOH fraction of the L. takesimense MeOH extract. HPLC quantification was performed on a Capcell Pak C18 column (5 µm, 4.6 mm × 250 mm) with a gradient elution of H₂O and 0.05% acetic acid in MeOH. The HPLC method was validated in terms of linearity, sensitivity, stability, precision, and accuracy. The quantitative level in plant material was determined as the following order: lamiuside C (4, 3.75 mg/g dry weight) > ecdysterone (1, 1.93 mg/g) > isoacteoside (2, 1.32 mg/g) > rutin (3, 0.97 mg/g).

Non-saponin constituents and their cytotoxicities from Trillium kamtschaticum / 中草药

Objective To investigate the chemical constituents and bioactivity of the moieties without hemostatic effects of Trillium kamtschaticum. Methods All compounds were isolated and purified by means of silica gel column chromatography, reverse phase C18 column chromatography, Sephadex LH-20, and recrystallization methods. Their structures were determined by physicochemical properties, and spectral data. The cytotoxic activity of selected compounds was also evaluated using the MTT method. Results Ten compounds were obtained from the moieties without hemostatic effects of T. kamtschaticum and their structures were identified as β-ecdysone (1), polypodine B (2), integristerone B (3), (9Z,15Z)-11,12,13-trihydroxyoctadeca-9,15-dienoic acid (4), (Z)-11, 12,13-trihydroxyoctadec-9-enoic acid (5), methyl (9Z,15Z)-11,12,13-trihydroxyoctadeca-9,15-dienoate (6), methyl (Z)-11,12,13- trihydroxyoctadec-9-enoate (7), 7,11-dimethyl-3-methylene-1,6-dodecadien-10β,11-diol-10-O-β-D-glucopyranoside (8), kaempferol- 3-O-α-L-arabinopyranosyl (1→6)-O-β-D-galacopyranoside (9), and 5-hydroxy-4-hydroxymethyl-2H-pyran-2-one (10), respectively. Compounds 1, 9, and 10 showed cytotoxic activities with IC50 values of (26.6 ± 1.3) μmol/L (DU145 cells), (16.2 ± 6.2) μmol/L (CEM cells), and (23.7 ± 1.2) μmol/L (HeLa cells), respectively. Conclusion Compounds 4—7 and 10 are obtained for the first time from Trillium genus, and compounds 1, 9, and 10 showed cytotoxic activities.

Monthly Dynamics of Physical and Chemical Indexes of Achyranthes Bidentatae Radix Stored in Simple and Cool Warehouses / 中国药学杂志

OBJECTIVE: To study the monthly dynamics of physical and chemical indexes in Achyranthis Bidentatae Radix(ABR, the dried root of Achyranthes bidentata Bl.) stored in simple and cool warehouses. METHODS: ABR was stored in simple and cool warehouses for 27 months. The color was observed. The water content was determined based on the drying method. The contents of β-ecdysone and 5-hydroxymethylfurfural (5-HMF) were determined by HPLC method. The accumulation of temperature difference between the simple and cool warehouses was evaluated with a relative temperature cumulation (RTC) method. The monthly dynamics of physical and chemical indexes of ABR was analyzed with RTC. RESULTS: As the extension of storage time, the ABR stored in the simple warehouse showed deeper color and harder texture, but the ABR stored in the cool warehouse still had soft texture without significant color change. The contents of β-ecdysone in ABR stored in the two warehouses both gradually decreased and dropped to lower than the limit of 0.030% ruled by China Pharmacopoeia when being stored for up to 27 months. The contents of 5-HMF of ABR stored in the two warehouses both increased and were higher for the sample in the simple warehouse than that in the cool warehouse. CONCLUSION: The concept of RTC is put forward and used to study the monthly dynamics of chemical constituents in traditional Chinese medicine during storage for the first time. The physical and chemical indexes of ABR varies during storage. Two years of storage time of ABR is suggested.

Anti-proliferative Activity of T-bet

T-bet is a critical transcription factor that regulates differentiation of Th1 cells from CD4+ precursor cells. Since T-bet directly binds to the promoter of the IFN-gamma gene and activates its transcription, T-bet deficiency impairs IFN-gamma production in Th1 cells. Interestingly, T-bet-deficient Th cells also display substantially augmented the production of IL-2, a T cell growth factor. Exogenous expression of T-bet in T-bet deficient Th cells rescued the IFN-gamma production and suppressed IL-2 expression. IFN-gamma and IL-2 reciprocally regulate Th cell proliferation following TCR stimulation. Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions. By using IFN-gamma-null mice to eliminate the anti-proliferative effect of IFN-gamma, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions. Since the anti-proliferative activity of T-bet may be influenced by IL-2 suppression in Th cells, we examined whether T-bet modulates IL-2-independent cell proliferation in a non-T cell population. We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-gamma promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells. Although the molecular mechanisms underlying anti-proliferative activity of T-bet remain to be elucidated, T-bet may directly suppress cell proliferation in an IFN-gamma- or an IL-2-independent manner.

Studies on chemical constituents in roots and rhizomes of Trillium tschonoskii (II) / 中草药

Objective: To investigate the chemical constituents in the roots and rhizomes of Trillium tschonoskii. Methods: The roots and rhizomes of T. tschonoskii were extracted with 70% ethanol and separated by chromatography on polyamide, silica gel, RP-C18, and Sephadex LH-20 columns. Chemical structures were identified by MS, 1D and 2D NMR experiments. Results: Twelve compounds were isolated and identified from the ethyl acetate extract from the roots and rhizomes of T. tschonoskii and n-butanol fractions were identified as β-ecdysone (1), pinnatasterone (2), polypodine B (3), methyl ferulorate (4), regaloside A (5), 4-hydroxy-benzoic acid (6), vanillic aldehyde (7), β-D-glucopyranosyl-(1→4)-O- [α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside (8), paris saponin V (9), paris saponin III (10), trillenoside A (11), and trillenoside C (12). Conclusion: Compounds 1, 2, 6, 7, 9, and 10 are isolated from the plants in this genus for the first time, and compounds 3-5, 11, and 12 are isolated from this plant for the first time.

Quantification of β-ecdysone in differents parts of Pfaffia glomerata by HPLC

Pfaffia glomerata (Spreng.) Pedersen, Amaranthaceae, is widely distributed in Brazil. Roots are considered as the world's greatest supplier and β-ecdysone is the most important compound extracted from roots of Pfaffia glomerata. So, the aim this study was analyze the presence of β-ecdysone in the inflorescences and stems and compared with the content from roots of Pfaffia glomerata and determine the best extractive method of β-ecdysone this plant. The crude extracts were obtained by Soxhlet method, reflux, maceration, percolation and turbolyse. Compound extracts were quantified by High Performance Liquid Chromatography (HPLC). The analysis were carried out a Phenomenex Column C18, 5 µm, 250x4,6mm, maintened at 30 ºC, gradient system using as mobile phase a mixture of methanol and water, flow rate 1,0 mL and detection at 245 nm. Results showed Soxhlet method with ethanol:water (90:10 v/v) presented the higher concentration of β-ecdysone in P. glomerata and inflorescences showed higher amount of this active substance (3,06%), compared with stems (2,37%) and roots (1,63%), showing that the inflorescences and plant stems may also be used as a rich source of β-ecdysone.

Is hexamerin receptor a GPI-anchored protein in Achaea janata (Lepidoptera: Noctuidae).

The process of uptake of hexamerins during metamorphosis from insect haemolymph by fat body cells is reminiscent of receptor-mediated endocytosis. Previously, we had identified a hexamerin-binding protein (HBP) and reported for the first time that uptake of hexamerins is dependent on the phosphorylation of HBP partly by a tyrosine kinase, which is, in turn, activated by 20-hydroxyecdysone (20E). However, the exact nature of HBP and the mechanism of interaction are still unknown. Here we report the possibility of HBP being a GPIanchored protein in the fat body of Achaea janata and its role in the tyrosine-kinase-mediated phosphorylation signalling. Digestion of fat body membrane preparation with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the hexamerin binding to HBP was inhibited after digestion. Hexamerin overlay assay (HOA) of co-immunoprecipitated in vitro phosphorylated HBP showed exclusive binding to ~120 kDa protein. Lectin-binding analysis of hexamerins revealed the presence of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GluNAc), whereas HBP showed the presence of GalNac alone. Mild chemical deglycosylation studies and binding interaction in the presence of sugars revealed that glycan moieties are possibly not involved in the interaction between HBP and hexamerins. Taken together, these results suggest that HBP may be a GPI-anchored protein, and interaction and activation of HBP is through lipid-linked non-receptor src tyrosine kinases. However, additional studies are needed to prove that HBP is a GPI-anchored protein.

Lethal and Sublethal Effects of Methoxyfenozide on the Development, Survival and Reproduction of the Fall Armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

The lethal and sublethal effects of the ecdysone agonist methoxyfenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith), were investigated by feeding a methoxyfenozide-treated diet to fifth instars until pupation in doses corresponding to the LC10 and LC25 for the compound. Larval mortality reached 8 percent and 26 percent in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12 percent for the LC10 and 60 percent for the LC25 was observed before pupation. Treated larvae exhibited lower pupal weights, higher pupal mortality, presence of deformed pupae, and more deformed adults than untreated larvae. The incorporation of methoxyfenozide into the diet had a significant effect on the timing of larval development. The development period for males and females was about seven days longer than the controls for both concentrations tested. In contrast, the compound affected neither pupae nor adult longevity. Finally, S. frugiperda adults that resulted from fifth instars treated with methoxyfenozide were not affected in their mean cumulative number of eggs laid per female (fecundity), nor percentages of eggs hatched (fertility), or the sex ratio. Our results suggest that the combination of lethal and sublethal effects of methoxyfenozide may have important implications for the population dynamics of the fall armyworm.

Effects of growth regulator and culture methods on rooting of Ajuga lobata and content of β-ecdysone / 中草药

Objective: To study the effects of indole butyric acid (IBA) and NAA at different concentrations and culture methods on the growth of Ajuga lobata root and content of β-ecdysone. Methods: Taking MS as basic medium supplemented with IBA (0.5, 1.0, and 2.0 mg/L) or NAA (0.3, 0.6, and 0.9 mg/L), the tissue culture was carried out. HPLC was used to determinate the content of β-ecdysterone. Results: The results showed that the roots could grew well on MS as basic medium supplemented with IBA 0.5-1 mg/L. The best culture condition for the highest β-ecdysone accumulation was MS medium supplemented with IBA 2 mg/L. The content of β-ecdysterone in the whole plant and the roots of A. lobata with tissue culture was twice times of hydroponics and about three times of cultivation. Conclusion: MS medium supplemented with IBA at the proper concentration could promote the root growth of A. lobata. The content of β-ecdysterone in the root of A. lobata is the highest in tissue cultures.

Ecdisteróide e iridóide glicosilado de Vitex gardneriana Schauer (Verbenaceae) / Ecdysteroid and iridoid glycoside from Vitex gardneriana Schauer (Verbenaceae)

O extrato metanólico das cascas do caule de Vitex gardneriana Schauer forneceu o ecdisteróide, 20-hidroxi-ecdisona e o iridóide glicosilado, aucubina. As estruturas foram caracterizadas por métodos químicos e espectroscópicos.

The methanolic extract of the stem bark of Vitex gardneriana Schauer afforded the ecdysteroid, 20-hydroxy-ecdysone and the iridoid glycoside, aucubin. Their structures were established on the basis of chemical and spectroscopic studies.

Construction of a Yeast Model for Screening Aedes albopictus Ecdysone Agonist Pesticides / 中国寄生虫学与寄生虫病杂志

Objective To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. Methods The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene——green fluorescence protein(GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site(pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcrip-tion level of GFP in the tebufenozide affected yeast and the control. Results In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tubufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tubufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. Conclusion The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

Inhibitory Effects of Extracted Components from Chinese Medicinal Herbs on Tyrosinase / 中华皮肤科杂志

Objective To develop an in vitro method to determine the effects of drugs on tyrosinase(TYR)activity and screen the inhibitory effects on TYR of14extracted components of Chinese medicinal herbs.Methods The activity of TYR was detected by revised Matsuda's method.Effects of14extracted com-ponents on tyrosinase system were observed in vitro.Results Ten of the14components showed dose-depen-dent down-regulation of tyrosinase activity and melanogenesis.The inhibitory activities of eugenol,curcumin and gallic acid were significantly higher than those of arbutin(P