Analytical Methods and Bioassays for Cytotoxicity and Antidiabetic Properties of Aquilaria crassna Leaf Extracts in HepG2 Cells.

Aquilaria crassna is a herbal plant that has recently been reported to possess several biological activities. A. crassna leaf extracts have been demonstrated to have a glucose-lowering effect in animal models. However, it is unclear what phytochemical compounds mediate this antidiabetic property. Here, we describe analytical methods for identifying such compounds from dried leaves by differential extractions with ethanol, butanol, ethyl acetate, and water, respectively. The phytochemical compounds in each fraction were further identified by gas chromatography-mass spectrometry. The cytotoxicity of these fractions was tested against a HepG2 cell line, while the rate of glucose utilization was determined using glucose oxidase assay. Lastly, the inhibitory effect on suppression of hepatic glucose production in HepG2 cells was determined by quantitative real-time PCR of genes encoding pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and liver glycogen synthase.

Metabolic disturbance and phytochemical changes in Andrographis paniculata and possible action mode of andrographolide

Objective:To explore the effect of gibberellic acid (GA3) and its inhibitor paclobutrazol (PBZ)on chemical composition and their pharmacological effects on Andrographis paniculata (Burm.f.) Wall.ex Nees,and to clarify action mode of andrographolide.Methods:The chemical composition was extracted by sequential extraction with hexane,dichloromethane,ethyl acetate and methanol,respectively.Andrographolide and its derivatives were evaluated by HPLC.Moreover,the metabolic profiling was analyzed by GC-MS.Inhibitory effect of crude extracts was tested against Staphylococcus aureus using agar well diffusion method.Mode of action was tested against mutant yeast by spotting assay.Andrographolide were tested for their mode of action against eukaryotes.Results:Among different solvents,dichloromethane gave the highest yield of crude (3.58％ DW),with the highest andrographolide content (8.3 mg/g DW).The effect of plant hormone (10 mg/L GA3 or PBZ) on phytochemical variations and bioactivity of Andrographis paniculata was demonstrated.It was found that PBZ promoted sesquiterpene compounds about 3.5 times over than GA3 treatment.But inhibitory effect of extracts against Staphylococcus aureus was highest in GA3 treated plants;andrographolide and 14-deoxy-11,12-didehydroandrographolide contents were significantly higher than those of water or PBZ.It was found that there were 11 strains involving in ergosterol biosynthesis,V-ATPase activity and homeostasis,and superoxide detoxification process.In this regard,andrographollde might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis.Conclusions:It is found that GA3 promotes andrographolide and 14-deoxy-11,12-didehydroandrographolide content while PBZ promotes sesquiterpene content.Andrographolide might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis.It might also affect mitochondria electron transport chain,leading to the occurrence of ROS,which can further harm cell organelles.However,the library screening is the first step to investigate mode of action of andrographolide.

A Phenylurea Cytokinin, CPPU, Elevated Reducing Sugar and Correlated to Andrographolide Contents in Leaves of Andrographis paniculata (Burm. F.) Wall. Ex Nees.

Cytokinins are phytohormones that play multiple roles to control plant growth and development. In this study, leaf biomass and the production of andrographolide compounds in a medicinal plant Andrographis paniculata were significantly increased after exogenously treating with the synthetic cytokinin cytokinin-1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) at 0 (water), 5, or 10 mg L-1 and observed the results for 24 h, 48 h, and 7 days of treatment. It was found that CPPU could significantly enhance new axillary bud formation and further promote branching 4.6-5.6-fold higher, resulting in higher fresh weight (FW) and dry weight (DW) than the control. Application of CPPU at 5 mg L-1 significantly promoted the highest contents of total reducing sugar at 2.5-fold in leaves and at 1.5-fold in roots. Although treatments of CPPU significantly affected the increasing contents of chlorophyll and carotenoid (1.2-1.6-fold), CPPU at 10 mg L-1 slightly caused leaf stress and chlorophyll reduction. Interestingly, 5 mg L-1 CPPU could enhance andrographolide content, an active anti-infectious compound in Andrographis paniculata (2.2-fold higher than the control) that reached the highest content at 24 h after treatment. This study suggested that CPPU should be suitable for field application to promote leaf yields and induce the production of useful pharmaceutical compounds in Andrographis paniculata.

T-DNA insertion alters the terpenoid content composition and bioactivity of transgenic Artemisia annua.

In this study, the interference of T-DNA insertion upon Agrobacterium-mediated transformation on the biochemical expression of the host genome is discussed. Plant extracts of transgenic Artemisia annua L. with or without an overexpressed famesyl pyrophosphate synthase gene have been investigated for their bioactivity and metabolic profile in comparison with wild type A. annua. The highest antimicrobial activity against Staphylococcus aureus, Bacillus subtilis and Candida albicans was observed in the T253 transgenic lines. Moreover, the crude extract from T253 showed higher antimalarial activity against the Plasmodium faciparum K1 strain than those of the others. The terpenoid constituents and antimicrobial properties of the plant samples were grouped by hierarchical clustering analysis. The clustering showed that squalene is a putative compound that might be involved in increasing the bioactivity of the transgenic line. In addition, T253 had a triterpene content that was about twice as great as that of the T253-2 line, which had a higher content of sesquiterpenes. However, both lines were transformed by the same FPS gene. These results suggested that the different bioactive properties observed in each transgenic line may be caused by variations in their terpenoid composition, which is affected by T-DNA insertion at different positions in the host plant.

Chemical and bioactivity evaluation of the bark of Neonauclea purpurea.

Bioassay-guided fractionation of the MeOH extract from the stem bark of Neonauclea purpurea used in traditional medicine, resulted in the isolation of 2 indole alkaloids, cadambine (1) and alpha-dihydrocadambine (2), as well as a quinolic compound, 2,6-dimethoxy-1,4-benzoquinone (3). Antimalarial activity evaluation showed that compounds 2 and 3 exhibited mild in vitro antimalarial activity against Plasmodium falciparum, the chloroquine-resistant strain K1 with IC50 values of 6.6 and 11.3 microM, respectively. Compounds 1 and 2 showed no cytotoxicity to monkey (Vero) cells, but compound 3 showed weak cytotoxicity with an IC50 value of 1.19 microM.