Study on chemical constituents of Wedelia trilobata / 中国中药杂志

A new taraxer-based triterpenoid ester, taraxer-14-en-30-al-3β-O-palmitate(1), was isolated from the whole plant of Wedelia trilobata, along with six known compounds, ent-kaur-16-en-19-oic acid(2), 16α-hydroxy-ent-kauran-19-oic acid(3), tara-xerol(4), β-amyrin(5), 1β-acetoxy-4α, 9α-dihydroxy-6β-isobutyroxyprostatolide(6), and stigmasterol(7). Their structures were elucidated with use of a combination of spectroscopic techniques(IR, HR-ESI-MS, 1 D, 2 D NMR data) and chemical methods.

Development and validation of a rapid high performance liquid chromatography - photodiode array detection method for estimation of a bioactive compound wedelolactone in extracts of Eclipta alba

Following optimization of extraction, separation and analytical conditions, a rapid, sensitive and simple reverse-phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed for the identification and quantification of wedelolactone in different extracts of Eclipta alba. The separation of wedelolactone was achieved on a C18 column using the solvent system consisting of a mixture of methanol: water: acetic acid (95: 5: 0.04) as a mobile phase in isocratic elution mode followed by photo diode array detection at 352 nm. The developed method was validated as per the guidelines of the International Conference on Harmonization (ICH). Calibration curve presented good linear regression (r²>0.998) within the test range and the maximum relative standard deviation (RSD, %) values for intra-day assay were found to be 0.15, 1.30 and 1.1 for low (5 µg/mL), medium (20 µg/mL) and high (80 µg/mL) concentrations of wedelolactone. For inter-day assay the maximum RSD (%) values were found to be 2.83, 1.51 and 2.06 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 2 and 5 µg/mL respectively. Analytical recovery of wedelolactone was greater than 95%. Wedelolactone in different extracts of Eclipta alba was identified and quantified using the developed HPLC method. The validated HPLC method allowed precise quantitative analysis of wedelolactone in Eclipta. alba extracts.

Desenvolveu-se método rápido, sensível e simples de Cromatografia Líquida de Alta Eficiência em fase reversa, utilizando-se arranjo de fotodiodo (HPLC-PDA), visando à separação, extração e às condições analíticas para a identificação e quantificação de wedelolactona em diferentes extratos de Eclipta alba. A separação de wedelolactona foi efetuada por meio de uma coluna C18, utilizando mistura de metanol:água:ácido acético (95:5:0.04) como fase móvel, em sistema de eluição isocrática, seguida de detecção por arranjo de fotodiodo a 352 nm. O método desenvolvido foi validado de acordo com as diretrizes da Conferência Internacional de Harmonização (ICH). As curvas de calibração apresentaram boa regressão linear (r²>0,998), dentro dos intervalos de teste, e os valores máximos de desvio padrão relativo (RSD,%) dos ensaios intra-dia foram 0,15, 1,30 e 1,1 para concentrações de wedelolactona baixa (5 µg/mL), média (20 µg/mL) e elevada (80 µg/mL) Para o ensaio inter-dia,os máximos de RSD (%) foram 2,83, 1,51 e 2,06 para as concentrações baixa, média e alta, respectivamente. O Limite de Detecção (LD) e o Limite de Quantificação (LOQ) foram de 2 e 5 µg/mL, respectivamente. A recuperação analítica de wedelolactona foi maior do que 95%. A wedelolactona em diferentes extratos de Eclipta alba foi identificada e quantificada pelo método de HPLC desenvolvido. O método de HPLC validado permitiu a análise quantitativa precisa de wedelolactona em extratos de Eclipta alba.

Alleviated affect of exogenous CaCl2 on the growth, antioxidative enzyme activities and cadmium absorption efficiency of Wedelia trilobata hairy roots under cadmium stress / 生物工程学报

In order to study the physiological mechanism of exogenous calcium on the toxicity of heavy metal cadmium (Cd) to Wedelia trilobata hairy roots, the effects of Cd alone, and in combination with different concentrations of Ca on growth, contents of soluble protein and malondialdehyde (MDA), activities of superoxide dismutase (SOD) and peroxidase (POD), Cd2+ absorption in W. trilobata hairy roots were investigated. Cd concentrations lower than 50 micromol/L enhanced the growth of hairy roots, while concentrations higher than 100 micromol/L inhibited growth, making the branched roots short and small, and also turning the root tips brown, even black. In comparison with the control (0 micromol/L Cd), the soluble protein content in hairy roots was found to increase when cultured with 10-50 micromol/L Cd, and decrease when exposed to a cadmium concentration higher than 100 micromol/L Cd. In addition, the activities of POD and SOD activity and MDA content were significantly higher than the control. Compared to the control (hairy roots cultured without 10-30 mmol/L Ca), 100 micromol/L Cd or 300 micromol/L Cd in combination with 10-30 mmol/L Ca resulted in increased growth, causing the main root and secondary roots thicker and also an increase in soluble protein content. On the contrary, MDA content and POD and SOD activities decreased. Quantitative analysis by Atomic Absorption Spectrophotometry showed that W. trilobata hairy roots can absorb and adsorb heavy metal Cd in the ionic form of Cd2+. The maximum content of Cd2+ absorbed by the hairy roots was obtained with a concentration 100 micromol/L Cd2+ while that of Cd2+ adsorbed by hairy roots was achieved with a concentration of 300 micromol/L Cd2+. The exogenous addition of 10-30 mmol/L Ca2+ was found to reduce the absorption, adsorption of Cd2+ and the toxicity of Cd significantly. This reduction in toxicity was caused by the reduction in the absorption of Cd and decreasing the lipid peroxidation through regulating the activities of antioxidant enzymes SOD and POD in the hairy roots.

iso-Kaurenoic acid from Wedelia paludosa D.C.

A recent reinvestigation of aerial parts of Wedelia paludosa D.C. is described and reports, for the first time, the isolation of iso-kaurenoic acid from this species.

Uma recente reinvestigação das partes aéreas de Wedelia paludosa D.C. é descrita e relata, pela primeira vez, o isolamento do ácido iso-caurenóico desta espécie.

Induction and in vitro culture of Wedelia trilobata hairy roots / 生物工程学报

To study the possibilities for improvement of the ornamental character and production of secondary metabolites by using Wedelia trilobata hairy roots, we investigated the induction of W. trilobata L. hairy roots and its consumption changes of carbon resource, nitrogen resource, phosphate and calcium in the medium during liquid culture. The results showed that hairy roots could be incited from the cut edges of leaf explants 7 days after inoculation with Agrobacterium rhizogenes ATCC15834 and could have an autonomous growth on the medium without phytohormones. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed hairy roots. The hairy root line grew very slowly in 0-7 days, very fast from 7 to 21 days. During the liquid culture of hairy roots, sucrose, NO3(-)-N, PO4(3-) and Ca2+ in the medium could be gradually absorbed and utilized with time. The content of NO3(-)-N in the medium was 5.8% of the initial amount at day 7, while sucrose content was about 50% of the initial amount. At day 35, the NO3(-)-N and sucrose content in the medium was 1.82% and 3.39% of the initial amount, respectively. In combination with Ca2+ consumption, PO4(3-) of the medium was rapidly absorbed and utilized. At day 7, the content of PO4(3-) in the spent medium was only 1.76% of the initial amount; but even at day 35, the content of Ca2+ in the spent medium was still 61.3% of the initial amount. The results presented here had provided the possibilities on improvement the ornamental character and how to prepare optimum medium for large scale cultivation and production of secondary metabolites from W. trilobata L. hairy roots.

High frequency axillary bud multiplication and ex vitro rooting of Wedelia chinensis (Osbeck) Merr.--a medicinal plant.

An efficient protocol was achieved for rapid propagation of Wedelia chinensis (Osbeck) Merr. through axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 8.87 microM) and indole-3-butyric acid (IBA; 2.46 microM) was optimal for axillary bud proliferation, which developed a mean of 8.3 shoots/node. Excision and culture of node segments from in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 days. Excision and culture of nodes in succession enhanced the number of shoots. Shoot multiplication did not exhibit decrease in the number of shoots even at 10th subculture. Nevertheless, the shoots exhibited a tendency towards stunted nature. But reduction of BA to 4.44 or 2.22 microM resumed normal growth of shoots. Half strength MS medium fortified with IBA (2.46 microM) induced the highest number of roots. All in vitro rooted shoots survived in field. Dipping of the basal end of shoots collected from multiplication medium in IBA (2.46 microM) solution for 7 days induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100%). Rooting ex vitro by direct transfer of shoots from multiplication medium exhibited 89.2 per cent survival. Use of commercial sugar and tap water and also the omission of in vitro rooting reduce the propagation cost 50-70 per cent. The protocol enables to harvest more than 50,000 plantlets within 150 days starting from a single node explant.