Bio-Guided Isolation of Prospective Bioactive Constituents from Roots of Clausena indica (Dalzell) Oliv.

Clausena indica fruits are routinely used for the culinary purpose as natural spices, whereas leaves and roots are folk medicine with various health benefits in southern China, South and Southeast Asia. In this study, the bioassay-guided fractionation by column chromatography yielded three pure compounds including dentatin, nordentatin, and clausine K and five active fractions (Re1-5) from C. indica roots. These known anticancer compounds were confirmed by X-ray diffraction, 1H-, 13C-nuclear magnetic resonance (NMR), and electrospray ionization tandem mass spectrometric (ESI-MS-MS) analyses. Meanwhile, the phytochemical constituents from fractions were identified by gas chromatography-mass spectrometry (GC-MS). The isolates, fractions' components and their biological activities were first time investigated on C. indica. By in vitro DPPH and ABTS scavenging assays, nordentatin (IC50 = 49.2 and 69.9 µg/mL, respectively) and the fraction Re4 (32.4 and 38.5 µg/mL, respectively) showed the strongest antiradical activities, whereas clausine K presented a moderate and dentatin had negligible antioxidant activity, respectively. The anti-α-amylase activity of C. indica root extracts was mainly attributed to the fraction Re2 which inactivated the enzymatic assay with IC50 of 573.8 µg/mL. Among tested samples, only nordentatin and clausine K were effective in the pancreatic elastase inhibition, however, their influences were trivial. Markedly, clausine K and Re4 performed the most remarkable tyrosinase inhibition with IC50 values of 179.5 and 243.8 µg/mL, respectively, which were in turn 4 and 3 times stronger than myricetin (IC50 = 735.6 µg/mL), a well-known tyrosinase inhibitor. This is the first report affirming clausine K to be a new strong tyrosinase inhibitor. Isolated compounds from C. indica roots were quantified by high-performance liquid chromatography (HPLC), of which, dentatin, nordentatin, and clausine K accounted for 14.74, 6.14, and 1.28 mg/g dry weight. The findings suggest that bioactive constituents from C. indica roots may be potentially employed for the development of antidiabetic, antiaging and cosmetic agents.

Inducing G2/M Cell Cycle Arrest and Apoptosis through Generation Reactive Oxygen Species (ROS)-Mediated Mitochondria Pathway in HT-29 Cells by Dentatin (DEN) and Dentatin Incorporated in Hydroxypropyl-ß-Cyclodextrin (DEN-HPßCD).

Dentatin (DEN), purified from the roots of Clausena excavata Burm f., has poor aqueous solubility that reduces its therapeutic application. The aim of this study was to assess the effects of DEN-HPßCD (hydroxypropyl-ß-cyclodextrin) complex as an anticancer agent in HT29 cancer cell line and compare with a crystal DEN in dimethyl sulfoxide (DMSO). The exposure of the cancer cells to DEN or DEN-HPßCD complex leads to cell growth inhibition as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To analyze the mechanism, in which DEN or DEN-HPßCD complex causes the death in human colon HT29 cancer cells, was evaluated by the enzyme-linked immunosorbent assay (ELIZA)-based assays for caspase-3, 8, 9, and reactive oxygen species (ROS). The findings showed that an anti-proliferative effect of DEN or DEN-HPßCD complex were via cell cycle arrest at the G2/M phase and eventually induced apoptosis through both mitochondrial and extrinsic pathways. The down-regulation of poly(ADP-ribose) polymerase (PARP) which leaded to apoptosis upon treatment, was investigated by Western-blotting. Hence, complexation between DEN and HPßCD did not diminish or eliminate the effective properties of DEN as anticancer agent. Therefore, it would be possible to resolve the conventional and current issues associated with the development and commercialization of antineoplastic agents in the future.

Simultaneous determination of nine analytes in Clausena harmandiana Pierre. by new developed high-performance liquid chromatography method and the influence of locations in Thailand on level of nordentatin and dentatin.

BACKGROUND: Clausena harmandiana Pierre. (CH) contains various bioactive analytes with pharmacological benefits. Most researches were focused on carbazole analytes determined by isocratic high-performance liquid chromatography (HPLC), only few were focused on coumarin analytes and harvested location. OBJECTIVE: To develop and validate gradient HPLC method to analyze the variance of nine target analytes contained in roots of CH grown naturally in four different provinces of Thailand. MATERIALS AND METHODS: The analytical method was undertaken by gradient HPLC with 3% tetrahydrofuran in acetonitrile, and 0.05% phosphoric acid in water as mobile phases, on Hypersil ODS column (4.0 × 250 mm, 5 µm), at flow rate 1.0 mL/min and detected at wavelength 280 nm. The method was validated for system linearity, limit of detection, limit of quantitation, precision and accuracy. RESULTS: The new-developed method was able to detect the nine target analytes in CH root. The validation showed the reliability of the method. All system suitability parameters were within the satisfied limits. The linear responses of method were observed at r (2) ≥ 0.999 for all analytes. The obtained amount of nine analytes showed the biodiversity of contents in different provinces. Of the nine target analytes, the level of nordentatin and dentatin in coumarin groups were considerably high in plants collected from one specific province of Thailand. CONCLUSION: This study has shown that the new-developed method is reliable, precise, accurate and sensitive to determine and quantify the nine target analytes in CH. Nordentatin and dentatin obviously show the higher level in one specific province of Thailand.