Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo
Introduction
Plant materials have long been used as traditional medicines for the treatment of a wide variety of ailments and diseases. Components of Tamarindus indica, a tree indigenous to India and South East Asia, have been used as a spice, food component, and snack. According to Thai traditional medicine, the fruit of T. indica is regarded as a digestive, carminative, laxative, expectorant and blood tonic. In addition, the seeds of T. indica are used as an anthelmintic, antidiarrheal, and an emetic, and the seed coat is used to treat burns and aid in wound healing as well as an antidysenteric (Farnsworth and Bunyapraphatsara, 1992).
Recently, Pumthong (1999) demonstrated the antioxidant activity of the seed coat extract of T. indica. The extract is composed of flavonoids including tannins, polyphenols, anthocyanidin, and oligomeric proanthocyanidins. Many of these flavonoids are also components of Pycnogenol®, a nutritional supplement which has been shown to have vasorelaxant activity, increase capillary permeability and participate in the cellular antioxidant network as indicated by its ability to regenerate the ascorbyl radical and to protect endogenous vitamin E and glutathione from oxidative stress (Packer et al., 1999, Rohdewald, 2002). Flavonoids found in various medicinal plants are natural antioxidants with free radical scavenging activity and they have also been shown to prevent free radical formation via inhibition of oxido-reductases (Middleton, and Kandaswami, 1986, Chen et al., 1993, Krol et al., 1995, Carlo et al., 1999).
Based on the reported antioxidant activity of the seed coat extract of T. indica which contains flavonoids among its major constituents, and the limited toxicological data available, the purpose of the present studies was to assess the anti-inflammatory potential of this extract and begin to access its safety. In vitro studies using T. indica seed coat extract were conducted to evaluate the modulation of nitric oxide (NO) production by RAW 264.7 macrophages using LPS and IFN-γ as stimulants. Confirmation of the effect in vivo was tested by orally exposing B6C3F1 mice to T. indica extract for 14 days and evaluating NO production by freshly isolated peritoneal macrophages following stimulation in vitro with lipopolysaccharide (LPS) and/or interferon gamma (IFN-γ), and in vivo or in vitro with 12-O-tetradecanoylphorbol-13-acetate (TPA). A 14-day toxicity study and studies to evaluate the effect of T. indica seed coat extract on components of innate and cellular immunity were performed to begin to assess safety.
Section snippets
Chemicals
The seed coat extract of T. indica was kindly provided by Dr. Maitree Suttajit (Chiang Mai University). Tamarind seeds were obtained from ripened tamarind fruits after removing the edible parts. The seeds were heated in a hot air oven at 140 °C, for 45 min, cooled and cracked to separate their outside brown layer. Only brown-red seed coats were collected and these were then ground into fine powder. In a separating funnel, 10 ml of 70% ethanol was added to 0.5 g of the ground tamarind seed coat.
Modulation of NO production by LPS & IFN-γ stimulated RAW 264.7 cells following in vitro exposure to seed coat extract of T. indica
To investigate the effect of the seed coat extract of T. indica on NO production, the accumulation of nitrite, the stable metabolite of NO, was measured in the culture media of RAW 264.7 cells using Greiss reagent. Resting RAW 264.7 cells were stimulated with LPS (5 μg/ml) and/or IFN-γ (10 ng/ml) to stimulate NO production. Cells were simultaneously treated with increasing concentrations of the seed coat extract of T. indica or with vitamin C (500 μm), vitamin E (141.3 μm), or beta-carotene
Discussion
Although individual components have not been identified, the seed coat extract of T. indica contains high amounts of polyphenolic flavonoids which are known to exhibit strong antioxidant scavenging activity against peroxyl radicals generated by ABTS/H2O2/peroxidase and ABTS/H2O2/myoglobin systems, hydroxyl radicals produced by ABTS/H2O2/FeCl3 (Feton reaction) and superoxide anions generated by hypoxanthine-xanthine oxidase (neotetrazolium) system (Pumthong, 1999). Due to the multiple phenolic
Acknowledgments
This work was funded in part by The Royal Golden Jubilee Ph.D. Program, The Thailand Research Fund.
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