Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo

https://doi.org/10.1016/j.fct.2003.12.001Get rights and content

Abstract

The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2–200 μg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) in a concentration-dependent manner. In vivo administration of T. indica extract (100–500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-γ induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity.

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.

References (43)

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