Research in Veterinary Science 91 (2011) 422425

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Research in Veterinary Science

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In vivo efcacy of tamarind (Tamarindus indica) fruit extract on experimental uoride exposure in rats
S. Dey , D. Swarup, Anju Saxena, Ananya Dan
Laboratory of Comparative System of Medicine, Division of Medicine, Indian Veterinary Research Institute, Izatnagar 243 122, UP, India

a r t i c l e

i n f o

a b s t r a c t
The study was undertaken to determine the efcacy of hydro-methanolic (1:1) extract of tamarind (Tamarindus indica L.) fruit pulp in removing body uoride burden. Thirty rats were divided into ve groups. Keeping no uoride group as the control, rats of no treatment, low dose, middle dose and high dose groups received sodium uoride orally at the rate of 200 mg per kg body weight daily for 14 weeks. Rats of low dose, middle dose and high dose group simultaneously received tamarind fruit pulp extract at three doses, viz. 25 (low), 50 (medium) and 100 mg (high) per kg body weight orally, respectively. Fluoride concentration in blood, urine and long bone of experimental rats was monitored to assess the efcacy of the extract. Mean serum uoride concentration in uoride exposed rats was 0.145 0.009 and 0.783 0.042 lg/ml on days 0 and 98. In comparison, uoride concentrations in tamarind treated rats were 0.179 0.021 and 0.633 0.015; 0.179 0.021 and 0.502 0.025 and 0.176 0.021 and 0.498 0.030 lg/ml in low, medium and high dose groups, respectively on day 0 and day 98 of the experiment. There was a signicant (p 6 0.01) increase in urinary uoride excretion from day 28 onwards. The mean uoride concentration in long bones of treated rats was signicantly lower than the values recorded in uoride exposed rats. These ndings suggest that concomitant use of tamarind fruit pulp extract can reduce uoride concentration in blood and bone and enhanced urinary excretion, indicating the ameliorative potential of fruits of tamarind in uoride toxicity. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 9 January 2010 Accepted 23 September 2010

Keywords: Ameliorative potential Fluoride excretion Rats Serum uoride Tamarindus indica fruit extract

1. Introduction Fluoride is a cumulative poison and prolonged ingestion of small but toxic doses of uoride causes in chronic uoride poisoning commonly known as uorosis. Cases of uorosis in man and animals have been reported globally. In India, uorosis has been encountered due to both natural (Dwivedi et al., 1997) and industrial sources (Patra et al., 2000). Clinically, uoride toxicity is manifested mainly by overt bony and dental lesions. The predominant site of uoride accumulation is bone and teeth. However soft tissues like liver, kidneys, heart and lung also accumulates uoride in the body (WHO, 2002). Therefore, vital organs like liver and kidneys are also susceptible to its toxic effects, pathological changes occur even before the development of overt clinical signs of uoride toxicity, and toxic effects of uoride can occur at exposure far lower than those producing clinical symptoms (Partanen, 2002). Amelioration of toxic effects of uoride in man and animal remained unresolved and controversial till date due to lack of safeeffective ameliorative agents that can remove F from the body
Corresponding author. Tel.: +91 581 2300587; fax: +91 581 2303284.
E-mail address: sahadeb_dey@hotmail.com (S. Dey). 0034-5288/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.09.013

and can ameliorate toxic effects as well (Osweiler, 1999). Chemical compounds such as aluminum sulfate (Radostits et al., 2000), ascorbic acid and boron (Elsar et al., 1979) have been reported effective in experimental uoride exposure with variable success (Coffey et al., 2007). These agents are generally not indicated for prolong use due to their toxic side effects, which underlines need for evaluating a nontoxic-safe agent that can reduce uoride burden in body and ameliorate toxic effects of uoride (WHO, 2002). In India, Ayurvedic system of medicine proposes a number of herbs that can ameliorate toxicity due to chemical agents. However, these herbs have been put to limited scientic evaluation to date and their use is mainly restricted to observable recovery. Tamarind (Tamarindus indica L.) is found all over India and has been used in the treatment of pain, diabetes, urinary problems, infection and stress in man and animals (Nadkarni, 1982). The traditional ethnoveterinary practitioners consider tamarind fruit as an excellent natural product that has immense therapeutic potential in many pathological conditions. Use of powdered tamarind fruit pulp for amelioration of metal poisoning is mentioned in the literature (Kirtikar and Basu, 2001). In this study, hydro-methanolic (1:1) extract of tamarind fruit pulp was tested for its potential to reduce body uoride residues in experimentally uoride exposed rats.

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2. Materials and methods 2.1. Source of plant material Fresh tamarind fruits were collected from the natural habitat around Bareilly during FebruaryMarch 2007. These were identied and authenticated from Botanical Survey of India, Central National Herbarium, Howrah, India (voucher No. CNH/1-1/2007 Tech 11) where voucher reference specimens were deposited. 2.2. Extract preparation The plant material (fruits of tamarind) was washed with distilled water to remove the dirt and air dried. The fruit kernel and seed were removed and fruit pulp was separated. The pulp was dried in shed and stored in air tight glass container. Ten grams of the pulp was mixed with 150 ml of solvent (methanol:water, 1:1) and kept at 25 C overnight. Thereafter, it was stirred in a magnetic stirrer for 1 h and ltered. The extract was dried in vacuo and stored refrigerated until used. 2.3. Calculation of recovery percentage For calculation of recovery percentage of T. indica fruit pulp, 2 g of test material was mixed with 100 ml aqueous methanol (1:1), stirred after overnight, ltered and dried at 100 C in an oven to remove the water. The percent dry matter was calculated as per the following formula

capillaries piercing through the outer canthus of the eye for estimation of uoride. 2.6.2. Urine Urine samples were collected on morning hour of day 0 and there after at 14 days interval directly into a clear plastic vial without any preservative for estimation of uoride. 2.6.3. Bone samples The animals were sacriced 24 h after the last dose of uoride administration. The muscle tendons and ligaments were removed to clean bones. Whole of the femur and radius-ulna were collected in polyethylene bags without any preservative. The bones were cleaned and processed (Mikaelian et al., 1999) for F estimation. 2.7. Analyses of uoride The uoride concentration in serum, urine and bone was estimated by ion specic potentiometry following the method of Cernik et al. (1970) with a slight modication using uoride ion specic electrode (Orion Model 96-09) and ISE meter (Orion Model 290A). The detection range for this instrument was 0.019 1900 ppm. Calibration was made using ve freshly prepared working standards. The results were expressed in parts per million (ppm). The accuracy and precisions of the measurements were maintained by repeated analysis of reference standard procured from Orion Research Inc. Laboratory, USA. 2.8. Statistical analysis The data were analysed statistically. Standard error of mean and p-values were used to determine whether there was any signicant difference among different treatment groups using Fishers t-test and one-way analysis of variance (ANOVA) following standard protocol (Snedecor and Cochran, 1994). 3. Results The concentration of uoride in serum, and bones of rats of different groups is presented in Fig. 1 and Table 2. In comparison to healthy control rats (no uoride group), rats of no treatment group had signicantly higher serum uoride concentration from day 14 onwards. Concomitant use of tamarind extract signicantly (p 6 0.001) reduced serum uoride concentration in rats. Preventive effects were more pronounced in rats of medium and high dose group receiving fruit pulp extract @ 50, 100 mg/kg body weight. The serum level of uoride in these groups remained signicantly lower than exposed untreated rats (no treatment group) from day 42 onwards. Rats, receiving T. indica fruit extract showed signicant increase in urinary uoride excretion from day 42 (high dose group), 56 (middle dose group) and 84 (low dose group) onwards. Maximum excretion was in high dose group, followed by middle dose and low dose group (Fig. 2). Fluoride excretion in urine of rats of middle dose and high dose group was 158.3% and 185.3% higher than corresponding values in no treatment group on day 98.

Recovery percentage X =Y 100

where X is the weight of the dried extract and Y the weight of the plant material taken for extraction. The recovery percentage was 60% in the present experiment. 2.4. Animals Thirty female rats (IVRI 2CQ) of 1213 weeks old, weighing about 160180 g, bred in the Laboratory Animal Research Division of the institute were used for experiment after obtaining permission from Institute Animal ethics Committee. The rats were housed in plastic cages with proper bedding and maintained on standard ration and ad libitum water. They were acclimatized for 30 days before starting the experiment. 2.5. Experimental protocol The rats were divided into ve groups of six animals each and subjected to following treatments for 98 days (Table 1). 2.6. Sampling 2.6.1. Blood Serum samples were collected from individual rats on morning hour of day 0 of the experiment and thereafter at 14 days intervals for 98 days from the orbital plexus using microhaematocrit

Table 1 Treatment schedule given to experimental rats. Treatment Sodium uoride (mg/kg b.wt.) as 1.0% aqueous solution once daily The extract (mg/kg b.wt.) Placebo No uoride 0 0 Distilled water No treatment 200 0 0 Low dose the extract 200 25 0 Middle dose the extract 200 50 0 High dose the extract 200 100 0

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1 Fluoride concentration(g/ml)
No Fluoride

0.75
No Treatment

0.5

Low Dose Middle Dose High Dose

0.25

0 0 14 28 42 56 70 84 98 Days of exposure

Fig. 1. Mean uoride concentration (lg/ml) in serum of different treatment groups over the whole experimental.

Fluoride excretion in urine (g/24 h)

40 No Fluoride 30 20 10 0 0 14 28 42 56 70 84 98 Days post exposure No Treatment Low Dose Middle Dose High Dose

Fig. 2. Fluoride excretion in urine (mean s.e.) of different groups over the experimental period.

Table 2 Fluoride concentration (lg/g) in bone of different groups of rats after 24 h of the last dose. Group Bone Femur A B C D E
* **

different doses was capable of reducing concentration of uoride in serum and bones with concomitant increase in excretion of uoride in urine. The medium and high dose groups were essentially equal in efcacy, with neither achieving levels seen in rats not treated with uoride. No published data are available to explain the therapeutic value of T. indica fruit pulp in the management of uorotic patient (Khandare et al., 2002). Tamarind has been used traditionally in India and elsewhere for varied purposes ranging from food spices to medicines. Pods (fruits) are rich in ascorbic acid and sugar and have been used in syrup juice and drinks and many food preparations as spices. Medicinally the fruit pulp is valued as cathartic, astringent, febrifuge antiseptic and refrigerant. The extract of tamarind fruit is reported to contain chemical constituents like pectin, 2,3,5, tri-o-methyl arabinose, 2,3,4,tri-o-methyl D xylose, 2,3,4,6,tetra-omethylgalactose, 2,3,6,tri-o-methyl D-glucose (Asolkar et al., 1992); organic acids mainly potassiumhydrogen tartarates as well as free acids such as tartaric, malic and citric acids; phenolic compounds (+)catechin, ()epicatichin, procyanidine, taxifolin, apigenin, eriodictyol, luteolin and naringenin (Sudjaroen et al., 2005). The exact mechanism by which tamarind extract interferes with uoride deposition in tissues is not yet clear. Presence of terpenoid like geraniol and limonene has been reported in aqueous methanol extract of T. indica fruit pulp (Sudjaroen et al., 2005). These chemical constituents contain free OH group in their structure which possibly interact with uoride with hydrogen bonding and help in removing the element from the body (Pandey et al., 1996). The results of the present study suggested that hydro-methanolic extract of tamarind fruit pulp possessed the potency to reduce body uoride burden with enhanced excretion of uoride in urine. However, further studies are required to establish the mechanism of interference and activity guided separation and characterization of compounds in T. indica hydro-methanolic fruit extract involved in removal of uoride from the body.

Acknowledgements
Tibia and bula 1151.32 5.04 3038.61 9.36 2772.54 7.04** 2293.37 12.06** 2136.52 9.44**

1002.2 4.26 2901.78 6.14 2708.82 11.32* 2214.15 9.36** 2019.93 8.70**

Differ signicantly (p 6 0.05) compared to group B. Differ signicantly (p 6 0.01) compared to group B.

The nancial assistance given in the form of Network project on Ethnoveterinary Medicine by The Indian Council of Agricultural Research is thankfully acknowledged. We acknowledge the technical assistance of Mr. Brijesh Tyagi in estimation of uoride in biosamples.

References In healthy rats (no uoride group), the mean uoride concentrations in long bones were 1002.2 4.26 and 1151.32 5.04 ppm in femur and tibia and bula, respectively. Exposure of uoride for 12 weeks led to an increased uoride concentration to 2901.78 6.14 ppm in femur and 3038.61 9.36 ppm in tibia and bula of no treatment group rats. The rats receiving tamarind extract showed signicantly (p 6 0.001) lower uoride accumulation in bones. The mean uoride concentrations in femur was estimated as 2708.82 11.32, 2214.15 9.36 and 2019.93 8.70 ppm in rats of low dose, middle dose and high dose groups respectively (Table 2). 4. Discussion and conclusions The ameliorative potential of the test extract was assessed on the basis of its effect on uoride concentration in serum, and long bones and uoride excretion in urine. Our study shows that simultaneous administration of T. indica methanolic fruit pulp extract at
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