Safety assessment of pomegranate fruit extract: Acute and subchronic toxicity studies

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Abstract

Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. Because of its potential for health benefits, pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years. The objective of the present study was to investigate potential adverse effects, if any, of a standardized pomegranate fruit extract in rats following subchronic administration. The extract was standardized to 30% punicalagins, the active anomeric ellagitannins responsible for over 50% of the antioxidant potential of the juice. The oral LD50 of the extract in rats and mice was found to be greater than 5 g/kg body weight. The intraperitoneal LD50 in rats and mice was determined as 217 and 187 mg/kg body weight, respectively. In the subchronic study, Wistar strain rats (10/sex/group) were administered via gavage 0 (control), 60, 240 and 600 mg/kg body weight/day of the extract for 90 days. Two additional groups received 0 and 600 mg/kg/day of the extract for 90 days, followed by a 28 day recovery phase. Compared to the control group, administration of the extract did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. The hematology and serum chemistry parameters that showed statistical significant changes compared to control group were within the normal laboratory limits and were considered as biological variations and not the toxic effect of the extract. Terminal necropsy did not reveal any treatment-related gross or histopathology findings. Based on the results of this study, the no observed-adverse-effect level (NOAEL) for this standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested.

Introduction

Pomegranate (Punica granatum L.) is one of the ancient fruit that is widely consumed as fresh fruit and juice. Use of pomegranate fruit dates back to Biblical times and reports of its therapeutic qualities have echoed throughout the millennia (Longtin, 2003). Pomegranate juice is a popular drink in the Middle East, and is also used in Iranian and Indian cuisine. In many traditional systems of health condition treatment, use of pomegranate for both internal and external conditions has been documented (Seeram, 2007). In traditional medicine pomegranate fruits have been used to treat acidosis, dysentery, microbial infections, diarrhea, helminthiasis, haemorrhage and respiratory pathologies (Vidal et al., 2003). In recent years several pomegranate-containing products have been widely marketed for health benefits around the world, including the United States (Cerda et al., 2003a).

The health benefits of pomegranate have been attributed to its wide range of phytochemicals. The phytochemicals found in pomegranate are predominantly polyphenols, including primarily hydrolysable ellagitannins, anthocyanins and other polyphenols. Ellagitannins found in the outer part of the fruit are largely responsible for the antioxidant activity of the pomegranate juice. It has been demonstrated that one of the ellagitannins, punicalagins (punicalagin anomers A and B) are responsible for over 50% of the antioxidant activity of the pomegranate juice (Gil et al., 2000). Since punicalagin is water soluble, commercial pomegranate juice obtained by pressing the fruit is found to contain significant amounts of this antioxidant. Tzulker et al. (2007) reported that the homogenates prepared from the whole fruit exhibited an approximately 20-fold higher antioxidant activity than the level found in the aril or seed sacs (fleshy or brightly colored cover of seed) juice. Depending on the cultivar of pomegranate and juice processing and storage methods, punicalagin content of the juice can vary from 1500 to 1900 mg/L (266–337 mg/6 oz) (Gil et al., 2000). For an individual weighing 60 kg and consuming one can of 6 ounce pomegranate juice, the punicalagin intake may range from 4.4 to 5.6 mg/kg. This suggests that the regular intake of pomegranate juice could result in high intake of this water-soluble hydrolysable ellagitannin. Punicalagin (CAS No.: 65995-63-3; Fig. 1), naturally abundant in pomegranate fruit and unique to pomegranate as a food source, is a proper chemical marker for the authentication, quality control and standardization of pomegranate products (Seeram et al., 2005b).

In recent years, several investigators have attempted to unravel the underlying mechanisms of beneficial effects of pomegranate. These investigations have focused mainly on the antioxidant, anti-inflammatory, and antibacterial potentials of pomegranate (Aviram et al., 2000, Aviram et al., 2002, Aviram et al., 2004, Cerda et al., 2003a, Cerda et al., 2003b, Lansky and Newman, 2007, De Nigris et al., 2007). Pomegranate juice and fruit extracts normalized to punicalagins and a total pomegranate tannin fraction (TPT) from the fruit have been shown to inhibit the proliferation of human cancer cells and modulate inflammatory subcellular signaling pathways (Adams et al., 2006, Seeram et al., 2005a, Afaq et al., 2005). The effects of pomegranate juice and extracts on molecular and cellular mechanisms has been researched on the systemic level, where benefits for cardiovascular, prostate, dental, and metabolic health have been shown in the clinical literature (Aviram et al., 2000, Aviram et al., 2002, Aviram et al., 2004, Pantuck et al., 2006, Menezes et al., 2006, Esmaillzadeh et al., 2006). Although increasingly claimed health-beneficial properties of pomegranate have been investigated to some extent, its adverse effects, if any, have not been systematically studied, partly because it is considered as a food. The objective of the present study was to investigate the adverse effects, if any, of a standardized pomegranate fruit extract following acute administration to rats and mice and subchronic administration to rats. The effects of the extract were investigated in a dose-response study.

Section snippets

Acute studies

The acute toxicity study was performed in compliance with Schedule “Y” and Good Laboratory Practices requirements of Govt. of India. In the acute studies, male and female Wistar strain rats and Swiss albino mice from Cadila Pharmaceutical Ltd. (Dholka, India) were used. In two separate experiments designed to determine oral LD50 of pomegranate fruit extract (POMELLA® Extract) in rodents, Wistar rats (6/sex/group; 10–11 weeks of age; body weight range: males 178–260 g, females 147–200 g) and Swiss

Acute toxicity studies

In the acute toxicity studies, oral LD50 of pomegranate extract in Wistar rats and Swiss albino mice was greater than 5000 mg/kg body weight. The 14-day observation period during the acute oral toxicity study and body weight measurements did not reveal any toxic effects in either species (data not shown). Necropsy at the end of the study did not reveal any gross pathological abnormalities in rats and mice. These observations from oral acute toxicity study suggest that the extract is practically

Discussion

In a previous study, Cerda et al. (2003a) investigated the effects of pomegranate extract (6% punicalagin) in female Sprague-Dawley rats following exposure to diet containing 20% of the extract for 37 days. The exposure to pomegranate extract resulted in intake of 4800 mg punicalagin/kg/day. A significant decrease in feed consumption and body weight of the animals during the early part of the study was noted. Except for a significant decrease in plasma urea and triglycerides, administration of

Conflict of interest

A conflicting interest exists when professional judgement concerning a primary interest (such as patient’s welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). It may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert

Acknowledgement

Verdure Sciences Inc. for supplying POMELLA® Pomegranate Extract for use in this study.

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