Acute and sub-chronic oral toxicity studies of hesperidin isolated from orange peel extract in Sprague Dawley rats

https://doi.org/10.1016/j.yrtph.2019.04.001Get rights and content

Highlights

  • Hesperetin-7-rhamnoglucoside (Hesperidin) was isolated and standardized from citrus fruit.

  • In acute oral toxicity (AOT), hesperidin showed median lethal dose (LD50) of 4837.5 mg/kg.

  • In 90 days sub-chronic toxicity, low observed adverse effect level was 1000 mg/kg.

  • Hesperidin showed a good safety profile in the animal toxicological evaluation.

Abstract

Citrus sinensis contains glycoside hesperetin-7-rhamnoglucoside (hesperidin) which harbor an array of therapeutic potentials including antioxidant, anticancer, and anti-inflammatory. However, a systematic examination of safety is needed before its utilization. Hence, the present investigation is aimed to evaluate acute and sub-chronic toxicity of hesperidin isolated from the citrus fruit. Hesperidin (73%) was isolated from a methanolic extract of dried peel of the citrus fruit, characterized using FTIR, and standardized by HPLC. Its acute oral toxicity (AOT) and sub-chronic toxicity studies were carried out in Sprague-Dawley rats. Hesperidin (5000 mg/kg) showed 10% mortality in AOT. In sub-chronic toxicity study, hesperidin (250 and 500 mg/kg) did not induce any abnormalities in body weight, food consumption, clinical signs, ophthalmological and neurological observations, urine analysis, hematology, clinical chemistry, organ weights, and gross pathology. However, hesperidin (1000 mg/kg) showed significant (p < 0.05) alterations in body and organ weights, hematology, clinical chemistry, and tissue histopathology. To conclude, hesperidin has median lethal dose (LD50) of 4837.5 mg/kg, and Low Observed Adverse Effect Level (LOAEL) at 1000 mg/kg for both male and female Sprague-Dawley rats. Thus, hesperidin isolated from citrus fruit showed a good safety profile in animal study.

Introduction

Natural products are an essential source of drugs for management of various diseases. Numerous evidence have suggested that herbal therapeutic moieties are better in terms of safety and efficacy compared to synthetic chemicals (Aniagu et al., 2004; Delaney, 2007; Joshua Allan et al., 2007; Saad et al., 2006). Therefore, with the development of new medical technology, research on isolated compounds from natural and biological resources have garnered much attention. Various natural substances have wide applications in an array of fields including medicine, functional health food, and home remedies (Jakkula et al., 2004; World Health Organization, 2002; Yang et al., 2009).

In developing countries, majority of the population rely on folk medicine obtained from natural sources for the treatment of various diseases. However, these plants contain diverse bioactive compounds that bear the potential to cause an adverse effect (Bent and Ko, 2004). In the Indian subcontinent, ‘Ayurveda’ has been widely used as an ‘alternative medicine’ for the past 3000 years or so. Recently, Ayurveda has attained more focus in the field of medicine due to its safety and frequency of success. Moreover, it has been shown to provide excellent clinical results with lower adverse effects than Western medicine (Corns, 2003; Mashour et al., 1998; Yin et al., 2013). But before these traditional medicines can be transformed into modern drugs, significant toxicological information is needed. However, in recent years, the toxicological studies of natural products have rarely been reported. Therefore, there is a need to conduct and document systematic safety studies which examine these substances for their possible toxic effects.

Citrus sinensis (L.) Osbeck, (family: Rutaceae), commonly known as Sweet orange, is a citrus fruit which is largely cultivated in India and various countries around the world. It serves as a major source of vitamin C, potassium, folic acid, and pectin. It is widely consumed as fresh fruit, and juice while the peel is often discarded as waste. According to a report by the United States Department of Agriculture, the global production of this citrus fruit is 82 million tons per year (United States Department of Agriculture, 2018) of which 34% is used for juice production which yields about 44% peel as a by-product (Rafiq et al., 2016). It is to be noted that these peels possess an array of secondary components including polyphenols, vitamins, amino acids, minerals, dietary fibers, essential oils, pectin, flavonoids, vitamin C, and carotenoids. Thus, these have a broad range of activities including antimicrobial, antioxidant, anticancer, diuretic, and stomachic (Grosso et al., 2013; Manthey and Grohmann, 2001; Wang et al., 2014). In traditional Chinese medicine, orange peels have been used as a tonic to treat disorders related to skin, immune system, digestive system, and vitamin deficiencies.

It has been reported that some of its promising biological properties can be attributed to the presence of flavonoids in citrus fruit peel extract (Rafiq et al., 2016). One of these is hesperidin (hesperetin-7-rhamnoglucoside) which is a flavonoid glycoside. The effects on experimental animals of various clinical and epidemiological studies have supported the role of hesperidin across multiple diseases. Hesperidin is reported to have therapeutic potential against various diseases including hyperlipidemia, diabetes, cancer, arthritis, infections, allergies, inflammation, hypertension, fibrosis, and oxidative stress (Akiyama et al., 2010; Jin et al., 2008; Kandhare et al., 2017; Kaur et al., 2006; Kawaguchi et al., 1997a; Lee et al., 2004; Shah and Patel, 2010; Shi et al., 2012). Hesperidin has been known to play a vital role in the prevention of diabetes-induced complications including nephropathy, neuropathy, cardiomyopathy, and brain damage (Ibrahim, 2008; Shah and Patel, 2010; Visnagri et al., 2014). Previous research showed that treatment with hesperidin attenuates sodium arsenite-induced toxicity (Pires Das Neves et al., 2004) and breast cancer in rats (Nandakumar et al., 2011). Hence, hesperidin isolated from citrus peel extracts received considerable attention for the management of various diseases. In view of the above, use of hesperidin for clinical management of various diseases is expected to increase. However, no cases of intoxication have been reported to date, and no adverse effect have been systematically studied after hesperidin ingestion.

Various animal models have played a significant role in the evaluation of safety of drugs and plant products (Delaney, 2007). Therefore, toxicological insult in rat and mice provide a good correlation of safety between the preclinical and clinical settings (Olson et al., 2000). In the past, various studies have been undertaken with high doses in mice for acute effects, and lower doses in rats for chronic effects to determine the clinically useable dose of potential drugs while abiding by the guidelines of Organization for Economic Cooperation and Development (OECD) (Jadeja et al., 2011; OECD, 1998; OECD, 2008). To our knowledge, toxicological evaluation of hesperidin isolated from citrus fruit extract has not been systematically studied yet. Hence, the present study was aimed to investigate the toxicological effects, if any, of hesperidin isolated from citrus peel extracts in rats by acute and sub-chronic administration using biochemical, hematological, and histopathological examinations in accordance with the OECD guidelines.

Section snippets

Collection and authentication

Fresh fruits of Citrus sinensis (L.) Osbeck, Rutaceae, were collected between November and December 2013 at Nagpur, India. These were authenticated by the Department of Botany, Agharkar Research Institute, Pune. Voucher specimen of plant materials are maintained in the laboratory. Macroscopic characteristics of the fresh and dried peels were noted. Chopped green peels were dried for 36 h in the sunlight, followed by shade drying. Dried peels were powdered in a mixer grinder, packed in paper

Isolation, and characterization of hesperidin

The methanol extract of citrus peels showed the presence of carbohydrates, glycosides, steroids, tannins, flavonoids, and polyphenols but absence of alkaloids. The percentage yield of methanolic extract was 60.24%. Isolated hesperidin is a light yellowish amorphous powder (yield 50.24%). With the ferric chloride test, it showed wine red color whereas, for Shinoda test it gave bright pinkish violet color. The melting point of isolated hesperidin was found to be 242–244 °C.

In the identification

Discussion

In developing countries, herbal medicines are the cynosure of scientific attraction due to their wide use as alternative medicines. However, these herbal remedies significantly affect the clinical outcomes when administered as a supplement with others, thus needing more careful monitoring (Stanley et al., 2005). In view of the increasing use of hesperidin as a pharmacological intervention for potential health benefits, and with the dearth of its toxicological documentation, there is a pressing

Conclusion

In summary, results of the present acute toxicity study showed that acute oral lethal dose of hesperidin was more than 5000 mg/kg with a median lethal dose (LD50) value of 4837.5 mg/kg. The sub-chronic oral toxicity study suggested that oral administration of hesperidin had Low Observed Adverse Effect Level (LOAEL) at 1000 mg/kg for both male and female Sprague-Dawley Rats. Thus, hesperidin isolated from citrus fruits showed a good safety profile in animal study and can be considered as an

Acknowledgments

The authors would like to acknowledge Dr. S.S. Kadam, Chancellor and Dr. K.R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, for providing necessary facilities to carry out the study.

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