Ulcer protective potential of standardized hesperidin, a citrus flavonoid isolated from Citrus sinensis

Free radicals play an important role in stomach ulcer formation. The present investigation validates the anti ulcer activity of hesperidin, isolated from Citrus sinensis (L.) Osbeck, Rutaceae, through the assessment of its antioxidant potential over stomach mucosal tissue by histological examination. Hesperidin was isolated from the dried peel of C. sinensis, and authenticated by TLC, IR and HPLC. The anti-ulcerogenic potential of this fruit was assessed using indomethacin and hypothermic restrain stress-induced ulceration models on rats at 150, 300 and 450 mg/kg dose orally. The parameters measured were gastric pH, volume, free and total acidity, ulcer index, and mucin, glutathione, super oxide dismutase, catalase and protein content. Hesperidin at 300 and 450 mg/kg dose showed significant (p < 0.01-0.001) increase in pH, decrease in acidity and ulcer index against indomethacin and hypothermic restrain stress, along with histological evidence of cytoprotection. Glutathione, super oxide dismutase, catalase and mucin levels increased significantly at 450 mg/kg (p < 0.05-0.001) after indomethacin ulceration, whereas hypothermic restrain stress only increased glutathione and mucin levels. Hesperidin prevents oxidative cell injury by significant rise of super oxide dismutase, glutathione and catalase levels in gastric mucosa. Hesperidin allowed the regeneration of ulcerated tissue, and prevented hemorrhagic injury of gastric mucosa. The potential anti-ulcer effect of hesperidin may be due to antioxidant, mucoprotective and cytoprotective activities. © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. Papiya Bigoniya et al. / Rev Bras Farmacogn 24(2014): 330-340 331 many health benefits (Gattuso et al., 2007). Citrus herbal products prepared from mature or immature citrus peels have been traditionally used for liver rejuvenation (Dan and Andrew, 1986). During the processing of citrus fruit in juice and other food industries, peels are the primary byproducts, normally discarded after consuming the fruits. Many studies have shown that bioactive flavonoids present in the citrus peel possess strong antioxidant, antiatherogenic, anti-viral, antiaggregation, anti-mutagenic, anti-ulcer and antitumor effects (Del-Rio et al., 1992; Zia, 2006; Parmar and Kar, 2008). Flavonoids are a group of about 4,000 naturally occurring compounds that have a wide range of biological effects, including anti-ulcer activity. Plants synthesize flavonoids as an adaptive response to stress conditions (infection, wounding, water stress, cold stress). In recent years flavonoids have attracted the interest of researchers because they have promising powerful antioxidant properties that protect the human body from free radicals by their hydrogen donating abilities (Jeong et al., 2007). They are important constituents of the human diet (a daily diet should provide approximately 1 g of flavonoids per day) and are also found in several medicinal plants used in folk medicine around the world. Many epidemiological studies have shown that consumption of plants rich in phenolic compounds is associated with a lower risk of degenerative diseases such as cancer (Harris et al., 2007), cardiovascular disease (Naruszewicz et al., 2007), immune dysfunction (Kale et al., 2008) and allergic (Sokmen et al., 2004) complications. Free radicals play an important role in ulcer formation and erosive lesions in the gastrointestinal tract. Flavonoids like naringin, quercetin, silymarin, anthocyanosides and sophoradin derivatives have been reported to have antiulcer properties (Borrelli and Izzo, 2000). Citrus sinensis (L.) Osbeck, Rutaceae, is a small, shallowrooted evergreen shrub or tree about 6-13 m high with an enclosed conical top and mostly spiny branches. Leaves are smooth, oval, dark green and glossy, with a distinctive smell often similar to the fruit. Fruits are reddish-green to yellowishgreen, round, 4-12 cm in diameter, and have a leathery peel 6 mm thick, tightly adherent, protecting the juicy inner pulp (Anonymous, 2006). Hesperidin (1) is a flavanone glycoside found abundantly in sweet orange and lemon, and is an inexpensive by-product of citrus products (Garg et al., 2001). Hesperidin may be associated with potential benefits for the prevention of diseases, like decreased capillary permeability, inflammation, microbial infection and carcinogenic effects. A number of researchers have examined the antioxidant activity and radical scavenging properties of hesperidin using a variety of assay systems (Orallo et al., 2004; Hirata et al., 2005; Cho, 2006). Rossi et al. (1957) reported the effect of hesperidin on histamine-induced gastric ulcers in guinea pigs. Hesperidin (1) and neohesperidin dihydrochalcone allowed reduction of cold restraint-induced ulcer, but was ineffective on absolute ethanol-induced ulcer (Suárez et al., 1996). Lee et al. (2009) reported the inhibitory effect of neohesperidin and poncirin, isolated from Poncirus trifoliata, against HCl/ethanol-induced gastric lesions. Furthermore, Ping-Wei San, a Chinese decoction containing hesperidin, reduced gastric ulceration induced by indomethacin (Cheng et al., 2011). These reports showed experimental evidence of the beneficial effects of hesperidin against peptic ulcer. The present investigation was carried out to validate the anti-ulcer activity of hesperidin isolated from C. sinensis assessing its antioxidative potential in stomach mucosal tissue, along with histological examination of stomach mucosa, to evaluate the regenerative and cytoprotective effect on damaged mucosa. The present investigation was carried out to test and validate the anti-ulcer activity of hesperidin isolated from C. sinensis on two gastric ulcer models. The ulcer models used were, indomethacin to induce oxidative damage by inhibition of in situ synthesis of cytoprotective prostaglandins in stomach mucosa; and cold restrain to induce physiological stress related to the autonomic nervous system. Materials and methods Collection and authentication The fresh fruits of Citrus sinensis (L.) Osbeck, Rutaceae, were collected from November between December, 2010 at Bhopal (M.P). The botanical identity was confirmed from Saifia College of Science, Peer Gate, Bhopal (M.P.), by Prof. Ziaul Hassan and sample stored for future reference with authentication no. 192/ BOT/ SAIFIA/ 2010. Macroscopic characteristics of the fresh and dried peels were noted. Chopped green peels were dried for 36 h under the sunlight, followed by shade drying. Dried peels were powdered in a mixture grinder, packed in paper bags and stored in an air tight container until use.