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Free radical-scavenging activities of Homalium species – An endangered medicinal plant of Eastern Ghats of India a

Ajay Kumar Mahapatra , Sweta Smita Pani & Atish Kumar Sahoo a

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Medicinal & Aromatic Plant Division, Phytotherapy Research Laboratory, Forest & Environmental Department, Regional Plant Resource Centre, Govt. of Odisha, Nayapalli, Bhubaneswar 751015, Odisha, India Published online: 10 Dec 2014.

To cite this article: Ajay Kumar Mahapatra, Sweta Smita Pani & Atish Kumar Sahoo (2014): Free radical-scavenging activities of Homalium species – An endangered medicinal plant of Eastern Ghats of India, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.987142 To link to this article: http://dx.doi.org/10.1080/14786419.2014.987142

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.987142

SHORT COMMUNICATION Free radical-scavenging activities of Homalium species – An endangered medicinal plant of Eastern Ghats of India Ajay Kumar Mahapatra, Sweta Smita Pani and Atish Kumar Sahoo* Medicinal & Aromatic Plant Division, Phytotherapy Research Laboratory, Forest & Environmental Department, Regional Plant Resource Centre, Govt. of Odisha, Nayapalli, Bhubaneswar 751015, Odisha, India

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(Received 6 August 2014; final version received 7 November 2014)

Three species of the genus Homalium – e.g. Homalium nepalense, Homalium tomentosum and Homalium zeylanicum (Flacourtiaceae) – are recorded in India which are confined to the Eastern Ghat hill ranges. While H. zeylanicum is the IUCN red-listed medium-sized tree, the other two are endangered species of medicinal significance. The antioxidant potential of leaf and bark of the plants was evaluated through successive extraction methods by using hexane, chloroform, ethyl acetate and methanol. The extracts were subjected to in vitro assays as DPPH, hydroxyl, nitric oxide and superoxide along with its biochemical estimation. Amongst all, the ethyl acetate extracts of bark is found to be most potent compared with that of the leaves. H. nepalense has the highest amount of total phenolic and flavonoid contents followed by H. tomentosum and H. zeylanicum, respectively, and significant antioxidant behaviour. Keywords: free radicals; Homalium; flavonoids; phenols; antioxidant; phytochemical

1. Introduction Reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as nitric oxide and nitrogen dioxide which are generated through endogenous processes, such as mitochondrial respiration, or by activation of polymorphonuclear leucocytes, arachidonic acid metabolism and enzymatic functions, are responsible for many disorders and human diseases such as infections, diabetes, arthritis, cardiovascular diseases, cancer, Alzheimer and AIDS (Seal 2012). The human organs produce these ROS as a functional part of the harmonic balance between several physiological processes (Gupta & Verma 2010). Antioxidant compounds such as phenolic acids,

*Corresponding author. Email: [email protected]; [email protected] q 2014 Taylor & Francis

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polyphenols and flavonoids scavenge free radicals and inhibit the oxidative mechanisms (Arshad, Naveed, et al. 2013). Antioxidants stabilise or deactivate free radicals before they attack targets in biological cells (Gulcin 2012). Plant-derived antioxidants, especially the polyphenols and flavonoids, have attracted medicinal attention as bioactive agents and increasingly used for the treatment of diseases and upkeep of human health (Manian et al. 2008; Arshad, Naghmana, et al. 2013). A large array of plants of ethnobotanical importance has been subjected to biological assay to explore their antioxidant value in the last two decades. In this study, three species of Homalium, namely Homalium nepalense (Wall.) Benth., Homalium tomentosum Benth., and Homalium zeylanicum (Gardner) Benth. (Flacourtiaceae), are screened to identify the biological activity of the leaves and bark used in folk medicine. They are deciduous medium-sized trees up to 30 –40 m tall, found in Odisha, Bihar, West Bengal, Western Ghats and north-eastern states of India. Under the genus Homalium, 33 species are found across the world and many of them have been reported for pharmacological activities. For instance, Homalium deplanchi has antileishmanial, antitrypanosomal and antitrichomonal activities (Desrivot et al. 2007) while Homalium panayanum was found to exert antibacterial activity against Gram-positive and Gram-negative bacteria (Chung et al. 2004). Similarly, Homalium cochinchinensis has antiviral property (Ishikawa et al. 2004), whereas the African species Homalium africanum is tested for filaricidal activity (Cho-Ngwa et al. 2010). Homalium letestui was studied for its antioxidative, cytotoxic, antileishmanial, antiplasmodial, anti-inflammatory, antinociceptive and antidiabetic activities (Okokon et al. 2006, 2007, 2012, 2013). So far, amongst the Indian species, only H. zeylanicum has been examined for its anthelmintic activity (Gnananath et al. 2012). However, in terms of phytochemical screening as well as pharmacological evaluation H. nepalense, H. tomentosum and H. zeylanicum are yet to be studied, notwithstanding ethnobotanical evidence of these plant uses for common ailments such as wound healing, joint pain and fever by tribes in eastern India. Local use of leaves and bark varies from treating wounds to containing malarial fever. This prompted the author’s interest in examining the radical-scavenging activity of H. nepalense, H. tomentosum and H. zeylanicum through in vitro studies which will enhance the scope for deriving potential agents of drug value from these species. 2. Results and discussions 2.1. Phytochemical analysis The ethyl acetate extract of leaves and bark of H. nepalense, H. tomentosum and H. zeylanicum plants tested positive for the presence of flavonoids, tannins and saponins, indicating that the ethyl acetate extract of leaves and bark of all species contain substantial polyphenols and flavonoids. 2.2. Total phenols Total phenolic contents of leaves and bark extract of three Homalium plants are presented in Table S1. The phenolic content was calculated using the standard gallic acid and is expressed in terms of gallic acid equivalent (GAE). The results show that the phenolic content of ethyl acetate extract of H. nepalense bark was found to be 209.01 mg/g, while leaf extract possesses 191.33 mg/g (expressed as GAE). This was followed by ethyl acetate extract of H. tomentosum leaves (185.02 mg/g). The methanolic extract of H. zeylanicum bark contains 194.04 mg/g which is almost comparable to the ethyl acetate extract of H. nepalense bark and leaves. High solubility of phenols in polar solvents such as ethyl acetate provides high concentration of these compounds in the extract, and the principle of this method is the reduction ability of functional phenol group. The reduction of phosphotungstate – phosphomolybdenum complex

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(Folin –Ciocalteu reagent) by phenolate ion will change its colour to blue (Elizabeth et al. 2007). The reduction of the complex will increase when the extract contains adequate amount of phenolic compounds. Thus, the colour will be darker and the absorbance will be higher (Arbianti et al. 2007). Phenolic contents are higher in the three Indian species of Homalium than H. letestui which is reported in west Africa (Basil & Raphael 2014), and the scavenging ability of phenolics is mainly due to the presence of hydroxyl groups. It is responsible for a wide range of therapeutic uses such as antioxidant, antimutagenic, anticarcinogenic, free radical-scavenging activities and also decreases cardiovascular complications (Yen & Chen 1995).

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2.3. Total flavonoid content Flavonoid, one of the most diverse and widespread group of natural compounds, is an important natural phenol. We reported the flavonoid content in terms of quercetin equivalents (QEs). The maximum flavonoid content was recorded in the ethyl acetate extract of H. nepalense leaves and bark (92.1 and 105.8 mg/g, expressed as QEs), respectively, and followed by ethyl acetate extract of H. tomentosum (83.3 mg/g) and H. zeylanicum bark (80.11 mg/g) (Table S1). The ethyl acetate bark extract of all the three species have more flavonoid contents as compared with all other extracts of bark and leaves. Flavonoids possess biological activities such as antiinflammatory, anti-carcinogenic and anti-atherosclerotic activities (Olajire & Azeez 2011) and may have contributed to the potency of the genus Homalium, for which it is used in herbal medicine. 2.4. DPPH radical-scavenging activity DPPH assay is one of the most widely used methods for screening the antioxidant activity of plant extracts. The method is based on the reduction of alcoholic DPPH solutions in the presence of antioxidant. A direct linear relationship was found between the concentrations of different plant extracts of H. nepalense, H. tomentosum and H. zeylanicum in the DPPH solution and the inhibition percentages with a correlation factor R 2 $ 0.988 (Biotek, SynergyH1MF, USA). The IC50 value of ethyl acetate extracts of H. nepalense leaf and bark was found to be 85 and 50 mg/ mL, respectively (Table S2). The ethyl acetate extract of the bark showed stronger scavenging activity than the extract of leaves which is comparable to the standard ascorbic acid (25 mg/mL). Of all solvents, the ethyl acetate extract of leaves and bark of H. nepalense, H. tomentosum and H. zeylanicum showed significantly higher inhibition percentage (stronger hydrogen-donating ability), and the phenolic constituents (phenolic hydroxyls) could be responsible for high antiradical properties of the ethyl acetate extract of Homalium, which was also reported for H. letestui, an African species (Basil & Raphael 2014). 2.5. Superoxide radical-scavenging activity In living organisms, the superoxide dismutases (SODs) cause the removal of O2 (Li et al. 1995). Superoxide generated in biological systems has high reactivity and short half-life due to which this assay becomes a difficult task. Antioxidant enzyme such as SOD converts the superoxide free radical anion to hydrogen peroxide. The superoxide anion radical is the major reactive oxygen species, and the toxic peroxidative products cause wide spread cellular injury. Out of the three Homalium species, H. nepalense showed strongest ability to neutralise SOD radicals. Superoxide radical-scavenging potential of the ethyl acetate extract of H. nepalense and H. tomentosum bark was determined to be in the concentration of 58 and 38 mg/mL (Table S2). The ethyl acetate extract of H. tomentosum leaves exhibited IC50 values of 86 mg/mL, which is comparable to the standard drug quercetin (IC50 values of 50 mg/mL). Scavenging of SOD is a

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measure of the antioxidant activity in the bark extract of H. nepalnese and H. tomentosum which was possible due to the presence of phenolic groups. 2.6. Hydroxyl radical-scavenging activity

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Hydroxyl radical, considered as one of the most reactive oxygen radical can destroy the biomolecules in the body, such as protein and DNA, causing mutagenesis, carcinogenesis and cytotoxicity (Tsai et al. 2001). It is the iron-salt-dependent degradation of dihydrogen peroxide that produces the highly reactive hydroxyl radical (Halliwell 2012). In our study, the ethyl acetate extract of H. nepalense and H. tomentosum bark showed strong potential to scavenge the hydroxyl radicals as the IC50 values were found to be 57 and 69 mg/mL (Table S2) comparable to standard drug ascorbic acid (60 mg/mL). The ethyl acetate extract of H. nepalnese, H. tomentosum and H. zeylanicum leaves and bark when added to the reaction mixture scavenged the hydroxyl radicals and prevented the degradation of deoxyribose. 2.7. Nitric oxide radical-scavenging activity The nitric oxide-scavenging activity in leaf and bark extracts were found to be concentration dependent (Table S2). The ethyl acetate extracts of H. nepalnese, H. tomentosum and H. zeylanicum bark were most potent and exhibited lowest IC50 value of 38, 36 and 50 mg/mL, respectively. Even the ethyl acetate extract of H. nepalnese leaves showed IC50 value of 40 mg/ mL, which is comparable to the standard drug-quercetin (60 mg/mL). An increased nitric oxide production in human body generates ROS and RNS, and these free radicals contribute to the pathogenesis of pain, inflammation, etc. (Arshad, Naveed, et al. 2013). The extract from Homalium species leaves and bark can be an important biological source to manage ROSinduced tissue damages. 3. Conclusion This study reported antioxidant activity, total phenolic and flavonoid contents of the leaves and bark part of three Indian Homalium species. In order to realise medicinal values from potential plant sources, it is important to measure the antioxidant activity using various radicals and oxidation systems. From the above-mentioned experimental design, it is established that leaves and bark of H. nepalense possess better antioxidant quality than other Homalium species that need further study and clinical tests. Bark of the plant was proved to be more potent than the leaves. Three Indian Homalium species have shown better scavenging activities than the reported West African species of H. letestui and further study on Indian species is essential for the isolation of active constituents, its structural determination and mode of action of the extracts. Supplementary material Experimental details relating to this article are available online, alongside Tables S1 and S2. Acknowledgements The authors wish to acknowledge the financial support of National Medicinal Plants Board (NMPB), New Delhi, India for funding the research.

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Free radical-scavenging activities of Homalium species--An endangered medicinal plant of Eastern Ghats of India.

Three species of the genus Homalium - e.g. Homalium nepalense, Homalium tomentosum and Homalium zeylanicum (Flacourtiaceae) - are recorded in India wh...
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