Anais da Academia Brasileira de Ciências (2018) 90(4): 3871-3878 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201820180285 www.scielo.br/aabc | www.fb.com/aabcjournal Antioxidant and antimicrobial potential of selected varieties of Piper betle L. (Betel leaf) CHAYANIKA SARMA1,2, PRASAD RASANE1,3, SAWINDER KAUR1,4, JYOTI SINGH1, JOGINDER SINGH5, YOGESH GAT1, UMAR GARBA6, DAMANPREET KAUR1 and KAJAL DHAWAN1 1Department of Food Technology and Nutrition, Lovely Professional University, GT Road, 144411 Phagwara, Punjab, India 2Indian Institute of Food Processing Technology, Pudukkottai Road, 613005 Thanjavur, Tamil Nadu, India 3Centre of Food Science and Technology, Banaras Hindu University, Lanka Road, 221005 Varanasi, Uttar Pradesh, India 4Sant Longowal Institute of Engineering and Technology, Campus Road, Sangrur, 148106 Longowal, Punjab, India 5Department of Microbiology, Lovely Professional University, GT Road, 144411 Phagwara, Punjab, India 6Department of Agro-Industry, Naresuan University, Phitsanulok-Nakhonsawan Road, Tapho Sub-District, Muang District, Phitsanulok 65000, Thailand Manuscript received on March 19, 2018; accepted for publication on July 2, 2018 ABSTRACT Piper betle L., is an evergreen perennial creeper belonging to family Piperaceae and is known to possess numerous medicinal properties. Current study focuses on evaluating antioxidant and antimicrobial potential of betel leaf. For the present study, distilled water, hexane, acetone and ethanolic extracts of two varieties of betel leaves: Meetha paan and Banarasi paan were used. Biochemical tests such as proximate analysis (moisture, ash, protein, lipids, minerals viz., sodium and potassium), antioxidant activity tests (DPPH radical scavenging activity, total phenolics, ascorbic acid, reducing power) and antimicrobial test (antibacterial and antifungal susceptibility test) against four pathogens viz., B. subtilis, E. coli, A. niger and S. cerevisiae were determined. Ethanolic extract had the highest antioxidant activity (89.46% inhibition), while the aqueous extract exhibited lowest antioxidant activity (62.03% inhibition). With increasing concentration (5, 10, 25 and 50 µg/mL), the reducing power of leaf extracts also increased. The ascorbic acid was not significant in Banarasi paan (5.21mg/100 g) and Meetha paan (5.20mg/100 g). The highest antibacterial activity of ethanolic extract (Banarasi paan) may be attributed to the presence of phytosterols in the leaf varieties. Antioxidant and antimicrobial potential study will help to build a database and promote the utilization of betel leaf as a medicinal herb. Key words: Piper betle, piperaceae, antioxidant, antimicrobial, biochemical. INTRODUCTION way of life, mankind so far has been dependent on natural resources for its every need. We have been Naturally occurring herbs are being used for ignoring the rich natural heritage inherited from our a long time in food and for medicinal purposes ancestors. This ignorance has aggravated numerous throughout the world. Although, modern approach health issues in our day to day lives such as digestive towards lifestyle has isolated us from the natural problems, aging problems, etc. However, as the Correspondence to: Prasad Rasane menace of synthetic medicines and food additives E-mail: [email protected] in the form of preservatives, coloring agents, An Acad Bras Cienc (2018) 90 (4) 3872 CHAYANIKA SARMA et al. and antioxidants kept on increasing, mankind Varanasi, Uttar Pradesh, India. The local names of is becoming increasingly aware of the natural different varieties taken for the present study were: resources and its benefits. Several researches are Meetha paan (Midnapore, West Bengal, India) and now being directed to explore natural herbs for Banarasi paan (Varanasi, Uttar Pradesh, India). The their nutraceutical, antimicrobial and nutritive leaf varieties were authenticated in the Department potential. One such herb of importance is Piper of Botany, Lovely Professional University, Punjab betle L., commonly known as betel leaf. (India). Betel leaf belongs to the family Piperaceae. It has more than 100 varieties, all over the world of SAMPLE PREPARATION which about 40 of them are found in India. It grows in dry, loam and clay soils that contain high amount With the help of a chilled mortar and pestle, fresh of detritus, maintaining a pH of 7-7.5. These heart leaves (10 g) were ground and dissolved in 100 mL shaped leaves are aromatic because of the presence of respective solvents i.e., distilled water, hexane, of essential oils and its taste ranges from sweet to acetone, and ethanol (Himedia, India). The extracts pungent (Pradhan et al. 2013). It is locally known were incubated in a shaker incubator (Remi Co., as ‘paan’ in Hindi (India) and is mostly consumed Model CIS 18 Plus, Mumbai, India) overnight at 28 in the form of mouth freshener or appetizer in India. ºC. Further, the samples were centrifuged at 10,000 The betel leaves are nutritive and possess an rpm for 10 min. All the prepared samples were insecticidal and antitumor activity (Gundala and stored at -20 ºC until further analysis. Aneja 2014), antioxidant activity (Jaiswal et al. PHYTOCHEMICAL SCREENING 2014), neuroprotective activity (Chan and Wong 2014), antidiabetic and antihelmintic activity (Shah Different biochemical assays were performed on et al. 2016), antimicrobial activity (Nouri and Nafchi both varieties of betel leaves extracted with hexane, 2014) and many more. The leaves also contain a ethanol, acetone and distilled water for checking the variety of biologically active components like presence of moisture, ash, carbohydrate, protein, hydroxychavicol, chavicol, piperbetol, chavibetol, piperol A, methylpiperbetol, and piperol. The key lipids, total soluble solid (TSS), vitamin C, total component of the leaf is a volatile oil known as phenolic, sodium and potassium. betle oil (Kumari and Rao 2015, Widawati and PROXIMATE ANALYSIS Riandi 2015). The present study was designed to evaluate Proximate analysis of samples was carried out the nutraceutical properties of two selected, according to Association of Offical Analytical popularly consumed varieties of Piper betle L., Chemists (AOAC) international methods (AOAC namely, Meetha and Banarasi. The antioxidant 2004). Moisture was determined by drying to a and antimicrobial potential of these varieties were constant weight at 105 °C. Ash content was carried analyzed. The database is aimed to help promote out at 550 °C (method 923.03). Crude protein (N the utilization of betel leaf in nutraceutical and functional food development. × 6.25) content was determined by the micro- Kjeldahl procedure (method 960.52). Crude lipid MATERIALS AND METHODS content was quantified by extracting the sample with petroleum ether in a Soxhlet apparatus. Total SAMPLE COLLECTION soluble solids (TSS) content for the extract was Healthy and young betel leaves were procured from determined with the help of hand refractrometer the local market of Midnapore, West Bengal and of range 0-32 ºBrix (Model ERMA) as described An Acad Bras Cienc (2018) 90 (4) BETEL LEAF: FUNCTIONAL PROPERTIES 3873 in Ranganna (2007). The potassium and sodium RESULTS AND DISCUSSION content of leaves was determined by the method NUTRITION AND PHYTOCHEMICAL PROFILE OF described in Ranganna (2007). BETEL LEAF DETERMINATION OF ANTIOXIDANT ACTIVITY The nutritional and phytochemical profile of the Free radical scavenging activity was determined betel leaf was analyzed and the result is shown using DPPH radical on the basis of scavenging in the Table I. The composition of lipids, vitamin ability of the extracts of betel on 2,2-diphenyl- C and total ash, including sodium and potassium 1-picrylhydrazyl (DPPH) free radicals (Jenitha content showed no significant (p>0.05) difference in and Anusuya 2016). The reducing power was both the varieties (Meetha and Banarasi) of leaves. determined by ferricyanide-ferric chloride There was slightly significant difference in the method as described by Shiban et al. (2012). Total carbohydrates and protein amongst the two varieties phenolic content of the sample was determined of the leaves. The results were in accordance with spectrophotometrically at 765 nm by using the findings of Shah et al. (2016). The presence of folin-ciocalteu’s reagent (Rasane et al. 2015). the five industry’ standard proximate composition Ascorbic acid content was determined using reveals the nutritive value of the leaves (Table 2,6-dichlorophenol indophenol dye (AOAC 2004). I). The essential oil contained in the leaves is known to possess antifungal, antibacterial and DETERMINATION OF ANTIMICROBIAL ACTIVITY antiprotozoan properties with the potential to kill or Antifungal and antibacterial susceptibility test inhibit the growth of disease causing bacteria. The significant vitamin and mineral content of leaves Two different betel leaf extracts were tested against also adds to its nutritive value (Guha 2006). Betel four microorganisms viz; Escherichia Coli (gram leaf oil contains a phenol called chavinol, which negative), Aspergillus niger (fungi), Bacillus has antiseptic properties. It was also reported by subtilis (gram positive), and Saccharomyces Dwivedi and Tripathi (2014) that, the chavinol in cerevisiae (yeast). Agar well diffusion method betel leaf is an aromatic compound responsible for was used for testing anti-microbial activity of the spicy odor of the leaves. Total soluble solids four