Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Extraction of betel leaves (Piper betle L.) essential oil and its bio-actives identification: Process optimization, GC-MS analysis and anti-microbial activity Mitali Madhumita a, ⁎ , Proshanta Guha a , Ahindra Nag b a Department of Agricultural and Food Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India b Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India ARTICLE INFO Keywords: Fresh and cured betel leaves Essential oil GC–MS analysis Antibacterial activity SEM ABSTRACT Betel leaf (Piper betle L.) var. Bangla is an essential oil (EO) rich plant belongs to Piperaceace family used as traditional herbal medicine. The extraction of EO from fresh and cured betel leaves is of high interest for in- dustrial application. Thus, the present study was aimed to optimize the EO extraction and its bio-chemical characterisation. Box-Behnken Design coupled with response surface methodology was employed to optimize the independent variables such as liquid to solid ratio (30:1-50:1 mL/g), extraction time (5–7 h), and particle size (0–20 mesh). Quadratic polynomial models were found to be highly significant (p < 0.05) obtaining fresh and cured betel leaf EO with a yield of 0.18% ± 0.01% and 0.22% ± 0.02% at optimal condition: 40:1 mL/g, 6 h, and 10 mesh. Gas chromatography mass spectrometry (GC–MS) analysis revealed thirty-three and thirty volatile compounds, representing 98.41% and 97.34% of the optimized fresh and cured leaf EO, respectively which have a varied range of biological activities and industrial applications. Eugenol, estragole, linalool, α-copaene, an- ethole, chavicol, and caryophyllene were found to be highly abundant in both EO with different percentages. Antibacterial activity was evaluated against Mycobacterium smegmatis, Staphylococcus aureus and Pseudomonas aeruginosa and the results demonstrated that cured leaf EO showed significantly higher antimicrobial activity against M. smegmatis than fresh leaf EO. Moreover, morphological study was carried out to investigate the extraction mechanism of fresh and cured betel leaves with the help of scanning electron microscopy (SEM). 1. Introduction Betel leaves (Piper betel L.), is a perennial traditional herbal plant belonging to Piperaceae family and originated in Malaysia. Over 700 species of Piper betel were discovered in both Northern and Southern hemispheres of the world and widely grown in most of the countries such as India, Sri Lanka, Malaysia, Indonesia, Philippines, and other Southeast Asian and East African countries (Umar et al., 2018). This plant is popularly known as Paan and widely cultivated as well as consumed in all over India (Guha, 2006). It is an evergreen aquatic root climbing vine with dorsiventral heart shaped leaves which are con- sumed regularly by a large number of the Asians. The leaves having high pungent palatable taste causes irritation in mouth to some con- sumers. Therefore, sometimes this pungency is removed by a special smoke treatment called curing. Cured betel leaves were prepared by local farmers of West Bengal within a closed chamber. In that chamber, fresh green betel leaves were treated with smoke at a temperature of 50ᵒC for 6 h and then cooled at room temperature for 12 h. The green betel leaves were converted to yellow leaves (i.e. called cured leaves) after incubating for 15–20 days (Guha, 2007). The cured leaves produce better taste, colour (light yellow to white) and other attractive orga- noleptic properties besides stimulating and refreshing effects. The leaf has got a good nutritive value particularly due to its high mineral contents (mainly calcium), vitamins and bioactive compounds like phenolic, flavonoid, essential oil (EO) etc. Betel leaves contain a vola- tile EO contributing most of its medicinal, organoleptic, and other de- sirable properties. Essential oil is a mixture of a large number of volatile compounds (secondary metabolites) having complex composition with https://doi.org/10.1016/j.indcrop.2019.111578 Received 19 April 2019; Received in revised form 14 July 2019; Accepted 16 July 2019 Abbreviations: EO, essential oil; HD, hydro distillation; BBD, Box-Behenken Design; RSM, response surface methodology; ANOVA, analysis of variance; FLEO, fresh leaf essential oil; CLEO, cured leaf essential oil; GC–MS, gas chromatography–mass spectrometry; HPLC, high-performance liquid chromatography; LC–MS, liquid chromatography with mass spectrometry; NMR, nuclear magnetic resonance spectrometry; FT-IR, Fourier transform infrared; d.b., dry basis; SEM, scanning electron microscopy; LB, lysogeny broth; Eq., equation; 3D, three dimensional; CFU, colony forming unit; SD, standard deviation ⁎ Corresponding author. E-mail address: mitalimadhumita5@gmail.com (M. Madhumita). Industrial Crops & Products 138 (2019) 111578 0926-6690/ © 2019 Elsevier B.V. All rights reserved. T