Betel (Piper betle L.) leaf essential oil extraction using steam distillation

— The highest essential oil content was obtained at distillation conditions as below raw material’s moisture content of 81.72%, distillation time of 5 hours, NaCl concentration of 15%, the ratio of raw materials and solvent of 1:3 (w/v), and the material size of 0.5mm <d <1.5mm. The quality of essential oils was then refined by using different method such as crystallization, anhydrous Na 2 SO 4 filtration and double distilling. By gas chromatographic mass spectrometry (GC-MS), the main chemical components in betel essential oil are identified includinging eugenol, aceteugenol, 4-allyl-1,2-diacetoxybenzene.


INTRODUCTION
Betel vine (Piper betle L.) belongs to genus Piper of the family Piperaceae. Leaves of Piper betle have several bioactivities properties and are used in traditional medicinal systems to cure indigestion, stomach ache, diarrhea, flatulence and to heal wounds, scales, burns, swelling etc. The leaves are nutritive and contain ant carcinogens, showing future opportunities in anticancer drugs. The main biochemical component betel leaf is essential oil and it contributes flavor to the leaf It is also reported that essential oil contributed to the medicinal property of betel vine [1]. The main ingredient in betel essential oil is phenols (Chavicol, Chavibetol, Eugenol, ..) along with some other phenolic compounds including beta-Caryophyllen, beta-Cadinen, 4-allyl-1,2-diaxetoxybenzen (27.51%) [2]. These phenol derivatives presented in betel leaf essential oil have multi biological effects such as antibacterial, antifungal, and antioxidant effects, etc. Betel leaf essential oil exhibits inhibitory activity against 3 strains of microorganisms: Gram (+) Bacillus subtillis, Aspergillus niger and Fusarium oxysporum molds with values of 100, 200 and 200 µg/mL, respectively [3]. In the previous research the essential oils of betel plants contain phenol group compounds such as safrol and eugenol which have insect repellent and insecticidal activity [4,5]. Compounds such as eugenol, limonene, terpinolene, citronellal, and camphor contained in volatile oils indicate rejection activity in mosquitoes and other biting insects, which can be used as an alternative mosquito repellent replace synthetic chemicals [6,7].
In this study, extraction essential oil from betel leaves (Piper betle L. ) by using steam distillation will be carried out. This objectif of study is evaluate different conditions for Piper betle L. essential oils extraction. The antibacterial and antioxidant activity of Piper betle L. essential oil are also evaluated in this study.

Materials
Preparation of material samples: The material samples used for this study were collected in the region of Hoc Mon, Ho Chi Minh city in Viet Nam. The betel leaves were washed and dried at room temperature. Agrinder (LC-1416B, Alaska, Vietnam) was used to change the size of materials.

Essential oil extraction
The betel leaf essential oil was extracted by the steam distillation method. An instrument has five main parts, including a 1000 mL volume flask containing solvent, which was distilled water in this case, another flask of 500 mL capacity was used to contain material of lemon peels. The system was heated by a VELP heating device (Model: ARE, Italy) placed under the solvent flask. A condensing equipment was placed on the top of 500 mL flask containing materials, in order to condense the steam to collect the essential oils.

Experimental design
The chemical compositions of betel leaves were firstly identified including: carbohydrates, Glycoside, Flavonoid, Phenol, Tannins, Saponin và Terpenoid. The moisture content, total solid content and color of raw material were also analysed.
In this study, four factors influencing betel essential oil distillation were investigated. The effect of raw material's moisture content on essential oil content was evaluated through different drying time of sample, which varies from 15 to 75 minutes of drying under temperature of 50ºC by using tray drying. Time of extraction varied at 60 minutes intervals from 60 to 360 minutes. Concentration of NaCl varied from 5 to 25%. A ratio of betel leaves and water varied from ½ to ¼ (w/v). All the experiments were repeated three times and average values were expressed.
The essential oil content was used to compared the efficiencies of different experiments. The obtained essential oil was dehydrated using different technique and calculated the content using the following equation: Chemical composition of essential oils was analysed using GC-MS method using THERMO SCIENTIFIC Trace GC Ultra -ISQ instrument. Antimicrobial activity of betel essential oil was evaluated through MIC value by using agar-well diffusion method. Antioxidant activity of betel essential oil was carried out using DPPH (1,1-diphenyl-2-picrylhydrazyl) method.

III. RESULTS AND DISCUSSION
Physicochemical characterisation of raw material  (Table 1). Glycosides and terpenoids are ingredients that make the acrid taste and pungent aroma of betel leaves. Phenol and sapiens are the components that make up the bioactive of betel leaves, especially in betel leaves contain many phenol derivatives, so betel oil has good anti-inflammatory, antifungal and antioxidant activity. Moisture content and total solid content of betel leaf are 81.72 ± 3.45 %wb and 2.27 ±0.18 %, respectively. The colour of betel leaf is present in Table 2. Effect of moisture content of raw material on essential oil content Essential oils are volatile compounds and may be evaporated along with steam. When increasing drying time, moisture content of the material decreases caused a reduction of essential oil content. At the moisture content of 81.72% (corresponding to the fresh sample), the highest concentration of essential oil was obtained. Table 5 shows that the content of essential oils varies with the distillation time. At 6h of steam distillation, the content of the essential oil tends to decrease. At 5h of distillation time, the content of essential oil is highest. This result is consistent with the results of Nguyen Nho Dung, 2011. The distillation time of 5h is used to conduct the further experiment.  /dx.doi.org/10.22161/ijaers.76.20  ISSN: 2349-6495(P) | 2456-1908(O) www.ijaers.com

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Effect of NaC concentrationl on essential oil content The betel essential oil content varies with the concentration of NaCl added ( Table 6). The essential oil content tends to decrease at the NaCl concentration of 20%. Using a high content of NaCl, the outer epidermis containing essential oils shrinks, preventing the diffusion of essential oils, results an decrease of essential oil content. At NaCl concentration of 15%, the highest content of essential oil. This result is consistent with the research results of Nguyen Thien Chi, 2016. NaCl concentration of 15% is used to conduct the further experiment.
Effect of raw material size and ratio of raw material/ solvent content on essential oil content  Table 7 shows that the essential oil content varies with the size of the material and the ratio of material/solvent. A grinned sample has a higher extracted essential oil than a non-grounded material. The contact surface between the grinded material and the solvent is larger which help the oil diffuse more easily. At the ratio of material/solvent at 1/ 3 (w/v), the highest content of essential oil is obtained. Chemical properties of betel essential oil The major components in betel essential oil is phenol compounds (> 77.59%)including aceteugenol (27,503%), 4-Allyl-1,2-diacetoxybenzene (27,391%), and Eugenol (22,693%). Other chemical components of betel essential oil are terpene compounds (terpinen-4-ol, -Muurolene, Germacrene D, -Muurolene, -Cadionol, ..). The phenol derivatives present in the essential oil have good biological effects such as anti-inflammatory, antibacterial and antioxidant.

IV. CONCLUSION
The suitable conditions for steam distillation of betel essential oil are as below the moisture content of raw materials of about 81.72% (fresh sample), distillation temperature of 100ºC, distillation time of 5 hours, NaCl concentration of 15%, the ratio of material/solvent of 1/3 (w/v) and the size of raw material varied from 0.5mm to 1.5mm.