Essential oil of Piper betle L. leaves: Chemical composition, anti-acetylcholinesterase, anti-β-glucuronidase and cytotoxic properties

Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx

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Essential oil of Piper betle L. leaves: Chemical composition, antiacetylcholinesterase, anti-β-glucuronidase and cytotoxic properties Swagata Karak, Jayashree Acharya, Sainiara Begum, Indira Mazumdar, Rita Kundu, Bratati De

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Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Piper betle L. Essential oil Acetylcholinesterase β-glucuronidase Cytotoxicity

The aroma and taste of different locally available varieties of Piper betle L. leaves vary due to their essential oil and other constituents. In this study, the essential oils were extracted from seven local varieties of P. betle L. leaves (Bangla, Bagerhati, Manikdanga, Meetha, Kalibangla, Chhaanchi and Ghanagete) with the aim to characterize the varieties on the basis of oil constituents and to study their enzyme inhibitory and cytotoxic properties. Forty five identified essential oil constituents were subjected to different multivariate statistical analyses e. g. PCA, PLS-DA (R2 = 0.40694; Q2 = 0.31199), OPLS-DA (R2X = 0.154; R2Y = 0.742; Q2 = 0.664) and sPLS-DA. The first three statistical models distinguished mainly Chhaanchi, Meetha, Manikdanga from the others. sPLS-DA model separated all the varieties. The biplot, VIP score plot and other loading plots obtained from the above mentioned models identified mainly the phenylpropanes and a few terpenes to be responsible for distinguishing the varieties chemically. The essential oils exhibited very good acetylcholinesterase inhibitory properties, suggesting their potential for enhancing memory function. The essential oils also inhibited β-glucuronidase, involved in excretion of xenobiotics and other materials from the body, and showed cytotoxic properties. The study suggested potential applicability of the P. betle L. leaf essential oils in medical and cosmetic sector.

1. Introduction

habits of people are changing with the change of life style. As a result, consumption of P. betle by local people is gradually decreasing and consequently the farmers are worst affected. The wastage may be minimized by extracting unsold leaves for EOs which may have promising industrial future (Guha, 2006). Essential oil components were analyzed from betel leaves collected from South India (Jirovetz et al., 1999), North India (Saxena et al., 2014), Eastern India (Suryasnata et al., 2016), other parts of India (Kumar et al., 2007; Bajpai et al., 2010), Sri Lanka (Mohottalage et al., 2007), Philippine (Rimando et al., 1986), Thailand (Tawatsin et al., 2006). Different bioactivities of betel leaf oils e.g. antifungal (Dubey and Tripathi, 1987; Prakash et al., 2010; Basak and Guha, 2015), antimicrobial (Sugumaran et al., 2011; Saxena et al., 2014), larvicidal (Wardhana et al., 2007), insecticidal (Tawatsin et al., 2006; Mohottalage et al., 2007), phytotoxic (Dubey and Tripathi, 1987) and antioxidant (Prakash et al., 2010) activities have been reported. Nanoemulsion (oil-in-water) of betel leaf essential oil showed antibacterial activity against selected food pathogens (Roy and Guha, 2018). Betel leaf essential oil could be applied as food preservative (Basak, 2018). The aim of the present study was to compare the essential oil composition and bioactivities e.g. acetylcholinesterase, βglucuronidase inhibitory and cytotoxic properties of different local

Essential oils are commercially important in perfumery, food and pharmaceutical industries because of their aroma. Bioactivities of different essential oils have been studied. Such oils have been screened for their antifungal, antimicrobial, and antiviral effects to a great extent. Essential oils and their components have also been reported to possess antioxidant, analgesic, digestive, anticarcinogenic, immunomodulatory, antiapoptotic, anti-angiogenic, semiochemical and other activities as reviewed recently (Koroch et al., 2007; Saad et al., 2013; Perricone et al., 2015). The aromatic leaves of Piper betle L. (Piperaceae), commonly known as betel leaf, are consumed as masticatory as well as for its medicinal importance in South East Asian countries. The essential oil (EO) present in the leaves is also consumed during chewing. The aroma and taste of such leaves varies with local varieties (Karak et al., 2016). Volatile oil content of betel leaves varies with varieties from 0.15% to 0.2% (Saxena et al., 2014). A large number of agricultural families depend on cultivation of betel vine in rural areas of India, particularly the state West Bengal, for their livelihood. But a huge quantity of the unsold perishable leaves are wasted (Guha, 2006). In addition, the chewing

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Corresponding author. E-mail address: [email protected] (B. De).

https://doi.org/10.1016/j.jarmap.2018.06.006 Received 8 August 2017; Received in revised form 6 June 2018; Accepted 20 June 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.

Please cite this article as: Karak, S., Journal of Applied Research on Medicinal and Aromatic Plants (2018), https://doi.org/10.1016/j.jarmap.2018.06.006

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varieties of P. betle leaves collected from West Bengal, a part of Eastern India.

Table 1 Volatile oil constituents identified in seven local varieties of P. betle leaf. Oil constituents

AIc

AIr

Mode of identification

MONOTERPENES

α-Thujene α-Pinene Camphene Sabinene Myrcene α-Terpinene β-Phellandrene 1,8-Cineole/Eucalyptol (E)-β-Ocimene γ-Terpinene Terpinolene Linalool Terpinen-4-ol α-Terpineol

928 934 948 971 991 1018 1029 1031 1052 1060 1086 1108 1179 1191

924 932 946 969 988 1014 1025 1026 1044 1054 1086 1095 1174 1186

AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI,

MS MS MS MS MS MS MS MS MS MS MS MS MS MS

SESQUITERPENES

δ-Elemene α-Copaene β-Elemene E-β-Caryophyllene β-Copaene γ-Elemene Aromadendrene α-Humulene γ-Muurolene Germacrene D β-Selinene α-Selinene Bicyclogermacrene α-Muurolene cis-β-Guaiene δ-Cadinene Palustrol Spathulenol Caryophyllene oxide Globulol Viridiflorol Cubenol α-Cadinol

1339 1377 1393 1421 1431 1435 1441 1455 1478 1483 1488 1497 1498 1502 1505 1526 1568 1580 1583 1585 1593 1642 1653

1335 1374 1389 1417 1430 1434 1439 1452 1478 1484 1489 1498 1500 1500 1492 1522 1567 1577 1582 1590 1592 1645 1652

AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI,

MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS

PHENYLPROPANES

Estragole /Methyl chavicol Chavicol Anethole/Isoestragole Safrole Chavicol, acetate Eugenol Methyl eugenol Eugenol acetate

1200 1260 1286 1290 1349 1368 1407 1532

1195 1247 – 1285 – 1356 1403 1521

AI, MS AI, MS MS AI, MS MS AI, MS AI, MS AI, MS

2. Materials and methods 2.1. Plant material Seven local varieties of betel leaves i.e., Piper betle L. var Bangla, Bagerhati, Manikdanga, Meetha, Kalibangla, Chhaanchi and Ghanagete were collected from betel baroujs located in specific areas of West Bengal, India, during late winter of 2014. Intermediary sized leaves were collected from more than 15 plants, for each variety considered here. The varieties were identified and validated. Voucher specimens [Accession no. 20018(CUH)/1,3,4,5,6,7 & 8] were deposited in the Herbarium of Department of Botany, University of Calcutta. 2.2. Chemicals and reagents Acetylcholineesterase enzyme (Source: electric eel), β-glucuronidase (ex. bovine liver), 4-nitrophenyl-β-D-glucuronide, MTT 3-(4, 5Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium Bromide and alkane standard solution (C8-C20) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5, 5´ Dithiobis (2-nitrobenzoic acid) (DTNB), Acetyl thiocholine iodide, myristic acid methyl ester (Methyl myristate), HPLC grade water, anhydrous Na2SO4, DMSO and methanol were bought from Merck Specialities Private Limited (Mumbai, India). HPLC-grade n-Hexane (95%) was procured from Spectrochem Pvt. Ltd. (Mumbai, India). Eugenol, MEM, FBS, Antibiotic-antimycotic solution was bought from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). 2.3. Sample preparation Freshly procured betel leaves were cleaned and cut into small pieces. Essential oil from the leaves of each local variety was extracted by hydro distillation in double distilled water (2.5 L) using Clevenger type apparatus. The essential oils collected were dried over anhydrous sodium sulphate and stored at 4 °C in the dark. Leaves collected from at least 15 plants of each variety were separated into four portions (2 kg and 3 X 500 g) for distillation of oil and analysis of chemical composition. For bioactivity analysis, oil extracted from leaf (2 kg) was used. 2.4. GC/MS analysis Profiling of the volatile oil constituents from the hydro distilled oil of 7 varieties of betel leaves was performed by GC–MS analysis. Essential oils were diluted with HPLC grade hexane (1: 200 V/V). GC–MS analysis was carried out using Agilent 7890 A GC [software driver version 4.01(054)] equipped with 5795C inert MS with Triple Axis Detector. HP-5MS capillary column [Agilent J & W; GC Columns (USA)] of dimensions 30 m x 0.25 mm x 0.25 μm was used. The analyses were done under the following oven temperature programme: injection at 60 °C (5 min), then temperature increase at 4 °C min to 220 °C and hold time 10 min (total 55 min run). The detector was operated on EI mode (70 V). The injection temperature was set at 230 °C; the MS transfer line at 280 °C; the ion source at 250 °C. Helium was used as the carrier gas with flow rate 1 ml / min (Carrier linear velocity 36.623 cm/ sec). Samples (1 μl) were injected via split less mode onto the GCColumn. Mass spectra ranging from 30 to 500 m/z were recorded. Essential oil constituents were identified by comparing the fragmentation patterns of the mass spectra with entries of mass spectra library NIST 2008 (fit and retrofit values higher than 75%) and. Arithmetic indices (AI) with respect to n-alkanes (C8 – C20) were calculated for comparison, using the following formula: AI(x) = 100 Pz+ 100 [(RT (x) – RT (Pz))/ RT (Pz + 1) – RT (Pz))], where ‘x’ is the unknown compound; ‘RT’ means retention time; ‘Pz’ is the alkane before x; ‘Pz + 1’ is the alkane after x (Adams, 2009). The relative amount of each

Index: AIc = Calculated Arithmetic Index; AIr= Reported Arithmetic Index; AI = Arithmetic Index; MS = Mass spectra.

constituent was expressed as percentage peak area, relative to the total peak area.

2.5. Assay for acetylcholinesterase inhibition Acetylcholinesterase (AChE) inhibition was measured following the method of Ellman et al. (1961) with a little modification as reported earlier from the laboratory (Acharya et al., 2016). In brief, oil (0.01 ml of different concentrations 0.5 - 0.8 mg/ml methanol), AChE (0.02 ml; 19.93 unit/ml buffer), buffer (1 ml, pH 8.0), 0.5 mM DTNB (0.01 ml), and 0.6 mM acetylthiocholine iodide solution (0.02 ml) were mixed. This reaction mixture was incubated at 37 °C for 20 min. Following incubation, optical density (OD) was measured immediately at 412 nm. Controls were devoid of test samples. The percentage of inhibition was calculated as: Inhibition (%) = [(Control OD – Sample OD)/ Control OD] × 100. One oil from each variety was analysed 4 times.

2

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Table 2 Relative area percentage of oil constituents. Local Varieties

Oil yield (vol/wt), [wt/wt] → Oil constituents ↓ α-Thujene α-Pinene Camphene Sabinene Myrcene α-Terpinene β-Phellandrene 1,8-Cineole/Eucalyptol (E)-β-Ocimene γ-Terpinene Terpinolene Linalool Terpinen-4-ol α-Terpineol Methyl chavicol Chavicol Anethole/Isoestragole Safrole δ-Elemene Chavicol, acetate Eugenol α-Copaene β-Elemene Methyl eugenol E-β-Caryophyllene β-Copaene γ-Elemene Aromadendrene α-Humulene γ-Muurolene Germacrene D β-Selinene α-Selinene Bicyclogermacrene α-Muurolene cis-β-Guaiene δ-Cadinene Eugenol acetate Palustrol Spathulenol Caryophyllene oxide Globulol Viridiflorol Cubenol α-Cadinol

Bangla

Bagerhati

Manikdanga

Meetha

Kalibangla

Chhaanchi

Ghanagete

(0.06), [0.07]

(0.1), [0.112]

(0.07), [0.08]

(0.08), [0.08]

(0.02), [0.02]

(0.08), [0.09]

(0.17), [0.19]

0.07 0.32

0.03

0.03

1.06 4.57 23.60 4.94 0.36 7.95 0.35 0.23 0.13 1.80 4.59 6.61 0.11 3.58

1.52 35.77 0.18 0.28 0.14 0.75 0.37 0.67

– – – – – – – ± ± – – ± – – – ± – – ± ± ± – ± ± ± ± ± ± ± ± ± ± – ± – – ± ± ± ± ± ± ± ± –

0.01 0.02

0.02 0.17

0.30 0.46

0.02

0.28

0.01

0.03

0.10 0.24 2.70

1.38 5.36 28.39

0.95 0.02 0.60 0.05 0.01 0.01 0.46 4.06 0.48 0.01

3.71 1.15 7.23 0.37 0.27 0.14 1.56 3.41 6.08 0.07

0.30

4.47

0.12 1.62 0.02 0.03 0.02 0.17 0.05 0.05

0.33 1.73 31.46

0.32 0.65 0.29 0.57

– ± ± – – – – ± ± – – ± – – – ± – – ± ± ± – ± ± ± ± ± ± ± ± ± ± – ± – ± ± ± – – ± ± ± ± –

0.00 0.01

0.16 0.13

0.15

0.26

0.04 0.02

0.03

0.35 1.31 4.62 0.49 0.37 1.44 0.09 0.07 0.03 0.41 0.87 1.02 0.03 1.17 0.15 0.45 13.17

0.19 0.18 0.09 0.15

4.64 0.51 5.11 33.06 4.27 0.61 3.48 0.22

0.72 1.20 3.05

1.44

0.55 40.03 0.18 0.44 0.16 0.15

– – – – – – – ± – – – ± ± – – – – ± ± ± ± – ± ± ± ± – – ± ± ± – – ± – – ± ± – ± ± – ± ± –

0.02 0.13

0.05

0.01 0.00

0.34 0.04 0.32 2.81 0.40 0.06 0.32 0.13

0.15 0.17 0.26

0.12

0.03 2.46 0.01 0.04 0.04 0.01

1.38 0.04

0.05 0.07 0.06 11.73 23.85 0.27 0.70 18.60 14.82 1.29 0.58 7.90 0.21 0.10 0.22 1.27 1.48 3.99 0.14 2.48 0.15 0.08 1.51 5.27 0.18 0.38 0.24 0.43 0.40

– ± ± – – – – ± ± – – ± ± ± ± ± ± – ± ± ± – ± ± ± ± ± ± ± ± ± ± – ± ± ± ± ± – ± – ± ± ± ±

0.01 0.04

0.06

0.36 0.02

0.38 0.20

0.06 0.04 0.03 1.34 5.57 0.06

0.06 0.02

0.34 8.46 5.99 0.46 0.37 2.69 0.11 0.04 0.06 0.60 0.62 1.48 0.07 0.96 0.04 0.04 0.75 2.26

0.34 0.61

0.51 22.16 20.40

0.31 13.23 0.40 0.38 2.51 4.30 5.87

0.90 0.30 1.76 24.46

0.10 0.40 0.18 0.15 0.25 0.21

0.43

– – ± – – – – ± ± – – ± ± – ± ± – – ± ± ± – – ± ± ± ± – ± ± ± – – ± ± – ± ± – – ± – – ± –

0.08 0.04 4.13 0.19 0.55 0.91 0.05 0.10

0.02 0.01

0.07 2.25 0.16 0.45 8.24

0.12 0.45

0.05 7.58 6.92

42.77 0.14 0.99 13.13 0.07

0.04 1.20 0.04 0.05

3.73 2.46 0.17 0.93

0.28 0.55 0.53

1.30 1.30 1.31 1.70

0.10 0.03

0.17

0.14 7.29

0.36 12.31

0.19

0.03

0.13

± – – ± ± ± ± – ± ± ± ± ± – – – – ± ± ± ± ± – ± ± ± ± – ± – ± ± ± – ± – ± ± – – – – – – –

0.04

0.59 0.12 0.27 0.66 0.01 0.38 0.03 0.04 1.21

12.03 0.02 0.15 2.54 0.01

0.16 0.07

1.13 6.07 23.02

0.60 0.37 0.06 0.15

0.58 9.66 0.37

0.20

1.05 2.11 5.91

0.20 0.21 0.28 0.03

3.21 0.19

0.08 6.68

1.13 43.97 0.25 0.59

0.02

0.18 0.33

– – – – – – – ± ± – – – – – – – – – ± ± ± – – ± ± ± – – ± ± ± – – ± ± – ± ± – ± ± – ± ± –

0.02 0.02

0.13 0.17 1.13

0.03 0.30 0.02

0.48 0.08 0.23

0.15 0.01 0.05 1.17 0.03 0.06 0.01 0.05

Not detected; Bold indicates important constituents and their corresponding area percentage.

2.6. Assay for β-glucuronidase inhibition

solution in a humidified incubator with 5% CO2.

β-Glucuronidase inhibition was measured following the method proposed by Kim et al., (1999) with modification. In brief, plant extract (0.34 ml) and β-glucuronidase (0.1 ml, 986.4 units/ml in 0.1 M buffer, pH 7.0) were incubated at 37 °C for 15 min. Following this pre-incubation, 0.06 ml of p-nitrophenyl- β-D-glucuronide (3.15 mg/ml in 0.1 M buffer) was added and the mixture was incubated at 37 °C for 50 min. OD value of the reaction mixture was measured at 405 nm and percentage inhibitions of the plant extracts were calculated as mentioned above. One oil from each variety was analysed 4 times.

2.8. Cytotoxicity assay Cytotoxicity and IC50 doses of the betel oil were determined by studying the cell viability by MTT assay. The assay was conducted according to Mosmann (1983) with slight modification. Cells (1 × 103) were seeded in a 96 well plate and incubated overnight. Then the cells were treated with different concentrations of essential oil obtained from P. betle ranging from 1 μl/well to 4 μl/well for 24 h. Then 100 μg of MTT (20 μl of 5 mg/ml MTT in PBS) was added to each well in dark and mixed well. This mixture was incubated in CO2 incubator for another 3 h. MTT was reduced to water insoluble formazan crystals by mitochondrial dehydrogenases of live cells. Media was removed from the wells, washed in PBS and the formazan crystals were dissolved with DMSO (80 μl) in dark. Absorbance was recorded in a microplate reader (BioRad) at 595 nm. From the absorbance data, cell death percentage was calculated with the given formula: Percentage of inhibition =

2.7. Cell maintenance Adherent human cervical cancer cell line HeLa (HPV18 positive) cells were obtained from NCCS, Pune, and maintained in Eagle’s MEM containing sodium pyruvate, glutamine and non essential amino acids supplemented with 10% FBS (Gibco) and 1x antibiotic-antimycotic 3

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Fig. 1. Eugenol content in varieties of P. betle L. leaf.

analysis (PCA), partial least squares discriminant analysis (PLS-DA) (validated by R2 and Q2 values), and orthogonal PLS-DA (OPLS-DA) showed distinct separation of varieties Meetha, Chhaanchi, and Kalibangla. OPLS-DA could also separate Manikdanga and Ghanagete. However, Bagerhati and Bangla could not be separated from each other. Sparse-PLS-DA (sPLS-DA), however, could separate all the varieties from each other on the basis of oil constituents (Fig. 3). In the next step attempts were made to characterize the local varieties on the basis of essential oil constituents. The biplot obtained from PCA, variable importance in projection (VIP) scores plot and variable influence in OPLS-DA model suggested that significantly important constituents for separation of varietal clusters were eugenol acetate, chavicol, chavicol acetate, methyl chavicol, safrole, E-β-caryophylline, terpinen-4-ol, and E-β-ocimene. sPLS-DA model selected the variables ranked by the absolute values of their loadings. Loading plot (Fig. 4) shows loadings 1 (for component 1) and loadings 2 (for component 2). Ten variables selected by this model included both phenylpropanes and terpenes for characterization of the varieties. The variables selected in loadings 1 were high in variety Chhanchi. Nine, out of ten, variables selected in loadings 2 were found to be highest in relative amount in Meetha. The tenth variable (eugenol acetate) was present in highest level in Ghanagete. Thus the different multivariate statistical analysis models indicated that mainly the phenylpropanes e.g. chavicol, chavicol acetate, eugenol acetate, methyl chavicol, safrole, eugenol and the terpenes e.g. E-βcaryophyllene, terpinen-4-ol were the important components for varietal variation. Chavicol was present in highest percentage in Meetha followed by Kalibangla and Bangla. The compound could not be detected in other varieties. Chavicol acetate could be detected in the essential oil of all the varieties, highest amount being present in Meetha and Kalibangla. Eugenol acetate also could be detected in all the varieties. Meetha contained lowest amount of this constituent. Safrole was present in high amount in Chhaanchi, followed by Manikdanga with significantly lower amount of this oil constituent. Eugenol was present in all the varieties. Interestingly the varieties Meetha (containing chavicol and methyl chavicol) and Chhaanchi (containing safrole) had low amounts of eugenol and eugenol acetate. E-β-Caryophyllene was highest in amount in Kalibangla. Highest level of terpinen-4-ol was detected in Chhaanchi. So Chhaanchi was distinctly different from other varieties due to high levels of safrole and terpinen-4-ol. Meetha

[(absorbance of control set - absorbance of treated set) / (absorbance of control set)] X100. Cell growth inhibition percentages in treated and control sets were calculated along with standard deviations. One oil from each variety was analysed 3 times. 2.9. Statistical analysis Each experiment was performed 3–5 times. Regression equations were prepared from the concentration of plant extracts and their corresponding inhibition percentages. Inhibition percentage is presented as mean ± standard deviation. IC50 value (sample concentration required to inhibit 50% enzyme activity) was calculated for each variety based on these regression equations. The data were also analysed by hierarchical cluster analysis and multivariate analyses such as PCA (Principal Component Analysis); PLS-DA (Partial Least Squares Discriminant Analysis); OPLS-DA (Orthogonal PLS-DA) and sPLS-DA (sparse PLS-DA) using Metaboanalyst 3.0 – a comprehensive tool suite for metabolic data analysis. 3. Results and discussions 3.1. Essential oil composition Forty five essential oil constituents identified from seven different local varieties of P. betle leaves included fourteen monoterpenes, twenty three sesquiterpenes and eight phenylpropanes (Table 1). On the basis of relative percentage area (Table 2) of each constituent with respect to total area, it was observed that the P. betle leaf oil had predominance of phenylpropanes as the major constituents. In varieties Bangla, Bagerhati, Manikdanga, and Ghanagete, eugenol acetate and eugenol were the major constituents. Eugenol content was measured quantitatively in each variety and compared (Fig. 1). Other varieties Meetha and Chhaanchi contained high proportion of other phenylpropanes such as chavicol and safrole respectively (Table 2). Hierarchical cluster analysis separated all the varieties based on 45 essential oil components, Chhaanchi and Meetha being distinctly different from the rest (Fig. 2). The data based on relative percent area were analyzed by multivariate exploratory approaches to characterize the varieties with more information. Two-dimensional score plot of principal component 4

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Fig. 2. Hierarchical cluster analysis of P. betle varieties based on normalized relative percentage areas of essential oil constituents (Distance measure: Euclidean; Clustering Algorithm: Ward).

et al., 2015). Known acetylcholinesterase inhibitor eserine had IC50 value 0.391 μg/ml (Nag and De, 2011). So essential oils obtained from P. betle leaves (except Chhaanchi) showed high acetylcholinesterase inhibitory properties. Eugenol, one of the major constituent of betel leaf essential oil was earlier reported to have acetylcholinesterase inhibition property with IC50 value 0f 0.48 mg/ml (Dohi et al., 2009). Some other constituents of betel leaf essential oil also individually inhibited acetylcholinesterase such as α-pinene (IC50 value = 0.022 mg/ml); 1,8-cineole/eucalyptol (IC50 value = 0.015 mg/ml); terpinen-4-ol (IC50 value = 3.2 mg/ml) and α-terpineol (IC50 value = 1.3 mg/ml) (Dohi et al., 2009). The total EOs obtained from betel leaves containing all the constituents, however, had much higher enzyme inhibitory activity than the individual components, suggesting synergism of all constituents.

was distinctly different from the other varieties due to high level of chavicol, methyl chavicol, and chavicol acetate. Kalibangla was distinct from others in chavicol acetate and E-β-caryophyllene levels. E-βOcimene was present in high amount in Bangla and Bagerhati. Thus the study shows the importance of the chemometrics approach for distinguishing oil constituents of different P. betle leaf oil. 3.2. Acetylcholinesterase inhibition Essential oil of all the varieties, except Chhaanchi, inhibited the enzyme acetylcholinesterase in a dose-dependent manner. A comparative account of their IC50 values (concentration of oil required for inhibition of enzyme activity by 50%) is presented in Table 3. The activity differed in different varieties. Ghanagete and Manikdanga varieties showed highest inhibitory activity. Acetylcholinesterase inhibitors are nowadays used for the treatment of Alzheimer’s disease (AD) so that there is no depletion of acetylcholine in the neural synapses. Acetylcholine is important for memory function (McGleenon et al., 1999). According to cholinergic hypothesis, to date, the agents which inhibit degradation of acetylcholine are used for the treatment of AD (Bishara

3.3. β-Glucuronidase inhibition Lipophilic xenobiotics are converted to more water soluble metabolites after glucuronidation, and thus, are more readily excreted in the urine or bile. Conjugation with glucuronic acid results in the 5

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Fig. 3. Segregation of P. betle varieties by multivariate statistical analyses: 3D sPLS-DA score plot of normalized relative percentage areas of essential oil constituents.

Fig. 4. Loading plots of ten variables selected by sPLS-DA model (a) loadings of component 1 (b) loadings of component 2.

Activities of all the EOs obtained from different varieties were significantly lower than that of the commercial β-glucuronidase inhibitor silymarin (IC50 value 794.62 ± 10.01 μg/ml) (Karak et al., 2017). Eugenol was also assayed for β-glucuronidase inhibition property. This phenolic compound inhibited the enzyme in a dose dependent manner with IC50 value 616.68 ± 8 μg/ml. Thus β-glucuronidase inhibition activity of eugenol was found to be higher than that of silymarin. β-Glucuronidase inhibition activity of EOs has not been studied much. EO of Cymbopogon citratus and some other members of the genus Citrus showed significant inhibition of β-glucuronidase, citral being the active constituent (Saleem et al., 2003). Another non-therapeutic use of β-glucuronidase inhibitors in cosmetic deodorant or antiperspirant composition for reducing the body odour has been invented. Body odours result from the decomposition of steroid esters by β-glucuronidase (Banowski et al., US patent, 2007). Specific β-glucuronidase inhibitors present in deodorants may prevent body odour. So the

inactivation of the toxic chemicals, carcinogens, steroid hormones, bile acids and bilirubin (Miners and Mackenzi, 1991). Hydrolysis of the glucuronide moiety, destined for excretion, can be carried out by βglucuronidase (Dutton, 1980), thus releasing harmful components. So inhibitors of β-glucuronidase (Kim et al., 1994) are considered for treatment of hepatic disorders. β-Glucuronidase inhibitors reduce carcinogenic potential of certain compounds normally excreted in bile after glucuronidation (Walaszek et al., 1984). β-Glucuronidase inhibitory activity of P. betle leaf oil obtained from seven different varieties were compared (Table 3). Initially the IC50 values of the essential oil obtained from Meetha and Ghanagete were calculated. As their IC50 values range from 5 mg/ml (in Meetha) to 14 mg/ml (in Ghanagete), so, the activity of all the varieties was compared at 7 mg/ml oil concentration. Bangla and Kalibangla varieties showed highest enzyme inhibition (97%–99%) followed by Meetha variety (95%). Chhaanchi variety showed lowest inhibition (9%) at the tested concentration. 6

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Table 3 Enzyme inhibitory and cytotoxic properties of essential oils. Bangla (A)

Bagerhati (B)

Manikdanga (C)

Meetha (D)

Kalibangla (E)

Chhaanchi (F)

Ghanagete (G)

Tukey Test#

Acetylcholinesterase Inhibition IC50 value (μg/ml)

0.36 ± 0.01

0.47 ± 0.01

0.11 ± 0.00

0.27 ± 0.00

0.35 ± 0.01

NA

0.10 ± 0.00

MTT Assay IC50 value (μg/ml)

7.33 ± 0.05

7.38 ± 0.16

7.84 ± 0.29

4.76 ± 0.01

3.79 ± 0.32

3.65 ± 0.00

12.74 ± 0.08

A-B**** A-C**** A-D** A-E** A-G**** B-C** B-D**** A-Bns A-C* A-D**** A-E**** A-F**** A-G**** B-C* B-D****

β-glucuronidase inhibition IC50 value (mg/ml) β-glucuronidase inhibition (%) at 7.5 mg/ml

–

–

–

5.26 ± 0.05

–

–

14.45 ± 0.25

98.98 ± 0.51

24.87 ± 1.53

24.87 ± 0.51

94.92 ± 2.54

97.80 ± 0.78

9.13 ± 2.04

14.5 ± 1.78

A-Bns A-C**** A-Dns A-Ens A-F**** A-G**** B-C**** B-D***

B-E**** B-G**** C-D**** C-E**** C-G****

D-Ens D-F**** D-G**** E-G**** F-G***

B-E**** B-F**** B-G**** C-D**** C-E**** C-F**** C-G****

D-E**** D-F**** D-G**** E-Fns E-G**** F-G****

B-Ens B-F**** B-Gns C-D*** C-E**** C-F**** C-G****

D-Ens D-F**** D-G**** E-F**** E-G**** F-Gns

NA: Not active; - not calculated. # P Value < 0.0001 (****); < 0.001 (***); < 0.01 (**); < 0.1 (*); Non significant (ns).

and β-glucuronidase inhibitory properties. Thus the present experimental findings suggest future applicability of P. betle essential oil as medicine and cosmetics after further research.

present findings regarding the β-glucuronidase inhibitory property indicated its potential therapeutic and non-therapeutic uses. 3.4. Cytotoxicity (antiproliferative study)

Acknowledgements

From the experiment, it was observed that essential oils obtained from different varieties showed varied level of cytotoxicity on the cervical cancer cell line (HeLa). Meetha, Kalibangla and Chhaanchi varieties were found to have a strong inhibitory (antiproliferative) activity at very low (3.65–4.76 μg/ml) concentrations, whereas Bangla, Bagerhati and Manikdanga showed activity at medium concentrations (7.33–7.8 μg/ml). Ghanagete variety exhibited somewhat higher IC50 value (12.74 μg/ml). So, on the basis of IC50 values, Ghanagete has the lowest activity. Highest activity was observed in Chhaanchi and Kalibangla followed by Meetha, Bangla, Bagerhati, and Manikdanga (Table 3). A positive inducer of cell death, doxorubicin (topoisomerase II inhibitor) was used as a standard and showed an IC50 of 2μM in HeLa cells. Among the seven varieties, the Chhaanchi showed antiproliferative activity at very low concentration indicating highest activity. P. betle leaf extract showed effect against the proliferation MCF-7 cells (Abrahim et al., 2012), in cancerous cell lines such as KB and HeLa cell line (Fathilah et al., 2010). Extracts of different species of Piper induced apoptosis in HeLa cells (Widowati et al., 2013). In this paper we report cytotoxic properties of seven betel leaf oils.

SK and BD acknowledge financial support from Department of Science and Technology, Govt. of West Bengal). All authors are grateful for financial support from Department of Science and Technology, Govt. of India (FIST programme), and UGC. References Abrahim, N.N., Kanthimathi, M.S., Abdul-Aziz, A., 2012. Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase. BMC Complementary and Alternative Medicine 12, 220. Acharya, J., Karak, S., De, B., 2016. Metabolite profile and bioactivity of Musa X Paradisiaca L. flower extracts. Journal of Food Biochemistry 40, 724–730. Adams, R.P., 2009. Identification of Essential Oil Components by Gas chromatography/ Mass Spectrometry, 4th ed. Allured Business Media, Illinois. Bajpai, V., Sharma, D., Kumar, B., Madhusudanan, K.P., 2010. Profiling of Piper betle Linn. Cultivars by direct analysis in real time mass spectrometric technique. Biomedical Chromatography 24, 1283–1286. Banowski B, Hoffmann D, Wadle A, Siegert P, Saettler A, Gerke T, 2007. β-glucuronidase inhibitors for use in deodorants and antiperspirants. Patent No. US 7,294,330 B2.. Basak, S., 2018. The use of fuzzy logic to determine the concentration of betel leaf essential oil and its potency as a juice preservative. Food Chemistry 240, 1113–1120. Basak, S., Guha, P., 2015. Modelling the effect of essential oil of betel leaf (Piper betle L.) on germination, growth, and apparent lag time of Penicillium expansum on semisynthetic media. International Journal of Food Microbiology 215, 171–178. Bishara, D., Sauer, J., Taylor, D., 2015. The pharmacological management of Alzheimer’s disease. Progress in Neurology and Psychiatry 19, 9–16. Dohi, S.I., Terasaki, M., Makino, M., 2009. Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils. Journal of Agricultural and Food Chemistry 57, 4313–4318. Dubey, P., Tripathi, S.C., 1987. Studies on antifungal, physico-chemical and phytotoxic properties of the essential oil of Piper betle. Journal of Plant Diseases and Protection 94, 235–241. Dutton, G.J., 1980. Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton, FL. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–90. Fathilah, A.R., Sujata, R., Norhanom, A.W., Adenan, M.I., 2010. Antiproliferative activity

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