Environmental Technology & Innovation 20 (2020) 101140

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Antidiabetic, Antithrombin and Cytotoxic bioactive

compounds in five cultivars of Piper betle L.
Subramaniam Yogeswari a , Kaipa Hima Bindu b , Subban Kamalraj a ,
∗
Veeramuthu Ashokkumar c , Chelliah Jayabaskaran a ,
a
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
b
Division of Floriculture and Medicinal crops, ICAR, Indian Institute of Horticultural Research, Hessaraghatta Lake
(PO), Bangalore, Karnataka 560 089, India
c
Department of Chemical Technology, Chulalongkorn University, Bangkok, Thailand

article info a b s t r a c t

Article history: Piper betle (L.) cultivars such as Bangla (B), Desawari (D), Kapoori (K), Sanchi (S) and
Received 1 June 2020 Meetha (M) were designed for the first time to investigate the comparative study on
Received in revised form 19 July 2020 anti-diabetic, anti-thrombin and anticancer activity by an in-vitro assay using differ-
Accepted 27 August 2020
ent organic solvent extracts. In this study, anti-thrombin activity was determined by
Available online 12 September 2020
thrombin substrate III and thrombin. Among the different extract tested, methanolic
Keywords: extract of Kapoori (KM) showed the maximum thrombin inhibition (80%) with IC50
Piper betle at 165.5 µg/ml. In addition, a significant result was obtained from anti-diabetic assay
Anti-diabetic by α -amylase (26.2 µg/ml) and α -glucosidase (96.8 µg/ml) enzyme inhibition in KM
Anti-thrombin with respective IC50 value followed by methanolic extract of Sanchi (SM). Moreover, the
Cytotoxicity cytotoxicity effect was observed in MM cultivar against HeLa cancer cell line using MTT
Betelvine and propidium iodide (PI) live/dead assay with IC50 of 26 µg/ml at 24 h incubation. Fur-
thermore, methanolic extract of five cultivars was analyzed for the presence of bioactive
secondary metabolites by chromatography technique, which revealed the presence of
phenol, terpenoid and alkaloid compounds. Additionally, Gas chromatography with Mass
spectroscopy (GC–MS) analysis results showed that the potentially bioactive substances
like, isoniazid, eugenol and n-hexadecanoic acid was recognized as an anti-thrombin,
anti-diabetic and anticancer properties. This study revealed that methanolic extract
showed the significant result in Kapoori, Sanchi and Meetha cultivars by in-vitro assays,
hence authenticating the traditional claim of the plant.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction

Medicinal plants are essential source of life-saving drugs for the mainstream of the ecosphere’s inhabitants. Plant life
designed the basis of a cultured traditional medication system that has been in way of life in countries like India and
China (Ramasubramania and Sreenivasulu, 2015). Piper betle (L.) generally identified as the betelvine is one of the notably
studied plants exhibiting potential medicinal resources. Betel leaves are considered in environmentally and economically
significant species in the family piperaceae residing of around 1200–2000 species. Betelvine cultivars are dioecious,
perennial, semi woody climber and leaves that have a powerful flavour commonly used as masticatory (Hewageegana
et al., 2011; Datta et al., 2011; Magdalene et al., 2014).

∗ Corresponding author.
E-mail address: cjb@iisc.ac.in (C. Jayabaskaran).

https://doi.org/10.1016/j.eti.2020.101140
2352-1864/© 2020 Elsevier B.V. All rights reserved.
S. Yogeswari, K.H. Bindu, S. Kamalraj et al. Environmental Technology & Innovation 20 (2020) 101140

Table 1
Plant material used in this study.
Sl. No. Accession No. Vernacular name Group
1. IIHR BV 33 GodiBangla (B) Bangla
2. IIHR BV 40 Dalvi (D) Desawari
3. IIHR BV 47 Swana Kapoori (K) Kapoori
4. IIHR BV 24 Halisahow Sanchi (S) Sanchi
5. IIHR BV 165 Meetha (M) Meetha

Growing economies and industrialization lead the environmental contamination as well as soil pollution (Sarma et al.,
2019), which may affect the economically important crop cultivars. Das et al. (2016b) reported that the secondary
metabolites are connected with soil and environmental factors on production and quality in betelvine. There are more
than 100 landraces and are known by vernacular names for betelvine and it has been clarified under five group’s cultivars
Bangla (B), Desawari (D), Kapoori (K), Sanchi (S) and Meetha (M) (Rawat et al., 1989). Other cultivars of betelvine such as
Chandrakala, Karpada local, Nahua, Balia, Desibangla, Dandabangla, Maghai were reported as an antioxidant activity with
the chemical composition (Das et al., 2016a). Further, rich polyphenol natural sources were reported in P. betle leaves for
health and environmental use (Kuppusamy et al., 2015).
Leaves of betelvine are generally used as a post-meal mouth freshener, it also improve digestive and to treat dysentery,
venereal sores, phthisis, syphilis and intestinal strangulation (Datta et al., 2011; Rintu et al., 2015). It has correlated with
a study demonstrates for that type 2 diabetes have higher amounts of reflux, indigestion and stomachache. The cause of
indigestion was reported as uncontrolled blood sugar level (Kim et al., 2010). Also, diabetes are described by the highest
amount of blood sugar which can lead complications in the organelles such as eyes, kidneys and cardiovascular system
(Abirami et al., 2014).
The disease of cardiovascular remains the main cause of illness and mortality in individual with diabetes. The 80% of
diabetic patients were died by cardiovascular and the risk of atherothrombotic actions in this population is comparable to
individual non-diabetics with a history of ischemic heart disease (Haffner et al., 1998). Several anti-thrombotic drugs are
yet available for the inhibition of intravascular thrombosis, but bleeding risk, narrow therapeutic space, the occurrence
of resistance and non-specific drug interactions are the reason of major concern (Bhatt and Topol, 2003; Chakrabarti and
Das, 2007). Therefore, anti-thrombotic drugs with least side effects and enhance efficiency are required. Connectively,
betelvine extract was reported as an anticoagulant activity (Jesonbabu et al., 2012).
As per 2019 record, about 18 million fresh cases of cancer occurred annually in worldwide and it initiated about 8.8
million deaths (Sciacovelli et al., 2020; GBD 2015 M Mortality and Causes of Death Collaborators, 2016). The common
types of cancer in females: breast, cervical cancer, etc.; in males: lung, stomach cancer, etc. (World Cancer Report, 2014).
HeLa cervical cancer cells were used widely to investigate the anticancer potential of various phytochemical/bioactive
molecules (Kim et al., 2012). Similarly, research studies noted that new-onset diabetes in person over 50 might also
be primary indication of pancreatic carcinoma. A rapid change in blood glucose levels in diabetics who earlier had
well-controlled diabetes can additionally be an indication of pancreatic carcinoma (Pannal et al., 2009).
Previous reports showed that extracts of betelvine leaves possess anti-microbial, anti-oxidant and cytotoxic activ-
ity (Murakami et al., 2000). Moreover, pre-clinical investigations have revealed that betel leaf contains anti-diabetic,
antiplatelet aggregation, cardiotonic, anticancer, anti-mutagenic, antiulcer, respiratory depressant and anthelmintic prop-
erties (Kumar et al., 2010; Chakraborty and Shah, 2011). However, no reports available on a comparative study of betelvine
cultivars such as Bangla (B), Desawari (D), Kapoori (K), Sanchi (S) and Meetha (M) for in-vitro biological studies. Therefore,
this study focused on in-vitro anti-thrombin, anti-diabetic and cytotoxicity activity from five different cultivars of betelvine
leaves.

2. Materials and methods

2.1. Plant sources

The fresh leaves of five different betelvine cultivars B, D, K, S and M were collected from Indian Institute of Horticultural
Research field in Hirehalli, Bangalore, Karnataka and India. Indian Council of Agricultural Research (ICAR) - Indian Institute
of Horticultural Research (IIHR) maintains large collection in germplasm repository of betelvine at Central Horticultural
Experimental Station (CHES) located at Hirehalli, authentic plant materials were sourced from the germplasm repository
and one cultivar from each of their groups have been used for studies (Table 1) (Rawat et al., 1989). All cultivars were
surface sterilized with running tap water followed by 0.1% mercuric chloride and sterile double distilled water separately.
Fresh leaves were sliced into small pieces and ground with liquid nitrogen individually. The powder was collected and
stored at −80 ◦ C for further experiments.
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2.2. Preparation of plant extracts

The 40 g of powdered betel leaves (B, D, K, S and M) were extracted separately with one-third volume of methanol,
petroleum ether, ethyl acetate and aqueous (sterile double distilled) at 37 ◦ C under shaking condition of 120 rpm for 24 h.
The extraction was continued until the powder was free of extracts and they were filtered using filter paper, concentrated
to dry in a reduced pressure at 40 ◦ C by a rotary evaporator. The aqueous extract was concentrated to dry powder by
lyophilization at −80 ◦ C. The residues were dissolved in 0.4% dimethyl sulfoxide (DMSO) and they were subjected to
in-vitro bio-assays.

2.3. In-vitro anti-thrombin activity

The anti-thrombin assay was carried out by a standard method (Batra et al., 2004). In a 96-well plate, the assay mixture
having 20 µl Thrombin Substrate III from Calbiochem (USA), 200 µl Tris-buffer (pH 7.5), 5 µl varying concentrations of
methanolic extract of five betelvine cultivars such as B, D, K, S and M (100, 200, 300, 400 and 500 µg/ml), 15 µl Thrombin
(1U/ml) from Sigma Chemicals Inc. (USA). The reaction mixture was kept in 37 ◦ C for 1 h 30 min. The absorbance was
measured at 450 nm and 390 nm of emission and excitation respectively in a fluorimeter (SpectraMax⃝ R
M5e, Molecular
Devices, Inc., USA). The effective cultivar was further tested with extract of petroleum ether, ethyl acetate and aqueous.
DMSO served as a control. The inhibition percentage was calculated by the following formula,
% of inhibition = [1 − As/Ac]×100
[ As — Absorbance of sample; Ac — Absorbance of control]

2.4. In-vitro cytotoxicity assay

Cervical cancer cell line (HeLa) was acquired from National Centre for Cell Science (NCCS), Pune, India. The Dul-
becco’s Modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 0.2% (v/v) of strepto-
mycin/penicillin solution were used for cell culture. Cells were cultured at 37 ◦ C with 5% CO2 , for maintenance, complete
medium was changed every 2–3 days.
For cytotoxicity assay, HeLa cells were seeded in 96 well plates at 1×104 cells per well by standard method (Mosmann,
1983). After 24 h incubation the cell supernatant was removed and 10 µl methanolic extract of B, D, K, S and M at
different concentrations (50, 75 and 100 µg/ml) added followed by 90 µl of fresh medium and incubated at 37 ◦ C in 5%
CO2 incubator. After 24 h, the old media was removed from the well and MTT (10 µl) was added to the respective well.
The plates were incubated for 4 h at 37 ◦ C in 5% CO2 incubator. Further, the supernatant was removed and DMSO (100
µl) was added. The absorbance was carried out by a microplate reader (570 nm). DMSO served as a control. The effective
cultivar was further tested for different time incubation period (24, 48 and 72 h) and different solvent extracts (aqueous,
ethyl acetate and petroleum ether). The inhibition percentage was calculated by the following formula,
% of cell inhibition = (Ac − As)/Ac×100
[ Ac — Absorbance of control; As — Absorbance of sample]

2.4.1. PI live/dead assay

HeLa cells were seeded in DMEM medium at 0.5 × 105 with 10% FBS in 24 well culture plate (Gini et al., 2016). The
cells were adhered for overnight and subsequently treated with IC50 concentration of methanolic extract of M cultivar
(26 µg/ml) dissolved in DMSO for 24 h. Following treatment, the cells were harvested and centrifuged for 3 min at 3000
rpm. The supernatant was discarded and the cells were washed with ice-cold PBS by twice. Further, the cell pellet was
dispensed with PBS followed by staining with 50 µg/ml of PI. Paclitaxel (12 nM) used as a positive control. The cells were
studied with fluorescence-activated cell sorting (FACS) analysis. The percentage of live and dead cells were calculated
using CytExpert 2.0 software.

2.5. In-vitro anti-diabetic activity

2.5.1. α -amylase inhibitory activity

α -amylase inhibitory assay was performed in a 96-well plate according to Ademiluyi and Oboh (2013) with some
modifications. The reaction mixture having 20 mM of phosphate buffer with pH 7.8 (50 µl), 1 U/ml of α -amylase (10 µl)
and 20 µl of various concentrations of methanolic extract of five different cultivars such as B, D, K, S and M (10, 20, 30,
50, 100 and 200 µg/ml) were pre-incubated at 37 ◦ C for 20 min. Then, 20 µl of substrate containing 1% soluble starch
(20 mM phosphate buffer, pH 7.8) was added and further incubated at 37 ◦ C for 30 min; 3,5-dinitrosalicylic acid (DNS)
color reagent (100 µl) was added and boiled for 10 min. The absorbance was carried out by a microplate reader (540 nm).
DMSO was served as a control and maltose standard curve was used by DNS method. One unit of amylase activity was
determined as the quantity of enzyme that liberated 1 µmol reducing sugar correspondent to maltose per minute in the
assay condition (Bernfeld, 1955). The inhibition percentage was assessed by the following formula,
% of inhibition = 1 − (AS/AC)×100
[ AC — Absorbance of control; AS — Absorbance of sample]
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2.5.2. α -glucosidase inhibitory assay

α -glucosidase inhibitory assay was carried out in 96-well plate followed by Shai et al. (2011) with some modifications.
The assay mixture having 100 mM phosphate buffer with pH 6.8 (50 µl), 1 U/ml of α -glucosidase (10 µl) and 20 µl
of various concentrations of methanolic extract of five different cultivars such as B, D, K, S and M (50, 100 and 200
µg/ml) were incubated at 37 ◦ C for 15 min. Then, 20 µl of 5 mM substrate (p-nitrophenyl alpha-D-glucopyranoside) was
added and incubated at 37 ◦ C for 20 min. Finally, 0.1 M of Na2 CO3 (50 µl) was added to stop the reaction. The released
p-nitrophenol absorbance was measured (405 nm) by a microplate reader. DMSO was served as a control. The inhibition
percentage was assessed by the following formula,
% of inhibition = 1 − (AS/AC)×100
[ AC — Absorbance of control; AS — Absorbance of sample]

2.6. Phytochemical analysis by qualitatively

Thin-layer chromatography (TLC) was achieved with 1 mm (20 × 20 cm) silica gel pre-coated plates (Merck). About
10 µl (50 µg) of methanolic extract of five different cultivars (B, D, K, S and M) were loaded on TLC plates using capillary
tube and then it was allowed to air dry. The plates were developed by the solvents chloroform: ethyl acetate: methanol
(6:3.5:0.5) v/v/v. The solvents were allowed to run until reached the solvent front and evaporate from the TLC plate.
The developed plates were sprayed using Dragendorff’s spray reagent (Harborne, 1988), Vanillin sulfuric acid and Ferric
chloride (Touchstone, 1992) separately to detect secondary metabolites. The Rf value was measured by the following
formula,
Rf value = Distance traveled by substance/Distance traveled by solvent

2.7. GC-MS analysis

Sample preparation
For GC–MS, each extract of betelvine cultivars was prepared separately by dissolving 1 mg/ml using HPLC grade
methanol, vortexes for 2 min and the extract was filtered by 0.2 µl filter. Further, 1 ml of each extract (25 µg/ml) was
added in respective GC vial for analysis. Methanol was completely dried using nitrogen gas. The sample was derivatized
with 30 µl pyridine and 60 µl of BSTFA: TMCS (99:1) and incubated at 60 ◦ C for 60 min. Derivatized samples 1 µl were
subjected to GC–MS analysis.
Analysis
The measurement of GC–MS was accomplished by a Shimadzu equipment furnished with Agilent GC 7890 A, 5975C
MS detector, electron impact ionization, single quadruple mass, data was acquired by AMDS software; column: DP 5 ms,
Dimensions: 30 m Length × 0.25 mm ID ×0.25µm film thickness, primary temperature 240 ◦ C for 2 min hold time, ram
temperature 50 ◦ C to 280 ◦ C for 5 min hold time, total run time 40 min, helium was used as a carrier gas, flow was 1.0 (13
ml/min), split flow was 1.5 ml/min, 1 µl sample was injected, scan mass ranges: 30 m/z–600 m/z and positive polarity.
The resulting spectrums were compared with the spectrum of known compounds available in the NIST (2011) library.

2.8. Statistical analysis

Data are given as mean ± S.E.M with triplicate values. Statistical comparisons were prepared by GraphPad Prism
software (5.03) and statistical software of SPSS version 11.5.

3. Results and discussion

3.1. Thrombin inhibition assay

Blood coagulations are involved several factors in the tissues and plasma, which includes the intrinsic and extrinsic
pathways play an important role in blood coagulation. Anticoagulation is described due to the thrombin, coagulation
serine proteases and factors Xa by the activation of serine protease inhibitor anti-thrombin III (Shanmugam and Mody,
2000). In this study, methanolic extract of five different cultivars (B, D, K, S and M) of betel leaves were tested for thrombin
inhibition activity. The IC50 value at 157.8 µg/ml in SM, 162.3 µg/ml in DM, 165.5 µg/ml in KM, 258.7 µg/ml in BM and
439.5 µg/ml in MM were recorded (Table 2). Maximum thrombin inhibition of 80% was observed in KM and SM; 70% in
BM and DM at 500 µg/ml (Fig. 1). Supportively, Jeng et al. (2002) reported that betelvine extracts showed scavenged ROS,
decreased platelet aggregation and production of thromboxane B2.
KM showing maximum thrombin inhibition, hence KM was further extracted with solvents viz., aqueous, ethyl acetate
and petroleum ether. The resulting extract was considered as KP, KE, and KA respectively and tested for its thrombin
inhibition potential. The IC50 value was observed at 165.5 µg/ml in KM, 303 µg/ml in KE and >500 µg/ml in KA (Fig. 2).
Thrombin inhibition assay was determined by dose-dependent manner for each extract. Relatively, Kee et al. (2008)
studied thrombin inhibition assay for several plants, from that methanolic extract was showed best thrombin inhibitor
compared to aqueous extract. Also, 80% anti-thrombin activity recorded in methanolic and methylene chloride extracts
in thirty plants from Central Florida were reported (Natalya et al., 2002). This results supporting the highest thrombin
inhibition activity of KM.
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Table 2
Biological activity with IC50 values from five cultivars of betel leaves.
Five cultivars of Piper betle IC50 Value (µg/ml)
Cytotoxicity Anti-Thrombin Anti-Diabetic
α -amylase α -glucosidase
Bangla (BM) >100 258.7 – >200
Desawari (DM) >100 162.3 – –
Kapoori (KM) 74.4 165.5 26.2 157.7
Sanchi (SM) 75.9 157.8 27.6 96.8
Meetha (MM) 26.0 439.5 30.3 –

(–): No IC50 value was found.

Fig. 1. In-vitro Thrombin Inhibitory effect of methanolic extract from different cultivars of Betel leaf.
(BM: Bangla; DM: Deshwari; KM: Kapoori; SM: Sanchi and MM: Meetha).

Fig. 2. In-vitro Thrombin Inhibitory effect of Kapoori leaves for different solvents extracts.
(KP: Kapoori-Petroleum ether extract; KE: Kapoori-Ethyl acetate extract; KA: Kapoori-Aqueous extract).

3.2. Cytotoxicity assay

In this study, methanolic extract of five different cultivars of betel leaves (B, D, K, S and M) were carried out to
determine the cytotoxicity against HeLa cell line at different concentrations by MTT assay. Significant cytotoxicity was
found in MM cultivar with 58% at 50 µg/ml for 24 h treatment followed by SM, KM, DM and BM cultivars with 55%, 50%,
41% and 27% at 100 µg/ml respectively (Fig. 3). The IC50 values recorded in the methanolic extract of five cultivars were
listed in the Table 2.
From this study, methanolic extract of M cultivar was further studied at different time incubation (24, 48 and 72 h)
and resulted with the IC50 value at 26 µg/ml for 24 h, 10.8 µg/ml for 48 h and 4.8 µg/ml for 72 h (Fig. 4). Significant
results showed in 24 h at 5 to 100 µg/ml concentration and both 48 and 72 h treated cells showed remarkable cytotoxic
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S. Yogeswari, K.H. Bindu, S. Kamalraj et al. Environmental Technology & Innovation 20 (2020) 101140

Fig. 3. Cytotoxicity of betel leaves on HeLa cancer cells.

HeLa cells were treated with methanolic extract of B, D, K, S, and M at different concentration for 24 h. (BM: Bangla; DM: Deshwari; KM: Kapoori;
SM: Sanchi and MM: Meetha).

Fig. 4. Cytotoxicity of Meetha betel leaves on HeLa cancer cells.

HeLa cells were treated with methanolic extract of Leaves of Meetha at different concentration for 24 h, 48 h and 72 h.

activity. Relatively, the ethanolic leaf extract of betelvine was tested for cytotoxicity using murine and different cancer
cell lines with time dependent manner (Roy and Vijayalaxmi, 2013).
Further, cytotoxic activity of different extracts namely aqueous, ethyl acetate and petroleum ether of M cultivar from
betel leaves were carried out against HeLa cells. The IC50 value was recorded in petroleum ether at 75 µg/ml, the activity
of ethyl acetate and water extracts were not determined (Fig. 5a). From this, methanolic extract of M cultivar showing
excellent cytotoxic activity compared to other extracts. Similar to our study, Sunila and Kuttan (2004) reported that P.
longum was showed cytotoxic effect against on Dalton’s lymphoma ascites (250 µg/ml) and Ehrlich ascites cancer (100
µg/ml) cells in methanolic extract. In another study, methanolic extract of P. crocotum exhibited cytotoxicity on breast
cancer cells (T47D) (Britanto et al., 2009). Thus, the leaf extracts of betelvine have cytotoxic potential and can be used for
the treatment of several diseases (Banerjee and Shah, 2014). Further, the IC50 concentration of MM cultivar was analyzed
by PI live/dead cell assay. It confirmed the cytotoxicity potential in MM cultivar with 59.38% compared to 69.66% of
Paclitaxel (Fig. 5b).
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Fig. 5a. Cytotoxicity of Meetha leaves against on HeLa cancer cells.

HeLa cells were treated with different crude extract of Meetha leaves at the different concentration for 24 h. (MP: Meetha Petroleum ether extract;
ME: Meetha Ethyl acetate extract; MA: Meetha Aqueous extract).

Fig. 5b. PI Live/Dead assay for the methanolic extract of M cultivar at 24 h treatment.
(a. Control; b. Methanolic extract of M cultivar (26 µg/ml); c. Paclitaxel (12 nM/ml).

3.3. Anti-diabetic assay

In the present study α -amylase and α -glucosidase inhibition activity of BM, DM, KM, SM and MM were studied. In
α -amylase inhibition assay, IC50 value was examined at 26.2 µg/ml in KM, 27.6 µg/ml in SM and 30.3 µg/ml in MM
cultivar. There is no IC50 value recorded in DM and BM cultivar. The KM cultivar showed 38% inhibitory activity followed
by SM (36%) and MM (32%) at 20 µg/ml respectively (Fig. 6). Moreover, the glucose reduction level from the cultivars of
betelvine KM, SM and MM were determined by absorbance of a sample with known maltose standard in the presence
of amylase enzyme. It resulted in the linearly increased order up to 200 µg/ml with increased inhibitor concentration
(Fig. S1). However, the methanolic extract of betelvine KM proven the potential anti-diabetic activity compared to SM
and MM.
In α -glucosidase inhibition assay, IC50 value of SM at 96.8 µg/ml, KM at 157.7 µg/ml and BM at >200 µg/ml were
recorded (Table 2). A cultivar of DM and MM not shown activity at a concentration of 50, 100 and 200 µg/ml. The KM
cultivar showed 63% inhibitory activity at 200 µg/ml followed by SM (50%) at 100 µg/ml (Fig. 7). Similar to our study,
appreciable inhibition activity for the methanolic extract of P. betle leaf was reported at 96.56 µg/ml in α -glucosidase
inhibition activity and 84.63 µg/ml in α -amylase inhibition activity (Sindhu et al., 2013). Arambewela et al. (2005) reported
that the anti-diabetic activity presence in P. betle leaf. The herbal drugs are used as complementary methods in existing
treatment for diabetes and its difficulties are rising worldwide and several plants in various countries are known to have
anti-diabetic properties (Hasani et al., 2008). The anti-hyperglycemic activity of the most effective plant was described
by the capacity of the phytoconstituents to raise glucose transportation and metabolism in muscle to activate insulin
secretion (Gray et al., 2000).
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Fig. 6. α -amylase enzyme activity for the methanolic extract of five betelvine cultivars.
(BM: Bangla; DM: Deshwari; KM: Kapoori; SM: Sanchi and MM: Meetha).

Fig. 7. α -glucosidase enzyme activity for the methanolic extract of five betelvine cultivars.
(BM: Bangla; DM: Deshwari; KM: Kapoori; SM: Sanchi and MM: Meetha).

3.4. Phytochemical analysis

Several polyphenolic compounds, triterpenoids and other chemical compounds exist in leaf extract of plants may
account for the observed anti-diabetic effect (Stalin et al., 2013). Betelvine leaves are used in much traditional medicines,
the anti-tumour property of phytochemicals like polyphenols and alkaloids (Abrahim et al., 2012). In this study, TLC was
performed to identify the phytochemicals present in different cultivars such as BM, DM, KM, SM, and MM. Bioactive
compounds were detected as red/brown spots for alkaloids with Rf value of 0.83 and 0.29 by Dragendorff’s reagent, navy
blue spots for phenol with Rf value of 0.61 by ferric chloride and blue spots for terpenoid with Rf value of 0.75 by vanillin
sulfuric acid (Fig. S2). The developed TLC plates showed the active principle of secondary metabolites existed in methanolic
extract of five different cultivars of betelvine.
In general phenolic compounds are responsible for their bioactive properties (eg. anticarcinogenic or antimutagenic,
antioxidant and anti-inflammatory) and also contribute to induce apoptosis by cell cycle arrest (Wu-Yang et al., 2009).
Vallianou et al. (2011) study found camphene example of terpenoid, which decreases triglycerides and plasma cholesterol
in hyperlipidemic rats. So, the mechanism of hyperlipidemia plays a vital role in heart disease. Alkaloids possess a wide
range of pharmacological properties as well as anti-hyperglycemic activities in piperine isolated from piperaceae family
(Qiu et al., 2014). Interesting results were recorded in the present study, MM cultivar showed good cytotoxicity effect and
rich in phenolic compound. Whereas, terpenoids and alkaloids present in KM and SM may responsible for the anti-diabetic
and anti-thrombin activity.
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Table 3
Bioactive compounds from methanolic extract of Piper betle by GC–MS analysis.
S. No. Name of the compounds Retention Time Purity (%)
MW and B D K S M B D K S M CAS No.
MF
1. Pyridine 79 8.11 8.18 8.12 8.73 8.09 26 47 17 44 48 110-86-1
(C5 H5 N)
2. Eugenol 164.20 15.98 15.71 15.70 15.97 15.99 89 93 75 89 94 97-53-0
(C10 H12 O2 )
3. n-Hexadecanoic acid 256.4 24.49 24.49 – 24.50 24.50 56 76 – 70 76 57-10-3
(C16 H32 O2 )
4. Octadecanoic acid 284.47 26.88 26.89 – 26.89 26.90 69 68 – 81 84 57-11-4
(C18 H36 O2 )
5. Isoniazid 137.13 – 11.42 11.46 – – – 63 82 – – 54-85-3
(C6 H7 N3 O)
6. 1,3-Benzodioxole, 162.18 – 14.63 – – – – 83 – – – 94-59-7
5-(2-propenyl)- (C10 H10 O2 )
7. Hexadecanoic acid, methyl 270.45 – 24.07 – – – – 43 – – – 112-39-0
ester (C17 H34 O2 )
8. 2-Pyrrolidinone, 1-methyl- 99.13 – – – 9.28 – – – – 33 – 872-50-4
(C5 H9 NO)
9. Dibutyl phthalate 278.34 – – – 24.46 24.46 – – – 20 52 84-74-2
(C16 H22 O4 )
10. Hydroquinone 110.11 – – – – 14.32 – – – – 26 123-31-9
(C6 H6 O2 )
11. Phenol, 206.32 – – – – 18.20 – – – – 31 96-76-4
2,4-bis(1,1-dimethylethyl) (C14 H22 O)

MW: Molecular weight; MF: Molecular formula.

3.5. Bioactive compounds

In order to find out the bioactive chemical compounds of methanolic extract from five different cultivars of P. betle
were analyzed by GC–MS (Fig S3a-e). Our results indicated that, the presence of isoniazid in KM cultivar with 82% of
purity. Supportively, isoniazid, was used for the anti-tuberculosis treatment (ATT) and clinical reports are highlighted
acute pulmonary tuberculosis at risk of emerging thromboembolic actions from patients and proven these can normalize
with sufficient ATT (Goncalves et al., 2009; Turken et al., 2002). Moreover, isoniazid and rifampicin were administered to
tuberculosis can decrease the difficulties of tuberculosis by its anti-inflammatory activity (Gandigawad et al., 2015) but
its anti-thrombin activity is yet unexplored. It was believed that the presence of isoniazid in KM cultivar, may responsible
for the anti-thrombin activity.
In addition, well-known compound eugenol was observed in all P. betle cultivars 94% in MM cultivar followed by
93% (DM), 89% (SM) and 75% (KM) (Table 3). Previous reports revealed that eugenol is a phenolic compound that possess
various biological activity, like anti-diabetic (Al-Trad et al., 2019), antitumor (Dervis et al., 2017) and anti-oxidant (Hamed
et al., 2012). Moreover, dibutyl phthalate (52%), n-hexadecanoic acid (76%) octadecanoic acid (84%) and phenol, 2,4-
bis(1,1-dimethylethyl)- (31%) were found in MM cultivar (Table 3). Significantly, dibutyl phthalate, n-hexadecanoic acid
and phenolic compounds were reported as an anticancer activity (Wójtowicz et al., 2017; Ravi and Krishnan, 2017;
Varsha et al., 2015). Therefore, dibutyl phthalate and n-hexadecanoic acid may play an important role in MM cultivar
for anticancer activity.

4. Conclusion

In conclusion, our finding showed Eugenol, n-Hexadecanoic acid, Dibutyl phthalate from MM cultivar and Isoniazid
from KM cultivar, are remarkable active chemical constituents and may play role in anticancer, anti-diabetic and anti-
thrombin activities. The quantification of individual phytoconstituents as well as pharmacological profile based on in-vivo
studies of M, K and S should be further investigated.

Supporting information

Supporting information such as Figure S1, S2 and S3 associated with this research article was attached.
9
S. Yogeswari, K.H. Bindu, S. Kamalraj et al. Environmental Technology & Innovation 20 (2020) 101140

CRediT authorship contribution statement

Subramaniam Yogeswari: Methodology, Visualization, Investigation, Writing-original draft. Kaipa Hima Bindu: Fund-
ing acquisition, Project administration, Plant resource. Subban Kamalraj: Data curation, Writing - review & editing.
Veeramuthu Ashokkumar: Writing - review & editing. Chelliah Jayabaskaran: Conceptualization, Supervision, Funding
acquisition, Project administration, Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by funding agency of ICAR-Extramural research grant, GOI, New Delhi, India (Scheme code:
ICAR0013, 2016). We acknowledge DST-FIST, UGC special assistance programme and DBT-IISc partnership program for
providing facilities and financial support. Authors are thankful to Dr. K.K. Upreti, Principal Scientist (Organic Chemistry),
Division of Plant Physiology & Biochemistry, IIHR, Bangalore for valuable discussion of all assays and author SY thankful
to Dr. T.H. Anantha Krishna, Department of Biochemistry, IISc, Bangalore for discussion of anti-thrombin assay.

Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.eti.2020.101140.

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