J Parasit Dis

https://doi.org/10.1007/s12639-018-1064-1

ORIGINAL ARTICLE

In-vitro and in silico efficacy of isolated alkaloid compounds

from Rauvolfia tetraphylla L. against bovine filarial parasite
Setaria cervi: a drug discovery approach
Dipti Ranjan Behera1 • Sunita Bhatnagar1

Received: 19 September 2018 / Accepted: 26 November 2018

Ó Indian Society for Parasitology 2018

Abstract Bioassay guided isolation from the leaves of third of the endemic population resides in South-East Asia
Rauvolfia tetraphylla L. resulted in the isolation and and one-third lives in India. This is also called a poor
characterization of three compounds of alkaloid in nature man’s disease prevailing in most of the slum areas (Dreyer
namely, Curan-17-oic acid (F1); 18, 19-Secoyohimban et al. 1997). Lymphatic filariasis or elephantiasis is the
(F2) and Reserpiline (F3). Macrofilaricidal activity of three fourth most common cause of disability worldwide. The
compounds was tested against bovine filarial parasite Se- filarial nematode W. bancrofti accounts for 91% of Lym-
taria cervi using in vitro assays and supported by in silico phatic Filariasis infections, while B. malayi and B. timori
docking analysis on glutathione-S-transferase (GST) are responsible for the remaining 9% in the Southeast Asia
enzyme of Wuchereria bancrofti. All the molecules region (WHO 2009). The control of human filariasis caused
inhibited GST enzyme to some extent 35.78%, 78.22% and by Brugia malayi, Wuchereria bancrofti and Onchocerca
64.21% respectively. Results were supported by molecular volvulus, currently relies on community-wide mass distri-
docking studies, which showed docking scores for com- bution of ivermectin and albendazole, either individually or
pound F1 (- 5.14), compound F2 (- 7.19) and compound in combination with diethylcarbamazine. Unfortunately,
F3 (- 7.2) on GST enzyme. Thus, in conclusion the these drugs are mainly microfilaricidal rather than macro-
in vitro and in silico studies indicated that isolated com- filaricidal, which means that repeated treatment is required
pounds are promising, inexpensive and widely available over many years, and the possibility that resistance to them
natural leads, which can be designed and developed into may develop is a cause for concern (Ardelli et al. 2005;
the macrofilaricidal drugs. Schwab et al. 2005) but the same when given for a longer
duration are full of side effects like nausea, vomiting and
Keywords Rauvolfia tetraphylla
Setaria cervi
head ache.
MTT
Glutathione-S-transferase
The antifilarial potential of crude extracts of Rauvolfia
Macrofilaricidal activity
In-silico docking tetraphylla plant has been analysed profoundly in our
previous studies that signified this plant being a promising
source of antifilarial principles (Behera and Bhatnagar
Introduction 2017). In the present study, R. tetraphylla L. one of the
most important medicinal plant species of Apocynaceae
Filariasis is one of the most important tropical diseases family was explored for the evaluation of macrofilaricidal
affecting more than 80 countries with 120 million infected potential and the same was also employed for the isolation
and more than 1.3 million people at risk globally. Two- of antifilarial principles by targeting bovine filarial parasite
Setaria cervi. The in vitro activity of isolated compounds
against adult worms of S. cervi, in silico docking on glu-
& Dipti Ranjan Behera tathione-S-transferase (GST) enzyme of W. bancrofti par-
[email protected]
asite model have been discussed here in detail.
1
Regional Plant Resource Centre, Nayapalli, Bhubaneswar,
Odisha 751015, India

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Materials and methods forest area of Regional Plant Resource Centre, Bhuba-
neswar, Odisha, India and voucher specimen (RPRC-
General experimental procedures 15010) was deposited to herbarium of Regional Plant
Resource Centre, Bhubaneswar, Odisha. The leaves were
Column chromatography was performed by using 60–120 washed thoroughly in running tap water and shade dried at
mesh silica gel (Sisco Research Laboratory, India) through room temperature. The dried leaves were grinded to make
a glass column of size 400 9 30 mm. Preparative TLC was powder; same was used for preparing the solvent extracts.
carried out on prep. TLC glass plates coated with silica gel
GF254 (Merck, Germany). UV spectral data were obtained Extraction and isolation
on a Shimadzu UV-1800 double beam spectrophotometer
(Tokyo, Japan). HPLC analysis was done on a Shimadzu— The crude extracts such as hexane, chloroform, acetone and
UFLC (Tokyo, Japan) system that includes a C18 column methanol were prepared by soxhlet extraction technique.
(150 mm 9 4.6 mm, 5 lm) with a biocompatible binary The crude methanolic extract (50 g dry weight) was frac-
pump (LC-20AD) plus a wavelength detector (SPD- tionated by liquid–liquid separation technique using sol-
M20A). IR spectra of samples in KBr discs were recorded vents such as Hexane, Chloroform and Ethyl acetate
on a Perkin Elmer-Spectrum-GX spectrometer (Perk- according to increasing polarity. The crude plant extract
inElmer, USA) with KBr pellets. GC–MS analysis of iso- was separately suspended in distilled water using separat-
lated compounds was carried out using GCMS-QP 2010 ing funnel (100 ml) and successively partitioned with
Ultra instrument (Shimadzu, Japan) that includes a head- hexane, chloroform and ethyl acetate in the order of
space auto sampler (AOC-20s) and auto injector (AOC- polarity. This afforded four partition fractions of active
20i). NMR experiments were carried out on a Bruker plant extract. The chloroform fraction (20 g) was subjected
Avance III 500 MHz spectrometer (Karlsruhe, Germany) to column chromatography over silica gel (60–120 mesh),
with pulsed field gradient and signals were referenced to twenty sub-fractions were collected using mobile phase
the residual solvent signals (CD3OD, at dH 3.31 and dC starting from hexane (100%), dichloromethane, chloro-
49.0 ppm). Melting point detection was done by a digital form, ethyl acetate and methanol in the ratio of 100, 90: 10,
melting point apparatus (BPL-53A, BP lab solutions, 70: 30, 50: 50 and 20: 80. Further, the sub-fractions were
India). TLC was carried out on silica gel 60 F254 TLC combined according to their similarity in Rf values deter-
sheets (Merck, Germany) and spots were detected by mined by TLC in mobile phase toluene: ethyl acetate:
spraying agents like DPPH (0.2% in CH3OH), Vanillin- methanol (3.6.1) and therefore resulting in five combined
H2SO4 in EtOH and Dragondroff’s reagent. sub-fractions. Further purification of sub-fractions was
achieved by preparative TLC method (prep. TLC silica gel
Collection of parasites plates, Merck) using mobile phase toluene: ethyl acetate:
methanol (3.6.1) and benzene: ethanol: ammonium
Setaria cervi, resembles the human bancroftian parasite in hydroxide (90: 10: 1). The compounds 1–3 were examined
its nocturnal periodicity, antigenic pattern and same using standard protocols for the identification of their
chemotherapeutic antifilarial drug response, has been used chemical nature and therefore phytochemical study was
as a model parasite for drug discovery research in filariasis carried out by the methods described by Sofowora (1993)
(Kaushal et al. 1987). The adult parasites (S. cervi) were and Trease and Evans (1989). Then the compounds 1–3
collected from peritoneal cavity of freshly slaughtered (40 mg) were filtered by syringe filters (PTFE membrane,
buffalo obtained from slaughter house of Nandan Kanan pore size 0.45 lm, Axiva) and purity was checked by TLC
Zoological Park, Bhubaneswar. These adult parasites were (Silica gel 60 F254, Merck) and HPLC (Shimadzu) using a
washed in PBS (1 9) and transferred immediately to C18 column (150 mm 9 4.6 mm, 5 lm) with mobile phase
RPMI-1640 medium supplemented with 5% (v/v) heat-in- CH3OH: C2H3N (60: 40 v/v) (Masoko and Eloff 2007;
activated Fetal bovine serum (Mathew et al. 2002). The Malinowska et al. 2005; Panwar and Guru 2011; Kardono
adult female parasites measuring average length et al. 1990).
80–100 mm and width 0.45 mm were used for the exper- Compound 1 [Curan-17-oic acid, 19, 20-dihydroxy,
imental purposes. methyl ester (19S)]: Amorphous solid; kmax 234, 278.5
and 303.5 nm; melting point: 255–265 °C; IR (KBr) mmax:
Plant sample collection and preparation 3360, 2930, 1687, 1623, 1437, 800, 620 cm-1. GC–MS
m/z [M ? H] ? 358; 1H NMR (500 MHz, CD3OD): d
The fresh leaves of R. tetraphylla were collected from 1.19 (3H, d, J = 6.2 Hz), 1.63–1.87 (3H, 1.82 (ddd,
medicinal germplasm garden, RPRC botanical garden and J = 13.8, 3.0, 2.3 Hz), 1.72 (ddd, J = 13.7, 6.8, 1.7 Hz),
1.71 (ddd, J = 13.7, 7.8, 6.9 Hz)), 1.92 (1H, ddd, J = 13.8,

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3.3, 2.5 Hz), 2.39 (1H, dt, J = 3.3, 2.3 Hz), 2.56 (1H, ddd, 30.50 (C-9), 60.00 (C-10), 32.63 (C-11), 56.67 (C-12),
J = 12.9, 7.8, 6.8 Hz), 2.70 (1H, d, J = 7.9 Hz), 2.88–3.07 73.63 (C-13), 134.37 (C-14), 53.10 (C-15), 107.63 (C-16),
(3H, 2.94 (ddd, J = 12.9, 6.9, 1.7 Hz), 3.01 (dd, J = 6.4, 21.23 (C-17), 107.43 (C-18), 128.30 (C-19), 15.84 (C-20),
2.3 Hz), 3.04 (d, J = 7.9 Hz)), 3.31 (1H, dd, J = 3.0, 154.63 (C-21), 136.83 (C-22), 167.43 (C-23), 98.70 (C-24),
2.5 Hz), 3.65 (3H, s), 3.70–3.80 (2H, 3.74 (d, J = 6.4 Hz), 93.90 (C-25), 149.61 (C-26), 148.97 (C-27), 51.58 (C-28),
3.75 (q, J = 6.2 Hz)), 6.54 (1H, td, J = 7.6, 1.1 Hz), 6.69 56.13 (C-29), 56.16 (C-30).
(1H, ddd, J = 7.8, 1.1, 0.4 Hz), 6.90–6.99 (2H, 6.94 (ddd,
J = 7.8, 7.6, 1.5 Hz), 6.93 (ddd, J = 7.6, 1.5, 0.4 Hz)). 13C- Macrofilaricidal assays on model parasite S. cervi
NMR (125 MHz, CD3OD): 53.3 (C-7), 62.15 (C-8), 58.06
(C-9), 44.64 (C-10), 34.45 (C-11), 40.95 (C-12), 79.36 (C- The isolated compounds alone and in combination with
13), 39.9 (C-14), 61.71 (C-15), 52.27 (C-16), 137.7 (C-17), Diethylcarbamazine (DEC) were prepared at concentra-
149.89 (C-18), 71.67 (C-19), 172.03 (C-20), 122.5 (C-21), tions 1.0, 0.5 and 0.25 mg/ml in dimethyl sulfoxide
109.8 (C-22), 17.19 (C-23), 120.92 (C-24), 129.58 (C-25), (DMSO) to determine the efficacy of the pure compounds
52.08 (C-26). on the target organism. In vitro worm motility inhibition
Compound 2 [18,19-Secoyohimban-19-oic acid, and MTT reduction assay was carried out by the standard
16,17,20,21-tetradehydro-16-(hydroxymethyl)-, methyl procedures described earlier (Murthy 1999; Comley et al.
ester]: Amorphous solid; kmax 227.5, 303.5 and 401.5 nm; 1989; Sahare and Singh 2013; Lakshmi et al. 2010). The
melting point: 240–250 °C; IR (KBr) mmax: 3390, 2927, experiment was conducted in two replicates with one adult
1707, 1624, 1210, 750, 470 cm-1.GC–MS m/z [M ? female worm per well in RPMI 1640 media supplemented
H] ? 354; 1H NMR (500 MHz, CD3OD): d 1.94 (3H, d, with 0.5% Fetal Bovine Serum. A set of control worms
J = 7.1 Hz), 2.16–2.41 (2H, 2.24 (ddd, J = 13.8, 9.8, (DMSO treated) were also maintained along with the
9.3 Hz), 2.35 (ddd, J = 13.8, 4.9, 4.2 Hz)), 3.11–3.26 (2H, treated worms for each doses to calculate inhibition per-
3.18 (ddd, J = 14.1, 8.9, 5.3 Hz), 3.17 (ddd, J = 14.1, 5.4, centage. The complete immobilization of the adult worms
1.4 Hz)), 3.42 (1H, dd, J = 9.3, 4.9 Hz), 3.54 (1H, ddd, with [ 50% inhibition of MTT reduction by treated para-
J = 13.8, 8.9, 5.4 Hz), 3.62–3.73 (4H, 3.70 (s), 3.68 (ddd, sites compared to untreated controls was considered as
J = 13.8, 5.3, 1.4 Hz)), 5.16 (1H, dd, J = 9.8, 4.2 Hz), 6.75 macrofilaricidal action (Mukherjee et al. 1998). The iso-
(1H, q, J = 7.1 Hz), 6.92 (1H, ddd, J = 7.8, 7.6, 1.2 Hz), lated compounds were also evaluated for the glutathione-S-
7.05–7.15 (2H, 7.08 (ddd, J = 7.8, 1.2, 0.5 Hz), 7.10 (ddd, transferase (ScGST) inhibition assay using the cytosolic
J = 7.8, 7.6, 1.3 Hz)), 7.40 (1H, ddd, J = 7.8, 1.3, 0.5 Hz), fraction of adult Setaria worms using standard procedure
8.44 (1H, s), 9.67 (1H, s). 13C-NMR (125 MHz, CD3OD): with some modifications (Habig et al. 1974; Ahmad and
d 58.23 (C-6), 20.50 (C-7), 134.44 (C-8), 38.00 (C-9), Srivastava 2008; Srinivasan et al. 2011). Protein content
50.80 (C-10), 107.64 (C-11), 21.90 (C-12), 101.80 (C-13), was estimated by Lowry’s method using bovine serum
139.84 (C-14), 127.37 (C-15), 136.07 (C-16), 146.43 (C- albumin (BSA) as standard (Lowry et al. 1951) to find out
17), 117.95 (C-18), 166.60 (C-19), 63.35 (C-20), 110.93 the changes in protein content of worm homogenate in
(C-21), 129.64 (C-22), 119.52 (C-23), 121.26 (C-24), 13.88 control and treated samples.
(C-25), 50.40 (C-26).
Compound 3 [Reserpiline/Oxayohimban-16-carboxylic Molecular docking study on target glutathione-S-
acid, 16, 17-didehydro-10,11-dimethoxy-19-methyl-, transferase enzyme
methyl ester, (3-beta, 19-alpha, 20-alpha)-]: Amorphous
solid; kmax 228 and 279.5 nm; melting point: 250–260 °C; GST(s) (E.C.2.5.1.18), a large family of multifunctional
IR (KBr) mmax: 3407, 2927, 1629, 1620, 1089, 800, dimeric enzymes responsible for detoxification of xenobi-
480 cm-1.GC–MS m/z [M ? H] ? 412 1H NMR otics have been a potential drug target for antifilarial drug
(500 MHz, CD3OD): d 1.18 (3H, d, J = 6.4 Hz), 1.82 (1H, development. There is a significant difference between the
ddd, J = 13.6, 2.6, 2.1 Hz), 1.96 (1H, ddd, J = 13.6, 10.3, classes of GSTs expressed in helminths and mammals that
3.7 Hz), 2.17 (1H, dddd, J = 10.3, 4.1, 3.0, 2.8 Hz), 2.46 confirmed by primary amino acid sequence alignment of
(1H, dd, J = 10.3, 6.6 Hz), 2.72–2.92 (5H, 2.87 (dd, helminth GSTs with mammalian GST classes that showed
J = 6.6, 3.0 Hz), 2.83 (ddd, J = 4.1, 3.7, 2.1 Hz), 2.79 non-mammalian GST classes are expressed in parasites, i.e.
(ddd, J = 14.4, 10.0, 3.9 Hz), 2.87 (ddd, J = 14.4, 4.0, less than 30% identity (Brophy and Pritchard 1994). The
1.9 Hz), 2.84 (ddd, J = 13.8, 10.0, 4.0 Hz)), 3.01 (1H, ddd, significant differences between the tertiary structure of the
J = 13.8, 3.9, 1.9 Hz), 3.54 (3H, s), 3.60 (3H, s), 3.72 (3H, helminth GSTs and that of the host enzymes make the
s), 3.91 (1H, dd, J = 10.3, 2.6 Hz), 5.02 (1H, qd, J = 6.4, GSTs promising chemotherapeutic targets (Lazdins and
2.8 Hz), 6.32 (1H, d, J = 0.5 Hz), 6.38 (1H, d, J = 0.5 Hz), Kron 1999; Brophy and Pritchard 1994; Campbell et al.
8.03 (1H, s). 13C NMR (125 MHz, CD3OD): d 40.73 (C-8), 2001). The GST enzyme of W. bancrofti (WbGST) was

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targeted for the insilico molecular docking analysis using

three isolated compounds (Compound 1, 2 and 3). As GST
enzyme of all the filarial nematode parasites shares struc-
tural similarity in their amino acid sequence (Veerapathran
et al. 2009) and due to a major cause of Filariasis (90%)
in tropical countries, the molecular docking target was
aimed at GST of W. bancrofti. The 3D structures of iso-
lated compounds were obtained from PubChem
(http://pubchem.ncbi.nlm.nih.gov) and visualized in BIO-
VIA Discovery Studio Visualizer and saved as *.pdb for-
mat for input into AutoDock 4.2 docking analysis software.
The 3D structure of pi class GST of W. bancrofti was
downloaded from Protein Data Bank (PDB id: 5D73)
(https://www.rcsb.org) and the structure optimization were
done using PyMol molecular visualization system. Auto-
Dock 4.2 docking software was used to perform molecular
docking simulation between WbGST and the compounds
isolated from R. tetraphylla. This is a web service devel-
oped in the Biomedical Research Foundation of the
Academy of Athens. Docking of the ligands was done by
default parameters. The compounds with lowest dock score
and high interaction with active sites was determined by
docking analysis that gave perfect idea about protein ligand
interaction (Azeez et al. 2012; Rajesh and Shamsudin
2017; Kalani et al. 2014).

Statistical analysis

Statistical analyses were carried out using Minitab 18

software. Results were expressed as mean ± S.D. (n = 3).
The data were subjected to one-way ANOVA and the
significance of the difference between control and treated
groups were determined by Dunnett’s multiple comparison
test and significance levels were calculated at p \ 0.001
and p \ 0.05.

Results

Isolated alkaloid compounds

Using column chromatography and preparative TLC, three

compounds were isolated from chloroform fraction of the
leaf extracts of R. tetraphylla and the compounds showed
positive result in Dragondroff’s test, Mayer’s test and
Wagner’s test that confirmed the alkaloid nature of the
compounds. The structures of the compounds were deter-
mined with the help of IR, GCMS and 1H and 13C NMR Fig. 1 Chemical structures of compounds 1–3 isolated from leaf
spectroscopy (Fig. 1). extracts of R. tetraphylla
Compound 1 obtained as amorphous solid, and it
showed retention time of 2.685 with 86.194% purity in Fragment ions m/z values 57, 144, 228, 358 that assigned to
HPLC. The mass spectrum analysis from GC–MS data the molecular formula C20H26N2O4. IR absorptions at 3360
showed compound with molecular weight 358 g/mol and

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and 2930 cm-1 suggested the presence of alcohol and alcohols and esters in the compound. The 13C NMR of the
alkane (C–H). Three stretches at 1687, 1623 and compound 1 showed 20 resonance spectrum values that are
1437 cm-1 corresponds to double bonds and carboxylic corresponding to carbon atoms attached with various
acid group. The presence of amines and aromatic ring was bonding pattern of the structure. The signals obtained at dC
confirmed by the stretches at 800 and 620 cm-1. The 1H 120.92, 122.5, 129.58, 149.89 and 172.03 showed carbon
NMR spectrum for compound 1 showed signals at dH atoms present in the aromatic benzene ring structure, dC
6.5–7.5, typical for an aromatic benzene ring system. The 44.64, 53.3 showed allylic, benzylic or keto group, dC
signals between dH 1.5 and 2.5 corresponding to allylic, 62.15, 61.71, 71.67 showed alcoholic and ester groups. The
benzylic or keto group in the compound. The signals signals obtained from 1H and 13C NMR of compound 1
obtained between dH 3.5 and 5.55 revealed the presence of were compared with the signals of compound Curan-17-oic

Fig. 2 a Macrofilaricidal effect

of compounds 1–3 alone,
b effects of compounds 1–3 in
combination with DEC on adult
parasites of S. cervi. Notes:
Vertical lines show
mean ± SD. All the values
obtained are statistically
significant at p \ 0.05 when
compared with their respective
controls. Parasites treated with
DMSO were taken as control
from which inhibition
percentage was calculated

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acid, 19, 20-dihydroxy, methyl ester (19S) and found carbon atoms in alcohol and ester groups, dC values at
resemblance in both GC–MS and NMR spectroscopy 56.13, 56.16 and 51.58 showed carbon atoms in allylic,
studies. benzylic and keto groups. Combined analysis of 1H and 13C
The molecular formula for compound 2 was established NMR data of compound 3 indicated that the resonance
as C21H24N2O3 by GC–MS study that showed Fragment values have resemblance with Reserpiline (Fig. 1) and it
ions m/z values: 43, 57, 71, 149, 279 and 352 with also confirmed by GC–MS analysis data.
molecular weight 352 g/mol. HPLC chromatogram for
compound 2 showed retention time 2.160 with 73.026% In-vitro macrofilaricidal activity
purity. IR data for compound 2 showed various bonding
pattern and functional groups such as 3390 cm-1 (Alcohol All compounds 1–3 showed significant adulticidal activi-
O–H), 1707 cm-1 (Carboxylic Acid C=O Stretch), ties with IC50 values 0.54, 0.21 and 0.44 mg/ml (Fig. 2a)
1210 cm-1 (Amine C–N Stretch), and 470 cm-1 (Aro- for compounds 1, 2 and 3 respectively in terms of MTT
matic C–H bend). The 1H NMR signals at dH 6.75, 6.92, reduction assay that clearly proved the mortality of the
7.57, 7.1 and 7.4 corresponding to aromatic protons in adult worms as shown in Table 1 in different time inter-
benzene ring structure. The signals at dH 8.44 and 9.67 vals. DEC showed completely less active with percentage
showed protons at heteroaromatic NH bonding system, dH inhibition value of 47.31 ± 6.6 at 1.0 mg/ml (Fig. 2b). The
at 1.94, 2.16, 2.35 showed protons at alkanes and keto combined effect of isolated compounds (F1, F2 and F3)
groups, dH at 3.42, 3.54, 3.65, 3.68 showed protons at and Diethylcarbamazine showed less effectiveness at the
alcohol and ester groups, dH at 5.16 showed vinylic concentrations 0.25, 0.5 and 1.0 mg/ml than the com-
hydrogen atom bonded with alkene carbon atom. The 13C pounds alone on Setaria worms. The combined effect was
NMR spectrum data exhibited carbon atoms at dC 168, studied for assessing the effectiveness of compounds and
155.5, 149.2, 144, 135.5, 126 and 120 corresponding to
aromatic carbon atoms, dC signals at 115.7, 109.7, 106.6 Table 1 Effect of compounds 1–3 on the motility of adult parasites
showed carbon atoms in alcohol and ester groups, dC sig- (S. cervi) in different time interval
nals at 72.5, 59.9, 56.6, 53.5, 51.3, 38.6, 34.3, 31.5, 21.8
Compounds and DEC Conc. (mg/ml) Motility scores (in hours)
and 18.7 corresponding to carbon atoms at benzylic, keto,
alkane groups. These evidences suggested that compound 2 1h 2 h 3 h 4 h 24 h
was a yohimban class of alkaloid compound that was F1 0.25 4 ? 4? 4? 4? 2?
identified as 18, 19-Secoyohimban-19-oic acid, F1 ? DEC 4? 4? 4? 4? 4?
16,17,20,21-tetrahydro-16-(hydroxymethyl)-methylester F1 0.5 4? 4? 4? 4? 2?
(Fig. 1) first time isolated from R. tetraphylla leaf extracts. F1 ? DEC 4? 4? 4? 4? 4?
Compound 3 was isolated as amorphous solid and its F1 1.0 4? 4? 4? 4? 1?
molecular formula was established as C23H28N2O5 based F1 ? DEC 4? 4? 4? 4? 4?
on the Fragment ions m/z values 216, 283, 311, 397, 412
F2 0.25 4? 4? 4? 4? 1?
with molecular weight 412 g/mol in GC–MS. It showed
F2 ? DEC 4? 4? 4? 4? 4?
sharp peak at retention time 2.167 with purity 83.436% in
F2 0.5 4? 4? 4? 4? 1?
HPLC. IR spectrum for 3 exhibited various bonding pattern
F2 ? DEC 4? 4? 4? 4? 4?
and functional groups such as 3407 cm-1 (Alcohol O–H
F2 1.0 4? 4? 4? 4? 1?
and Amine N–H Stretch), 1089 cm-1 (Alcohol O–H
F2 ? DEC 4? 4? 4? 4? 3?
stretch), 800 cm-1 (Amine N–H bend) and 480 cm-1
F3 0.25 4? 4? 4? 3? 1?
(Aromatic C–H stretch). IR spectra show numerous sharp
F3 ? DEC 4? 4? 4? 4? 4?
bands between 500 and 1500 cm-1 which are assigned to
F3 0.5 4? 3? 3? 2? 1?
deformation and stretching vibrations of the alkaloid ring
F3 ? DEC 4? 4? 4? 4? 4?
system. The 1H NMR spectrum for compound 3 showed
F3 1.0 4? 2? 1? 1? 1?
three signals at dH 6.32, 6.38 and 8.03 corresponding to
hydrogen atom linked to aromatic carbon typically for a F3 ? DEC 4? 4? 4? 4? 3?
benzene ring. The dH signals at 3.54, 3.6 and 3.72 showed DECa 0.25 4? 4? 4? 4? 4?
protons at three methoxy groups, dH signals at 1.18, 1.82, 0.5 4? 4? 4? 4? 4?
1.96 showed protons at methylene groups. The 13C NMR 1.0 4? 4? 4? 4? 4?
spectrum data exhibited carbon signals at dC 134.37, 128.2, All results are repeated three times for all groups
136.83, 149.61, 148.97, 167.53, 154.63 corresponding to Motility scoring: 4? (Highly motile), 3? (Motile), 2? (Sluggish), 1?
aromatic carbons present in a benzene ring structure. The (non-motile) and Nil (Dead)
a
dC values at 60.00, 56.67, 98.7, 93.9 and 73.63 showed Standard drug used as control

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DEC standard drug, as DEC alone is ineffective in killing high binding affinity, stability and activity of these isolated
adult parasites thus a combined effect may enhance the compounds.
power of standard drug. But unfortunately DEC in com-
bination with compounds 1–3 showed very low activity.
The macrofilaricidal effect was further analysed by tar- Discussion
geting the glutathione-S-transferase enzyme of S. cervi
adult worms. This study showed that GST enzyme of adult Filariasis imparts a huge socio-economic burden in tropical
parasites was significantly inhibited by the treatment of and sub-tropical countries. According to WHO, Filariasis is
compounds 1–3 with percentage inhibition of 35.78%, one of the debilitating vector borne disease second to
44.95% and 64.21% for compound 1, 2 and 3 respectively Malaria in Asian countries. The conventional drugs such as
at concentration of 1.0 mg/ml (Table 2). The protein con- DEC, albendazole and ivermectin are more microfilaricidal
tent estimation in each treated and control worm samples than macrofilaricidal and also possess side effects. The
denoted their activity in vitro condition in a specific time. development of a natural safer drug is therefore urgently
needed that would surely effective against adult parasites
In-silico molecular docking of filarial disease. In this regard, a promising ethno-
medicinal plant R. tetraphylla was explored for effective
The present study revealed that Reserpiline with highest antifilaricidal principles and computational study was
binding affinity of - 7.2 kcal/mol, second is the 18, undertaken for the evaluation of affinity of isolated com-
19-Secoyohimban with binding affinity of - 7.19 kcal/mol pounds with antifilarial drug target i.e. filarial GST
and then Curan-17-oic acid with binding affinity of enzyme.
- 5.14 kcal/mol (Table 3). These three compounds In this study, isolation of active principles resulted into
showed interaction by forming hydrogen bonds with amino three compounds namely, Compound F1 i.e., Curan-17-oic
acid residues of target GST molecule. The binding site acid, 19, 20-dihydroxy, methyl ester (19s) has been
amino acid residues that took part in bond formation are reported first time from R. tetraphylla plant leaf extract.
TYR-106, TYR-7, LEU-50, ASN-203, THR-102 (Fig. 3). Previous reports on isolation and characterization of com-
This strong hydrophobic interaction may be the reason of pounds by gas chromatography and mass spectroscopy

Table 2 Effect of compounds 1–3 on the GST specific activity of adult parasite (S. cervi)
Compounds Conc. (mg/ml) GST specific activity (lM/ml/min) % Inhibition Protein content (mg/ml)

F1 0.5 59.89 ± 2.3** 15.6% 2.13 ± 0.31*

1.0 45.57 ± 1.8** 35.78% 2.39 ± 0.02*
F2 0.5 59.24 ± 2.2** 16.51% 2.51 ± 0.51*
1.0 39.06 ± 3.11** 44.95% 2.39 ± 0.06*
F3 0.5 58.59 ± 0.88** 17.43% 2.46 ± 0.02*
1.0 25.39 ± 1.32** 64.21% 2.06 ± 0.06*
Control – 70.96 ± 1.21 – 1.67 ± 0.02
GST specific activity of S. cervi after treatment with compounds were compared with control and expressed as mean ± standard deviation
(n = 3). Protein content was simultaneously estimated and also presented as mean ± standard deviation (n = 3). **Indicates p \ 0.001 and
*indicates p \ 0.05 proved by one-way ANOVA followed by Dunnett’s two sided multiple comparison test as compared to control

Table 3 Molecular docking analysis of ligands 1–3 with target GST enzyme (WbGST)
Ligands Binding energy Ligand efficiency Inhibition Electrostatic Hydrogen bond Hydrophobic interaction
(kcal/mol) (kcal/mol) constant energy

Curan-17-oic acid - 5.14 - 0.2 171.22 0.17 LEU50, TYR7, TYR7, PHE8, PHE8
LEU50
18, - 7.19 - 0.28 5.41 - 0.11 LEU50, THR102, LEU13, LEU13, HIS98,
19-Secoyohimban THR102 LEU13
Reserpiline - 7.2 - 0.24 5.25 - 0.08 TYR106, ASN203, PHE8, PHE8, LEU13,
TYR106, HIS98 LEU13, PHE8

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analysis of methanolic leaves extract of Adiantum capillus-

veneris revealed Curan-17-oic acid compound that shows
antimicrobial and anti-inflammatory activities (Hussein
et al. 2016). GC–MS analysis of methanolic extract of
Eucalyptus citriodora also exhibited Curan-17-oic acid and
bioactivities such as antidiabetic and insecticidal properties
reported from for that compound (Sahi 2016). Compound
F2 i.e., 18, 19-seco-yohimban has also been reported from
plants such as Sesamum radiatum essential oil of the
leaves, methanol extract of Eichhornia Crassipes (water-
hyacinth), methanol extract of Basella alba leaf (Ogunlesi
et al. 2010; Shanab et al. 2010; Baskaran et al. 2015).
Yohimbine has been found active on endocrine and
reproductive systems. Yohimbine has also been found to
exhibit cardiovascular activity (Henauer et al. 1984).
Compound F3 identified as Reserpiline i.e., one of the well
known alkaloid earlier reported from root bark and stem
bark of Rauvolfia vomitoria, roots of Rauvolfia serpentina
and from chloroform and methanol extract of leaves of R.
tetraphylla (Poisson 1959; Srivastava et al. 2006; Maurya
et al. 2013). Reserpiline has antipsychotic, antigastric
secretion, antihypertension activities. In the present study
this compound has studied first time for antifilarial property
on S. cervi.
The in silico molecular docking study was performed to
determine the binding affinity of three isolated compounds
(F1, F2 and F3) with the target enzyme pi-class GST of W.
bancrofti. Earlier studies on GST enzyme of W. bancrofti
revealed that WbGST shares 98% identity with B. malayi
GST, 79% identity with O. volvulus GST and 75% identity
with D. immitis GST. This report has confirmed by mul-
tiple sequence alignment of amino acid sequences of
WbGST with other filarial nematode GSTs (Veerapathran
et al. 2009). Thus to enumerate the interaction mechanism
of pi-class glutathione-S-transferase (detoxification
enzyme) of W. bancrofti (nematode parasite) with the
selected significant ligands from the plant R. tetraphylla,
molecular docking was undertaken We have also attempted
the same for our isolated molecules and results are
promising.
Previous reports on docking of 58 phytochemicals with
nematicidal, anthelmintic and GST substrate activities
shows that only Curcumin, Vanillin and Brucine have
highest binding affinity of - 8.4343, - 7.0792 and
- 4.2539 kcal/mol respectively with GST enzyme (Azeez
et al. 2012). It has also been reported that the molecular
docking of reference drugs DEC and Ivermectin with
Fig. 3 Molecular docking study of three isolated compounds against
target GST enzyme. a Compound 1 binding affinity with filarial GST,
nematode GST enzyme reveals the binding energy as
b compound 2 binding affinity with filarial GST and c compound 3 - 4.9 kcal/mol and - 8.4 kcal/mol respectively (Kalani
binding affinity with filarial GST. Stronger binding energy leads to et al. 2014). This study also stated about the toxicity of
better antifilarial efficacy of drugs these reference drugs by examining through MetaDrug tool
(Thomson Reuters, USA).

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