Original Article
Pyrroloquinazoline Alkaloids from Tissue
Cultures of Adhatoda vasica and their
Antioxidative Activity
Bharat Singh1* and Ram Avtar Sharma2
1
2
Institute of Biotechnology, Amity University Rajasthan, Jaipur – 303002, India.
Department of Botany, University of Rajasthan, Jaipur 302004, India.
ABSTRACT
Address for
Correspondence
Institute of
Biotechnology, Amity
University Rajasthan,
Jaipur, 303 002, India
E-mail:
bharatsingh217@
gmail.com
Adhatoda vasica (L.) Nees (Acanthaceae) is a well known plant drug
in Ayurvedic medicine, used for the treatment of different types of
diseases and disorders. The tissue cultures of this plant species were
established on Murashige and Skoog culture medium by using
various concentrations of growth hormones (0.5-5.0 mg/L αnapththaleneacetic acid + 0.5-3.5 mg/L 6-benzylaminopurine). The
concentration of vasicine acetate was found higher (0.51 0.519%)
in six week old callus than in vivo (0.47 0.556%) plant material
(roots). The antioxidant activity was evaluated by using DPPHradical scavenging, reducing power and superoxide anion scavenging
models. We found that 2-acetyl benzyl amine showed maximum
antioxidant activity against various antioxidant models, including
DPPH-radical scavenging (40.59 ± 0.774%), reducing power (461.55
± 0.402%) and superoxide anion scavenging (52.54 ± 0.553%) than
other isolated alkaloids at 10-30 μM concentrations. Our observed
results suggest that the pyrroloquinazoline alkaloids of A. vasica
demonstrated strong antioxidant activity, which could be used as
potent drugs against different types of oxidative stresses.
Keywords: HPLC; DPPH-radical scavenging; Reducing power;
Superoxide anion scavenging models.
INTRODUCTION
Ayurveda, the ancient Indian
therapeutic system is renowned as one of the
major systems
of
alternative
and
complementary medicine. The thorough
knowledge about the medicinal plant is
mandatory for all who is working in the field
of Ayurveda, in order to identify and select
the appropriate plant for a cure of specific
disease1. Vasicine, isolated from A. vasica
is used in the preparation of Vasaca, a well
known drug in the Ayurvedic system of
medicine in India. The drug is recommended
for a range of ailments viz, bronchitis,
asthma, jaundice, diseases of respiratory
system, diphtheria, gonorrhea and uterotonic
abortifacient2. The whole plant has been
American Journal of Phytomedicine and Clinical Therapeutics
www.ajpct.org
Bharat et al_________________________________________________ ISSN 2321 – 2748
used worldwide as an herbal remedy for
treating cold, cough, whooping cough,
respiratory
tract
infections,
chronic
bronchitis, asthma as sedative expectorant,
antispasmodic, anthelmintic, rheumatism,
and rheumatic painful inflammatory
swellings3,4.
Phytochemical studies of Adhatoda
vasica revealed that the aerial parts contain
viz. vasicine, vasicinone5,6,7,8, vasicine
acetate and 2-acetyl benzyl amine9,10,11. The
other compounds like as adhatodine,
vasicoline, vasicol12, vasicinolone and
deoxyvasicinone have also been reported
from A. zeylanica Medic.13. Adhatoda
species have antimicrobial and several other
important activities14,15,16,17. However, there
are no published reports available on
isolation of pyrroloquinazoline alkaloids
from the roots and tissue cultures of A.
vasica, nor are there any reports of
antioxidant activity. Therefore, the study
was carried out.
MATERIALS AND METHODS
Plant Materials
The plant materials of Adhatoda vasica
were collected (Feb, 2012) from the Aravali
Hilly areas of Jaipur and botanical
authentication was confirmed by Professor R.
S. Mishra. The voucher specimens were
deposited in the Herbarium, Department of
Botany, University of Rajasthan, Jaipur, India
(sheet no. RUBL - 200431).
General Experimental Conditions
The melting points of purified
compounds were recorded on capillary
Toshniwal melting point apparatus and are
uncorrected. The spectral data were obtained
on the following instruments: ir, NICOLET –
AVATAR 330 FTIR; ms, Hewlett Packard
HP 5930 A; gc–ms, equipped with a HP 5933
data system, direct inlet at 70 eV; uv, Perkin Elmer, model–200; nmr, Bruker AM 400
system at 400 and 100 MHz. Adsorbents as
AJPCT[2][3][2014]403-412
silica gel 60 (230-400 mesh, Merck) used for
column chromatography and silica gel G
(Merck) for preparative thin layer
chromatography.
Extraction and Characterization
Shade-dried powdered plant roots
(10.0 kg) were soaked in ethanol (10.0 l) for
15 days and then filtered. The extraction
was repeated three times. The filtrate was
concentrated under reduced pressure at 40
°C to gum. This crude- gum (98.563 g) was
treated with 5.0% HCl (250 mL), warmed
for 30 min and filtered. The filtrate was
basified with ammonia (pH 10.0) and then
successively fractionated with hexane (300
mL × four times) and chloroform (300 mL ×
four times). The chloroform-soluble fraction
(yield 20.678 g) was used for isolation of
pyrroloquinazoline alkaloids. The five
pyrroloquinazoline alkaloids were purified
and
characterized
by
column
chromatography over silica gel (200 g)
using CHCl3: MeOH: EtOAc mixtures with
increasing polarity. Total 10 fractions (A-J)
were collected from chloroform-soluble
fraction.
Vasicine (1) and vasicinone (2): A
portion of fraction A–B, were combined
(5.432 g) and rechromatographed on silica
gel and purified by preparative TLC.
Solvent system used as -MeOH: H2O (60–
40, v/v), Rf ~0.70, crystallized with
methanol, odorous volatile compound,
detection on TLC by Dragendorff’s reagent,
yielded 1, vasicine (556 mg), mp 210-211
°C, C11 H12N2O, UVλmax 270 (0.857 nm),
HPLC solvent system–MeOH: H2O (4: 6),
RT (min) 3.12, IR (KBr)v: 3416.2 (O–H
stretching), 3065.3 (Ar C–H stretching),
2930.2 (C–H stretching), 1613.4 (C=N
stretching), 1584.1 (C=C stretching), 1299.6
(C–N stretching), 1164.7 cm-1 (C–O
stretching). MS showed (M) + peak at m/z
188, 1H NMR (DMSO): δ 2.66 (s, 2H, CH2),
3.23 (s, 2H, CH2), 4.23 (s, 2H, CH2), 5.23
Bharat et al_________________________________________________ ISSN 2321 – 2748
(s, 1H, OH), 7.42-7.83 ppm (m, 4H, Ar –
H). Fractions C–D pooled together (3.836
g), rechromatographed on silica gel and
purified by preparative TLC with
development by CHCl3–MeOH–EtOAc
(85–15–10 v/v), Rf ~0.62, crystallized with
methanol, positive to Dragendorff’s reagent,
yielded to compound 2 (vasicinone, 833
mg), mp 198-200 °C, C11H10N2O2, ([α]D23
97=0.9). UV (MeOH)max log(ε) 273 nm. IR
(KBr) Vmax 3169, 1683, 1463 cm-1, 1H NMR
(CDCl3, 200 MHz): δ 2.20-2.45 (m, 1H),
2.60-2.80 (m, 1H), 3.90-4.15 (m, 1H), 4.154.30 (m, 1H), 5.27 (H), 7.40-7.60 (m, 1H),
7.65-7.85 (m, 2H), 8.31 (d, J = 6H = 1H).
13
C NMR (CDCl3 50 MHz) δ 29.4, 43.5,
72.0, 121.1, 126.7, 126.8, 134.4, 148.6,
160.1, 160.6. MS (m/z): 202, 185, 174, 146,
130, 119, 102, 90, 76, 63, 55. Anal: C 65.34:
H4.99: N13.85 calculated for C11H10N2O2,
found C. 65.18, H5.06, and N13.77.
Vasicine acetate (3) and 2-acetyl
benzyl amine (4): Fractions E–H pooled
together (2.248 g), rechromatographed on
silica gel and purified by means of
preparative TLC. Compound 3, vasicine
acetate (749 mg), solvent system: CHCl3:
MeOH: EtOAc (60:20:10, v/v), Rf ~0.54,
mp 122 °C, C13H14N2O2, positive to
Dragendorff’s reagent, white powder, IR
data: max cm−1 (400-4000) 1720 (acetate),
1629 (> C=N) 1579, 1498, 1406 (aromatic
system), 1385, 1345, 1241 (acetate), 1162,
1129, 1105, 1072, 933, 903, 767, 722
(aromatic system). 1H NMR data for the
active molecule were the following: δ 7.14
(2H, m, H–5 and H–6), 6.96 (1H,brs, H–7)
6.84 (1H, bs, H–8), 4.72, 4.80 (each 1H, d, J
= 14.5 HZ, H–9), 2.09 (3H, s, −OCOCH3),
3.52, 3.70 (1H each, m, H–1), 2.25, 2.55
(1H each, m, H–2), 5.19 (1H, brt, H–3). 13C
NMR: δ 50.7 (C–1), 28.0 (C–2), 70.2 (C–3),
163.9 (C–3a), 134.0 (C–4a), 129.5 (C–5),
126.6 (C–6), 126.2 (C–7), 120.3 (C–8),
116.3 (C–8a), 46.8 (C–9), 167.3 (–
OCOCH3), 23.3 (–OCOCH). MS: [M]+ m/z
AJPCT[2][3][2014]403-412
230. Compound (4) was purified by
preparative TLC (solvent system: hexaneethyl acetate, 8:2 v/v), Rf ~0.65, yielded 2acetyl benzyl amine (446 mg), white
powder, tested positive for primary amine.
The IR spectrum gave the following data:
Max cm−1 = (500-4000) 3400 (−NH2), 2928,
1685 (Ar CO–), 1628, 1576, 1498, 1459 and
1406 (aromatic system), 1188, 775
(aromatic system). 1H NMR data for the
active molecule were the following: δ 7.91
(2H, m, H–1 and H–3), 7.52 (2H, m, H–2
and H–4), 4.42 (1H, brs, −CH2NH2), 2.6
(3H, s, COCH3). The 13C NMR spectrum of
the active molecule was the following: δ
129.1 (C–1), 127.8 (C–2), 132.6 (C–3),
128.2 (C–4), 136.1 (C–5), 137.6 (C–6), 54.3
(−CH2NH2), 197.6 (–COCH3), 26.6 (–
COCH3). MS: [M] + m/z 147, m/f–C9H9NO.
Vasicinolone (5): The fractions I-J
combined
together
(3.345
g),
rechromatographed on silica gel and
purified by preparative TLC (solvent
system: CHCl3: MeOH, 8–2 v/v),
crystallized with methanol, pale yellow
crystals, mp 278-280 °C, positive
to
Dragendorff’s reagent, Rf ~0.59, UV λmax
270 (0.832) nm, yielded to the compound V,
vasicinolone (198 mg). HPLC solvent
system–CHCl3: MeOH (8: 2 v/v), RT (min)
3.12, IR (KBr)υ: 3221.0(O-H ), 3021.0 (Ar
C–H), 2926.1 (C–H), 1680.8 (C=O), 1590.5
(C=N), 1541.3 (C–C ), 1216.2 cm-1 (C–N).
MS showed (M) + peak at m/z 218.1H NMR
(DMSO): δ 2.48 (s, 2H, CH2), 4.31 (s, 2H,
CH2), 5.29 (s, 1H, OH), 7.21 (s, 1H, Ar–
OH), 7.22–7.67 ppm (m, 3H, Ar–H).
Tissue Culture
The unorganized callus tissue of A.
vasica was induced by roots. The roots were
surface sterilized (size 2-4 mm) with 0.1%
(w/v) HgCl2 solution for 1-1.5 min and then
rinsed three times with sterilized distilled
water. To initiate callus from root explants,
the explants were grown on MS culture
Bharat et al_________________________________________________ ISSN 2321 – 2748
medium18 supplemented with different
concentrations of growth regulators: 0.5-5.0
mg/L α-napththaleneacetic acid (NAA) and
0.5-3.5 mg/L 6-benzylaminopurine (BAP).
The culture medium was also supplemented
with 3.0% sugar. The root parts started
differentiated tissue formation after 20-25
days of inoculation. These cultures were
incubated at 25 1 C with 60% relative
humidity under room light conditions (300
Lux). The callus tissue sample was
transferred onto the fresh MS medium after 45 weeks intervals. The callus tissue was
harvested at the transfer age of 2, 4, 6, 8
weeks and the growth index was calculated
(GI = Final dry weight of callus-Initial dry
weight of callus/Initial dry weight of callus;
0.98 in 2 weeks; 3.65 in 4 weeks; 4.89 in 6
weeks; 4.67 in 8 weeks old cell cultures). The
compounds were isolated as per the above
mentioned methods.
HPLC Analysis
A Shimadzu (Japan) gradient HPLC
instrument equipped with two LC–8A
pumps, a model 7725 I manual injector
valve (Rheodyne). Solvents were filtered
through a Milipore system and analysis was
performed on a Merck Hibar C18 column
(250 mm x 4.0 mm I. D. 10 μm). Mobile
phase was optimized by varying percentage
of acetonitrile in phosphate buffer and peak
purity and similarity results of compounds
1-5 (isolated and standard) detected by using
photodiode detector, these resulted in the
following operating conditions: MeOH–H2O
(4.0: 6.0, v/v) and CHCl3–MeOH (8.0: 2.0,
v/v)10,19.
DPPH-radical Scavenging Activity
The free radical scavenging activity of
pyrroloquinazoline
alkaloids
was
determined by DPPH assay20. The reaction
takes place when a solution of DPPH was
prepared in methanol (150 µM). Then 0.5
ml of DPPH solution was added to 0.5 mL
AJPCT[2][3][2014]403-412
of isolated pyrroloquinazoline alkaloids at
different concentrations (10, 20, 30 µM).
The reaction mixture was then shaken
vigorously and incubated at room
temperature for 1 h; absorbance values were
measured at 517 nm in a spectrophotometer
against methanol as blank (negative control)
and (±)–α–tocopherol as positive control.
The inhibition percentage was calculated of
DPPH–radical scavenging activity as
follows:
Inhibition (%) =
(Absorbancecontrol - Absorbancesample)
___________________________________
Absorbance control × 100
Reducing Power
Reducing
power
of
pyrroloquinazoline alkaloids was measured
using the established protocol21. Varied
concentrations
of
pyrroloquinazoline
alkaloids (10, 20, 30 µM) were mixed with
phosphate buffer (pH 6.8) and potassium
ferric cyanide. The reaction mixture was then
allowed to stand at 52 °C for 15 min. A
portion of trichloroacetic acid was added to
the reaction mixture and finally centrifuged
for 15 min at 1000 g. The upper layer of
reaction mixture (1.0 mL) was mixed with
distilled water (1.0 mL) and FeCl3 (1.0 mL)
for 15 min and at last the absorbance was
measured at 700 nm in a spectrophotometer.
The inhibition percentage was calculated of
reducing power activity as follows:
Inhibition (%) =
(Absorbance control – Absorbance sample)
____________________________________
Absorbance control × 100
Superoxide Anion Scavenging Activity
The determination of superoxide
scavenging activity of pyrroloquinazoline
alkaloids was performed by using the method
Bharat et al_________________________________________________ ISSN 2321 – 2748
of Robak and Gryglewski22. The superoxide
radicals were generated in phenazine
methosulfate – nicotinamide adenine
dinucleotide systems by nicotinamide adenine
dinucleotide oxidation and assayed by
nitroblue
tetrazolium
reduction.
The
generated superoxide radicals were incubated
with
different
concentrations
of
pyrroloquinazoline alkaloids and (±)-αtocopherol (10, 20, 30 µM). The chemical
reaction was started with addition of 1.0 mL
of phenazine methosulfate (110 µM) solution
to the reaction mixture. The reaction mixture
was incubated at room temperature for 10 min
and the absorbance was measured at 560 nm
against
blank
sample
by
using
spectrophotometer. The inhibition of
superoxide anion scavenging activity was
calculated as follows:
Inhibition (%) =
(Absorbance control – Absorbance sample)
____________________________________
Absorbance control × 100
RESULT AND DISCUSSION
The tissue cultures of A. vasica were
established by using different concentrations
of NAA (0.5-5.0 mg/L) and BAP (0.5-3.5
mg/L). The maximum growth of tissue was
achieved at 3.5 mg/L NAA and 1.25 mg/L
BAP (Fig. 1). Growth of A. vasica cell
cultures was increased up to 6 weeks and later
on started to decrease up to 8 weeks old cell
cultures. The growth index was maximum
(4.89) in 6 weeks while minimum (0.98) in 2
weeks old cell cultures (0.98 < 3.65< 4.89 >
4.67).
The five pyrroloquinazoline
alkaloids were isolated and characterized
from the chloroform-soluble fraction of A.
vasica roots (1, 556 mg; 2, 833 mg; 3, 749
mg; 4, 446 mg; 5, 198 mg; Fig. 2). The
pyrroloquinazoline alkaloids were also
estimated from the chloroform-soluble
fraction of 2, 4, 6, 8 weeks old callus. The
AJPCT[2][3][2014]403-412
vasicine acetate accumulated in higher
concentration (0.51 0.519%) in 6 weeks old
callus tissue than in vivo (roots) plant material
(0.47 0.556%). The quantities of isolated
alkaloids were compared in both in vivo and
in vitro studies and found that in vitro cells
produced higher quantities of alkaloids (Table
1).
Present investigation revealed the
comparison of antioxidative activity of
pyrroloquinazoline alkaloids (10-30 µM) and
(±)-α-tocopherol (10-30 µM) using different
antioxidative models such as DPPH-radical
scavenging, reducing power and superoxide
anion scavenging. The DPPH-radical has
been used widely to test the potential of
phytocompounds as free radical scavengers of
hydrogen donors. 2-acetyl benzyl amine
demonstrated
maximum
DPPH-radical
scavenging activity (40.59 ± 0.774%) than
other pyrroloquinazoline alkaloids and
positive control showed 65.34 ± 0.509% of
inhibition respectively (Fig. 3). The
transformation of Fe3+–Fe2+ was monitored in
presence of pyrroloquinazoline alkaloids. The
reducing power activity 2-acetyl benzyl
amine was observed as 461.55 ± 0.430%
much higher than other isolated compounds
(Fig. 3). The maximum superoxide anion
scavenging activity was showed (52.54 ±
0.553%) by the 2-acetyl benzyl amine in
comparison to other pyrroloquinazoline
alkaloids (Fig. 3).
The
accumulation
of
pyrroloquinazoline alkaloids was estimated in
callus tissue and also compared with
accumulation of phytochemicals in roots of A.
vasica. Manipulation of physical parameters
and nutritional elements in a culture is
perhaps the most fundamental approach for
optimization of culture productivity23,24. The
total protein and carbohydrate values
increased slowly up to day 12-14 but later
reached a sharp peak at day 30–35 day and
then gradually diminished to the initial
inoculum
value.
The
microscopical
Bharat et al_________________________________________________ ISSN 2321 – 2748
observations suggest that much increase in
cell dry weight (6 weeks old) is due to
accumulation of starch grains, which
subsequently disappear during the growth
cycle25.
The DPPH scavenging activity is
totally based on the decolorization of DPPH
by the antioxidant compounds26. The 2-acetyl
benzyl amine is a primary amine and the
primary amines are extremely potent radical
scavengers capable of reacting directly with
carbon centered radicals and interacting the
auto-oxidation cycle earlier than primary and
secondary antioxidants. The primary
antioxidants consist mainly of hindered
aromatic amines. They scavenge and destroy
the chain propagating peroxy and alkoxy
radicals before they can react with the
polymer27,28. Therefore, DPPH-free radical
scavenging effect of chloroform-soluble
fraction and isolated pyrroloquinazoline
alkaloids might be attributed to a direct role in
the trapping of free radicals by donating
hydrogen atom. Similarly, superoxide anion
radical is also considered as a one of the
strongest reactive oxygen species among the
free radicals21.
CONCLUSION
From the observation it may be
concluded that the roots of A. vasica are the
good source of natural antioxidants and might
be useful in treating the diseases associated
with oxidative stress. The data obtained from
literature as well as results reveal the great
potential of A. vasica for therapeutic
treatment, in spite of the fact that they have
not been completely investigated. Therefore,
more studies need to be conducted to search
for new compounds.
ACKNOWLEDGEMENTS
Authors like to thank to Professor
Naveen Sharma, National Institute of
AJPCT[2][3][2014]403-412
Ayurveda, Jaipur, for this research work and
also for the Professor R. S. Mishra,
Department of Botany, University of
Rajasthan, Jaipur, for identification of plant
materials.
DECLARATION OF INTEREST
The authors report no conflict of
interest. The authors alone are responsible for
the content and writing of this research paper.
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Table 1. Quantity of isolated pyrroloquinazoline alkaloids from A. vasica Nees in vivo and in
vitro
Percentage of isolated compounds (w/w) ± SD
Isolated
Compounds
I
II
III
IV
V
In vivo
0.35 0.212
0.44 0.413
0.47 0.556
0.24 0.661
0.31 0.784
In vitro (Age of callus in weeks)
2
0.071 0.721
0.064 0.437
0.081 0.444
0.076 0.228
0.0232 0.187
4
0.099 0.469
0.084 0.555
0.11 0.616
0.091 0.319
0.071 0.228
6
0.32 0.111
0.24 0.813
8
0.31 0.808
0.25 0.718
0.51 0.519
0.36 0.777
0.096 0.559
0.089 0.353
0.18 0.448
0.16 0.331
Isolated compounds: I, vasicine; II, vasicinone; III, vasicine acetate; IV, 2- acetyl benzyl amine;
V, vasicinolone. Values are mean of triplicate readings
Figure 1. A. vasica (a) view of whole plant; (b) callus cultured on MS culture medium
supplemented with 3.5 mg/L α-napththaleneacetic acid + 1.25 mg/L 6-benzylaminopurine
AJPCT[2][3][2014]403-412
Bharat et al_________________________________________________ ISSN 2321 – 2748
Figure 2. Structures of isolated pyrroloquinazoline alkaloids
from A. vasica Nees
AJPCT[2][3][2014]403-412
Bharat et al_________________________________________________ ISSN 2321 – 2748
Figure 3. Antioxidant activity of isolated pyrroloquinazoline alkaloids from A. vasica Nees
AJPCT[2][3][2014]403-412