Identification of an anti-inflammatory marker in Coccinia indica and a validated HPLC method
for its standardization
Yogita Bansal, Parabhdeep Singh, Junaid Niazi, Rajesh K. Goel and Gulshan Bansal*
Department of Pharmaceutical Sciences and Drug Research, Punjabi University,
Patiala – 147 002, India
* Corresponding author
Phone: +91-175-3046255 (Office)
Fax: +91-175-2283073
E-mail: gulshanbansal@rediffmail.com; gulshanbansal@gmail.com
1
Title: Identification of an anti-inflammatory marker in Coccinia indica and a validated HPLC
method for its standardization
Running Title: Chromatographic standardization of Coccinia indica
Abstract
Background: Coccinia indica (Family Cucurbitaceae) is traditionally used as anti-inflammatory
herb and various phytoconstituents are also characterized. However, neither the constituent
responsible for this activity of the plant is known nor any analytical method is reported for its
standardization.
Methods: Ethanolic extract of fresh leaves of C. indica (CIEE) was fractionated. The extract and
fractions were evaluated for anti-inflammatory activity using carageenan-induced rat paw
edema model. The pure fractions were characterized through spectral analyses. An HPLC-UV
method was developed and validated for quantification of the marker for standardization of the
CIEE.
Results: Anti-inflammatory activity of CIEE was comparable to diclofenac. The pure fractions
were characterized as vanillin and p-hydroxybenzaldehyde. Vanillin was equipotent to CIEE
whereas p-hydroxybenzaldehyde was found inactive. No synergism was found for the two
constituents. The HPLC method developed for quantification of vanillin was proved to be linear,
precise, accurate, sensitive and robust over the concentration range of 0.2-200 µg/ml. The
content of vanillin in CIEE was found to be 9.86% w/w.
Conclusions: The anti-inflammatory activity of CIEE was found to be due to vanillin. The HPLC
method was validated for standardization of the CIEE with respect to vanillin taken as antiinflammatory marker.
Key words: Coccinia indica; vanillin; p-hydroxybenzaldehyde; anti-inflammatory; HPLC;
standardization.
2
INTRODUCTION
Coccinia indica (syn. C. grandis, C. cordifolia, Bryonia cordifolia, Family Cucurbitaceae) is
commonly known as Scarlet Gourd or Ivy Gourd. It is a climbing, perennial herb which grows
abundantly in India, Sri Lanka, Malaysia and Tropical Africa.1 In the Ayurveda and Unani systems
of medicine, the plant is indicated for treatment of inflammation, diabetes mellitus, asthma,
cough, bronchitis, skin eruptions, tongue sores, earache and stomatitis.2,3 The aqueous and
alcohol extracts of C. indica leaves are known to possess anti-inflammatory4, hypoglycaemic5,
hypolipidaemic6, hepatoprotective7, antioxidant8 and antiparasitic9 activities. Various
phytoconstituents known to present in C. indica include cephalandrol, tritriacontane, lupeol, βsitosterol, cephalandrine A, cephalandrine B, stigma-7-en-3-one, taraxerone, taraxerol,
alkaloids and fatty acids.10-12 However, despite the ethnic use of this plant as potent antiinflammatory herb, no attempt has been reported by any research group to identify the
constituent responsible for its anti-inflammatory activity. Moreover, there is no analytical
method available for standardization of C. indica. Hence, the present study reveals the
phytoconstituents responsible for anti-inflammatory activity of ethanol extract of fresh leaves
of C. indica (CIEE) and a validated HPLC-UV method for standardization of CIEE with respect to
the isolated anti-inflammatory marker.
MATERIALS AND METHODS
Plant material and preparation of the extract (CIEE)
The leaves of C. indica were collected from Punjabi University, Patiala in the month of July and
were identified and authenticated by Department of Botany, Punjabi University, Patiala (India)
with sample being deposited in Herbarium having voucher number 49406. The fresh leaves
(500 g) were washed in running water, chopped and soaked in 1.5 L of 95% ethanol for 24 h.
The extract was filtered and residue re-suspended in 95% ethanol (1.5L) for 48 h. The extract
was filtered, the two extracts pooled and the solvent was distilled on a rotary evaporator to
obtain the extract as greenish black semisolid mass. The extract was stored at 0-4oC and
dissolved in desired solvent, whenever needed. The extract was subjected to qualitative
chemical examination for detection of phytoconstituents like alkaloids, flavonoids, proteins,
saponins, sterols, etc.
Drug, chemicals and instrumentation
Carrageenan was purchased from Sigma-Aldrich (Bangalore, India) and diclofenac procured as
gift sample from Crystal Pharmaceuticals Ltd. (Ambala, India). The HPLC grade solvents and the
other chemicals (AR grade) were purchased from Loba Chem (Mumbai, India). Milli-Q water
(Milli–Q water purification system, Synergy, Millipore, USA) was used for preparation of all
solutions. IR spectra were recorded as KBr pellets on a Perkin-Elmer Fx spectrophotometer. 1HNMR, 13C-NMR, DEPT-NMR and HSQC spectra were run on Bruker Avance II 400 NMR
spectrometer using CDCl3 as solvent. Mass spectra were recorded in +ESI mode on Vg Micro
Mass 7070F mass spectrometer. Anti-inflammatory activity was evaluated with Plysethometer
(Labco, India). The HPLC analysis was carried out using Waters HPLC system (Milford, USA)
consisting of 515 binary pumps, 2487 dual wavelength UV detector, Rheodyne manual injector
and Spherisorb ODS2 (250 mm x 4.6 mm, 5 µ) column. The data were acquired and processed in
Empower Pro® Operating System.
3
Animals
Wister rats (150-200g) of either sex were procured from Haryana Agriculture University (Hisar,
India) and housed in Central Animal House, Punjabi University (Patiala, India). They were
exposed to 12 h light/dark cycle and given standard laboratory feed and water ad libitum, both
being withdrawn 12 h prior to experiment. The experimental protocol involving use of animals
for the study was approved by the duly constituted Institutional Animal Ethical Committee in
accordance with the guidelines of Committee for the Purpose of Control and Supervision of
Experimental Animals (CPCSEA), Ministry of Environments and Forests, Government of India
(Reg. No. CPCSEA/107/1999).
Fractionation of CIEE
The CIEE (2 g) was column chromatographed over silica gel (100-200 mesh) with isocratic
elution using mobile phase composed of toluene and ethyl acetate (97:3 %v/v) at a flow rate of
about 1 ml/min. The eluent was collected in 25 ml portions. TLC profiling of each portion was
performed using the same mobile phase and portions showing fractions with same Rf values
were pooled. The pooled portions showing single fraction were then subjected to TLC again in
randomly selected five solvent systems (cyclohexane:ethyl acetate :: 99:1, 98:2, 97:3, 96:4 and
95:5) to ascertain their purity. The portions giving single spot in all these solvent systems were
deemed pure and the pure fractions isolated in appreciable amounts from these portions were
characterized through spectral techniques.
HPLC method
The fractions F2 (characterized as vanillin) was optimally resolved from other peaks in the CIEE
on a Spherisorb® ODS2 C18 column (250 mm x 4.6 mm i.d., 5 µm) with mobile phase composed
of methanol and water (55:45 % v/v) flowing at a rate of 1 ml/min. The injection volume was
fixed at 20 µl and eluent was detected at 231 nm. The mobile phase was filtered through 0.45µ
nylon membrane (47 mm) and degassed by sonication. The HPLC method was validated for
standardization of CIEE taking F2 as marker. Stock solutions of F2 (1 mg/ml) in the mobile phase
and of CIEE (20 mg/ml) in methanol were prepared were used to prepare varied standard
working solutions of F2 and CIEE for validation of the method. Each solution was filtered
through 0.45µ nylon membrane (13 mm) before injection. The method was validated for
specificity, precision, robustness, LOD, LOQ, linearity and accuracy in accordance with ICH
guidelines.13 The CIEE was standardized with respect to vanillin (the marker) by analyzing its
solution (20 mg/ml) by the validated HPLC method. The content of vanillin was calculated and
reported as % w/w in the extract.
Linearity
Standard solutions of vanillin of concentrations 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 µg/ml were
prepared, analyzed in order of increasing concentration and calibration curve was generated to
determine the slope, intercept and correlation coefficient (r2). The standard solutions were
prepared in triplicate to generate three calibration curves. The percent relative standard
deviations (%RSD) of the slope, intercept and correlation coefficient were calculated.
Precision
The intra-day precision was determined by analyzing (n=6) three different concentrations (2, 20
and 100 µg/ml) of vanillin on same day while for inter-day precision, the same three
concentrations were analyzed (n=6) on three different days and it was expressed as %RSD of
each concentration calculated.
4
Accuracy
A diluted CIEE was mixed with equal volume of mobile phase to produce CIEE solution serving
as unfortified extract. The dilute CIEE was also mixed separately with equal volume of vanillin
standard solution of concentrations 10, 50 and 100 µg/ml so that vanillin concentrations in the
dilute CIEE was fortified by 5, 25 and 50 µg/ml, respectively. The unfortified and fortified
extracts were prepared in triplicate and accuracy was expressed as % recovery of vanillin from
fortified extracts with respect to the unfortified extract.
LOD and LOQ
These were determined by using standard deviation method through equations, LOD = 3.3 σ/S
and LOQ =10 σ/S, respectively. Where σ was standard deviation of the response and S was
slope of the calibration curve.
Robustness
Various parameters of the optimized chromatographic conditions like mobile phase, λmax, flow
rate and column were varied a little deliberately to establish robustness of the developed HPLC
method. A standard solution of vanillin (50 µg/ml) was analyzed (n=3) by the optimized as well
as each varied chromatographic condition to calculate % recovery and retention time (Rt) of
vanillin through each chromatographic condition.
Anti-inflammatory activity
The activity was evaluated using carrageenan-induced rat paw edema model.14 Animals were
divided randomly into 11 groups with 6 animals in each group. Edema was induced on the right
hind paw by subplantar injection of 0.1 ml of 1% solution of carrageenan sodium in 0.9% NaCl.
Edema was measured before injection of carrageenan (zero time) and at 5, 15, 30, 45 and 60
min after the injection using plythesmometer. CIEE, F2, F3 (in different doses) and diclofenac
were administered 30 min before carrageenan challenge in treated groups. The antiinflammatory activity was calculated as percent inhibition of paw edema using the formula: %
inhibition = 100-[(edema volume in treated/oedema volume in control) x 100]. The statistical
significance of the observed data was determined by One Way Analysis of Variance (ANOVA).
The results were reported to be statistically significant at various p values.
RESULTS
Fractionation of CIEE
The phytochemical screening of CIEE revealed the presence of phytosterols, phenolic
compounds, triterpenoids and flavonoids. Five fractions (F1-F5) from CIEE were isolated through
column chromatography. The first three fractions (F1-F3) were found to be pure while each of
the other two fractions (F4 and F5) was found to contain three components. The fraction F1
(semisolid, 1.5% w/w of CIEE) eluted at Rf 0.81 as a yellow colored spot that did not turn brown
in iodine chamber suggesting it to be weakly unsaturated or completely saturated molecule.
The fraction F2 was noted as a brown spot in iodine chamber at Rf 0.72 and isolated in good
yield (10% w/w of CIEE) as brownish yellow liquid which crystallized to light brown solid (m.p.
820C). The fraction F3 was eluted at Rf 0.51 and isolated as yellowish crystals (118-1190C) in
sufficient yield (4.3% w/w). The F1 was not characterized due to insufficient spectral data owing
to its very low yield.
5
Table 1: Spectral data of fraction F2
Spectral technique and data
Structural
interpretation
-1
IR; cm (assignment)
1668 (C=O stretching) and
-CHO group
2854 (Fermi resonance in
aldehydes)
1299 and 1200 (C-O
Aryl alkyl ether
stretching)
linkage
3175 (=C-H stretching); 1591, Phenyl ring
1511 and 1461 (skeletal
bands); 731 (out of plane C-H
bending)
1H-NMR;
(Number of protons, signal multiplicity, δ
value)
1H, s, δ 9.83
3H, s, δ 3.93
1H, d (8Hz), δ 7.04 and
2H, m, δ 7.42
13C-NMR;
Aldehydic proton
–OCH3 group
Three aromatic
protons (two are
ortho to each other
(δ value)
190.96
151.76, 147.22, 130.02,
127.62, 114.46 and 108.86
56.2
-CHO group
Aromatic (Phenyl)
carbons
Methoxy carbon
DEPT (for only primary and tertiary carbons); (δ value)
δ 190.96
-CHO group
δ 108.86, 114.46, and 127.62 Phenyl carbons
bearing hydrogens
Methoxy carbon
δ 56.2
HSQC; correlations between 1H-NMR and 13C-NMR
signals
1H-NMR
13C-NMR
3.9
56.23
7.42
108.86
7.43
127.62
4-Hydroxy-37.04
114.46
methoxy
9.8
190.96
benzaldehyde
6
Table 2: Spectral data of fraction F3
Spectral technique and data
Structural
interpretation
-1
IR; cm (assignment)
1670 (C=O stretching) and
-CHO group
2879 (Fermi resonance in
aldehydes)
3750-3500 (O-H stretching),
Phenolic group
1284 (C-O stretching) and
1390 (O-H bending)
3178 (=C-H stretching); 1596
Phenyl ring
and 1459 (skeletal bands);
834 (out of plane C-H
bending)
1H-NMR;
(Number of protons, signal multiplicity, δ
value)
1H, s, δ 9.87
1H, s, δ 6.47 (Disappeared
upon D2O exchange)
2H, sta, δ 7.82-7.85
2H, sta, δ 6.98-7.01
13C-NMR;
Aldehydic proton
–OH group
para disubstituted
phenyl ring
(δ value)
δ 191.32
δ 161.66, 132.55, 129.81 and
116.02
δ 161.66 and 129.81 (low
intensity)
-CHO group
Phenyl group
Quaternary carbons
DEPT 45 and 135 (for only primary and tertiary
carbons); (δ value)
δ 190.96
-CHO group
δ 132.55 and 116.02
Phenyl carbons
bearing hydrogens
δ 161.66 and 129.81 (Absent) Phenyl carbons
bearing no hydrogens
ast,
symmetrical triplets which is characteristics of 1,4-disubstituted phenyl ring15
Characterization of F2 and F3
Based on the IR, 1H-NMR, 13C-NMR, DEPT and HSQC spectral data (Tables 1 and 2), the fractions
F2 and F3 were proposed to be 4-hydroxy-3-methoxy-benzaldehyde (vanillin) and p-hydroxy
benzaldehyde, respectively. The two fractions were detected as [M+1] ions at m/z 153 and m/z
7
123 in +ESI mass spectra which supported the structural propositions. Further, the melting
points of F2 and F3 were found to be same as those of the reference standards of vanillin and phydroxy benzaldehyde confirming the structural assignments.
HPLC method
An HPLC method for quantification of F2 (vanillin) was developed for standardization of CIEE.
The optimum resolution of vanillin from other phytoconstituents in CIEE was achieved by
mobile phase composed of methanol and water (55:45% v/v) flowing at a rate of 1 ml/min on
C18 column (4.6 mm × 250 mm, 5 µ, Spherisorb®). The injection volume was fixed at 20 µl and
eluent was detected at 231 nm. The method was found excellently linear for vanillin in
concentration range of 0.2-200 µg/ml. The mean (n=3) slope, intercept and r2 were found to be
140411, 7879 and 0.9999 with %RSD of 0.606, 4.758 and 0.005, respectively. The %RSD for
inter-day and intra-day precision at each test concentration was always <1.2 and 1, respectively
(Table 3) suggesting the method to be precise. The recovery of vanillin from each fortified
sample was 99.80-102.02% with %RSD of <1 (Table 3) which suggested that method was
accurate to quantify vanillin in CIEE. Insignificant changes in calculated vanillin contents (0.324.44 %) were noted upon deliberate changes in various chromatographic conditions with
respect to the optimized conditions (Table 4) which indicated that the method were robust
enough to small changes. The LOD and LOQ were found to be 0.073 µg/ml (%RSD 3.77) and 0.2
µg/ml (%RSD 2.79), respectively. The method was applied successfully for quantification of
vanillin in CIEE. The content of vanillin in CIEE was found to be 9.86 % w/w of CIEE. The
chromatograms of vanillin standard and CIEE obtained by using validated HPLC method are
shown in Figure 1.
Table 3. Precision and recovery studies
Precision
Actual
conc.
(µg/ml)
2
20
100
Measured concentration (µg/ml)
Mean ± SD; %RSD
Intra-day (n = 6)
Inter-day (n = 3)
2.015 ± 0.013; 0.64
1.97± 0.02; 1.11
20.33±0.08; 0.41
20.42±0.14;
0.71
99.71±0.57; 0.57
100.00±0.59;
0.59
Accuracy
Added
conc.
(µg/ml)
5
25
50
Calculated
conc.
(µg/ml)
Mean ± SD; %RSD (n =
3)
4.99± 0.01; 0.21
25.17±0.18; 0.71
51.01±0.18; 0.35
Recovery (%)
99.80
100.68
102.02
8
Table 4. Robustness studies
Robustness
Rt (min)
Parameter
Mean Difference
w.r.t.
standard
Optimized conditions
5.15
---------Flow rate 1.1
4.91
0.24
Flow rate 0.9
5.26
0.11
Mobile phase
5.24
0.09
Water(55):Methanol(45)
Mobile phase
4.97
0.18
Water(45):Methanol(55)
λ 226 nm
5.05
0.10
λ 236 nm
5.06
0.09
Flow rate 0.9, λ 226
5.33
0.18
Flow rate 0.9, λ 236
5.39
0.24
Flow rate 1.1, λ 226
4.92
0.23
Flow rate 1.1, λ 236
4.96
0.19
Column Kromasil
4.14
1.01
Column Inertsil
4.36
0.79
Content of vanillin
(µg/ml)
Mean ± SD; % RSD
49.83 ± 0.03; 0.071
47.78 ± 0.27; 0.58
49.56 ± 0.22; 0.45
49.68 ± 0.41; 0.82
%
Difference
in content
of vanillin
--------4.11
0.56
0.32
49.19 ± 0.12; 0.24
1.30
50.65 ± 0.37; 0.74
49.32 ± 0.25; 0.51
49.33 ± 0.24; 0.50
49.32 ± 0.05; 0.11
48.47 ± 0.08; 0.16
48.29 ± 0.24; 0.51
47.62 ± 0.31; 0.55
48.55 ± 0.24; 0.63
1.63
1.04
1.01
1.04
2.73
3.09
4.44
2.58
Figure 1. HPLC chromatograms of blank (A), vanillin standard (50 µg/ml) (B) and CIEE (C)
9
3.3 Anti-inflammatory activity
Inflammation induced by carrageenan during the first 5 min was noted to be rapid which slows
down during 5-30 min and it reaches the maximum at 30 min (Fig. 2). Subsequently, it
regressed to normal paw volume at 120 min. The early steep increase in inflammation may be
attributed to the increased vascular permeability by inflammatory mediators like histamine,
serotonin and bradykinin while the subsequent slow increase in inflammation is due to
overproduction of prostaglandins.16 CIEE inhibited the inflammation during the first 5 min
which suggested that the extract may be eliciting its effect through inhibition of histamine
release. It is further in consonant with the ethnic use of C. indica in asthma, cough and
bronchitis. The inflammatory activity of carrageenan has also been demonstrated to be
accompanied by elevation in the free radicals level17 and hence the anti-inflammatory activity
of CIEE can also be associated with its antioxidant activity.8 CIEE showed significant activity at
various doses (200, 500 and 1000 mg/kg) and the response was statistically significant with
respect to control (Fig. 3). The CIEE at dose of 1000 mg/kg was found to be equiefficacious to
diclofenac (at 25 mg/kg). The activity of F2 was evaluated at doses of 50 and 100 mg/kg. These
doses were selected on the basis of its about 10% w/w content in CIEE. The activity of F2 at 50
mg/kg was found similar to that of CIEE at 500 mg/kg (Table 5). The same correspondence was
noted between activity of vanillin at 100 mg/kg and that of CIEE at 1000 mg/kg (Fig. 4). It
indicated that anti-inflammatory activity of CIEE was due to F2. The F3 exhibited lesser activity at
various doses (50, 100 and 200 mg/kg) with respect to the extract, F2 and diclofenac (Fig. 6).
Moreover, F3 and F2 did not show any synergism in the activity (Table 5) which supported the
earlier contention that only F2 was responsible for the anti-inflammatory activity of CIEE.
Table 3. Anti-inflammatory activities of diclofenac, CIEE, F2, F3 and combined fractions.
Group
Statistical
Time (min)
Dose
significance
(mg/kg)
5
15
30
45
60
a
Diclofenac
Vanillin
25
50
Vanillin
100
CIEE
CIEE
200
500
CIEE
1000
PHB
50
PHB
100
43.36±2.37 54.01±1.64 62.11±3.70 54.33±1.71 40.89±0.71
42.75±2.25 37.20±3.12 30.83±2.16 16.25±1.15 12.49±1.24 SS (P=0.011)
NSS
28.44±1.08 53.65±2.13 50.56±3.07 48.46±2.11 40.41±1.11
(P=0.299)
16.80±1.76 25.00±1.39 37.50±0.98 22.90±2.19 20.00±1.56 SS (P=0.003)
28.45±2.08 37.38±3.24 30.11±1.47 26.46±2.02 12.40±1.21 SS (P=0.003)
NSS
28.50±2.67 53.77±2.14 49.80±1.48 47.14±0.76 40.27±1.14
(P=0.267)
04.60±0.18 12.50±0.98 25.00±1.57 10.80±1.65 06.70±0.41 SS (P=0.008)
SS
16.51±1.76 20.34±1.63 28.51±2.63 13.67±1.13 10.30±1.02
(P=<0.001)
18.71±1.73 23.24±2.07 29.25±1.79 17.21±1.73 12.27±1.08 SS (P<0.001)
SS (P=0.003)
28.63±1.42 37.32±2.29 30.66±3.12 17.12±1.26 12.78±1.32
PHB
200
Vanillin+PH
50+25
B
a SS, Statistically significant; NSS, Not statistically significant.
10
Figure 2. Response of carageenan induction on paw volume in rats (A) and anti-inflammatory
effect of CIEE (B), of vanillin (C) and of p-hydroxybenzaldehyde (PHB) on carragenan-induced
paw volume. Results are expressed as mean±SEM (n=6).*p<0.05 for all treatment versus
control.
CONCLUSIONS
The CIEE was found to have anti-inflammatory activity comparable to diclofenac. The two
fractions F2 and F3 were characterized as vanillin and p-hydroxybenzaldehyde in Coccinia indica
perhaps for the first time. The activity of vanillin was equivalent to that of CIEE whereas phydroxybenzaldehyde was inactive. There was no synergism in effect of vanillin when given
along with p-hydroxybenzaldehyde. Hence, the activity of CIEE was attributed to vanillin in it
and later was taken as anti-inflammatory marker for development of a HPLC-UV method. The
method was developed and validated for standardization of the plant with respect to vanillin.
The method proved linear, precise, accurate, sensitive and robust for determination of vanillin
over the concentration range of 0.2-200 µg/ml.
ACKNOWLEDGEMENTS
The authors are thankful to Punjabi University, Patiala (India) for financial assistance and
infrastructure required for the study and S. Avtar Singh, Regional Sophisticated Instrumentation
11
Centre, Panjab University, Chandigarh (India) for carrying out spectral analyses of the isolated
fractions.
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