Anti-inflammatory and ulcerogenic activity of newer phytoisolates of Swertia alata C.B. Clarke

Article abstract of DOI:10.1080/14786419.2020.1775224

The present study was intended to evaluate the in?vitro (COX-1/COX-2) and in?vivo anti-inflammatory and ulcerogenic activity of newer phytoconstituents isolated from the aerial parts of Swertia alata C.B. Clarke (Gentianaceae). For isolation of newer phyt

Full text of DOI:10.1080/14786419.2020.1775224

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Anti-inflammatory and ulcerogenic activity of
newer phytoisolates of Swertia alata C.B. Clarke
Sakshi Bajaj, Sharad Wakode, Avneet Kaur, Shivkanya Fuloria & Neeraj
Fuloria
To cite this article: Sakshi Bajaj, Sharad Wakode, Avneet Kaur, Shivkanya Fuloria & Neeraj
Fuloria (2020): Anti-inflammatory and ulcerogenic activity of newer phytoisolates of Swertiaꢀalata
C.B. Clarke, Natural Product Research, DOI: 10.1080/14786419.2020.1775224
To link to this article: https://doi.org/10.1080/14786419.2020.1775224
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1775224
Anti-inflammatory and ulcerogenic activity of
newer phytoisolates of Swertia alata C.B. Clarke
Sakshi Bajaja, Sharad Wakodeb, Avneet Kaurb, Shivkanya Fuloriac and
Neeraj Fuloriac
aDepartment of Pharmacognosy and Phytochemistry, Delhi Institute of Pharmaceutical Sciences
b
and Research, University of Delhi, New Delhi, India; Department of Pharmaceutical Chemistry,
Delhi Institute of Pharmaceutical Sciences and Research, University of Delhi, New Delhi, India;
cPharmaceutical Chemistry Unit, Faculty of Pharmacy, AIMST University, Kedah Darul Aman, Malaysia
ABSTRACT
ARTICLE HISTORY
Received 16 January 2020
Accepted 17 May 2020
The present study was intended to evaluate the in vitro (COX-1/
COX-2) and in vivo anti-inflammatory and ulcerogenic activity of
newer phytoconstituents isolated from the aerial parts of Swertia
alata C.B. Clarke (Gentianaceae). For isolation of newer phytocon-
stituents, the ethanolic extract of aerial parts of S. alata was
subjected to column chromatography using mixture of petroleum
ether and chloroform in various concentrations, which yielded
two phytoisolates characterised as nonacosyl triacontanoate
(SA-3) and 8-O-glucpyranosyl-(2-acetyl)-1,3-dihydroxy-5-methoxy-
xanthone (SA-9). Identification of compounds was based on
KEYWORDS
Phytochemistry; partitioned;
chemical methods;
chromatographic;
phytoconstituents
1
melting point, UV, FTIR, H-NMR, 13C-NMR and mass spectrometric
data. The isolates were screened for in vitro COX-1/COX-2
inhibitory activity, in vivo anti-inflammatory and ulcerogenic activity.
Among the two compounds, SA-3 was found to be more effective
than SA-9. The ulcerogenic study revealed significant gastric
tolerance of SA-3 and SA-9 in comparison to indomethacin.
1. Introduction
Swertia (family Gentianaceae) is a large genus of herbs distributed in the mountainous
regions of tropical area at an altitude of 1200–3600 m. Genus Swertia is well known for its
medicinal plant species (Patro et al. 2005; Jiang et al. 2016). Swertia alata C.B. Clarke is a
CONTACT Sakshi Bajaj
sakshibajaj84@gmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1775224.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
S. BAJAJ ET AL.
branched herbaceous plant with yellowish green flowers (Natrajan and Prasad 1972). It is
widely used in the Indian system of medicine (ISM). The S. alata plant is generally used as
a substitute of Swertia chirata in many herbal formulations (Bajaj and Wakode 2017). S.
alata is known to possess oleanolic acid, swertisin (Pant et al. 2000), swertiamarin (Negi
et al. 2011), swertianin (Khetwal et al. 1997), methyl swertianin, methylbellidifolin (Khetwal
et al. 1997), bellidifollin (Bajpai et al. 1991), swertiaperennine and decussatin (Karan et al.
2005), but still very limited data is available over aerial parts of S. alata.
Facts suggest chronic inflammatory diseases are associated with cardiovascular dis-
eases, inflammatory bowel disease and acceleration of ageing, that are the major
cause for morbidity and mortality in the world (MacNee, Rabinovich, and Choudhury
2014; Mason and Libby 2015; Alatab et al. 2020). The NSAIDs are one of the most
widely prescribed drugs in the world and are extensively used to alleviate clinical
cases especially for pain and inflammation (Brooks et al. 1999). However, these drugs
are well-known to induce gastric/intestinal ulceration, anemia, platelet inhibition, delay
ulcer healing, and in some cases increase the risk of myocardial infarction (Hippisley-
Cox and Coupland 2005; Lanas et al. 2005; Craig et al. 2008).
Steroidal anti-inflammatory drugs prevent or suppress inflammation but do not
attack the root cause of the disease, and the prolonged use of these compounds can
inhibit the synthesis of the inducible isoform of nitric oxide synthase enzyme and
cause pituitary-adrenal suppression, hyperglycemia, glycosuria and an increased sus-
ceptibility to infections and peptic ulcers (Ogirala et al. 1991).
Despite recent advances, an adequate remedy for NSAID-induced gastropathy remains
elusive. Discovery of potent NSAIDs with very low or no gastrointestinal (GI) side effects is
the area of prime interest. Use of most commonly prescribed drug from analgesics such
as aspirin nowadays have been limited due to their potential side effects like severe gastric
disorders. With the introduction of cyclooxygenase-2 (COX-2) inhibitors problem of GI side
effects is overcome up to certain level (Chhabria and Doda 2005; Dutt et al. 2007).
The World Health Organization has stressed over the need for development of drugs
from plant origin owing to their inexpensive, accessibility (particularly to rural people of
developing countries) and less/no side effects. Hence, development of drug having anti-
ulcerogenic property from plant source without compromising the efficacy and safety
would be expected to benefit millions of suffering humanity. The Ayurvedic and Yunani
system highlights the astringent, stomachic, sedative and anti-inflammatory potential of a
large genus called Swertia belonging to the Gentianaceae family (Negi et al. 2011; Mehta
et al. 2017). But, evaluation of anti-inflammatory potential of isolates of S. alata is still due.
Hence, based on the aforementioned findings, an attempt has been made to isolate and
characterise the phytoconstituents from S. alata and evaluate against in vitro (COX-1/COX-
2) and in vivo anti-inflammatory and ulcerogenic activity.
2. Results and discussions
2.1. Structure elucidation of compounds
2.1.1. Nonacosyl triacontanoate (SA-3)
SA-3 (Figure 1) was obtained as white crystalline powder from elution of the column
with chloroform:petroleum ether (2:3), recrystallised from methanol, 80 mg (0.06%) and

NATURAL PRODUCT RESEARCH
3
Figure 1. Structure of nonacosyl triacontanoate.
having melting point 78–79 ꢀC. UV (MeOH) max: 257 nm; FTIR: (KBr, ꢀmax, cmꢁ1): 2917,
1
2848 (CH stretching), 1735 (C⩵O stretch) cmꢁ1. H-NMR (DMSO-d6, 300 MHz): 0.85 (t,
J¼ 6.9 and 6.9, attributed to 6H of 02 terminal methyl units), 1.16–1.25 (m, attributed to
110H of 55 methylene groups), 1.61 (t, J¼ 6.9, attributed to 2H of methylene unit at pos-
ition 29), 2.32 (t, J¼ 6.9 and 6.9, attributed to 2H of methylene unit at position 31). 13C-
NMR (DMSO-d6, 300 MHz): 14.21 (C1 and C59), 29.26 (C-29), 32.02 (C-31), 76 (methylene 54
carbon units), 164 (carbonyl carbon). LC-ESI-MS m/z (Relative intensity): No parent ion peak
at m/z: 859; characteristic peaks fragment ion peaks at m/z: 423, 395, 367, 283, 255, 227]þ
(Fragmentation pattern given in Supplementary Material Figure S1).
Chemical Identification: Compound SA-3 (80 mg) was acid hydrolysed using 2 M
H2SO4 and confirmed the presence of ester group and steryl alcohol.
The Compound SA-3, identified as nonacosyl triacontanoate, was obtained as white
amorphous powder. It was acid hydrolysed and responded positively for steryl alcohol
and ester group. The presence of sterol nucleus was confirmed by acid hydrolysis
using 2 M H2SO4. The IR spectra of SA-3 exhibited characteristics IR absorption bands
at 2917 cm–1, 2848 cm–1 (due to CH stretching) and carbonyl group at 1735 (due to
C⩵O stretch). Its þ ve ion ESI mass spectra showed no molecular ion peak, which indi-
cated saturated nature of molecule. The generation of the prominent ion peaks at m/z
423 [C29H59O]þ signaled the presence of steryloxy group. Signals at 395 [C27H55O]þ,
367 [C25H51O]þ, 283 [C19H39O]þ, 255 [C17H35O]þ, 227 [C15H31O]þ suggested the pres-
ence straight chain saturated steryl moiety (Supplementary Material Figure S1). The
1H-NMR spectra of compound SA-3 displayed a 6 proton triplet at d 0.85 (J ¼ 6.9 and
6.9 Hz) assigned to two terminal methyl groups, one triplet at d 1.61 (J ¼ 6.9 Hz)
assigned to two hydrogen protons present on H-29 unit, one triplet at d 2.32 (J ¼ 6.9
and 6.9 Hz) assigned to two hydrogen protons present on H-31 unit and remaining
protons appeared as broad multiplet in the range of d 1.16–1.25, assigned to remain-
ing 55 methyl units. The 13C-NMR spectral data of SA-3 exhibited important signals for
two methyl carbons at d 14.21 (C-1 and C-59), signal at d 29.26 for (C-29), signal at
32.02 for (C-31 as signal at 164 is assigned to carbonyl carbon) and remaining 55 car-
1
bon signals at d 76. The IR data, mass fragmentation pattern, H- and 13C-NMR chem-
ical shifts of the fatty acid ester were compared with related compounds viz steryl
alcohol and ester group. On the basis of spectral data analysis and acid hydrolysis,
chemical identification of compound SA-3 has been formulated as nonacosyl triaconta-
noate. This is a new ester reported for the first time in S. alata species.
2.1.2. 8-O-b-D-Glucopyranosyl-(2-acetyl)-1,3-dihydroxy-5-methoxy-xanthone (SA-9)
SA-9 (Figure 2) was obtained as yellow amorphous powder from elution of the col-
umn with chloroform:petroleum ether (8:2), recrystallised from methanol, 88 mg

4
S. BAJAJ ET AL.
Figure 2. Structure of 8-O-b-D-glucopyranosyl-(2-acetyl)-1, 3, dihydroxy-5-methoxy-xanthone.
(0.073%) and having melting point 121 ꢀC. FTIR: (KBr, ꢀmax, cmꢁ1): 3394 (OH), 2919,
1
2861 (CH), 1736 (C⩵O), 1564, 1601 (C⩵C); H-NMR (DMSO-d6, 300 MHz) (in d): 2.31
0
(s, 3H, C⩵ O CH3), 3.79 (s, 3H, OCH3), 3.78 (H50), 3.82 (H40), 3.95 (CH3 ), 4.02 (H20),
4.64(s, 1H, H2), 6.68 (s, 1H, H4), 7.04 (d, 1H, J ¼ 4.5, H10), 7.42 (d, 1H, H7, J ¼ 3.3),
7.48–7.49 (d, 1H, J ¼ 3.3, H7), 8.6 (OH) and 13C-NMR (DMSO-d6, 500 MHz): 76.60–77.44
(C10, C20, C30, C40, C50, C13), 24 (C60), 105–109 (C2, C4, C7, C6, C9, C10), 155–160 (C1,
C3, C11, C5, C12, C8), 165 (C60), 180 (C⩵O); LC-ESI-MS m/z (Relative intensity): 478
(Mþ), 477 (M – 1), 257 (Fragmentation pattern given in Supplementary Material
Figure S2).
The compound SA-9 identified as 8-O-b-D-glucopyranosyl-(2-acetyl)-1,3-dihydroxy-5-
methoxy-xanthone was obtained as yellow amorphous powder. It gave positive tests
for carbohydrate. Its IR spectrum displayed characteristic absorption band for hydroxyl
groups at 3394 cm–1 (OH), 2919 cm–1, 2861 cm–1 for CH stretching, 1736 cm–1 for C⩵
O stretching and 1564, 1601 and 1146 cm–1 for C⩵C stretching. Its þ ve ion FAB mass
spectra showed molecular ion peak at m/z 478 [C22H22O12]þ corresponds to M þ 1,
and 477 corresponds to M – 1. The generation of the prominent ion peaks at m/z 299
[C15H9O7]þ, 257 [C14H9O5]þ showed the presence of xanthone nucleus. Fragment ion
peak at 179 [C7H14 O5]þ suggested the presence of glucopyranosyl moiety. The peak
at 165 [C6H11O5]þ and other fragmented peaks suggested the presence of gucopyra-
nosyl substituted xanthone (Supplementary Material Figure S2).
1
The H-NMR spectra of compound SA-9 displayed a singlet at d 2.31 assigned to
three protons of methyl group (H-70). Another singlet at d 3.79 was assigned to proton
of methoxy protons, hydroxy protons showed a broad signal at d 8.6. The aromatic
protons displayed their signals from d 6.68 to 7.49. The protons of glucose moiety
showed their signals at d 3.78, 3.82, 3.95, 4.02 and 7.04 glucopyranosyl protons. The
13C-NMR spectral data of SA-9 exhibited important signals for carbonyl group at d 165
and 180 for carbonyl of xanthone and ester group, respectively. Remaining carbons
1
signaled between d 76.6 to 77.44, 105 to 109 and 155 to 160. The H- and 13C-NMR
chemical shifts of the fatty acid ester were compared with related compounds viz
steryl alcohol and ester group. On the basis of spectral data analysis and acid hydroly-
sis, chemical identification of compound SA-9 has been formulated as 8-O-b-D-gluco-
pyranosyl-(2-acetyl)-1,3-dihydroxy-5-methoxy-xanthone. This is a new ester reported
for the first time in S. alata species. The spectral characterisation of molecular structure

NATURAL PRODUCT RESEARCH
5
of isolated compounds SA-3 and SA-9 was also confirmed and supported by standard
literature (Tapondjou et al. 2003; Brahmachari et al. 2004).
Based on phytochemical tests and characterisation of spectral data, the presence of
a newer phytoisolates nonacosyl triacontanoate (SA-3) and 8-O-glucopyranosyl-(2-ace-
tyl)-1,3-dihydroxy-5-methoxy-xanthone (SA-9) was revealed in aerial parts of S. alata.
This study has enhanced the knowledge base related to phytochemical composition
of S. alata.
2.2. In vitro COX-1 and COX-2 inhibitory assay
The enzyme that catalyses the formation of prostaglandin GS2 (PGG2) from arachidonic
acid followed by its conversion into PGH2 is designated as COX, prostaglandin-endo-
peroxide synthase, or prostaglandin H synthase. There are two mammalian isozymes
encoded by different genes: the constitutive COX-1 and the inducible COX-2. Both
enzymes are the major pharmacologic targets of NSAIDs (Taketo 1998). Facts suggest
that COX-1 and COX-2 catalyse the biosynthesis of PGH2 from arachidonic acid substrate.
Inhibition of COX-1 results in some undesirable side-effects, whereas COX-2 inhibition
provides therapeutic effects in pain, inflammation, cancer, glaucoma, Alzheimer’s and
Parkinson’s disease (Blobaum and Lawrence 2007). The COX-2 is an inducible enzyme,
whereas COX-1 is constitutive that is present even in the absence of inflammatory condi-
tions. In addition to pro-inflammatory prostaglandins, COX-1 is responsible for the syn-
thesis of those prostaglandins that are necessary to maintain the integrity of gastro-
intestinal mucosa. A higher inhibition of COX-1 increases the tendency of a drug to
induce gastric ulcers and related complications (Yang et al. 2017).
The inhibitory activity of isolated compounds (SA-3 and SA-9) was evaluated
against ovine COX-1 and human recombinant COX-2 using enzyme immunoassay kit
(Supplementary Material Figure S3) and IC50 (mM) values were determined
(Supplementary Material Table S1). The property of indomethacin for nonselective
inhibition of COX-1 and COX-2 enzyme justifies its suitability as a positive control for
the present study (Takeuchi et al. 2010; Guo et al. 2013). The IC50 values of indometh-
acin for COX-1 and COX-2 were observed as 53.00 mM and 36.56 mM, respectively. The
results of the in vitro COX-1 and COX 2 inhibitory studies revealed that the isolated
compounds (SA-3 and SA-9) potentially inhibit COX-2 (IC50 ¼ 98.54–52.36 mM) over
COX-1 (120.3–79.89 mM). Whereas, isolated compound SA-3 (COX-1/COX-2 ¼ 79.89/
52.36) were found to be potent inhibitor of COX-2 than SA-9 (COX-1/COX-2 ¼ 120.3/
98.54) (Supplementary Material Figure S4). To understand the inhibitory activity, the
isolated compounds were further evaluated for their in vivo anti-inflammatory activity.
2.3. In vivo activity
2.3.1. Anti-inflammatory activity
Anti-inflammatory activity of the phytocompounds (SA-3 and SA-9) was evaluated in
Wistar albino rat by carrageenan-induced rat paw edema method (Winter et al. 1962).
Carrageenan-induced paw edema is a standard assay for acute inflammation that is
effectively employed to evaluate the phytoisolate against anti-inflammatory activity

6
S. BAJAJ ET AL.
(Crunkhorn and Meacock 1971). The biological data of the selected compounds is
shown in Supplementary Material Table S2, which clearly implies that the isolated
compounds exhibit varying degree of anti-inflammatory activity ranging from 37.27%
to 59.54%. The results were compared with standard anti-inflammatory drug such as
indomethacin (8 mg/kg) which showed effective inhibition (65.90%) at 3 h. The findings
of the present study revealed that the phytoisolates SA-3 and SA-9 show dose-
dependent anti-inflammatory activity by suppressing the rat paw edema.
It was observed that at a higher dose of 8mg/kg, SA-3 showed significant decrease
in edema volume after 3 h. Edema is produced by a sequential release of inflammatory
mediators such as histamine, serotonin, kinnins, prostaglandins and bradykinins which
leads to fluid accumulation (Sedgwick and Willoughby 1989). Edema is characteristic of
acute inflammatory response (Brattsand et al. 1982). The release of histamine or sero-
tonin occurs in the first phase (up to 1 h) and the second phase (over 1 h) is associated
with the production of bradykinins (Wallace 2002). Statistical analysis revealed that SA-3
(2, 4, 8 mg/kg) significantly inhibited paw edema at 3 h after treatment (p< 0.05). These
results support the possible role of SA-3 in inhibition of lysosomal enzyme secretion and
arachidonic acid release from membranes by inhibiting lipoxygenase cyclooxygenase,
and phospholipase A2. Such arachidonic acid inhibition by inflamed cells could reduce
endoperoxides, prostaglandins, prostacyclin and thromboxanes from the lipoxygenase
pathway as well as hydroperoxy and hydroxy eicosatetraenoic acids and leukotrienes
from the cyclooxygenase pathway (Gabor 1986). Indeed, previous studies have shown
that this compound plays a pharmacological role in inflammation (Laavola et al. 2016).
2.3.2. Ulcerogenic activity
It has been reported that NSAIDs are inadequately dissolvable in gastric acid and stay
in contact with the stomach wall for a more extended period, thus produces a highly
dangerous local concentration. This manifests in local irritation of the stomach wall
after ulceration (Kumar and Mishra 2006). Both compounds SA-3 and SA-9 possessing
in vivo anti-inflammatory activity when screened for their ulcerogenic activity, exhib-
ited significant ulcerogenic potential compared to the control treated group at a dose
of 12 and 24 mg/kg. A strong correlation between potency of NSAID’s as inhibitor of
prostaglandin synthesis and ulcerogenic activity has been observed (Boyle et al. 1982).
The resultant data (Supplementary Material Table S3 and Figure S5) revealed that
tested compounds SA-3 and SA-9 showed better GI safety profile with the ulcer score
of 2.0 0.35 and 2.44 0.56, respectively, in comparison to standard drug indometh-
acin 2.7 0.27. Further, SA-3 possesses significantly less ulcerogenic potential as com-
pared with SA-9 at a dose of 16 mg/kg. Most potent compound SA-3 showed severity
index less than the standard drug indomethacin. Hence, this compound may ascertain
to have better safety margin on gastric mucosa than indomethacin (Singh et al. 1992).
3. Experimental
3.1. General experimental procedure
The melting points of isolated compounds were uncorrected and determined on
Visual Melting Range apparatus. Ultraviolet spectra were recorded in methanol (MeOH)

NATURAL PRODUCT RESEARCH
7
on a Shimadzu UV-160A UV Visible Recording Spectrophotometer (Shimadzu Scientific
Instruments, Kyoto, Japan). IR spectra were recorded as KBr pellets on Nicolet FTIR
spectrophotometer. MS were recorded by effecting electron impact ionisation at 70 eV
1
on an ESIMS analyst QS TOF (Canada) mass spectrometer. H- and 13C-NMR spectra
were scanned on Bruker DRX-300,500 NMR (300 MHz) instrument in CDCl3 and D2O
using tetramethyl silane (TMS) as the internal standard and coupling constants (J val-
ues) are expressed in Hertz (Hz). Silica gel G (Qualigen, 60-120 mesh) was used for col-
umn chromatography. TLC was performed on plates coated with silica gel G (E.
Merck, Germany).
3.2. Plant material and extraction
The dried plant material was supplied by Almas Pharmaceutical Ltd., Uttar Pradesh
and identified by Dr. H.B Singh, Scientist F and Head, Raw Material Herbarium and
Museum, NISCAIR (National Institute of Science Communication and Information
Resources) Pusa Gate, New Delhi. The voucher specimen (NISCAIR/RHMD/2013/2185/
190) of the test drug has been deposited in the herbarium of NISCAIR for future refer-
ence. In the present study, the aerial parts were carefully collected and air dried under
shade. The air dried materials were reduced to coarse powder. The coarse powdered
material (1.2 kg) was subjected to exhaustively extraction with 95% ethanol in a
Soxhlet apparatus (50 h). The extract was concentrated in rotary evaporator to obtain
dried greenish brown colored 52.63 g (4.385%) residue.
3.3. Isolation and purification
The residue was chromatographed on a silica gel-based column chromatography. The
residue was dissolved in the minimum amount of ethanol and absorbed on column
grade silica gel (60–120 mesh, stationary phase) to obtain slurry, dried in air to free
flow and subjected to silica gel-based column loaded in petroleum ether. The column
was eluted successively using solvent system of petroleum ether and chloroform and
offered two phytoconstituents SA-3 and SA-9. The structures of the compounds were
established by spectroscopic method and by comparison with the previous reported
works. All spectroscopic data are available in Supplementary Material Figures S6–S13.
3.4. Animals
The animals (Wistar albino rats) used in the study were procured from Animal House
Center and were divided and housed in different cages at 25–28 ꢀC, under well main-
tained hygienic and environmental conditions with relative humidity of 50%–65%,
under 12-h light and dark cycles. All animals were acclimatised for a week before use.
All experimental work was conducted after receiving the approval from Institutional
Animal Ethics Committee (IAEC) via protocol no. IAEC/2015-I/Prot no. 09, 10 and IAEC/
2016-I/Prot no. 10, Delhi Institute of Pharmaceutical Sciences and Research, New Delhi.

8
S. BAJAJ ET AL.
3.5. In vitro activity
3.5.1. In vitro COX-1 and COX-2 inhibitory assay
The isolated compounds were screened for their in vitro COX-1 and COX-2 enzymatic
activity using enzyme immunoassay kit (catalog No. 560131, Cayman Chemicals Inc.,
Ann Arbor, MI, USA). Enzymatic assay was performed as per the manufacturer’s assay
instructions and literature procedure (Herrera-Salgado et al. 2005). The efficiencies of
the test compounds that causes 50% inhibition of COX-2 was calculated as IC50 from
the concentration response curve. The compounds were further tested for their in vivo
anti-inflammatory activity.
3.6. In vivo activity
3.6.1. Anti-inflammatory activity
The anti-inflammatory activity of isolated compound was evaluated on Wistar albino
rat by carrageenan-induced rat paw edema method as described earlier (Winter et al.
1962; Turner and Hebborn 1971). The animals were divided into six groups, each with
five animals. They were starved overnight with water prior to the day of experiment.
The acute hind paw edema was produced by injecting 0.1 mL of freshly prepared 1%
(wt/vol) carrageenan solution in normal saline locally into the plantar region of the
left hind paw of rats of each group. Isolated compounds were administered orally at
the dose of 2, 4 and 8 mg/kg body weight (b. wt) while the two other groups received
standard drug indomethacin (8 mg/kg, b. wt) and normal saline. The compounds with
three dose levels, indomethacin and normal saline were administered orally 1 h prior
to injection of carrageenan. The rat paw volume up to the ankle joint was measured
using Plethysmometer (Ugo-Basyl, Italy) at the interval of 3 h (0, 1, 2 and 3 h) after
injection of carrageenan and the percentage inhibition of edema in the various treated
groups was calculated using the following formula:
ꢀ
ꢁ
vs
vc
% Inhibition ¼ 1 ꢁ
ꢂ 100
where,
vs ¼ paw volume in sample treated group
vc ¼ paw volume in control group.
3.6.2. Acute ulcerogenic activity
The ulcerogenic activity of the isolated compound was performed according to previ-
ous method (Cioli et al. 1979). Each study group consisted of five Wistar albino rats.
The animals were fasted for 18 h before the administration of the test compound,
while water was given continuously. The dose quantity was made three times (6, 12,
24 mg/kg) of the administered dose for anti-inflammatory studies (2, 4, 8 mg/kg). The
control group received only normal saline. After 6 h of the drug administration the
rats were sacrificed, stomach was removed and opened around the greater curvature.
Inner lining was washed properly with distilled water followed by normal saline. The
mucosal damage was examined, and number of ulcers and severity index was

NATURAL PRODUCT RESEARCH
9
calculated on a scale of 0–3, where: 0 ¼ no lesions; 0.5 ¼ redness; 1.0 ¼ spot ulcers;
1.5 ¼ hemorrhagic streaks; 2.0 ¼ ulcers > 3 but ꢃ5; 3.0 ¼ ulcers > 5.
3.7. Statistical analysis
Experimental data are expressed as mean. Statistical difference between the treated
and control group was evaluated by one-way analysis of variance (ANOVA) followed
by Turkey’s test as a post ANOVA (Graph Pad Prism 5, San Diego, CA, USA) to deter-
mine the statistical significance. The significance level was #p < 0.05, ##p < 0.01 and
ꢄ
ꢄꢄ
ꢄꢄꢄ
###p < 0.001, when compared to control and p < 0.05, p < 0.01 and p < 0.001,
when compared to control.
4. Conclusion
In view of phytochemical tests and interpretation of spectral data, the present study
concludes the presence of phytoconstituents SA-3 and SA-9 in aerial parts of S. alata.
This is the first report of isolation of these compounds from this species. Ayurvedic
knowledge bolstered by modern science is necessary to isolate, characterise and
standardise the dynamic constituents from herbal sources for anti-inflammatory and
anti-ulcer activity.
Acknowledgement
The corresponding author is thankful to DIPSAR, AIRF JNU, IIT Delhi, India and AIMST University,
Malaysia for providing necessary facilities for successful completion of the study.
Disclosure statement
The authors declare that they have no conflict of interest.
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