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ORIGINAL PAPER
Gastroprotective effect of standardized extract of Amukkarachoornam on experimental gastric ulcer in rats
Kartik Chandra Patra • K. Jayaram Kumar •
Dheeraj Kumar Ahirwar
Received: 10 February 2012 / Accepted: 4 July 2013
� The Japanese Society of Pharmacognosy and Springer Japan 2013
Abstract Amukkara choornam ethanolic extract (ACE)
was investigated for phytochemical screening, content of
total phenolics and flavonoids, in vitro radical scavenging
activity (RSA), quantification of various antiulcer marker
compounds (i.e., eugenol, piperine, trans-caryophyllene,
and withaferine A) by a validated HPTLC method, and
evaluated for its in vivo gastroprotective ability against
ethanol (EtOH)-induced and pylorus ligation (PL)-induced
ulcer models in rats. Phytochemical screening revealed the
presence of flavonoids, saponins, phenols, bitter principles,
and steroids. Total phenolic and flavonoid content was
found to be 61.12 ± 0.72 mg GAE/g of ACE and
24.06 ± 1.07 mg RE/g of ACE, respectively; this was
found to be very high in plant extracts showing very good
antioxidant and antiulcerogenic effect. RSA of ACE
appeared significantly (p \ 0.05) lower than that of
ascorbic acid (AA), but higher than that of ranitidine
(RAN). In vivo the pretreatment of rats with RAN
(100 mg/kg) and 50, 100, and 200 mg/kg doses of ACE
significantly reduced the ulcer index in a dose-dependant
manner in both the models by blocking lipid peroxidation
and by significant increases in superoxide dismutase and
catalase activity. But rats treated with AA (200 mg/kg) did
not have any effect on the ulcer induced by EtOH or PL as
it has very good in vitro and in vivo antioxidant activity.
HPTLC analysis showed the presence of 0.198 ± 0.01 lg/
g, 0.754 ± 0.06 mg/g, 3.50 ± 0.04, and 0.854 ± 0.04 lg/
g of eugenol, piperine, trans-caryophyllene, and withafer-
ine A per gram of Amukkara choornam (AC). So the an-
tiulcerogenic activity of ACE might be due to a possible
synergistic antioxidant, supported by the holistic approach
of polyherbal formulations, i.e., systematism, multi-target
and multi-channel owing to their complex chemical con-
stituents and antihistaminic-like effects.
Keywords Amukkara choornam � Antiulcer � Antioxidant
� HPTLC � Eugenol � Piperine � trans-Caryophyllene �Withaferine A
Introduction
Plants and plant-derived products have been a part of
health care systems since ancient civilizations. In India,
texts like Charak Samhita and Sushruta Samhita document
the use of plants and polyherbal formulations for health
care in about 1,000 years b.c. [1]. According to Ayurvedic
text, a combination of substances was used to enhance the
desired action and eliminate unwanted side effects [2]. In
recent years, there has been an increased inclination
towards the use of herbal formulations owing to the trend
towards the use of natural sources and a healthy lifestyle.
Moreover, the complexity, side effects, and costly treat-
ment associated with the allopathic medicines have caused
both the health care practitioners and the majority of world
populations to turn towards alternative therapies, especially
herbal medicines [3]. Herbal medicines are used in several
disorders like diabetes, hypertension, asthma, and ulcers.
K. C. Patra (&)
SLT Institute of Pharmaceutical Sciences, Guru Ghasidas
University (Central University), Bilaspur, Chattisgarh 495009,
India
e-mail: [email protected]
K. Jayaram Kumar
Department of Pharmaceutical Sciences, Birla Institute of
Technology, Mesra, Ranchi, Jharkhand 835215, India
D. K. Ahirwar
School of Pharmacy, Chouksey Engg. College, Bilaspur 495001,
India
123
J Nat Med
DOI 10.1007/s11418-013-0792-x
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The growing use of botanicals by the public is forcing
moves to evaluate the health claims that accompany these
agents and to develop standards of quality and manufacture
[4].
Gastric ulcers are the most common gastrointestinal
disorder in clinical practice and they arise because of
various factors [5]. Even though the etiology of gastric
ulcers is still debated, it is accepted that ulcers are caused
by the to net imbalance in mucosal offensive and defensive
factors [6]. Ulcer therapy is now mainly focused on lim-
iting the deleterious effects of offensive acid secretion, but
the search for new safer drugs has rekindled the interest in
natural drugs possessing this activity. Considering the
several side effects (arrythmias, impotence, gynecomastia,
and hematopoeitic changes) of modern medicine [7], tra-
ditional drugs possessing fewer side effects should be
looked for as a better alternative for the treatment of peptic
ulcer.
Amukkara choornam (AC) is a popular polyherbal sid-
dha formulation, composed of herbs and spices, used for
gastric ulcer, spleen enlargement, leucorrhea, hiccup,
anemia, tuberculosis, and kappa diseases [8]. But there is
no scientific evidence for its antiulcer activity. The pres-
ence of ulcer protective plant materials in this formulation
has been investigated [9–15]. Documented reports suggest
that eugenol, piperine, trans-caryophyllene, and withafer-
ine A are marker components of AC having antiulcer
activity through various mechanisms [16–24]. Several
chromatographic methods have been reported for the
quantification of these bioactive principles [25–33] in
various crude drugs either as single or multicomponent
analyses. However, there is no evidence for simultaneous
estimation of these active compounds in any crude drug or
any polyherbal formulation. In the last two decades, high-
performance thin layer chromatography (HPTLC) emerged
as an efficient tool for the phytochemical evaluation of
herbal drugs [34]. Considering the therapeutic importance
of AC, we developed a simple HPTLC method for the
simultaneous quantification of eugenol, piperine, trans-
caryophyllene, and withaferine A in AC. HPTLC is the
method of choice for the analysis of these compounds
because several samples can be run simultaneously using
small quantities of mobile phase like HPLC. A mobile
phase of pH 8 and above can be employed. This facilitates
the repeated detection (scanning) of a chromatogram with
the same or different parameters [35–37]. Also the simul-
taneous assay of several components in a multicomponent
formulation is possible [38, 39].
There is evidence concerning the participation of
reactive oxygen species in the etiology and pathophysi-
ology of human diseases such as neurodegenerative dis-
orders, inflammation, viral infections, autoimmune
pathologies, and digestive system disorders (e.g.,
gastrointestinal inflammation and gastric ulcer). A great
number of spices and aromatic herbs contain chemical
compounds exhibiting antioxidant properties [40]. These
properties are attributed to a variety of active phyto-
chemicals including vitamins, carotenoids, terpenoids,
alkaloids, flavonoids, lignans, simple phenols, and phe-
nolic acids [41].
The present study thus aimed to standardize the AC
chemically for the content of antiulcer marker compo-
nents such as eugenol, piperine, trans-caryophyllene, and
withaferine A by a validated HPTLC method and to
evaluate biologically for antiulcer activity along with
effects on the antioxidant enzymes to justify its antiulcer
action.
Materials and methods
Chemicals and reagents
Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and
reference standards eugenol (purity 99.98 %, w/v), piper-
ine (purity 99.98 %, w/w), and trans-caryophyllene (purity
99.98 %, w/v) were purchased from Sigma Aldrich,
Kolkata, India. Withaferine A (purity 95 %, w/w) was
obtained from Natural Remedies Ltd., Bangalore, India.
Gallic acid, rutin, and Folin–Ciocalteu reagent were pur-
chased from Merck (Darmstadt, Germany). All other
chemicals used in the experiments were of analytical grade.
Composition of AC
AC [manufactured by SKM Siddha and Ayurvedic Medi-
cines India (P) Ltd, Erode] contains the fine powders of
Syzygium aromaticum Linn. (Myrtaceae; flower bud; 10
parts), Cinnamomum wightii Blume. (Lauraceae; Flower;
20 parts), Elettaria cardamomum Maton. (Zingiberaceae;
fruit; 40 parts), Piper nigrum Linn. (Piperaceae; Fruit; 80
parts), Piper longum Linn. (Piperaceae; fruit;160 parts),
Zingiber officinale Rosc. (Zingiberaceae; Rhizome; 320
parts), Withania somnifera Dun. (Solanaceae; Root; 640
parts), and cane sugar (1280 parts).
Preparation of extract and standard solution
Air-dried powder of AC (20 g) was extracted with 100 mL
of EtOH for 20 min with the help of a sonicator at room
temperature and concentrated under reduced pressure to
yield 7.25 % w/w (ACE). For assay in the linear working
range, the sample was diluted as necessary. Standard
solutions of eugenol, piperine, trans-caryophyllene, and
withaferine A were prepared with EtOH at concentrations
of 0.0025, 1, 0.0046, and 1 lg/lL, respectively.
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Preliminary phytochemical screening
ACE was subjected to qualitative chemical screening for
the identification of the various major classes of active
chemical constituents. Test for flavonoids: 2 mL of the
extract was filtered and 1 mL of the filtrate was mixed
with dilute NaOH; a golden yellow precipitate confirmed
the presence of flavonoids. Test for phenols: 2 mL of the
extract was mixed with 3 mL 5 % ferric chloride and
five drops of potassium ferricyanide; a dark green pre-
cipitate confirmed the presence of phenols. Test for
steroids/saponins: 1 g of the extract was mixed with
10 mL of warm distilled water; persistent frothing indi-
cated the presence of saponins. In addition, the Lieber-
mann–Burchard test was performed. To 100 mg of
extract, 2 mL of acetic anhydride was added; the mixture
was thoroughly stirred, heated for 2 min on a water bath,
and allowed to stand at room temperature. When 2 mL
of sulfuric acid was gently added to 0.7 mL of the
supernatant acetic anhydride layer, the upper layer gave
a blue to green color confirming the presence of steroidal
saponins [41].
Determination of total phenolics contents
The total phenolic content was determined by the Folin–
Ciocalteu reagent method [42]. A known concentration of
0.5 mL of ACE aqueous solution was added to 2.5 mL of
10 % Folin–Ciocalteu reagent (v/v) and 2.0 mL of 7.5 %
Na2CO3. The reaction mixture was incubated at 45 �C for
40 min, and the absorbance was measured at 765 nm with
a UV–Vis spectrophotometer (Shimadzu UV-1800, Kyoto,
Japan). Gallic acid was used as a standard and treated like
that of sample. All tests were carried out in triplicate and
the results were expressed as gallic acid equivalents
(GAE).
Determination of total flavonoid content
Total flavonoid content was determined by the method
described by Djeridane et al. [43]. Flavonoids react with
aluminum chloride to form a flavonoid–aluminum com-
plex, which shows maximum absorption at 430 nm.
Rutin solutions in the concentration of 2–20 lg/mL were
prepared. One milliliter of each strength of rutin solution
was mixed with 1 mL of 2 % aluminum chloride solu-
tion. After incubation for 15 min at room temperature,
the absorbance of the reaction mixture was measured at
430 nm. The same procedure has been followed for the
ACE for the measurement of absorbance. The amount of
flavonoid was determined by the calibration equation
method and expressed as rutin equivalents (mg/g dry
weight) and all tests were carried out in triplicate.
DPPH radical scavenging activity
The free radical scavenging activity of ACE was evaluated
using the stable radical DPPH, according to the method of
Grzegorczyk et al. [44]. For this purpose, solutions con-
taining 2, 5, 10, 20, 50, 100, 150, 200, 250, and 300 lg/mL
of ACE were prepared in methanol. One milliliter of these
solutions was added to 1 mL of a 0.1 mM methanolic
solution of DPPH and allowed to stand for 30 min at
27 �C. The absorbance of the sample was measured at
517 nm with a UV–Vis spectrophotometer (Shimadzu UV-
1800, Kyoto, Japan). DPPH radical scavenging activity
(RSA), expressed as a percentage, was calculated using the
following formula: RSA (%) = ADPPH - (Asample - Acon-
trol)/ADPPH 9 100 where ADPPH is the absorbance of DPPH
solution without sample extract, Asample is the absorbance
of sample extract mixed with DPPH solution, and Acontrol is
the absorbance of the sample extract tested without DPPH.
DPPH RSA of ACE was compared with ascorbic acid (AA)
and ranitidine (RAN) used as standards, with the same
concentration of sample.
HPTLC analysis of AC
The HPTLC system (CAMAG, Muttanz, Switzerland)
consisted of (1) a TLC scanner connected to a PC running
WinCATS software under MS DOS. (2) A Linomat IV
automatic sample applicator; CAMAG (Muttenz, Switzer-
land) using a 100-lL syringe and connected to a nitrogen
tank. (3) A TLC chamber: glass twin trough chamber
(20 9 10 9 4 cm3); CAMAG. (4) HPTLC plates:
20 9 10 cm2, 0.2 mm thickness precoated with silica gel
60 F254; E. Merck (Darmstadt, Germany). (5) Experimental
conditions: temperature 25 ± 2 �C, relative humidity
40 %, and solvent system toluene/ethyl acetate (9:3, v/v).
Calibration curves
Standard solutions of eugenol (2.5–32.12 ng/spot), piperine
(1–12 lg/spot), trans-caryophyllene (4.6–55.2 ng/spot),
and withaferine A (1–12 lg/spot) were applied in triplicate
on precoated silica gel 60 F254 HPTLC plates (E. Merck) of
uniform thickness of 0.2 mm. The plates were developed in
a solvent system of toluene/ethyl acetate (9:3, v/v) in a
CAMAG twin-trough chamber up to a distance of 7 cm.
After development, the plate was dried in air and scanned
at 260 nm using the absorbance reflectance mode by a
CAMAG Scanner 3 and WinCATS software for eugenol,
piperine, trans-caryophyllene, and withaferine A. The peak
areas were recorded. Respective calibration curves were
prepared by plotting peak area vs. concentration of euge-
nol, piperine, trans-caryophyllene, and withaferine A
applied.
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Simultaneous quantification of eugenol, piperine, trans-
caryophyllene, and withaferine A in various AC
samples
Suitably diluted sample solutions (8 lL) were applied in
triplicate on a precoated HPTLC plate with the CAMAG
Linomat IV Automatic Sample Spotter. The band length
was 5 mm and the space between two bands was 5 mm.
The plate was developed and scanned at 260 nm. The peak
areas and absorption spectra were recorded. To check the
identity of the bands, the UV absorption spectrum of each
standard was overlayed with the corresponding band in the
sample track. Overlaying the absorption spectra at the start,
middle, and end position of the band enabled one to check
the purity of the bands in the sample extract. The amount of
eugenol, piperine, trans-caryophyllene, and withaferine A
in AC sample was calculated using the respective calibra-
tion curves.
Method validation
Linearity, limits of detection (LOD), and quantification
(LOQ)
The linearity of the area under the curve for the prepared
standards was assessed by means of linear regression
regarding the amounts of each standard and the area of the
corresponding peak on the chromatogram. Linearity was
also confirmed for the ethanolic extract of AC. After
chromatographic separation, the peak areas obtained were
plotted against the extract concentrations by linear
regression. LOD and LOQ were experimentally verified by
diluting known concentrations of eugenol, piperine, trans-
caryophyllene, and withaferine A until the average
responses were approximately three or ten times the stan-
dard deviation of the responses for six replicate determi-
nations. For the determination of LOD and LOQ, different
dilutions of the standard solutions of eugenol, piperine,
trans-caryophyllene, and withaferine A were applied along
with EtOH as the blank and determined on the basis of
signal-to-noise ratio.
Accuracy
The accuracy of the method was determined by analyzing
the percentage recovery of the main constituents from the
AC extract. The samples were spiked with three different
amounts of standard compounds before extraction. The
spiked samples were extracted three times and analyzed
under the previously established optimal conditions. The
obtained average contents of the target compounds were
used as the ‘‘real values’’ to calculate the spike recoveries.
Precision
Precision of the method was checked by repeated scanning
(n = 7) of the same spot of eugenol, piperine, trans-
caryophyllene, and withaferine A seven times each. Vari-
ability of the method was studied by intra- and interday
precision.
Robustness
For the determination of the method’s robustness, chro-
matographic parameters such as mobile phase composition
and detection wavelength were varied to determine their
influence on the quantitative analysis. Inter- and intraday
variability were studied for the samples, by injecting the
same concentration of the sample on three different days
and the standard error mean was calculated.
Study of antiulcer activity
Animals
Albino rats of the Wistar strain weighing between 160 and
200 g were obtained from the Departmental Animal House,
GGU, Bilaspur. They were kept in the departmental animal
house at 26 ± 2 �C and relative humidity 44–56 %, light
and dark cycles of 10 and 14 h, respectively, for 1 week
before and during the experiments. The animals described
as fasted were deprived from food but allowed free access
to water. All the treatments (except EtOH) dissolved in
water were administered orally in a volume of 10 mL/kg in
all the experiments. These experiments were conducted
after getting approval from the Institutional Animal Ethical
Committee (IAEC) duly constituted according to CPCSEA
(Control and Prevention of Cruelty and Supervision in
Experiments on Animals) guidelines of the Government of
India.
Acute toxicity
Acute toxicity study was performed according to the
Organisation for Economic Co-operation and Development
(OECD) guideline. Different doses (50–2,000 mg/kg, p.o.)
of ACE were administered to groups of rats and observed
continuously for 1 h, then at half-hourly intervals for 4 h,
for any gross behavior changes further up to 72 h, and up to
14 days for any mortality. No mortality was observed.
Experimental procedures
The animals were divided into 13 groups each consisting
of six rats. Whereas for 5 days groups 1 (normal) and 2
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(control) received vehicle 10 mL/kg, groups 3, 4, and 5
(test) were given 50, 100, and 200 mg/kg of ACE and
groups 6 (RAN) and 7 (AA) given reference drug RAN
and AA at the dose of 100 and 200 mg/kg (highest
dose), respectively. All the doses were calculated with
respective body weights of animals and administered
orally. Afterwards, groups were subjected to induction of
ulcer by pylorus ligation (PL), except in group 1, which
served as the normal group. Groups 8 to 13 followed
same treatment protocol as followed in groups 2 to 7,
respectively, and ulcer was induced by administering
EtOH.
Study of antiulcer and antioxidant activity using PL
method
The method of Shay rat ulcer was adopted. The rats were
kept for 48 h fasting and care was taken to avoid
coprophagy. After the pretreatment period of 1 h animals
were anesthetized using pentobarbitone (35 mg/kg, i.p.),
the abdomen was opened, and PL was done without
causing any damage to its blood supply. The stomach was
replaced carefully and the abdomen wall was closed in two
layers with interrupted sutures. After 4 h of PL, stomachs
were dissected out and cut open along the greater curva-
ture. Ulcer index (UI) was calculated by adding the total
number of ulcers per stomach and the total severity of
ulcers per stomach [45].
The fundic part of the stomach was homogenized
(5 %) in ice cold 0.9 % saline with a Potter–Elvehjem
glass homogenizer for 30 s. The homogenate was then
centrifuged at 8009g for 10 min followed by centrifu-
gation of the supernatant at 12,0009g for 15 min and the
obtained homogenate was used for the estimations of
lipid peroxide (LPO), catalase, and superoxide dismutase
(SOD) [46, 47].
Study of antiulcer and antioxidant activity
using EtOH-induced ulcer methods
On the 5th day, 1 h after final dose of treatment, the gastric
ulcers were induced in rats by administering 96 % EtOH
(5 mL/kg) after overnight fasting [48] and after 1 h ani-
mals were killed by cervical dislocation and stomach was
incised along the greater curvature and examined for
ulcers. The stomach was then weighed and processed for
antioxidant marker estimations as mentioned in the previ-
ous section.
LPO was estimated by the standard method of Okh-
awa et al. [49] and expressed as nanomoles of mal-
ondialdehyde (MDA) formed per minute per milligram
of protein. SOD activity was estimated by the inhibition
of nicotinamide adenine dinucleotide (reduced)–
phenazine methosulfate–nitro blue tetrazolium reaction
system as adapted by Kakkar et al. [50] and the results
are expressed as units (U) of SOD activity per milligram
of protein. Catalase (CAT) was estimated by the method
of Aebi [51] and results are expressed as micromoles of
H2O2 consumed per minute per milligram of protein.
Statistical analysis
Statistical analysis was performed using one-way
ANOVA followed by Tukey’s multiple comparison test.
Data are expressed as mean ± standard deviation of the
mean.
Results and discussion
Herbal medicines have maintained their importance irre-
spective of the availability of modern medicines for
socioeconomic, cultural, and historical reasons. Further,
unpredictable side effects of the long-term use of com-
mercially available drugs compel one to search for a drug
possessing antioxidant and antiulcer properties. In this
context, ACE was phytochemically studied for the content
of various classes of constituents, total phenolic and fla-
vonoid content, HPTLC-assisted quantification of major
phytoconstituents, evaluated for its in vitro antioxidant
activity, and investigated for its in vivo antiulcerogenic
activity.
Preliminary phytochemical screening
The qualitative phytochemical screening of the extract
gave positive results for the presence of flavonoids, sapo-
nins, phenols, bitter principles, and steroids. The presence
of these phytochemicals may be responsible for the anti-
secretory, cytoprotective, and gastroprotective actions of
ACE, the mechanisms of which are unknown. Therefore, it
can be concluded that this formulation has great potential
to be used as a gastroprotective drug. Work is in progress to
isolate and purify the active principle responsible for the
gastroprotective activity.
Total phenolic content
Total phenolic content by Folin–Ciocalteu’s assay reveals
high total phenolic content (61.12 ± 0.72 mg GAE/g of
ACE) which is much higher than the phenolic content of
various plant extracts showing very good antiulcerogenic
effect. The higher amount of total phenolic content detec-
ted in ACE may be responsible for this extract’s potential
as an antioxidant.
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Total flavonoids content
The amount of total flavonoids assessed in ACE was found
to be 24.06 ± 1.07 mg/g of ACE; this content appears
higher than that of the flavonoids content detected in var-
ious citrus fruits having potential antioxidant properties
[52].
DPPH radical scavenging activity
Free radical scavenging activity of ACE was tested using
the DPPH radical scavenging assay and compared with AA
and RAN used as standards. From the analysis, we con-
cluded that the RSA of AA, ACE, and RAN against DPPH
radicals increased in a dose-dependant manner and
respectively reached maximum inhibitions of 85.30 ±
4.10, 71.12 ± 3.10, and 53.36 ± 3.20 % for the same
concentration (300 lg/mL) as shown in Fig. 1. The EC50
values calculated from the graph show that the RSA of
ACE was significantly (p \ 0.05) lower than that of AA,
but higher than that of RAN.
HPTLC analysis
In the present study, we quantified four marker compounds,
viz. eugenol, piperine, trans-caryophyllene, and withafer-
ine A, in AC by a TLC densitometric method using silica
gel HPTLC. The developed method was validated as per
the International Conference on Harmonisation (ICH)
guidelines (Table 1, 2, 3). Mobile phase consisting of tol-
uene/ethyl acetate (9:3, v/v) gave a sharp and well-defined
peak at Rf values 0.73, 0.28, 0.33, and 0.39 for eugenol,
piperine, trans-caryophyllene, and withaferine A. Well-
defined spots were obtained when the chamber was satu-
rated with the mobile phase for 15 min at room tempera-
ture. The chromatogram also shows many other peaks apart
from the four standards studied (Fig. 2).
The HPTLC method was validated by defining the lin-
earity, LOQ, LOD, precision, accuracy, and robustness. A
good linearity was achieved in the concentration ranges of
2.5–32.12 ng/spot for eugenol, 1–12 lg/spot for piperine,
4.6–55.2 ng/spot for trans-caryophyllene, and 1–12 lg/
spot for withaferine A. The correlation coefficients for the
references shown in Table 1 confirm the linearity of the
method. Instrumental precision was checked by repeated
scanning of the same spot of eugenol (25 ng), piperine
(10 lg), trans-caryophyllene (46 ng), and withaferine A
(10 lg) seven times each. The repeatability of sample
application and measurement of peak area were expressed
in terms of coefficient of variance (%CV) and found to be
0.19 and 0.11 for eugenol, 0.23 and 0.13 for piperine, 0.18
and 0.14 for trans-caryophyllene, 0.21and 0.16 for with-
aferine A, respectively, as shown in Table 1. Standards of
eugenol (25 ng/spot), piperine (10 lg/spot), trans-caryo-
phyllene (46 ng/spot), and withaferine A (10 lg/spot) wereFig. 1 Radical scavenging activity levels of ACE against DPPH
radical
Table 1 Method validation parameters for the quantification of eugenol, piperine, trans-caryophyllene, and withaferine A
Parameters Eugenol Piperine trans-Caryophyllene Withaferine A
Precision (% CV)
Repeatability of application (n = 7) 0.19 0.23 0.18 0.21
Repeatability of measurement (n = 7) 0.11 0.13 0.14 0.16
Rf value 0.73 ± 0.02 0.28 ± 0.01 0.33 ± 0.01 0.19 ± 0.01
Conc. range 2.5–32.12 ng/spot 1–12 lg/spot 4.6–55.20 ng/spot 1–12 lg/spot
Linearity (correlation coefficient) 0.996 ± 0.04 0.998 ± 0.04 0.999 ± 0.03 0.999 ± 0.03
Limit of detection 0.12 ng 0.01 lg 0.10 ng 0.02 lg
Limit of quantification 0.76 ng 0.06 lg 0.30 ng 0.12 lg
Recovery (%) 97.64 ± 0.95 95.43 ± 1.07 96.57 ± 0.80 94.73 ± 0.97
Robustness Robust Robust Robust Robust
The Rf values are the mean of 5 replicates ±SD (standard deviation)
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spotted both at intraday (spotting each concentration five
times within 24 h) and interday (spotting each concentra-
tion four times during 5-day intervals separated by at least
24 h) intervals to check the precision. The results are
shown in Table 2. The results are expressed as % relative
standard (% RSD) and standard error (SE) that indicated
high precision.
Serial dilutions of eugenol, piperine, trans-caryophyl-
lene, and withaferine A were analyzed by TLC. The LOD
and LOQ were obtained with the signal-to-noise ratio of 3
and 10. LOD represents the lowest concentrations of
eugenol, piperine, trans-caryophyllene, and withaferine A
that can be detected, whereas the LOQ represents the
lowest concentrations of eugenol, piperine, trans-caryo-
phyllene, and withaferine A that can be determined with
acceptable precision and accuracy. The LOD and LOQ are
shown in Table 1. This indicated that the new method
exhibited a good sensitivity for the quantification of
eugenol, piperine, trans-caryophyllene, and withaferine A
from AC. In order to obtain more accurate regression, the
lower limit of linearity was adjusted to be higher than the
LOQ. The concentrations of eugenol, piperine, trans-
caryophyllene, and withaferine A in sample solutions were
within the range of linearity.
The recovery was used to evaluate the accuracy of the
method. The percentage recovery as well as average per-
centage recovery was calculated. Recovery studies were
done in triplicate on the AC extract by accurately spiking
with the highest concentration of reference solution from
linearity data just prior to the extraction. The percentage
recovery for eugenol, piperine, trans-caryophyllene, and
withaferine A are shown in Table 1. The high recovery
values (more than 94 % for all standards) indicate a satis-
factory accuracy of the proposed method. Likewise, the
accuracy was independent of both the compound concen-
tration and the chemical structure.
Finally, the robustness of the method was also assessed.
Minor modifications of the initial mobile phase gradient
(from 27 to 38 % solvent B instead of 33 %) and detection
wavelength (±5 nm) had no effect on the peak resolution
and detection of the compounds. Hence the developed
method is robust.
The proposed method has been successfully applied to
the analysis of AC. The sample was extracted as described
above and analyzed by TLC. The content of each com-
pound was determined by the corresponding regression
equation and the results are summarized in Table 4. The
results indicated that all four compounds were detected in
the samples, the most abundant compound being piperine.
Therefore, this HPTLC method can be regarded as
selective, accurate, precise, and robust. The method is very
adaptable because of the precision and repeatability for the
compound herbal formulations like AC, which is a major
finding. For the first time, a simple, accurate, and rapid
TLC method was developed for the simultaneous
Table 2 Intra- and interday precision of HPTLC method (n = 6)
Marker compound Amount Intra-day precision Inter-day precision
SD of areas RSD (%) SE SD of areas RSD (%) SE
Eugenol 25 ng/spot 1.60 0.11 0.18 1.91 0.08 0.24
Piperine 10 lg/spot 1.54 0.09 0.32 1.23 0.13 0.16
trans-Caryophyllene 46 ng/spot 1.74 0.12 0.22 1.75 0.07 0.17
Withaferine A 10 lg/spot 1.46 0.06 0.19 1.46 0.05 0.22
Fig. 2 Chromatogram of Amukkara choornam toluene extract at
260 nm, showing the following constituents: 5 piperine, 6 trans-
caryophyllene, 7 withaferine A, 10 eugenol
Table 3 Marker compounds quantified from Amukkara choornam using HPTLC method
Sample Eugenol (lg/g) Piperine (mg/g) trans-Caryophyllene (lg/g) Withaferine A (lg/g)
ACE 0.198 ± 0.01 0.754 ± 0.06 3.50 ± 0.04 0.854 ± 0.04
All values are expressed as mean of five replicates ±S.D (Standard Deviation)
J Nat Med
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determination of four major compounds from AC. It was
found that AC has a unique TLC chromatogram, thus
making this method useful for maintaining the quality and
batch-to-batch consistency of this important siddha prepa-
ration (Amukkara choornam) as well as in plants containing
these active compounds.
Study of antiulcer and antioxidant activity
Plant extracts containing a wide variety of antioxidants
such us phenolic and flavonoid compounds are some of the
most attractive sources of new drugs and have been shown
to produce promising results in the treatment of gastric
ulcers [53]. In the present study steps were taken for the
determination of total phenolics and antioxidant status of
ACE by DPPH scavenging activity. The result shows that
the high amount of total phenolics is responsible for the
excellent DPPH radical scavenging activity. So in this
connection an antioxidant (AA) and a routine standard
(RAN) were taken as standards for the present study. ACE
at dosages of 50, 100, and 200 mg/kg body weight, twice a
day for 5 days prevented the formation of acute gastric
ulcers in a dose-related manner. The oral administration of
ACE at 50–200 mg/kg in pylorus ligature decreased the
index of gastric lesion by 16.60 ± 1.01–8.60 ± 0.84,
respectively (23.32–60.27 % protection) in comparison to
control 21.64 ± 1.16. Studies suggest that the EtOH
damage to the gastrointestinal mucosa starts with micro-
vascular injury, namely disruption of the vascular perme-
ability, edema formation, and epithelial lifting [54].
Administration of ACE 1 h before the induction of gastric
lesions by EtOH showed significant activity, and decreased
the total UI by 13.30 ± 1.30–3.80 ± 0.50, respectively
(23.32–60.27 % protection) (Fig. 3). Results for ACE are
comparable to RAN at the dose of 100 mg/kg. Whereas
pretreatment with ascorbic acid (200 mg/kg) had no effect
on EtOH-induced ulcer lesion and expressed an UI near to
that of the control group, our results are in agreement with
those described by Galati et al. [55] and Alimia et al. [56].
So the AA-treated group was considered for the compari-
son of antioxidant enzyme status.
The imbalance of aggressive (gastric juice, pepsin) and
protective factors (mucosal blood flow, bicarbonate
secretion, the secretion of mucosa, integrality of the cel-
lular membrane, cell regeneration, prostaglandin and other
hormones) is considered the major mechanism leading to
ulcer formation. In general antiulcer drugs inhibit acid
secretion, protect the mucosa, and inhibit Helicobacter
pylori. We designed two different experimental models to
investigate the effect and mechanism of ACE on gastric
ulcer formation, namely EtOH-induced gastric ulcer and
PL-induced gastric ulcer. Alcohol can cause the lesion of
gastric mucosa, reinforcement of aggressive factors, and
weakens the protective factors, leading to ulcer formation.
PL can lead to the accumulation of gastric juice in the
stomach, damaging the balance of aggressive and protec-
tive factors and, therefore, leading to ulcer formation. Our
present study results clearly demonstrate that ACE has a
good preventive and therapeutic action on gastric ulcers.
ACE dose-dependently protected against the gross dam-
aging action of EtOH and PL on gastric mucosa of animals.
The PL-accumulated secretions and the related ulcers
confirm gastric acid output to be the root cause of gastric
ulcers [57]. The treatment with ACE was found to inhibit
the PL-accumulated secretions.
Table 4 Effect of ACE on the antioxidant parameters in the stomach of pylorus ligated rats
Normal Control 50 mg 100 mg 200 mg RAN AA
CAT 7.95 ± 0.25 4.87 ± 0.32 5.76 ± 0.27ns 6.45 ± 0.41* 7.1 ± 0.38** 7.54 ± 0.45*** 2.85 ± 0.23
SOD 6.12 ± 0.15 2.23 ± 0.37 3.78 ± 0.26ns 4.48 ± 0.52** 5.56 ± 0.43*** 5.75 ± 0.62*** 1.56 ± 0.12
LPO 3.56 ± 0.28 11.2 ± 0.34 10.12 ± 0.48ns 9.5 ± 0.53* 4.12 ± 0.16*** 3.81 ± 0.22*** 7.89 ± 0.26
All values are expressed in mean ± SEM (n = 6)ns P [ 0.05, * P \ 0.05, ** P \ 0.01, *** P \ 0.001
Fig. 3 Effect of ACE on pylorous ligated (PL)-induced and ethanol
(EtOH)-induced ulcers
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Studies have shown alterations in the antioxidant status
following ulceration, indicating that free radicals seem to
be associated with the PL-induced [58] and EtOH-induced
[59] ulceration in rats. In the present study, administration
of ACE at the doses of 50, 100, and 200 mg/kg in PL was
found to decrease lipid peroxidation and increase SOD and
catalase as compared to the control group, thus leading to
oxidative stress (Table 4). EtOH administration was found
to decrease lipid peroxidation and increase SOD and cat-
alase, as compared to the control group (Table 5).
Results in the present study indicate similar alterations
in the antioxidant status after PL- and EtOH-induced
ulcers. Preventive antioxidants, such as SOD and catalase
enzymes, are the first line of defense against reactive
oxygen species. Adminstartion of ACE resulted in a sig-
nificant increase in the SOD and catalase levels as com-
pared to the control animals, which suggests its efficacy in
preventing free radical-induced damage. Lipid peroxida-
tion is a free radical-mediated process, which has been
implicated in a variety of disease states. It involves the
formation and propagation of lipid radicals, the uptake of
oxygen, and rearrangement of double bonds in unsaturated
lipids which eventually results in destruction of membrane
lipids. Biological membranes are often rich in unsaturated
fatty acids and bathed in oxygen-rich metal-containing
fluid. Therefore, it is not surprising that membrane lipids
are susceptible to peroxidative attack [60]. The study has
revealed a significant decrease in lipid peroxidation by
ACE in both the experimental models, which suggests its
protective effect. The present study shows that AA has a
great antioxidant activity in vitro but its in vivo effect
appears weak. On the contrary RAN has a weak antioxidant
effect in vitro and a large antiulcerogenic activity in vivo.
But ACE has an intermediate antioxidant activity in vitro
and a potent antiulcerogenic activity in vivo. This can be
rationalized by the following facts. Various mechanisms
are involved in the antiulcer activity of herbal extracts such
as inhibiting gastric acid secretion or stimulating the
mucosal defense mechanism by increasing the mucus
production and protecting the surface epithelial cells, or
interfering with the PG synthesis [61]. AC is a polyherbal
formulation composed of herbs and spices as mentioned
earlier. A literature survey reveals that various components
in this formulation act as antiulcer agents by various
mechanism: for example, Withania somnifera acts as an
antiulcer agent via its antistressor activity [62], Cinnamo-
mum wightii acts via its urease activity [63], Elettaria
cardamomum significantly inhibited gastric lesions induced
by EtOH and aspirin but not those induced by PL [64],
Zingiber officinale acts by acid and pepsin secretory inhi-
bition [65], and Syzygium aromaticum act by stimulating
the synthesis of mucus, an important gastroprotective fac-
tor [66]. Further HPTLC analysis reveals that there is a
high content of eugenol, piperine, and trans-caryophyllene
in the formulation. These active constituents act by
reducing the volume of gastric juice, gastric acidity, and
pepsin activity and by stimulating mucus secretion [67–
70]. Phytochemical screening reveals that flavonoids, sap-
onins, phenols, bitter principles, and steroids are also
present. The possible mechanisms involved for these con-
stituents are radical scavenging activity and the antihista-
minic effect of flavonoids; preventing the formation of
ulceration of the gastric mucosa by maintaining the
integrity of mucus membranes for saponins and volatile oil
[71]; and antioxidant potential, promoting tissue repair,
anti-Helicobacter pylori effects of tannins [72]. In the
present study ranitidine was used as a positive antiulcero-
genic standard having the ability to block the H2 receptor
and preventing the stomach from producing excess acid.
On the basis of the aforementioned possible mechanisms
attributed to various plant ingredients and phytoconstitu-
ents in comparison with ranitidine’s antiulcerogenic pro-
priety, we suggest that ACE exhibited a powerful
antiulcerogenic effect through the possible synergistic
effect supported by the holistic approach using polyherbal
formulations, i.e., systematism, multi-target, and multi-
channel, owing to their complex chemical constituents and
antihistaminic-like mechanisms.
Conclusion
The present study clearly shows scientific evidence sup-
porting the use of AC as a potential antiulcer agent by
traditional siddha practitioners. Phytochemical screening,
total polyphenol and flavonoid estimation, in vitro antiox-
idant study, and in vivo antiulcer study reveal that the
formulation has antioxidant and antiulcer properties. An
Table 5 Effect of ACE on the antioxidant parameters in the stomach of ethanol-treated rats
Normal Control 50 mg 100 mg 200 mg RAN AA
CAT 7.95 ± 0.25 5.02 ± 0.44 6.58 ± 0.18* 6.97 ± 0.52** 7.32 ± 0.23*** 7.62 ± 0.35*** 2.76 ± 0.31
SOD 6.12 ± 0.15 2.43 ± 0.46 4.32 ± 0.33* 5.08 ± 0.74** 5.25 ± 0.35*** 5.86 ± 0.29*** 1.59 ± 0.14
LPO 3.56 ± 0.28 6.8 ± 0.39 4.85 ± 0.18ns 3.92 ± 0.68** 3.73 ± 0.41*** 3.47 ± 0.60*** 7.56 ± 0.54
All values are expressed in mean ± SEM (n = 6)ns P [ 0.05, * P \ 0.05, ** P \ 0.01, *** P \ 0.001
J Nat Med
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HPTLC method was developed for the quantification of
antiulcer marker compounds such as eugenol, piperine,
trans-caryophyllene, and withaferine A (Table 5). The high
content of phenolics is responsible for the moderate anti-
oxidant activity in vitro and potent antiulcerogenic activity
in vivo. Therefore, we conclude that the antiulcerogenic
activity of ACE involves antioxidant/ranitidine-like path-
way(s); however, other mechanisms cannot be excluded.
The present study therefore supports the use of AC by
traditional siddha practitioners for the treatment of ulcer
with holistic approaches using polyherbal formulations,
i.e., systematism, multi-target, and multi-channel, owing to
their complex chemical constituents.
Acknowledgments The authors are grateful to the Department of
Pharmaceutical Sciences BIT, Mesra, Ranchi, India for providing the
necessary facilities to carry out this research.
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