Pharmaceutical Biology
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Page 1 of 34
Evaluation of an ethnomedicinal formulation containing Semecarpus kurzii and
Hernandia peltata used for the management of inflammation
Sonali Das2, Hemanta Mukherjee1, SK Milan Ahmed3, Pallab K. Haldar3, Asit B. Mandal2,
Ambikesh Mahapatra4, Pulok K Mukherjee3, Sekhar Chakraborty1 and Debprasad
Chattopadhyay1*,
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ICMR Virus Unit, I.D. & B.G. Hospital, GB 4, 1ST Floor, 57, Dr. Suresh C Banerjee Road,
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Beliaghata, Kolkata 700010, India
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Directorate of Seed Research, Kushmaur, P.O.Kaithauli, Maunath Bhanjan 275101, U.P.
India
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Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India
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Department of Chemistry, Jadavpur University, Kolkata, India
Dr. D. Chattopadhyay M.Sc., Ph.D.
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Corresponding Author*
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Fellow, British Soc Antimicrob Chemother (England)
Assistant Director (Scientist)
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ICMR Virus Unit Calcutta
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ID & BG Hospital, GB 4, First Floor
57 Dr Suresh C Banerjee Road
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Pharmaceutical Biology
Beliaghata, Kolkata 700 010, India
Tel: 0091 33 2353 7425/7424 [Telefax]; 9433331293 (Mob)
E. Mail: debprasadc@yahoo.co.in; debprasadc@gmail.com
Keywords: Ethnomedicines, Antiinflammatory activity, Analgesic activity, Traditional use.
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Pharmaceutical Biology
Abstract
Context: Scientific validation of an ethnomedicinal formulation containing Semecarpus kurzii
leaves (SKL) and Hernandia peltata stem-bark (HPB), traditionally used in ailments related
to inflammation, pain, fever and skin problems.
Objective: To validate in vivo and in vitro analgesic and antiinflammatory activities of
methanol extract (ME) of SKL, HPB and their combination.
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Materials and methods: The analgesic activity was tested by acetic acid induced writhing
reflex and tail flick methods in Swiss albino mice, while the anti-inflammatory activity was
studied by carrageenan and dextran induced paw edema, and cotton pellet-induced granuloma
in Wistar rats at doses of 0-500 mg/kg body weight. The in vivo vascular permeability, in
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vitro membrane stabilizing activity in human RBC, and denaturation of egg protein were
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examined to know the possible mode of action.
Results: Significant (p<0.01) analgesic and antiinflammatory activity was observed in
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combination of SKL and HPB extracts at 250mg/kg each. Moreover, the SKL extract alone
significantly inhibit acetic acid-induced vascular permeability (64.4%) at 500mg/kg, while in
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combination (250mg/kg each) the inhibition was 69.49% (P<0.01). Furthermore, SKL in
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combination with HPB (at 0.25 mg/ml each) could prevent RBC haemolysis (61.91%) and
inhibition of protein denaturation (76.52%) like indomethacin.
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Discussion and Conclusion: The SKL and HPB extracts alone (500mg/kg) and in
combination (250mg/kg each) had significant analgesic and antiinflammatory activity,
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probably by inhibiting the release of certain inflammatory mediators and membrane
stabilization due to flavonoids, triterpene and related phytochemicals present in the extracts.
Thus, this combination may provide the scientific rationale of its folk use.
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Introduction
The use of traditional medicines and medicinal plants for primary health care has been widely
observed in most developing countries (UNESCO, 1996), including India. India has been
blessed with four riches biodiversity zones, and one such richest tropical biodiversity zone is
the Andaman & Nicobar Island, situated about 1200 km away from the mainland India. The
tropical rain forest of Andaman & Nicobar Islands harbour about 2500 angiospermic plants,
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of which 52 are used as medicaments by its aboriginal populations viz. Great Andamanese,
Onge, Jarawas and Sentinels of Andaman group of Islands, and Nicobarese and Shompen of
the Nicobar group of Islands. These tribes used diverse herbal formulations containing
several endemic or extra-Indian plant species as time tested remedies (Dagar & Dagar, 1991)
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for ailments ranging from cancer to common cold. Furthermore, many plant species of these
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islands possess unique bioactive compounds due to congenial environment which bear scope
for new drug development.
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An ethnomedicinal formulation containing Semecarpus kurzii leaf (SKL) and Hernandia
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peltata stem bark (HPB) at 1:1 ratio was used by Onge, Nicobarese and Shompen tribes of
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Andaman and Nicobar Islands for fever, pain, and skin ailments (Mandal et al., 2000). S.
kurzii (Anacardiaceae), locally known as Bara Bhilawa, is an endemic species to South
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Andaman Islands (Mandal et al., 2000), whose leaves are used for treating skin ailments and
pain (Bhargava, 1983; Chakraborty & Vasudeva Rao, 1988; Dagar & Dagar, 1991); resin for
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painful wound, fever, allergy, and helminthic infections (Das et al., 2006). On the other hand,
H. peltata (Hernandiaceae) locally known as jack box is a littoral tree of the seashores, used
to treat wounds, tumour, headache, epilepsy and fits (Bhargava, 1983; Chakraborty &
Vasudeva Rao, 1988; Dagar & Dagar, 1991). Until now there were no pharmacological
studies on these ethnomedicinal remedy of Andaman and Nicobar Islands, either alone or in
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Pharmaceutical Biology
formulation. Thus, the present study was undertaken to evaluate the in vivo analgesic and
antiinflammatory efficacies and possible mode of action of these two plants alone and in
combination (1:1), as used by the ethnic community.
Materials and methods
Plant materials, preparation of extract(s) and traditional formulation
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The SKL and HPB were collected from rain forest of South Andaman and Great Nicobar
Islands, in August, December and April of 2007- 2008 to rule out the seasonal variation. The
plants were identified and authenticated by Dr Sreekumar, Botanical Survey of India (BSI),
Port Blair, and the voucher specimens (No. ABM 071BSI/PB: 2008 and ABM 072BSI/PB:
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2008) were deposited in the BSI, Port Blair. The plant parts were shade dried, separately
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powdered, passed through 40-mesh sieve and stored for future use. The powdered materials
(200 g) of respective plant parts were extracted with 95% methanol for 72 h at room
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temperature (Arunachalam et al., 2002). The whole extract was separately collected,
repeatedly filtered (Whatman No. 1 filter paper), and solvent evaporated to dryness under
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reduced pressure in an Eyela Rotary Evaporator (Japan) at 40-45°C. The concentrated
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extracts of SKL (yield 8.9 ± 0.13%) and HPB (9.15 ± 0.11%) were stored in a desiccator for
further study. A weighted amount of the dried extract(s) were dissolved in 2% (w/v) Tween-
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80 and diluted in sterile distilled water; while the formulation was made by mixing the dried
extract of SKL and HPB (1:1). The formulation was dissolved in 2% (w/v) Tween-80 diluted
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in sterile distilled water for pharmacological studies, whenever required.
Standardization of the extract(s) was done by physicochemical constant, fluorescence
analysis and phytochemical tests (WHO, 1998; Arunachalam et al., 2009). Physicochemical
parameters like total ash, acid insoluble ash content, water content and the property of
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powdered drug in different solutions under UV312
nm
and visible light was determined
following WHO Guideline, 1998. Preliminary phytochemical studies are performed by
standard methods (Chhabra et al., 1984; Arunachalam et al., 2009) to know the chemical
groups present within the extracts.
HPLC instrumentation and experimental conditions
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HPLC analysis was done by the method of Kontogianni et al., 2009, using a Shimadzu liquid
chromatograph consisting of binary LC‐20AD pumps, coupled with a SPD-M20A photodiode array detector. Injection was done through a Rheodyne 7725 injector equipped with a
20 L loop, and a reverse phase C18, 250 X 4.6 mm, 5µm, Zorbax column (Agilent, USA).
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Both the column and HPLC system were kept at ambient (25°C) conditions. The mobile
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phase was 1% acetic acid in HPLC grade water: methanol (10:90) for SKL and 1% acetic
acid in HPLC grade water: methanol (35:65) for HPB with the flow rate of 1 ml/min. The
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absorbance was monitored at 210 nm with the injection volume of 20 L.
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The stock solutions were prepared by dissolving 10 mg of pure ursolic acid and chlorogenic
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acid (Sigma, USA) separately in 10 ml of methanol. All the samples were filtered through
0.45 m membrane, and 20 l of this sample solution was injected into the HPLC system for
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analysis. Identification of Ursolic acid and Chlorogenic acid was based on their retention time
and comparison of their UV spectra. Quantitative analysis was achieved by five point
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calibration curves for Ursolic acid and Chlorogenic acid, at concentrations of 5–1000 g/ml
and from the equation Y= 6987.2 X + 60941 for ursolic acid and Y= 6977.3 + 60943 for
Chlorogenic acid respectively, where Y represents the area of the extract and X represents the
concentration of either Ursolic acid or Chlorogenic acid. The regression coefficient values
were 0.9985 for Ursolic acid and 0.9945 for Chlorogenic acid. Real samples were diluted
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Pharmaceutical Biology
accordingly to fit the dynamic linear range of the regression line, when necessary. All
measurements were performed in triplicates.
Animals
Healthy inbreed Swiss albino male mice (20 ± 2 g) and adult Wistar male rats (150-180 g)
were housed at 23 ± 4° C, of relative humidity 60-70% in the animal house facility of the
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Pharmaceutical Technology Department, Jadavpur University, Kolkata and were fed with
standard pellet diet and water ad libitum. All the animals were acclimatized for one- week
before the experiments, and all experiments were carried out according to the Institutional
Animal Ethics
committee
07.07.2008).
(Permission
No.
JU/Pharm/007/2008; dated
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Drugs and chemicals
guidelines
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Paracetamol, morphine sulfate, diclofenac disodium, carrageenan, dextran powder and all
other analytical grade chemicals were purchased from the respective manufacturers.
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Acute toxicity study
The acute toxicity of SKL and HPB extracts alone and in combination was conducted on 13
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groups of mice (n=6) at oral (p.o.) doses of (200-2000) mg/kg body weight including a
control group. While the same in Wistar rats at 200, 500, 1000 and 2000, using 9 groups of
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animals (n=6). Simultaneously, the acute toxicity of SKL and HPB extracts was tested in
Wistar Rats at 200, 500, 1000 and 2000 mg/kg body weight orally along with a control group.
The animals were observed for toxic symptoms and death within 24h (6, 12, 18 and 24 h) and
then daily for next 14 days (OECD, 2000). The study showed neither any observable toxic
effects (agility, muscular tonus, tremors, convulsions, problem in breathing, and water or
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food intake) nor any death following treatment, and hence, the procedure was repeated up to
5.0 g/kg. On the basis of toxicity data the dose regimen for further tests were selected as 400
and 500 mg/kg of extracts alone and 250 mg/kg (each) in combination.
Analgesic activity
Acetic acid-induced writhing tests
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The writhing test was performed according to the method of Koster et al., 1959. Briefly,
Swiss albino mice of either sex were divided into seven groups of six animals each. The first
group served as control; the second group received paracetamol (50 mg/kg), while the 3rd to
6th group received SKL and HPB extract at 400 and 500 mg/kg respectively, and the last
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group was administered with a combination of SKL and HPB (250 mg/kg each) as i.p. After
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30 min of drug administration, the animals were orally fed with 1% v/v fresh acetic acid
solution (10 ml/kg), and immediately counted the numbers of writhing or stretches
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(abdominal contraction, trunk twist response and extension of hind limbs) for 10 min. A
reduction in the writhing number compared to the control was considered as evidence of
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analgesia (Pradeepa et al., 2012). The percentage inhibition of writhing was calculated as: %
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Inhibition= C-T/C X 100. Where C is the mean number of writhes produced by the control
group and T represents the mean number of writhes produced by the test groups.
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Tail flick test
Swiss albino mice of either sex weighing 20 ± 2g were divided into seven groups of five
animals each. The tail of each mouse was placed on the nichrome wire of an analgesiometer
(Techno Lab, Lucknow, India) and the time taken by the animal to withdraw (flick) its tail
from the hot wire was taken as a reaction time. The extract of SKL, and HPB at 400 and 500
mg/kg and the combination of SKL and HPB (250 mg/kg each) were injected i.p., using
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Pharmaceutical Biology
morphine sulphate (5 mg/kg) as standard drug. Analgesic activity was measured after 30 min
of administration of test and standard drug (Das et al., 2011) and the percentage inhibition
was calculated by the same formula as above.
Anti-inflammatory activity
Carrageenan-induced rat paw edema (acute model)
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Wister male rats were divided into seven groups of six animals (n=6) each. The extracts of
SKL and HPB at 400 and 500 mg/kg body weight were administered orally to the animals of
groups I to IV, while Vth group received a combination of SKL and HPB (250 mg/kg each),
60 min prior to carrageenan injection. The VIth group serves as vehicle control, and the VIIth
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group received diclofenac disodium (20 mg/kg) as drug control, for assessing comparative
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pharmacological significance. Edema was induced by sub-plantar injection of 0.05 ml of
fresh carrageenan suspension (1% w/v) in normal saline into the right hind paw of each rat
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(Winter et al., 1962). The paw volume (linear circumference) was measured at 0 h and at 1 to
5 h after carrageenan injection by a plethysmometer (Oyemitan et al., 2008). The
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antiinflammatory activity was evaluated using the ratio of the changes in paw diameter in
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treated and untreated group by the formula: Anti-inflammatory activity (%) = (1- D/ C) X
100; Where D is the change in paw diameter in treated group and C is the change in paw
diameter in untreated group.
Dextran-induced rat paw edema (sub-acute model)
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The hind paw edema in each rat was induced by sub-plantar injection of 0.1 ml of 1% dextran
solution (Das et al., 2011) in the right foot. Paw volumes were measured 30 min before and
after dextran injection. The treatment of extracts (test), control (vehicle), and standard drug
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was the same as described for carrageenan model. The percentage inhibition of edema was
calculated by the method of Arunachalam et al., 2002.
Cotton pellet induced granuloma (chronic model)
The rats were divided into seven groups of six animals each, and the animals were
anaesthetized after shaving of the fur. Sterile preweighed cotton pellets (10 mg) were
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implanted in the axilla region of each rat through a single needle incision (D’Arcy et al.,
1960). The extracts of SKL and HPB were administered orally at 400 and 500 mg/kg to the
groups’ I - IV, and a combination of SKL and HPB (250 mg/kg each) to group V. The group
VI serves as vehicle control, while group VII received diclofenac disodium (20 mg/kg), for
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consecutive seven days from the first day of implantation. On 8th day the animals were
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anaesthetized, cotton pellets were removed surgically and made free from extraneous tissues.
To obtain constant weight, the pellets were incubated at 370C for 24 h and dried at 600 C. The
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granuloma formation was measured by the increase in dry weight of the pellets (Winter &
Porter, 1957).
Acetic acid-induced vascular permeability in mice
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The vascular permeability was tested by the method of Whittle (1964). Briefly, mice were
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divided into seven groups, six per group (n=6). Group I served as vehicle control, groups II V were treated orally with 400 and 500 mg/kg of SKL and HPB extract; group VI with a
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combination of SKL and HPB (250 mg/kg each), and group VII received indomethacin (20
mg/kg). One hour after the treatment, 0.2 ml of 0.2% Evans’ blue in normal saline was
injected intravenously to each mouse through tail vein and 30 min later, each mouse was
injected i.p. with 0.2 ml of 0.6% acetic acid in normal saline. After 1 h, the animals were
sacrificed, and the abdominal cavity was exposed and washed with 5 ml normal saline to
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Pharmaceutical Biology
collect the content in a sterile test tube. The content was centrifuged to eliminate
contaminants, and the absorbance (A) of the solution was measured by a spectrophotometer
at 590 nm. The vascular permeability effects, expressed as the absorbance (A590), represented
the total amount of dye leaked into the intraperitoneal cavity.
Membrane stabilizing activity
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This was assessed by hypotonic solution-induced human erythrocyte haemolysis (Shinde et
al., 1999). Whole blood collected from a healthy volunteer (DC) in a heparinized tube was
washed thrice with isotonic buffer (154 mM NaCl in 10 mM sodium phosphate buffer, pH
7.4) for 10 min at 3000 g. The test sample consisted of RBC (0.50 ml) in 5 ml hypotonic
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solution (50 mM NaCl in 10 mM sodium phosphate buffer saline, pH 7.4) containing the
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extracts (0.2 - 1.0 mg/ml) alone and in combination, or indomethacin (0.1 mg/ml). The
control tube contain RBC (0.5 ml) mixed with hypotonic-buffered saline. The mixtures were
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incubated for 10 min at room temperature, centrifuged (3000 g for 10 min) and the
absorbance of the supernatant was measured at A540 nm. The percentage inhibition of
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haemolysis or membrane stabilization, tested in triplicate, was calculated as % Inhibition of
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haemolysis = 100 x {OD1 - OD2/ OD1}, Where OD1 is the Optical density of hypotonic buffer
saline and OD2 is the Optical density of test sample in hypotonic solution.
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Effect on in vitro protein denaturation
Test solution (1ml) containing 100-1000
g/ml of extracts, alone or in combination or
indomethacin (100 g/ml) was mixed with 1 ml of egg albumin (1 mM) and incubated at 27 ±
1° C for 15 min. The mixture was then placed at 70° C in a water bath for 10 min to induce
denaturation of albumin. After cooling, the turbidity was measured at 590 nm (Mizushima,
1966) and the average of three sets was taken. The percentage inhibition of denaturation was
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calculated relative to the control as % inhibition of denaturation = (Abs of control - Abs of
treated)/Abs of control X 100 (Ramalingam et al., 2010).
Statistical Analysis
The results were expressed as mean, mean ± S.D. and SEM (Standard Eror Mean). The
significance between the two groups was evaluated by Student’s t-test, and the significance of
difference among the various treated and control group were analyzed by one-way Anova
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followed by Dunnett’s t-test (Woodson, 1987).
Results
In this study analgesic and anti-inflammatory activity of ME of SKL and HPB and their
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combination (1:1), used as ethnomedicinal formulation, was tested by in vivo and in vitro
screening methods.
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Standardization of sample and physicochemical study of extract
The ME of plant parts collected in different session showed that the post rainy session
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samples (August) had the minimum amount of water, total ash and acid insoluble ash content,
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with maximum yield (Table 1), and thus selected for further study. Under visible and UV312
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light the colour of powdered SKL extracts was yellowish to light brown; while it was grey
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to light brown in HPB extract. Preliminary phytochemical screening of the SKL extract
revealed the presence of flavonoids, triterpene, tannins, saponins and glycosides; while HPB
extract contains alkaloid, flavonoid, steroid and tannin.
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The quantification by HPLC analysis revealed that, ursolic acid (triterpene) in the ME of
SKL was 0.39 % (w/w) while the amount of chlorogenic acid in HPB extract was 0.46 %
(w/w). The ursolic acid in SKL and chlorogenic acid in HPB extract is unknown as one of the
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main principle of the plants, hence we have quantified the leaf extract of S. kurzii for ursolic
acid and bark extract of H .peltata for chlorogenic acid as marker compound (pure Ursolic
acid and chlorogenic acid, Sigma). Specimen HPLC chromatograms of the standard ursolic
acid and ME of SKL are represented in Figure 1 (a and b), while standard chlorogenic acid
and ME of HPB are represented in Figure 1 (c and d).
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Acute toxicity
Toxicity study conducted over 14 days showed no observable toxic effects (convulsion,
ataxia, agility, dyspnoea, water or food intake, diarrhoea or diuresis) or any death, signifying
that both SKL and HPB extracts possess good safety profile. However, slightly reduced
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motor activity, ataxia and hyperventilation was observed in mice at 5 g/kg orally. The median
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lethal dose (LD50) of SKL and HPB extract(s) was found to be 3.6 g/kg and 3.2 g/kg
respectively in mice. In rats, the LD50 of SKL and HPB extract(s) was 4.0 g/kg and 3.6 g/kg
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orally, respectively. Thus, the dose regimen for further study was selected as 400 and 500
mg/kg alone, and 250 mg/kg (each extract) in combination.
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Analgesic activity:
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Effect of acetic acid induced writhing test
The results of acetic acid induced writhing test with SKL and HPB extracts (400 and 500
mg/kg), and their combination (250 mg/ml each) i.p. presented in Table 2, showed that the
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inhibition of writhing reflexes was 59.87% at 400 mg/kg and 64.70% at 500 mg/kg of SKL.
While it was 61.66% at 400 mg/kg, and 73.08% at 500 mg/kg of HPB extract (p < 0.01).
Interestingly, the combination of SKL and HPB extracts (250 mg/kg each) produced
maximum inhibition (78.04%), which was nearly similar to the paracetemol treated group
(81.95%).
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Effect of tail flick induced test
The results of tail flick test, presented in Table 2, showed that the SKL extract had reaction
time of 9.25 sec (57.24%) at 400 mg/kg and 7.67 sec (64.53%) at 500 mg/kg i.p doses. While
the reaction times in vehicle control and morphine sulfate (5 mg/kg) group was 21.63 sec and
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5.20 sec (75.95%), respectively. The HPB extracts at 400 mg/kg showed reaction time as
9.11 sec with 57.88% inhibition; while at 500 mg/kg doses it was 6.51 sec (69.90%
inhibition). Thus, significant (P < 0.01) activity was noted with both the extracts at 500
mg/kg alone and in combination of SKL and HPB (250 mg/kg each), with maximum
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inhibition of 71.70% at 6.12 sec, close to morphine (75.95%).
Antiinflammatory activity
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Effect of carrageenan induced paw edema
The results of antiinflammatory activity of SKL and HPB extracts and their combination,
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presented in Table 3, showed that SKL at 500 mg/kg exhibited 65.08% inhibition of paw
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edema; while at 400 mg/kg the inhibition was 56.94%. Here the standard drug diclofenac
disodium (20 mg/kg) produced 74.23% inhibition of edema volume after 3 h of treatment.
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The HPB extract exhibited 43.05% inhibition at 400 mg/kg and 52.20% at 500 mg/kg,
respectively. However, the combination of SKL and HPB extracts (at 250 mg/kg each)
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produced highest inhibition (72.54%) which is highly significant (P < 0.01), compared to the
drug control group.
Effect of dextran induced paw edema
In dextran-induced paw edema test the maximum inhibition (74.36 %) of edema swelling was
noted with 20 mg/kg of diclofenac disodium, followed by SKL extract at 400 mg/kg
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Pharmaceutical Biology
(55.37%) and 500 mg/kg (64.24%), respectively. However, HPB extract showed significant
(P < 0.05) inhibition (50.63%) of paw edema at 500 mg/kg dose only. Here again, the
combination of SKL and HPB extracts (250 mg/kg each) revealed highest inhibition
(72.15%). All these data are significant (P < 0.01) with respect to the control groups,
indicating the possible antiinflammatory activity of these extracts (Table 3).
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Effect of cotton pellet induced granuloma
The results presented in Figure 2 showed that SKL and HPB extracts significantly inhibited
the granuloma weight. The SKL extract produced 54.40% inhibition at 500 mg/kg (p < 0.01)
compared to diclofenac disodium (60.77%) at 20 mg/kg (p < 0.05). However, 49%
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inhibitions of granuloma weight was recorded with 500 mg/kg HPB extract, while the
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combination of SKL and HPB extracts produced 57.55% inhibition, nearly equal to
diclofenac disodium.
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Inhibition of acetic acid-induced vascular permeability in mice
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Effect of the oral administration of SKL and HPB extracts (400 and 500 mg/kg) on acetic
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acid-induced vascular permeability in mice was presented in Figure 3. Results showed that
SKL at 400 and 500 mg/kg inhibited 54.23% and 64.4% vascular permeability, compared to
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indomethacin (70.33%). While HPB extract at 400 and 500 mg/kg produced 42.37% and
52.54% inhibition of vascular permeability. Here again the highest inhibiton (69.49%) was
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recorded when both the extracts were combined. All these data are significant at 5% level (P
< 0.05).
Membrane stabilizing activity
To validate the in vivo data further we conducted the non-specific membrane stabilization
study with both the extracts alone and in combination. The results presented in Table 4,
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showed that the SKL and HPB extracts can protect the erythrocyte membrane against lysis
induced by hypotonic solution. Both extracts at 0.5 mg/ml significantly (P < 0.01) protect the
erythrocyte membrane (59.45% and 54.79% inhibition) against lysis, compared to
indomethacin (63.69% inhibition) at 0.1 mg/ml. While the combination of SKL and HPB
achieved 61.91% protection in half the concentration (0.25 mg/ml) of each extract, which is
highly significant (p < 0.01) and nearly equal to the standard drug.
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Inhibition of protein denaturation
The inhibitory effect of SKL and HPB extracts on protein denaturation, presented in Figure 4,
showed that both the extracts (200 and 400 g/ml) moderately inhibited (34.64 - 51.37%) the
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denaturation of egg albumin. However, at 800 g/ml extracts produced 65.64% and 63.80%
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inhibition of egg albumin denaturation, compared to indomethacin (86.36%) at 100 g/ml.
Here again the combination of SKL and HPB (400 µg/ml each), produced 76.52% inhibition
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(P < 0.01), similar to indomethacin (86.36%).
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Discussion
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We have studied the in vivo analgesic and antiinfammatory activity along with the vascular
permeability, in vitro membrane stabilization of RBC and inhibition of protein denaturation
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of the standardized extracts of two ethnomedicinal plants S. kurzii and H. peltata, alone and
in combination (as formulation), used by some ethnic community of Andaman and Nicobar
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Islands, India. The importance of this study lies on their ethnomedicinal use and endangered
nature of one of its component S. kurzii, which have not been validated yet. The HPLC
analysis of the ME of both the plants showed the presence of ursolic acid and chlorogenic
acid as one of the component, and both these compounds are known to have analgesic and
antiinflammatory activity (Liu, 1995; Azza et al., 2011). Nevertheless our findings for the
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Pharmaceutical Biology
first time scientifically validated this combination for analgesic and antiinflammatory
activity, which is probably due to several group of compounds including chlorogenic acid and
ursolic acid.
The analgesic activity tested by acetic acid- induced writhing model, indicated that the
numbers of writhing movements were significantly less in the mice treated with SKL and
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HPB extracts alone and in combination, comparable to untreated group. The effect of the
extracts when compared to paracetemol, suggests that both the extracts might have peripheral
analgesic effect. Furthermore, the analgesic effect tested by the tail flick test was comparable
to that of morphine treated control, suggesting central analgesic effect.
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Formation of edema by a complex array of enzyme activation, mediator release, cell
migration, extravasations of fluid, tissue breakdown and repair is one of the cardinal sign of
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inflammation (Vane & Bolting, 1995). We used the most acceptable carrageenan induced
paw edema model, as carrageenan helps to release the chemical mediators in an orderly and
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biphasic manner (Di Rosa, 1972; Sur et al., 2002), where the vasoactive histamine and 5HT
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(bradykinin and serotonin) released in the early phase and prostaglandins (kinin) in the late
phase (Heller et al., 1998), resulting in increased vascular permeability, leading to the
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accumulation of fluid in the tissues to form edema (White, 1999). On the other hand, dextranmediated inflammation was reduced probably due to antihistaminic effects of the extracts, as
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dextran cause inflammation through the release of histamine and serotonin (Ghosh et al.,
1963). Carrageenan- and dextran-induced paw edema test revealed that SKL and HPB
extracts possesses significant anti-inflammatory activity at 400 and 500 mg/kg oral dose,
while the combination of both the extract (at 250 mg/kg) showed much better activity
(p<0.01) at 3 h, comparable to diclofenac disodium. Thus, the ability of SKL and HPB
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extracts alone or in combination to reduce the edema volume, suggests that the
phytochemicals of these extracts, including ursolic acid and chlorogenic acid, may block the
release of some mediators like histamine, bradykinin, serotonin and prostaglandins.
Furthermore, the decrease in the cotton-pellet induced granuloma weight by the extracts is
due to the inhibition of proliferative phage of inflammation (Rajavel et al., 2009), as the
inflammatory response induced by the cotton pellet can modulate the release of mediators
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leading to the tissue proliferation and granuloma formation (Stoeck et al., 2004).
We use the acetic acid induced vascular permeability test, a capillary permeability assay, to
confirm the antiinflammatory potential of the tested extracts. The acetic acid cause dilation of
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blood vessels and increased permeability through the release of histamine, prostaglandins and
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leukotrienes by stimulating mast cell (Brown & Roberts, 2006). During inflammation these
mediators increase vascular permeability, while acetic acid cause an immediate sustained
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reaction over 24 h and its inhibition by the test extracts alone or in combination, suggests that
SKL and HPB may effectively suppress the exudative phase of acute inflammation.
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Furthermore, the non-specific RBC membrane stabilization test showed the protective effect
on hypotonic saline induced RBC lysis, an index of antiinflammatory activity (Oyedapo &
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Famurewa, 1995). When RBC is exposed to hypotonic medium, it undergoes haemolysis and
oxidation of haemoglobin (Augusto et al., 1982; Ferrali et al., 1992) due to excessive
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accumulation of fluid within the cell. Such injury renders the RBC more susceptible to
secondary damage through free radical induced lipid peroxidation (Ferrali et al., 1992). This
is consistent with the observation that the breakdown of bio-membranes induces free radical
generation, which in turn enhance cellular damage (Maxwell, 1995). Thus, compounds with
membrane-stabilizing property can protect cell membrane against injurious substances (Liu et
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al., 1992; Perenz et al., 1995; Shinde et al., 1999) by interfering the release of phospholipases
to trigger the formation of inflammatory mediators (Aitadafoun et al., 1996). Here the
significant (p<0.01) membrane stabilizing activity of SKL and HPB extracts alone and in
combination, suggests that the extracts might inhibit the release of chemical mediators in
inflammatory events.
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Moreover, we have studied the denaturation of proteins, as it is a well known cause of
inflammation (Mizushima, 1966), and several anti-inflammatory drugs inhibit thermally
induced protein denaturation dose dependently (Grant et al., 1970). Here, the ability of the
tested extracts to bring down thermal denaturation of protein is possibly a contributing factor
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for its anti-inflammatory activity, probably due to the presence of bioactive flavonoids and
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related compounds like chlorogenic acid and ursolic acid, as reported with other plants
(Middleton et al., 2000; Havsteen, 2002).
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The contemporary literature revealed that the Malaysian species of H. peltata Meisn, used by
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the Samoan healers, has anti-hypertensive (Singh, 1986; Dittmar, 1991), antitumor (Pernet,
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1971), piscicide (Nishino & Mitsui, 1973) and anti-arteriosclerosis (Ebel & Roth, 1987)
activity. Chemically H. peltata Meisn of Malaysia is reported to contain benzylisoquinoline
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alkaloid hebridamine (Chalandre et al., 1986) that inhibit chloroquine-resistant Plasmodium
falciparum (Angerhofer et al., 1999), and artherosclerosis (Lakshmi et al., 2009). Moreover
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the lignans like podophyllotoxins (deoxypodophyllotoxin and deoxypicropodophyllin)
isolated from H. peltata Meisn have anticancer activity (Pettit et al., 2004) while hernanol,
benzopyran, epiaschantin, deoxypodorhizone inhibit Neisseria gonorrhoeae (Pettit et al.,
2004). Interestingly another plant of the formulation, S. kurzii, is reported to contain 9hydroxy-cis-12-enoic (isoricinoleic) acid and fatty acids (Farooqui et al., 1985). However, the
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phytochemical study of this formulation revealed the presence of flavonoids, tannins,
saponins, glycosides, alkaloid and steroid, and the HPLC revealed the presence of ursolic
acid and chlorogenic acid as one of their compounds.
Conclusion
In conclusion, this study suggests that the traditional formulations of SKL and HPB in
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combination (250 mg/kg each) may offer beneficial effects in the management of
inflammatory conditions, as both the extracts significantly inhibited writhing reflexes, paw
oedema, vascular permeability, membrane-stabilization and protein denaturation, probably
due to the flavonoids like chlorogenic acid, triterpenes like ursolic acid and related
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phytophores present in both the extracts. Further studies involving the purification of the
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chemical constituents and the investigations on biochemical pathways are required to
establish the exact mechanism of its action. However, these findings endorsed the vernacular
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medicinal use of both these unique plants to treat ailment related to inflammatory conditions.
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Acknowledgements
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Financial support from Central Agricultural Research Institute is highly acknowledged.
Declaration of interest statement
There is no conflict of interest in any form between the authors.
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References
Aitadafoun M, Mounieri C, Heyman SF, Binistic C, Bon C, Godhold J. (1996). 4Alkoxybenzamides as new potent phosholipase A2 inhibitors. Biochem Pharm, 51, 737742.
Angerhofer CK, Guinaudeau H, Wongpanich V, Pezzuto JM, Cordell GA. (1999).
Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J Nat
rP
Fo
Prod, 62, 59-66.
Augusto O, Kunze KL, Montellano PR. (1982). Nphenylprotoporphyrin formation in the
haemogolobin-phenylhydrazine reaction. J Bio Chem, 257, 6231-6241.
Arunachalam G, Chattopadhyay D, Mandal AB, Sur TK, Mandal SC. (2002). Evaluation of
ee
antiinflammatory activity of Alstonia macrophylla wall ex A.DC. leaf extract.
Phytomedicine, 9, 632-635.
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Arunachalam G, Bag P, Chattopadhyay D. (2009). Phytochemical and phytotherapeutic
ev
evaluation of Mallotus peltatus (Geist.) Muell. Arg. var acuminatus and Alstonia
macrophylla Wall ex A. DC: Two ethnomedicine of Andaman Islands, India. J
ie
Pharmacog Phytother, 1, 01-13.
w
Azza EM, Yieldez B, Mahmoud K, Abdullatif M, (2011). Chlorogenic acid as potential antiinflammatory analgesic agent: an investigation of the possible role of nitrogen-based
On
radicals in rats. International Journal of Pharmacology & Toxicology Science, 1, 24-33.
Bhargava N. (1983). Ethnobotanical studies of the tribes of Andaman and Nicobar Islands,
ly
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 20 of 34
India. I. Onge. Econ Bot. 37, 110-119.
Brown JN, Roberts J. (2006). Histamine, bradykinin, and their antagonists, In: Gilman AG,
Hardman JG, Limbird LE, Goodman and Gilman’s the Pharmacological Basis of
Therapeutics, 11th ed. McGraw Hill Co: New York. 645-667.
20
URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu
Page 21 of 34
Chakraborty T, Vasudeva Rao MK. (1988). Ethnobotanical studies of the Shompen of Great
Nicobar Islands. J Econ Taxon Bot, 12(1), 43-54.
Chalandre MC, Cabalion P, Guinaudeau H. (1986). Étude des Hernandiacées. XII. Dimères
aporphine–benzylisoquinoléine originaux isolés de Hernandia peltata. Canadian J Chem,
64, 123-126.
Chhabra SC, Viso FC, Mshin EN. (1984). Phytochemical screening of Tanzanian medicinal
rP
Fo
plants. J Ethnopharmacol, 11, 157-179.
D’Arcy PD, Howard EM, Muggleton PW, Townsend SB. (1960). The antiinflammatory
action of griseofulvin in experimental animals. J Pharm Pharmacol, 12, 659-665
Dagar HS, Dagar JC. (1991). Plant folk medicines among Nicobarese of Katchal Island,
ee
India. Econ Bot, 45, 114-119.
rR
Das S, Haldar PK , Pramanik G, Panda SP, Bera S. 2011. Evaluation of Analgesic and AntiInflammatory Activity of Diospyros Cordifolia Extract, Afr J Trad Comp and Alt Med, 8
(1), 11-14.
ev
Das S, Sheeja TE, Mandal AB. (2006). Ethnomedicinal uses of certain plants from Bay
ie
Islands. Indian J Traditional Knowledge, 5(2), 207-211.
w
Dittmar A. (1991). The effectiveness of Hernandia species (Hernandiaceae) in traditional
Samoan medicine and accordining to scientific analysis. J Ethnopharmacol, 33, 243-251.
On
Di Rosa M. (1972). Biological properties of carrageenan. J Pharm Pharmacol, 24, 89-102.
Ebel S, Roth H. (1987). Lexikon der Pharmazie. Thieme, Stuttgart, 10, 704S, 217.
ly
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmaceutical Biology
Farooqi JA, Jamal S, Ahmad I. (1985). Isoricinoleic acid in Semecarpus kurzii seed oil. J
American Oil Chemists' Society, 62(12), 1702-1703.
Ferrali M, Signorni C, Ciccoli L, Comporti M. (1992). Iron release and membrane damage in
erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil.
Biochem J, 285, 295-301.
21
URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu
Pharmaceutical Biology
Ghosh MN, Banerjee RH, Mukherjee SK. (1963). Capillary permeability increasing property
of hyaluronidase in rat. Indian J Physiol Pharmacol, 7, 17-21.
Grant NH, Alburn HE, Kryzanauskas C. (1970). Stabilization of serum albumin by antiinflammatory drugs. Biochem Pharmacol, 19, 715-722.
Havsteen BH. (2002). The biochemistry and medical significance of the flavonoids.
Pharmacol Therapeut, 96(2-3), 67-202.
rP
Fo
Heller A, Koch T, Schmeck J, Acker VK. (1998). Lipid mediators in inflammatory disorders.
Drugs, 55, 487-496.
Kontogianni VG, Exarchou V, Troganis A, Gerothanassis LP. (2009). Rapid and novel
discrimination and quantification of oleanolic and ursolic acids in complex plant extracts
ee
using two-dimensional nuclear magnetic resonance spectroscopy-Comparison with HPLC
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methods. Analytica Chimica Acta, 635(2), 188–195.
Koster R, Anderson M, De Beer EJ. (1959). Acetic acid for analgesics screening. Federation
Proceed, 18, 412-417.
ev
Lakshmi V, Pandey K, Mishra SK, Srivastava S, Mishra M, Agarwal SK. (2009). An
ie
Overview of family Hernandiaceae. Records of Nat Prod, 3(1), 1-22.
w
Liu GT, Zhang TM, Wang BE, Wang YW. (1992). Protective action of seven natural
phenolic compounds against peroxidative damage to biomembranes. Biochem Pharmacol,
On
43, 147-152.
Liu J. (1995). Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol, 49 (2),
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52
53
54
55
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58
59
60
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57-68.
Mandal AB, Chattopadhyay D, Coomar T. (2000). Rare and endangered flowering plants of
Bay Islands with special reference to endemics and extra Indian Taxa. Indian Forester,
126 (4), 389 -396.
Maxwell SRJ. (1995). Prospects for the use of anti-oxidant therapies. Drugs, 49, 345-361.
22
URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu
Page 23 of 34
Middleton E, Kandaswami C, Theoharides TC. (2000). The effects of plant flavonoids on
mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol
Rev, 52, 673-751.
Mizushima Y. (1966). Screening test for anti-rheumatic drugs. Lancet, 2, 443.
Nishino C, Mitsui T. (1973). Lignans from Hernandia ovigera. Tethedron Let, 4, 335-338.
OECD Guideline 425 (2000). Acute Oral Toxicity. Environmental Health and Safety
rP
Fo
Monograph series on Testing and Assessment No. 24.
Oyemitan IA, Iwalewa EO, Akanmu MA and Olugbad TA. (2008). Antinociceptive and antiinflammatory effects of essential oil of Dennettia Tripentala G. Baker (Annonaceae) in
Rodents, AJTCAM, 5(4), 355-362.
ee
Oyedapo OO, Famurewa AJ. (1995). Anti-protease and membrane stabilizing activities of
rR
extracts of Fagra zanthoxiloides, Olax subscropioides and Tetrapleura tetraptera. Internat
J Pharmacog, 33, 65-69.
ev
Pernet R. (1971). Revue des Hernandiacees. Planta Med, 20, 314-319.
Perez RM, Perez S, Zavala MA, Salazar M. (1995). Anti-inflammatory activity of the bark of
ie
Hippocratea excelsa. J Ethnopharmacol, 47, 85-90.
w
Pettit GR, Meng Y, Gearing RP, Herald DL, Pettit RK, Doubek DL, Chapuis J-C, Tacket LP.
(2004). Antineoplastic agents. 522. Hernandia peltata (Malaysia) and Hernandia
On
nymphaeifolia (Republic of Maldives). J Nat Prod, 67(2), 214-220.
Pradeepa K, Krishna V, Venkatesh, Girish Kumar K, Santosh Kumar SR, Joy H Hoskeri,
ly
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmaceutical Biology
Gnanesh AU. 2012. Antinociceptive activity of Delonix elata leaf extract, Asian Pac J
Trop Biomed S229-S231
Rajavel R, Sivakumar T, Jagadeeswaran M, Malliga P. 2009. Evaluation of analgesic and
anti-inflammatory activities of Oscillatoria willei in experimental animal models. J Med
Plants Res, 3(7), 533-537.
23
URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu
Pharmaceutical Biology
Ramalingam R, Bindhu Madhabi B, Ravinder Nath A, Duganath N, Udaya Sri E, Banji D.
(2010). In vitro antidenaturation and antibacterial activity of Zizyphus oenoplia. Der
Pharmacia Lettre, 2(1), 87-93.
Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf VO. (1999).
Membrane stabilizing activity- a possible mechanism of action for the anti-inflammatory
activity of Cedrus deodara wood oil. Fitoter, 70, 251-257.
rP
Fo
Singh YN. (1986). Traditional medicine in Fiji: Some herbal folk cures used by Fiji Indians. J
Ethnopharmacol, 15, 57-88.
Stoeck M, Riedel R, Hochhaus G, Ha¨ fner D, Masso J, Schmidt B, Hatzelmann A, Marx D,
Bundschuh D. 2004. In Vitro and in Vivo Anti-Inflammatory Activity of the New
ee
Glucocorticoid Ciclesonide. J Pharmacol Exp Therap, 309(1), 249–258.
rR
Sur TK, Pandit S, Bhattacharya DK, Ashok Kr CK, Mohanlakshmi S, Chattopadhyay D,
Mandal SC. (2002). Studies on anti-inflammatory activity of Betula alnoides bark.
Phytother Res, 16(7), 669-671.
ev
UNESCO, 1996. Culture and health, orientation texts-world decade for cultural development
ie
1998-1997. Document CLT/DEC/PRO-1996, Paris, France, pp: 129.
w
Vane JR, Bolting RM. (1995). New insights into the mode of action of anti-inflammatory
drugs. Inflammation Res, 44, 1-10.
On
Wagner H, Bladt S. (1996). A Thin Layer Chromatography Atlas. Second ed. Verlag, Berlin,
Heidelberg, Germany, Plant Drug Analysis. (WRONGLY CITED)
ly
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 24 of 34
White M. (1999). Mediators of inflammation and inflammatory process. J Allergy Clin
Immunol, 103, 5378-5381.
Whittle BA. (1964). The use of changes in capillary permeability in mice to distinguish
between narcotic and nonnarcotic analgesics. Brit J Pharmacol, 22, 246–253.
24
URL: http:/mc.manuscriptcentral.com/nphb Email: pezzuto@hawaii.edu
Page 25 of 34
Macroscopic and Microscopic Examination: Quality Control methods for medicinal plant
materials, WHO, Geneva, (1998).
Winter CA, Risley EA, Nuss GW, (1962). Carrageenan induced oedema in hind paw of rat as
assay for antiinflamatory drugs. Exp Biol Med, 111, 544-547. (In Correct Jour Name---)
Winter CA, Porter CC. (1957). Effect of alteration in side chain upon anti-inflammatory and
liver glycogen activities in hydrocortisone esters. J Americ Pharmacol Soc, 46, 515.
rP
Fo
Woodson RF. (1987). Statistical Methods for the Analysis of Biomedical Data, Probalility
and Mathematical Statistics. UK: Wiley, Chichester, 315-316.
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