International Journal of PharmTech Research

CODEN (USA): IJPRIF ISSN: 0974-4304

Vol.2, No.1, pp 283-291, Jan-Mar 2010

Antioxidant and Gastroprotective activities of

Rhodomyrtus tomentosa (Ait.)Hassk.
Geetha K. M.1*, Sridhar C.2 and Murugan V.1
1
Department of Pharmacology, Dayananda Sagar College of Pharmacy, Bangalore, India.
2
C.E.S. College of Pharmacy, Kurnool, Andhra Pradesh, India
*
E-mail- geethakm@yahoo.com
Phone: +919900508674, 08026664534

ABSTRACT: The present study was aimed at evaluating the antioxidant and antiulcer potential of the extracts of
Rhodomyrtus tomentosa leaves (RTLE). The leaves and young buds of Rhodomyrtus tomentosa (Family: Myrtaceae) are
used in folk medicine for the treatment of abdominal pain, diarrhoea, dysentery and externally for lavage of wounds.
Antiulcerogenic activity of the petroleum ether, chloroform, ethyl acetate, aqueous alcoholic (70%) and water extracts
of RTLE (100 and 200mg/kg b.w., orally) were evaluated by employing ethanol induced acute gastric ulcer model. The
invitro antioxidant activity of the extracts were assayed using 2,2-Diphenyl-1-1-picrylhydrazylhydrate (DPPH), 2,2
Azinobis-3–ethylbenzthiazoline-6-sulfonic acid radical cation (ABTS), nitric oxide radical and hydrogen peroxide
scavenging assays Their reductive abilities were also assessed using ascorbic acid as standard. There was a significant
dose dependent decrease in ulcer index in the rats treated with aqueous alcoholic, ethyl acetate and water extracts and
was comparable to the standard drug Omeprazole (2mg/kg.b.w.). However, the petroleum ether and the chloroform
extracts did not exhibit significant antiulcer activity in the doses used. The extracts also exhibited significant free radical
scavenging activity in the invitro antioxidant models studied. These results suggest that the aqueous alcoholic, ethyl
acetate and the water extracts of Rhodomyrtus tomentosa possesses potential antiulcer activity in the doses used which
may be attributed to their antioxidant mechanism.
Key words: Antiulcer activity, Antioxidant, Rhodomyrtus tomentosa leaves, 2,2-diphenyl-1-1-picryl-hydrazylhydrate
(DPPH) radical, Absolute alcohol induced ulcer model.

INTRODUCTION antioxidants such as flavonoids, tannins, coumarins,

The etiology of gastroduodenal ulcers is influenced by xanthenes and more recently, procyanidins have been
various aggressive and defensive factors such as acid- shown to scavenge radicals in a dose-dependent
pepsin secretion, parietal cell, mucosal barrier, mucus manner and therefore are viewed as promising
secretion, blood flow, cellular regeneration, and therapeutic drugs for free radical pathologies 2. Mucosal
endogenous protective agents (prostaglandins and damage can be easily produced by the generation of
epidermic growth factors). The reactive oxygen exogenous and endogenous active oxygen and free
species generated by the metabolism of arachidonic radicals3.
acid, platelets, macrophages and smooth muscle cells The process of lipid peroxidation is mediated by the
may contribute to gastric mucosal damage. Therefore, interaction of hydroxyl radicals with the cell
by scavenging the free radicals, the reactive oxygen membrane, subsequently producing lipid-derived free
metabolites might be useful by protecting the gastric radicals such as conjugated dienes and lipid
mucosa from oxidative damage or by accelerating hydroperoxides. These radicals are known to be
healing of gastric ulcers1. Antioxidants act as radical extremely reactive products that cause oxidative
scavengers, inhibit lipid peroxidation and other free damage. Ethanol increases superoxide anion and
radical-mediated processes, and therefore protect the hydroxyl radical production and lipid peroxidation in
human body from several diseases attributed to the the gastric mucosa 4.
reactions of radicals. Numerous substances have been Ethanol-induced gastric damage may be due to a direct
suggested to act as antioxidants. Various phenolic action on the gastric epithelium causing lipid
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 284

peroxidation, but this damage can be prevented by with food and water ad libitum. The animals were
prostaglandin administration. Several studies have deprived of food for 24 h before experimentation, but
shown antioxidant activity of flavonoids and other had free access to drinking water. The study was
natural compounds 5. conducted after obtaining institutional ethical
Rhodomyrtus tomentosa (Ceylon hill gooseberry) of committee clearance bearing the number
family Myrtaceae is a flowering plant, native to DSCP/PhD/PCol /IAEC/08/08-09.
southern and southeastern Asia, from India, east to
southern China, Taiwan and the Philippines, and south Drugs and Chemicals
to Malaysia.The buds and young leaves have a Chemicals of standard grade were purchased from S.D
beneficial effect in colic, diarrhoea, dysentery, Fine Chemical Pvt. Lt. India, Merck Specialties Pvt.
abscesses, furanculosis, haemorrhage, and the Lt. and Omeprazole, as standard drug was supplied by
concentrated decoction of the leaves have been used as Dr.Reddy Laboratory, Hyderabad.
an antiseptic wash for wounds, impetigo and
abscesses.6 Acute Toxicity Studies
Hence, based on the ethnopharmacological Six rats were fasted overnight and were administered a
information, the main objective of the study was to single oral dose (2000 mg/kg, b.w.) of RTLE. After the
investigate the antioxidant activities of the various administration of the extract, food was withheld for
extracts of the leaves of Rhodomyrtus tomentosa using further 3–4 h. Animals were observed individually at
various invitro methods and to study the antiulcer least once during the first 30 min after dosing,
activity using ethanol induced acute gastric ulcer periodically during the first 24 h and daily thereafter
model. for a period of 14 days. Once daily, cage side
observations including changes in skin and fur, eyes
MATERIALS AND METHODS and mucous membrane (nasal) and respiratory rate,
Collection of Plant Material circulatory (heart rate and blood pressure), autonomic
The leaves of Rhodomyrtus tomentosa were collected (salivation, lacrimation, perspiration, piloerection,
in the month of April / June from Ooty district of urinary incontinence, and defecation) and central
Tamil Nadu. The plant was identified, confirmed and nervous system (ptosis, drowsiness, gait, tremors and
authenticated at Survey of medicinal Plants & convulsion) changes were observed. Mortality, if any,
Collection Unit, Department of AYUSH, Ministry of was determined over a period of 2 weeks.
Health and Family Welfare, Govt. of India, Emerald
by Field Botanist, Dr. S. Rajan. Selection of the Dose of the Extract
The leaves were cut into small pieces and shade dried. LD50 was done as per OECD guidelines for fixing the
The dried material was then ground separately into dose for biological evaluation. The LD 50 of the extracts
coarse powder by a mechanical grinder. The resulting as per OCED guidelines falls under class four values
coarse powder was then used for extraction. with no signs of acute toxicity at 2000 mg/kg b.w.
The antiulcer evaluation of RTLE was carried out at
Preparation of Extracts 7 the dose of 100 and 200mg/kg b.w.
The powdered leaves were subjected to successive hot
extraction in soxhlet apparatus with various solvents in Evaluation of Antiulcer Activity
the increasing order of polarity, such as Petroleum The antiulcer activity of RTLE was evaluated using
ether, chloroform, ethyl Acetate, aqueous alcohol ethanol induced Gastric ulcer model.
(70%) and water. The extracts were filtered, and dried
under vacuum in a rotary flash evaporator and the Absolute alcohol induced ulcer 9
yields of Petroleum Ether, Chloroform, Ethyl Acetate, The animals were divided into four groups consisting
aqueous alcohol and water extracts were 0.4%, 11%, of six animals each. Group I received absolute alcohol
15%, 18% and 16% respectively. alone (1ml of absolute alcohol, p.o.). Groups II and III
received petroleum ether extract of RTLE orally at the
Phytochemical screening 8 dose of 100 and 200 mg/kg b.w, respectively for seven
Preliminary phytochemical screening of the powdered days. Group 1V received Omeprazole 2mg/kg b.w.
leaves extracts was performed to detect the presence of RTLE and the standard drug Omeprazole were also
alkaloids, phenolics, flavonoids, saponins, administered orally 30 mins before the oral
carbohydrates, steroids and terpenoids. administration of 1 ml of absolute alcohol. Sixty mins
later, the animals were sacrificed and their stomachs
excised and the gastric contents were aspirated.
Animals Stomachs were removed and kept immersed in 10%
Albino rats of Wistar strain of either sex weighing formalin for 5 mins. Each stomach was incised along
between 150-200g were used. They were housed in the greater curvature and observed or linear
standard cages at room temp. (25±2 oC) and provided haemorrhagic lesions in the glandular region. The
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 285

length (mm) of each lesion was determined at 10x ABTS (54.8mg) was dissolved in 50 ml of distilled
magnification with a pair of dividers and each length water to 2 mM concentration and potassium
was summed up per stomach. The sum of length of all persulphate (17mM, 0.3 ml) was added. The reaction
lesions for each stomach was used as ulcer index.(UI) mixture was left to stand at room temperature
Stomachs were again immersed in 10% formalin for overnight in dark before usage. To 0.2 ml of various
24 h and histopathological examinations were carried concentrations of the extracts or standards was added,
out and the slides were photographed. The percentage 1.0 ml of distilled DMSO and 0.16 ml of ABTS
inhibition was calculated by the following formula: solution to make a final volume of 1.36 ml.
% inhibition = UI control - UI treated x 100 Absorbance was measured spectrophotometrically
UI control after 20 mins at 734 nm. IC 50 values of the crude
The same procedure was repeated with the extracts was calculated and compared with that of
Chloroform, ethyl acetate, aqueous alcohol and the ascorbic acid and rutin which were used as standards.
water extracts and the results were tabulated.
Reductive ability 12
Histopathological evaluation of absolute alcohol, Measurement of reductive ability of RTLE was
induced ulcers investigated in the Fe3+ to Fe2+ transformation in the
The stomachs of the animals were immersed in 10% presence of the extract. One millilitre of the plant
formalin solution for histopathological examinations extract (10-100µg/ml), 2.5ml of phosphate buffer and
after assessing the ulcer index. After the standard 2.5 ml of 1% potassium ferricyanide were incubated at
processing the wet ulcerated tissues were embedded in 50°C for 30 mins and 2.5ml of 10% trichloroacetic
paraffin and cut into thick sections. The sections were acid were added to the mixture and centrifuged for 10
stained with haematoxylin – eosin and mounted with mins at 3000rpm. 2.5 of the supernatant was diluted
Canada balsam. Histopathological observations were with 2.5 ml of water and shaken with 0.5 ml of freshly
carried out for parameters such as oedema, prepared 0.1% ferric chloride. The absorbance was
inflammation, infiltration, and erosions. measured at 700 nm. Ascorbic acid and rutin were
used as standards. All tests were performed in
Statistical Analysis triplicate and graph was plotted with the average of
Values were expressed as mean ± SEM. The three determinations.
experimental mean values were compared statistically
with that of vehicle control mean values using One Nitric oxide scavenging activity13
Way Analysis of Variance (ANOVA) followed by Sodium nitroprusside spontaneously generates nitric
Dunnett’s multiple comparison test. The analysis was oxide in aqueous solution. Nitric oxide generated in
carried out using Graph Pad Prism software V.4. P this manner is converted into nitric acid and nitrous
values less than 0.5 were considered to be statistically acids in contact with dissolved oxygen and water. The
significant. liberated nitrous acid is estimated using Griess reagent
which forms a purple azodye in the presence of a test
ANTIOXIDANT ACTIVITY compound likely to be the scavenger and the amount
DPPH assay10 of nitrous acid will decrease. The degree of decrease in
The antioxidant activity of the extracts of the plant was the formation of purple azodye will reflect the degree
assessed by the invitro method by 1,1-Diphenyl-2- of scavenging. The reaction mixture (3ml) containing
picryl-hydrazyl (DPPH) assay. 10mM sodium nitroprusside in phosphate buffered
To 200 µl of DPPH solution, 10 µl of each of the test saline , and the extract and the reference compounds at
sample or the standard solution was added separately different concentrations (500, 250, 125, 62.5, 31.2,
in wells of the microtitre plate. The final concentration 15.1,7µg/ml) were incubated at 25°C for 150 mins.
of the test and the standard solutions used were 1000, After incubation 0.5 ml of the reaction mixture
500, 250, 125, 62.5, 31.25 and 15.625 and 7.8 µg /ml. containing nitrite ion was removed and 1ml of
The plates were incubated at 37°C for 30 mins and the sulfanilic acid reagent was added, mixed well and
absorbance of each solution was measured at 490 nm allowed to stand for 5 mins for completion of
using Elisa reader against the corresponding test diazotisation. Then 2ml of Naphthyl ethylene diamine
blanks. IC50 values were calculated and compared with dihydrochloride was added and allowed to stand for 30
that of ascorbic acid and rutin which were used as mins in diffused light. A pink coloured chromophore
standards. was formed. The absorbance of these solutions was
measured at 540 nm against the corresponding blank
ABTS assay11 solutions and IC50 values were determined. IC50 value
Free radical scavenging activity of ABTS [2,2’-Azino- is the concentration of the sample required to inhibit
bis (3–ethylbenzthiazoline-6-sulfonic acid)] radical 50% nitric oxide radical.
cation was measured using the spectrophotometric
method
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 286

Scavenging of Hydrogen peroxide 14 a protection index of 45.33%, 58.66 and 40.66%

Hydrogen peroxide is the least reactive molecule respectively and at 200 mg/kg b.w. the extracts showed
among reactive oxygen species and is stable under protection index of 48%, 62% and 55.33%
physiological pH and temperature in the absence of respectively. The results were comparable to
metal ions. It can be generated through a dismutation Omeprazole which reduced the ulcer index
reaction from superoxide anion by superoxide significantly. The rats treated with absolute alcohol
dismutase It can generate the hydroxyl radical ion in showed loss of gland architecture with erosion of the
the presence of metal ions and superoxide anion. 30 epithelial layer and evident edema and infiltration by
mg of each of the extracts and the standards, ascorbic inflammatory cell. The rats treated with the aqueous
acid and rutin were dissolved in 10 ml of methanol. alcoholic extract at 100 mg/kg, b.w showed no
These solutions were serially diluted with methanol to ulceration but intactness of gastric epithelium was not
obtain lower dilutions. A solution of hydrogen restored. However at 200 mg/kg the rats showed
peroxide (20mM) was prepared in phosphate buffered significant regenerative changes. Minimal oedema and
saline (PBS). Various concentrations of 1 ml of the infiltration were seen in one area. Omeprazole treated
extracts or standards in methanol were added to 2 ml groups showed no ulceration in gastric mucosa, glands
of hydrogen peroxide solutions in PBS. The were regular and minimal inflammation was observed.
absorbance was measured at 230nm after 10mins Peptic ulcer and gastritis have multi-etiopathogenetic
against a corresponding blank solution that contained factors. It is widely accepted that a major underlying
the extracts in PBS without hydrogen peroxide and IC factor of this disorder is the generation of free radicals.
50 values were calculated. There is substantial evidence that oxygen derived free
radicals play an important role in the pathogenesis of
RESULTS AND DISCUSSIONS the injury of various tissues including the digestive
In the qualitative phytochemical analysis, system.15,16 Many reports have demonstrated that most
carbohydrates, alkaloids, terpenoids, steroids, tannins, injury of gastric mucosa can be reduced by pre-
saponins, flavonoids and proteins were found to be treatment with scavengers of reactive oxygen species. 17
present in the leaf extracts of the plant. Fixed oils and In folk medicine, the leaves of Rhodomyrtus
fats were present in all the extracts except aqueous tomentosa have been used for the treatment of ulcers,
extract. Flavonoids in varying proportions were wounds, etc. The petroleum ether extract exhibited
present in all the extracts except petroleum ether moderate antioxidant activity in the ABTS method but
extract. did not exhibit significant activity in the DPPH, H2O2
All the extracts of RTLE at 500, 1000 and 2000 mg/kg and the N2O methods. The chloroform extract
b.w. dose level did not show any significant exhibited potent antioxidant activity in all methods
behavioural alterations and toxicity in the initial h (1 st except the DPPH method. The aqueous alcoholic
four h). However, invariably in all the extracts slight extract possessed potent antioxidant activity in all the
irritability was noticed. Moreover extracts produced methods. The water extract exhibited potent
some dose dependent sedation in the initial hours, 72 antioxidant activity in all the methods except the
hrs observations showed no mortality. Therefore it was hydrogen peroxide method.
concluded that for upto 2000mg/kg b.w there is no The results of our study also proved that the crude
toxicity and hence for further pharmacological studies aqueous alcoholic, ethyl acetate and the water extracts
the biological evaluations were carried out at doses of of Rhodomyrtus tomentosa leaves possess significant
100 and 200 mg/kg antiulcer activity against experimentally induced acute
Administration of absolute alcohol induced gastric ulcer model. However the petroleum ether and
haemorrhagic gastric lesions in the gastric mucosa of the chloroform extracts did not possess significant
the control group. The ethyl acetate, aqueous alcoholic antiulcer activity in the doses used. Hence it can be
and the water extracts of Rhodomyrtus tomentosa suggested that the antiulcer activity of the extract may
reduced these lesions as evidenced by a significant (P< be attributed to its antioxidant activities. Further in
0.01) reduction in the ulcer index when compared with vivo antioxidant studies are in progress to study the
the control group. At 100 mg/kg b.w. the ethyl acetate, exact mechanism of action.
aqueous alcoholic and water extracts of RTLE showed
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 287

Table 1: Effect of Rhodomyrtus tomentosa leaf extracts on Absolute alcohol induced gastric ulcers in rats

Treatment % ulcer
Dose (mg /kg b.w) Ulcer index inhibition
Solvent Control - 30 ±0.56 -
100 28.7 ± 0.45 4
NS
Petroleum Ether extract 200 28.2 ± 0.39 9.3
100 28.0 ± 0.2NS 10
Chloroform extract 200 27.5 ± 0.46* 13.33
100 16.4 ± 0.74** 45.33
Ethyl acetate extract 200 15.6 ± 0.74** 48
100 12.4 ± 0.50** 58.66
Aqueous alcoholic
extract 200 11.4 ± 0.74** 62
100 17.8 ± 1.02** 40.66
Water extract 200 13.4 ± 0.4** 55.33
Omeprazole 2 7.2 ± 0.4** 76

Data are expressed as mean SEM., n=6 in each group.

F values F (6,35) = 238.
NS-not significant, *P<0.05, **P<0.01
(One way ANOVA followed by Dunnett’s test)

Table 2: Invitro antioxidant activity of various extracts of Rhodomyrtus tomentosa in different methods

IC50 values ± SEM (µg/ml) by methoda

Extract / Standard Nitric Oxide H2O2 DPPH ABTS
Pet Ether Extract 130.66 ± 1.76 188.0 ± 1.15 >500 18.0 ±1.15
Chloroform Extract 83.0 ± 3.60 91.33 ± 3.5 >500 14.0 ± 1.15
Ethyl Acetate
Extract 74.66 ± 2.40 76.0 ± 1.15 11.0 ± 1.73 6.66 ± 0.66
Aqueous Alcoholic 77.0± 1.0 70.0 ± 1.76 5.33 ± 0.66 15 ± 1.73
Water Extract 90.0 ± 4.6 141.33 ± 2.40 10.66 ± 0.66 18.0 ±1.15
Rutin 65.0 ± 0.57 35.33 ± 1.33 2.66 ± 0.66 0.52 ± 0.05
Absorbic Acid - 180.66 ± 1.76 3.90 ± 0.57 11.25 ± 0.49
a- Average of three determinations, mean ± SEM
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 288

1.4

1.2
Pet Ether
1
Absorbance (nm)

Chloroform
0.8 Ethyl Acetate
Aqueous Alcoholic
0.6 Water
Rutin
0.4
Absorbic Acid
0.2

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Concentration (mg/ml)

Figure 1: Reductive ability of the various extracts of Rhodomyrtus tomentosa leaves

Figure 2: Section of ulcerated stomach of rats treated with RTLE (200mg/Kg b.w) in absolute alcohol induced
ulcer model
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 289

Figure 3: Section of ulcerated stomach obtained from rats of solvent control group treated with absolute alcohol

Figure 4 Section of ulcerated stomach of rats treated with RTLE (100mg/Kg b.w) in absolute alcohol induced
ulcer model
Geetha K. M.et al /Int.J. PharmTech Res.2010,2(1) 290

Figure 5: Section of ulcerated stomach of rats treated with Omeprazole(2mg/Kg b.w) in absolute alcohol induced
ulcer model

ACKNOWLEDGEMENT 6. Agro’s Dictionary of medicinal plants by Narayan

The authors are thankful to the Management and Das Prajapati, U. Kumar, Agrobios ( India)., 288
Principal of Dayananda Sagar College of Pharmacy, 7. Harborne J.B., Phytochemical methods, A guide to
Bangalore for providing the facilities for the research modern techniques of plant analyses, 2nd ed. Chagman
work. and Hall New York, 1984, 120
8. Kokate C.K., Purohit A.P., Gokale S.B.,
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