Anti-Inflammatory Effects of Rothmannia longiflora Fruit Extract
1Ezugwu,
A. C.*; 1Njoku, O. U.; 2Ozoemena, N. F.; 1Joshua,
P. E.; 3Nwachi,
U. and 1Nwodo, O. F. C.
1Department of Biochemistry, University of Nigeria, Nsukka, Nigeria.
2Department of Anatomy, University of Nigeria
Teaching Hospital, Enugu, Nigeria.
3Department of Biochemistry, College of Medical
Sciences, Ebonyi State University, Nigeria.
ABSTRACT:
The anti-inflammatory activity of the fruit
extract of Rothmannia longiflora
was carried out by evaluating its inhibitory effect on Phospholipase
A2 and Prostaglandin Synthetase. As R. longiflora
was found to have febrifugal and analgesic properties by reducing oedema formation; the ability of the extract to inhibit the
synthesis of prostaglandin which is one of the mediators of inflammation, was
utilized as the model for anti-inflammation. R. longiflora was found to significantly
(p<0.05) inhibit the activity of both Phospholipase
A2 and Prostaglandin Synthetase activities. The
results of this study shows the anti-inflammatory activity of R. longiflora
extract and suggest further investigations into the effects of specific
constituent(s) of the plant on inflammation.
KEYWORDS: Rothmannia longiflora; Inflammatory; Prostaglandin Synthetase; Phospholipase A2.
INTRODUCTION:
The use of plants as a source of relief for
illness is as old as mankind, with recorded practices dating back at least 4000
years (Christophersons et al., 1991). Rothmannia longiflora (Family Rubiaceae)
commonly referred to as Uri (Igbo Eastern Nigeria) is a shrub or small tree
and occasionally herbs or climbers which grow in old farms of secondary forest
and thickets (Joffe, 2005). The fruit of the plant is
widely distributed in tropical and sub-tropical regions of the globe and
sometimes they are found in cold regions. They abound in Ghana through Nigeria
to Congo. R. longiflora
is also found from east of Gambia to Sudan and Kenya, and South of Tanzania and
Angola (Coates, 2002). The fruits are ingredients of common anti-inflammatory
remedies used locally (Abubakar et al., 2007).
Inflammatory reactions in humans are usually
characterizes by pains, swelling and fever and a major inflammatory disease
(rheumatoid arthritis) is one of the most distressing and disabling syndromes
encountered in medical practice and although it is one of the oldest disease,
there is no drug leading to a permanent cure. The side effects of existing
steroidal and non-steroidal anti-inflammatory drugs have led to increasing
efforts in search of novel compounds especially from plants which would possess
long acting anti-inflammatory activity with minimum side effects.
In the present study, we have examined the
anti-inflammatory potential of R. longiflora using its ability to inhibit the synthesis
of inflammatory mediators like prostaglandin. Aspirin and indomethacin
inhibited the synthesis of prostaglandins (example PGF and PGE series) from arachidonic acid (Vane, 1971); therefore any agent that
acts in a similar manner should be considered as an anti-inflammatory agent.
MATERIALS
AND METHODS:
Plant Material:
Mature fruits of R.
longiflora were harvested from Nkpologu in Uzo-uwani Local
Government Area of Enugu State and authenticated by Mr. M. C. Eze of the Department of Botany, University of Nigeria, Nsukka, Enugu State, Nigeria. Voucher specimen of the
leaves are retained as reference in the herbarium unit of the department of
Botany, University of Nigeria, Nsukka.
Animal Material:
Ox-testes were purchased from the local slaughter house
in Nsukka market and processed as the source of
prostaglandin synthetase.
Chemicals:
All chemicals used in this study were of analytical
grade and they were used as such. They were products of May and Baker, England
and Merck, Darmstadt, Germany. All laboratory reagents were freshly prepared.
Preparation of plant extract:
Fresh fruits of R.
longiflora were extracted using chloroform and
methanol in the ratio of 2:1(v/v) (Folch et al., 1957). The fruits were
pulverized and soaked for 24 hours with a mixture of 1260ml of chloroform and
630ml of methanol. The extract was filtered first through calico, and then
through a (Whatman No.4) filter paper. Next, 360ml of water was added to 1800ml
of the filtrate and shaken to give upper and lower layers; methanol-water
filtrate and chloroform filtrate respectively. The upper aqueous methanol layer
was drawn out and lyophilised. The concentrated
portion gave 5.95% of methanol fraction. Bambara oil
was also extracted using the Folch method.
Preparation of Phospholipase A2:
Specimen (enterobacter) in
agar slant in bijou bottles was incubated overnight at 37°C and then inoculated
on nutrient agar slant already prepared. The inoculated slants were incubated
for 24 hour at 37°C. The nutrient broth used was prepared by dissolving 13g of
nutrient broth in 1000ml of distilled water, homogenized in a water bath for 10
min and 5ml dispensed into two clean bijou bottles. The broth was autoclaved at
121°C for 15 min. The broth was allowed to cool to normal room temperature, and
then the organisms in the slant above were aseptically inoculated into the
broth and incubated for 24hr at 37°C. Bacterial cells were harvested by
centrifuging at 3000rpm for 15min at 4°C, then washed in 0.02M Tris-HCl buffer, pH 7.4 and sonicated.
The sonicate was then centrifuged at 3000 rpm for
30min at 4°C. The supernatant was decanted and stored at 15°C and was used as
an enzyme preparation (Nwodo and Eze,
2001).
Preparation of Prostaglandin Synthetase (PGS):
The method of Nugteren et al. (1966) was used in the isolation
of the enzyme from beef seminal vesicle (the source of prostaglandin synthetase). The frozen beef seminal vesicle (50g) obtained
was thawed and freed of fat and connective tissues. Then the seminal vesicle was
sliced and homogenized in 40ml of 0.02M Tris-HCl
buffer, pH 7.6 for 2min at 4°C with a blender. The homogenate was centrifuged
at 6000 rpm for 10min at 4°C. The supernatant was decanted and centrifuged
twice and the resulting supernatant was then used as the crude enzyme
preparation.
Anti-inflammatory test:
Effect of Rothmannia longiflora fruit extract on Phospholipase A2 activity:
The method of Vane (1971) was used. Aliquots (0.1ml) of
re-suspended fresh human red blood cell (HRBC), 0.2ml of CaCl2 (2mM) and 0.2ml
of enzyme preparation from enterobacter and varying
concentrations of the extract were incubated for one hour. The incubates were
centrifuged at 3000rpm for 10min. Samples of the supernatant were diluted with
10ml normal saline and the absorbance of the solutions read at 418nm using
distilled water as blank.
The formula below was used to calculate the maximum
enzyme activity:
O.D.
of test sample 100
% Maximum enzyme activity = -------------------- X
---------
O.D.
of control 1
Percentage inhibition = 100 - % Maximum
enzyme activity.
Effect
of the extract on Prostaglandin Synthetase activity:
The resultant supernatant of the crude
enzyme preparation above was used as the source of the enzyme. The method of
Yoshimoto et al. (1970) was used for
the determination of the effect of the extract on prostaglandin synthetase activity. Thus, increase in absorbance was
monitored at 278nm due to formation of PGB from PGE2 and the difference in
absorbance reading determines the degree of the activity of the extract.
The cofactor solution was prepared by mixing
33mM hydroquinone, 2mM glutathione and 40μM haemoglobin
in the ratio of 1:1:8. Thus, 8mg solution of prostaglandin synthetase
was weighed into each test tube and 1.5ml of cofactor solution was also added,
then the mixtures were allowed to pre-incubate for 2 min at 37°C. The reaction
was started by adding 0.2cm3 of the substrate (bambara
oil) and allowed to proceed for 2 min at 37°C. Varying concentrations of the
extract and buffer were added to make the volume up to 2.5cm3. The
mixtures were incubated for 2 min and the reaction was terminated by the
addition of 0.5ml of citric acid.
The reaction mixtures were then extracted
twice with 5ml ethyl acetate and centrifuged at 2,500 Χ g for 10 min. For each
extraction, 4ml of the top organic layer was pipetted
out into a clean test tube. The combined ethyl acetate extract (8ml) was
evaporated to dryness using sand bath. The dried residue was then dissolved in 2ml
methanol and 0.5ml of 3M KOH solution was also added and was allowed to stand
for 15 min. The absorbance of test solution was read at 278nm. The experiment
was compared with the results of the blank (boiled enzyme) and that of the
positive control (indomethacin).
To calculate the enzyme activity =
Data analysis
Data obtained from this study were analyzed
statistically and expressed as mean ± SD. The results were compared using one
way ANOVA and regarded as significant at p<0.05.
RESULTS:
Acute toxicity studies:
From the acute toxicity and mortality (LD50)
tests for the extract, no death was recorded in the two stages of the test
using intraperitoneal (i.p.)
route. Therefore the LD50 was greater than 5g/kg.
Effect of extract on egg
albumin-induced oedema in rats:
Data from Table 1 shows that the paw volume taken
immediately after fasting the animals, and soon after the injection of the egg
albumin was assumed to be at time zero (t = 0), while the mean paw volumes for
the various groups were taken at different time intervals. The oedema swellings induced by egg albumin in rats were
significantly (p<0.05) inhibited by the extract especially after the third
hour. However, the oedema reduction for the R. longiflora
treated groups was more than that observed for the standard anti-inflammatory
drug, indomethacin.
Effect of extract on
erythrocyte membrane-stability:
The extract at different concentrations significantly
(p<0.05) protected the rat erythrocyte membrane against lysis
induced by hypotonic solution. However, at a concentration of 0.2mg/ml, indomethacin (standard drug) produced 52.0% inhibition of
RBC haemolysis as compared with 33.0% produced by the
extract at the same concentration (Table 2).
DISCUSSION:
The results of this study showed that Rothmannia longiflora
fruit extract possesses anti-inflammatory property as it significantly
(p<0.05) inhibited oedema induced by egg albumin
in rats. Although the reduction of the oedema took
effect after the third hour. This report agrees with earlier reports of Ely et al. (2006) which showed that
suppression of inflammation at one hour post injection of phlogistic
agent tends to show that the agent could be antihistamine, whereas reduction of
inflammation at three hours and beyond, shows that the agent could have caused
inhibition of arachidonic acid pathway.
In inflammatory reactions, there is increased vascular
permeability which leads to exudation of fluid rich in plasma proteins
including immunoglobulins, coagulation factors and
cells into the injured tissues with subsequent oedema
at the site (http://www.Afri.Biotech, 2006). Oedema
results from the action of inflammatory mediators such as histamine, serotonin,
prostaglandin and bradykinin at the site of a local
inflammatory insult (Harriot et al., 2004).
So the ability of the extract to reduce the size of oedema produced by egg albumin at the third hour suggests
that it was active at the later phase of the oedema
and not at the early phase.
Furthermore, the extract did show membrane stabilizing
effect, as it offered significant (p<0.05) protection of the erythrocyte
against lyses induced by hypotonic solution but the mechanism of action was not
known. The erythrocyte membrane resembles lysosomal
membrane and such, the effect of drugs on the stabilization of erythrocyte
could be extrapolated to the stabilization of lysosomal
membrane (Oyedapo et
al., 2004). Also according to Nwodo and Eze (2001), when lytic enzyme
activity and membrane-stabilization were investigated with Abrus precatorius, it was found that the
extract stabilizes membrane. This may suggest that the anti-inflammatory
properties of this extract may be due to the inhibition of enzymes or
processes, which may enhance the release of lytic
enzymes.
Therefore, as the mechanism of action of Rothmannia longiflora in
stabilizing membrane was not known, further studies will be undertaken to
determine whether R. longoflora
fruit extract will be able to inhibit these enzyme that may enhance the release
of lytic enzyme.
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