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90

Youmbie et al. World Journal of Pharmacy and Pharmaceutical Sciences

ANTI-INFLAMMATORY AND ANTINOCICEPTIVE EFFECTS OF

THE STEM BARK AQUEOUS EXTRACT OF RAUWOLFIA

VOMITORIA (APOCYNACEAE)

Youmbie Djanche Duplex Bonheur1, Dzeufiet Djomeni Paul Désiré

1, Nkwengoua

Ernestine2, Mezui Christophe

3, Dimo Théophile

1*.

1Department of Animal Biology and Physiology, Faculty of science, University of Yaounde I

P.0. Box 812, Yaounde, Cameroon.

2Department of Organic Chemistry, Faculty of science, University of Yaounde I P.0. Box

812, Yaounde, Cameroon.

3Department of Biological Sciences, Higher Teachers’ Training College, University of

Yaounde I P.0. Box 047, Yaounde, Cameroon.

ABSTRACT

Rauwolfia vomitoria (Apocynaceae) has been used traditionally in

Cameroon and other countries as remedy against gastritis, chest pain,

epilepsy, carious teeth, rheumatic pain, headache and inflammation.

This study was aimed to investigate the anti-inflammatory and

antinociceptive activities of the stem bark aqueous extract of

Rauwolfia vomitoria which is empirically used in Cameroon to treat

rheumatic pain, so as to justify its traditional use in the above

mentioned pathological conditions. Anti-inflammatory activity test was

done on female wistar rats at three differents doses (100, 200 and 300

mg/kg body weight) by using carrageenan, serotonin, histamine,

dextran and formalin paw edema tests. The antinociceptive activity test

was performed employing tail immersion test on rats, hot plate method,

acetic acid induced writhing test, and formalin-induced pain test on

swiss albino mice on both sexes at the doses of 100, 200 and 300 mg/kg body weight. The

extract significantly reduced the oedema induced by carrageenan within 30 min with 300

mg/kg body weight being the most potent. The extract at the same dose also significantly

inhibited the oedema induced by serotonin, histamine, dextran and formalin with the

maximum percentage of inhibition of 36.89, 32.75, 39.67 and 38.35% respectively. The

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Article Received on

24 April 2015,

Revised on 18 May 2015,

Accepted on 10 June 2015

*Correspondence for

Author

Pr. Dimo Théophile

Department of Animal

Biology and Physiology,

Faculty of Science,

University of Yaounde I,

P.0. Box 812, Yaoundé,

Cameroon.


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extract significantly reduced the number of writhing with the percentage of inhibition of

56.63 % at the dose of 300 mg/kg. In the hot plate and tail immersion tests, aqueous extract

increased significantly the latency reaction time from 11.58 s to 16.62 s (44.27%) and from

8.31 s to 12.41 s (48.31%) respectively at the dose of 300 mg/kg. It also significantly

inhibited pain and inflammation induced by formalin in neurogenic (37.64%) and

inflammatory (46.44%) phases at the dose of 300 mg/kg. Qualitative phytochemical

screening revealed presence of alkaloids and flavonoids, which are involved in anti-

inflammatory and antinociceptive activities. The results indicated that aqueous extract might

have central and peripheral analgesic properties as well as anti-inflammatory activities.

KEYWORDS: Rauwolfia vomitoria; anti-inflammatory activity; antinociceptive activity;

inflammation; pain.

INTRODUCTION

The majority of the population in developing countries relies on traditional herbal medicine

as the primary source of treatment for illnesses. About 25% of the drugs prescribed

worldwide come from plants, 121 such active compounds being in current use from 252

drugs considered as basic and essential by the World Health Organization, 11% are

exclusively of plant origin and a significant number are synthetic drugs obtained from natural

precursors.[1]

Alkaloids form a class of natural products with increasing interest[2]

since

Reserpine, was firstly isolated from Rauwolfia serpentine in 1952 and was widely used as an

anti-hypertensive drug.[3]

Interest in herbal medicine as a path to drug development increased

greatly in the early1980s.[4]

Nowadays, with the limitation of conventional medicine such as

no access to large percentage of population and high cost,[5]

natural products which are

accessible need to investigate.

Inflammation is a pathophysiological response of living tissue injuries that leads to the local

accumulation of plasmatic fluid and blood cell. Although it is a defence mechanism, the

complex events and mediators involved in the inflammatory reaction can be induced,

maintained or aggravated to many diseases. Inflammation is usually associated with pain as

secondary process resulting from the releases of antinociceptive mediators.[6]

Therapy of

inflammatory diseases is usually targeted at the inflammatory processes. Thus, many non-

steroidal anti-inflammatory agents (NSAIDS) have been prepared and marketed.[7]

According

to the fact that Rauwolfia vomitoria has been used traditionally in Cameroon and other

countries as remedy against gastritis, chest pain, epilepsy, carious teeth, rheumatic pain,


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headache and inflammation,[8,9]

this plant has been chosen to be investigated. Keeping in this

view, the present study has been undertaken to investigate the anti-inflammatory and

antinociceptive potential of stem bark aqueous extract of Rauwolfia vomitoria on

experimentally induced inflammation and pain in rats and mice in order to authenticate some

of its traditional uses.

MATERIALS AND METHODS

2.1. Plant material

2.1.1. Plant collection and identification

Stem bark of Rauwolfia vomitoria was collected in Kepche village, Bangou city, West region,

Cameroon, in August 2010. The plant was identified at the National Herbarium Yaounde,

Cameroon by comparison with voucher specimen No16887/HNC.

2.1.2. Preparation of aqueous extract of Rauwolfia vomitoria

The fresh stem bark of plant was cut into pieces, air-dried away from solar radiation

(Temperature between 20-25oC) for two weeks after which they were pulverized using

warning mechanical blender. 1.5 kg of powder obtained was stored in air tight container for

further use. 500 g powder were macerated in 4.5 l of distilled water for 24 h and the filtrate

obtained was evaporated in an incubator at 45oC and 33.70 g of dark brown solid extract was

obtained (yield of 6.74 %). The plant extract were dissolved in distilled water and

administered to rats and mice. Basing on a preliminary screening test, carrageenan-induced

inflammation, the doses of 100, 200 and 300 mg/kg body weight were selected.

2.1.3. Preliminary qualitative phytochemical analysis of Rauwolfia vomtoria

The aqueous extract was analyzed by the procedures described byTrease in 1983 [10, 11]

for the

presence of metabolites as follow: Saponins (Frothing test: 0.5 g extract + 5 ml warm

distilled water. Frothing persistence means saponins present). Tannins (2 ml extract + 10 ml

of distilled water filtered). 2 ml of filtrate + 2 ml FeCL3, blue-black precipitate indicated the

presence of tannins. Reducing Sugar (2 ml extract + 2 drops of Molisch’s reagent + 2 ml

H2SO4). A reddish violet ring indicated the presence of carbohydrates. Glycosides (2 ml

extract + 10 ml H2SO4 + 10% NaOH + 5 ml of Fehling solution). Glycosides are indicated by

a brick red precipitate. Alkaloids (2 ml extract + 2 N Hydrochloric acids + 6 drops

Dragendoffs reagents. Orange red precipitate indicated the presence of alkaloids). Flavonoids

(2 ml extract + 1 ml of 50% methanol solution + Metal magnesium + 6 drops H2SO4. Red

color was observed for flavonoids and orange color for flavones). Volatile oils (2 ml extract +


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0.1 ml NaOH + 6 drops of HCl. White precipitate indicated volatile oils). Terpenoids (2 mg

extract + 0.5 ml of acetic anhydride + 0.5 ml of chloroform + H2SO4. Red-violet color

indicated the presence of terpenoids). Steroids (2 ml of acetic anhydride + 2 ml extract + 2 ml

H2SO4. The color changed from violet to blue or green in some samples indicating the

presence of steroids). Anthraquinones (0.5 g extract + 5 ml of chloroform, filtered. Filtrate +

10% of ammonia solution. A pink violet or red color in the ammonical layer indicates the

presence of anthraquinones). Proteins (2 ml of protein solution + 1 ml of 40% NaOH solution

+ 2 drops of 1% CuSO4 solution. A violet color indicated the presence of peptide linkage of

the molecule). Amino acids (2 ml extract + 2 ml of Ninhydrin reagent. Purple color indicated

the presence of amino acids). Tri-terpenoids (5 ml extract + 2 ml of chloroform + 3 ml of

H2SO4. Reddish brown coloration of the interface was showed to form positive result for the

tri-terpenoids). Anthocyanins (100 mg extract + 3 ml of distilled water, filtered). 1 ml filtrate

+ 2M HCl and 2M NH4OH. Pink-red color that turned blue-violet indicated the presence of

anthocyanins. Coumarins (100 mg extract + 3 ml of distilled water, filtered). 1 ml of the

filtrate + 1 ml of 10% NaOH. The formation of a yellow color indicated presence of

coumarins). Resins (10 ml extract + cupper acetate solution. Green color indicates the

presence of resins).

The metabolites proportion were characterized as strongly present (+++), present (++),

weakly present (+), and absent (−) when the test result was negative.

2.2. Animals

Female wistar rats weighting 90-120 g were used for anti-inflammatory tests and 120-140 g

for tail immersion as well as Swiss albino mice (18-25 g) of both sexes were used for

antinociceptive tests. Animals were bred in plastic cages under standard light (from 6.a.m to

6. p. m) and temperature (22o C) in the animal house of the Laboratory of Animal Biology

and Physiology, Faculty of Science of the University of Yaounde I. The animals were feed

with standard food and water ad libitum and fasted for 16 hours (with free access to water)

before anti-inflammatory and analgesic tests. The ethical guidelines for investigation were in

conformity with the guidelines of the Cameroon National Ethical Committee on the use of

laboratory animals for scientific research (CEEC Council 86/609).


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2.3. Anti-inflammatory activity

2.3.1. Carrageenan- induced rat paw oedema

The method used in this test has been described by Winter in 1962[12]

. Adult wistar rats used

for this experiment were fasted for 16 hours; these female animals were randomly divided

into five groups of five in each. Different substances were administered per os before

inflammation was induced. One group received distilled water (control) and another received

Diclofenac (5 mg/kg, drug reference). The other three groups received each, aqueous extract

at the doses of 100, 200 and 300 mg/kg. The linear circumference was drawn with permanent

marker on the rat’s right hind paw. Acute inflammation was induced by injecting carrageenan

(1%, 0.1 ml) into right hind limb of each rat under the subplantar aponeuvrosis. Measurement

of paw size was done by mean of volume displacement technique using plethysmometer

37140 Ugo basile, Italia immediately before carrageenan injection and 0.5, 1, 2, 3, 4, 5 and 6

h after carrageenan injection. Carrageenan- induced paw oedema was used to determine

active fractions of aqueous extract the percentage of inhibition in this anti-inflammatory test

were obtained for each group using the following ratio:

(Vt─Vo) control─ (Vt─Vo) treated

%I ═ χ 100

(Vt─Vo) control

Where, %I= Percentage of Inhibition; Vt= Average volume for each group;

Vo= Average volume obtained for each group before treatment

2.3.2. Histamine and Serotonin induced paw oedema

The anti-inflammatory activity of aqueous extract was tested with two phlogostic agents

(histamine, serotonin). The paw oedema was induced in the rats by subplantar injection of

freshly prepared histamine (0.1 ml of10-3

g/ml), and serotonin (0.1 ml of 10-3

g/ml) solutions,

respectively.[13, 14]

The paw volume was recorded at 1 h after histamine injection and 30 min

after serotonin injection.[15, 16]

The drugs ( aqueous extract, Promethazin and Cortencyl) were

administered orally 1 h before eliciting paw oedema and the percentage of inhibition

evaluated as above mentioned in carrageenan induced rat paw oedema test.

2.3.3. Dextran induced paw oedema

The treatment of animals and measurement of paw oedema was done as in histamine and

serotonin tests. Aqueous extract (100, 200 and 300 mg/kg), Cyproheptadin (2 mg/kg, drug

reference) and distilled water (control) were orally administered to the different groups of rats


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1 h before the injection of destran (0.1 ml, in 0.9 % NaCl). The measurement of oedema

volume 0.5, 1 and 2 h after destran injection.[17, 18]

2.3.4. Formalin induced paw oedema

The paw oedema was induced by subplantar injection of 0.1 ml of 2% formalin in to right

hind paw of rats; 1 h after treatment with aqueous extract (100, 200 and 300 mg/kg),

Diclofenac (5 mg/kg) and distilled water (control). The oedema volume was measured after

1, 2, 3 and 4 h [19]

.

2.4- Analgesic activity

2.4.1. Writhing test

The mice (18-25 g) of both sexes were randomly divided into seven groups of five in each.

The total number of writhing and stretching following intraperitoneal administration of acetic

acid solution (1%, 10 ml/kg) was recorded over a period of 30 min. Different substances were

administrated per os 30 min before acetic acid injection. One group received distilled water

(control) and another received Morphine (5 mg/kg, drug reference). Three groups received

each aqueous extract at the doses of 100, 200 and 300 mg/kg body weight respectively. One

group received Naloxon + Morphine and another Naloxon + extract at the dose of 300 mg/kg

[20, 21]. The number of writhing was recorded and permitted to express the percentage of

protection or inhibition (PI) using the following ratio:

Control mean – Treated mean

PI ═ χ 100

Control mean

2.4.2. Hot plate test

The method proposed by Walter in 1992[22]

with modification to suit experimental needs was

used in this test. Mice were kept in glass (cylinder open at both ends) on plate such that, mice

have direct contact with the hot plate (Ugo basile) maintained at constant temperature of

55±0.5°C, mice were randomly divided into seven groups. Each mouse (five per group) acted

as its own control. The reaction time was recorded with a stopwatch. The unit of latency

reaction time was in seconds (s). Before the treatment the reaction time of each mouse

(licking of the forepaws or jumping out of the plate) was determined at 0 and 10 min. The

average of the two readings was obtained as the initial time (It) for each animal. The cut-off

time (i.e. time of no response was put at 55 s). The reaction time following the administration

(Ft) of the extract (100, 200 and 300 mg/kg), Morphine (5mg/kg, p.o.), Naloxone + extract (1


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mg/kg i.p. + 300 mg/kg), Naloxone + Morphine (1 mg/kg i.p. + 5 mg/kg, p.o.) and distilled

water (p.o.), was measured at 0.5, 1, 2, 3, 4, 5 and 6 h after a latency period of 30 min. The

percentage of inhibition (PI) was evaluated using the ratio:

Ft ─ It

PI ═ χ 100

It

2.4.3. Tail immersion test

The method proposed by Vogel in 1997[23]

has been used. The rat’s tail at the length of 3 cm

from end was submerged in hot water inside the water bath at constant temperature of

55±0.5°C. Within a few minutes the rat reacted by withdrawing the tail. The rats of sex

female were randomly divided into seven groups. Each rat (five per group) acted as its own

control. The reaction time was recorded with a stopwatch. The unit of latency reaction time

was in seconds (s). Before the treatment the reaction time of each rat (violent withdrawal of

the tail) was determined at 0 and 10 min. The average of the two readings was obtained as the

initial time (It) for each animal. The cut-off time was put at 15 s. The reaction time following

the administration (Ft) of the extract (100, 200 and 300 mg/kg), Morphine (5 mg/kg, p.o.),

Naloxone + extract (1 mg/kg i.p. + 300 mg/kg), Naloxone + Morphine (1 mg/kg i.p. +5

mg/kg, p.o.) and distilled water (p.o.), was measured at 0.5, 1, 2, 3, 4, 5 and 6 h after a

latency period of 30 min. The percentage of inhibition (PI) was evaluated as above mentioned

in hot plate test:

2.4.4. Formalin test

The method used in this test has been described by Patriziain 2000[24]

. Mice of both sex were

randomly divided into eight groups of five in each, formalin (1.4%, 20 μl) was injected into

the sub-plantar of the right hind paw of the animals, the duration of paw licking was

measured for 0-5 min (neurogenic phase) and 15-30 min (inflammatory phase), after

administration of formalin. One group received distilled water (control) and the others two

groups received each Morphine and Diclofenac (5 mg/kg each) respectively. Three groups

received each aqueous extract at the doses of 100, 200 and 300 mg/kg respectively. One

group received Naloxon (1 mg/kg, i.p.) 15 min before administration per os of Morphine and

another in the same procedure received Naloxon + aqueous extract at the dose of 300 mg/kg.

This permitted to calculate the percentage of analgesic activity as previously in writhing test.


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2.5. Statistical analysis

All values are presented as mean ± S.E.M of five rats or mice. Differences between means

were assessed by one-way analysis of variance (ANOVA), followed by dunnett’s test using

Graph pad prism 5.03. The level of significance was set at *P< 0.05,**P< 0.01, ***P< 0.001.

RESULTS

3.1. Qualitative phytochemical analysis of Rauwolfia vomitoria

Phytochemical screening of aqueous extract of Rauwolfia vomitoria revealed the presence of

various bioactive components of which flavonoids, tannins, alkaloids, saponins and phenolic

compounds were the most prominent. The result of phytochemical test has been summarized

in the table 1.

Table 1: Preliminary phytochemical analysis of the aqueous extract

No Constituents Class Aqueous extract

1 Saponins +++

2 Tannins +++

3 Sugar -

4 Glycosides +

5 Alkaloids +++

6 Flavonoids +++

7 Volatile oils ++

8 Terpenoids and terpenes ++

9 Steroids ++

10 Anthraquinones ++

11 Proteins ++

12 Amino Acids -

13 Anthocyanines ++

14 Resins +

15 Coumarins -

16 Lipide +

17 Phenol +++

18 Acid -

Strongly present: +++; present: ++; weakly present: +; absent: −.

3.2. Anti-inflammatory activity of R. vomitoria

3.2.1. Effects of aqueous extract of R. vomitoria.on carrageenan-induced oedema

The effects of aqueous extract of R. vomitoria on carrageenan-induced oedema are reported

in table 2. It is showing that, aqueous extract at a dose of 100 mg/kg significantly presents the

inhibition of oedema with the highest percentage of 34.78% (3h). The extract at the dose of

200 mg/kg is observed with significantly inhibition of 37.96% (4h). The extract at the dose of


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300 mg/kg is also observed with significantly inhibition of 34.37% (0.5 h) which is

maintained up to 6th

h with the maximum of 56.48% of inhibition (4h). Aqueous extract was

significant during the three phases of inflammation. The anti-inflammatory effects induced by

Diclofenac (standard drugs) progressively reached a maximum of 57.87% (3h).

Table 2: Influence of Rauwolfia vomitoria extract on carrageenan-induced rat hind paw

oedema.

Treatment Dosage

(mg/kg)

Volume of inflammation (ml)

(Percentage of inhibition)

0.5 h 1 h 2 h 3 h 4 h 5 h 6h

Control - 0.13±0.01 0.21±0.01 0.29±0.03 0.32±0.03 0.43±0.02 0.50±0.03 0.53±0.03

Extract 100 0.12±0.02

(6.25)

0.20±0.02

(5.71)

0.25±0.02

(14.58)

0.21±0.02*

(34.78)

0.28±0.02*

(34.26)

0.34±0.02*

(30.64)

0.44±0.02

(15.97)

Extract 200 0.11±0.01

(10.94)

0.18±0.01

(16.19)

0.20±0.03

(31.25)

0.208±0.02*

(35.40)

0.35±0.02*

(37.96)

0.33±0.03*

(33.87)

0.39±0.03

(30.41)

Extract 300 0.10±0.01

(6.74)

0.14±0.02*

(35.24)

0.20±0.03*

(34.03)

0.19±0.03*

(40.99)

0.19±0.02***

(56.48)

0.31±0.02*

(37.09)

0.36±0.02

(31.55)

Diclofenac 5 0.09±0.00

(29.69)*

0.14±0.01*

(32.38)

0.20±0.01*

(30.55)

0.164±0.02**

(49.07)

0.18±0.03***

(57.87)

0.36±0.03

(27.01)

0.40±0.02

(22.81)

n=5. Results are expressed as mean ± S.E.M. Percentage of inhibition are in brackets. The

statistical analysis was performed on absolute data. The extract independent of the dose used,

significantly began reducing the oedema 30 min following oral administration of the extract.

*p˂0.05, **p˂0.01 and ***p˂0.001, significantly different compared to control.

3.2.2. Effects of aqueous extract of R. Vomitoria on rat paw oedema induced by histamine

and serotonin

Table 3 shows that, inflammation induced by injecting histamine (0.1 ml) into right hind limb

of rats under the subplantar aponeuvrosis presents variations of inflammation volume after 1

h which is 0.45 ml (control) and decrease to 0.26 ml to those receiving Promethazin (1

mg/kg). Aqueous extract (300 mg/kg) has significantly inhibited the oedema–induced by

histamine with the percentages of 36.89% and Promethazin (drug reference) has significantly

inhibited oedema of 42.67%. Following the table 4 it is observed that, inflammation induced

by injecting serotonin (0.1 ml) into right hind limb of rats under the sub-plantar aponeuvrosis

presents variations of inflammation volume after 30 min which is 0.35 ml (control) and

decrease to 0.21 ml (Cortencyl, 5 mg/kg). Serotonin increase paw volume with the control

whereas aqueous extract of R. vomitoria (300 mg/kg) decreased these volume of

inflammation significantly (32.75%)and Cortencyl (drug reference) has also significantly

inhibited oedema of 40.23%.


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Table 3: Influence of Rauwolfia vomitoria extract on histamine-induced rat hind paw

oedema

Treatment Dosage

(mg/kg)

Histamine

Volume of inflammation

(ml) (Pourcentage of

inhibition)% 1 h

Control 0.45±0.02 -

Extract 100 0.37±0.03 (18.67)

Extract 200 0.36±0.03 (20.89)

Extract 300 0.28±0.03* (36.89)

Promethazine 1 0.26±0.02** (42.67)


statistical analysis was performed on absolute data. *p˂0.05, and **p˂0.01, significantly

different compared to control.

Table 4: Influence of Rauwolfia vomitoria extract on serotonin-induced rat hind paw

oedma

Treatment

Dosage

(mg/kg)

Serotonin

Volume of inflammation

(ml) (Pourcentage of inhibition)%

0,5 h

Control - 0.35±0.03 -

Extract 100 0.32±0.04 (8.62)

Extract 200 0.28±0.02 (20.11)

Extract 300 0.23±0.01* (32.75)

Cortancyl 5 0.21±0.02** (40.23)


statistical analysis was performed on absolute data. *p˂0.05, and **p˂0.01, significantly

different compared to control.

3.2.4. Effects of aqueous extract of R. vomitoria on dextran induced oedema

Fig.1 shows that, the maximum inflammatory effect, caused by dextran (1%), occurred 2

hours after injection. Aqueous extract at the doses independent of 100, 200 and 300 mg/kg

significantly inhibited inflammation volume with 30.53, 33.59 and 38.17 % (2h)

respectively.Whereas Cyproheptadin significantly inhibited at 0.5, 1 and 2 h.


100


0.5h 1h 2h

0.0

0.1

0.2

0.3 Control

E 100 mg/kg

E 200 mg/kg

E 300 mg/kg

CPH**

**

**

***

******

Duration of test (h)

Mea

n vo

lum

e of

inf

lam

mat

ion

(m

L)

Fig. 1. Influence of Rauwolfia vomitoria extract on dextran-induced rat hind paw

oedema

n=5. Results are expressed as mean ± S.E.M. The statistical analysis was performed on

absolute data. p˂0.05, **p˂0.01 and ***p˂0.001, significantly different compared to control.

CPH: Cyproheptadin. E: Extract (mg/kg).

3.2.5. Effects of aqueous extract of R. vomitoria on formalin-induced oedema

Fig.2 shows that subplantar injection of formalin produced an inflammation which increased

progressively from 0.38 ml to 0.73 ml (Control, 4 h). The aqueous extract of R. vomitoria at

the dose of 300 mg/kg and Diclofenac significantly exhibited 38.35 and 45.48% inhibition of

inflammation (4 h) respectively.

Fig. 2. Influence of Rauwolfia vomitoria extract on formalin-induced rat hind paw

oedema


101



absolute data. p˂0,05 and **p˂0.01, significantly different compared to control. DICLO:

Diclofenac. E: Extract (mg/kg).

3.3. Analgesic Activity of R.vomitoria

3.3.1. Effects of aqueous extract on pain induced by acetic acid (Writhing test)

Table 5 shows that aqueous extract of R. vomitoria (300 mg/kg) reduced the intensity of

acetic acid-induced abdominal contraction in mice with the percentages of 56.63%. Morphine

(drug reference) has reduced of 65.66% and mice receiving Naloxon + morphine and

Naloxon + aqueous extract 300 mg/kg are 19.56% and 17.51% respectively. The effect of the

highest dose (300 mg/kg) (p˂0.001) was compare to Morphine.

Table 5: Influence of Rauwolfia vomitoria extract on mouse writing induced by acetic

acid

Treatment Dosage (mg/kg) Number of writing

per 30 min

Pourcentage of

inhibition (%)

Control - 146.20± 3.12

Extract 100 121.60±6.81 (16.83)

Extract 200 119.00±8.17 (18.60)

Extract 300 63.40±7.05*** (56.63)

Morphine 5 50.20±6.84*** (65.66)

Naloxone +Morphine 1 + 5 117.60±8.85 (19.56)

Naloxone +Extrait 1 + 300 120.60±6.19 (17.51)


statistical analysis was performed on absolute data. ***p˂0.001, significantly different

compared to control

3.3.2. Effects of aqueous extract on pain induced by hot plate test

Table 6 shows that plant extract increase latency reaction time induced by hot plate. The

maximum percentage of protection is 44.27% (3h) at the dose of 300 mg/kg, in group

receiving morphine, the maximum percentage of protection is 56.39% (2 h). The mice

receiving naloxon + morphine, the percentage has been reduced to 8.90% (2 h) and those

receiving naloxon + 300 mg/kg, it has been reduced to 11.28% (2 h).


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Table 6: Influence of Rauwolfia vomitoria extract on hote plate-induced pain

Treatment Dosage

(mg/kg)

Duration of test

0 h 1/2h 1h 2h 3h 4h 5h 6h

Control - 11.70±0.69 11.64±0.74 11.70±0.53 11.68±0.68 11.58±0.46 11.58±0.47 11.68±0.77 11.62±0.71

Extract 100 11.57±0.68 11.96±0.64

(3.33)

12.06±0.72

(4.24)

13.46±0.77

(16.34)

12.54±0.64

(8.38)

13.16±0.72

(13.74)

13.40±0.65

(15.82)

12.62±0.61

(9.08)

Extract 200 11.61±0.61 15.14±0.62*

(30.40)

15.34±0.58*

(32.13)

15.50±0.59*

(33.51)

13.68±0.51

(17.83)

13.22±0.55

(13.87)

15.82±0.64*

(36.26)

13.52±0.64

(16.42)

Extract 300 11.52±0.60 15.04±0.70*

(30.54)

13.70±0.65

(18.92)

15.92±0.51*

(38.19)

16.62±0.54**

(44.27)

13.32±0.68

(15.63)

13.68±0.49*

(18.75)

15.10±0.56*

(31.08)

Morphine 5 11.65±0.60 15.68±0.54*

(34.59)

15.26±0.58*

(30.99)

18.22±0.57***

(56.39)

15.20±0.60*

(30.47)

15.24±0.64*

(30.82)

16.86±0.62**

(44.72)

13.04±0.61

(11.93)

Naloxone+

Morphine 1+5 11.57±0.63

13.10±0.57

(13.22)

13.12±0.56

(13.40)

12.60±0.73

(8.90)

11.98±0.58

(3.54)

12.12±0.62

(4.75)

13.54±0.69

(17.03)

14.49±0.53

(06.53)

Naloxone+

Extract

1+300 11.70±0.70 12.00±0.56

(2.56)

11.74±0.68

(0.34)

12.56±0.78

(7.35)

13.02±0.51

(11.28)

12.58±0.51

(7.52)

13.16±0.58

(12.48)

12.40±0.58

(5.98)

n=5. Results are expressed as mean ± S.E.M. Percentage of inhibition are in brackets. The unit of reaction time is in seconds. The statistical

analysis was performed on absolute data. *p˂0.05, **p˂0.01 and ***p˂0.001, significantly different compared to control.

3.3.3. Effects of aqueous extract on pain induced by tail immersion

Table 7 shows that, aqueous extract of stem bark of R. vomitoria significantly increase latency reaction time of pain by rat’s tail immersion in

hot water at the dose of 300 mg/kg with a maximum percentage of protection of 48.31% (3 h). Naloxon has reduced the effects of Morphine

from 52.47% to 8.80% (2 h). It has also reduced the effects of extract at the dose of 300 mg/kg from 48.31% to 13.93% (3 h).


103


Table 7: Influence of Rauwolfia vomitoria extract on tail immersion-induced pain

Treatment Dosage

(mg/kg)

Duration of test

0h 1/2h 1h 2h 3h 4h 5h 6h

Control - 8.30±0.57

8.31±0.59 8.36±0.62 8.36±0.70 8.31±0.60 8.35±0.69 8.27±0.69 8.39±0.62

Extract 100 8.33±0.65 8.94±0.62

(7.27)

8.76±0.67

(5.14)

9.52±0.70

(14.24)

8.87±0.59

(6.51)

9.83±0.66

(18.00)

9.94±0.70

(19.31)

9.50±0.57

(14.02)

Extract 200 8.36±0.65 11.11±0.69*

(32.89)

11.10±0.63*

(32.72)

10.98±0.64*

(31.39)

9.90±0.66

(18.44)

10.54±0.67

(26.12)

11.19±0.66*

(33.85)

10.10±0.61

(20.81)

Extract 300 8.37±0.61 10.39±0.61

(24.09)

10.18±0.68

(21.67)

11.39±0.63*

(36.05)

12.41±0.68**

(48.31)

9.26±0.62

(10.58)

10.05±0.60

(20.07)

10.29±0.61

(25.33)

Morphine 5 8.37±0.57 10.43±0.64

(24.66)

11.48±0.64**

(37.18)

12.76±0.60***

(52.47)

10.23±0.68

(22.25)

11.48±0.70**

(37.18)

12.01±0.66**

(43.51)

10.35±0.59

(23.63)

Naloxone+

Morphine

1+5

8.37±0.64

9.22±0.62

(10.15)

9.37±0.59

(11.97)

9.64±0.61

(15.22)

9.15±0.68

(9.29)

8.94±0.71

(6.76)

9.21±0.67

(10.08)

9.43±0.62

(12.69)

Naloxone+

Extract 1+300 8.34±0.62

8.91±0.71

(06.88)

9.49±0.70

(13.83)

9.07±0.59

(8.80)

9.50±0.67

(13.93)

9.57±0.67

(14.77)

9.41±0.69

(12.87)

9.24±0.62

(12.77)

n=5. Results are expressed as mean ± S.E.M. Percentage of inhibition are in brackets. The unit of reaction time is in seconds. The statistical

analysis was performed on absolute data. *p˂0.05, **p˂0.01 and ***p˂0.001, significantly different compared to control.

3.3.4. Effects of aqueous extract on pain induced by formalin

The aqueous extract of R. vomitoria produced inhibition on formalin- induced biphasic pain responses (neurogenic and inflammatory pain) in

mice (Fig 3). Extract at the dose of 300 mg/kg has significantly inhibited pain with the percentage of 21.59% at the neurogenic phase and

46.44% at inflammatory phase. Morphine (drug, reference) significantly attenuated the pain responses of two phases (59.50% and 47.74%),

whereas Diclofenac significantly inhibited only the inflammatory phase. The aqueous extract (300 mg/kg) was compared to Diclofenac and

Morphine (drugs references).


104


0 - 5 min (Neurogenic phase ) 15 - 30 min (Inflammatory phase )0

20

40

60

80

100

Control

E 100 mg/kg

E 200 mg/kg

E 300 mg/kg

Diclofenac

MPN

NLX+ MPN

NLX +E 300

*

*****

***

***

**

Duration of test (seconds)

Lic

kin

g t

ime (

s)

Fig.3. Influence of Rauwolfia vomitoria extract on formalin-induced pain


absolute data. *p˂0.05, **p˂0.01 and ***p˂0.001, significantly different compared to

control. MPN: Morphine. NLX: Naloxone. E: Extract

DISCUSSION

Inflammation is the body defense mechanism. The acute inflammation is characterized by

vasodilatation, exudation of plasma release of various inflammatory mediators (cytokines,

growth factor and emigration of leukocytes), infiltration of mononuclear cells and

proliferation of fibroblaste.[25]

The effects of aqueous extract of R. vomitoria were evaluated

in inflammation induced by carrageenan, dextran, histamine, serotonin, and formalin.

Inflammation induced by carrageenan, which is a classical model in acute inflammation to

evaluate anti-inflammatory activities of Non Steroidal Anti-inflammatory Drugs

(NSAIDs),[26]

involves three distinct phases of the release of the mediators including

serotonin and histamine in the first phase (0-2h), kinins in the second phase (3h) and

prostaglandin in the third phase (>4h).[27]

In this study, aqueous extract of R. vomitoria has

significantly inhibited oedema induced by carrageenan. These results suggest that R.

vomitoria possess bioactive substances acting in 1st, 2

nd and 3

rd phase of inflammation.

Probably extract has inhibited the production of histamine, serotonin, prostaglandin, kinin

and bradykinin. These results are similar to those obtained by Dimo in 2005[19]

with aqueous

extract of Kalanchoe crenata. Furthermore, our results have also demonstrated that


105


Cyproheptadin used as standard drug inhibited significantly oedema dextran-induced; this

probably as antihistaminic and antiserotonic effects. Our extract significantly inhibited paw

oedema dextran-induced which is known to cause inflammation through both histamine and

serotonin liberated from mast cells.[28]

Thus, our extract might act probably through inhibition

of histamine and serotonin. Promethazin and Cortencyl are drugs references to treat

inflammation induced by histamine and serotonin respectively. These significant results

suggest that, during the first phase of inflammation, anti-inflammatory activities of extract

were essentially due to the inhibition of the synthesis of serotonin and histamine and/or to the

reduction of the production of prostaglandin. These results corroborated those previously

obtained by Agathe in 2009[29]

while working with methanol extract of S. Birrea in acute

inflammation in rats. Anti-inflammatory activities of Diclofenac (5 mg/kg) were significant

(4h). It is well known that, NSAIDs as Diclofenac exert anti-inflammatory activity by the

inhibition of cyclooxygenase and suppresse production of chemical substances

(prostaglandin, histamine and serotonin) which are involved to increase vasodilatation and

vascular permeability.[30]

The extract significantly inhibited formalin-induced paw oedema

similary as Diclofenac. This result indicates that stem bark aqueous extract of R. vomitoria

may act preferentially by inhibition of cyclooxygenase pathway. The strongly presence of

flavonoids and alkaloids observed in aqueous extract might account for such pharmacological

activities: They might prevent inflammation through blocking the metabolic pathway of

arachidonic acid.[31,32]

Dimo[19]

had also shown in 2005 that n-butanol fraction of Kalanchoe

crenata Andr. Inhibited inflammation induced by formalin in rat.

The anti-nociceptive activity of aqueous extract of R. vomitoria was evaluated in mice using

acetic acid-induced writhing, hot plate and formalin and in rats using tail immersion test.

Intraperitoneal injection of acetic acid induced pain is manifested by writhing and stretching.

Acetic acid induced pain is due to release of mediators such as PGE2, PGG2.[33]

The number

of writhing and stretching decreases from control to groups treated. Aqueous extract of R.

vomitoria might play a role in inhibition of prostaglandin syntheses and/or sticking

neurogenic compound pain induced. Morever, hot plate test and rat’s tail immersion test

considered as thermical stimulus inducing nociceptive responses (which are characterized by

licking forepaw or jumping out and the violent withdrawal of tail respectively) are models of

evaluating central analgesic effects of the drugs.[34]

The increasing of latency reaction time is

the manifestation of protection against pain. However, those stimuli produced pain through

nerves up to hypothalamus and thalamus. Our aqueous extract significantly inhibited pain


106


induced by hot plate and tail immersion in hot water (55±0.5°C); these results suggest that

aqueous extract of R. vomitoria could act as opioid agonist in central nerves system as

demonstrated by Asongalem in 2004.[35]

Furthermore, in the presence of naloxon, analgesic

effects of aqueous extract as well as morphine have been reduced in both hot plate and tail

immersion tests, which are specific tests to study central analgesic activities. Knowing that

Naloxon is an antagonist of opioid and it has an affinity with µ receptors,[36]

in experimental

models, Naloxon acts by antagonising opioid action produced during pain or stress.[37]

These

observations could partially explain the reductions of the effects of analgesic activities of

aqueous extract or Morphine in present of Naloxon in these both tests. In fact, the

modification of central analgesic activities of aqueous extract by naloxon could be due to the

fact that, bioactive substances of extract (tannins, saponines, terpenes, terpenoids, alkaloids)

may fixe to the opioid receptors mean while by inducing the reduction of electric nerves

activity and/or inhibiting the release of the neuromediators. The similar result has been

previously obtained by Asongalem in 2004[35]

while working with aqueous extract of

Erigeron floribundus in mice induced by acetic acid writhin test. Formalin test was used in

this study to understand the mechanism of analgesic effects of the drugs. The formalin

induced biphasic nociceptive response; thus, the neurogenic phase seems to attribute to direct

chemical stimulation of receptors, whereas the inflammatory phase is due to the regulation of

the central system: In fact, at the level of hypothalamus and thalamus, the stimulation of fibre

C release substance P and kinin which participate in neurogenic phase while serotonin,

histamine, bradykinin, nitric oxide and prostaglandin are involved in the inflammatory phase

[38, 39]. The aqueous extract of R. vomitoria 300 mg/kg has significantly reduced pain induced

during the neurogenic and inflammatory phases of nociceptive responses. It might be due to

its central analgesia and anti-inflammatory actions. This is in accordance with the results

obtained by Adeyemi in 2004[40]

on the methanolic extract of Acanthus montanus. Diclofenac

has also significantly inhibited pain induced by formalin only in the inflammatory phase

while aqueous extract inhibited at both phases. Thus, the aqueous extract might be both

analgesic and non steroidal anti-inflammatory drug (NSAID). The aqueous extract might

contained flavonoids, terpenes and terpenoids which are reported to possess analgesic and

non steroidal anti-inflammatory activites,[41,42,43,44,45,46,47]

and have the ability to inhibit

phospholipase A2 and thereby ultimately blocking the metabolism of arachidonic acid and

Cox-II enzymes.[48,49,50]

Morphine has also inhibited neurogenic and inflammatory phases. It

is well known that only drugs with central action like opioid has effect in neurogenic and

inflammatory phase. Thus, plant extract might also have alkaloid compounds as Reserpine,


107


which has been reported in a study of comparative effects of Rauwolfia vomitoria and

chlorpromazine on social behaviour and pain,[51]

which might act as opioids. The similar

result was obtained by Mahnaz in 2012[52]

while study the evaluation of effects of

hydroalcoholic extract of M. parviflorum.

Results of the present study show that the plant extract produced antinociceptiove effect

against both neurogenic and inflammatory phases of formalin induction. The fact that the

extract at the doses tested produced analgesia in antinociceptive models is indicative that it

possesses both central and peripheral antinociceptive effects and the mechanism of action of

the extract could, in part, be related to lipooxygenase and/or cyclooxygenase and/or

arachidonic acid cascade pathways and/or opioid receptors.

Phytochemical investigation of the stem bark of aqueous extract of Rauvolfia vomitoria

indicates the presence of matabolites such as protein, amino acid, sugars, tannins, saponins,

glycosides, alkaloids, flavonoids, volatile oils, terpenes&terpenoids, steroids, anthraquinones,

anthocyanins, coumarins, resins. The investigation on the stem bark of aqueous extract of

Rauwolfia vomitoria from Nigeria by Borokini in 2012[53]

didn’t indicated the presence of

triterpenes and sterols. This shows that our extract has more phytochemicals. The

phytochemicals like phenolic compounds, alkaloids, flavonoids and tannins might be

responsible for these pharmacological activities.

CONCLUSION

The present study on anti-inflammatory and antinociceptive activities of stem bark aqueous

extract R.vomitoria provides useful information about anti-inflammatory and antinociceptive

activities, whereas the mechanism action of antinocceptive will be peripheral and central.

Moreover, the aqueous extract of R. vomitoria presents anti-inflammatory activity during the

three phases of inflammation. These activities can be attributed to the presence of the plant’s

various phytochemicals. These experimental findings support the traditional use of this plant

for the treatment of various ailments especially against inflammatory and pain conditions.

However, further investigations are required like the evaluation of chronic inflammation,

hepatoprotective, its toxicity and isolation of the active constituents to elucidate observed

effects and possible mechanisms of action of the plant extract.


108


ACKNOWLEDGMENT

The authors wish to thank the Rector of the University of Yaounde I for valuable financial

assistance. They are also express sincere gratitude to Mrs Kepgang Bernadette, traditional

medicine practitioner for her implication in collecting the plant material.

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