Evaluation of Anti-Inflammatory and Membrane-Stabilizing Property of Methanol Extract of Rothmannia longiflora Fruit

 

*¹Ezugwu, A. C.; ¹Njoku, O. U.; ²Ozoemena, N. F.; ¹Nwodo, O. F. C.  and ¹Joshua, P. E.

¹Dept. of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

²Department of Anatomy, University of Nigeria Teaching Hospital, Enugu, Nigeria.

*Corresponding Author E-mail: ofcnwodo@yahoo.com and parkeselisco@yahoo.co.uk

ABSTRACT:

This study reports the anti-inflammatory and membrane-stabilizing property of methanol extract of Rothmannia longiflora fruits. The egg albumin-induced rat paw oedema was utilized as the model for inflammation. The probable mode by which R. longiflora mediates its effects on inflammatory conditions was studied on human blood cells (RBC) exposed to hypotonic solution. The results of the study revealed that the extract possesses anti-inflammatory property. Rothmannia longiflora was found to significantly (p<0.05) reduce the oedema induced by the phlogistic agent in rats in a dose-related manner; the reduction was during the later phase of the inflammation. Also, the extract was shown to exhibit membrane-stabilizing property, as it significantly (p<0.05) reduced the levels of haemolysis of RBC exposed to hypotonic solution. The acute toxicity studies of methanol extract of Rothmannia longiflora fruit in mice revealed that it has high safety profile, as there was no death recorded in two stages of the test using intraperitoneal (ip) route.

 

 

 

INTRODUCTION:

Rothmannia longiflora (Rubiaceae), known as Uri (in Igbo language) 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 (Joffe, 2005)

 

Rothmannia longiflora is used in ethnomedicine to treat numerous ailments. In Nigeria, the roots are used to treat bowel complaints. In the Democratic Republic of Congo, the root infusion is used as a treatment for throat abscesses, tooth ache and leprosy (Joffe, 2005). In West Africa, the leaf pulp is used as an enema against kidney pain and diarrhoea with blood. The leaf juice is used to enhance labour. In Sierra Leone, the leaves are used to treat itching skin diseases and the fruit pulp is emetic. In DR Congo, the seed is used to treat ulcers (Joffe, 2005). R. longoflora is considered to have febrifugal property and a decoction of the leaves, twings, bark and roots is applied internally and externally in lotion washes and baths (Coates, 2002). The fruits of R. longiflora are recorded as edible and the juice from the fruits is heated and applied to wounds and burns to speed up the healing process (Coates 2002).

 

Phytochemical studies revealed that R. longiflora contains alkaloids, flavonoids, saponins, glycosides, tannins, steroids and terpenoids. The presence of 4-oxonicotinamide-1-(1’-β-D-ribofuranoside) in the fruits, branches and leaves has also been reported (Jansen, 2005). Literature survey however, revealed scanty information that suggests the usefulness of the fruit extract of R. longiflora in the control of inflammation. This study therefore, reports the anti-inflammatory properties of the methanol extract of R. longiflora fruit.

 

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 was retained as reference in the Herbarium Unit of the Department of Botany, University of Nigeria, Nsukka.

 

Laboratory animals:

Wistar albino rats (150-200g) and Swiss albino mice (18-25g) of either sex obtained from the Animal Centre House of the Faculty of Biological Sciences, University of Nigeria, Nsukka, were used for the various studies. They were kept in a well-ventilated environment, had free access to food and water ad libitum. They were housed in steel-top, plastic-bottom cages.

Chemicals:

All chemicals used in this study were of analytical grade. They were products of May and Baker, England and Merck, Darmstadt, Germany.

 

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 24hr 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.

 

Anti-inflammatory test:

The test was carried out using a phlogistic agent-induced rat hind paw oedema as a model for acute inflammation (Winter et al.,1962). The phlogistic agent employed in this study was fresh egg albumin. Adult Wistar albino rats of either sex (150-200g) were used after a 12hr fast (Akah and Nwambie, 1994). Animals were deprived of water only during the experiment. Inflammation of the hind paw was induced by injection of 0.1ml of fresh egg white into the sub-plantar surface of the right hind paw of the rat. The right hind paw volumes of the rats were measured by volume displacement using LETICA Digital Plethysmometer (LE 7500). Paw volumes were measured immediately before the administration of the phlogistic agent (zero time) and at 1hr intervals for a period of 5hrs.

 

The extract (100, 200 and 400mg/kg) suspended in normal saline were administered intraperitoneally, one hour before inducing inflammation. Control group received equivalent amount of normal saline and the reference group administered 25mg/kg indomethacin.

 

Average oedema at every interval was assessed in-terms of difference in volume displacement after injecting the phlogistic agent and zero time volume displacement of the injected paw (Vt–Vo) (Akah and Njike, 1990).

 

Membrane-stabilizing activity:

(A) Preparation of erythrocyte suspension:

Whole blood was obtained with heparinized syringes from rats through cardiac puncture. The blood was washed three times with isotonic buffered solution (154mM NaCl) in 10mM Sodium Phosphate buffer (pH 7.4). The blood was centrifuged each time for 10 minutes at 3000 rpm.

 

(B) Hypotonic solution-induced rat erythrocyte haemolysis:

The blood samples were centrifuged at 3000rpm for 10minutes and the supernatant was discarded. Then a volume of normal saline equivalent to 2–volume of the supernatant was used to re-suspend the red blood cell pellet for use.  A total of sixteen (16) test tubes were used; eight (8) for the main test and the remaining eight (8) as the blank; for each main test.

 

The first test tube contained 0.1ml of resuspended blood sample (RBS) and 2.4ml of normal saline. Test tube two contained 0.1ml of RBS, 1.9ml of normal saline and 0.5ml of distilled water. Test tube three to eight contained the same volume of RBS and distilled water but varying volumes of extract and normal saline. The volume of each test tube was 2.5ml. The blank contained everything in the test solution except the RBS.

 

The percentage inhibition of haemolysis or membrane stabilization was calculated according to modified method Shinde et al., (1999).

 

To calculate the % inhibition of haemolysis:

 

Where OD₁ = Absorbance of Isotonic solution

OD₂ = Absorbance of Test samples (extract)

OD₃ = Absorbance of hypotonic solution

 

Acute toxicity study

Acute toxicity study was determined by the method of Lorke (1984). In the first stage, mice were divided into three groups (n=3) and each group received intraperitoneal doses of 10, 100 and 1000mg/kg of the extract respectively. After 24 hr observation for any behavioural change(s) and death; subsequent doses of 1600, 2900 and 5000mg/kg of the extract were administered to the different mice respectively and observed for another period of 24 hr.

 

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 (ip) 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.

 

Table 1:Effect of Extract on Egg albumin-Induced Oedema and Average Inhibition of Inflammation of the Rat Paw

Treatment	Dose (mg/kg)	∆ Paw volume (oedema) ml and average oedema
		Zero	30 (Mins)	1hr	2hrs	3hrs	4hrs	5hrs
Extract	200	0.36±0.006	0.60±0.021 (0.24)	0.86±0.020 (0.50)	1.06.0.000 (0.70)	0.95±0.021 (0.59)	0.67±0.000* (0.31)	0.50±0.021* (0.14)
Extract	400	0.44±0.000	0.67±0.020 (0.23)	0.81±0.000 (0.37)	1.05±0.021 (0.61)	0.77±0.021* (0.33)	0.51±0.021* (0.07)	0.47±0.021* (0.03)
Indomethacin	25	0.80±0.021	1.05±0.021 (0.25)	1.28±0.000 (0.48)	1.14±0.021 (0.34)	1.00±0.000 (0.20)	0.90±0.020* (0.10)	0.86±0.021* (0.06)
Normal Saline	–	0.25±0.021	0.51±0.000 (0.26)	0.77±0.021 (0.52)	0.99±0.000 (0.74)	1.00±0.000 (0.75)	1.01±0.000 (0.76)	1.02±0.020 (0.77)

* Reduction in oedema is significant at p<0.05 compared to the control. Values of oedema shown are mean ± SD (n=5).

Values in parenthesis are Average oedema calculated relative to the paw volume at time zero.

 

Table 2: Effect of the methanolic fruit extract of R. longiflora on rat erythrocyte haemolysis

Test Tube	RBC (ml)	Normal Saline (ml)	Extract (mg/ml)	Indomethacin (mg/ml)	Distilled H2O (ml)	OD (418nm)	% Inhibition of Haemolysis
1	0.1	2.4	–	–	–	0.030 ± 0.001	–
2	0.1	1.9	–	–	0.5	0.090 ± 0.001	–
3	0.1	1.8	0.1	–	0.5	0.075 ± 0.005	25
4	0.1	1.7	0.2	–	0.5	0.070 ± 0.001	33
5	0.1	1.5	0.4	–	0.5	0.060 ± 0.001	50
6	0.1	1.3	0.6	–	0.5	0.058 ± 0.001	53
7	0.1	1.7	–	0.2	0.5	0.059 ± 0.000	52
8	0.1	1.5	–	0.4	0.5	0.045 ± 0.001	60
9	0.1	1.3	–	0.6	0.5	0.040 ± 0.001	83

Each value represents the mean ± SD of six experiments.

 

 

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.

 

REFERENCES:

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6.       Http://www.Afri. Biotech, 2006. Retrieved April, 2008.

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10.     Nwodo, O. F. C. and Eze, S. O. (2001). Anti-inflammatory effects of Abrus precatorius seed extract. Nigerian journal of Biochemistry and Molecular Biology, 16(3): 95 – 98.

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12.     Winter, C. A, Risley E. A. and Nuss G. W. (1962). Carrageenin-Induced oedema in right hind limb paw of the rat as an assay for anti-inflammatory drugs Proceedings of Sociological and Experimental Biology, 111: 544-557.

 

 

Received on 10.05.2010        Modified on 23.07.2010

Accepted on 11.08.2010        © AJRC All right reserved