http://www.knowledgebasepublishers.org/maincjms.html Comprehensive Journal of Medical Sciences Vol. 2(1), pp. 9 -13 , June 2014 ISSN-2135-6848 Copyright © 2014 Knowledgebase Publishers.

BLEEDING AND CLOTTING TIME EFFECT OF

ETHANOLIC EXTRACTS OF CHROMOLAENA ODORATA VERSUS OCIMUM GRATISSIMUM TREATED ALBINO RATS.

Akomas, S.C and Ijioma S.N

Department of Veterinary Physiology, Pharmacology, Biochemistry and Animal Health, College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, Nigeria

Corresponding Author Email: ijiomasolo@yahoo.co.uk

Accepted June, 2014

In view of the traditionally acclaimed wound healing and hemostatic properties of Chromolaena odorata and Ocimum gratissimum respectively, this study investigated the effects of ethanol extracts of the leaves of both plants on bleeding and clotting times in albino rats. The extracts were administered to the rats by oral gavage at doses of 150mg/kg and 300mg/kg body weight daily for 14 days, while a non- treated group and another group treated with Aspirin (5mg/kg) served as control. At the end of the treatment period, bleeding and clotting times were determined for each rat. The ethanol extract of Chromolaena odorata caused a significant (p<0.05) decrease in bleeding and clotting times, while ethanol extract of Ocimum gratissimum significantly (p<0.05) increased bleeding and clotting times in a manner similar to that of Aspirin, a standard antiplatelet and anti-hemostatic agent. These results suggest that Chromolaena odorata leaves possess strong hemostatic properties and may be useful in wound healing and management of bleeding problems, while Ocimum gratissimum possess antiplatelet and anti-hemostatic properties and may be useful in the managing thrombosis and removal of blood clots from the system. KEY WORDS: Bleeding time, Clotting time, Chromolaena odorata, Ocimum gratissimum

INTRODUCTION Hematological parameters have been associated with health indices and are of diagnostic significance in routine clinical evaluation of the state of health (Saliu et al., 2012)

[1]. Bleeding time is the interval from

oozing of blood after a cut or injury till arrest of bleeding while clotting time is the time interval from oozing of blood after a cut or injury till the formation of clot (Sembuligan and Prema, 2010)

[2]. These two

parameters, although considered by many as obsolete provides enough information about platelet activation and function and may serve as a means of accessing clinical conditions such as disseminated intravascular coagulation, Von willebrand disease, thrombocytopenia, end stage liver failure and uremia.

Several green leafy vegetables and weeds abound in tropical Africa that could be used in the management of hematological abnormalities (Saliu et al., 2012)

[1]. Ocimum gratissimum and

Chromolaena odorata have traditionally been indicated. Ocimum gratissimum is a shrub which grows up to 1.9m high with stems that are branched and leaves

which measure up to 10x5cm. The plant is used extensively in the traditional system of medicine in many

Akomas and Ijioma. 10

countries. In Nigeria, it is used in the treatment of epilepsy, diarrhea, wounds, fever and fungal infections (Ochugbo et al., 2009)

[3]. In Brazil, the decoction of Ocimum gratissimum roots is used as a sedative for

children. In Kenya, the leaves extract is used to treat catarrh, abdominal pains, ear infections, coughs, convulsion, menstrual problems, pneumonia, and as an agent for the arrest of bleeding (Olusegun et al., 2008)

[4].

Chromolaena odorata on the other hand, is a rapidly growing perennial herb with multi-stemmed shrub and grows up to 2.5m tall in open areas. Available literature reveals that the plant contains carcinogenic pyrrolizidine alkaloid and can cause toxicity and allergic reactions in cattle (http://en.Wikipedia.org/wiki/chromolaena)

[5]. However there is report that the extract from the leaves is

used traditionally to treat skin wounds (Hataichanok et al., 2013) [6]

. In view of the traditionally acclaimed hematological effects of Ocimum gratissimum and Chromolaena

odorata, this study was designed to study comparatively, the effects of the ethanol extracts of the leaves of both the plants on bleeding and clotting times in rats as a means of establishing/validating this traditional claim.

MATERIALS AND METHODS Collection and preparation of plant material Fresh leaves of Chromolaena odorata and Ocimum gratissimum were collected from a farm settlement in Ozuitem, Bende Local Government Area, Abia State, Nigeria and were identified by Dr. M. C. Dike of the Forestry Department, College of Natural and Environmental Sciences, Michael Okpara University of Agriculture, Umudike, Abia state, Nigeria. The leaves were dried under shade in the laboratory for 7 days after that, the dried leaves were ground to powder using an electric blender. Extraction was done using the method of Akah et al., (2009)

[7]. In this method 35g of each ground plant sample was introduced into

the extraction chamber of the soxhlet extractor and extraction was done using ethanol as solvent for 48 hours with temperature maintained at 58

0C. Each extract was afterwards dried at low temperature (40

0C)

to obtain a solid extract of each plant material. Chromolaena odorata gave a yield of 35% (12.3g) while Ocimum gratissimum gave a yield of 29% (10.2g). The extracts were kept in a refrigerator for preservation and used when needed.

Animals 70 Albino swiss mice of both sexes(25-30g) and 42 albino rats(130-160g), obtained from the Animal house unit of the Department of Physiology, Pharmacology, Biochemistry and Animal Health, College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, were used for the study. The Mice were used for the acute toxicity studies while the Rats were used for the bleeding and clotting times work. All the animals were fed with standard animal feed and water ad libitum and handled in accordance with the NIH guidelines for Care and Use of Laboratory Animals (Pub. No.85-23, Revised 1985), as described by Akah et al., (2009)

[7].

Acute toxicity study For Chromolaena odorata ethanol extract, 35 mice of both sexes were divided into 7 groups of 5 mice each and were assigned graded intraperitoneal (I.P.) doses of 500, 1000, 2000, 2500, 3000, 4000 and 5000mg/kg, as well as 500, 1000, 1500, 2000, 2500, 3000, 3500mg/kg Ocimum gratissimum ethanol leaf extract were assigned to the second set of 35 mice also divided into 7 groups of 5 mice each. After administration, the animals were allowed free access to feed and water and the number of deaths in each group was noted at the end of 24 hours. LD50 value for each extract was calculated using Karber’s formular described by Enegide et al 2013

[8] as:

LD50 = LD100 -∑ (Dd x Md) ∕ N Where:

LD50 = Dose that killed 50% of animals in a group

11. Akomas and Ijioma LD100= Dose that killed all animals in a group ∑(Dd x Md) = Summation of all products of dose difference and mean deaths N = Number of animals in each group

Bleeding and clotting times 42 Albino rats of both the sexes (130-160g) were divided into 6 groups of 7 rats each. Each group was housed in a separate aluminum cage. Group 1 served as the normal control and received daily oral dose of normal saline (0.2ml), group 2 received Aspirin (5mg/kg), groups 3 and 4 received 150 and 300 mg/kg of Chromolaena odorata ethanol leaf extract respectively, while groups 5 and 6 received 150 and 300mg/kg ethanol leaf extract of Ocimum gratissimum. Treatments were done daily via the oral route using oral gavage and lasted for 21 days. At the end of the period, bleeding time was determined for each animal using Duke’s method while clotting time was determine by Ivy’s reported by Ibu and Adeniyi, (1989)

[9]. For bleeding time, the tip of the tail of each rat was cut to cause bleeding. A stopwatch was

started as soon as animal began to bleed. A blotting paper was used to wipe off blood every 15 seconds. As soon as bleeding ceased the stopwatch was stopped and the time recorded as bleeding time for that particular animal. For the clotting time, a drop of blood from the tail of each rat was placed on a clean glass slide and a stopwatch was started at the same time. A pin was passed across the drop of blood once every 15 seconds. As soon as threads of fibrin were noticed, the stopwatch was stopped and the time recorded as the clotting time for that particular rat.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) using student’s t-test at 95% level of significance in accordance with Steele and Torrie, (1980)

[10].

RESULTS Acute toxicity result A lethal dose (LD50) Values of 2700mg/kg for Chromolaena odorata and 2150mg/kg for Ocimum gratissimum indicates that both extracts could be toxic at higher doses. Toxicity symptoms and deaths were observed in some groups within the 24 hours period of acute toxicity study. With this result, 150 and 300mg/kg body weight were chosen as doses to be administered to the treated rats for the bleeding and clotting time studies. The rationale was to conform to dosage forms as in traditional practice where plants are taken in little quantities.

Table I: Effects of Chromolaena odorata and Ocimum gratissimum on bleeding time in Rats

Group Treatment Bleeding time ±SEM Minutes

1 Control(0.2 ml, normal saline) 4.54 ± 0.80

2 Aspirin, 5mg/kg 8.00 ± 1.20*

3 C. odorata, 150mg/kg 2.95 ± 0.70*

4 C. odorata, 300mg/kg 2.74 ± 0.81*

5 O. gratissimum, 150mg/kg 5.20 ± 1.30*

6 O. gratissimum, 300mg/kg 8.22 ± 1.40*

*P<0.05 versus control

Effects of Chromolaena odorata and Ocimum gratissimum on Bleeding and Clotting Times Oral administration of Aspirin (5mg/kg) for 21 days caused a significant (P< 0.05) change in the mean bleeding and clotting times. Aspirin increased bleeding time from 4.54±0.80 minutes in control group to

Akomas and Ijioma. 12 8.00 ± 1.20 minutes (Table 1) and clotting time from 2.64±0.51 minutes in the control group to 3.74±0.63 minutes (Table 2). Ethanol extract of Chromolaena odorata significantly (P<0.05) lowered bleeding and clotting times in rats when compared to control. In contrast to the effect of Aspirin, 150mg/kg of C. odorata lowered bleeding and clotting times to 2.95±0.70 and 1.80± 0.42 minutes respectively, while 300mg/kg lowered same to 2.74±0.81 and 1.54±0.33 minutes respectively.

Ethanol extract of Ocimum gratissimum exhibited a dose dependent increase in bleeding and clotting times when compared to control. The effect was opposite to that of C. odorata. 150mg/kg of O. gratissimum increased bleeding and clotting times to 5.20 ±1.30 and 3.22±0.92 minutes respectively while 300mg/kg increased same to 8.22±1.40 and 4.20±0.96 minutes respectively.

Table 2: Effects of Chromolaena odorata and Ocimum gratissimum on Clotting time in Rats

Group Treatment Clotting time ± SEM Minutes

1 Control (0.2 ml, normal saline) 2.64 ± 0.51

2 Aspirin, 5mg/kg 3.74 ± 0.63*

3 C. odorata, 150mg/kg 1.80 ± 0.42*

4 C. odorata, 300mg/kg 1.54 ± 0.33*

5 O. gratissimum, 150mg/kg 3.22 ± 0.92*

6 O. gratissimum, 300mg/kg 4.20 ± 0.96*

*P<0.05 versus control

DISCUSSION The toxicity observed during the 24 hours acute toxicity study of ethanol extract of Chromolaena odorata (LD50 = 2700 mg/kg) and Ocimum gratissimum (LD50 = 2150mg/kg) suggests that both extracts could be toxic at doses beyond effective limits. The level of toxicity observed may be due to the presence of toxic phytochemical components in the plants. This finding agrees with Nweze and Eze (2009)

[11], who studied

the phytochemistry of Ocimum gratissimum and reported that the plant contains toxic substances such as saponins and cardiac glycosides. His findings revealed that these toxic substance are however present in small quantities and could be tolerated. Carcinogenic pyrrolizidine alkaloids present in Chromolaena odorata leaf extract may have accounted for its level of toxicity. The relative abundance of toxic substances including alkaloids, lactones, tannins, saponins and steroids accounts may also have accounted for toxicity observed in Chromolaena odorata leaf extract (Prasad et al., 2005)

[12].

The significant (p<0.05) reduction in bleeding and clotting times in rats treated with Chromolaena odorata ethanol extract suggest that the extract contain strong principles with hemostatic property and may have achieved these bleeding and clotting times lowering effects by stimulating increased formation and activation blood of platelets. Available literature reports that increased number of platelets usually favors the blood clotting process (Guyton et al., 1996)

[13] and lowers both bleeding and clotting times.

The effect of C. odorata was significantly (p<0.05) different from that of Aspirin(5mg/kg), a standard drug which usually increases bleeding and clotting times. Results obtained therefore justifies the traditional claim that C. odorata promotes wound healing, since stopping of bleeding may be the first line of action in the physiology of wound healing. The antibiotic property of C. odorata ethanol leaf extract reported by Irobi, (1997)

[14] may be a complementary mechanism and attests more to the wound healing properties of

the extract which was reported by Hataichanok, (2013) [6]

. Like Aspirin, Ocimum gratissimum extract increased bleeding and clotting times in the treated rats. This

result suggests that the extract contains agents with antiplatelet, fibrolytic and antihemostatic properties. The extract may have achieved this activity due to its effect on the blood platelets counts. Inyang et al., (2001)

[15] reported that the prolonged consumption of Ocimum gratissimum leaves significantly reduces

platelets counts due to the effects of its phytochemical saponins and glycosides content. The increase in bleeding and clotting times observed after the administration of ethanol extract of Ocimum gratissimum attests to the anti-haemostatic and fibrinolytic effect of the extract and tends to agree with its traditional use as an agent which removes blood clots during puerperium but fails to agree with the claim that it stops bleeding. However further work can be done to find out if topical application of the extract to cuts can induce vascular spasm which is a stage hemostatic mechanism.

13. Akomas and Ijioma CONCLUSION Ethanol extract of Chromolaena odorata leaves has been found to reduce bleeding and clotting times and may be useful in managing bleeding problems and accelerating wound healing. On the other hand Ocimum gratissimum leaf extract increased bleeding and clotting times. It is obvious therefore that Chromolaena odorata has greater potential for reducing bleeding and clotting time. Further work may be carried out to buttress these findings. The antiplatelet and fibrolytic activity of Ocimum gratissimum may be useful in the management of thrombosis and removal of blood clots in women after delivery.

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