**4. Discussion**

182 Toxicity and Drug Testing

There was also a significant increase in the values of Neutrophil count (1.08 0.7 in 750 mg/kg/d) versus control (0.53 0.02). However, no major difference was noted between the

Clinical chemistry analysis of the rats indicate increased ASAT and ALAT ratios in the 1500 mg/kg/d dose, compared to control values (Table 7). In addition, significant increased in

Table 7. Mean clinical chemistru values of rats after repeated (6 weeks) orad dosing with

**Dose Mg/kg/d ASAT (U/L) ALAT (U/L) ALB (g/dl) TRIG (mg/dl) UREA (mg/dl)**

Control 132

93

3.4

120

52

12

33

24

0.5

0.38

80

0.20

88

4.1

68

5.9

152

1196

26

750 202

29

1500 373\*

186\*

3.7

112

56

6.4

24

28

0.6

0.80

70

0.18

83

6.7\*

59

5.9

147

1219

77

3000 313

140

3.3

104

57

3.9

24

16

0.5

0.58

148

0.10

76

4.8

56

5.9

151

1267

63

23

0.1

n = 7, Values presented as mean standard deviation

15

3

1.2

4

3.0

0.02

ASAT = Aspartate aminotransferase; ALAT = Alanine aminotransferase; ALB = Albumin; TRIG = Triglyceride; CAL = Calcium; T.PROT =

Total protein; PHOS = Phosphate; CREAT = Creatine; T. BILI = Total bilirubin ; D. BILI = Direct bilirubin; T.CHO = Total Cholesterol; HDL

= High density lipoprotein; LDL = Low density lipoprotein; K = Potassium; NA = Sodium; AMY = Amylase

Statistcal analysis: ANOVA, Dunnett's test

\*Mean values of groups is significantly different from control at p<0.05.

0.1

19

.02

7

0.6

6

0.4

2.4

63

26

0.2

17

3

1.8

5

5.9

0.2

0.2

25

.02

19

0.7

15

0.4

4

94

9

0.2

13

2

4

15

11

0.2

0.1

40

.03

10

0.5

7

0.3

5

36

4

94

3.5

115

56

14

39

40

0.6

0.42

81

0.12

69

3.4

55

5.7

146

1301

0.1

15

3

3

10

6

0.3

0.1

30

.02

10

0.7

9

0.2

3

49

 **CALC (mg/dl) T.PROT (g/L) PHOS (mg/dl)**

**CREAT (mg/dl) T.BIL (mg/dl)**

**URICAC**

**D.BIL** 

**T.CHO** 

**HDL** 

**LDL** 

**K** 

**NA** 

**AMY** 

**(mol/l)** 

**(mg/dl)**

**(mg/dl)**

**mg/dl)** 

**(mg/dl)**

**(mmol/l)**

**(mmol/l)** 

**(U/L)** 

aqueous extract leave of *V.amygdalina.*

HDL levels were noted in the same dose level when compared to controls.

control and assayed groups.

**3.8 Biochemical analysis** 

As herbal medicine become more popular especially in rural areas, pharmacological evidences to understand the action of these medicine and the underlying mechanisms, to support the proper and safe use of these medicine are indispensable

Our ethnopharmacology survey showed that medicinal plants are still widely used by the population in the area where the study was conducted. Several types of preparations of plants were used. The plants grow over an extended area and are used by healers separated by long distances.

In the current study, the analgesic effect of the leave extract of *Vernonia amygdalina* was assessed using three nociceptive animal models. In the writhing response model, acetic acid is injected into the peritoneal cavity of mice. The acid causes nociception in the abdomen due to the release of various substances that excite pain nerve ending (Raj, 1996). According to previous reports this assay is commonly used in mice to detect both central and peripheral analgesic efficacy of agents (Dewey, 1970; Fukawa et al 1980), *V amygdalina* showed an ability to diminish the numbers of the writhing episode in a dose-dependent manner. The results of the writhing test alone did not ascertain whether antinociceptive effects are central or peripheral.

The formalin test is considered a model for chronic pain (Duduisson and Dennis, 1977). In this test, animals present two distinct nociceptive behavior phases, which probably involve different stimuli. The first phase initiates immediately after formalin injection and lasts 3 to 5 mins, resulting from chemical stimulations of nociceptor. The second phase initiates 15 to 20 mins after formalin injection, lasts 20 to 40 mins and seems to depend on a peripheral mechanism as well as a central one. While substance P and bradykinins are involved in the first phase, histamine, 5HT, prostaglandins and bradykinin are involved in the second phase. The effect of extract was significant in both phases. Since the mechanism of the analgesic effect of *V. amygdalina* is apparent in these two models, it can however be speculated that this effect may be linked to processes in the prevention of sensitization of the nociceptor, down-regulation of the sensitized nociceptor and/or blockade of the nociceptor at peripheral and/or central levels.(Ferreira, 1990). Another possible mechanism may be that the extract blocks effect or the release of endogenous substances, including prostaglandin E2 (PGE2) and PGF2 that excites pain nerve ending which is found in writhing response test model of mice (Deraedt, et al, 1980).

The extract fails to exhibit antinociceptive effect in the tail-flick test, as values obtain were not significantly different from control animals. Pethidine (50mg/kg p.o.) the reference drug used exhibited significant antinociceptive effect in rats. It is known that the tail-flick (thermal nociceptive) response appears to be a spinal reflex sensitive to opioid -agonists and non-thermal tests to opioid -agonists (Abbott, 1988; Furst et al, 1988), furthermore thermally-induced pain is also mediated by A and C fibers. The data in the present study suggest the involvement of opioid receptors in the analgesic activity and a decrease activity of A and C fibers against inflammatory-induced activation but not thermallyinduced activation (Puig and Sorkin, 1980).

Aqueous extracts of *V. amygdalina* were found to have *in vivo* activities against *P. berghei* in mice. At 200 mg/kg the antiplasmodial activity were comparable to CQ treated mice. Empirically, this plant is used in decoction alone, other plants may be added to reduce the side effect of nausea that result from the herb's bitter taste (15).

The acute oral toxicity results from the *V. amygdalina* extract (3.32 0.15 g/kg p.o) indicate that the extract may be safe based on the chemical labeling and classification of acute

Herbal Medicine in the Treatment of Malaria:

2004a, 91: 301-308.

32, 448.

174.

chap 9

18(12)1723-1725.

2005;4:539–544.

Press, Oxford, 1990, pp.59–72.

Institutes of Health, Bethesda, MD, 1978.

Uganda 1. *J Ethnopharmacol* 2000;70: 281–300.

rabbits. *Plant Med Res* 1992;1:6–10.

and its inhibition. *Eur J Pharmacol* 1980;51:17–24.

*Vernonia amygdalina*: An Overview of Evidence and Pharmacology 185

Akah PA, Okafor CI: Hypoglycaemia effect of *Vernonia amygdalina* Del in experimental

Amacher,D.E. et al (1998) The relationship among microsomal enzyme induction, liver

Asongalem, E.A., Foyet, H.S., Ngogang, J., Folefoc, G.N., Dimo, T., Kamchouing, P.

Bailey, S. A., Zidell, R. H., and Perry R. W., (2004) Relationships between organ weight and

Deraedt R, Jougney S, Falhout M: Release of prostaglandin E and F in an algogenic reaction

Dewey, W.L., Harris, L.S., Howes, J.F., Nuite, J.A. The effect of various neurohumoral

Dioka C, Orisakwe OE, Afonne OJ, *et al*.: Investigation into the haematologic and

Dubuisson, D, Dennis SG: The formalin test: a qualitative study of analgesic effects of morphine, meperidine and brain stem stimulation in rats and cats. *Pain* 1977;4:161–174. Dubuisson, D., Dennis, S.G. The formalin test: a qualitative study of analgesic effects of

Ferreira SH: A classification of peripheral analgesics based upon their mode of action. In:

Fukawa K, Kawano O, Hibi M, Misaki M, Ohba S, Hatanaka Y: A method for evaluating

Greaves,P., (2000) *Histopathology of Preclinical Toxicity studies,* 2nd ed., Elsevier, Amsterdam,

*Guide for the Care and Use of Laboratory Animals*, revised. NIH publication No. 83-23. National

Hamill FA, Apio A, Mubiro NK, Mosango M, *et al.*: Traditional herbal drugs of Southern

Homsy J.K.E., Kabatesi D., Mubiru F., Kwamya L., Tusuba C., Kasolo S., Mwebe D., Ssentamu L., Okello M., King R., Evaluating herbal medicine for the management of Homsy J.K.E., King R., Balaba D., Kabatesi D. Traditional health practitioners are key to

Huffman MA, Koshimizu K, Ohigashi H: Ethnobotany and zoopharmacognosy of *Vernonia* 

Iwalewa EO, Adewunmi CO, Omisote NO, *et al*.: Pro-and antioxidant effects and

the medicinal properties of plant. *Proc Nutr Soc* 2003;62:371–381.

scaling up comprehensive care for HIV/AIDS in sub-Saharan Africa. AIDS, 2004

*amygdalina*, a medicinal plant used by humans and chimpanzees. In: *Compositae: Biology Utilization. Proceedings of the International Compositae Conference* (Caligary PDS, Hind DJN, eds.). The Royal Botanical Garden, Kew, UK, 1996, pp. 351–360. Huffman MA: Animal self-medication and ethno-medicine exploration and exploitation of

cytoprotective potentials of nine edible vegetables in southwest Nigeria. *J Med Food* 

analgesic agents in rats. *J Pharmacol Methods* 1980;4:251–259.

and phenylquinous tests. *J Pharmacol.Exp.Ther* 1970, 175: 435 – 442

hepatotoxic effects of rinbacin in rats. *J Health Sci* 2002;48:393–398.

weight and histological change in rat toxicology studies, *Food Chem.Toxicol*. 36, 831,

Analgesic and anti-inflammatory activities of *Ergeron floribundus*. *J Ethnopharmacol*

body/brain weight in the rat: what is the best analytical endpoint? *Toxicl. Pathol*.,

modulators on the activity of morphine and the narcotic antagonists in the tail-flick

morphine, meperidine and brain stem stimulation in rats and cats. *Pain* 1977, 4:161-

*Migraine: A Spectrum of Ideas* (Sandler M, Collins GM, eds.). Oxford University

systemic toxicity on oral LD50 values, recommended by the organization for Economic Cooperation and Development (Walum et al,1986). It has, however, been reported, that the median lethal dose is not an absolute value but is an inherently variable biologic parameter that cannot be compared to constants such as molecular weight or melting point (Oliver, 1986). The adverse signs of gaiting, reduction in stereotypic activities and deaths were however seen in high doses.

 In the sub-chronic study, the hematologic parameter shows a decrease in the RBC counts and an increased neutrophil in the treated groups. The serum chemistry parameter shows an increase in the direct and total bilirubin value. In several organs, mainly heart and liver, cell damage is followed by increased levels of a number of cytoplasmic enzymes in the blood, a phenomenon that provides the basis for clinical diagnosis of heart and liver diseases . For example, liver enzymes are usually raised in acute hepatotoxicity but tend to decreased with prolonged intoxication due to damage to the liver cells (Orisakwe et al, 2004). In this study, the extract did not exert significant effects on the serum chemistry parameters, the increased in bilirubin levels were probably due to the decrease RBC values.

 Since the traditional healers reported use of the drug as prophylactics against malaria, male rats were exposed to the extract for 6 weeks. No extract-related deaths occurred, the clinical condition of the animals, body weight gain, and food consumption were unaffected. Clinical pathology parameters (hematology, serum chemistry) exhibited no treatment-related effect. Organ weight changes can be sensitive indicators of target organ toxicity, and significant changes in organ weights may occur in the absence in changes in other pathology parameters (Bailey,S.A., 2004 ), for example, increased liver weight associated with hepatic cytochrome P450 induction is a common finding in toxicology studies. Liver weight increases of up to 20% relative to control without microscopic evidence of hepatocellular hypertrophy or changes in serum chemistries (Amacher, et al., 2000). Similarly modest doserelated changes in kidney weight commonly occur in toxicology studies without histopathologic evidence of cellular alteration (Greaves, P. 2000).

In conclusion, the results of this study showed analgesic activity of the extract with clear and significant antiplasmodial effects in mice, no indication of toxicity in rats, incidental findings below or above standard reference levels were all within control values based on historical reference ranges. This might explain the pharmacological basis for the successes in pain and malaria treatment claimed by traditional healers who use *V. amygdalina.*
