Trypanocidal effect of ethanolic leaf extract of khaya nyasica




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IN VIVO TRYPANOCIDAL EFFECT OF ETHANOLIC LEAF EXTRACT OF Khaya nyasica Stapf
V Mugoyela and S Mung’ong’o


Summary
The ethanolic and aqueous crude extracts of Khaya nyasica Stapf. (Meliaceae) leaves and stem bark were screened for their trypanocidal effects against Trypanosoma brucei brucei in mice. At a single 25mg dose, the ethanolic leaf extract demonstrated trypanocidal activity by reducing parasitaemia from day 8 post treatment. There was no observed reduction of parasitaemia in solvent treated and untreated mice. The best trypanocidal effect was achieved at the same single 25mg dose when treatment commenced simultaneously with inoculation of trypanosomes by delaying development of parasitaemia by three days and reducing it from day 9 post treatment. Further bio-guided fractionation studies of ethanolic fractions is recommended to evaluate the observed trypanocidal effect of the plant and identify the active principle.
Key words: Khaya nyasica, Trypanocidal, Parasitaemia,

T.b. brucei.
Introduction
Trypanosomiasis or sleeping sickness is a group of closely allied diseases of man and animals caused by infection with protozoan species of the genus Trypanosoma. The East African sleeping sickness is caused by a parasite named Trypanosoma brucei rhodesiense and the West African type also known as Gambian sleeping sickness is caused by T.b. gambiense. Worldwide, 20,000 new cases of African trypanosomiasis are reported each year. The prevalence of the infection ranges between 250,000 to 300,000 people worldwide.(1,2) In the sixties the disease remained rare and was considered eradicated but it re-emerged in the 1980’s as a result of socio-economic changes that ranged from lack of integration of screening and treatment to exclusion of any vertical control system that was formerly in place.(3) Treatment of African human trypanosomiasis is limited to a small number of old, expensive, and toxic medicines (suramin, pentamidine, nitrofurans and arsenicals), some of which would not pass today’s standards for drug safety.(4) Recent drug developments have provided DL--difluoromethylornithine (DFMO) or eflornithine as the only new trypanocidal medicine.(5) The medicine is used for the late stage Gambian sleeping sickness. Thus, there is need for development of new drugs and formulations to combat this fatal disease. As such, one avenue of search for medicines against African trypanosomiasis is screening of plant extracts for their trypanocidal effects. Of recent, several plants have been screened for their trypanocidal activity both in vivo and in vitro.(6,7) Khaya species namely K. grandifoliola and K. senegalensis were among of the plants that demonstrated trypanocidal activity against T. b. brucei in vitro. Various parts of Khaya species have been reported to have a variety of curative effects. In Tanzania, K. nyasica is found widely distributed in forests along the coast.
Correspondence to: Mugoyela V, Box 64013, Muhimbili University College of

Health Sciences, Dar es Salaam, Tanzania


Dept. of Medicinal Chemistry, School of Pharmacy
Todate, several species of khaya have been screened for trypanocidal activity, K.nyasica Stapf.ex Baker f. (Meliaceae) has not been screened for antitrypanosomal activity.(6,7) Thus in this paper we report preliminary in vivo antitrypanosomal activity of the ethanolic leaf extract of the plant against T.b. brucei, a morphologically indistinguishable relative of T.b. rhodesiense and T.b. gambiense.
Materials and Methods
Plant material
Plant material was collected in Tanzania in May 1998, from Pugu forest in the Dar es Salaam region. The plant was identified by a botanist from the Department of Botany, University of Dar Es Salaam and a voucher specimen was kept there.
Animals
Male and female albino mice weighing 17- 24g were obtained from a small animal unit of the Muhimbili University College of Health Sciences.
Parasites
The population of trypanosomes used in this study were T.b. brucei IL4159 (Iltat1.1) blood stabilates initially derived from T.b.brucei IL3303 (Iltat1.1) from the International Livestock Research Institute (ILRI) Kenya in January 1999. The T.b.brucei IL4159(Iltat1.1) blood stabilates were kept under liquid nitrogen at Temeke Veterinary Center in Dar es Salaam.
Extraction
An amount of 100g of each of powdered dried leaves and stem bark of K. nyasica was separately added to 500ml of distilled water and boiled for 4 hours and filtered while hot through a Whatman No.1 filter paper. The resulting extract was concentrated on a water bath to dryness and then freeze-dried. Ethanolic extracts were prepared by adding 500 ml of 90% v/w ethanol in water, to 20 g of powdered leaves or stem bark and the mixture left at room temperature for 72 hours. The resulting extract was filtered through a Whatman No. 1 filter paper. The extract was concentrated using a rotary evaporator at 400C until a gummy paste was produced and stored in the refrigerator.
Parasites resuscitation
A sample of T.b.brucei IL4159(Iltat1.1) blood stabilate was resuscitated by mixing with 0.4ml Phosphate buffered-Saline-Glucose (PSG) solution (PH 8, ionic strength 0.145, Batch No. 401 ILRI) in a 1ml syringe. The presence of viable T.b.brucei was checked under a light microscope at X250 magnification. Trypanosome population for the experiment was obtained from mice infected by injecting intraperitoneally (i.p.) with the resuscitated parasites in 0.2ml of Phosphate buffered-Saline-Glucose and maintained by passaging.
Infection
A total of 18 mice (17-24 g) were grouped into 6 groups of 3 mice each and put in different metal cages. They were fed and given drinking water freely. A drop of blood from the tail of a pre-infected mouse by T.b.brucei was diluted with 0.5 ml of buffered Phosphate-Saline-Glucose solution. Before infecting other mice, the presence of 4-5 viable parasites per one microscope field was confirmed under a light microscope at X400 magnification. Each experimental animal was then infected with T.b.brucei through i.p. injection of 0.1ml of buffered solution containing parasites. Parasitaemia was monitored everyday by microscopic examination of wet smears of tail blood.
In vivo tests
Treatment with the plant extracts was started immediately after animals became parasite positive on the third day after infection. Three different doses of 25mg, 50mg and 75mg from each extract were prepared by dissolving in 0.5ml, 1.0ml, 1.5ml of ethanol respectively and administered intraperitoneally as single doses. Six groups of mice were involved in testing each extract. Mice in group 1-3 received single 25mg, 50mg and 75 mg doses, respectively. Mice in group 4 received a single 25mg dose simultaneously with parasite inoculation. Mice in group 5 received 0.5ml, 1.0ml and 1.5ml of ethanol, respectively (as solvent control) and group 6 remained untreated. Parasitaemia was then examined daily by microscopic examination of a drop of tail vein blood.

The number of swarming trypanosomes per microscope field was counted using a Tally Counter. For each parasitaemia determination, several fields were examined by counting the number of parasites and a mean recorded. The number of trypanosomes per field was then matched with figures in a table designed by ILRI (appendix 1) and the estimated number of trypanosomes per millilitre of blood was obtained. The process of examining parasitaemia continued daily until death of experimental animals.


Results
In this study it was found that aqueous extracts from leaves, stem bark and ethanolic extract from stem bark had no effect on parasitaemia in mice since it resulted in the death of mice within 4-5 days after treatment. On the other hand, mice treated with ethanolic leaf extract at a single 25mg dose (group 1) had parasitaemia reduced from day 8 to 9 post treatment. This was followed by recurrence in parasitaemia killing the mice by day 13 (Table 1). Mice treated with a single 50mg dose (group 2) of ethanolic leaf extract showed a slight reduction in parasitaemia from day 7 to 8 post treatment and followed by an increase in parasitaemia that killed the mice by day 11. Mice in group 3 died within 4-5 days post treatment without showing a reduction in parasitaemia. Table 1 shows that mice in group 4 in which treatment at a single 25mg dose commenced simultaneously with infection, had a delay in development of parasitaemia for 3 days compared to mice in group1. Further, results in group 4 indicate a slow increase in parasitaemia followed by reduction from day 9 and then a subsequent recurrence by day 11. Results in the control group 5 with solvent treated and group 6 with untreated mice show a consistent increase in parasitaemia causing death within 4-5 days after infection. However all mice were dead by the 15th day post treatment time.

Table 1. Parasitaemia in infected mice treated with a single dose of the K. nyasica ethanolic leaf extract and the controls




PTT

Mean number of swarming trypanosomes per microscope field

Ethanolic leaves extract

Controls

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

m1

m2

m3

m1

m2

m3

m1

m2

m3

m1

m2

m3

m1

m2

m3

m1

m2

m3

0

3

4

4

3

4

6

3

3

4

0

0

0

2

2

2

5

3

2



4

5

4

1

3

10

4

3

5

0

0

0

3

2

5

5

3

2



12

13

14

2

10

50

20

19

13

0

0

0

28

19

22

28

32

20



28

26

32

6

15

D

29

34

19

4

0

1

40

>100

50

50

48

D



36

28

40

12

19




56

D

D

28

2

16

50

D

D

D

70






42

32

46

20

24




D







12

18

18

D










D






48

49

43

36

33













38

20

28





















36

35

D

28

70













46

32

48





















0

15




18

56













26

22

36





















0

13




21

D













24

0

D





















6

D




40
















D

0
























12







D



















9
























25




























15






















13.

32




























28






















14.

D




























30






















15.































D























PTT= Post treatment time; D = dead; Swarming >10, estimated trypanosomes ml-1 of blood is 5x105; Swarming 1-10, estimated trypanosomes ml-1 of blood is 104-5x105; Swarming >100, estimated trypanosomes per ml-1 of blood is 5x106.

Appendix 1


SCORE

Trypanosomes/Field*

Estimated Parasitaemi

A(trypanosomes ml-1)

6+ Swarming

>100

> 5 x 106

5+ “

>10

> 5 x 105

4+ “

1 – 10 per field

104 – 5 x 105

3+ “

1 per 2 fields –1 per 10 fields

5 x 103 – 5 x 104

2+ “

1 – 10 per preparation

103 - 104

1+ “

1 per preparation

102 - 103


* Magnification = X 250

Discussion

This work is a preliminary study which was conducted to screen for trypanocidal activity of the aqueous and ethanolic extracts of K. nyasica leaves and stem bark. The effects were compared between the treated and untreated mice as controls. Only the ethanolic leaf extract of the plant demonstrated best trypanocidal effect in mice at a single 25mg dose. This study showed interesting results because among the four different types of extracts that is aqueous leaf and stem bark extracts, ethanolic leaf and stem bark extracts, only the ethanolic leaf extract was found to be active on T.b brucei in mice. Freiburghaus et al showed that different parts of a plant could demonstrate varying activities(1). Results of this study demonstrated that a single 25mg dose of ethanolic leaf extract (Table1) caused reduction of parasitaemia from day 8 post treatment. This reduction was followed by emergence and concomitant steady rise in parasitaemia from day 10 until death of the mice by day 14.

The ethanolic leaf extract that was given simultaneously with infection at the same single 25mg dose seemed to delay development of parasitaemia up to 3 days when compared to other groups of mice (Table1). This observation is similar to what has been reported in the literature on effects of Morinda lucida leaf extract against T.b.brucei in mice.(8) Further, this study revealed a much slower rate of increase and reduction of parasitaemia from day 9 to 10 and then recurrence and increase in parasitaemia until death of the experimental animals. This has demonstrated the ability of ethanolic leaf extract to protect infected mice against T.b.brucei. Mice treated with a single 50mg dose of ethanolic extract, showed a slight reduction of parasitaemia, however they died earlier than those treated with half of the dose (goup1mice). At a higher single 75mg dose, there was no observed reduction in parasitaemia. However at this dose, immediately after administration of the extract, animals exhibited certain toxic reactions including dizziness (mouse circling at same position), increase in respiration rate and temporary paralysis for few minutes but they did survive and death was recorded starting the second day post treatment. At the moment, some questions from this study need to be addressed. Although the study indicated that the ethanolic leaf extract reduced parasites from the blood, relapse was prompt and eventually led to death of the mice. Post-chemotherapy recurrence of parasitaemia in animals infected with trypanosomes has been reported in literature.(9) This could be explained in terms of re-invasion of blood by trypanosomes from the central nervous system or rapid metabolism of the active principle(s). Another question which need to be addressed is failure to reduce parasitaemia at higher doses. Could perhaps a reason be that the plant is more toxic at a higher dose. A similar trend of activity was observed by Okanla et al in a study of in vivo trypanocidal effect of an aqueous leaf extract of Acalypha hispida.(10) At this juncture in our study it is difficult to speculate the reasons for the differences in the effect of aqueous vis-à-vis ethanolic extraction on trypanosomes in mice. However these results corroborate the findings of Freiburghaus et al that clearly indicated that different solvent extracts of the same plant may exhibit different trypanocidal activity.(1) Nevertheless in this study the difference in activity between aqueous and ethanolic extracts can also be explained in terms of the different methods of extraction and drying used. Higher temperatures used in aqueous extraction and concentration could have caused decomposition of the active principles. Furthermore, the active principles may be more soluble in ethanolic than aqueous media.

Solvent-treated mice did not show signs of toxicity and died within 4-5 days revealing high levels of parasitaemia similar to the untreated mice. This study showed that the solvent had no therapeutic effect regardless of the volume administered. With these results, any conclusive attribution of activity and toxicity shown should be deferred until isolated compounds can be tested. The actual trypanocidal principle in the extracts of K. nyasica was not determined. This aspect requires further study.


Conclusion


The results of this study showed that the ethanolic extract of K. nyasica leaf had some trypanocidal effects and the best effect was observed when treatment commenced simultaneously with infection. However no concrete conclusion on the effectiveness of the extract, can be made now until isolated active compounds are tested.


Acknowledgements
We appreciate the technical training extended to one of us (V.M.) at ILRI on how to handle the trypanosomes. We are also indebted to ILRI for providing us with trypanosome strains that were used in the study and Temeke Veterinary Centre for storing the trypanosome strains. This study was supported by a grant from the Research and Publications Committee, Muhimbili University College of Health Sciences.

References



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  2. World Health Organization (WHO) 1994; Press Release WHO/73.

  3. Louis FJ, Simarro, PP, Lucas P. Sleeping sickness: a century of evolution of control strategies. Bulletin de la Société de Pathologie Exotique 2003; 95: 331-336.

  4. Fairlamb AH. Future prospects for the chemotherapy of human Trypanosomasis. 1. Novel approaches to the chemotherapy of trypanosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 1990; 84: 613-617.

  5. Van Nieuwenhove S, Schechter PJ, Declercq J, Boné G, et al. Treatment of Gambiense sleeping sickness in the Sudan with oral DFMO (dl-- difluoromethylornithine), an inhibitor of ornithine decarboxylase; first field trial. Transactions of the Royal Society of Trop Med & Hyg 1977; 79: 692- 698.

  6. Atawodi SE, Bulus T, Ibrahim DA, et al. In vitro trypanocidal effect of Methanolic extract of some Nigerian savannah plants. African Journal of Biotechnology 2003; 2: 312-321.

  7. Owolabi OA, Makanga B, Thomas EW, Molyneux DH, et al. Trypanocidal potentials of African wood plants; in vitro trial of Khaya grandifoliola seed extracts against Trypanosoma brucei brucei. Journal of Ethnopharmacology 990;30: 227-231.

  8. Basson W, Page ML, Myburgh DP. Human trypanosomasis in southern Africa. South Africa Medical Journal 1977; 51: 453-457.

  9. Evans DA, Brightman CAJ. Pleomorphism and the problem of recrudescent pasasitemia following treatment with salicylhydroxamic acid (SHAM) in African trypanosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 1980; 74: 601-604.

  10. Okanla EO, Owayale JA, Akinyanju JA. Trypanocidal effect of an aqueous extract of Acalypha hispida leaves. Journal of Ethnopharmacology 1990; 29: 233-237.


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