267
I.A. Ibrahima,
S.A. Khalidb,
Sawsan A. Omer” and S.E.I. Adam”
(Accepted August 27. 19911
Brown Misex chicks were fed diets containing 2% and 5% Azadirar&a indict te& from their 7th to 35th day of age. Thereafter. the chicks were fed control diet for 2 weeks, A depression in body weight gain and efficiency of feed utihzation was observed in chicks fed A. ~&XJ leaf when compared with the control. The main clinicopathalogical changes were increases in lactic dehydrogenase, glutamic oxalaacetic transaminase and alkaline phosphatase activities and uric acid and bilirubin concentrations and decreases in the total protein levels in serum. Changes in the values of erythrocyte count, haemoglobin concentration. packed cell volume, mean corpuscularvolume and mean corpuscular haema$lobtn were remarkable and assaciated with yellow discoloration on the legs and combs and hepatonephropathy. Tissue recovery was incomplete 2 weeks after removal from the experimental diets. Key wurdts: ~~~d~r~~h~u &d&a;
toxicity; chicks; antimalarial agent; clinicopathology
Introdlldon
Recently, there has been a revival of interest in herbal anti-malarial preparations to combat the problem of chloroquine resistant Plasmodium falciprum strains. The current interest in Azadira&a indica A. Juss., (Meliaceae) as a potential antimalarial candidate demonstrates the validity of the traditional medicinal approach. The leaf extract has been widely used in Sudan and other African countries as an antimalarial agent. However, a number of cases of toxicity arising from over”~onsum~tion of A. &dim leaf extract have been reported in the Sudan after the dramatic outbreak of malaria as a consequence of the 19X7 flood disaster, due to the severe shortages in drug supplies and alleged chloroquine resistance (Khalid, unpublished data). The principal danger in the traditional use of A. indica extracts appears to be the lack of sufficient toxicological investigations on both the crude extract and the anCorrespande*rre io: S.A. Khahd, Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, P.O. Box 1996, Khartoum, Sudan.
t~ma~arial bioactive substan~e(s~. Although there are a number of reports addressing various pharmacological activities (e.g., Van Der Nat, 1987; Prakash et ah, 1986; Abatan and Makinde, 1986) and voluminous literature is available on the chemistry of A. indica (Connolly, 1983), very meager data exist regarding the toxicology of this taxon (Ali and Salih, 1982; Ali, 1987). Particular attention has been directed recently to the a~tirnal~r~a~ activity of A. in&a (Tella, 197% Obih and Makinde, 1985; Qbaseki and Jegede-Fadunsin, 1956). The in vitro antimalarial activity of the main bioactive agents has been demonstrated by Khahd et al. (1986) and its detailed chemiea1 structure has already been estabhshed as nortriterpenoidal derivative, gedunin (Khalid et related anal., 1989). Another structurally timalarial compounds known as nimbolide has been identified by Rochanakij et al. (1985). However, gedunin seems to be the most potent antimalarial and yet the least cytotoxic compound among all the limonoids so far isolated from A. indiea (Khalid et al., in preparation). We consider the selection of chicks as a biological model for this toxicological studies as
037~~~~41/92/~~S.O~ 0 t992 Etsevier Scientific Publishers treland Ltd Primed and Published in ireland
quite appropriate since they are very susceptible malarial infection. The adequacy of chicks as an vivo model for testing the antimalarial activity plant extracts has long been demonstrated Spencer et al. (1947).
to in of by
Materials and Methods Chicks and diets Thirty-six l-day-old Brown Hisex chicks of either sex were housed in the premises of the Faculty of Veterinary Science, University of Khartoum under illumination at night and early morning with feed and water provided ad libitum. At the age of 7 days, the chicks were allotted to three groups of 12 each. Group 1 chicks were controls and fed a starter diet. Leaves of A. indica were collected from the Mogran area in Khartoum and a herbarium specimen was deposited at the Faculty of Pharmacy, University of Khartoum. Leaves were dried in the shade for 2 weeks, ground, added to the mash and fed at dietary concentration of 2% (group 2), or 5% (group 3). Feeding was continued for 4 weeks. Thereafter, the test diets were withdrawn and replaced by the control mash for 2 weeks (recovery period). Mean body weights, weight gain and feed conversion ratios (kg feed/kg gain) were determined for each group weekly. Three chicks from each group were killed for pathologic examination and blood samples were collected at weeks 1, 3, 4 and 6. Histopatho~ugica~ methods Chicks sacrificed were examined for gross lesions. Tissues were fixed in IO%, formal saline, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (H and E). Chemical methods Serum samples were analysed for glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), total bilirubin and uric acid using commercial kits (Boehringer Mannheim GmbH Diagnostica, F.R.G.). The concentration of serum total protein was measured by refractometry.
The erythrocyte count (RBC), packed cell volume (PCV), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) were estimated by standard methods (Schalm, 1965). Statistical analysis Statistical significance was assessed by Student’s t-test (Mendenhall, 1971). Results Effect on growth The effect of 2% or 5% dietary A. indica leaf on body weight, weight gain and feed conversion ratio in Brown Hisex chicks is given in Table I. The results were compared with those in the control group. Statistical analysis of the data indicated that A. indica leaf fed in the diet at 2% or 5% for 4 weeks significantly depressed body weight (P < 0.05-0.02~, weight gain fP < 0.0.5-0.01) and efficiency of feed utilization (P < 0.05-0.001) in chicks of groups 2 and 3 when compared to the control chicks of group 1. At the end of the recovery period, the body weight of chicks in group 3 was lower (P < 0.01) than that of birds in groups I and 2. Efficiency of feed utilization in the control group was significantly higher (P < 0.001) than the test groups 2 weeks after removal from the experimental diet. Yellow discoloration on the combs and legs of chicks in groups 2 and 3 appeared on day 7 and became intense during the period between days 20 and 28. Following withdrawal of the test diets, the intensity of yellow colour gradually decreased and it was no longer seen on the comb by the end of the recovery period. No other clinical signs were observed. Changes on serum constituents The results of serum chemistry for the chicks which consumed 2’%,(group 2) or 5% (group 3) of the basic diet are presented in Table 2. Significant differences (P < 0.05-0.501) in uric acid, bilirubin, total protein, GOT, ALP and LDH values were observed between the test and control
269
TABLE
1
GROWTH
CHANGES
FED A. iNDlCA
IN CHICKS
LEAF
Feeding
Group No
Recovery Week 2
Week
458 f 11.8 (P < 0.01)
219 * 9.4 (P < 0.05) 214 f 2.9
336 f 11.6 (P < 0.05) 275 f 10.6 (P < 0.05) 265 zt 12.9
61.5 f 3 N.S. 56.5 + 2.5 N.S. 52.5 f 3.2
87.2 (P < 68.3 N.S. 15.0
83.6 (P < 55.6 (P < 57.9
f
122. 5 zt 6 (P < 0.01) 85 f 4 (P < 0.001) 44.2 f 7
43.4 f 3 (P < O.OOl) 95.0 f 5
2.4 f 0.2 N.S. 3.1 * 0.3 (P < 0.001)
2.1 (P 3.8 (P 5.2
Week 2
I
131 zlz 6 (P < 0.05)
165 N.S.
2
95 f 8 (P < 0.05) 86 f 5.9
151 * 4.1 (P < 0.02) 139 f 3.5
Mean weight gain 46 zt 2.9 1 N.S. 36 f 3.1 2 (P < 0.01) 28 zt 4 3
3
I
Week 4
Week 3
Week 1
l
253 f N.S.
5.9
14.7
f
4 0.05) zt 3 f
2
360 + 8.2 (P < 0.001) 310 f 8.5
5 0.01) f 3 0.01) f 4
502 zt I.5 N.S. 4.55 f 18 (P < 0.01) 392 f 7
(P < 0.001) 81.7 f 3.5
Mean feed conversion ratio 1
2.3 zt 0.1 (P < 0.01)
2.2 * 0.2 N.S.
2.3 z+z0.1 (P < 0.02)
2
3.1 f N.S.
0.09
2.8 f 0.1 (P < 0.05)
3.0 f 0.15 (P < 0.001)
3
2.4 f
0.12
3.2 zt 0.3
3.8 f
Group
1 = control;
TABLE
group
2 = 2”/u; group
0.12
f < f < 5.1 f
2.0 (P 4.9 (P
3 = 5%; N.S. = not significant;
0.12 0.001) 0.3 0.001) 0.4
7.5 f 0.4
P = level of signilicance
f < * < f
0.1 0.001) 0.2 0.001) 0.4
below or above control
values.
2
SERUM
CHANGES
Group
IN CHICKS
GOT
FED A. INDICA
GPT
LEAF
LDH
FOR 4 WEEKS ALP
T.P.
Bilirubin
Uric acid
0.48 zt 0.03 (P < 0.001) 0.79 l 0.02 (P < 0.01) 0.62 f 0.04
4.3 f
0.1
(P 6.1 (P 6.7
< f < j,
0.001) 0.2 0.001) 0.18
0.47 f 0.02 N.S. 0.66 f 0.04 (P < 0.001) 0.8 f 0.04
4.1 (P 6.8 (P 7.2
f 0.16 < 0.001) f 0.18 < 0.001) f 0.2
NO.
Feeding
2 3 Recovery 1 2 3 Group
f
18.7 zt 0.5
1138 zt 35
280 zt 12
3.2 f 0.4
42.3 zt 0.5
(P < 0.03) 16.3 zt 0.4 N.S. 17.3 f 0.4
N.S. 1195 f 37 (P e 0.02) 1347 rt 38
(P < 0.001) 340 * 13 (P < 0.001) 362 f 11.8
(P 2.1 (P 2.3
18.9 f 0.6 (P c 0.05) 16.9 ztz 0.5
1140 f N.S. 1040 f
377 l 11 (P < 0.01) 309 * I3
2.9 f N.S. 2.6 f
N.S. 17.1 f
(P c 0.01) 1350 f 41
(P < 0.05) 338 f 10.5
(P < 0.05) 2.3 f 0.2
15.7 (P < 42.7 (P c
1
0.3 0.001) f 0.9 0.001)
15.9 f 0.2 N.S. 17.4 f 0.3 N.S. 17.6 f 0.2
1 = control;
group
0.5
2 = 2%; group
40 38
3 = 5%; N.S. = not sign&ant;
< * < f
0.05) 0.6 0.02) 0.3
0.3 0.5
P = level of significance
below or above control
values.
270
groups. At the end of the 2-week recovery period, the concentration of serum uric acid was higher (P < 0.01) and the activity of ALP was lower (P < @OS-0.01) in the test groups than the control group. The values of total protein and GOT returned to normal and LDH activity was higher in group 3 than group 1.
The values of Hb, PCV and RBC were lower (P < 0.05-0.01) in the test groups than the control group (Table 3). MCV did not change but MCH and MCHC were higher (P < 0.001) in group 3 than groups 1 and 2. At the end of the recovery period, the value of RBC, PCV and Hb were within the normal range.
The spleen was enlarged in some birds, the yellow liver showed fatty cytoplasmic vacuolation and focal necrosis of the hepatocytes (Fig. I) and lymphocytes nodules at the portal tracts. In the kidney, there was degeneration or necrosis of the epithelial cells of the convoluted tubules and shrinkage of the glomerular tufts (Fig. 2). The blood vessels of the heart and intestines were congested and petechia1 or ecchymotic haemorrhages with or without small erosions on the intestinal mucous membrane were seen. At the end of the recovery period, the damage to the vital organs was reduced but some of the cells of the renal tubules and the hepatocytes had not completely returned to normal. No lesions were seen in the controt chicks.
Pathological findings
Discussion
In chicks fed dietary 2% (group 2) or 5% (group 3) A. indica leaf, there was yellow discoloration on the combs, shanks, reflected skin, breast and thigh muscles, fiver and body adipose tissue. This was more marked in birds fed 5% dietary A. indica leaf at weeks 3 and 4.
To date no research has been done to deiineate toxic effect of neem (A. indim) products in chicks. As the length of time on experimental diets (2% and 5% A. indica leaf meals) increased from 1 week to 4 weeks, the difference in chick weights among
TABLE
3
HAEMATOLOGICAL Group
Feeding 1 2 3
RBC (x IO6 mm’)
CHANGES
IN CHICKS
FED A. i/VDlCA
Hb (g/100 ml)
PCV (‘XI)
2.12 ziz 0.06
29.0 i
(P < 0.05) 1.9 f 0.5 (P f O.OI) 1.73 ct 0.03
(P < 0.05) 265 i 0.2 (P < 0.01) 25.3 f 0.5
0.4
2.20 zt 0.05 N.S. 2.23 * 0.04 N.S. 1.99 f 0.04
29.2 f. 0.6 N.S. 27.0 i 0.4 (P < 0.05) 25.5 f 0.1
9.1 f
0.1
LEAF
FOR 4 WEEKS
MCV
MCH
(firn’)
(*;Pg)
MCHC (‘%I)
136.8 z+z2.9
42.9 f
8.3 l 0.2 (P < 0.05) 8.6 zt 0.15
N.S. i39.5 f 3.1 N.S. 146.2 + 2.5
MS. 45.3 f I.1 (P < 0.001) 54.3 f 0.9
N.S. 32.5 f 0.4 (P < 0.00~) 37.2 f 0.7
9.2 f N.S. 9.0 f N.S. 8.8 f
137.0 N.S. 135.7 (P < 120.8
43.1 zt 1.3 N.S. 40.5 f 0.9 N.S. 43.2 f 1.2
31.6 f 0.5 N.S. 31.9 rt 0.3 (P < 0.05) 33.4 * 0.2
(P < 0.059
1.2
31.4 xk 0.6
Recovery
1 2 3
Group No. I = control; control values.
Group
0.2 0.1 0.3
No. 2 = 2%; Group
f 0.2 f 2.7 0.02) + 0.29
No. 3 = 5%; N.S. = not significant;
P = level of significance
below or above
271
Fig. I. Necrosis
of the hepatocytes
in a chick fed So/u dietary
A. indica leaf for 28 days.
the groups became increasingly larger demonstrating that the levels of the test diets are responsible for reduction in body weight. The fact that weight gain and feed conversion ratio were decreased in the test chicks suggest that A. indict leaf contains toxic substance(s) that impaired growth.
Fig. 2. Degeneration
and necrosis
of the cells of the renat tubules
H and E, x 100
The elevated activity of LDH and GOT and decrease in the concentration of total protein in the serum of Brown Hisex chicks fed A. indica leaf for 4 weeks indicate severe Iiver damage. The increase in the concentration of bilirubin and in the activity of ALP associated with the development of yellow discoloration of the legs, combs, re-
in a chick fed 5% A. in&u
for 28 days. f-t and E, x 100.
272
fleeted skin, liver and adipose tissue of the birds on A. in&a leaf meal are suggestive of hepatic excretory dysfunction. The intensity of yellow discoloration might, however, suggest that the severe damage of the kidney resulted in retention of some pigments in the circulation. The process of pigments degradation, absorption and excretion in chicks is nat well known (Sturkie, 1965). The pigment fraction of A. indica leaves consists mainly of quercetin-based flavonoid glycosides and carotenoids (Khalid, unpubi~shed results) along with chlorophylls. Some information exists about the process of flavonoids interaction with isolated enzymes, cell constituents and membranes and the deposition of these pigments in organs, tissues and biological fluids (Griffiths, 1982). However, there is a high degree of variation regarding the pharmacokinetics and site of deposition of the flavonoids and their metabolites. This variation depends to a large extent on the route of administration and the intestinal microflora involved during oral administration. Bihary excretion has been established as a major route of excretion of many tlavonoids (Griffiths, 1982). However, very limited information is available concerning the subsequent fate of the conjugate entering the intestine. Some of the flavonoids such as quercetin-based glycosides which is present in A. in&a leaf extract were not detectable in the urine after oral administration to man (Griffiths, 1982). Therefore, the deposition of quercetin-based glycosides and/or their metabolites is quite feasible interpretation for the discoloration encountered during the present experiments, The increase in the activity of GOT and in the concentration of uric acid and hypoprotcinaem~a are suggestive of nephrotoxic properties of neem bioactive constituent(s) and/or their respective metabolites. It has been shown that A. indicu leaf extract causes a hepatobiliary diseases by raising 5’-nucleotidase (5’-NT) to abnormally high levels (Komolafe et al., 1988). The possible role of mixed-function oxidases (MFO) in the hepatobiliary toxicity of A. indica has been investigated by Komolafe et ai. (1988). Their results tend to indicate the involvement of MFO and that the metabolites, and not the parent constituents of the extract, are responsible for the toxicity involved.
In the present study, significant increases in serum uric acid concentration corroborate the findings of Omer (1990) and Ayed et al. (1989) confirming changes in glomerular filtration. Analysis of the parameters suggest that uric acid concentration would be useful dia~ostically because of uniform changes along with pathologic features of renal toxicity. The hepatonephrotoxicity was severe enough to account for the reduced growth rate in the test chicks and that associated lesions were consistent and incompletely reversible at the end of a 2-week recovery period. There were increases in MCV and MCH and decreases in Hb, RBC and PCV in chicks fed A. indica leaf for 4 weeks. These findings suggest macrocytic anaemia. We conclude that concentrations of 2% and 5% of A. &dim leaf in the basal diet are toxic to Brown Hisex chicks and that toxicity is mainly associated with hepatonephrotoxic lesions.
This work was supported by Band Aid (U.K.) grant: ‘Species of Sudanese Meliaceae as a Source of Novel Antimalarial Agents’. Thanks are due to the NEF (U.S.A.) for administering this project. References Abatan, M.O. and Makinde, M.J. (1986) Screening Azudiracru indica and Pisum safivum for possible antimalarial activities. Journai of E~~~opha~~ac~logy
17, 85-93.
Ali, B.H. (1987) The toxicity of Azudirachtu indicu in goats and guinea pigs. Yete,inarj, and ~~~ ToxicobgOy 29, 15- 19. Ali, B.H. and Safih, A. (1982) Suspected toxicity of Az#diracZ$a indica in sheep. Yeierinary Records 1t2, 494. Ayed. I.A.M., Dafalla, B. and Adam, S.E.I. (t989) Effects of various levels of dietary vitamine E on broiler chicks. Veterinary and Human Toxicology 31, 50. Connolly, J.D. (1983) Chemistry of the limonoids of the Meliaceae and Cneoraceae. In: P.G. Waterman and M.F. Grundon (Eds.), Chemisrry and Chemical Taxonomy of the Rurales. Academic Press, London and New York. Grifiths, L. (1982) Mammalian metabolism of flavonoids. In: J.B. Harborne and T.M. Mabry (Eds.), The Flavonoids Advances in Research. Chapman and Hall. London and New York. Khafid, S.A., Farouk, A., Gary, T.M. and Jensen, J.3. (L986) Potential anti malarial candidates from African piants: an in vitro approach using ~~as~~~~~ fair&vwm. ..fownai of Erhnophartnacology
i 5, 201-209.
Khaiid, S-A., Duddeck, H. and Gonzalez-Sierra, M. t1983) Isolation and characterization ofan ant~rna~r~aI agent ofthe neem tree /Iz~&~c&a indica. ~a~~~ufa~~a~~~uf Products 52, 922-927. Komolafe, O.O., Anyabuike. CF. and Obaseki, A.O. (19881 The possible role of mixed-function oxidases in the he~atabiliary toxicity of Aradirarhru indicu. Flturerapiu 59, 109-113. Mendenhall, W.S. ( 197 I) Inrroduction 10 Prahahilir)? and Swisries. 3rd Edn Wadsworth Publishing Co, Inc., CA. Obaseki, 0. and Jegeda-Fadunsin. HA. (1986) The antimalarial activity of A-_ndira~hra inu’ifdl. ~~r~~~r~~if~17, 247-251. Ubih, P.Q. and Makinde, J.M. (1985) Effect of ~~u~~r~e~~~u indira on Ffasmad~~m berg&i in mice. Africorr hmai of Medicine and Medicaf Sciences 14, 5I-54.
Prakash, A.D. and Mat&r, R. (1986) Interceptive plants. P&z@iu Me&c@ 52, 432. Spencer. CF., Koniuszky, F.R., Rogers, E.F., Shavei. J., Jr., Easton. N.R., Kaczka, E.H., Kuehl, F.A., Jr.. Pbiliips, RF., Walti, J., Folkcrs, K., Malanga, C. and Seeler. A.O. (1947) Survey of plants for antimalarial activity, L!~_).x& 10, 145-175. Tella, A. (1976) ‘The effects of A-_adiracfztu indica in acute Plasmodium berghei malaria. Wesr African Jowwf as Pharmacology and Drug Research 3, 80.
Van Der Nat, J.M., Klerx, J.P.A.M. . Van Dijk, H., De Silva, K.T.D. and Labadie, RI’. { 1987) Immunomodulatory activity of an aqueous extract of A:adiratfa in&u stem bark. Joirrn& of ~r~~ap~a~rnacafagy 19, I 25- 13I.
I.A. Ibrahima,
S.A. Khalidb,
Sawsan A. Omer” and S.E.I. Adam”
(Accepted August 27. 19911
Brown Misex chicks were fed diets containing 2% and 5% Azadirar&a indict te& from their 7th to 35th day of age. Thereafter. the chicks were fed control diet for 2 weeks, A depression in body weight gain and efficiency of feed utihzation was observed in chicks fed A. ~&XJ leaf when compared with the control. The main clinicopathalogical changes were increases in lactic dehydrogenase, glutamic oxalaacetic transaminase and alkaline phosphatase activities and uric acid and bilirubin concentrations and decreases in the total protein levels in serum. Changes in the values of erythrocyte count, haemoglobin concentration. packed cell volume, mean corpuscularvolume and mean corpuscular haema$lobtn were remarkable and assaciated with yellow discoloration on the legs and combs and hepatonephropathy. Tissue recovery was incomplete 2 weeks after removal from the experimental diets. Key wurdts: ~~~d~r~~h~u &d&a;
toxicity; chicks; antimalarial agent; clinicopathology
Introdlldon
Recently, there has been a revival of interest in herbal anti-malarial preparations to combat the problem of chloroquine resistant Plasmodium falciprum strains. The current interest in Azadira&a indica A. Juss., (Meliaceae) as a potential antimalarial candidate demonstrates the validity of the traditional medicinal approach. The leaf extract has been widely used in Sudan and other African countries as an antimalarial agent. However, a number of cases of toxicity arising from over”~onsum~tion of A. &dim leaf extract have been reported in the Sudan after the dramatic outbreak of malaria as a consequence of the 19X7 flood disaster, due to the severe shortages in drug supplies and alleged chloroquine resistance (Khalid, unpublished data). The principal danger in the traditional use of A. indica extracts appears to be the lack of sufficient toxicological investigations on both the crude extract and the anCorrespande*rre io: S.A. Khahd, Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, P.O. Box 1996, Khartoum, Sudan.
t~ma~arial bioactive substan~e(s~. Although there are a number of reports addressing various pharmacological activities (e.g., Van Der Nat, 1987; Prakash et ah, 1986; Abatan and Makinde, 1986) and voluminous literature is available on the chemistry of A. indica (Connolly, 1983), very meager data exist regarding the toxicology of this taxon (Ali and Salih, 1982; Ali, 1987). Particular attention has been directed recently to the a~tirnal~r~a~ activity of A. in&a (Tella, 197% Obih and Makinde, 1985; Qbaseki and Jegede-Fadunsin, 1956). The in vitro antimalarial activity of the main bioactive agents has been demonstrated by Khahd et al. (1986) and its detailed chemiea1 structure has already been estabhshed as nortriterpenoidal derivative, gedunin (Khalid et related anal., 1989). Another structurally timalarial compounds known as nimbolide has been identified by Rochanakij et al. (1985). However, gedunin seems to be the most potent antimalarial and yet the least cytotoxic compound among all the limonoids so far isolated from A. indiea (Khalid et al., in preparation). We consider the selection of chicks as a biological model for this toxicological studies as
037~~~~41/92/~~S.O~ 0 t992 Etsevier Scientific Publishers treland Ltd Primed and Published in ireland
quite appropriate since they are very susceptible malarial infection. The adequacy of chicks as an vivo model for testing the antimalarial activity plant extracts has long been demonstrated Spencer et al. (1947).
to in of by
Materials and Methods Chicks and diets Thirty-six l-day-old Brown Hisex chicks of either sex were housed in the premises of the Faculty of Veterinary Science, University of Khartoum under illumination at night and early morning with feed and water provided ad libitum. At the age of 7 days, the chicks were allotted to three groups of 12 each. Group 1 chicks were controls and fed a starter diet. Leaves of A. indica were collected from the Mogran area in Khartoum and a herbarium specimen was deposited at the Faculty of Pharmacy, University of Khartoum. Leaves were dried in the shade for 2 weeks, ground, added to the mash and fed at dietary concentration of 2% (group 2), or 5% (group 3). Feeding was continued for 4 weeks. Thereafter, the test diets were withdrawn and replaced by the control mash for 2 weeks (recovery period). Mean body weights, weight gain and feed conversion ratios (kg feed/kg gain) were determined for each group weekly. Three chicks from each group were killed for pathologic examination and blood samples were collected at weeks 1, 3, 4 and 6. Histopatho~ugica~ methods Chicks sacrificed were examined for gross lesions. Tissues were fixed in IO%, formal saline, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (H and E). Chemical methods Serum samples were analysed for glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), total bilirubin and uric acid using commercial kits (Boehringer Mannheim GmbH Diagnostica, F.R.G.). The concentration of serum total protein was measured by refractometry.
The erythrocyte count (RBC), packed cell volume (PCV), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) were estimated by standard methods (Schalm, 1965). Statistical analysis Statistical significance was assessed by Student’s t-test (Mendenhall, 1971). Results Effect on growth The effect of 2% or 5% dietary A. indica leaf on body weight, weight gain and feed conversion ratio in Brown Hisex chicks is given in Table I. The results were compared with those in the control group. Statistical analysis of the data indicated that A. indica leaf fed in the diet at 2% or 5% for 4 weeks significantly depressed body weight (P < 0.05-0.02~, weight gain fP < 0.0.5-0.01) and efficiency of feed utilization (P < 0.05-0.001) in chicks of groups 2 and 3 when compared to the control chicks of group 1. At the end of the recovery period, the body weight of chicks in group 3 was lower (P < 0.01) than that of birds in groups I and 2. Efficiency of feed utilization in the control group was significantly higher (P < 0.001) than the test groups 2 weeks after removal from the experimental diet. Yellow discoloration on the combs and legs of chicks in groups 2 and 3 appeared on day 7 and became intense during the period between days 20 and 28. Following withdrawal of the test diets, the intensity of yellow colour gradually decreased and it was no longer seen on the comb by the end of the recovery period. No other clinical signs were observed. Changes on serum constituents The results of serum chemistry for the chicks which consumed 2’%,(group 2) or 5% (group 3) of the basic diet are presented in Table 2. Significant differences (P < 0.05-0.501) in uric acid, bilirubin, total protein, GOT, ALP and LDH values were observed between the test and control
269
TABLE
1
GROWTH
CHANGES
FED A. iNDlCA
IN CHICKS
LEAF
Feeding
Group No
Recovery Week 2
Week
458 f 11.8 (P < 0.01)
219 * 9.4 (P < 0.05) 214 f 2.9
336 f 11.6 (P < 0.05) 275 f 10.6 (P < 0.05) 265 zt 12.9
61.5 f 3 N.S. 56.5 + 2.5 N.S. 52.5 f 3.2
87.2 (P < 68.3 N.S. 15.0
83.6 (P < 55.6 (P < 57.9
f
122. 5 zt 6 (P < 0.01) 85 f 4 (P < 0.001) 44.2 f 7
43.4 f 3 (P < O.OOl) 95.0 f 5
2.4 f 0.2 N.S. 3.1 * 0.3 (P < 0.001)
2.1 (P 3.8 (P 5.2
Week 2
I
131 zlz 6 (P < 0.05)
165 N.S.
2
95 f 8 (P < 0.05) 86 f 5.9
151 * 4.1 (P < 0.02) 139 f 3.5
Mean weight gain 46 zt 2.9 1 N.S. 36 f 3.1 2 (P < 0.01) 28 zt 4 3
3
I
Week 4
Week 3
Week 1
l
253 f N.S.
5.9
14.7
f
4 0.05) zt 3 f
2
360 + 8.2 (P < 0.001) 310 f 8.5
5 0.01) f 3 0.01) f 4
502 zt I.5 N.S. 4.55 f 18 (P < 0.01) 392 f 7
(P < 0.001) 81.7 f 3.5
Mean feed conversion ratio 1
2.3 zt 0.1 (P < 0.01)
2.2 * 0.2 N.S.
2.3 z+z0.1 (P < 0.02)
2
3.1 f N.S.
0.09
2.8 f 0.1 (P < 0.05)
3.0 f 0.15 (P < 0.001)
3
2.4 f
0.12
3.2 zt 0.3
3.8 f
Group
1 = control;
TABLE
group
2 = 2”/u; group
0.12
f < f < 5.1 f
2.0 (P 4.9 (P
3 = 5%; N.S. = not significant;
0.12 0.001) 0.3 0.001) 0.4
7.5 f 0.4
P = level of signilicance
f < * < f
0.1 0.001) 0.2 0.001) 0.4
below or above control
values.
2
SERUM
CHANGES
Group
IN CHICKS
GOT
FED A. INDICA
GPT
LEAF
LDH
FOR 4 WEEKS ALP
T.P.
Bilirubin
Uric acid
0.48 zt 0.03 (P < 0.001) 0.79 l 0.02 (P < 0.01) 0.62 f 0.04
4.3 f
0.1
(P 6.1 (P 6.7
< f < j,
0.001) 0.2 0.001) 0.18
0.47 f 0.02 N.S. 0.66 f 0.04 (P < 0.001) 0.8 f 0.04
4.1 (P 6.8 (P 7.2
f 0.16 < 0.001) f 0.18 < 0.001) f 0.2
NO.
Feeding
2 3 Recovery 1 2 3 Group
f
18.7 zt 0.5
1138 zt 35
280 zt 12
3.2 f 0.4
42.3 zt 0.5
(P < 0.03) 16.3 zt 0.4 N.S. 17.3 f 0.4
N.S. 1195 f 37 (P e 0.02) 1347 rt 38
(P < 0.001) 340 * 13 (P < 0.001) 362 f 11.8
(P 2.1 (P 2.3
18.9 f 0.6 (P c 0.05) 16.9 ztz 0.5
1140 f N.S. 1040 f
377 l 11 (P < 0.01) 309 * I3
2.9 f N.S. 2.6 f
N.S. 17.1 f
(P c 0.01) 1350 f 41
(P < 0.05) 338 f 10.5
(P < 0.05) 2.3 f 0.2
15.7 (P < 42.7 (P c
1
0.3 0.001) f 0.9 0.001)
15.9 f 0.2 N.S. 17.4 f 0.3 N.S. 17.6 f 0.2
1 = control;
group
0.5
2 = 2%; group
40 38
3 = 5%; N.S. = not sign&ant;
< * < f
0.05) 0.6 0.02) 0.3
0.3 0.5
P = level of significance
below or above control
values.
270
groups. At the end of the 2-week recovery period, the concentration of serum uric acid was higher (P < 0.01) and the activity of ALP was lower (P < @OS-0.01) in the test groups than the control group. The values of total protein and GOT returned to normal and LDH activity was higher in group 3 than group 1.
The values of Hb, PCV and RBC were lower (P < 0.05-0.01) in the test groups than the control group (Table 3). MCV did not change but MCH and MCHC were higher (P < 0.001) in group 3 than groups 1 and 2. At the end of the recovery period, the value of RBC, PCV and Hb were within the normal range.
The spleen was enlarged in some birds, the yellow liver showed fatty cytoplasmic vacuolation and focal necrosis of the hepatocytes (Fig. I) and lymphocytes nodules at the portal tracts. In the kidney, there was degeneration or necrosis of the epithelial cells of the convoluted tubules and shrinkage of the glomerular tufts (Fig. 2). The blood vessels of the heart and intestines were congested and petechia1 or ecchymotic haemorrhages with or without small erosions on the intestinal mucous membrane were seen. At the end of the recovery period, the damage to the vital organs was reduced but some of the cells of the renal tubules and the hepatocytes had not completely returned to normal. No lesions were seen in the controt chicks.
Pathological findings
Discussion
In chicks fed dietary 2% (group 2) or 5% (group 3) A. indica leaf, there was yellow discoloration on the combs, shanks, reflected skin, breast and thigh muscles, fiver and body adipose tissue. This was more marked in birds fed 5% dietary A. indica leaf at weeks 3 and 4.
To date no research has been done to deiineate toxic effect of neem (A. indim) products in chicks. As the length of time on experimental diets (2% and 5% A. indica leaf meals) increased from 1 week to 4 weeks, the difference in chick weights among
TABLE
3
HAEMATOLOGICAL Group
Feeding 1 2 3
RBC (x IO6 mm’)
CHANGES
IN CHICKS
FED A. i/VDlCA
Hb (g/100 ml)
PCV (‘XI)
2.12 ziz 0.06
29.0 i
(P < 0.05) 1.9 f 0.5 (P f O.OI) 1.73 ct 0.03
(P < 0.05) 265 i 0.2 (P < 0.01) 25.3 f 0.5
0.4
2.20 zt 0.05 N.S. 2.23 * 0.04 N.S. 1.99 f 0.04
29.2 f. 0.6 N.S. 27.0 i 0.4 (P < 0.05) 25.5 f 0.1
9.1 f
0.1
LEAF
FOR 4 WEEKS
MCV
MCH
(firn’)
(*;Pg)
MCHC (‘%I)
136.8 z+z2.9
42.9 f
8.3 l 0.2 (P < 0.05) 8.6 zt 0.15
N.S. i39.5 f 3.1 N.S. 146.2 + 2.5
MS. 45.3 f I.1 (P < 0.001) 54.3 f 0.9
N.S. 32.5 f 0.4 (P < 0.00~) 37.2 f 0.7
9.2 f N.S. 9.0 f N.S. 8.8 f
137.0 N.S. 135.7 (P < 120.8
43.1 zt 1.3 N.S. 40.5 f 0.9 N.S. 43.2 f 1.2
31.6 f 0.5 N.S. 31.9 rt 0.3 (P < 0.05) 33.4 * 0.2
(P < 0.059
1.2
31.4 xk 0.6
Recovery
1 2 3
Group No. I = control; control values.
Group
0.2 0.1 0.3
No. 2 = 2%; Group
f 0.2 f 2.7 0.02) + 0.29
No. 3 = 5%; N.S. = not significant;
P = level of significance
below or above
271
Fig. I. Necrosis
of the hepatocytes
in a chick fed So/u dietary
A. indica leaf for 28 days.
the groups became increasingly larger demonstrating that the levels of the test diets are responsible for reduction in body weight. The fact that weight gain and feed conversion ratio were decreased in the test chicks suggest that A. indict leaf contains toxic substance(s) that impaired growth.
Fig. 2. Degeneration
and necrosis
of the cells of the renat tubules
H and E, x 100
The elevated activity of LDH and GOT and decrease in the concentration of total protein in the serum of Brown Hisex chicks fed A. indica leaf for 4 weeks indicate severe Iiver damage. The increase in the concentration of bilirubin and in the activity of ALP associated with the development of yellow discoloration of the legs, combs, re-
in a chick fed 5% A. in&u
for 28 days. f-t and E, x 100.
272
fleeted skin, liver and adipose tissue of the birds on A. in&a leaf meal are suggestive of hepatic excretory dysfunction. The intensity of yellow discoloration might, however, suggest that the severe damage of the kidney resulted in retention of some pigments in the circulation. The process of pigments degradation, absorption and excretion in chicks is nat well known (Sturkie, 1965). The pigment fraction of A. indica leaves consists mainly of quercetin-based flavonoid glycosides and carotenoids (Khalid, unpubi~shed results) along with chlorophylls. Some information exists about the process of flavonoids interaction with isolated enzymes, cell constituents and membranes and the deposition of these pigments in organs, tissues and biological fluids (Griffiths, 1982). However, there is a high degree of variation regarding the pharmacokinetics and site of deposition of the flavonoids and their metabolites. This variation depends to a large extent on the route of administration and the intestinal microflora involved during oral administration. Bihary excretion has been established as a major route of excretion of many tlavonoids (Griffiths, 1982). However, very limited information is available concerning the subsequent fate of the conjugate entering the intestine. Some of the flavonoids such as quercetin-based glycosides which is present in A. in&a leaf extract were not detectable in the urine after oral administration to man (Griffiths, 1982). Therefore, the deposition of quercetin-based glycosides and/or their metabolites is quite feasible interpretation for the discoloration encountered during the present experiments, The increase in the activity of GOT and in the concentration of uric acid and hypoprotcinaem~a are suggestive of nephrotoxic properties of neem bioactive constituent(s) and/or their respective metabolites. It has been shown that A. indicu leaf extract causes a hepatobiliary diseases by raising 5’-nucleotidase (5’-NT) to abnormally high levels (Komolafe et al., 1988). The possible role of mixed-function oxidases (MFO) in the hepatobiliary toxicity of A. indica has been investigated by Komolafe et ai. (1988). Their results tend to indicate the involvement of MFO and that the metabolites, and not the parent constituents of the extract, are responsible for the toxicity involved.
In the present study, significant increases in serum uric acid concentration corroborate the findings of Omer (1990) and Ayed et al. (1989) confirming changes in glomerular filtration. Analysis of the parameters suggest that uric acid concentration would be useful dia~ostically because of uniform changes along with pathologic features of renal toxicity. The hepatonephrotoxicity was severe enough to account for the reduced growth rate in the test chicks and that associated lesions were consistent and incompletely reversible at the end of a 2-week recovery period. There were increases in MCV and MCH and decreases in Hb, RBC and PCV in chicks fed A. indica leaf for 4 weeks. These findings suggest macrocytic anaemia. We conclude that concentrations of 2% and 5% of A. &dim leaf in the basal diet are toxic to Brown Hisex chicks and that toxicity is mainly associated with hepatonephrotoxic lesions.
This work was supported by Band Aid (U.K.) grant: ‘Species of Sudanese Meliaceae as a Source of Novel Antimalarial Agents’. Thanks are due to the NEF (U.S.A.) for administering this project. References Abatan, M.O. and Makinde, M.J. (1986) Screening Azudiracru indica and Pisum safivum for possible antimalarial activities. Journai of E~~~opha~~ac~logy
17, 85-93.
Ali, B.H. (1987) The toxicity of Azudirachtu indicu in goats and guinea pigs. Yete,inarj, and ~~~ ToxicobgOy 29, 15- 19. Ali, B.H. and Safih, A. (1982) Suspected toxicity of Az#diracZ$a indica in sheep. Yeierinary Records 1t2, 494. Ayed. I.A.M., Dafalla, B. and Adam, S.E.I. (t989) Effects of various levels of dietary vitamine E on broiler chicks. Veterinary and Human Toxicology 31, 50. Connolly, J.D. (1983) Chemistry of the limonoids of the Meliaceae and Cneoraceae. In: P.G. Waterman and M.F. Grundon (Eds.), Chemisrry and Chemical Taxonomy of the Rurales. Academic Press, London and New York. Grifiths, L. (1982) Mammalian metabolism of flavonoids. In: J.B. Harborne and T.M. Mabry (Eds.), The Flavonoids Advances in Research. Chapman and Hall. London and New York. Khafid, S.A., Farouk, A., Gary, T.M. and Jensen, J.3. (L986) Potential anti malarial candidates from African piants: an in vitro approach using ~~as~~~~~ fair&vwm. ..fownai of Erhnophartnacology
i 5, 201-209.
Khaiid, S-A., Duddeck, H. and Gonzalez-Sierra, M. t1983) Isolation and characterization ofan ant~rna~r~aI agent ofthe neem tree /Iz~&~c&a indica. ~a~~~ufa~~a~~~uf Products 52, 922-927. Komolafe, O.O., Anyabuike. CF. and Obaseki, A.O. (19881 The possible role of mixed-function oxidases in the he~atabiliary toxicity of Aradirarhru indicu. Flturerapiu 59, 109-113. Mendenhall, W.S. ( 197 I) Inrroduction 10 Prahahilir)? and Swisries. 3rd Edn Wadsworth Publishing Co, Inc., CA. Obaseki, 0. and Jegeda-Fadunsin. HA. (1986) The antimalarial activity of A-_ndira~hra inu’ifdl. ~~r~~~r~~if~17, 247-251. Ubih, P.Q. and Makinde, J.M. (1985) Effect of ~~u~~r~e~~~u indira on Ffasmad~~m berg&i in mice. Africorr hmai of Medicine and Medicaf Sciences 14, 5I-54.
Prakash, A.D. and Mat&r, R. (1986) Interceptive plants. P&z@iu Me&c@ 52, 432. Spencer. CF., Koniuszky, F.R., Rogers, E.F., Shavei. J., Jr., Easton. N.R., Kaczka, E.H., Kuehl, F.A., Jr.. Pbiliips, RF., Walti, J., Folkcrs, K., Malanga, C. and Seeler. A.O. (1947) Survey of plants for antimalarial activity, L!~_).x& 10, 145-175. Tella, A. (1976) ‘The effects of A-_adiracfztu indica in acute Plasmodium berghei malaria. Wesr African Jowwf as Pharmacology and Drug Research 3, 80.
Van Der Nat, J.M., Klerx, J.P.A.M. . Van Dijk, H., De Silva, K.T.D. and Labadie, RI’. { 1987) Immunomodulatory activity of an aqueous extract of A:adiratfa in&u stem bark. Joirrn& of ~r~~ap~a~rnacafagy 19, I 25- 13I.
