Vol. 14(5), pp. 107-112, June, 2020
DOI: 10.5897/AJPP2019.5001
Article Number: D6B976163870
ISSN: 1996-0816
Copyright ©2020
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJPP
African Journal of Pharmacy and
Pharmacology
Full Length Research Paper
Phytochemical screening and toxicological effects of
Amblygonocarpus andongensis aqueous stem bark
extract in wistar albino rat
Abdullahi Aliyu Ebbo1, Abdullahi Teleh Elsa2, Emmanuel Udok Etuk3,
Muhammad Jengebe Ladan4 and Saganuwan Alhaji Saganuwan5*
1
Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, Usmanu Danfodiyo University,
P. M. B. 2346,Sokoto, Sokoto State, Nigeria.
2
Department of Veterinary Surgery and Diagnostic Imaging, College of Veterinary Medicine,
Federal University of Agriculture, P. M. B. 2373,Benue State, Nigeria.
3
Department of Pharmacology, College of Health Sciences, Usmanu Danfodiyo University, P. M. B. 2346, Sokoto,
Sokoto State, Nigeria.
4
Department of Chemistry, Faculty of Science, Usmanu Danfodiyo University, P. M. B. 2346, Sokoto, Sokoto State,
Nigeria.
5
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
Federal University of Agriculture, P. M. B. 2373, Makurdi, Benue State, Nigeria.
Received 15 January, 2019; Accepted 9 December, 2019
Amblygonocarpus andongenesis is being used as antipsychotic. Hence, it may portend high risk of
toxicosis. In view of this, phytochemical screening and toxicological effects of aqueous stem bark
extract of the plant was studied in wistar albino rats. Qualitative screening of the stem bark extract
revealed the presence of alkaloid, hydrolysable tannin, saponin, glycoside, cardiac glycoside, saponin
glycoside and anthraquinone glycoside. Quantitative screening revealed 4.04% alkaloid, 4.94% saponin
and 14.38% true tannins. Subchronic dose (900 mg/kg) caused increased and decreased significant
levels of basophils and eosinophils (P<0.05) respectively. Serum biochemistry revealed increased
alkaline phosphatase level (P<0.05). A mild periportal and perivascular lymphocytic infiltrations of the
liver and moderate focal lymphocytic infiltration of the menninges (focal nonsupurative meningitis)
which are suggestive of mild toxicity of A. andongensis were observed. Hence, the plant is toxic at dose
level of 900 mg/kg body weight when administered for a period of 28 days.
Key words: Toxicity; Amblygonocarpus andongensis, phytochemicals, stem bark, brain, liver, rat.
INTRODUCTION
Amblyogonocarpus andongensis (Fabaceae) known in
Hausa as Kolon itche, is a tall tree of 6 to 25 m in height
*Corresponding author. E-mail: pharn_saga2006@yahoo.com. Tel: +2347039309400.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
108
Afr. J. Pharm. Pharmacol.
having wide crown with glabrous leaves (2-5) and 5-9
pairs of leaflets distantly placed. The flower is white or
yellow, fragrant, paired with dense axillary Eduardo et al.,
2000, recimose. It has small calyx and pod is tetragonal
and glossy (Shahina, 1989). It is widely spread in tropical
Africa and usually found in moist and sandy habitats
(Exell and Torre, 1989). Roger (2000) also reported the
plant to be an economic tree that can be found in the
west central Africa. Paul et al. (2000) had reported the
presence of this tree in Nigeria. Osemeobo and Ujor
(1999) also reported the presence of this tree in Nigeria
and particularly in the Northern part of the country.
The tree was described to have clear potential
therapeutic and economic values (Paul et al., 2000;
Roger, 2000). It has activities against venomous stings,
bites, diarrhea, dysentery, constipation, pulmonary
problems and fish poisons (Burkill, 1985). The plant has
long-standing history of antipsychotic effect reported in
wistar albino rats. The plant was reported to be mildly
toxic in rats, caused weight loss and reduced mortality
caused by amphetamine in wistar rats (Ebbo et al., 2008;
2009). The plant showed dose dependent potential
antiosychotic effect in Wistar rats (Ebbo et al., 2010). In
spite of all these claims, there is no documentation on
phytochemical analysis, haematogical and biochemical
effects of A. andongensis in wistar albino rat. This is
because traditional medicine in Africa is not codified but
verbally passed unto apprehentice as folk medicine
(Ohaeri, 1989).
MATERIALS AND METHODS
Collection of the plant materials
The plant samples were obtained from Anka, the headquarter of
Anka Local Government Area of Zamfara State, Nigeria and
identified by Mallam Umar Denge a botanist in the Herbarium of the
Department of Biological Science, Usmanu Danfodiyo University,
Sokoto where a voucher specimen has been deposited.
Preparation of aqueous extract
Stem barks of the plant were obtained, dried under shade to a
constant weight and pounded into fine powder using pestle and
mortar. One hundred gram of the pulverized bark of A. andongensis
powder was placed in a conical flask and 500 ml of distilled water
added and shaken manually for 6 h, but allowed to stand for 18 h
and filtered into beaker using Whatman (No. 1) filter paper. The
filtrate was concentrated at 50°C and kept in the refrigerator until
ready for use (Eduardo et al., 2000).
Phytochemical analyses
Phytochemical analyses of the plant were carried out using
aqueous bark extract of A. andongensis for the presence of
alkaloids, cardiac glycosides, saponins, tannins, steroids,
flavonoids, anthraquinones, total glycosides, and reducing sugars
using the methods of Trease and Evans (1989), Haborne (1973),
and Edeoga et al. (2005).
Experimental animals
Twelve adult Wistar albino rats were obtained from Zoological
Garden of Usmanu Danfodiyo University Sokoto Nigeria and
acclimatized for two weeks. Laboratory animal care was provided to
the animals according to the NIH (1985) guidelines and procedures
were followed in this study as directed by the Department of
Pharmacology, College of Health Sciences, Usman Danfodiyo
University Sokoto and approved by research ethics committee.
Sub chronic administration of aqueous extract
The rats of either sex weighing 156.75±10.26 g and divided into two
groups of 6 each were used for the study. Six out of the twelve rats
received 30% of the limit dose (3000 mg/kg) of oral A. andongensis
aqueous stem bark extract for a period of 4 weeks. The control rats
were administered water only. Grower’s marsh® and water were
provided ad libitum.
Haematology
Blood sample (0.5 ml) was collected from the tail vein at the end of
4th week from all the rats into ethylene diammine tetraacetic acid
(EDTA) bottles for haematological and biochemical analyses. Full
blood cells count was done using the method of Baker (1985).
Brain, kidney, liver, heart and skeletal muscle harvested from each
rat under the effect of anaesthetic ether for histopathology (Rivera
et al., 2001).
Biochemistry
Another blood sample (2.0 ml) was collected from the tail vein for
serum biochemistry. Total protein was determined using biuret
method (Tietz, 1995). Albumin was determined using bromocresol
green method (Doumas 1971). But conjugated bilirubin and total
bilirubin were determined using the method of Jendrassik and Grof
(1938). Whereas serum glutamic aspartate amino transferase and
alanine aminotransferase were determined using the method of
Reitman and Frankel (1957).
Statistical analysis
The data on haematological and biochemical parameters were
expressed as mean±S.D. Test for significance was performed at
5% level between mean parameters of the control and experimental
rats using student ‘t’ test unpaired (Petrie and Watson, 2002).
RESULTS
Results of phytochemical screening carried out on
aqueous stem bark extract of A. andongensis revealed
the presence of alkaloid, hydrolysable tannins, saponin,
flavonoid, cardiac glycoside, saponnin glycoside and
anthraquinone glycoside. Whereas flavonoid and
flavonols were absent. The results of quantitative screening
Ebbo et al.
109
Table 1. Qualitative and quantitative screening of A.
andongensis aqueous stems bark extract.
Secondary metabolites
Alkaloid
Tannins (hydrolysable)
Saponin
Flavonoid
Flavonols
Glycoside
Cardiac glycoside
Saponin glycoside
Anthraquinone glycoside
Result
4.012%
14.38%
4.94%
+
+
+
+
+ = presence, - = absence.
Figure 1. Brain showing moderate lymphocytic infiltration of meninges (focal non-suppurative
meningitis) (x 400).
of A. andongensis aqueous stem bark extract revealed
4.01, 4.94 and 14.38% of alkaloid residue, saponin and
true tannins (Table 1).
Haematology showed significant increased and
decreased (P < 0.05) levels of eosinophils and basophils
in the control and experimental animals respectively. But
packed cell volume (PCV), white blood cells (WBC), red
blood cells (RBC), lymphocytes, neutrophils and
monocytes were not significantly affected (P>0.05).
Biochemistry revealed significant increased level of
alkaline phosphatase (P<0.05). But all other investigated
parameters such as total bilirubin, conjugated bilirubin,
total protein, albumin amino transferase and aspartate
amino transferase were not increased significantly
(P>0.05).
Histopathology
revealed
mild
periportal
and
perivascular lymphocytic infiltration of liver (Figure 2) and
moderate lymphocytic infiltration of the meninges (focal
nonsuppurative meningitis) of the brain (Figure 1). But
kidney had no visible lesion (Figure 3).
DISCUSSION
The presence of alkaloid in the extract corroborates the
report of Ajao et al. (2018) indicating that alkaloid has
been responsible for antipsychotic effect of the plant.
However, the presence of hydrolysable tannins, saponin,
cardiac glycoside, saponin glycoside, flavonoid and
anthraquimone glycoside in the plant, agrees with the
report of Aliyu and Yusuf (2013 indicating that the plant
contains phytotoxic principles. This agrees with the report
of Akinloye et al. (2003) indicating that tannin and saponin
are antinutritional substances. These substances can
cause haemolysis and nutrient malabsorption (Conning,
1993). The presence of alkaloid residue (4.01%), saponin
(4.94%) and true tannins (14.38%) in the stem bark of A.
andongensis (Table 2) may suggest the ability of the
plant to cause both beneficial and toxic effects in animals.
Saponin cause haemolysis, nutrient malabsorption and
abnormal haemopoeisis (Moody et al., 2003), the plant
may be useful in treatment of polycythemia vera in human.
110
Afr. J. Pharm. Pharmacol.
Figure 2. Liver shows mild peripotal and perivascular lymphocytic infiltrations (x 400).
Figure 3. Kidney shows no visible lesion (x 400).
Table 2. Effects of A. andongensis aqueous stem bark extract on haematological and biochemical
parameters of wistar albino rats.
Indices
Packed cells volume (%)
Red blood cells (%)
White blood cells (%)
Lymphocytes (%)
Neutrophils (%)
Monocytes (%)
Eosinophils (%)
Basophils (%)
Total bilirubin (g/dl)
Conjugated bilirubin (µg/dl)
Total protein (g/dl)
Albumin (g/dl)
Aspartate amino transferase (g/L)
Alanine amino transferase (g/L)
Alkaline phosphatase (g/L)
Control
50±0.98
4.96±0.33
7.55±0.35
70.5±1.38
21.5±1.0
5±0.59
3±0.26
0.0±0.0
0.27±0.01
0.14±0.0
6.75±0.2
3.4±0.05
39.5±2.67
21±2.63
31.25±1.69
Values are expressed as mean ± S.D., n = 6, a = statistically significant (P<0.05).
Experimental
48.33±1.72
6.11±0.23
6.33±0.59
73.5±2.17
19.7±1.86
5.7±0.49
1.0±0.37a
0.16±0.17a
0.25±0.02
0.18±0.02
7.4±0.28
3.71±0.23
41±0.22
22±0.53
42.2±3.04a
Ebbo et al.
The presence of tannin may be responsible for its
antiulcer (Oluranti et al., 2012) and antidiarrchoeic
activities (Ugwah et al., 2014). Saganuwan (2009) had
earlier attributed the pharmacological property of plants
to its chemical principles. The presence or absence of
such principles may depend on the methods of
extraction. However, the higher content of true tannins
may be responsible for its astringent activity. Tannins are
capable of turning animal hides into leather by binding
proteins to form water-insoluble substance that are
resistant to proteolytic enzymes (WHO, 1998). Since
tannin contains phenol, it may have antimicrobial activity.
Quattrouichi (2016) had also reported that bark, roots and
seeds are poisonous, whereas fruits are used as tonic,
stimulant, anticonvulsant, and anti-inflammatory agent.
But powdered pods are used to treat skin diseases, and
leaf extract is stomachache. Decoctions of root are
emetic, vermifuge, antipyretic, antimalarial, anticolic,
anticough and anti-dote for food poisoning. Powder from
the pod is used as fish poison and insecticide
(Quattrouichi, 2016). Significant increased level of
eosinophils in the control animals cannot be explained
however, the basophilia observed in the experimental
rats may be attributable to stress or the presence of
glucocorticoid principle in the test plant. The eosinopenia
observed in the present study agrees with the report of
Tvedten (1989) indicating that eosinopenia is usually due
to stress or exogenous glucocorticoid treatment. Hence,
the plant may have glucocorticoid principle. However, the
significant increased level of basohilia observed in the
experimental extract treated group as compared to the
control group may also suggest antilipaemic property of
the plant. This agrees with report of O’keefe et al. (1987)
indicating that basophilia in the absence of an
eosinophilia has been associated with altered lipid
metabolism. Basophil is one of the sources of heparin
which enhances the activity of lipoprotein lipase along
blood vessels in clearing fat from blood (Tvedten, 1989).
Invariably A. andongensis may have ability to cause
decreased blood cholesterol level. However, the
observed basophilia in the experimental animals further
agrees with the report of Tvedten (1989) indicating that
basophilia is caused by the same process causing an
eosinophilia or is associated with lipaemia. But the
significant (P<0.05) difference between control value and
the experimental value of alkaline phosphatase may be
associated with age or the injected aqueous extract of A.
andongensis. The extract may have mild deleterious
effect on the liver as shown by mild peripotal and
perivascular lymphocytic infiltrations of liver (Figure 2).
However, since there is no significant increased level of
alanine aminotransferase and aspartate aminotransferase,
the increased level of alkaline phosphatase may also be
of bone origin. Serum alkaline phosphatase is commonly
increased in animals less than 6 to 8 months (Willard et
al., 1989) perhaps because of bone formation. Mild
111
periportal and perivascular lymphocytic infiltrations of the
liver (Figure 2) and moderate focal lymphocytic infiltration
of the meninges (Figure 1) are suggestive of the ability of
the plant to cause liver and brain damage. Hence there is
need to investigate the intensity of long-time effects of A.
andongensis aqueous stem bark extract on vital organs
of wistar albino rats. Information obtained from such
investigation may give a lead to understanding of
mechanism of its toxicological action. However, Nwinyi et
al. (2006) reported that the plant is relatively safe when
large dose is administered for short period of time.
Conclusion
Qualitative analysis of the aqueous stem bark extract
revealed the presence of alkaloid, hydrolysable tannin,
saponin, glycoside, cardiac glycoside, saponin glycoside
and anthraquinone glycoside. But quantitative analysis
revealed 4.04% alkaloid residue, 4.94% saponin and
14.38% true tannins. The plant caused eosinopenia,
basophilia, increased alkaline phosphatase, mild
periportal and perivascular lymphocytic infiltration of the
liver and non-suppurative meningitis which are
suggestive of mild toxicity. But the extract has no evident
toxicity effect on kidney.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
REFERENCES
Ajao AA, Alimi AA, Olatunji OA, Balogun FO, Saheed SA (2018). A
synopsis of anti-psychotic medicinal plants in Nigeria. Transactions of
the Royal Society of South Africa 73(1):33-41.
Akinloye O, Ahmed AS, Ajagbona OP, Olorede BR (2003).Some
biochemical effects of various doses of aqueous seed extracts of
Cassia occidentalis in rabbits. Bioscience Biotechnology Research
Communications 15(1):85-90.
Aliyu BS, Yusuf HK (2013). An assessment of the toxicity of some
plants used locally as aphrodisiac in northern Nigeria and their
efficacy in enhancing erectile dysfunction in cats. Planta Medica
79(13):8.
Baker FJ (1985). The full blood cells count. In: Baker F J, Silverton R E,
Kilshaw D, Shannon R, Egglestone S, Guthine D L, Mackenzie J C,
th
eds. Introduction to Medical Laboratory Technology, 6
ed.
Butterworth and Company Limited, London. pp. 320-330.
Burkill HM. (1985).The useful plants of west tropical Africa Royal
Botanic Gardens. London, 3, 101.
Conning DM (1993). The basic issues in: Anderson, D. and Conning, D.
nd
M. eds Experimental Toxicol. 2 ed., p 1-3.
Doumas BT, Watson WA, Biggs H B (1971). Albumin-Bromocresol
green method. Clinical Chemistry 56:31-87.
Ebbo AA, Elsa AT, Etuk EU, Ladan MT, Saganuwan SA (2008). Weight
reducing and antiamphetamine effects of Amblygonocarpus
andongensis aqueous stem bark extract in wistar albino rats.
Research in Biosciences 4(2):39-43.
Ebbo AA, Elsa AT, Etuk EU, Ladan MJ,Saganuwan SA (2010).
Antipsychotic effect of aqueous stem bark extracts of
Amblygonocarpus andongensis in Wistar albino rats. Journal of
112
Afr. J. Pharm. Pharmacol.
Medicinal Plants Research 4(11):1033-1038.
Edeoga HO, Okwu DE, Mbaebie BO (2005). Phytochemical
constituents of some Nigerian medicinal plants. African Journal of
biotechnology 4(7):685-688.
Eduardo G, Irene I, Kline N S (2000). Amblygonocarpus andongensis.
Journal of Ethnopharmacology 70:329-333.
Exell T (1989). Notes on Amblygonocarpus andongensis. In: Savanna
plants, An Illustrated Guide by Shahina G. p. 9.
Haborne JB (1973). Physiochemical Methods: Guide to Modern
Techniques of Plant Analysis. 3rd ed. London: Capman and Hall, p.
279.
Jendrassik L, Grof P (1938). In-vitro determination of total and direct
bilirubin in serum. Journal of Biochemistry 299:81-88.
Moody JD, Segun FT, Aderounmu O, Omotade OO (2003). Terminalia
catapper leaves harvested at different stages of growth. Nigerian
Journal of Natural Products and Medicine 7:3032.
National Institute for Health, NIH (1985). Respect for life. National
Institute for Health Science. NIH, No: 85-93.
Nwinyi FC, Ajoku GA, Aniagu SO, Kubmarawa D, Enwerem N, Dzarma
S, Inyang US (2006). Pharmacological justification for the
ethnomedicinal use of Amblygonocarpus andongensis stem bark in
pain relief African Journal of Biotechnology 5(17):1566-1571.
Ohaeri JU (1989). African Traditional medicine: A stage in the people’s
history. African traditional medicine: A stage in the people’s history.
African Notes 12(2):24-28.
Oluranti AC, Michael UO, Jane UOC, Ayembe NA. (2012).
Ethnobotanical studies of medicinal plants used in the management
of peptic ulcer disease in Sokoto State, North Western Nigeria.
International Research Journal of Pharmacy and Pharmacology
2(9):225-230.
O'Kefee DA, Couto CG, Burke-Schwartz C, Jacobs RM (1987).
Systemic mastocytosis in 16 dogs. Journal of Veterinary Internal
Medicine 1:75-80.
Osemeobo GJ, Ujor G (1999). Non-wood forest products in Nigeria.
Project Technical Report F.A.O. Document Repository, p. 30.
Paul PS, Robert F, Zoab Z (2000). Chipya and Mateshe in Kasanka
national park, F. A. O. p. 20.
Petrie A, Watson P (2002). Hypothesis tests 1 - the t-test: comparing
one or two means. In: Petrie A, Watson P (eds). Statistics for
Veterinary and Animal Science Blackwell Science Limited, United
Kingdom, pp. 78-88.
Quattrouichi U (2016). Dictionary of Medicinal and Poisonous Plants:
Common Names, Scientific Names, Eponyms, Symptoms and
Etymology. 5thy Vol.,P. 3960.
Reitman S, Frankel S (1957). Quantitative in-vitro determination of
glutamic-pyruvic transaminase in serum. American Journal of Clinical
Pathology 28:56-66.
Roger B (2000). Trees on the March, the dispersal of economic tree in
the prehistory of west central Africa. F.A.O. Document Repository, p.
45.
Rivera-Posada JA, Pratchett M, Owens L (2001). Injection of
Acanthaster planci with thiosulphate-citrate-bile-sucrose agar
(TCBS). II.Histopathological changes.
Diseases of Aquatic
Organisms 97:95-102.
Saganuwan SA (2009). Tropical plants with antihypertensive,
antiasthmatic and antidiabetic value. Journal of Herbs, Spices and
Medicinal Plants 15(1):24-44.
Shahina G (1989). Savanna plants. An Illustrated Guide, p.35.
Tietz NW (1995). Total protein determination. Clinical Guide to
rd
Laboratory Tests, 3 ed. W. B. Saunders, Philadelphia, pp. 518-519.
th
Trease GE, Evans WC (1989). A Textbook of Pharmacognosy. 13 ed.
London: Bailliere-Tindall Ltd.
Tvedten H (1989). Leucocyte disorders. In: Small Animal Clinical
Diagnosis by Laboratory Methods (Willard, M. D., Tvedten, H. and
Turnwald, G. H. eds.) W.B. Saunders, Philadelphia, USA, pp. 57-85.
Ugwah OM, Ugwah-Ogciniofor CJ, Abubakar K, Okorie NA, Njan AA.
(2014). Anti-diarrhoeal activities of aqueous stem bark extract of
Amblygonocarpus andongensis (Wel. Ex Oliv.) Exell & Torre.
Nigerian Journal of Pharmaceutical Sciences 13(2): 1-11.
WHO (1998). Determination of tannins. Quality Control Methods for
Medicinal Plant Materials, Geneva, Switzerland, p. 44.
Willard MD (1989). Gastrointestinal, pancreatic and hepatic disorders.
In: Willard M D, Tvedten H, Turnwald G H (eds). Small Animal
Clinical Diagnosis by Laboratory Methods. W. B. Saunders,
Philadelphia, USA, pp. 189-228.
Saganuwan Saganuwan