Content uploaded by Blessing C Didia
Author content
All content in this area was uploaded by Blessing C Didia on Feb 04, 2015
Content may be subject to copyright.
Asian Journal of Medical Sciences 4(1): 55-60, 2012
ISSN: 2040-8773
© Maxwell Scientific Organization, 2012
Submitted: January 19, 2012 Accepted: February 17, 2012 Published: February 25, 2012
Corresponding author: C.W. Paul, Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences,
University of Portharcourt, Nigeria, Tel.: +2348035486046
55
The Effect of Methanolic Extract of Moringa oleifera Lam Roots on the
Histology of Kidney and Liver of Guinea Pigs
C.W. Paul and B.C. Didia
Department of Human Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences,
University of Portharcourt, Nigeria
Abstract: Moringa oliefera lam has Horseradish tree, Drumstick tree and Ben oil tree, as its English names.
Principal Constituents of the roots include an active anti-biotic principle, pterygo-spermin, two alkaloids viz;
moringine and Moringinine contained in the root bark. The aim of the study is to investigate effect(s) of
methanolic extract of Moringa oleifera lam root on Histo-architechture of Liver and Kidney, considering
objectives such as determining if the effect of the extract is dose and time-dependent, and determination of
LD50. ’LD50 ip’ of 223.61 mg/kg was determined using modified Lorke’s (1983) method. Twenty four (24)
guinea pigs were used for the study. They were acclimatized and randomly distributed into groups A-C and
control. They were given daily intra-peritoneal injection of methanolic extract of Moringa oleifera lam root was
done for three (3) weeks. Doses of 3.6, 4.6 and 7.0 mg/kg were given to groups A, B and C respectively. Four
pigs; one from each group were sacrificed on 8th, 15th and 22nd days. Tissues collected were immediately
prepared histologically for haematoxylene and eosin stain. The photomicrographs were observed under the
microscope with magnifications of X400 and X200. Histological sections of group A revealed that kidney
sections did not differ from the control group, while liver sections had balloon degeneration. For guinea pigs
in group B, histological sections of liver showed balloon degeneration, while kidney sections showed mild
tubular damage with interstitial inflammations. For guinea pigs in group C, histological sections of liver had
balloon degeneration with microvessicular steatosis and sections of kidney had infiltration of interstitium with
inflammatory cells as well as tubular Lumina filled with amorphous eosinophilic materials. Histological
sections of liver and kidney of guinea pigs after eight weeks of cessation of treatment did not show normal
histo-architecture. Histological sections of all treated groups had ballooning degeneration of the liver, means
that hepatotoxicity is not dose-dependent, but time-dependent. Concerning the kidneys, sections from group
B showed mild tubular damage with tubular cast and interstitial inflammation while group C had infiltration
of interstitium by inflammatory cells and amorphous eosinophilic materials In the Lumina of the tubules.
Methanolic extracts of Moringa oleifera lam roots was found to distort the histo-architecture of both liver and
kidneys of guinea pigs. All the reversal groups retained histo-architectural distortions.
Key words: Histo-architecture, methanolic, Moringa oleifera lam
INTRODUCTION
Moringa oleifera lam has Horseradish tree,
Drumstick tree and Ben oil tree, as its English names.
While the local names in Nigeria include; Fulani; Gawara,
Konamarada, Kini maka. Hausa; Zogall, Zogalla-gandi,
Bagaruwar. Ibo; Ikwe oyibo. Yoruba; Ewe ile, Ewe
igbale. The moringa tree grows mainly in semi-arid,
tropical and sub tropical areas, it is native to the southern
foothills of the Himalayes, and possibly Africa and the
middle East (Duke James, 1982). Currently it is widely
grown in Africa, central and South America, Sri-lanka,
India, Mexico, Malaysia and the Philippines.
The tree itself is rather slender with drooping
branches that grows to approximately 10 m in height. The
bark is thick, soft, corky and deeply fissured. The leaves,
usually tripinnate: the leaves elliptic and the flowers,
while fragrant in large panicles; the pods; pendulous,
greenish, triangular and ribbed with trigonor’s winged
seeds. The fruit is long, slender and more or less straight,
up to 30 cm long by 2.5 cm across, triangular in cross
section and has attracted the name drumstick in India.
Principal constituents: The Moringa oleifera lam root
contains an active anti-biotic principle, pterygo-spermin.
The root bark contains two alkaloids (total alkaloid 0.1%)
viz. moringine which is identical with benzyl amine and
moringinine belonging to the sympathomimetic group of
bases. It also contains traces of an essential oil with a
pungent smell. Phytosterol, waxes and resins. An alkaloid
Asian. J. Med. Sci., 4(1): 55-60, 2012
56
named spirochin, has been isolated from roots (Chopra
and Chopra, 1957). The roots also contain phyto-
chemicals like; 4-("-L-rhamnopyranosyloxy)-
benzylglucosinolate and benyl-glucinolate (Menhnaz,
2009-2011).
Pterygospermin (in concs of 0.5-3 kg/cc) inhibits the
growth of many gram positive and gram negative bacteria,
in higher concs (7-10 kg/cc) is active against fungi. It is
stable in the presence of blood and gastric juice but breaks
down in the presence of pancreatic juice. Its effect is
counteracted by thiamine and glutamic acid but reinforced
bypyridoxine (http: //www.himalayahealthcare.com/ herb
finder/h_ moringa.htm).
The aim of the study was to investigate effect(s) of
methanolic extract of Moringa oleifera lam root on Histo-
archtechture of Liver and Kidney. Objectives were; To
determine if methanolic extracts of Moringa oleifera lam
root has effect(s) on the histology of Liver and Kidney of
guinea pigs. And to determine whether the effects are
dose-dependent and/or time-dependent.
Different parts of Moringa oleifera lam have different
pharmacological actions and toxicity profiles which have
not yet been completely defined. It has been used over the
years for different ailments, out of which only few were
investigated. Moringa oleifera is one of the leading names
recently in plants and drug research. A large number of
reports on the nutritional qualities of Moringa now exist
in both the scientific and the popular literature. However,
the outcome of well controlled and well documented
clinical study are still clearly of great value (Mazumder
et al., 1999).
Moringa oleifera lam has vast medicinal properties
and every part is said to have beneficial properties
(Garima et al., 2011). It has been described as having
medical and health importance such as Abortifascient
(Nath et al., 1992; Tarafder, 1983), Aphrodisiaec (Fuglie,
1999), Birth Control (Shukla et al., 1988, 1989; Faizi
et al., 1988). Nikkon et al. (2009) worked on
Benzylcarbamothioethionate from root bark of Moringa
oleifera Lam, and its toxicological effects. They
concluded that histopathology of the liver, kidney, heart
and lung did not reveal acute toxicity. The bark of the tree
may cause violent uterine contractions that can be fatal
(Bhattacharya et al., 1978). Methanolic extract of
Moringa oleifera root was found to contain 0.2%
alkaloids. Effects of multiple weekly doses (35, 46, 70
mg/kg, respectively) and daily therapeutic (3.5, 4.6, and
7.0 mg/kg, respectively) intraperitoneal doses of the crude
extract on liver and kidney function and hematologic
parameters in mice have been studied. The results indicate
that weekly moderate and high doses (>46 mg/kg body
weight) and daily/therapeutic high doses (7 mg/kg) of
crude extract affect liver and kidney function and
hematologic parameters, whereas a weekly dose (3.5
mg/kg) and low and moderate daily/therapeutic doses (3.5
and 4.6 mg/kg) did not produce adverse effects on liver
and kidney function (Mazumder et al., 1999). LD50 and
lowest published toxic dose (TDLo) of root bark extract
Moringa oleifera Lam. are 500 and 184 mg/kg,
respectively, when used intraperitoneally in rodents
(mice). Changes in clotting factor, changes in serum
composition (e.g., total protein, bilirubin, cholesterol),
along with enzyme inhibition, induction, or change in
blood or tissue levels of other transferases have been
noted (Woodard et al., 2007).
However, the interior flesh of the plant can also be
dangerous if consumed too frequently or in large amounts.
Even though the toxic root bark is removed, the flesh has
been found to contain the alkaloid spirochin, which can
cause nerve paralysis (Morton, 1991).
Nikkon et al. (2009) considered the new compound,
benzylcarbamothionate Isolated from the chloroform
soluble fraction of the ethanolic extract of the root bark of
the Moringa oleifera Lam. The acute toxicity studies of
the extract on long Evan’s rats were carried out using four
groups of animals. The haematological parameters,
biochemical study and histopathalogy of the liver, kidney,
heart and lung did not reveal acute toxicity. They
concluded that both chloroform soluble fraction and
compound I of Moringa oleifera Lam. Had no toxic
effects in the experimental model.
Kumar et al. (2010) Compared the hepato-protective
activity of leaves and roots of moringa olerifera lam
against carbon tetrachloride induced hepatotoxicity in
albino rats. The hepatoprotective activity of the both
extracts were tested using histopathological parameters.
The liver section of methanolic extracts of Moringa
oleifera Lam (leaves and roots) treated groups clearly
showed normal hepatic cells and central veins, which are
comparable with silymaria treated group of animals. The
potent hepotoprotective activity of leaves of Moringa
oleifera was confirmed from this study.
METHODOLGY
The internationally accepted principles for laboratory
animal use and care were adopted, and Ethical Clearance
for research was obtained from the University of Port
Harcourt ethical committee and the protocols were strictly
adhered to.
‘LD50 ip’ of 223.61 mg/kg was determined using
Lorke’s (1983) method and as used by (Azikwe et al.,
2007; Azikwe et al., 2009; Bassey et al., 2009) was
adapted for the LD50 study. Twenty four (24) Male
guinea pigs (Boars),weighing between 200 and 500 g,
were purchased and housed in plastic cages with steel
nettings and solid bottom, in the animal house of the
faculty of basic medical sciences, University of Port
Harcourt. The acclimatization period was for two (2)
weeks. The animals were weighed and randomly
Asian. J. Med. Sci., 4(1): 55-60, 2012
57
distributed into groups A-C and control. They were also
weighed on weekly basis. To identify the animal groups,
fur dye was applied. Daily intra-peritoneal injection of
methanolic extract of Moringa oleifera lam root was done
for three (3) weeks. Doses of 3.6, 4.6 and 7.0 mg/kg, were
given to groups A, B and C, respectively. Four Boars; one
from each group (groups A, B, C and control) were
sacrificed on 8th, 15th and 22nd days. They were weighed,
anesthetized and dissected, tissues collected were
immediately fixed in formalin, prepared histologically for
haematoxylene and eosin stain. The photomicrographs
were observed under the microscope with magnifications
of X400 and X200.
Fresh roots of Moringa oleifera lam was collected
from Port Harcourt in January, the roots were identified in
the Department of Plant Science and Biotechnology,
University of Port Harcourt. Cold suscinate extraction
was done using methanol. The extract was weighed and
stored in sub-zero temperature in the refrigerator. The
extracts were administered once daily, slowly, and intra-
peritoneally to the healthy guinea pigs, using insulin
syringes and needles.
RESULTS
Histological findings of control and experimental groups:
CControl group (Administered with 0 mg/kg of
plant extracts): Histological sections of liver and
kidney from control group showed normal
histological features which served as reference points
for comparing with experimental groups.
C Guinea pigs treated with 3.5 mg/kg of extract:
Histological sections of the kidney of guinea pigs
treated with 3.5 mg/kg of extract did not differ from
the control group (normal histo-architecture). While
histological sections of liver treated with 3.5 mg/kg
of plant extract had balloon degeneration.
C Guinea pigs treated with 4.6 mg/kg of extracts:
For guinea pigs treated with 4.6 mg/kg of plant
extracts; histological sections of liver showed balloon
degeneration, while kidney sections showed mild
tubular damage with interstitial inflammations.
C Guinea pigs treated with 7.0 mg/kg of extract: For
guinea pigs treated with 7.0 mg/kg of extract,
histological sections of liver had balloon
degeneration with microvessicular steatosis and
sections of kidney had infiltration of interstitium with
inflammatory cells as well as tubular Lumina filled
with amorphous eosinophilic materials.
C Reversal group: Histological sections of liver and
kidney of guinea pigs after eight weeks of cessation
of treatment did not show normal histo-architecture.
Fig. 1: A Photomicrograph of guinea pig kidney from the
control group showing normal glomeruli and tubules.
Magnification X400 H & E
Fig. 2: A Photomicrograph of guinea pigs kidney treated with
3.5 mg/kg of extract showing mild tubular damage
(tubulointerstitial nephritis). Magnification. X400. H &
Fig. 3: A photomicrograph of guinea pig kidney treated with
3.5 mg/kg of extract showing mild glomerular damage
(glomerulo-nephritis). Magnification X400 H & E
DISCUSSION
Histological sections of guinea pig’s kidneys treated
with methanolic extracts of Moringa oleifera lam roots
(Fig. 1); Fig. 2 and 3 were collected from guinea pigs
treated with 3.5 mg/kg of plant extract, Fig. 2 was
collected early in the study and it revealed little or no
damages, Fig. 3 exhibited mild glomerular damage
(glomerulonephritis)
Asian. J. Med. Sci., 4(1): 55-60, 2012
58
Fig. 4: A photomicrograph of guinea pig kidney treated with
4.6 mg/kg of extract showing glomerular damages
(glomerulo-nephritis). Magnification X400 H & E
Fig. 5: A photomicrograph of guinea pigs kidney treated with
4.6 mg/kg of extract showing tubular and severe
glomerular damagesand inflammation (glomerulo-
nephritis). Magnification X400 H & E
Fig. 6: A photomicrograph of guinea pig kidney treated with
7.0 mg/kg of extract showing severe glomerula damage
and interstitial inflammation. Mag. X400. H & E
and venous congestion. Figure 4 and 5 were collected
from guinea pigs treated with 4.6 mg/kg of plant extract.
Figure 4; the sample was collected early in the study and
it revealed distortion of glomeruli as well as venous
congestion. Figure 5 represents the later part of the study
and it showed glomerular and tubular inflammation.
Figure 6 Represent the group that was treated with 7.0
Fig. 7: A photomicrograph of Guinea Pigs liver from the
controlgroup (0 mg/kg of plant extract) Magnification X
400 H & E
Fig. 8: A photomicrograph of guinea pig liver treated with 3.5
mg/kg of plant extracts showing normal hepatocytes
with contiguous zone of cells with balloon-
degeneration. Magnification X400 H&E
mg/kg of plant extract, the findings are; glomerular,
tubular and interstitial damages in both early and later
parts of the study. The pattern of distortion is such that
lower doses (3.5 and 4.6 mg/kg) affect mainly the
glomeruli, while the higher dose (7.0 mg/kg) has a global
effect on the kidney tissues (affecting; glomeruli, tubules,
and interstitial spaces surrounding the tubules). These
findings mean that the toxicity of methanolic extract of
Moringa oleifera roots to the guinea pig’s kidneys is both
time-dependent and dose-dependent. The reversal group
retained features of distortion of histo-architecture of
kidney tissues, which means that injuries inflicted on the
kidney tissues are irreversible. The findings are in keeping
with Woodard et al. (2007), but contradict some previous
studies (Mazumder et al., 1999; Nikkon et al., 2009).
Concerning the liver, Fig. 8 represents liver tissues
collected from guinea pigs treated with 3.5 mg/kg of plant
extract in the later stage, it revealed normal hepatocytes
with contiguous zones of ballooning degeneration
(microvessicular steatosis). Photomicrographs made from
liver tissues treated with 3.5 mg/kg in the early stage of
the study were found to be essentially normal
Asian. J. Med. Sci., 4(1): 55-60, 2012
59
Fig. 9: A photomicrograph of guinea pigs livertreated with 4.6
mg/kg of extract showing balloon- degeneration of
hepatocytes. Mag. X400.H & E
Fig. 10: A photomicrograph of guinea pig liver treated with 7.0
mg/kg of extract showing balloon degeneration
(macrovessicular steatosis). Magnification X400 H
& E
Fig. 11: A photomicrograph of guinea pigs liver treated with
4.6 mg/kg showing congestion of central vein and
microvessicular steatosis. Mag. X400 H & E
(comparable to the control group, Fig. 7). Figure 9 and 11
were collected from the guinea pig group that was
exposed to 4.6 mg/kg of plant extract; Fig. 9 was prepared
from the early part of the study and it revealed congestion
of the central vein, while Fig. 11 was collected in the later
stage of the study, it revealed balloon degeneration of
Fig. 12: A photomicrograph of guinea pig liver treated with 7.0
mg/kg showing macrovessicular steatosis.
Magnification X400. H & E
the hepatocyte (microvessicular steatosis). Figure 10 and
12 were collected from guinea group treated with 7.0
mg/kg of plant extract. Figure 10 was prepared early in
the study and it revealed ballooning degeneration of the
hepatocytes (macrovessicular steatosis), while, Fig. 12
was prepared in the later stage of the study, it showed
ballooning degeneration of the hepatocyte
(macrovessicular steatosis) and distortion of the central
vein. Microvessicular steatosis (also called fatty change,
fatty degeneration or adipose degeneration) which is
abnormal retention of lipids within a cell in vesicles that
displace the cytoplasm. This is synonymous with liver
tissues exposed to lower doses (3.5 and 4.6 mg/kg) of the
plant extract. Whereas, Macrovessicular steatosis (which
is development of vesicles large enough to distort the
nucleus in addition to features of microvessicular
steatosis) is characteristic of liver tissues treated with 7.0
mg/kg of plant extract. The fatty degeneration is made
possible because liver is the primary organ of lipid
metabolism, so is mostly associated with steatosis. These
spaces were occupied by lipids in life but histological
fixation caused the lipids to be dissolved, hence only
empty/clear spaces are seen in the photomicrographs. The
reversal group showed feature that are similar to the
control group, which means that, the destructive effects
of the plant extract on the histo-architecture of the
liver is reversible. The result of our study contradicts
Mazumder et al. (1999) and Nikkon et al. (2009).
CONCLUSION
Although the consumption of different parts of
Moringa oleifera lam including the roots for various
purposes has been widely accepted, Methanolic extracts
of Moringa oleifera lam roots was found to distort the
histo-architecture of both liver and kidneys of guinea pigs.
These effects are time-dependent and dose-dependent.
The liver and kidney of guinea pigs in the reversal group
retained histo-architectural distortions.
Asian. J. Med. Sci., 4(1): 55-60, 2012
60
REFERENCES
Azikwe, C.C.A., L.U. Amuzu, P.C. Unekwe,
M.J. Nwankwo, S.O. Chilaka and O.J. Afonne, 2007.
Anticoagulation and antithrombotic effect of triclisia
dictyophylla, moonseed. Niger. J. Nat. Prod. Med.,
11: 26-29.
Azikwe, C.C.A., L.U. Amuzu, P.C. Unekwe,
P.J.C. Nwosu, M.C. Ezeani, M.I. Siminialayi, S.O.
Obidiya and J.E. Arute, 2009. Antidiabetic fallacy of
vernonia amygdalina (bitter leaves) in human
diabetes. Asian Pa. J. Trop. Med., 2(5): 54-57.
Bassey, A.S., J.E. Okokon, E.I. Etim, F.U. Umoh and
E. Bassey, 2009. Evaluation of the in vivo
antimalarial activity of ethanolic leaf and stembark
extracts of Anthocleista djalonensis. Ind. J.
Pharmacol., 41(6): 258-261.
Bhattacharya, J., G. Guha and B. Bhattacharya, 1978.
Powder microscopy of bark-poison used for abortion:
Moringa pterygosperma gaertn. J. Indian Forensic.
Sci., 17: 47-50.
Chopra, R.N. and I.C. Chopra, 1957. Ind. J. Med. Res.
Med., 15: 123.
Duke James, A., 1982. Handbook of energy crops:
Moringa oleifera. From the produce center for new
crops Website.
Faizi, S., B.S. Siddiqui, R. Saleem, K. Aftab and F.
Shaheen, 1988. Bioactive compounds from the leaves
and pods of Moringa oleifera. New Trends in Natural
Products Chemistry, pp: 175-183.
Fuglie, L.J., 1999. The Miracle Tree: Moringa Oleifera:
Natural Nutrition for the Tropic. Church world
service, Dakar. Revised in 2001 and Published as
Miracle Tree: The Multiple Attributes of Moringa,
pp: 68, 172. Retrieved from: http://.echotech.org/
bookstore/advanced-search-result.php?
Garima, M., S. Pradeep, V. Ramesh, K. Sunil, S. Saurabh,
K.K. Jha and R.L. Khosa, 2011. Traditional Uses,
phytochemistry and pharmacological properties of
Moringa oleifera plant: An overview. Der Pharmacia
Lettre, 3(2): 141-164.
Kumar, C.S., B. Balanaurugam, S. Murugeswaran,
P. Natarajan, S.P. Sharavanan, S. Petchimuthu and
T.S. Murugan, 2010. Hepatoprotective activity of
leaves and roots extracts of Moringa oleifera Lam.
Int. J. Med. Res., 1(2): 90-93.
Lorke, D.A., 1983. A new approach to practical acute
toxicity testing. Arch. Toxicol., 54: 275-287.
Mazumder, U.K., M. Gupta, S. Chakrabarti and D. Pal,
1999. Evaluation of hematological and hepatorenal
functions of methanolic extract of Moringa oleifera
Lam. Root Treated Mice. Indian J. Exp. Biol., 37:
612-614.
Menhnaz, K., 2009-2011. Nutrition Information. John
Hopkin, Ratty Donovan.
Morton, J.F., 1991. The horseradish tree, Moringa
pterygosperma (Moringaceae)-A boon to arid lands?
Econ. Bot., 45: 318-333.
Nath, D., N. Sethi, R.K. Singh and A.K. Jain, 1992.
Commonly used Indian abortifacient plants with
special reference to their teratogenic effects in Rats.
J. Ethnopharmacol., 36: 147-154.
Nikkon, F., S. Hassan, K.A. Salam, M.A. Mosaddik,
P. Khondkar, M.E. Hague and M. Rahman, 2009.
Benzylcarbamothioethionate from root bark of
Moringa oleifera Lam. and its toxicological
evaluation Boletin Latinoameticano y del caribe de
plantas medicinales y Aromaticas, 8(2): 130-138.
Shukla, S., R. Mathur and A.O. Prakash, 1988.
Biochemical and physiological alteration in female
reproductive organs of cyclic rats treated with
aqueous extract of Moringa oleifera lam. Acta
europaea fertilits, 19: 225-232.
Tarafder, C.R., 1983. Ethnogynaecology in relation to
Plants: 2 plants used for abortion. J. Econ. Taxon.
Bot. 4(2): 507-516.
Woodard, D. and R. Stuart, 2007. The Vermont Safe
Information Resources, Inc. Material Safety Data
Sheet Collection. Retrieved from: http://hazard.com/
msds/tox/f/q77/q479.html.
