1. Indian Journal of Natural Products and Resources
Vol. 3(4), December 2012, pp. 541-546
Prophylactic and curative effects of Moringa oleifera Lam. pods in
CCl4 damaged rat liver
H M Nanjappaiah1,2 and Shivakumar Hugar1,2*
1
P G Department of Pharmacology, B.L.D.E.A’s College of Pharmacy, Bijapur-586 103, Karnataka, India
2
SCS College of Pharmacy, Harapanahalli-583 131, Karnataka
Received 22 July 2011; Accepted 25 August 2012
The present research work was designed to establish the possible preventive and curative hepatoprotective efficacy of
70% hydro-alcoholic extract of pods of Moringa oleifera Lam. at different dose levels (100, 250 and 500 mg/kg) on CCl4
induced liver injury in rats. Antihepatotoxic potential was assessed by the estimation of biochemical markers, viz. SGPT,
SGOT, ALP, ACP and bilirubin (Direct & Total). In addition liver GSH and MDA levels and histopathological examination
were also studied. The dose dependent significant reduction of biochemical markers and bilirubin levels were seen in rats
subjected to both pre and post treatments of test extract. Though, the extract at a dose of 100 mg/kg exhibited considerable
reduction in concentration of ACP, TB and DB in pre and post treatment models, the results were found to be statistically
not significant. CCl4 poisoned rats showed significant decrease in hepatic GSH level and elevation of MDA
(Malondialdehyde) level compared to normal control group. Pre and post treatment of extract dose dependently reversed the
CCl4 mediated altered hepatic GSH and MDA levels. The histopathological study supported the hepatoprotective activity of
the test extract. In conclusion, the findings of the present study suggest that M. oleifera pods extract possesses potent
prophylactic and curative hepatoprotective efficacy against CCl4 rendered liver injury in rats.
Keywords: Drumstick, Hepatoprotective, Liver injury, Moringa oleifera pods, Prophylactic.
IPC code; Int. cl. (2011.01)  A61K 36/00, A61K 131/00, A61P 1/16
Introduction vegetable, spice, for cooking and cosmetic oil3.
Liver diseases are large public health problem in The leaves, fruits, flowers and immature pods of this
the world1. Towards these pathologies modern tree are used as a highly nutritive vegetable in many
medicine does not find any curative treatments2. countries, particularly in India, Pakistan, Philippines,
Hence, searching the safe and potent remedies from Hawaii and many parts of Africa4-6. All the parts of
the herbal origin for the treatment of hepatic disorders the tree are used in folk medicine practices for
has become most fascinating and desired area of the treatment of various diseases such as UTI,
research for the pharmacologists. Literature review HIV-AIDS, external sores and ulcers, diabetes,
showed that some of the Indian medicinal plants used cancer, gastritis, diarrhoea, liver diseases7, etc.
traditionally in the management of liver disorders The plant is reported to possess anti-inflammatory,
have been scientifically investigated and reported for antioxidant, antiulcer, anticancer, antihyperlipidaemic
their measurable hepatoprotective effects against and cardiotonic properties8-13. A study on ethanol and
various experimental animal models. However, still aqueous extracts of whole pods and its parts, i.e. coat,
more numbers of medicinal plants are needed to be pulp and seed revealed that the blood pressure
screened for their hepatoprotective efficacy. lowering effect of seed was more pronounced with
Moringa oleifera Lam. (Family  Moringaceae) comparable results in both ethanol and water extracts
commonly known as Drumstick is one such plant indicating that the activity is widely distributed14.
cultivated for different purposes such as medicine, In vivo hepatoprotective activity of the M. oleifera
leaves, flowers, roots and seeds have been already
—————— documented in the literature15-18. In vitro
*Correspondent author: antihepatotoxic property has also been reported from
Address: Department of Pharmacology,
B.L.D.E.A’s College of Pharmacy, Bijapur-586 103, Karnataka its fruits19. However, comparative studies on
E-mail: shivkumarhugar@yahoo.com; Phone: +919448404102 prophylactic (preventive) and curative potential of

2. 542 INDIAN J NAT PROD RESOUR, DECEMBER 2012
pods on CCl4 damaged rat liver has not been Subramanya nagar, Bangalore-21 (237/CPCSEA),
investigated so far. Hence, the present investigation India. After randomization into various groups and
was undertaken. before initiation of experiment, the animals were
acclimatized for a period of 10 days. Animals were
Materials and Methods housed in polypropylene cages and maintained under
Plant materials standard environmental conditions such as
The pods of M. oleifera (Plate 1) were collected temperature (26 + 2°C), relative humidity (45-55%)
from the surrounding gardens of the Harapanahalli, and 12 h dark/light cycle. The animals were fed with
Karnataka, India and they were authenticated by rodent pellet diet (Golden Mohur Lipton India Ltd.)
Professor K. Prabhu, Department of Pharmacognosy, and water ad libitum. The study protocol was
S.C.S. College of Pharmacy, Harapanahalli. approved from the Institutional Animal Ethics
A voucher specimen has been deposited in the Committee (IAEC) before commencement of
museum of the college. experiment.
Preparation of extract
Determination of acute toxicity (LD50)21
Fresh mature pods were shade dried at room The acute toxicity of test extract was determined in
temperature, coarse powdered and extracted with 70% female albino mice following fixed dose method of
hydro-alcohol by Soxhlet’s extraction method. CPCSEA, (OECD, guideline No. 420, Annexure-2d).
Thereafter, the extract was concentrated using rotary The mice weighing 20-25g were fasted overnight
flash evaporator to obtain semisolid crude extract. prior to experiment and based on the LD50 cutoff
The percentage yield of the extract was found to be value 1/25th, 1/10th and 1/5th screening doses of the
7.8. The extract was stored in airtight container in extract were selected for the hepatoprotective study.
refrigerator below 10°C. Appropriate concentrations
of stock solutions was prepared using distilled water Evaluation of hepatoprotective activity on CCl4 rendered
and used for the following studies: hepatic damage in rats22
01. Preliminary phytochemical investigations. Albino rats of Wistar strain weighing 150-200 g
02. Acute toxicity studies in mice. were allocated to 08 groups of six each as shown
03. Evaluation of hepatoprotective activity against below:
CCl4 induced liver injury in rats.
Preliminary phytochemical screening
Group 1: Control - Untreated.
Preliminary phytochemical tests were conducted on Group 2: CCl4 control - Injected with fresh
test extract to detect the presence of phytochemicals mixture of equal volume of CCl4 and liquid paraffin
by following the standard methods described by by 3 i.p. injections at a doses of 2 ml/kg body wt.
Trease and Evans20.
Pre treatment of M. oleifera pods extract (Prophylactic study)
Experimental animals
Male albino Wistar rats (150-200 g) and female Group: 3, 4 and 5 were given orally 100, 250 and
albino Swiss mice (20-25 g) were used in the 500 mg/kg, respectively for 14 consecutive days and
experiments. They were procured from Sri injected intraperitonially CCl4 (2 ml/kg in equal
Venkateshwar Enterprises, 4304, 13th main 2nd cross, volume of liquid paraffin) on days 12, 13 and 14.
Plate 1 Moringa oleifera: a-Twig bearing pods, b-Dried pods

3. NANJAPPAIAH & HUGAR: CURATIVE EFFECTS OF MORINGA OLEIFERA IN CCL4 DAMAGED LIVER 543
Post treatment of M. oleifera pods extract (Curative study) Acute toxicity (LD50)
Group: 6, 7 and 8 were given orally 100, 250 In acute toxicity studies, test extract of title
and 500 mg/kg, respectively for 14 consecutive plant did not produce any mortality of the animals
days and injected intraperitonially CCl4 on days at dose of 2000 mg/kg. Hence, 2500 mg/kg was
1, 2 and 3. fixed as LD50 cutoff value as per fixed dose method
During the period of treatment the rats of CPCSEA.
were maintained under normal diet and water. The screening doses selected for the hepatoprotective
Twenty four hours after the last treatment i.e. on activity of pod extracts of the plant were:
15th day, the blood samples were collected 100 mg/kg-1/25th dose of 2500 mg/kg b.w.;
by puncturing the retro-orbital plexus under 250 mg/kg-1/10th dose of 2500 mg/kg b.w.;
the influence of light ether anaesthesia and 500 mg/kg-1/5th dose of 2500 mg/kg b.w.
allowed to coagulate for 30 min at 37°C. Plasma
was separated by centrifugation at 3000 rpm for Effect of M. oleifera pods extract on biochemical parameters
15 min and used for estimation of biochemical in CCl4 damaged rat liver
parameters such as SGPT, SGOT, ALP, ACP The CCl4 challenged animals exhibited significant
and bilirubin (Total and Direct) using ready elevation of serum marker enzymes SGPT,
Erba Diagnostics Mannheim GmbH – Germany SGOT, ALP, ACP and increased concentration
kits by STAR 21 PLUS semi autoanalyser. of bilirubin (Total & Direct) indicating
Then all the rats were euthanized and their hepatocellular damage when compared with
liver tissues were excised, rinsed in ice cold normal control rats. The dose dependent significant
normal saline to remove adhered blood. A reduction of elevated SGPT, SGOT, ALP, ACP,
section from the median lobe was preserved in total and direct bilirubin levels monitored in
10% formalin for histopathological studies. Rest rats subjected to both pre- and post-treatment
of the liver tissues were subjected to with 70% hydro-alcoholic extract of M. oleifera
homogenization and the resulted liver pods. Though, the extract at doses of 100 mg/kg
homogenate was used for the estimation of exhibited considerable reduction in concentration
GSH and LPO following the methods described of ACP, total and direct bilirubin in pre- and
by Van Dooran et al and Uchiyama & Mihara, post-treatment modes, but the results were found to
respectively23,24.
be statistically not significant. The results are
represented in Table 1.
Results
Phytochemicals Effect of test extract on liver GSH content
Results of the preliminary phytochemical In CCl4 poisoned rats a significant decrease
screening revealed the presence of in GSH level was observed compared to normal
carbohydrates, glycosides, flavonoids, alkaloids control group. Pre- and post-treatment with the
and tannins. test extract significantly attenuated CCl4 induced
Table 1  Preventive and Curative effects of Moringa oleifera pods extract on biochemical markers
Groups SGPT SGOT ALP ACP TB DB
Control 117.98 ± 4.13 145.1 ± 4.67 291.25 ± 1.33 30.45 ± 1.27 0.370 ± 0.007 0.283 ± 0.012
CCl4 control 250.00 ± 2.33 350.0 ± 6.16 560.80 ± 6.60 55.60 ± 1.22 0.639 ± 0.004 0.551 ± 0.007
Preventive (mg/kg)
100 220.30±1.87*** 302.87±2.87*** 486.10 ±3.87*** 43.01 ± 3.13ns 0.520 ± 0.09ns 0.490 ± 0.017ns
250 180.13±3.14*** 270.13±4.90*** 349.80 ±4.90*** 35.01 ± 4.70*** 0.498 ± 0.07* 0.349 ± 0.03**
500 120.70±3.80*** 157.19±4.83*** 301.10 ±3.83*** 33.07 ± 2.87*** 0.376 ± 0.03*** 0.285 ± 0.03***
Curative (mg/kg)
100 223.19±2.10*** 300.30±5.13*** 470.83 ±4.10*** 40.08 ± 4.10* 0.517 ± 0.001ns 0.483 ± 0.02ns
250 189.27±4.17*** 269.83±5.31*** 335.90 ±3.30*** 33.70 ± 3.10*** 0.510 ± 0.003** 0.360 ± 0.07**
500 125.60±5.17*** 153.93±3.87*** 310.90 ±2.87*** 32.90 ± 3.10*** 0.365 ± 0.01*** 0.275 ± 0.01***
Results are Mean ± SE, n = 6, * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to CCl4 control.
ns: Statistically not significant.

4. 544 INDIAN J NAT PROD RESOUR, DECEMBER 2012
decrease of GSH content in dose dependent Discussion
manner compared to CCl4 intoxicant group. The aim of current investigation was to study the
The results are presented in Table 2. prophylactic (preventive) and curative effects of
M. oleifera pods extract on CCl4 poisoned liver damage
Effect of test extract on liver LPO content in rats. Liver injury induced by CCl4 is perhaps widely
The effect of test extract on CCl4 mediated used experimental model for the screening of
LPO was examined through monitoring the levels hepatoprotective agent25. Several mechanisms
of MDA (Malondialdehyde). The extract of underlying this toxicity have been suggested. CCl4 the
title plant on pre- and post-treatments dose inactive metabolite is biotransformed by cytochrome
dependently reversed the CCl4 intoxicated P–450 enzymes to produce the trichloro methyl free
elevation of hepatic MDA level. Though, there radical (CCl3•) that causes lipid peroxidation and there
was considerable decrease in LPO content by produce liver damage26-28.
monitored in rats with pre- and post-treatments The dose dependent significant reduction of CCl4
of test extract at a dose of 100 mg/kg, but the rendered elevated plasma activities of SGPT, SGOT,
results were found to be non-significant statistically. ALP, ACP, total and direct bilirubin levels in rats
The results are tabulated in Table 2. pre-and post-treated with 70% hydro-alcoholic extract
of pods demonstrated their ability to restore the
Effect of M. oleifera pods extract on histopathological profile normal functional status of the poisoned liver, and
Histopathological profile of liver from also to protect against subsequent CCl4 liver injury.
CCl4 (Hepatic control group) intoxicated rats The development of hepatotoxicity induced by
reveal hepatic globular architecture disrupted, CCl4 challenge was exacerbated following the
hepatic cells has shown various degree of depletion of glutathione. Therefore, in the current
fatty degeneration like ballooning of hepatocytes, study glutathione level was measured to observe the
fatty cyst, infiltration of lymphocytes, preventive and curative effects of M. oleifera pods in
proliferation of Kupffer cells and congestion of experimental animals. The results obtained from
liver sinusoids. Pre and post treatment of test the present study clearly demonstrated that CCl4
extract was confirmed by histopathological intoxication has significantly reduced the glutathione
examination of liver sections. Administration of level compared to normal control animals. Rats on
test extract at the dose of 500 mg/kg exhibited pre- and post-treatment with pods extract (100, 250
a significant improvement of the hepatic architecture and 500 mg/kg, doses) have clearly restored the levels
and areas of Kupffer cell proliferation and of glutathione significantly in a dose related manner.
sinusoid appeared normal compared to 100 and The CCl4 damaged liver toxicity was associated
250 mg/kg. Histological studies of liver sections with marked increase in liver MDA level. The MDA
are shown in Plate 2 (a-d). elevation has been well accepted reliable marker of
Table 2  Preventive and Curative effects of Moringa oleifera pods extract on GSH and LPO levels
GSH LPO
Groups Absorbance
% increase Absorbance mean ± SEM % inhibition
mean ± SEM
Control 0.783 ± 0.057 0.225 ± 0.008
CCl4 control 0.251 ± 0.032 0.527 ± 0.112
Preventive(mg/kg)
100 0.458 ± 0.039 ns 78.48 0.382 ± 0.015** 33.21
250 0.603 ±0.044*** 140.23 0.313 ± 0.009*** 45.27
500 0.658 ±0.026*** 162.15 0.280 ± 0.014*** 51.04
Curative (mg/kg)
100 0. 428 ± 0.06 ns 87.25 0.392 ± 0.017* 31.46
250 0.623 ± 0.058*** 148.20 0.319 ± 0.012*** 44.23
500 0.663 ± 0.032*** 164.14 0.319 ± 0.012*** 48.42
Results are Mean ± SE, n = 6, * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to CCl4 control.
ns: Statistically not significant.

5. NANJAPPAIAH & HUGAR: CURATIVE EFFECTS OF MORINGA OLEIFERA IN CCL4 DAMAGED LIVER 545
Plate 2 (a-d)  Histological studies of liver sections treated with Moringa oleifera pods extracts: a-Normal control, b-Toxic control,
c-Pre-treatment of pod extract at 500 mg/kg, d-Post-treatment of pod extract at 500 mg/kg
lipid peroxidation. MDA elevation is a result of activity of cytochrome P–450, thereby favouring liver
oxidative stress demonstrated here through the regeneration23. On this basis, it is suggested that
decrease of GSH level in liver. Hence, in the present flavonoids content in the test extract (evident by
study MDA level was also estimated to evaluate preliminary phytochemical screening) could be the
prophylactic and curative properties of extract of the reason for contributing hepatoprotective ability
title plant. Results given in the Table 2 clearly through inhibition of cytochrome P–450 aromatase31.
indicated that CCl4 intoxicated rats showed significant
increase in the MDA level compared to normal Conclusion
control group. Rats on pre- and post-treatment with In conclusion, the findings of the present study
M. oleifera pods extract (100, 250 and 500 mg/kg, suggest that pods extract of the M. oleifera possesses
doses) has significantly decreased the MDA level in a equally potent prophylactic and curative
dose dependent manner. hepatoprotective efficacy against CCl4 rendered liver
The findings of the present investigation were also injury in rats.
consistent with previous reports, where different parts
of M. oleifera treatment (but only pre-treatment Acknowledgements
mode) shown to protect liver from hepatoxicity The authors are thankful to Sri Sha. Bra.
caused by ethanol, diclofenac and antitubercular Chandramoulishwara Shivacharya Swamiji,
drugs18,29,30. President and Sri. T.M. Chandrashekhariah, Secretary,
The mechanism by which M. oleifera pods extract T.M.A.E. Society, Harapanahalli, Karnataka,
exhibited hepatoprotective activity is not clear from for providing necessary facilities to carry out
the present study. However, the important factor in research work. We are also thankful to Principal
the hepatoprotective property of any drug is the & Management of BLDEA’s College of
ability of its constituents to inhibit the aromatase Pharmacy, Bijapur.

6. 546 INDIAN J NAT PROD RESOUR, DECEMBER 2012
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