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Pharmacogn. J.
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Original Article
Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016 255
Anti-anemia Eect of Standardized Extract of Moringa Oleifera
Lamk. Leaves on Aniline Induced Rats
Abdul Mun’im1*, Meidi Utami Puteri1, Santi Purna Sari3, Azizahwati4
1Department of Pharmaceutical Sciences, Laboratory of Pharmacognosy, Faculty of Pharmacy, Universitas Indonesia, Depok, 16424, INDONESIA.
3Department of Pharmaceutical Care, Laboratory of Pharmacology-Toxicology, Faculty of Pharmacy, Universitas Indonesia, Depok, 16424, INDONESIA.
4Department of Pharmaceutical Care, Laboratory of Pharmaceutical Analysis, Faculty of Pharmacy, Universitas Indonesia, Depok, 16424, INDONESIA.
ABSTRACT
Background: The leaves of Moringa (Moringa oleifera Lamk.) have been
empirically used as anti-anemia for pregnant woman. Objectives: In the
present study, the effect of ethanolic extract of moringa leaves was evaluated
on the hematology parameters (hemoglobin, red blood cell count, hematocrit),
on aniline induced white female rats. Materials and Methods: Thirty
female rats strain Sprague-Dawley were divided into six groups: normal,
anemia and ferrous fumarate group, and three groups of Moringa leaves
extract. All groups, except normal group, induced by aniline at dose
0.005 ml/ g bw intraperitonially. On fth day, normal and anemia groups
were given 0.5% CMC, ferrous fumarate group was given ferrous fumarate
orally and three other groups were given Moringa leaves extract at doses
198 mg, 396 mg, and 792 mg/200 g bw until the eleventh day. Results:
The results showed that as Moringa leaves extracts increased the level
of hemoglobin, red blood cell count, hematocrit, and total iron content in
blood. Moreover, Moringa leaves extract at dose 792 mg/200 g bw can
improve red blood cell’s morphology and increased the level of hemoglobin
and red blood cell count signicantly (p<0.05). Conclusion: The ethanolic
extract of Moringa leaves showed anti-anemia on aniline-induced rats.
Key words: Anemia, Aniline, Erythrocyte, Moringa oleifera L, Total iron.
Correspondence:
Abdul Mun’im,
Department of Pharmaceutical Sciences,
Laboratory of Pharmacognosy, Faculty of Pharmacy,
Universitas Indonesia,
Depok, 16424, INDONESIA.
Ph no: +62 85216104550
Email: munimabdoel@gmail.com
DOI : 10.5530/pj.2016.3.14
INTRODUCTION
According to data from Center Bureau of Statistic Bureau, Republic of
Indonesia in 2007, maternal mortality due to birth in Indonesia reached
out to 228 per 100,000 of natality. Based on the data, Maternal Mortality
Rate (MMR) in Indonesia is still the highest in Asia and bleeding occupies
the highest percentage of maternal mortality (28%). It is known that anemia
and chronic energy deciency in pregnant women are a major cause of
bleeding.1
Anemiais a decrease in number of red blood cells (RBCs) or less than
the normal quantity of hemoglobin in the blood. WHO recorded an
average of 45% of pregnant women in developing countries are anemic.
In Indonesia, there were 63.5% of pregnant women with anemia.2,3
e consequences of anemia in pregnant women include: increased
maternal and prenatal mortality and increased numbers of preterm birth
and/or low birthweight.4 ere are iron supplements as a therapeutic
agent for anemia but the limitations of bioavailability and side eects
of synthetic drugs encourage the development of the use of herbs as an
alternative therapy for anemia.
Moringa oleifera Lamk. is the most widely cultivated species of a mono-
generic family, the Moringaceae, that is native to the sub-Himalayan
tracts of India, Pakistan, Bangladesh and Afghanistan. It is a perennial
which for centuries has been advocated for traditional medicinal and
industrial uses. All parts of the Moringa tree are edible and have long
been consumed by humans. It is known that the Moringa has many
benets based on its nutrition. e ratio of grams per gram, Moringa
leaves dry powder contains 25 times more iron than spinach, in which
iron is one of the therapeutic agent for anemia.5 e aim of this study
was to evaluate the anti-anemia eect of Moringa leaves extract aniline-
induced rats.
MATERIALS AND METHODS
Plant materials
Fresh leaves of Moringa were collected from Botanical Garden of
National for Food and Drugs Control Agency Republic of Indonesia,
Bogor, East Java, Indonesia. e plant was authenticated by LIPI, Bogor,
Indonesia. e specimen was deposited in Herbarium of Laboratory of
Pharmacognosy, Faculty of Pharmacy Universitas Indonesia.
Preparation of the extract
e plant material was air-dried at room temperature. e dried leaves
were grounded to powder, then approximately 3 kg dried of the powder
were macerated with 70% ethanol for 24 h at room temperature (28oC ±
2°C). Extract was ltered, and the residue was re-macerated two times.
e extract was concentrated using rotary vacuum evaporator at a tem-
perature of 50oC.
Determination of loss on drying
e extract (1 g) was heated at a temperature of 105oC for 30 min in
oven. at 105oC until the weight is stable. Before each weighing, weigh the
bottle in the closed position is allowed to cool in a desiccator to room
temperature.
Determination of total ash content
e extract (2 g) was weighed carefully and then put in a crucible. en,
the crucible was ignited slowly until charcoal was eliminated, cooled,
and had constant weight. Determination levels of acid-insoluble ash:
ash obtained in the determination of the total ash content was boiled
ABDUL MUN’IM et al.: Anti-anemia moringa leaves extract
256 Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016
with 25 ml of dilute sulfuric acid for 5 min. Acid insoluble part was
collected and ltered through ash-free lter paper and then washed with
hot water and was incandescent to keep the weight. Ash content that
does not dissolve in acid calculated on the dried material.
Determination of total iron (Fe) content
e iron content in extract and blood were analysed using atomic
absorption spectrophotometry. e sample (extract or blood) was prepared
by wet destruction method. Sample was added 10 ml of concentrated
HNO3 and has been le for one day. en the sample was heated until the
white smoke appeared then added ve drops of HClO4 slowly. Sample
solution has been allowed to cool and then ltered using lter paper and
was diluted into a 50 mL volumetric ask with distilled water. e absor-
bance was then measure using spectrophotometer.
Phytochemical screening of the extracts
Phytochemical constituent such as alkaloid, avonoid, terpene, saponin,
glycoside and anthraquinone in the extracts was performed by using
standard analytical procedures with slight modication.6 e analysis
also was performed with TLC using specic sprayreagents. Spray
reagents used were Dragendor for alkaloid; AlCl3 for avonoid; and
FeCl3 for tannin.
Experimental design
e experiments were carried out in 30 female albino rats of the species
Ratus norvegicus, Sprague Dawley Strain, weighing 150–200 g. e study
was approved by the Institutional Animal Ethical Committee (Registration
no.634/02/a/CPCSEA). e animals were acclimatized for 2 week under
laboratory conditions. Completely randomized design (CRD) is used in
this study. e rats were divided into six groups with ve animals in each
groups (n=5) as follows:
• Group I (normal group)–received 0.5% CMC 3 ml/200 g bw/day
p.o. for 6 days.
• Group II(negative control)–received 0, 5% CMC 3 ml/200 g bw/day
p.o. for 6 days.
• Group III (positive control)–received Ferro Fumaras Suspension
175,5 mg/200 g bw/day p.o. for 6 days.
• Treatment group IV received Moringa leaves extract at doses 198
mg/200 g bw/day p.o. for 6 days
• Treatment group V received Moring leaves extract at doses 396
mg/200 g bw/day p.o. for 6 days
• Treatment group VI–received Moringa leaves extract at doses 792
mg/200 g bw/day p.o. for 6 days.
Induction of anemia
e experiment was performed using aniline (Wako, Japan) for induc-
ing anemia. Aniline suspension was given by injection intraperitoneally
(0.003 ml/gbw/day). All the animals, except the normal group were
treated with the injection of aniline for 2 days, along with the normal
diet. On the h day, blood of animals were collected for evaluation.
en normal and negative control were given 0.5% CMC, positive
control was given ferrous fumarate and three other groups were given
Moringa leaves extract at doses 198 mg, 396 mg, and 792 mg/200 g bw/
day until the eleventh day. e blood was collected from the rat’s eye.
Assessment parameters
In this study, three parameter of anemia were observed. ere are the
shape of red blood cells used light microscope, the hematology exami-
nation (hemoglobin levels, the number of erythrocytes and hematocrit
levels) using hematology analyzer, and the calculation of total iron (Fe)
concentration in blood using atomic absorption spectrophotometer.
Statistical Analysis
e data were presented in terms of mean and standard deviation. e
statistical signicance was evaluated by ANOVA and Mann-Whitney.
Dierences at pvalue of less than 0.05 was considered statistically
signicant.
RESULTS AND DISCUSSION
e ethanol extract of Moringa leaf extract obtained in the form of thick
and sticky, brown, aromatic odor, sour taste, and have solid extract in the
amount of 27.32%. Screening of the chemical constituent of Moringa leaf
extract obtained that extract contains of avonoids, alkaloids, saponins, tan-
nins, and glycosides. e Moringa leaves contained of avonoid, alkaloid,
phenol, steroid and glycosides. is results is similar with previous study.7
e results indicated that the determination of non-specic parameters
showed that extract of Moringa leaves have averaged 21.18% percent dry-
ing, total ash content of 1.70%, and acid insoluble ash content of 0.28%. To-
tal iron content in Moringa extract of the averageis 19.38 mg/kg of extract.
Hemolytic anemia with aniline as an induction model is chosen because
it is easier and cheaper than the other induction model, such as genetical
induction model or using the other medicines.8 Anemia occurred
accordance with the eects caused by aniline induction, which came
from methemoglobinemia and hemolytic anemia. ey can be occurred
because an active metabolite from aniline which is phenylhydroxylamine
oxidized oxyhemoglobin to methemoglobin and hydrogen peroxide
(H2O2). H2O2 could cause oxidation of SH groups that are important in
protein. Beside that, it also causes the lipid peroxidation on the red blood
cells’s membrane which lead the membrane to lysis.8-10
is study used six groups of female rats which are consisted of ve rats
per group. e dose variation which is used in this study is aimed to
know which dose of Moringa leaves that has optimal eect. In this study,
three parameters of anemia are observed. ere areobservation of the
shape of red blood cells, the calculation of the blood hematology (hemo-
globin levels, the numbers of erythrocytes, and hematocrit levels), and
the calculation of total iron (Fe) concentrationin blood.
e examination with hematology analyzer sometimes could not reect
the real situation therefore the conrmation of the red blood cells’s shape
is needed for cross checking. e red blood cells’s shape is observed by
using light microscope. is is a qualitative observation, which only
observes the shape of the red blood cells and compares the preparations of
peripheral blood smears between the treatment groups.e observation
was done on the 11th day aer treatment of the Moringa leaves extract.
Figure 1 shows that the red blood cells’shape on anemia control group
and dose 1 group has not recovered yet, because the size was smaller
than the normal group. On the ferrous fumarate group and the group
of dose 3 showed that the red blood cells’shape has already recovered
to be normal. Meanwhile, the red blood cell’s shape group of dose 2 has
not completely recovered. Based on these observations, researcher
conclude that on the anemia control group and the group of dose 1, it
had still mycrocitic anemia where the shape of red blood cells are hap-
pened to be smaller than normal. erefore, on the ferrous fumarate
group and the dose 3 group show that there was an inuence ingestion of
the ferrous fumarate suspension and Moringa leaves extract on the red
blood cells’shape. It can be seen from the shape which is back to normal.
e group of dose 2 of Moringa leaves extract recover the shape of red
blood cell, but it is not signicant.
e second and third parametersare the observation of hematology
value and total iron content. Detailed results can be seen at Table 1.
ABDUL MUN’IM et al.: Anti-anemia moringa leaves extract
Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016 257
e table shows the increasing percentage of hematology parameter and
total iron content in blood. e treatment of ferrous fumarate increased
erythrocyte, hematocrit and total iron.
e increase anemia parametersin positive group is caused by ferrous
iron which is presented in ferrous fumarate. It was absorbed eciently
by the rats and lead an increasing of red blood cells production.
e iron content in the Moringa leaves acts as main nutrient in hemato-
poiesis process in the spinal cord, because Moringa leaves were reported
iron-rich.11 Beside that, the protein and amino acids content in Moringa
leaves are also act as hematopoietic growth factor. e leaves reported
high content of protein and amino acids.12-13 It plays vital role in managing
proliferation and blood cells dierentiation. Vitamin C content in
Moringa leaves extract also increase iron absorption in body.14 In addition
to the dose-treatment groups, hemoglobin and erythrocytes levels are
also increased in anemia control group which did not get Moringa leaves
extract treatment. e increased is due to the normal process of the body,
which would boost the eritopoesis process (the formation and maturation
of erythrocytes) that happen when body cells are deprived of oxygen.15
CONCLUSION
e results showed that as the dose of Moringa leaves increasing, the level
of hemoglobin, erythrocyte, hematocrit, and total iron content in blood
of rats are also increased. Moreover, Moringa leaves extract at dose
792 mg/200 g bw/day can improve erythrocyte’s morphology and increase
the level of hemoglobin and erythrocyte of rats signicantly (p< 0,05).
ACKNOWLEDGEMENTS
is study was supported nancially by the Directorate General of High-
er Education, Ministry of National Education Republic of Indonesia, via
Hibah BOPTN Desentralisasi 2013.
CONFLICT OF INTEREST
All co-authors have seen and agree with the contents of the manuscript
and there is no nancial interest to report. We certify that the submission
is original work and is not under review at any other publication.
ABBREVIATION USED
RBCs: red blood cells; CMC: Carboxymethyl cellulose; CRD: Complete-
ly randomized design.
REFERENCES
1. Center Bureau for Statistic Republic of Indonesia. Demography and Health
Surveyin Indonesia: supporting document. 2008.
2. Mason J, Rivers J, Helwig C. Recent trends in malnutrition in developing
regions: Vitamin A deciencies, anemia, iodine deciency, and child under-
weight. Food Nutr Bull. 2005;26(1):57-162.
3. Saifudin. Buku Acuan Nasional Pelayanan Kesehatan Maternal dan Neonatal.
Ed I, Yayasan Bina Pustaka, Jakarta. 2006.
4. Fahey JW. Moringa oleifera: A Review of the Medical Evidence for Its Nutri-
tional, Therapeutic, and Prophylactic Properties. Part 1. Trees Life J. 2005;1:5.
Table 1: Increase in the percentage of hemoglobin levels, erythrocyte,
hematocrit and average total iron content in female rats after extract
administration
Group Percentage increase (%)
Erythrocyte Hemoglobin Hematocrit Total Iron
Normal 4.42 1.98 4.14 2.39
Anemia 8.48 8.78 18.75 33.43
Positive 28.25 26.67 50.08 52.92
Dose 1 30.63 25.26 46.35 40.04
Dose 2 33.83 36.13 53.82 26.69
Dose 3 40.32 34.97 60.38 59.51
Figure 1: Peripheral blood smears of the white female rats after extract administration.
ABDUL MUN’IM et al.: Anti-anemia moringa leaves extract
258 Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016
11. Barminas JT, Charles M, Emmanuel D. Minreal composition of non-conventional
leafy vegetables. Plants Food Hum Nutr. 1998;53(1):29-36.
12. Afuang W, Siddhuraju P, Becker K. Comparative nutritional evaluation of raw,
methanol extratcted residue and methanol extracts of moringa (Moringa oleifera
Lam.) leaves on growth performance and feed utilization in Nile tilapia (Oreo-
chromis niloticus L.). Aquacul Res. 2003;34(13):1147-59.
13. Makkar HPS, Becker K. Nutritional value and antinutritional components of
whole and ethanol extracted Moringa oleifera leaves. Animal Feed Sci Technol.
1996;63(1):211-28.
14. Katzung BG, Masters SB, Trevor AJ. Basic and Clinical Pharmacology, 10th ed,
San Fransisco: Mc.Graw Hill. 2006; 489-93.
15. Sherwood L. Fisiologi manusia dari sel ke sistem edisi edisi 2. Indonesian
translation fromHuman physiology:from Cells to system by Brahmm U.P. Jakarta:
EGC. 2001.
5. Kasolo JN, Bimenya GS, Ojok L, Ogwal-Okeng, JW. Sub-acute toxicity evalu-
ation of Moringa oleifera aqueous and ethanol extracts in Swiss Albino Rats.
Inter J Med Plant Res. 2012;1:75-81.
6. Mun’im A, Katrin, Azizahwati, Andriani A, Mahmudah KF, Mashita M. Screening
of α-glucosidase inhibitory activity of some Indonesian medicinal plants. Inter J
Med Arom Plant. 2013;3(2):144-50.
7. Anwar F, Latif S, Asraf M, Gilani AH. Moringa oleifera: A food plant with multiple
medicinal uses. Phytotherapy Research. 2007;21(1):17-25.
8. Ciccoli L, Ferrali M, Rossi V, Signorini C, Alessandrini C, Comporti M. Hemolytic
drugs aniline and dapsone induce iron release in erythrocyte and increase the
free iron pool in spleen and liver. Toxicol Letter. 1999;110(1-2):57-68.
9. Umbreit J. Methemoglobin-it’s not just blue: a concise review. Amer J Hematol.
2007;83(2):138-44.
10. Murray R, Granner DK, Mayes PA Rodwell VW, eds. Harper’s Biochemistry, 25th ed.
London, Prentice Hall International Inc. 2000.
PICTORIAL ABSTRACT
• Moringa leaves extract signicantly increased the level of haemoglobin.
• Moringa leaves extract increased the level of red blood cell count.
• Moringa leaves extract increased the level of total iron content in the
blood.
ABOUT AUTHORS
Abdul Mun’im: Completed his PhD degree at Institute of Applied Biochemistry, University of Tsukuba, Japan.
Currently, he focused his research on Indonesia’s traditional medicinal plants for anti-metabolic syndrome, such
anti-diabetes, anti-dyslipidaemia and anti-hypertension, via inhibition of angiotensin converting enzyme activity.
Meidi Utami Puteri: Finished her bachelor in pharmacy, at Faculty of Pharmacy, Universitas Indonesia. Now
sheis a master student at the Faculty of Medicine, University of Tsukuba (Japan). Her master research focused
on experimental pathology.
Santi Purna Sari: Finished her master program at Faculty of Pharmacy, University of Gadjah Mada, Yogyakarta,
Indonesia. Her research in master program wasabout Pharmacokinetic and pharmacodynamics interaction
between herbal products with conventionaldrugs. She focused her research on pharmacologicalactivity and
toxicology of natural productsin vitro and in vivo.
Azizahwati: Finished her master at Faculty of Medicine, Universitas Indonesia. She is Chairman of Laboratory
of Pharmaceutical Analysis. Currently, she focused her research on organic sulfur compound from Indonesia’s
Traditional Medicinal Plants, and their hepatoprotective activities.
SUMMARY
