Chapter 2

Review of Related Literature

A study was conducted to determine the effectiveness of Malunggay (Moringa
oleifera) in lowering the blood glucose in swiss mice. The experimental mice were
weighed. Before the experiment, their blood glucose was measured using a glucometer.
They were fed with condensed milk through gavage method for five (5) consecutive days.
Blood glucose measurements after the procedure revealed high blood glucose amongst all
the mice. Different concentrations of Malunggay (Moringa oleifera) leaf extracts were
prepared and were used to treat the mice. Data analysis revealed that when subjected to
different levels of Malunggay (Moringa oleifera) leaves extract to the mice, there was a
significant difference in the mean blood glucose level. The findings confirmed that the
Malunggay (Moringa oleifera) leaf extract has a hypoglycemic property that can be used
in treating diabetes [1].
Another study was conducted to test the effects of taking tea prepared from
Malunggay (Moringa oleifera) on the blood sugar levels in humans. There were two
groups involved in the study one group included people with normal fasting blood sugar
levels (60-120 mg/dl), and the other included those with hyperglycemic fasting blood
sugar levels (>120 mg/dl). Malunggay tea was administered to both groups. After two (2)
hours of intervention, there was no significant change in the fasting blood glucose level
of the people in the normal group. However, there was a significant drop in the fasting
blood glucose level among hyperglycemic individuals. The results indicate the benefit of
Malunggay in the management of hyperglycemia [2].
Another investigation was conducted to determine the effect of Malunggay on

blood glucose in male Wistar rats divided into four (4) groups with six (6) rats per group.
Dry leaf powder of Malunggay was extracted with water and lyophilized. Calculated
amounts of lyophilized aqueous leaf extracts of Malunggay were constituted in distilled
water to give doses of 250, 500, and 100 mg/kg body weight. Each dose is given to one
(1) group, the fourth group being the control group receiving only 1.0 ml distilled water.
The blood glucose levels of the rats, measured through the use of glucometer and test
strips, were recorded before the administration of aqueous leaf extracts. The aqueous leaf
extracts were administered to each experimental group orally, once daily, with the use of
a metal cannula attached to a 2 ml syringe, and the administration lasted for 56 days. The
rats were fasted for twelve (12) hours and the blood glucose was determined. Results
showed that all concentrations of the extract resulted in significant lowering of blood
glucose concentrations in a non-dose dependent pattern. This led the investigators to
conclude that the Malunggay aqueous leaf extract possessed blood glucose properties [3].
A different study was done to determine the effects of Oral Administration of
Moringa oleifera Lam on Glucose Tolerance in Goto-Kakizaki and Wistar Rats. Together
in this study, the researchers determined via High Performance Liquid Chromatography
that a high concentration of a certain polyphenol, quercetin-3-glycoside (Q3G), is present
in M. oleifera leaf powder to which they have attributed the glucose intolerance
ameliorating effect to. The researchers speculated that the hypoglycemic effect of MO
might be due to an inhibition of glucose uptake with Q3G and slowing gastric emptying
with fiber in MO leaf powder, although other active components existed in M. Oleifera.
The proponent suggests further studies are needed to identify the certain active
components of MO for glucose intolerance ameliorating actions [4].

In a study conducted by Giridhari, et. Al, (2011), sixty screened diabetic subjects,
who were taking sulfonylureas as oral anti-hyperglycemic, was divided into a control and
experimental group. Inclusion criteria such as BMI between 20-25kg/m2, involved in
sedentary activity and age group between 40-58 years were taken into account. The
subjects were given thirty tablets which they are supposed to take twice a day during a
15-day period, one for breakfast and one for dinner. They were also advised to take a
standardized diet during the study period. Glycated hemoglobin was measured after 3
months. There was no significant decrease in HbA1c in the control group over 90 days.
However, the reduction from a mean of 7.81 to 7.4 was observed in the experimental
group. This highly significant decrease emphasized that supplementation with Moringa
oleifera leaf tablets had a positive effect in lowering blood glucose over time [5].
-glucosidase inhibitory effect of Moringa oleifera was observed in a study
conducted by B. Chanathong & S. Adisakwattana. The aim of their study was to
investigate the effect of the leaf extract of Moringa oleifera on inhibition of glucosidase, pancreatic -amylase, pancreatic lipase, and pancreatic cholesterol esterase
activities. Determination of the inhibition of cholesterol micellization formation, and bile
acid binding capacity of the extract were also conducted. Concentration of glucose was
determined by glucose oxidase method after performing -glucosidase inhibition assay.
The results indicated that the extract inhibited both but was a more specific inhibitor of
intestinal sucrase than intestinal maltase. With the study, they have hypothesized that
phenolic compounds, flavanoids and condensed tannins contained in Moringa oleifera
contribute to its -glucosidase inhibitory effect [6].
Acarbose exerts its activity in the intestinal tract. The action of acarbose depends

on an inhibition alpha-glucosidases involved in the degradation of ingested disaccharides,

oligosaccharides, and polysaccharides, but not monosaccharides. This leads dose
dependently, to a delayed digestion of the above carbohydrates. The result is that
absorbable monosaccharides originating from carbohydrates are released more slowly
and hence more slowly taken up into blood. Absorption of monosaccharides is not
affected. In this way, acarbose reduces the postprandial rise in blood glucose, the bloodglucose fluctuations in the course of the day become truncated, and the mean bloodglucose level is reduced. Acarbose lowers abnormally high levels of glycosylated
haemoglobin [7].
Acarbose has an antihyperglycaemic effect, but does not itself induce
hypoglycaemia. If acarbose is prescribed in addition to drugs containing sulfonylureas or
metformin or in addition to insulin, a fall of blood glucose levels into the hypoglycaemic
range may necessitate a suitable decrease in the sulfonylurea, metformin or insulin dose.
In individual cases hypoglycaemic shock may occur [7].
Subjects should receive a challenge dose of 75 g of sucrose on the day prior to
drug treatment. The sugar may be given as a solution, 75 g in 150 mL water. The sucrose
challenge should follow an overnight fast. Following the administration of sucrose, blood
should be sampled for serum glucose for up to 4 hours. Drug treatment will take place on
the following day. On the drug treatment day, drug should be given together with 75 g of
sucrose. Blood should be sampled for serum glucose for up to 4 hours after
acarbose/sucrose administration. The literature suggests that the maximum reduction of
serum glucose following acarbose administration upon sucrose challenge occurs within
the first hour. Therefore, we recommend intensive sampling during the first hour post-

dosing to adequately capture the maximum reduction in serum glucose levels [8].
The blood sugar lowering effect of Moringa oleifera lam was tested in albino rats
by a group of researchers in Nigeria. In this study, albino rats of both sexes weighing 80170g were used. The subjects were housed for 14 days with free access to water and feed
before the experiment commenced. Diabetes was induced by slow intraperitioneal
injection of 1% solution of alloxan (120 mg/kg body weight) dissolved in water. After
this, the subjects were fasted for 12 hours. At the end of fasting, blood glucose levels
were taken from the subjects. Only albino rats with blood glucose levels above 100 mg/dl
were used. The rats were divided into three groups. Group 1 received normal saline,
group 2 received tolbutamide and group 3 received the moringa extract. Blood samples
were taken from the diabetic rats from the tail vein at 0, 1, 3, and 6 hours after
administration of the extract. The extract produced significant hypoglycemic effect,
giving a percentage of reduction in blood sugar levels of 31.22, 40.69 and 44.96% for
100, 200, and 300 mg/kg dosed of extract at 6 hours of administration while tolbutamide
exhibited 46.75% reduction of blood sugar levels. From the obtained results, the
researchers concluded that Moringa oleifera is comparable with the reference drug
tolbutamide [9].
Experimental models and methods
This part of the chapter of the study presents each experimental methods and models
that will be used in the study and the specificity of the animal to be used. This shall serve
as the basis of appropriateness and accuracy of the study.

Animal model. Animal models of diabetes are therefore greatly useful and
advantageous in biomedical studies because they have characteristics which are
associated with humans, the results of gathered from these animals may be an indicator of
a much larger species such as humans. They also present studies that can be correlated as
human diabetes. Most of the available models are based on rodents because of their small
size, short generation interval, easy availability and economic considerations. Non-rodent
models of diabetes are may be needed for valuable supplement to rodents for both
practical and physiological reasons with respect to humans.
Various types of animal models of type2 diabetes derived either spontaneously or
induced by treating with chemicals, or dietary or surgical manipulations and combination.
Sprague-Dawley Rats and Wistar albino rats are candidates for hyperglycemic studies
(Srinivasan & Ramarao, 2007).
Acclimatization. Research animals transported from outside the institution are
expected to experience mild to moderate stress. This stress results from changes in the
environment, fluctuations in temperatures during transportation, short-term food and
water deprivation, noise, or other physical aspects of shipping. Changes may be observed
from the laboratory animals from the place of purchase to the experimentation room,
these changes includes elevated heart rate and weight loss, as well as elevated
concentrations of adrenaline, noradrenaline, glucose, cortisol, free fatty acids, and betahydroxybutyrate. Carbohydrate, protein, and lipid metabolism (both lipolysis and
lipogenesis) are altered, and plasma osmolality, albumen, protein, and packed-cell
volume increase. These will be observed continuously if improper care for animals is
given (University of Toledo, 2011).

The acquisition of rodents from other establishments is subject to transportation

according to the IACUC Protocol for transportation. Upon arrival, quarantine must also
be conducted to the animals to make sure appropriateness. Boston University requires an
acclimatization period of three full days (72 hours) prior to any use of any survival
experiments (BU IACUC, 2013).
Drug-induced diabetes. Streptozotocin is an antibiotic derived from Streptomyces
achromogenes and structurally is a glucosamine derivative of nitrosourea. It causes
hyperglycemia mainly by its direct cytotoxic action on the pancreatic beta cells. The
evidences are accumulating on the mechanisms associated with diabetogenecity of STZ.
Its nitrosourea moiety is responsible for beta cell toxicity, while deoxyglucose moiety
facilitates transport across the cell membrane (Sharad, 2010).
STZ when injected neonatally or immediately after birth, rats develop type 2 diabetes
in the adult age. Single injection of STZ at the dose range of 80-100 mg /kg of STZ (iv or
ip or sc) to one or two or five day old Wistar or Sprague-Dawley neonatal rats has been
reported to produce type 2 diabetic conditions. The neonatal STZ rats are considered to
be better tools for the elucidation of the mechanisms associated with regeneration of the
beta cells, the functional exhaustion of the beta cells and the emergence of defects in
insulin action (Srinivasan, 2007).
Determination of blood glucose levels. Tail tipping is used to obtain blood samples
from the experimental animals to be able to determine the blood glucose levels. Excision
of a few millimeters from the end of a mouse's tail and partial amputation of the mouse's
tail are minor surgical procedures must be performed in accordance with Public Health
Service guidelines and policies for survival rodent surgery (Jackon Lab., 2001).

Standard tail tip excision is used when diameter of the cut end of the tail is 2 mm or
less (infant mice). The use sterile equipment for all procedures is highly advisable. It is
most practical to use a new sterile #10- or #15 scalpel blades. Sterilizing a 3 inch or 4
inch ssharp surgical scissors or a safety razor blade using steam or dry heat can be used in
tail tipping. The scissors must be allowed to cool for 90 seconds before use in order to
avoid burning the tail. The use chlorhexidine, glutaraldehyde or Betadine solution as a
chemical disinfectant in the tips of the tail (Jackon Lab., 2001).
In the practice of medicine, for persons with diabetes blood glucose levels are
monitored via glucose strips. It provides immediate feedback on the effects of daily
activities such as taking medication, exercise, or eating on blood glucose levels. Blood
glucose monitoring helps to evaluate glycemic control and is especially useful in
identifying hypoglycemia and hyperglycemia. Accuracy of glucose meter systems, in
particular for glucose values greater than 75 mg/dl. Todays sets are implied to have 99%
accuracy compared to the last version of only 95% (AADE, 2013).
Toxicity testing of Moringa oleifera (Malunggay). Acute toxicity tests can provide
preliminary information on the toxic nature of a material for which noother toxicology
information is available. According to FDA, it may determine possible target organs and
can be the determination of the effectivity of a certain study.
In most acute toxicity tests, each test animal is administered a single (relatively high)
dose of the test substance, observed for 1 or 2 weeks for signs of treatment-related
effects, then necropsied. Some acute toxicity tests (such as the "classical" LD50 test) are
designed to determine the mean lethal dose of the test substance. The median lethal dose
(or LD50) is defined as the dose of a test substance that is lethal for 50% of the animals in

a dose group. LD50 values have been used to compare relative acute hazards of industrial
chemicals, especially when no other toxicology data are available for the chemicals.
However, many important observations of toxicity are not represented by LD50 values or
by slopes of dose-response curves for lethality. An example stated would be about the
information about morbidity and pathogenesis may have more toxicological significance
than mortality, and these endpoints also should be evaluated in short term toxicity tests.
According to Food Drug Adminstration (2011), Acute toxicity studies (LD50) may be
measured using method of Lorke (1989).
The current level of awareness and usage of health supplements is high among adults in
metro manila. More adults surveyed in 2008 were aware and were users of health
supplements than in 1998. For both survey periods, there is a strong belief on the positive
effects of health supplements. Lastly, patients who were diagnosed with diabetes ranks 3rd
in percentage usage of health supplements as of 2008.
Source:
http://www.fnri.dost.gov.ph/images/stories/7thNNS/health/health%20survey_results.pdf
References:

1. Calapatia, R. The Efficacy of Moringa oleifera (Malunggay) Leaf Extracts in

Lowering Blood Glucose in Swiss Mice. [updated 2010; cited 2014 Aug 16].
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and Hyperglycemic Patients. No date. [cited 2014 Aug 16]. Available from
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Reducing Activities of the Oral Administration of Aqueous Leaf Extract of
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4. Ndong, M., Uehara, M., Katsumata, S. & Suzuki, K. Effects of Oral

Administration of Moringa oleifera Lam on Glucose Tolerance in Goto-Kakizaki
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