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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 studyone 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

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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 oleiferaLam 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]

.

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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

oleiferaleaf 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

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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 blood-

glucose fluctuations in the course of the day become truncated, and the mean blood-

glucose 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-

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dosing to adequately capture the maximum reduction in serum glucose levels[8]

.

The blood sugar lowering effect ofMoringa oleiferalam was tested in albino rats

by a group of researchers in Nigeria. In this study, albino rats of both sexes weighing 80-

170g 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.

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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 beta-

hydroxybutyrate. 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).

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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).

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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 oleif era (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

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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].

Available from

http://region3.dost.gov.ph/index.php?option=com_content&view=article&id=415:

the-efficacy-of-moringa-oleifera-malunggay-leaf-extracts-in-lowering-blood-

glucose-in-swiss-

2. Ples, M. & Ho, H. Comparative Effects ofMoringa oleiferaLam. Tea on Normal

and Hyperglycemic Patients. No date. [cited 2014 Aug 16]. Available fromhttp://www.fitnessiz4u.com/Health_International_Blood_Sugar.pdf

3. Oyewo, E., Adeleke, E., Fakunle, B. & Iniaghe, M. Blood Glucose and Lipid

Reducing Activities of the Oral Administration of Aqueous Leaf Extract of

Moringa oleiferain Wistar Rats. Journal of Natural Sciences Research [Internet].

2013. [cited 2014 Aug 16]. Available from

http://www.iiste.org/Journals/index.php/JNSR/article/view/5723/5843

http://www.fnri.dost.gov.ph/images/stories/7thNNS/health/health%20survey_results.pdfhttp://www.fnri.dost.gov.ph/images/stories/7thNNS/health/health%20survey_results.pdf

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4. Ndong, M., Uehara, M., Katsumata, S. & Suzuki, K. Effects of Oral

Administration ofMoringa oleiferaLam on Glucose Tolerance in Goto-Kakizaki

and Wistar Rats. Journal of Clinical Biochemistry and Nutrition [Internet]. 2007

Apr 25 [cited 2014 Aug 16]. 40(3): 229-233. Available from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275769/. doi

10.3164/jcbn.40.229

5. Giridhari, V., Malathi, D. & Geetha, K. Anti Diabetic Property of Drumstick

(Moringa oleifera) Leaf Tablets. International Journal of Health and Nutrition

[Internet]. 2011 Feb 22 [cited 2014 Aug 17] 2(1): 1-5. Available from

http://www.asciencejournal.net/asj/index.php/IJHN/article/view/68/pdf_27

6. Adisakwattana, S. & Chanathong, B. -glucosidase Inhibitory Activity and Lipid

Lowering Mechanisms of Moringa oleifera Leaf Extract. Eurpean Review for

Medical and Pharmacological Sciences. 2011 [cited 2014 Aug 16]. 15: 803-808.

Available from http://www.europeanreview.org/wp/wp-content/uploads/999.pdf

7.

Glucobay Product Information [Internet]. Australia: Bayer. 2009 Oct 12 [cited2014 Aug 16]. Available from

http://www.bayerresources.com.au/resources/uploads/PI/file9350.pdf

8. Darft Guidance on Acarbose. Food and Drug Administration. 2009 Jul [cited 2014

Aug 17]. Available from

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformatio

n/Guidances/UCM170242.pdf

9. Edoga, C.O., Njoku, O.O., Amadi, E.N. & Okeke, J.J. Blood Sugar Lowering

Effect of Moringa Oleifera Lam in Albino Rats. International Journal of Science

and Technology [Internet]. 2013 Jan [cited 2014 Aug 16]. Available from

http://ejournalofsciences.org/archive/vol3no1/vol3no1_16.pdf