Abstract

Diabetes is a disease which affects 5% of Philippine population. This is a

worrying prevalence rate. Our search for means of fighting this affection made us to
develop an herbal medicine from ethanolic extract of Terminalia catappa leaves. During
the experience we carried out, Winstar Rat received, inject induced, a solution of glucose
(1.0 cc/mL). As treatment, the hyperglycaemic rabbits were given, Terminalia catappa
leaf extract (1.0 cc/mL). Before administration of glucose overload, the animals had a
basal glycaemia of Mean = 127.1333333. The not treated rats’ glycaemia increased and
reached 194.8 mm/dL. The injectable administration of Terminalia catappa leaf extract
(1.0 cc/mL) lowers blood sugar at 182 mm/dL for 30%, 182 mm/dL for 60%, and 159
mm/dL for 90% concentration after 7 days. At 90% with 1.0 cc/ml, it induces a
significant decrease in blood glucose. The both three concentration exerts on
hyperglycaemic rats a significant basal glucose-lowering effect.The Terminalia catappa
leaf extract appears like an antidiabetic and produces its hypoglycaemic effect mainly
through alkaloids, sterols or triterpens.

Keyterms: diabetic, ethanolic extract, Terminalia catappa, Winstar Rat, hyperglycemic

1
 Chapter I
Introduction

Background of the Study

Terminalia catappa L. belongs to the family Combretaceae. T. catappa is

used primarily as an ornamental shade, and salt-tolerant street tree, but the leaves provide
food for the Tasar silkworm, and the seeds are edible like almonds with sim Malay
peninsula and through the Canary Islands this tree is known as the tropical almond been
claimed to have therapeutic effects for liver related diseases. In India, it is used as cardiac
stimulant. Its leaves are widely used as a folk medicine in Southeast Asia for the
treatment of dermatitis and hepatitis. More and more pharmacological studies have
reported that the extract of T. catappa fruits have anticancer, antioxidant,
antitranscriptase, anti-inflammatory, antidiabetic effects and hepatoprotective activities
components and related mechanisms remain unknown. In the present work,
phytochemical, antimicrobial and invitro antioxidant activities were carried out from
catappa bark methanol extract. Antimicrobial activity of T. catappa bark extract was
investigated against bacterial and fungal cultures whereas inviro antioxidant activity was
performed with DPPH method (Praveena (2014).

Tropical Almond Terminalia catappa is a fast-growing deciduous or semi-

evergreen tree, usually growing about 15 meters tall with specimens up to 40 meters
recorded. The canopy has a pagoda-like habit when young, but this becomes less
noticeable as the tree ages and the branches elongate and droop at the tips. The bole can
be straight or twisted, it is generally set with short woody spines, can be 100 - 150cm in
diameter and is often buttressed at the base with buttresses up to 3 meters high. An
important, multi-purpose tree, providing food, medicines and a host of other
commodities. This is one of the most common trees of many tropical coasts. It is widely
cultivated in the tropics, as a shade tree, for ornament and for its edible seeds. It is often
planted in avenues as a shade-tree, for which it is suitable because of its very regular
shape (Praveena 2014).

2
 Plants grow best in the lowland tropics at elevations below 800 meters. It grows
best in areas where the mean maximum and minimum annual temperatures are within the
range 21 - 32°c, though it can tolerate 10 - 36°c. It prefers a mean annual rainfall in the
range 1,500 - 3,400mm, though can tolerate from 700 - 4,200mm. Succeeds in any
moderately fertile, well-drained soil in a sunny
position. Prefers sands and loamy sands, though it
also does well on silts, loam, and even clays if the
drainage is good. Prefers soil that is neutral to
moderately alkaline and rich in bases; however it
will also grow in strongly acid soils. A particularly
useful plant for coastal gardens, being tolerant of salt spray, established plants are
drought tolerant. Prefers a pH in the range 5 - 7, but tolerates 4.3 – 8 (Ken Fern 2019)..

Plants are fast growing, often in excess of 1 meter a year when young. Trees can
yield two crops a year in some areas, and in places have been known to exhibit more or
less continuous fruiting. A tree may yield around 5 kg of kernels per year. There has been
some selection in cultivating forms with larger seeds. The tree sheds its leaves all at once,
quite suddenly, usually twice a year (January or February and July or August). Unlike
most tropical trees, the leaves turn first yellow, then vivid red before falling, giving a
well-marked 'autumn color'. The inconspicuous flowers have a sweet, delicate fragrance.
Various species of biting and stinging ants have been found inhabiting hollow twigs on
this tree (Tropical Plants Database, Ken Fern. tropical.theferns.info. 2019-08-28).

Wistar rats encompass a number of rat

substrains that have been derived from a common
lineage over the past 50+ years. The Wistar rat,
Wistar Hannover (Wistar HAN) rat, and Wistar
Unilever (WU) rat are outbred rat strains, while the
Wistar Kyoto and Wistar Furth rats are inbred strains.
The outbred Wistar and Wistar HAN strains have been used widely in Europe for
preclinical safety assessments, including 2-year carcinogenicity bioassays. Although
other Wistar substrains are commonly used in specialized carcinogenesis and safety

3
studies, these strains offer neither a substantial history of use in chronic carcinogenicity
bioassays nor a significant publicly available database of historical control pathology and
clinical pathology data (D.L. McCormick 2017).

Diabetes is a metabolic disease which exists everywhere in the world and interests
approximately 6% of the world population. Côte-d’Ivoire has about 5% of diabetics
(Djédjé, 2002). This prevalence rate places the diabetes like most frequent of endocrinien
diseases (Gentilini, 1993). This disorder concerns genetic and exogenic factors (viral,
chemical) and damages the β cells of Langerhans (Kadja, 1998). As a result, the body
becomes unable to produce insulin, a pancreatic hypoglycaemic hormone. This disorder
is characterized by polyuria (frequent and abundant urines), glycosuria (presence of
glucose in urines) and hyperglycaemia (glucose rate on an empty stomach higher than 1.2
g/l in plasma blood and confirmed in at least two occasions). Diabetes comes with other
complications (kidney, eye) and appears as a major cause of disability and death (Dièye
et al., 2008). In modern medicine, no satisfactory effective therapy is still available to
cure diabetes (Paris and Amarnath, 2004). Currently, diabetes therapy is based on the use
of hypoglycaemics (sulfonamides, biguanides, insulin), on hygieno-diet measures and
exercises (Reichard et al., 1993). Even if the injections of insulin or other products make
it possible diabetic to remain in life, the diabetes requires a long treatment, which the
patients have of the evil to support. In the search of means of fighting, man recognized
and used the medicinal properties of many cultivated or wild plants and many drugs to
combat this worrying affection.

Objectives

Specifically the study aims to:

1. Determine the active principles of T. catappa leaves; and

2. Test the antiglycemic activity of T. catappa leaf extract to Rattus norvegicus (Rat)

4
 Chapter II

Review of Literature

Glucose is derived from thediet and utilized by cells with the help of insulin.
Insulin insuffi-ciency elevates the blood glucose and causes hyperglycemia, whichleads
to diabetes. The ethanolic leaf extracts (300 mg/kg and500 mg/kg) decreased blood
glucose levels in diabetic rats. A similar effect has been found in treatment with different
extracts ofT.catappafruits (Nagappa et al., 2003), which confirm the anti-diabetic effects
ofT.catappa(Malviya et al., 2010). The secretion of insulin by beta cells of the pancreas
was affected by diabeticagents. On the other hand, the lower concentration of insulin
inthe toxin treated rat group was due to damage to the pancreatic cells, which were not
able to produce insulin due to DNA alkylation arising from the production of carbonium
ions (Wright et al.,1999). STZ-induced diabetes not only affects the pancreas, but
alsocauses damage to the liver, the kidneys and cardiac cells (Biswaset al., 2011). Related
studies have reported that the induction ofdiabetes in animals by beta cell destruction,
through the produc-tion of free radicals, leads to the alkylation of DNA and a conse-
quent increase in the blood glucose concentration (Takasu et al.,1991). In the present
study, there was a substantial increase inserum insulin levels of diabetic animals
afterT.catappa(300 mg/kg and 500 mg/kg) treatment. Hence, it might be recognized
thatsecondary metabolites of this plant extract stimulate the beta cells.

Ever since the dawn of civilization, medicinal plants have been of great
importance to the health of individuals, communities in Nigeria, and the whole world.
Man has continually investigated tropical and subtropical medicinal plants in order to
assess the importance of developing natural, sustainable, and affordable drugs and
cosmetics. The genus Terminalia L. are perennial shrubs or trees of the Combretaceae,
and nearly 200 species are identified. The genus is distributed in tropics and sub tropics
regions, a few species are found in Africa, northern Australia, Pakistan, India, Sri Lanka
and many other south Asian countries. Tropical almond (Terminalia catappa L.) is a
large tropical tree growing up to 35 m with an upright, symmetrical crown and horizontal
branches. This plant has been studied extensively. The bark is rich in tannins, the fruit is
bitter, acrid,astringent, an aphrodisiac. The leaves contain several flavonoids including

5
kaempferol and quercetin, saponins, and phytosterols. Fallen leaves of T. catappa have
been widely used in traditional medicine for the treatment of liver diseases, headache,
colic, and as a diuretic and cardiotonic T. catappa extracts have shown antidiabetic,
actinociceptive antiparasitic, antibacterial, and antioxidant activities, while the aqueous
extract of the bark is used traditionally to treat dysentery and diarrhea.

Traditional medicine has been practiced from many centuries, especially in India
tribal and rural areas people were used to treat several diseases, due to availability and
low cost and negligible side effects it occupies great importance in many formulations
and ayurvedic drugs. Nature has provided a source of medicinal agents for thousands of
years and an impressive number of modern drugs have been isolated from natural
sources, many based on their use in traditional medicine1 . There has been an increasing
the incidence of multiple resistances in human pathogenic microbial strains , largely due
to the indiscriminate use of synthetic antimicrobial drugs commonl treatment of
infectious diseases2 . The development of bacterial resistance to presently available
antibiotics has necessitated the search for new antibacterial agents, to overcome this
problem studies have been conducted with the various medicinal plants, screening
antimicrobial activity as well as for the discovery of new antimicrobial compounds3-4
and antioxidant activity.

Besides saponin, another group of phytochemicals, such astriterpenoid, quinon,

phenolic are also a common plant active compound that can be found in TCL and has
been reported to promote various activities like antistress, growth promotion, appetite
stimulation, tonic, and immune booster (Citarasu, 2010; Chakraborty et al., 2012;
Chakraborty et al., 2014). Meanwhile, tannins and flavonoid which were a presence in
TCL are the main group of plant phenolic compound that act as primary antioxidants or
free radical scavengers. According to Chansue and Assawawongkasem (2008), tannin
from T. catappa extract with water has the ability as an antibacterial substance for
ornamental fish. The tannin could inhibit the growth of bacteria in intestinal of fish by
binding iron and form a chelate. On the one hand, flavonoid is one of the most
widespread groups of phytochemicals in the plant that was shown to improve
reproduction of Japanese Medaka (Oryzias latipes) (Kiparissis, 2001). It acts as

6
antimicrobial properties on fish pathogen (Rattanachaikunsopon & Phumkhachorn,
2007), and as a growth promoter on juvenile Pargus major (Ji et al., 2007), Carassius
auratus (Ahilan. B et al., 2010), Catla catla (Kaleeswaran et al., 2011). Thus, the presence
of secondary metabolites in TCL might play a role in the survival and blood profile of
Betta sp.

Gao et al. identified various phytoconstituents from the fruits, seeds, and barks of
the T. catappa. The fruit has 1.95 g of protein, 12.03 g of carbohydrate, and 1.21 g of ash.
β-carotene (2,090 μg) and vitamin C (138.6 mg) are present in high amounts. The
mesocarp of fruits dehydrated by the sun having ash, protein, glucose, moisture, tannin,
carbohydrate, and oil with 3,434.5 kcal/kg calorific value is very essential for its nutritive
value. The seed is composed of fixed oil (51.2%), olein (54%), and stearin (46%). The
seeds yield 4.13% moisture, 4.94% crude fiber, 23.78% crude protein, 4.27% ash,
51.80% fat, and 16.02% carbohydrate; the total calorific value is 548.78 kcal. The bark
contains glycoside, cardiac tannins, volatile oils, saponin, steroid, glycosides, and
phenols. Classified in the oleic-linoleic acid group, the oils contain huge levels of
unsaturated fatty acids, exclusively oleic (up to 31.48%) and linoleic (up to 28.93%).
More recently, Mininel et al. isolated punicalagin (polyphenol), its derivatives, and other
several compounds in the leaves of T. catappa.

The leaves of T. catappa contain 1-degalloyl-eugeniin, 2,3-(4,4’,5,5’,6,6’-

hexahydroxy-diphenoyl)-glucose, chebulagic acid, gentisic acid, corilagin, geraniin,
granatin B, kaempferol, punicalagin, punicalin, quercetin, tercatain, tergallagin, terflavin
A, and terflavin B. The phytoconstituents like flavonoids, carotenoids, and phenolic
compounds may be responsible for the traditional use of this plant.

7
 Chapter III

Methodology

The following method was adopted from the existing research articles. This
research is entitled The Effects of Terminalia catappa L. Leaves Extract on the Water
Quality Properties, Survival and Blood Profile of Ornamental fish (Betta sp) Cultured.

Plant Material

Terminalia catappa’s dried cut brown leaves were collected on different areas
within Butuan City. In purpose of eliminating the extraneous matter, the collected T.
catappa’s leaves were washed with deionized water and immediately undergo an air-dried
process or dried in an oven at 40 degrees Celsius for 12 hours. T. catappa’s powder (8
opening/cm passed) was obtained using a mill. The powder is weighed using top loading
balance. Then put the powder in 1000 ml beaker with 95% ethanol. The powder was
extracted with ethanol 95% for three days (100 g/L). After filtration, the extract was
evaporated by using water bath and stored at 4 degrees Celsius until used as a crude
extract.

Phytochemical Tests

The Terminalia catappa leaf extract was brought to Department of Science and
Technology for the test of components.
Table 1. Animals and Experimental set up
Experimental Set-up

3 Winstar Rat 3 Winstar Rat 3 Winstar Rat 3 Winstar Rat 3 Winstar Rat
Control Group Experimental Group (Diabetic)
No treatment of T. catappa L. extract T. catappa L. extract T. catappa L. extract
T. catappa L. extract 30% 60% 90%
The preliminary phytochemical tests such as alkaloid, saponin, steroid,
triterpenoid, quinon, phenolic, tannin, and flavonoid were performed to detect the
presence of possible phytochemicals in the extract of T. catappa leaves.

Animals are divided into five groups. Group 1 is the control group, group 2, 3, 4
and 5 serve as the experimental group that administered with the same volume of
diabetic. Group 2 receives no treatment of T. catappa L. extract, group 3 is treated with

8
30% T. catappa L. extract, group 4 is treated with 60% T. catappa L. extract, group 5 is
treated with 90% T. catappa L. extract, and. The rats are monitored every 24 hours.
Before and during the experiment rats were fed with standard diet (Gold Mohr, Lipton
India Ltd). After randomization to various groups and before initiation of experiment, the
rats were acclimatized for a period of 7 days under standard environmental conditions of
temperature, relative humidity, and dark/light cycle. Animals described as fasting were
deprived of food and water for 16 hours ad libitum Ahmed S. M. et.,al 2005.

Statistical Analysis

The administration of T. cattapa L. extract is done once after the rats are diabetic.
Statistical analysis was carried out by one way ANOVA using the standard statistical
software package of social science (SPSS) version 12.0. P0.05 was considered as the
level of significance Divya, N. et.,al (2018).

Chapter IV

Results and Discussion

9
Results

Table 2. Terminala catappa powder were soaked in different sizes of beaker with
specific amount of 95% ethanol and mass of the powder. For Beaker 1 (1000 ml) with the
550 soaked in 600 ml of 95% of ethanol that produces 15.67 ml of Terminalia catappa
extract. Beaker 2 which is 500 ml soaked 144.19 g with 400 ml 95% of ethanol produces
10.86 ml of extract and Beaker 3 (1000 ml) soaked 600 g with 95% of ethanol which
produces 18.29 ml.

Table 2. Terminalia catappa Leaf Extract Produced

Volume of Beaker Mass of Terminalia Volume of 95 % Volume of Terminalia
catappa Leaf Powder Ethanol catappa Leaf Extract
Beaker 1 ( 1000ml) 550 g 600 ml 15.67 ml
Beaker 2 (500 ml) 144.19 g 400 ml 10.86 ml
Beaker 3 ( 1000 ml) 600 g 700 ml 18.29 ml

Table 3. Phytochemical screening

test of Terminalia catappa leaves Saponin is considered as anti-nutritional in
extract
Phytochemicals Results field aquaculture. However, previous experiments
Alkaloid -
stated that saponin had several beneficial effects on
Saponin +
Steroid - some Rats such as Winstar Rat (Stadlander, 2012). It
Triterpenoid +
Quinon +
is also revealed that low level of saponin was
Phenolic + improved the effectiveness of hyperglycemic, feed
Tanin +
Flavonoid + conversion efficiency, protein utilization and reduced
(+) Present oxygen consumption. Also, saponin had various
(-) Absent
effects on the physiological status such us, increasing
the number of RBC, hemoglobin, hematocrit, oxygen
uptake and binding capacity in Perch (Anabas Testudineus) (Roy & Munshi, 1989).
Besides saponin, another group of phytochemicals, such astriterpenoid, quinon, phenolic
are also a common plant active compound that can be found in TCL and has been
reported to promote various activities like antistress, growth promotion, appetite
stimulation, tonic, and immune booster (Citarasu, 2010; Chakraborty et al., 2012;
Chakraborty et al., 2014). Meanwhile, tannins

10
and flavonoid which were a presence in TCL are the main group of plant phenolic
compound that act as primary antioxidants or free radical scavengers. According to
Chansue and Assawawongkasem (2008), tannin from T. catappa extract with water has

11
the ability to lower glucose level. On the one hand, flavonoid is one of the most
widespread groups of phytochemicals in the plant that was shown to improve
reproduction of Japanese Medaka (Oryzias latipes) (Kiparissis, 2001). It acts as
antimicrobial properties on Rats pathogen (Rattanachaikunsopon & Phumkhachorn,
2007). Thus, the presence of secondary metabolites in TCL might play a role in the
diabetic resistance of Winstar Rat.

Table 4. illustrate the baseline of

250
250
glucose level in Winstar Rat before and
200
200
after inducing diabetic and Terminalia
150
150
100
100 catappa Leaf Extract. There are five groups
50
50 used in this research and each group
00 involved five samples to be administrated.
T1
T1 T2
T2 T3
T3
First three group are for experimental group
and the two groups are for control group.
The glucose level of WR before the
injecting diabetic T1 mean = 126.6, for group 1, mean = 126.2 for group 2, mean = 128.6,
for group 3, mean = 119.6, for group 4, and mean = 124.8, for group 5 with σ mean =
625.8. Glucose level of WR after injecting diabetic, T2 mean = 198.8, for group 1, mean
= 194.0, for group 2, mean = 192.8, for group 3, and mean = 194.8, and mean = 194.4,
with σ mean = 780.4. Glucose level of WR in five (5 days) after injecting Terminalia
catappa Leaf Extract with 1.00 cc/mL, N = 15.0, T3 mean = 182, std. deviation =
4.52769, for group 1, mean = 182, std. deviation = 4.52769, for group 2, and mean = 159,
std. deviation = 3.53553, for group 3, with σ mean = 523, N = 15.0, The effectiveness of
the Terminalia catappa Leaf Extract, respectively P < 0.05.

Figure 1. Induces the data on the changes of glucose level from T1 through T3.
From T1= 126.6 mean, before injecting diabetic its glucose level was at low level shown
in T1, after injecting diabetic the glucose level raises shown in T2 = 198.8 mean, after the
administration of TCLE the glucose level is slightly decreases T3 = 182 mean. In figure 2
shows also the abrupt increase of glucose level after injecting diabetic shown in T2 = 194
mean, and slightly decrease of glucose level after injecting TCLE in T3 = 182 mean.

12
 185
Figure 3 illustrate the baseline glucose level
250
180 in T1 < 150, after inducing 1.0 cc/mL
200
175
170
150 diabetic glucose level rises T2 > 150 and in
165
100
160 T3 = 159 mean, there is an abrupt decrease
155
50
150 of glucose level after injecting TCLE.
145 0
30% Extract
T1 60% Extract
T2 90% Extract
T3 Figure 4 shows the significant difference
between three concentrations and its effect
to the glucose level of the WR. In 30% and
60% has slightly effect compare to 90% has great effect to the glucose level in WR.

Discussion

In the light result of this study on Tropical Almond ( Terminalia catappa ) Leaf
Extract as Effective Anti-Hyperglycemic Activities in Rattus rattus (Rat), this study
indicates that Terminalia catappa leaves extracts have anti-diabetic activity ( Armed
2005). The 30% and 60% concentration of Terminalia catappa leaves extracts exhibited
significant anti hyperglycemic activities in diabetic-induced hyperglycemic WR without
any changes in its body setting, although rats become active. In 90% concentration is
results that abrupt decrease of glucose. The mean says that more concentration induced in
hyperglycemic WR is more effective.

According to Chakraborty & Hancz (2011), phytochemicals such as alkaloids,

flavonoids, pigments, phenolics, and terpenoids are secondary metabolites in plants. The
phytochemicals contained in the plant may have the ability to decrease diabetic in Rat.
Further, the phytochemical may improve the stability of glucose level in Rat. Some
previous studies have been conducted to evaluate the use of phytochemicals from various
parts of plants extract to enhance the survival rate of Winstar Rat caused by the diabetic
mellitus (Akinwande et al., 2011). The researcher performed a primary validation of the
traditional medical practices, by looking for the chemical groups that explain the
antidiabetic effect of the herbal medicine “KCTC”. Thus Terminalia catappa leaves were
chemically screened and yielded alkaloids, flavonoids, polyphenols, saponosides, sterols
and triterpenes. Among these compounds, the alkaloids, sterols and triterpens can be
incriminated in the antidiabetic activity of the plant. Alkaloids would be used as

13
stimulatives of the hepatic glycogenogenesis (Neuwinger, 1996). Sterols and triterpens
are recognized for their properties to decrease the rate of blood glucose (Nacoulma,
1996). Alkaloids, Sterols or triterpens highlighted in the leaves of the plant would be
responsible for the observed antidiabetic effect.

This experiment was conducted to study the antidiabetic activity of Terminalia

catappa leaves in rats as well as to provide an introductory approach for the evaluation of
its traditional preparation in order to scientifically validate the therapeutic preparation of
the plant in the control of diabetes. The results show that the effect of herbal medicine
(KCTC) at 1.0 cc/mL. The effect of herbal medicine was more prominent when the dose
used is higher: the herbal medicine exerts dose-dependant hypoglycemic effect and
appears like an antidiabetic.

Chapter V

Conclusion and Recommendation

Conclusion

The aqueous ethanolic extract from leaves of Terminalia catappa exerts a dose-
dependent hypoglycemic effect. It has a hyperglycaemic activity at lower concentration
90% concentration and a normoglycaemic activity, at higher doses (≥ 30% and ≥ 60%).
The dose of 90% concentration is the most effective among the administered doses.
Indeed, at 90%, the Terminalia catappa reduces hyperglycaemia and brings back
glycaemia to its normal value of about mean = 4.19697 with P < 0.05, after 7 days of

14
experiment. The plant proves to be an antidiabetic. Subsequent studies are needed to
deepen the mechanism of action and identify the active principle of Terminalia catappa
leaves which have a significant hypoglycaemic effect and appear like an antidiabetic due
to chemical components as alkaloids, sterols and triterpens.

Recommendations

1. Increase the number of rats to be treated.

2. Increase the mass of leaves powder to prepare enough extract.

3. Increase the volume extract.

4. Useful for future research.

5. Include the mass of each rat to observe the changes of its body weight during
administration.

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Appendix

ANOVA

Glucose level (T3)

Sum of Squares df Mean Square F Sig.

Between Groups 1454.800 2 727.400 32.717 .000

Within Groups 266.800 12 22.233

Total 1721.600 14

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