Antihyperglycemic Effect of Calabash Tree (Cresentia cujete) Fruit Pulp Solution Injection on Female

rabbits (Oryctolagus cuniculus)

Amigable, Khy T.
Sagario, Thessa Neca J.

2020-2021
 CHAPTER I
INTRODUCTION
Background of the study
Calabash tree is a tropical tree planted in tropical regions. It belongs to the family of Bignoniacea and
is scientifically known as Crescentia cujete. It is native to Central America. It is propagated either by seed or
by stem cuttings. It grows up to 6-10 m high with multiple trunks, simple lobe shaped leaves and gourd-like
fruit. The branches have simple elliptical leaves clustered at the anode. The greenish flowers arise from the
trunk or main branches and bloom at night.

Calabash fruit is a seasonal fruit that develops after pollination by bats. It appears at the end of dry
season or beginning of rainy season. The fruit is large up to 12-14 cm in diameter. It is globulose with a
smooth hard green woody shell. It takes about 6-7 months to ripen and eventually falls to the ground. The
fruit has a white pulp that has medicinal applications and small flat seeds which are embedded in the pulp

In Nigeria, the tree is widely grown in the Northern states  but little in other parts of the
country.  Virtually, all parts of the tree have been found to be  useful. The wood is used for tool handles, ribs
in boat  building and cattle yokes; and the gourd for cups,  containers and musical instruments. The fruit is
reported  to have medicinal application (Michael, 2004; Burkill, 1985; Plant Database, 2004).  
 
 The fruit of Crescentia cujete Linn. also has a reputation of  being used as traditional medicine to cure
many diseases and  other health related issues in the Philippines. Its effects include laxative, diuretic,
purgative, anti-inflammatory,  hypertension and diarrhea.

The fruit pulp of Crescentia cujete is traditionally used in folk medicine for the treatment of a variety of
respiratory conditions and gastrointestinal disorders. Due to the lack of a comprehensive phytochemical de
scription of the fruit of this plant, its active compounds and rational quality control parameters have not yet
been described.  Calabash is currently important in folk medicine, since extracts of leaves, fruit, stem bark,
flowers and seeds have been used for the treatment of various illnesses (Gentry, 2011; Wang et al., 2010).

The pulp of the fruit of C. cujete mixed with sugar or honey is used as a medicine for alleviating respiratory
conditions such as cold, cough and asthma, for the treatment of digestive system ailments (stomach pains,
intestinal parasites) and in case of infertility (Das et al., 2014; Olaniyi et al., 2018). The fruit of this plant has
been used since the eighteenth century in traditional Mexican medicine to prepare a tonic for the treatment of
different respiratory infections, asthma, bronchitis, tu berculosis and breast pain (Valladares and Rios, 2007).
In Panama, the fruit is used as febrifuge, laxative, emetic, vermifuge, and for inducing abortion. In Vietnam,
a fruit decoction is taken orally as an expectorant, antitussive, laxative and for the treatment of urethritis and
stomach disorders (Kaneko et al., 2012; Wang et al., 2010).

This plant is known as Miracle Tree in the Philippines due to its various medicinal properties as
reported by several testimonies. Studies have shown the efficacy of the fruit and leaf extracts against cough,
cold and other respiratory ailments. Other studies also indicate the potential use of the extracts of this plant
against inflammation, tumors and cancer. Phytochemical screening of the leaf and fruit extracts obtained
from solvent partitioning of this plant has been done through several detection tests. Studies showed presence
of secondary metabolites such as flavonoids, alkaloids, glycosides, saponins, tannins and terpenoids The
analyzed extract also eluted other compounds including methyl salicylate and 3-phenyl-2-propenoic acid,
also known as trans-cinnamic acid. This trans-cinnamic acid is known for its potential use as an anti-
inflammatory agent and in cancer treatment. Despite the reported medicinal uses, the fruit pulp is not
considered edible.
 Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and is
associated with various complications, ranging from chronic to acute cases, causing considerable damage
affecting different organs of the body, such as the renal, retinal, cardiovascular, and neurological systems,
resulting in microvascular complications. The main cause that underlines hyperglycemia is either an absolute
or relative lack of insulin secretion and/or its action on the target tissue (Alberti and Zimmet, 2003, Skyler et
al., 2017). In 2017, the global prevalence of DM was 11.4% with a treatment rate of 48.2% and a mortality
rate of 2.7% (Ge, Chen, & Chen, 2017). The International Diabetes Federation report recorded 425 million
people aged 20–79 years with diabetes in 2017, with an expected increase to 629 million by 2045
(International Diabetes Federation, 2017).
Prediabetes is a condition where blood sugar level is higher than normal but not yet high enough to be
classified as type 2 Diabetes. There is the presence of impaired fasting glucose (IFG) and/or impaired glucose
tolerance (IGT) that has not yet reached the thresholds for diagnosable type 2 Diabetes mellitus (T2DM). 1
Globally, the number of people with IGT is estimated at 280 million, projected to go up to 398 million by
2030.
A number of laboratory tests can be utilized to diagnose pre-diabetes. Among these include an oral
glucose tolerance test (OGTT) and a glycosylated hemoglobin (A1C) test. An OGTT with a 2-hour post-
prandial blood glucose level between 140 to 199mg/dl can be considered pre-diabetes. A1C, on the other
hand, is a blood test that gives the average amount of glucose in the blood over the past 3-4 months. An A1C
of 5.6% or below is normal. In pre-diabetes, A1C levels range between 5.7%-6.4%, and if the A1C is 6.5%
or above, a person has diabetes.
Progression to type 2 Diabetes mellitus may not be inevitable for those with prediabetes. Thus, aside
from diet and exercise, alternative treatments are currently being explored and ongoing trials are made to
prevent individuals from developing diabetes. Calabash (Crescentia cujete) has been an important source of
folk medicine for years now. Its indication includes diabetes. The phytochemical composition of Crescentia
cujeteincludes cyanhidric acid, alkaloids, iridoids, pectins, tartaric acid, citric acid, sterols, essential oil
(allicine, nerolidol), malic acid, peptides (insulin), proteins (bixine) and triterpenes which are all believed to
possess anti-diabetic properties.
In particular, cynhindric acid was found to stimulate insulin release while the alkaloids of Calabash
were noted to be involved in glycogenesis. In addition, iridoids were noted to reverse high glucose and
obesity-induced beta cell dysfunction in the pancreas. and can also stimulate insulin secretion. Pectin was
shown to increase the activity of glycogen synthase, increase hepatic glycogen and decrease blood sugar
level. Furthermore, citrate from citric acid is known to inhibit phosphofructokinase which helps regulate
glycolysis and eventually, the citric acid cycle.
Present study reviews of Basellarubra’santidiabetic property diabetes is a serious, chronic metabolic
disease characterized by an increase in blood sugar levels associated with long term damage and failure or
organ functions, especially the eyes, the kidneys, the nerves, the heart and blood vessels. Impaired glucose
tolerance test and impaired fasting blood glucose test are intermediate conditions in the transition between
normality and diabetes.
The oral hypoglycemic drugs used in the management of diabetes are effective in regulating blood
glucose, synthetic drugs have certain limitations and side effects such as severe hypoglycemia, lactic
acidosis, digestive discomfort, headache and dizziness accord.
Medicinal plants have been proposed as rich yet unexploited potential sources for antidiabetic drugs,
even though used since ancient times for the treatment of diabetes mellitus. Many of the synthetic drugs were
discovered either directly or indirectly from the plant source. As such with Basellarubra’santidiabetic
property.
A local study on calabash using its decoction resulted in significant lowering of FBS levels in
alloxan-induced diabetic rats. Acute and subacute toxicity testing of the decoction proved that it is safe.
Reproductive toxicity in animals did not produce any abnormality. All these studies were done using animal
subjects. Considering all these information about calabash, this pilot study among animals sought to
determine the effect of ED dose of calabash fruit decoction (Crescentia cujete) on the blood sugar levels of
prediabetic individuals. This study will further explore and validate the potential blood glucose lowering
effect of calabash. In this study, the calabash tree (Cresentia cujete) Fruit Pulp we determine the presence of
its phytochemicals,and antihyperglycemic effect on on Female rabbits (Oryctolagus cuniculus).
The guiding principle of the study is to affirm the hypoglycemic effects of the calabash fruit and to
determine the appropriate dosage to which it is effective. Another is to check the direct toxic effect of the
fruit. This is to ensure complete safety, before the use of Calabash fruit will be introduced and promoted to
the public.

REVIEW OF RELATED LITERATURE

Progression to type 2 Diabetes mellitus may not be inevitable for those with prediabetes. Thus, aside
from diet and exercise, alternative treatments are currently being explored and ongoing trials are made to
prevent individuals from developing diabetes. Calabash (Crescentia cujete) has been an important source of
folk medicine for years now. Its indication includes diabetes. The phytochemical composition of Crescentia
cujete includes cyanhidric acid, alkaloids, iridoids, pectins, tartaric acid, citric acid, sterols, essential oil
(allicine, nerolidol), malic acid, peptides (insulin), proteins (bixine) and triterpenes which are all believed to
possess anti-diabetic properties. In particular, cynhindric acid was found to stimulate insulin release while the
alkaloids of Calabash were noted to be involved in glycogenesis. 6 In addition, iridoids were noted to reverse
high glucose and obesity-induced beta cell dysfunction in the pancreas. 7 and can also stimulate insulin
secretion.8 Pectin was shown to increase the activity of glycogen synthase, increase hepatic glycogen and
decrease blood sugar level.9,10, Furthermore, citrate from citric acid is known to inhibit phosphofructokinase
which helps regulate glycolysis and eventually, the citric acid cycle.11

Calabash fruit shells are used for various purposes, such as food containers, musical instruments, food
plates, bowls and pipes. The white sponge pulp inside the shell contains numerous flat seeds. The fruit itself
can be mixed with milk, heated and use in the treatment of colds and asthma. Mature fruits are also
considered as laxative. Some traditional healers have recommended that the food may be ingested to force
menses, birth and after birth (Arvigo and Balick, 2010), or trigger abortions, the latter has been observed in
cattle.
There is evidence that microorganisms are resistant to global threats due to the wider use of
antibiotics (Elisha, Twaij, Ali, Tarish, Al-omari and Karim, 2000). The recent emergence of antibiotics
resistant strains increased the urgency of increasing the range of intractable bacterial infections and the
search for new strategies to combat infections. Unlike synthetic drugs, herbal antibiotics are not associated
with many side effects and have tremendous therapeutic potential in the treatment of many infectious
diseases (Iwu, Duncan and Okunzi,2008; Sieradzki, Roberts, Haber and Tomasz 2010). Plants are readily
abound in our environment and about 80% of the modern medicines come directly or indirectly from plants.
Surprisingly, the large quantity of modern drugs originated in less than 15% of plants known to have been
pharmacologically tested out of estimated 500,000 high- growth plant species on earth (Farnsworth, 2011). l
The fruit pulp of Crescentia cujete is traditionally used in folk medicine for the treatment of a variety of
respiratory conditions and gastrointestinal disorders. Due to the lack of a comprehensive phytochemical de
scription of the fruit of this plant, its active compounds and rational quality control parameters have not yet
been described.  Calabash is currently important in folk medicine, since extracts of leaves, fruit, stem bark,
flowers and seeds have been used for the treatment of various illnesses (Gentry, 2011; Wang et al., 2010).

The pulp of the fruit of C. cujete mixed with sugar or honey is used as a medicine for alleviating respiratory
conditions such as cold, cough and asthma, for the treatment of digestive system ailments (stomach pains,
intestinal parasites) and in case of infertility (Das et al., 2014; Olaniyi et al., 2018). The fruit of this plant has
been used since the eighteenth century in traditional Mexican medicine to prepare a tonic for the treatment of
different respiratory infections, asthma, bronchitis, tu berculosis and breast pain (Valladares and Rios, 2007).
In Panama, the fruit is used as febrifuge, laxative, emetic, vermifuge, and for inducing abortion. In Vietnam,
a fruit decoction is taken orally as an expectorant, antitussive, laxative and for the treatment of urethritis and
stomach disorders (Kaneko et al., 2012; Wang et al., 2010).
However, detailed chemical studies on the active compounds of the fruit of C. cujete are scarce; a literature
survey revealed that there are few studies addressing its phytochemical composition in a rather general way,
such as the presence of tannins, phenols, flavonoids, saponins, alkaloids and hydrogen cyanide (Ejelonu et
al., 2011). Additional stu dies showed the presence of iridoids (such as aucubin and ningpo genin), n-alkyl
glycosides, lignan glycosides (acanthoside D), and ben zoyl derivatives (Kaneko et al., 2012; Wang et al.,
2010)

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and is

associated with various complications, ranging from chronic to acute cases, causing considerable damage
affecting different organs of the body, such as the renal, retinal, cardiovascular, and neurological systems,
resulting in microvascular complications. The main cause that underlines hyperglycemia is either an absolute
or relative lack of insulin secretion and/or its action on the target tissue (Alberti and Zimmet, 2003, Skyler et
al., 2017). In 2017, the global prevalence of DM was 11.4% with a treatment rate of 48.2% and a mortality
rate of 2.7% (Ge, Chen, & Chen, 2017). The International Diabetes Federation report recorded 425 million
people aged 20–79 years with diabetes in 2017, with an expected increase to 629 million by 2045
(International Diabetes Federation, 2017).
Acute as well as chronic elevation in blood glucose levels can result in the release of reactive oxygen
species (ROS) via autoxidation side reactions. ROS, such as superoxide anion radicals, hydrogen peroxide,
and hydroxyl radicals, can produce more serious complications, such as lipid peroxidation, enhancing
glucose resistance when the body’s antioxidants are unable to scavenge the ROS (Lien et al., 2008, Maritim
et al., 2003, Yang et al., 2011). In vitro, cell-based studies have revealed microvascular complications that
are mediated via oxidative stress and ROS production in elevated glucose conditions (Barthelmes et al.,
2014, Wolff, 1993, Xu et al., 2014).

The calabash tree is widely distributed in the Caribbean  region, Mexico, Northern and Southern
American and  later introduced to tropical Africa from Senegal to  Cameroon then to other parts of Africa
(Micheal, 2004).  In Nigeria, the tree is widely grown in the Northern states  but little in other parts of the
country.  Virtually, all parts of the tree have been found to be  useful. The wood is used for tool handles, ribs
in boat  building and cattle yokes; and the gourd for cups,  containers and musical instruments. The fruit is
reported  to have medicinal application (Michael, 2004; Burkill, 1985; Plant Database, 2004).  
 
In South East Africa, the fruit of Crescentia cujete had been recorded edible. Most of cujete consumers
consume only the pulp throwing away the shell and the seeds, while some consume the pulp and the seeds
throwing away the empty shell (Burkill, 2010). 
 Hydrothermal carbonization is a thermochemical conversion process (wet pyrolysis) that can convert
wet biomass  into valuable products [1]. It is a process that utilizes high temperature to convert raw biomass
into a solid coal-like  product called hydrochar characterized by high carbon content and calorific value [2].
It is a carbonaceous solid  organic material which can be potentially used as solid fuel, as precursor of
activated carbon used in wastewater  pollution, or in soil remediation [1, 3, 4]. 

Crescentia cujete L., also known as calabash (Family Bignoniaceae), is an evergreen tree originating
from tropical  America but is now widely distributed in tropical regions [5]. This plant is known as Miracle
Tree in the Philippines  due to its various medicinal properties as reported by several testimonies [6, 7].
Studies have shown the efficacy of  the fruit and leaf extracts against cough, cold and other respiratory
ailments [8, 9]. Other studies also indicate the  potential use of the extracts of this plant against inflammation
[10], tumors and cancer [11, 12].  

Phytochemical screening of the leaf and fruit extracts obtained from solvent partitioning of this plant
has been done  through several detection tests. Studies showed presence of secondary metabolites such as
flavonoids, alkaloids,  glycosides, saponins, tannins and terpenoids [11,12]. However, results of another
study showed presence of mostly  benzene and its derivatives only. The analyzed extract also eluted other
compounds including methyl salicylate and  3-phenyl-2-propenoic acid, also known as trans-cinnamic acid
[13]. This trans-cinnamic acid is known for its  potential use as an anti-inflammatory agent and in cancer
treatment [14]. Despite the reported medicinal uses, the  fruit pulp is not considered edible. 

In this study, preliminary characterization of the fruit extracts and fruit pulp of C. cujete L. was
determined. The  crude methanolic (MeOH) extract and pure compound, trans-cinnamic acid isolated from
the fruit pulp of C. cujete were characterized using gas chromatography- electron ionization- mass
spectrometry (GC-EI-MS). Lyophilized  fruit pulp was characterized by energy dispersive X-ray
spectroscopy (EDX). The fruit pulp was subjected to  hydrothermal carbonization (HTC) and the resultant
hydrochars were analyzed using Fourier transform infrared  spectroscopy (FTIR), scanning electron
microscopy (SEM) and thermogravimetric analyzer (TGA). To the best of  our knowledge, this is the first
reported study to provide chemical characterization of the fruit pulp of C. cujete L.  following these
optimized analyses.
The Mayan civilization became vastly prominent in southern Mexico and other South  American counties
around 2000 BC. The tropical regions inhabited were an ideal environment  for parasites and other infectious
bacteria [1]. When diseases occurred, local Mayan healers  often took advantage of the variety of medicinal
plant resources provided in the local rain  forest [2]. Mayan healers used herbal remedies to cure many
different diseases, often relating  the disease with the plant to be used. For example, red plants were used for
rashes, blood  disorders, and burns; blue plants were used for neural disorders; and yellow plants were 
associated with the liver and spleen. Often white plants were avoided because white was  associated with
death [3].  
Calabash Crescentia cujete L. (family Bignoniceae) is a tree found in the West Indies, tropical  America, and
tropical areas of the Old World [4]. Calabash blooms during the month of June  and its fruits grow and ripen
slowly as they remain on the tree for six to seven months. During  this time, the fruit changes from green to
yellow and is harvested during the dry season from  December to May [5].  
The hard outer shell of the calabash fruit has been used for food containers, bowls, tobacco  pipes, and as
musical tools [6]. The white spongy pulp inside the shell contains numerous flat seeds [6]. The fruit itself can
be mixed with milk, heated, and consumed for treating colds and  asthma [7]. Ripe fruit has also been regarded as a laxative [8].
Traditional Mayan healers have  recommend that the fruit may be ingested to force menses, birth, after birth
[9, 10]
, or trigger  abortions [11]. The latter has been observed in cattle [12, 13].  
The purpose of this study was to determine if the constituents of the calabash fruit as it is  available to the
consumer, would directly influence the contractility of uterine smooth muscle  tissue in vitro following the
application of either an aqueous seed extract or raw fruit pulp  juice. Our specific objectives were to
characterize the resulting contractile waveform force and  frequency responses and compare them to standard
uterine positive contractile agents, namely  oxytocin and acetylcholine. The results from this investigation
might then advance our  knowledge base about the claims within traditional medicine and provide some
credibility to  the Mayan medicine man’s prescriptive outcomes. 

The pulp of calabash fruit (Crescentia cujete) has been studied for its nutritive and anti-nutritive
compositions in both the wet and dry samples. The values of the ash; 3.74% (dry), ether extract; 4.38% (wet)
crude protein; 7.67% (wet) and 10.01% (dry), crude fibre; 4.88% (dry), carbohydrate; 15.65% (wet) and
68.13% (dry) and dry matter; 31.32% (wet) and 87.48% (dry) are quite reasonable for fruits. Both the wet
and dry samples have very high concentrations of the mineral elements. Sodium (Na) has the highest values
of 3.20% (wet) and 0.32% (dry), while calcium has the least values of 0.12% (wet) and 0.06% (dry). The
pulp has very high values of Thiamin; 1.50 ug g-? (wet) and 0.93 ug g-'(dry). Both the wet and dry pulp
samples have reasonable values of phytochemicals and are free from HCN toxicity. The pulp can make
useful contributions to human nutrition and health. Calabash tree is a tropical tree planted in tropical regions.

It belongs to the family of Bignoniacea and is scientifically known as Crescentia cujete. It is native to
Central America. The tree is widely distributed to Caribbean region, Mexico, Northern, Southern America
and later introduced to tropical Africa after Senegal to Cameroon then to other parts of Africa (Michael,
2004). 
It is propagated either by seed or by stem cuttings. It grows up to 6-10 m high with multiple trunks,
simple lobe shaped leaves and gourd-like fruit. The branches have simple elliptical leaves clustered at the
anode. The greenish flowers arise from the trunk or main branches and bloom at night (Gilman, 1993). 

Calabash fruit is a seasonal fruit that develops after pollination by bats. It appears at the end of dry
season or beginning of rainy season. The fruit is large up to 12-14 cm in diameter. It is globulose with a
smooth hard green woody shell. It takes about 6-7 months to ripen and eventually falls to the ground
(Gilman, 1993). The fruit has a white pulp that has medicinal applications and small flat seeds which are
embedded in the pulp (Michael, 2004; Burkill, 1985).

The fruit of Crescentia cujete Linn. is widely used by locals throughout certain parts of the
Philippines who claim to have  observed some of the health effects of its fruit such as antimicrobial,
anthelmintic, and anti-cholesterol. This study was done to investigate  some of these health effects.
Phytochemical screening was done to support the results of the following tests: toxicity test by calculating
LC50 using brine shrimp lethality test; antioxidant screening using thin-layer chromatography; and
purgative test by anthelmintic assay using  Eudrilus eugeniae as test organisms. Phytochemical screening
revealed the presence of alkaloids, flavonoids, cardiac glycosides, reducing  sugars, saponins, tannins,
phytosterols, and terpenoids in the fresh C. cujete fruit. BSLT results showed that ethyl acetate extract is
the most  toxic and exhibits the highest bioactivity having LC 50 of 1.50ppm during the first 6 hours of
treatment. Thin-layer chromatography exhibited  the presence of phenolic antioxidants in C. cujete fruit
crude ethanolic extract. Anthelmintic assay shows that ethyl acetate extract at  20,000ppm has the least
time to paralyze and kill the test organisms, 1.39 minutes and 2.39 minutes, respectively. This effect is also
observed  to be dosage dependent and is significantly comparable with the positive control Levamisole
(Latigo-50) as referred to the statistical analysis  using ANOVA and Tukey Test. The fruit of C. cujete
Linn. is cytotoxic and bioactive, it is also a potential source of components which  exhibit antioxidant and
anthelmintic effects. 

The importance of plant-derived medicines  in modern medicine is often underestimated [3]. Plant 
derived substances have recently become of great interest  owing to their versatile applications. Medicinal
plants are the  richest bio-resource of drugs of traditional systems of  medicine, modern medicines,
 nutraceuticals, food  supplements, folk medicines, pharmaceutical intermediates  and chemical entities for
synthetic drugs [4]. 

The selection of the plant species to be studied is crucial for  the reason that this step does not
guarantee that the selected  plant species exhibit a specific biological activity. Through  theoretical
evaluation such as medicinal folklore,  ethnobotany, or primary experimentation, this selection is  made.
The fruit of Crescentia cujete Linn. was the result of  this selection which is based primarily on the fact
that not  much experimentation has been done for this plant species.  The fruit of Crescentia cujete Linn.
also has a reputation of  being used as traditional medicine to cure many diseases and  other health related
issues in the Philippines. Its effects  include laxative, diuretic, purgative, anti-inflammatory,  hypertension
and diarrhea.  
CHAPTER II
MATERIALS AND METHODS
Research Design
This study used experimental design in determining the effects of Antihyperglycemic effect of
Calabash Tree (Cresentia cujete) Fruit Pulp Solution Injection on rabbits (Oryctolagus cuniculus).
Research Settings
The Calabash Tree (Cresentia cujete) Fruit Pulp will be collected at different areas of Ozamiz City
and Bonifacio, Misamis Occidental. Calabash Tree (Cresentia cujete) Fruit Pulp was then pulverized at the
Medina College Chemistry Laboratory, using a blender and sieve.
The administration of Calabash Tree (Cresentia cujete) Fruit Pulp Solution Injection to rabbits and
extractions of blood sample for sugar testing using glucometer was conducted at Medina College, Chemistry
Laboratory Barangay Maningcol, Ozamiz city, Misamis Occidental
Research Subject
Rabbits (Oryctolagus cuniculus) commonly used for research as like in basic and biomedical
research, as models for diseases and disorders of the eyes, skin, heart, and immune system, as well as asthma,
cystic fibrosis, and diabetes.
The sample rabbits are picked randomly at Ozamiz city, Misamis Occidental.
Research Procedure
Plant Material Collection
Calabash Tree (Cresentia cujete) Fruit Pulp Solution Injection will be collected at different areas of
Ozamiz City and Bonifacio, Misamis Occidental. The collected Calabash Tree (Cresentia cujete) Fruit Pulp
is wash thoroughly with Distilled water to remove all dirt that accumulated at the surface. Calabash Tree
(Cresentia cujete) Fruit Pulp was then pulverized at the Medina College Chemistry Laboratory, using a
blender and sieve until it forms as solution or powder and there is more moisture.
Phytochemical Procedure
The 200 grams of air dried materials was used and macerated in 300 ml or sufficient 95% ethyl
alcohol to completely submerge the material. It was stoppered and soaked for the next 24 to 48 hours. After
which, filtered to a Buchner preferably with gentle suction. The flask and the plant material were rinsed with
fresh portion of alcohol. The washing was combined with the first filtrate, and the plant residue was
discarded. The filtrate extract was kept for phytochemical tests.

Confirmatory Test for Flavonoids

Test for Flavonoids (Antihyperglycemic)
To one milliliter of the crude stock extract, a few drops of diluted sodium hydroxide was added. An
intense yellow color appeared in the plant crude extract, which became colorless on the addition of a few
drops of diluted acid which indicates the presence of flavonoids.

Confirmatory Test for Tannis

Ferric Chloride Test
3ml of 5% w/v ferric chloride solution was added to 3 ml of extract. The presence of blue-black color
indicates the presence of tannins and phenols.

Preparation of Tea
Human adult tea preparation= 20g/200ml x 10 % for animal
Rabbit administration of tea 2g/200ml
The 800 grams of air dried and pulverized alugbati tea leaves, was weighed into 2g grams and put
into a tea bag. The packed alugbati tea leaves was then used and administered into 11-20 female rabbits to
test its antihyperglycemic effect.. On the first day, 10 pieces of 2 grams pulverized alugbati in a tea bag was
prepared and dissolved into a 20ml of warm water and it was administered orally into 11-20 rabbits, till the
last day of experimentation.
Animal Preparation,
The twenty rabbits that are subject for this study were purchased from GOOD HARVEST, P. Burgos
St. cor Pingol Drive, Ozamiz City, Misamis Occidental. The 20 rabbits will be caged separately and labeled
*1*2 *3 *4 *5 *6 *7*8 *9 *10 *11 *12 *13*14 *15 *16 *17 *18 *19*20. Before the initial dose, the 20
rabbits wasacclimatised for 10 days. By the time the rabbits were owned they were a grouped into three: the
positive group, 1-5 the usual diet, 5-6 rabbit induced sugar treated with metformin and negative group, 11-20
female rabbit which received and was treated with alugbati tea leaves.
Preparation of syrup
Sucrose Diluent 40%
Ingredients : 400.00g/L
40 grams of Sugar was suspended in 60 ml of distilled water. Boiled until the sucrose was completely
dissolved. If required make the volume to100 ml with distilled water.

Inducing Sugar to Sample Animal

The positive 6-10 and 11-20 negative female rabbits was fed with cogon grass and was given
sucrose syrup instead of pure water as their beverage. Dispensed in suitable clean container like syringe to
induce the sugar, until the last day of the experiment.
Metformin Treatment
The positive group 6-10 female rabbit feed with was treated with metformin 80mg/kl, until the last
day of the experiment.

Alugbati Administration
The 11-20 female rabbit was treated with alugbati tea leaves 2g/20ml, until the last day of
experiment.
Sugar-blood Sampling
A lancet was used to make small abrasion to subject test animals enough to withdraw blood
appropriate for the standard amount of blood in a sugar strips to test blood glucose level in a glucometer.
Glucose test was collected after one hour of feeding and inducing sugar of the sample animals. After one
hour glucose test was performed again, and the 6-10 female rabbit was treated with metformin and 11-20
female rabbit was treated withalugbati tea leaves 2g/20ml repeated every one hour for four times, until the
last of experiment.

REFERENCES

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Banda, M., Nyirenda, J., Muzandu, K., Sijumbila, G., & Mudenda, S. (2018). Antihyperglycemic and
Antihyperlipidemic Effects of Aqueous Extracts of Lannea edulis in Alloxan-Induced Diabetic Rats.
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compared to aspartame in alloxan-induced diabetic mice,

Journal of Integrative Medicine,

 Volume 18, Issue 6, 2020,

Obafemi, T. O., Olaleye, M. T., & Akinmoladun, A. C. (2019). Antidiabetic property of miracle fruit
plant (Synsepalum dulcificum Shumach. & Thonn. Daniell) leaf extracts in fructose-fed streptozotocin-
injected rats via anti-inflammatory activity and inhibition of carbohydrate metabolizing enzymes. Journal of
Ethnopharmacology, 112124. doi:10.1016/j.jep.2019.112124

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JC Jose, G Oyong, MD Ajero, I Chiong, E Cabrera, MCS Tan

Malaysian Journal of Analytical Sciences 24 (1), 134-145

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