Page 1 of 16
British Journal of Healthcare and Medical Research - Vol. 10, No. 4
Publication Date: August 25, 2023
DOI:10.14738/bjhmr.104.15156.
Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria
umbellata (abeere) on Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-
126.
Services for Science and Education – United Kingdom
Haematological Effects of Methanol and n-Hexane Seed Extracts
of Hunteria umbellata (abeere) on Alloxan Induced Diabetic
Wistar Rats
Obia, Chika
Department of Biochemistry, Michael Okpara University of Agriculture Umudike,
PMB 7267 Umuahia, Abia State, Nigeria and Coca-Cola Hellenic Bottling Company,
Owerri, Imo State, Nigeria
Nwuke, P Chinedu
Department of Biochemistry, Michael Okpara University of
Agriculture Umudike, PMB 7267 Umuahia, Abia State, Nigeria
Okereke, Goodluck Obioma
(ORCID: 0000-0002-7393-3819)
Department of Chemistry, Food Science and Technology, Centre for Food Technology and
Research, Benue State University, Makurdi, PMB 102119 Makurdi, Benue State, Nigeria
ABSTRACT
There is growing research studies in identifying local medicinal plants with
potentials in replacing the orthodox medicines and synthetic drugs that are now
expensive, less effective with side effects when used in treatments of chronic
diseases like diabetes mellitus. The chemical compositions, and effects of Methanol
and n-Hexane extracts of Hunteria umbellata (HU) seeds (folkloric remedies for
diabetes, obesity, anaemia etc.) on haematological parameters of alloxan induced
diabetic wistar rats, were investigated. Thirty albino rats were divided into six
groups- (1) Diabetic rats without treatment (Negative control); (2) Diabetic rats,
treated with glibenclamide; (3) Diabetic rats treated with 250mg/kg body weight of
methanol extract of HU seeds;(4) Diabetic rats treated with 500mg/kg body weight
of methanol extract of HU seeds; (5) Diabetic rats treated with 250mg/kg body
weight of n-hexane extract of HU seeds; (6) Diabetic rats treated with 500mg/kg
body weight of n-hexane extract of HU seeds. Treatments of the induced diabetic
rodents lasted for 14 days before they were sacrificed and blood was collected for
biochemical evaluation. From the results, significant (P≤ 0.05) high white blood
cells, platelets and mean corpuscular volume were observed in group 2. Reducing
sugars (in high amounts), flavonoids, tannin, alkaloids, saponins and 14 important
compounds were found present in the HU seed extracts. The acute toxicity study of
the extracts showed no death at doses of up to 500 mg/kg body weight after 24 h
and for 7 days of observation. Treatments with glibenclamide, and extracts of HU
seeds at various doses significantly increased (P<0.05) the red blood cells (RBC),
packed cell volume and hemoglobin but significantly (P<0.05) reduced the white
blood cells and platelets of the Haematological parameters. Thus, Methanol and n- Hexane extracts of HU seeds possess anti-hyperglycemic effects on the haematology
due to their high contents of phytochemicals.
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British Journal of Healthcare and Medical Research (BJHMR) Vol 10, Issue 4, August- 2023
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Keywords: Diabetic treatment, Hunteria umbellata, phytochemicals, haematology, acute
toxicity.
INTRODUCTION
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting
from defects in insulin secretion, insulin action, or both (WHO, 2016; Kumar et al., 2021). It is
one of the fastest-growing diseases of the 21st century, with a disproportionate burden on low
and middle-income countries (Pragya, 2015; IDF, 2021). The chronic hyperglycemia of diabetes
is associated with long-term damage, dysfunction, and failure of various organs, especially the
eyes, kidneys, nerves, heart, and blood vessels (Kesavadev et al., 2017). Several pathogenic
processes are involved in the development of diabetes. These range from autoimmune
destruction of the beta-cells of the pancreas with consequent insulin deficiency to
abnormalities that result in resistance to insulin action. The blood is a vital fluid, which contains
the red blood cells (RBC), white blood cells (WBC) and platelets suspended in the serum in
homeostatic concentrations. The blood is important for pulmonary and tissue respiration, as a
medium of endocrine and neurohumoral transmissions, biotransformation and metabolic
excretion (Adebayo et al., 2005), nutritional and immunological processes, as well as
homeostatic responses (Oze, 1992).
Over the years, diabetes mellitus has been the problem of the world, and the disease is ranked
one of the top killers in the world (Kesavadev et al., 2017). It is the major cause of heart failure,
stroke, sexual dysfunction, nephropathy, retinopathy, vascular dysfunction, hypertension,
coronary artery disease, hearing impairment, different forms of cancer, and non-healing of
wounds leading to skin complications, amputation of limbs and feet (Forbes et al., 2013; Padhi
et al., 2020; Kumar et al., 2021). The global prevalence of diabetes mellitus estimated at 2.8%
in 2000 (171 million people) is expected to rise to 9.9% (552 million) of adult population in
year 2030 if no urgent efforts are directed at stemming the tide of this global public health
burden (Whiting et al., 2011; Padhi et al., 2020). It is also predicted that by 2030 India, China
and United States will have the largest number of diabetes sufferers (Pragya, 2015). In 2016,
Nigeria had the highest number of people with diabetes with 3.9 million and there has been a
progressive increase since then (Adeleye, 2021). Today, the average household in Nigeria has a
diabetes scare and that craves for urgent effective prevention, treatment and management in
order to avert crash of health and socio-economic development in the country.
However, many synthetic drugs have been developed for treatment of diabetes mellitus, but
still they have not provided a complete cure, and worst still, long-term use of these expensive
synthetic drugs causes severe side effects (Pragya, 2015; Kumar et al., 2021). Thus,
revolutionized research studies are on top gear to provide alternative remedies that are
effective, non-toxic and affordable for treatment of diabetics. In the history of man, traditional
treatments have been an extremely valued sources of medicines. Of course, World Health
Organization (WHO) has listed a total of 21,000 plants, as valid sources of potent medicines in
the world (Kumar et al., 2021). Among them, more than 400 plants are available for the
treatment of diabetes due to their composition of bioactive compounds. Unfortunately, only a
small number of these plants have undergone scientific and medical evaluation to assess their
efficacy and this has limited their wide utilization in formulation of drugs. The traditional
medicinal plants are used due to the high cost of orthodox drugs and low potency of some
orthodox drugs.
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
Hunteria umbellata is a good source of bioactive compounds, carbohydrate, protein and
minerals (Adeneye and Adeyemi, 2009; Adeneye et al., 2012). The fruit is yellow in colour,
smooth, about 5–25cm and consists of two separates globose mericaps (3–6 cm long), 8 – 25
seeds embedded in a gelatinous pulp. In African folk medicine, different parts of Hunteria
umbellata plant are highly valued in the local treatment of gastric ulcers, liver diseases, diabetes
mellitus and obesity (Boone, 2006; Adeneye et al., 2012a). In Germany, extracts of Hunteria
umbellate are used for reducing the heart rate, as an aphrodisiac, to decrease blood pressure
and reduce blood lipids (Boone, 2006; Ahajumobi and Anderson, 2022). Among the Southwest
Nigerians, the plant is reputed for its anti-helminthic, anti-inflammatory, uterotonic and
antidiabetic activities (Falodun et al., 2006). Cold decoction made from the plant seeds is also
reputed for the management of obesity and hyperlipidaemia among the Yoruba herbalists in
Nigeria (Adeneye and Adeyemi, 2009; Ogunlana et al., 2021; Ahajumobi and Anderson, 2022).
Also, extracts of the seeds of the plant have been reported to be very active against micro- organisms such as Escherichia coli, Proteus spp. and Staphylococcus aureus (Adeneye et al.,
2012a). Hunteria umbellata seeds extract contains secondary metabolite like tannins, cardia
glycosides, flavonoids, saponins, alkaloids, anthraquinones, polyphenols and terpenoids (Igbe
et al., 2009; Adeneye et al., 2012; Oboh et al., 2018). The extract reduces the blood glucose
levels, cholesterol levels and triglycerides (Adeneye et al., 2011; Adeneye and Crooks, 2015).
Hunteria umbellata (HU) significantly elevates red blood cell, Packed cell volume, and
Hemoglobin levels (Ogunlana et al., 2021; Ahajumobi and Anderson, 2022), which strongly
suggests that HU could be useful in the management of anemia.
The aim of this study was to assess the haematological effects of Methanol and n-Hexane
extracts of Hunteria umbellata seeds on diabetes mellitus using alloxan induced diabetic wistar
rats as test animals.
MATERIALS AND METHODS
Materials
Procurement of Sample Material:
The fresh fruits of Hunteria umbelleta (HU) were purchased from Aba in Aba North local
government area of Abia State. They were botanically authenticated at the Department of Plant
Science and Biotechnology, Michael Okpara University of Agriculture Umudike, Nigeria.
Methods
Preparation of Sample Material:
The fruits were washed with distilled water and peeled to obtain the seeds. The Hunteria
umbelleta (HU) seeds were rinsed in clean tap water and air dried in an oven (Model, DHG
9101.1 USA) at 250C for 7 weeks. The dried seeds were pulverized into powder using an electric
blender (Model serial NO. 4745, Christy and Noris Ltd. England) and stored in a dry container
at room temperature.
Preparation of Methanol Hunteria umbelleta (HU) Seed Extract:
Hunteria umbelleta (HU) seed powder (841g) was poured into the material chamber of the
soxhlet extractor. Then extraction using methanol (solvent) of 1600 mL at a temperature of
60oC took place for 48 h. The solvent (methanol) in the Hunteria umbelleta (HU) seed extract
was recovered by evaporating at 40oC using a rotary evaporator to obtain a crude extract. The
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Hunteria umbelleta (HU) seed extract was packaged in a sealed zip lock and stored in
refrigerator for further analysis.
Preparation of n-Hexane Hunteria umbelleta (HU) Seed Extract:
Hunteria umbelleta (HU) seed powder (841g) was poured into the material chamber of the
Soxhlet extractor. Then extraction using n-hexane (solvent) of 1600 mL at a temperature of
60oC took place for 48 h. The solvent (n-hexane) in the Hunteria umbelleta (HU) seed extract
was recovered by evaporating at 40oC using a rotary evaporator to obtain a crude extract. The
Hunteria umbelleta (HU) seed extract was packaged in a sealed zip lock and stored in
refrigerator for further analysis.
Determination of Phytochemicals in the HU Seed Extracts:
The phytochemical screening was done on the methanol and n-hexane HU seed extracts using
GCMS-QP 2010 SHIMADZU instrument (Trease and Evans, 1989; Shale et al., 1999; Evans et al.,
2000; Abate, 2002; Sawadogo et al., 2006; Agim et al., 2017). The constituents tested include:
Alkaloids, Tanins, saponins, phenolic compounds, cardiac glycoside, flavonoids and steroids.
The HU seed extract (3 g) was dissolved in aqueous solvent of methanol. To analyze the sample,
the column oven temperature and Injector temperature were set at 800°C and 200°C
respectively. The flow control mode was maintained in linear velocity with a split injection
mode split ratio of 20. The column flow was 1.46 mL/min with a helium carrier gas of
99.9995% purity. The column oven temperature program was set as follows: The temperature
was set at 80°C with 2 minutes hold time by the rate of 10. The temperature was 300°C with
10 minutes hold time. The column at 5 minutes was used with a length of 30 mL and diameter
of 0.25 mm and its film thickness was 0.25 m. The ion source temperature for MS condition
was 200°C and interface temperature was 240°C. Starting m/z (Mass to charge) ratio was 40
and ending with m/z ratio of 700. (40-700 m/z).
Identification of the Phytochemical Constituents:
The unknown phytochemical components present in the extracts, were identified by
comparing their individual mass spectral peak values with the data base of National Institute
of Standard and Technology (NIST) which holds about 62,000 patterns. Then, the
phytochemical was identified based on the hits returned after comparing the unknown peak
value and the chromatogram from GCMS against the known chromatogram, peak value from
the NIST library data base. Subsequently, the detail about their molecular formula, molecular
weight, structures was obtained.
Animal for the Experiment:
Sixty-six sexually mature male Wistar rats (10-12 weeks old; weights-150-153g) obtained
from the laboratory animal house of the College of Veterinary Medicine, Michael Okpara
University of Agriculture, Umudike, Nigeria, were used for the experiment. Thirty of the rats
were assigned to six groups of five rats each, in metabolic cages equipped to separate feaces
and urine. The rats had exactly 12 h of light and 12 h of darkness in a day. National Research
Council guidelines on the care and use of laboratory animals (National Research Council, 2010)
were strictly adhered to. The rats had free access to food (Standard pellet diet, from Vital feed
Ltd. Aba, Abia State, Nigeria) and water at will for an acclimatization period of 21 days prior to
experiment. Baseline blood glucose and body weights of the rats were taken after
acclimatization period. The study was approved by Animal Experimentation Ethics Committee
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
of Michael Okpara University of Agriculture, Umudike, Nigeria. The seven groups of the rodents
(5 rats in each group) were obtained on the basis that the difference in mean body weight of
each group did not exceed 5g (AOAC, 1995). The rats were housed in the animal house of the
Department of Biochemistry, Michael Okpara University of Agriculture, Umudike. The animals
were acclimatized for a period of 21 days prior to the experiment. Rats were allowed free
access to water throughout the study and were maintained on a 12 h light: 12 h dark phase
schedule.
Experimental Design
The studies on diabetes mellitus were carried out using the Completely Randomized Design
(CRD). Thirty rats were randomly assigned to six groups of five rats each based on treatments
for them. There were six treatments, each replicated five times. The rats were the replicates
while the different diets/treatments differentiated the groups. Then, for the Acute Toxicity
Test (LD50), thirty-six rats (nine rats in phase one and nine rats in phase two for Methanol HU
seed extract; nine rats in phase one and nine rats in phase two for n-Hexane HU seed extract)
were used for the experiment
Induction of Diabetes Mellitus
The rats were fasted for 17 hours before induction of diabetes mellitus. Diabetes mellitus was
induced by a single intraperitoneal injection of 160 mg/kg body weight of Alloxan
monohydrate (Pari and Venkateswaran, 2002). Alloxan was dissolved in 0.9% normal saline
as vehicle. Therapeutic measures were adopted to ensure the survival of the rats by
administration of glucose to tide over hypoglycemic phase. The blood glucose levels were
assessed on days 3 and 6 following administration of alloxan. The blood samples were obtained
by sequential snipping of the tail. Animals with blood glucose level greater than 200 mg/dl
were considered diabetic after 2 days of induction of alloxan and were used for the experiment.
All animals were allowed free access to water and pellet diet.
Experimental Groups of the Animals for the Diabetes Mellitus studies
The six groups of the rats were treated as follows:
1. Group 1 (Negative control- diabetic rats without treatment)- allowed on animal feed
and water ad libitum;
2. Group 2 (Positive control- diabetic rats)- were given oral hypoglycemic drug
(Glibenclimide at 500 mg/kg body weight) and allowed on animal feed and water ad
libitum;
3. Group 3 (Test group- diabetic rats)- rats were orally administered 250mg/kg body
weight of methanol HU seed extract and allowed on animal feed and water ad libitum;
4. Group 4 (Test group- diabetic rats)- rats were orally administered 500mg/kg body
weight of methanol HU seed extract and allowed on animal feed and water ad libitum;
5. Group 5 (Test group-diabetic rats)- rats were orally administered 250mg/kg body
weight of n- hexane HU seed extract and allowed on animal feed and water ad libitum;
6. Group 6 (Test group- diabetic rats)- rats were orally administered 500mg/kg body
weight of n- hexane HU seed extract and allowed on animal feed and water ad libitu
Note: The rats were all sacrificed after 14 days of the study for biochemical analysis.
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Composition of the Animal Feed
The composition of rodent pelleted chow was 50% as carbohydrate, 21% as protein, 5% as fat,
15% as moisture and 9% mineral. The diets were formulated using AIN-93G (American
Institute of Nutrition) method (National Research Council, 1995).
Biological Studies
Acute Toxicity Test LD50:
Acute toxicity tests for the methanol and n-hexane seed extracts were done according to the
method of Lorke (1983) as follows:
Phase 1:
In this phase, nine test animals were divided into three groups of three animals each. Each
group of animals were administered different doses (10, 100 and 1000 mg/kg body weight) of
test substance respectively. The animals were placed under observation for 24 hours to
monitor their behaviors as well as if mortality will occur.
Phase 2:
In this phase, nine test animals were distributed into three groups of three animals each. The
animals were administered higher doses (1600, 2900 and 5000 mg/kg body weight) of test
substance and then observed for 24 hours for behavioral change as well as mortality.
LD50 was calculated by the formula;
LD
Where; D0 is the highest dose that gave no mortality
D100 is the lowest dose that produced mortality
Blood Sample Collection
At the 14th day, the animals were fasted overnight, anaesthetized with chloroform and
sacrificed. Blood was collected through sharp cardiac puncture. Blood samples were collected
into EDTA containers (whole blood for hematological analysis).
Determination of Blood Glucose Concentration (mg/dl)
The one touch glucose monitoring meter and test strips were used as described in the
operational manual. A drop of whole blood (0.04 mL) was placed on a strip inserted into the
glucometer. The glucometer automatically separated serum from blood cells and determined
the blood glucose, which was displayed on the glucometer and the reading taken.
Determination of Hematological Parameters
Hematological indices were determined by standard method as described by Ochei and
Kolhatkar (2008). The indices examined were Packed Cell Volume (PCV), Red Blood Cells
(RBC), White Blood Cells (WBC) and Platelets. The standard procedures used are described
below.
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
Packed Cell Volume (PCV)
This was determined using standard haematological procedure as described by Ochei and
Kolhatkar (2008). Well mixed anticoagulated blood (0.5 ml) was aspirated into a capillary tube
with one end sealed with plasticin. The tube was spun in a haematocrit centrifuge for 5 minutes
and then read off in a PCV reader. The values obtained were recorded as the PCV values.
Red Blood Cells
This was determined using standard haematological procedure as described by Ochei and
Kolhatkar (2008). Well mixed anticoagulated blood was diluted 1: 20 with 10% Na2CO3
solution. The mixture (0.5ml) was loaded into an improved Neubauer counting chamber.
Appropriate squares (5 significant squares) were counted and added up to determine the total
red cell count.
White Blood Cells (Total White Blood Cell Count)
This was determined using standard haematological procedure as described by Ochei and
Kolhatkar (2008). Well mixed anticoagulated blood was diluted 1: 20 with Turk solution (2%
Glacial acetic acid) in a test tube. This was loaded into an improved Neubauer counting
chamber. Appropriate squares (4 significant squares) were counted, added up and divided by
2 to determine the total white cell count.
Platelets
This was determined using the method described by Cheesbrough (2000). The blood sample
was diluted 1: 20 with 2% ammonium oxalate. The diluted sample was loaded into the
Neubauer counting chamber with the aid of a pasteur pipette. The platelets were counted from
appropriate five squares on the chamber under a microscope and summed up to obtain the
platelet values.
Mean Corpuscular Volume (MCV)
Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC)
were determined by calculation.
Where MCV = Mean Corpuscular Volume
PCV = Packed Cell Volume
RBC = Red Blood Cells
Where MCHC = Mean Corpuscular Hemoglobin Concentration
Statistical Analysis
Data obtained in this study were expressed as means of triplicate values. Then, the
phytochemical data were subjected to statistical analysis. Analysis of variance (ANOVA) were
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determined using SPSS Version 21.0 and the differences between the mean values were
evaluated at p<0.05 using Duncan’s multiple range test.
RESULTS
Table 1: The Median Lethal Dose (LD50) of the methanol HU seed extract
Dose mg/kg D/T Physical Observation
Phase 1
10 0/3 Scratching of the mouth within a minute
100 0/3 Scratching of the mouth and calmness after a while
1000 0/3 Calmness and slow movement
Phase 2
1600 0/3 Scratching of the mouth and a little restlessness
2900 3/3 Restlessness, Agitation, Climbing on other rats and
death within 1hr
5000 3/3 Restlessness, Agitation and death within 1hr
D = Number of deaths; Number of albino rats used per experiment = 3
The acute toxicity test for the methanol HU seed extract shown in Table 1 revealed that in phase
1 of the study, no death of animal occurred at a dose of up to 1000 mg/kg body weight and there
were no observable signs of toxicity. In phase 2, treatment of the animals with doses from 2900
to 5000 mg/kg body weight caused the death of all three animals with signs of toxicity.
Table 2: The Median Lethal Dose (LD50) of the N-hexane HU seed extract
Dose mg/kg D/T Physical Observation
Phase 1
10 0/3 Scratching of the mouth and calmness within a minute
100 0/3 Scratching of the mouth and calmness after a while
1000 0/3 Calmness and slow movement
Phase 2
1600 0/3 Calmness and slow movement
2900 0/3 Calmness and slow movement
5000 0/3 Calmness and slow movement
D = Number of deaths = 0; T = Number of albino rats used for the experiment = 3
The acute toxicity test of the n-hexane HU seed extracts as shown in Table 2, revealed that in
phase 1 of the study, no death of a rat occurred at a dose of up to 1000 mg/kg body weight, and
there were no observable signs of toxicity. In phase 2, treatment of the animals with doses of
up to 5000 mg/kg body weight did not cause any death or produce any signs of toxicity.
Table 3: Results for GC-MS Profiling of Methanol HU Seed Extract
Compound/
species
probabilit
y
Retentio
n time
Molecular
formula
Molecula
r weight
Compound
Nature
Activity
Benzenemethanol, αα- Dimethyl
48.2% 3.849 C9 H12 O 136 Colourless
aromatic
alcohol.
Acts as a
solvent, an
antioxidant
and a
fragrance.
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
Spiro-9,9'-bis(2-
hydroxyfluorene)
29.9% 3.913 C25H16O2 348 Extremely
weak basic
(essentially
neutral)
Potentially
toxic
compound.
Vismiaquinone 44.8% 5.013 C21 H20 O5 352 - -
Dichloroacetaldehyde 98.9% 7.550 C2 H2 Cl2 O 112 Colourless
organochlorin
e compound
Anti-fungal
activity
Trichloromethane 96.9% 8.022 C HCl3
118
Colourless,
volatile liquid
with an ether- like odour.
Acute
chloroform
toxicity
results in
impaired
liver
function,
cardiac
arrhythmia,
nausea and
central
nervous
system
dysfunction
.
1,3-Diphenyl-2,4,5-
trioxoimidazolidine
15.6% 8.569 C15H10N2O3 266 - -
2-Norpinanol, 3,6,6-trimethyl 8.78% 9.651 C10H18O 154 - -
Nonanamide, N'[(3-methoxy- 4[(trimethylsilyl)oxy]
phenyl]methyl]-8-methyl
29.5% 10.012 C21H37NO3S
i
379 - Harmful by
ingestion
n-Hexadecanoic acid 94.5% 13.906 C16H32O2 256 Saturated
fatty acid
Anti- bacterial
activity
5,5'-Di(benzyloxycarbonyl)-
3,3',4,4'-tetramethyl-2,2'-
dipyrrylmethane
54.5% 15.826 C29H30N2O4 470 - Synthesis of
anti- oxidant
Succinic acid, 2-pentyl 2,2,3,3-
tetrafluoropropyl ester
98.0% 16.746 C12H18F4O4 302 Alpha, omega,
dicarboxylic
acid
Atheiminic
activity
Glycerol tricaprylate 97.9% 17.398 C27H50O6 470 - Fragrant
ingredient
Vanadium,
(pentamethylcyclopentadieny
l)
(Tetraphenylcyclobutadiene
93.6% 18.527 C38H35V 542 Hard, silvery,
grey,
malleable
transition
metal
It is used for
pre- diabetes
and
diabetes
Acetic acid, 4,4,
6a,8a,11,12,14b-heptamethyl- 13-oxodocosahydropicen-3-ly
ester
98.9% 18.638 C31H50O3 470 Carboxylic
acid, clear,
colorless
organic fluid
Anti- bacterial
Key: (_) Not present
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The GC-MS spectra analysis in Table 3 revealed the presence of different compounds present in
the methanol HU seed extract, as well as their retention time, molecular formula, molecular
weight, compound nature and their different activities.
Table 4: Results for GC-MS Profiling of N-Hexane HU Seed Extract
Compound/species Probability Retentio
n time
Molecular
formulae
Molecula
r weight
Compound
Nature
Activity
1α- Hydroxyallobetulane
97.2% 5.455 C 50H 50O2 442 Cyclic
dimeric
esters
Anti-fungal
activities
Benzoic acid, 2,4-
dimethoxy-6-[(4-
methoxyphenyl])
95.1% 6.133 C 19H 20O6 344 Aromatic
carboxylic
acid
Anti- microbial
activity
Benzenepropanoic
acid
97.4% 6.776 C9H8BrNO4 273 - -
5α-Cholestane- 3β,4β,6α, 7 α,8 β,
15α,16β,26-octaol
41.8% 7.027 C 27H 48O8 500 Esterases
that lyses
choline
based esters
Neurotrans
miters toxic
by ingestion
5,9,23-
Tricontatrienoic acid,
methyi ester
11.101 C 31H 56O2 460 - -
Octadecanoic acid,
10,11,13,14-
tetrakis[(trimethylsilyl
)oxy]
93.0% 17.759 C31H 70O6Si4 650 Esters of
fatty acid
Anti- bacterial
activity
Nonanamide, 5-
hydroxy-5-methyl-2-
(2-methylpropyl)-N- benzyl- 91.6% 18.387 C21H 35NO2 333 - -
D-Ribose 90.1% 18.725 C 5H 10O5 150 Water
soluble
pentose
sugar
Coding,
decoding,
regulation
and
expression
of gene
1(3H)-
Isobenzofuranone,
3,3'-(4-hydroxy-1,-3-
phenylene)bis[3-(4-
hydroxyphenyl)
81.3% 20.017 C 34H 22O7 542 Natural oil Anti-oxidant
and
antimicrobia
l activity
Spirostan-3,11-diol
diacetate(ester)
4.06% 20.314 C 31H 48O6 516 Steriod
saponies in
plant
Anti- bacterial
activity
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
1H-Indole-3-acetic
acid, 1-(tert- butyldimethylsilyl)-5-
[(tert- butyldimethylsilyl)
oxy]
98.4% 20.541 C28H51NO3Si3 533 Naturally
occurring
plant
hormone of
the auxin
class
-
Hex-5-ynoic acid,
methyl ester
96.8% 23.055 C 7H 10O2 126 - -
Sebacic acid, dodecyl
pentafluorobenzyl
ester
85.4% 23.940 C29H43F5O4 550 Naturally
occurring
dicarboxylic
acid
derivatives of
oil castol oil
-
2,4-Cyclopentadiene- 1,2,3,4-
tetracarboxylic acid, 5-
(2-quinolinyl)-,
tetramethyl ester
32.7% 24.854 C22H 19NO8 425 - -
Key: (_) = Not present
The GC-MS spectra analysis in Table 4 revealed the presence of different compounds present in
N-hexane seed extract of Hunteria umbelleta as well as their retention time, molecular formula,
molecular weight, compound nature and their different activities.
Table 5: Effect of methanol and n-Hexane seed extract on the haematological profile of
Alloxan-induced diabetic Wistar rats
GROUP RBC PCV (%) HB(g/dL) WBC(×10
9
/L)
PLT(×103
/
mL)
MCV(f/L) MCH(p/L) MCHC(g/
dL)
1 5.99±0.
21ab
39.00+±.
00ab
12.90±0.
95 a
12.95±1.
56b
131.00±6.
89b
65.09±1.
06bc
21.50±0.
82a
33.05±1.
76a
2 6.40±0.
27b
41.33±1.
52ab
13.83±0.
20a
13.22±2.
68b
118.50±2.
05b
64.55±0.
41abc
21.62±0.
78a
33.49±1.
01ab
3 6.70±0.
12bc
42.33±1.
52 c
15.80±0.
43b
9.15±0.6
7
a
117.10±5.
77 b
63..17±1.
13ab
23.58±0.
36 c
37.33±0.
75 c
4 5.18±1.
13c
46.66±1.
15 d
16.46±0.
15b
10.86±0.
40ab
110.13±3.
23b
63.72±0.
14abc
22.49±0.
55abc
35.30±0.
86bc
5 5.19±0.
07a
38.66±2.
08a
13.66±0.
15a
10.59±0.
63a
113.63±0.
90b
66.13±2.
96 c
23.38±0.
35 bc
35.41±1.
94bc
6 6.40±0.
8
ab
38.33±0.
57a
13.76±0.
35a
11.12±0.
42ab
116.10±2.
00b
64.82±0.
55 abc
23.28±0.
55bc
35.910±0
.56 c
Values represent the mean +SD for n=3. Values in the same column bearing the same alphabets
are not significantly different from each other (P<0.05).
Key: Group 1= Diabetic control (untreated rats), Group 2= Positive control (Standard drug
(Glibenclamide, 150mg/kg body weight); Group 3= (methanol extract low dose, 250mg/kg
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body weight), Group 4= n(methanol extract high dose, 500mg/kg body weight); Group 5=( n- hexane extract low dose 250mg/kg body weight) Group 6=( n- hexane extract high dose,
500mg/Kg body weight). Red blood cell (RBC), Pack cell volume (PCV), Haemoglobin (HB),
White blood cell (WBC), Platelets (PLT), MCV, MCH, MCHC.
The result of the effect of aqueous extract of Hunteria Umbellata on the haematological
parameters of Alloxan induced diabetic rats are shown in Table 5. The result of the methanol
/n-hexane extract of Hunteria Umbellata had significant effect on RBC, Hb, MCHC, MCH, PCV,
MCV, WBC. Group 1 (Negative control) were significantly (P<0.05) high in WBC, PLT, MCV while
RBC, PCV, Hb, MCH, MCHC were significantly (P<0.05) low.
However, WBC, PLT, MCV decreased significantly in all treatment groups except WBC in group
3 (treated with 500 mg of Glibenclamide, a standard known drug) and MCV in group 6 (treated
with 250 mg/kg body weight of n-hexane). Also, RBC, PCV, HB, MCH, MCHC increased
significantly in all treatment groups except RBC in (group 6), PCV in group 6, and group 7. When
compared to the group 2 (Diabetes or Negative control).
DISCUSSION
The qualitative phytochemical screening of Methanol and n-hexane seed extracts of Hunteria
Umbellata indicated significant contents of secondary metabolites such as phenols, alkaloids,
flavonoids, oxalate, saponins, combined and free reducing sugars, tannins, sterols and balsams.
The phytochemicals exert a wide range of antidiabetic activities (Kong et al., 2021; Ramirez- Alarcon et al., 2021; Bahrampour et al., 2023; Yedjou et al., 2023). The Gas Chromatography
Mass Spectrophotometry analysis of Methanol and n- hexane seed extracts of Hunteria
umbellata revealed the presence of different compounds present, including their retention
time, molecular formula, molecular weight, compound nature and their different activities.
Acute Toxicity Test LD50 (Locke's Method, 1983)
Safety assessment of pharmaceuticals and foods are very crucial prior to their approval for
human uses (Erhirhie et al., 2018). LD50 is the amount of a substance, or material given at once
which causes death of 50% of a group of test animals. It is usually expressed as the amount of
chemical administered (eg. mg per 100 g for smaller animals) or (mg per kg for bigger subjects)
of the weight of the test animal. LD50 is important for drugs, food and accidental domestic
poisoning. In general, the smaller the LD50 value, the more toxic the chemicals are; the larger
the LD50 value, the lower the toxicity (Chinedu et al., 2013). Of course, extrapolation of these
animal data into humans may guarantee potential hazards of these extracts to humans.
Assessment of hematological parameters can not only be used to determine the extent of
deleterious effect of extract on the blood of an animal, but it can also be used to explain blood
relating functions of a plant extract or its product (Yakubu et al., 2007).
Haemotoxicity sets in when there is elevation of these blood components beyond their
reference ranges. There was a significant reduction (P<0.05) in WBC levels of diabetic rats
treated with aqueous extract of Hunteria umbellata when compared with the untreated Wistar
rats. The study shows that there is a significant increase (P<0.05) in the level of RBC, HB and
PCV of group 3 and the treated groups, (250 mg/kg body weight of the methanol extract of
Hunteria umbellata; 500 mg/kg body weight of the methanol extract of Hunteria umbellata)
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Obia, C., Nwuke, P. C., & Okereke, G. O. (2023). Haematological Effects of Methanol and n-Hexane Seed Extracts of Hunteria umbellata (abeere) on
Alloxan Induced Diabetic Wistar Rats. British Journal of Healthcare and Medical Research, Vol - 10(4). 111-126.
URL: http://dx.doi.org/10.14738/bjhmr.104.15156
when compared to the negative control. But there was a decrease in the 250 mg/kg body weight
of n-hexane extract of Hunteria umbellata and 500 mg/kg body weight of n-hexane extract of
Hunteria umbellata. This may be as a result of anemia or the onset of glycosylation process in
the untreated diabetic rats. There was a significant decrease (P<0.05) in the PLT concentration
of the diabetic rats when treated with the extracts of Hunteria umbellata. There was a
significant reduction (P<0.05) in the MCV except the group 6 treated with (250 mg/kg body
weight of the n- hexane extract) values.
There was a significant increase (P<0.05) in the MCH when treated with the extracts of Hunteria
Umbellata (i.e. group 3- group 6) when compared to the negative control. Also, there was a
significant increase (P<0.05) in MCHC when treated with extracts of Hunteria Umbellata on the
diabetic rats. The results obtained shows significant values of WBC; therefore, it is clear that an
increase in the number of WBC is a normal reaction of rats to foreign substances, which alters
their normal physiological processes. Platelets play a major role in the development as well as
in the stability of atherosclerotic plaques and as a consequence, anti-platelet agents have been
used clinically in patients at risk for myocardial ischemia, unstable angina and acute myocardial
infarction (Albers, 1995; George, 2000). Therefore, the high dose (500 mg/kg body weight) of
the Hunteria umbellata extracts were useful in reducing the platelets which in turn might be
useful in reducing the cardiovascular diseases as some studies suggested various mechanisms
by which flavonoid exert its antiplatelet property by lowering intracellular Ca2+ levels;
alteration in the metabolism of cAMP, and thromboxane A2 (Roy et al., 1999; Kang et al., 2001).
The haemoglobin content, RBC and PCV also significantly increased stimulate erythropoietin
release in the kidney which is the humoral regulators of RBC production (Degruchy, 19756;
Polenakovic and Sikole, 1996). The result of the study suggested that Hunteria Umbellata
extract studied showed positive haematological activities in rats and can be recommended on
the management of anemia and immunity dependent disorders.
CONCLUSION
Methanol and n-Hexane extracts of Hunteria umbellata (at both 250 mg and 500 mg per kg body
weight) exhibited effective positive antidiabetic properties, significant physiological and
biochemical functions in treatment of diabetic wistar rats, induced with alloxan. The methanol
and n-hexane seed extracts of Hunteria umbellate had significant composition of
phytochemicals such as phenols alkaloids, flavonoids, oxalate, saponins etc. The acute toxicity
test for methanol HU seed extract showed its safe nature at dose of up to 1600 mg/kg body
weight; while that of n-hexane HU seed extract showed no sign of toxicity at dose of up to 5000
mg/kg body weight. Therefore, these methanol and n-Hexane extracts of Hunteria umbellata
possess the potentials of management of diabetes mellitus in animals and humans better than
the standard drug (Glibenclamide); and thus, could serve as alternatives or replacements to
orthodox antidiabetic drugs that are associated with long-term side effects. Utilization of
medicinal plants (such as Hunteria unbellata) in the management of diabetes mellitus should
be encouraged in Africa.
RECOMMENDATION
Further studies should be undertaken on the effects of the mechanism of action of Methanol
and n-Hexane extracts of Hunteria Umbellata seeds on alloxan induced diabetic male wistar
rats. The results of such study can be used to verify the efficacy and potency of the results of
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this present study. Attention should be drawn to proper utilization of medicinal plants in the
treatment of diseases in Africa.
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