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South African Journal of Botany EVALUATION OF THE ANTIDIABETIC AND

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 South African Journal of Botany
EVALUATION OF THE ANTIDIABETIC AND TOXICOLOGICAL STUDIES OF
HIPPOCRATEA VELUTINA (Afzel.)
--Manuscript Draft--

Manuscript Number:

Article Type: Research Paper

Section/Category: Pharmacology

Keywords: Hippocratea Velutina; Diabetes mellitus; haematological and biochemical parameters

Corresponding Author: Farouk Oladoja, B.Pharm, M.sc ,

Olabisi Onabanjo University Faculty of Pharmacy
NIGERIA

First Author: Farouk Oladoja, B.Pharm, M.sc ,

Order of Authors: Farouk Oladoja, B.Pharm, M.sc ,

EMMANUEL S. IROKOSU, MBBS, M.sc

MARCUS DUROJAYE AYOOLA, P.hD

OLUWAFEMI EZEKIEL KALE, P.hD

MURTALA A. AKANJI, P.hD

Oladapo Elijah Oyinloye, P.hD

OGUNLEYE T. BEATRICE, B.sc

Samuel Akinniyi Odewo, P.hD

Abstract: Objective

The study evaluated the anti-diabetic potentials of the methanol leaf extract of
Hippocratea velutina Afzel. (Celastraceae) and its toxicity profile with a view to
justifying its antidiabetic ethnomedicinal claim and establish its safety.

Methods

Acute and sub acute toxicity tests of the plant extract were carried out using a modified
OECD test guidelines. The effects of the extract on glucose, haematological and
biochemical components were assessed using standard procedures. Its antidiabetic
activity in streptozotocin-induced diabetic rats at 50, 150 and 300 mg/kg for 28 days
was assayed while glibenclamide (5 mg/kg) and distilled water were positive and
negative controls, respectively. Histopathological examination of vital organs such as
liver and pancreas of rats treated with the extract at 200, 400 and 800 mg/kg was also
carried out. The results obtained were subjected to statistical analysis using Analysis of
Variance (ANOVA), followed by the Dunnett multiple comparison test, with p< 0.05
being taken as significant.

Results

Preliminary phytochemical screening of the plant leaf extract showed the presence of
tannins, flavonoids, saponins, alkaloids, terpenoids, deoxy-sugars, and
anthraquinones. The extract had LD 50 that was greater than 2000 mg/kg in mice. It
had no toxic effects on the haematological and biochemical components of blood
samples of the animals used but caused significant blood glucose level reduction in
normal rats at 150 and 300 mg/kg. In streptozotocin-induced diabetic rats, the extract
elicited a non dose dependent antidiabetic effect on day 7 of the study at the tested
doses of 50, 150 and 300 mg/kg that was significantly higher than that of glibenclamide
(10 mg/kg). On days 14, 21 and 28 however, the activity of the extract at all the tested
doses and glibenclamide were comparable. The extract did not affect the histology of
the liver, brain, kidney and pancreas at 200 mg/kg but caused slight and severe effects
on these organs at 400 and 800 mg/kg, respectively.

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 Conclusion : The study concluded that Hippocratea velutina possessed significant
antihyperglycaemic activity and is non toxic to haematological and biochemical
components. It is also safe in animal organs at low dose but could have deleterious
effects at high concentrations.

Suggested Reviewers: Olufunsho Awodele, P.hD Pharmacology

Associate Professor, University of Lagos College of Medicine
awodeleo@gmail.com
a renowned toxicolgist

Ismail Ishola, P.hD Pharmacogy

Senior Lecturer, University of Lagos College of Medicine
oishola@cmul.edu.ng
a great reviewer

Ibrahim Oreagba, P.hD Pharmacology

Professor, University of Lagos College of Medicine
iaoreagba@cmul.edu.ng
A great Pharmacologist

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Cover Letter

Farouk A.Oladoja,
Department of Pharmacology and Toxicology,
Faculty of Pharmacy
Olabisi Onabanjo University
Ago-iwoye, Ogun-State.
14th, April 2022

Dear Editor, SAJB

We are pleased to submit an original research article entitled " EVALUATION OF THE ANTIDIABETIC

AND TOXICOLOGICAL STUDIES OF HIPPOCRATEA VELUTINA (Afzel.) ” for consideration for

publication in the South African Journal of Botany.

We discovered that herbal medicines are safe and cheaper alternatives to combat one of the world’s highly

prevalent disease -Diabetes. However, till now there have been no scientific reports on the antidiabetic

effect of the leaves of HIPPOCRATEA VELUTINA (Afzel.) widely used to treating diabetes locally and this

manuscript builds on this fact to justify its use.

This manuscript showed the great and incredible antidiabetic and toxicological effects of this novel plant and
it is worthy of note that this research is arguably the first scientific publication on this novel plant.

This manuscript is appropriate for publication in the South African Journal of Botany .because it is part of the
significant scope of the journal.

This manuscript has not been published and is not under consideration for publication elsewhere. Therefore,
we have no conflicts of interest to disclose.

Thank you for your consideration!

Sincerely,

Farouk A.Oladoja
Lecturer, Department of Pharmacology and Toxicology
Olabisi Onabanjo University.
adedeji.oladoja@oouagoiwoye.edu.ng
+2347065153035.
Highlights

HIGHLIGHTS

Hippocratea velutina is an effective antidiabetic agent without toxic effects on different body
organs especially at low doses
Hippocratea velutina has a very high hypoglycaemic activity at elevated doses and may not
be safe for human consumption at high concentrations
Hippocratea velutina elicits a non-dose dependent anti-hyperglycaemic effect
Hippocratea velutina has leucopoetic and possible immunomodulatory effects
Hippocratea velutina has a possible triglyceride lowering effect
Manuscript File (original) Click here to access/download;Manuscript File (original);HV
Manuscript.docx
Click here to view linked References

1 EVALUATION OF THE ANTIDIABETIC AND TOXICOLOGICAL STUDIES OF

1
2 2 HIPPOCRATEA VELUTINA (Afzel.)
3
4
5
6 3 FAROUK A. OLADOJA*1, EMMANUEL S. IROKOSU2, MARCUS D. AYOOLA3,
7
8 4 OLUWAFEMI E. KALE2, MURTALA A. AKANJI2, OYINLOYE O. ELIJAH1,
9
10
5 OGUNLEYE T. BEATRICE2, ODEWO A, SAMUEL4
11
12
13 1
14 6 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Olabisi Onabanjo
15 7 University, Ago-Iwoye, Ogun-State. Nigeria.
16
2
17 8 Department of Pharmacology and Therapeutics, Faculty of Basic Medical Science, Olabisi
18
9 Onabanjo University. Ago-Iwoye, Ogun-State. Nigeria.
19
20 3
21 10 Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University Ile-Ife
22 11 Osun-State. Nigeria.
23
4
24 12 Forest Research Institute of Nigeria, Ibadan. Oyo-State. Nigeria
25
26 13 Mailing Address: Department of Pharmacology and Toxicology, Faculty of Pharmacy,
27
28 14 Olabisi Onabanjo University. Ago-Iwoye, Ogun-State. Nigeria.
29
30 15 Telephone number +2347065153035.
31
32 16 Corresponding e-mail: adedeji.oladoja@oouagoiwoye.edu.ng
33
34 17 ABSTRACT
35
36
37 18 Objective: The study evaluated the anti-diabetic potentials of the methanol leaf extract of
38
39
19 Hippocratea velutina Afzel. (Celastraceae) and its toxicity profile with a view to justifying
40
41
42 20 its antidiabetic ethnomedicinal claim and establish its safety.
43
44
45 21 Methods: Acute and sub acute toxicity tests of the plant extract were carried out using a
46
47
48
22 modified OECD test guidelines. The effects of the extract on glucose, haematological and
49
50 23 biochemical components were assessed using standard procedures. Its antidiabetic activity in
51
52 24 streptozotocin-induced diabetic rats at 50, 150 and 300 mg/kg for 28 days was assayed while
53
54
55 25 glibenclamide (5 mg/kg) and distilled water were positive and negative controls, respectively.
56
57 26 Histopathological examination of vital organs such as liver and pancreas of rats treated with
58
59
60 27 the extract at 200, 400 and 800 mg/kg was also carried out. The results obtained were
61
62 1
63
64
65
 28 subjected to statistical analysis using Analysis of Variance (ANOVA), followed by the
1
2 29 Dunnett multiple comparison test, with p< 0.05 being taken as significant.
3
4
5
6
30 Results: Preliminary phytochemical screening of the plant leaf extract showed the presence
7
8 31 of tannins, flavonoids, saponins, alkaloids, terpenoids, deoxy-sugars, and anthraquinones.
9
10 32 The extract had LD50 that was greater than 2000 mg/kg in mice. It had no toxic effects on the
11
12
13 33 haematological and biochemical components of blood samples of the animals used but caused
14
15 34 significant blood glucose level reduction in normal rats at 150 and 300 mg/kg. In
16
17
18 35 streptozotocin-induced diabetic rats, the extract elicited a non dose dependent antidiabetic
19
20 36 effect on day 7 of the study at the tested doses of 50, 150 and 300 mg/kg that was
21
22
23 37 significantly higher than that of glibenclamide (10 mg/kg). On days 14, 21 and 28 however,
24
25 38 the activity of the extract at all the tested doses and glibenclamide were comparable. The
26
27
39 extract did not affect the histology of the liver, brain, kidney and pancreas at 200 mg/kg but
28
29
30 40 caused slight and severe effects on these organs at 400 and 800 mg/kg, respectively.
31
32 41 Conclusion: The study concluded that Hippocratea velutina possessed significant
33
34
35 42 antihyperglycaemic activity and is non toxic to haematological and biochemical components.
36
37 43 It is also safe in animal organs at low dose but could have deleterious effects at high
38
39
40 44 concentrations.
41
42
43 45 Keywords: Hippocratea Velutina, Diabetes mellitus, haematological and biochemical
44
45 46 parameters.
46
47
48 47 Abbreviations: HV – Hippocratea velutina, NC – Negative control, DU – Diabetes
49
50
51 48 Untreated, Gli – Glibenclamide.
52
53
54 49
55
56
57 50
58
59
60 51
61
62 2
63
64
65
 52
1
2
3 53 1.0. INTRODUCTION
4
5
6 54 Diabetes mellitus is a long-term disorder of metabolism of carbohydrates, proteins, and fats
7
8
9 55 arising from deficiency of insulin, insulin resistance, or both, leading to hyperglycemia
10
11 56 (Tfayli et al., 2009). It is one of the most disturbing and common chronic diseases of our
12
13
14 57 times, causing life threatening, disabling and costly complications as well as reducing life
15
16 58 expectancy (Aamir et al., 2020). This cankerworm is now dubbed as a ‘silent epidemic’
17
18
19 59 which claims over 4 million lives every year, with this scourge knowing no socioeconomic
20
21 60 boundaries despite extensive efforts by the National and International organizations and
22
23
61 cutting-edge research, about 11 % of world's population is expected to suffer from diabetes
24
25
26 62 (and its complications) by year 2045 (Kowluru and Mohammad, 2022). In conventional
27
28 63 medicine, the treatment goal of diabetes mellitus seeks to achieve optimal glycaemic control
29
30
31 64 with insulin, oral hypoglycemic agents, and other non-pharmacological therapies (Ogurtsova
32
33 65 et al., 2017). However, serious unwanted effects of synthetic drugs (Adebajo et. al., 2007)
34
35
36 66 and the complications of diabetes mellitus have led to the increased investigations of plants.
37
38 67 (Pepato et. al., 2001; Adebajo et al., 2007, 2009, 2013) used ethnomedicinally to lower blood
39
40
41
68 glucose. Herbal medicine has gained popularity in the past decade and there has been a
42
43 69 growing demand for herbal products among diabetic patients because of its low cost,
44
45 70 perceived effectiveness, and little or no side effects (Kumar et al., 2006).
46
47
48
49
71 Hippocratea velutina Afzel (Celastraceae) is known as mawolule among the ‘Yoruba’ tribe of
50
51 72 South-West Nigeria and dawe among the ‘Mende’ people of Sierra Leone with wide
52
53 73 distribution in Guinea and Cameroon. It is commonly used for treating headaches and fever
54
55
56 74 in Sierra Leone (Burkill, 1995) while it is ethnobotanically used in Nigeria to lower blood
57
58 75 glucose levels in diabetic patients.
59
60
61
62 3
63
64
65
 76 To our knowledge, no pharmacological activities or isolated compounds from this plant have
1
2 77 been reported or published. This study was therefore designed to establish its antidiabetic
3
4
5 78 ethnomedicinal claim, safety and screen it for its secondary metabolites.
6
7
8 79 2.0. METHODOLOGY
9
10
11 80 2.1 Chemicals and Reagents
12
13
14 81 Glibenclamide (Nigeria-German Chemicals Plc, Ota, Nigeria), streptozotocin (Sigma-Aldrich
15
16
17 82 Chemical Co., St. Louis, MO, USA). All other chemicals and reagents were of pure analytical
18
19 83 grade.
20
21
22 84 2.2 Plant Material and Extraction
23
24
25 85 Hippocratea velutina leaves were collected in Oshogbo, Osun State, Southwest Nigeria. A
26
27
28 86 voucher specimen number UIH 23137 of the plant prepared by Dr. S. A. Odewo was
29
30 87 deposited at the Botany Department, University of Ibadan, Ibadan. Nigeria. The leaves were
31
32
33 88 air-dried, pulverized and 1.0 kg of the powdered leaf was extracted with methanol using
34
35 89 Soxhlet apparatus and concentrated in-vacuo to give 10.4 % w/w yield.
36
37
38 90 2.3 Phytochemical Screening
39
40
41
91 Preliminary phytochemical screening of the methanolic extract of HV was evaluated for the
42
43
44 92 presence of alkaloids, flavonoids, cardiac glycosides, tannins, reducing sugar, saponin, and
45
46 93 anthraquinones using standard methods (Harborne, 1973; Sofowora, 1993).
47
48
49
94 2.4 Animals
50
51
52
53 95 Adult mice (18-20 g) and Wistar rats (190-200 g) were procured from the Laboratory Animal
54
55 96 Center of College of Medicine, University of Lagos, Lagos, Nigeria. They were allowed to
56
57
97 acclimatize for two weeks (at the animal house, Faculty of Pharmacy, Olabisi Onabanjo
58
59
60 98 University, Shagamu) and maintained under standard conditions (temp. 27±3° C, relative
61
62 4
63
64
65
 99 humidity 65%). They were fed on a standard pellet diet (Vital Feed Nig. Ltd.) and water was
1
2 100 given ad libitum. The study conforms to the Guide for the Care and Use of Laboratory
3
4
5 101 Animals published by the United States National Institute of Health (NIH publication N. 85-
6
7 102 23, revised 1996) for experimental animal studies. Ethical clearance for the use of animals for
8
9
10 103 this research was obtained from the College of Medicine of the University of Lagos, Health
11
12 104 Research Ethics Committee (CMUL/HREC), and approval to carry out the study was given
13
14
15 105 with permission number (CMUL/ACUREC/02/22/997).
16
17
18 106 2.5 Oral acute toxicity test
19
20
21 107 According to the Organization for Economic Cooperation and Development (OECD)
22
23 108 guidelines for testing chemicals. Five groups of overnight fasted mice (n=5) were used.
24
25
26 109 Control group of mice received distilled water while the treatment groups were given (orally)
27
28 110 HV methanol extract suspended in distilled water at 5, 50, 500, and 2000 mg/kg. The
29
30
31 111 administration was done only once at the start of the experiment, and the mice were
32
33 112 monitored for gross morphological, physiological, behavioural changes and mortality
34
35
113 continuously for the first 4 hours after dosing and subsequently daily for 14 (OECD, 1992).
36
37
38
39 114 2.6 Intraperitoneal acute toxicity test
40
41
42 115 The same procedure for oral acute toxicity test above (section 2.5) was carried out except that
43
44 116 the intraperitoneal route of administration was used. The Median Lethal Dose, LD50 was
45
46
47 117 estimated according to the method of Finney (1985) (Finney, 1985).
48
49
50 118 2.7 Sub-chronic toxicity study
51
52
53 119 Twenty-five rats were randomized into five groups (n=5). Group A was given distilled water
54
55 120 for 90 days and taken as the control while groups B, C and D were orally administered with
56
57
58 121 HV extract at 200, 400, and 800 mg/kg, respectively daily for 90 days. Blood samples were
59
60
61
62 5
63
64
65
 122 collected on day 90 for biochemical and haematological analysis using the method described
1
2 123 by Yakubu et al., (2006) (Yakubu et al., 2006).
3
4
5
6 124 2.8 Effect of extract on normoglycaemic rats
7
8
9 125 Groups of 5 normal rats were fasted overnight and orally given either extract (50, 150, 300
10
11 126 mg/kg), distilled water (negative control), or glibenclamide (10 mg/kg, positive control) for
12
13
14 127 21 days. Their Fasting Blood Sugar (FBS) level was checked on days 0, 1, 7, 14, and 21 of
15
16 128 administration and recorded (Lanjhiyana et al., 2011).
17
18
19 129 2.9 Effect of extract on streptozotocin-induced diabetic rats
20
21
22 130 Diabetes was induced in rats by intraperitoneal injection of streptozotocin (55 mg/kg)
23
24
25 131 dissolved in freshly prepared 0.1 mMol/L cold sodium citrate buffer (pH 4.5). Rats with
26
27 132 fasting blood glucose (FBG) level ≥ 11.0 mmol/L (200 mg/dL) 6 days after the
28
29
30 133 administration of streptozotocin were considered diabetic. They were divided into groups of
31
32 134 five rats each and treated as shown below:
33
34
35 135 Group I: Normal rats + distilled water,
36
37
38 136 Group II: Diabetic rats + distilled water,
39
40 137 Group III: Diabetic rats + HV extract (50 mg/kg),
41
42
138 Group IV: Diabetic rats + HV extract (150 mg/kg),
43
44
45 139 Group V: Diabetic rats + HV extract (300 mg/kg) and
46
47 140 Group VI: Diabetic rats + glibenclamide (10 mg/kg).
48
49
50 141 Fasting blood sugar levels were checked on days 0, 1, 3, 7, 10, 12 and 14, 21, and 28 of
51
52 142 administration. After the study, blood and tissue samples were collected using the method
53
54
55 143 described by Yakubu et al. (2006) (Yakubu et al., 2006).
56
57
58 144
59
60
61
62 6
63
64
65
 145 2.10 Effect of extract on Haematological parameters
1
2
3 146 Blood sample (1.5ml) was used for haematolgical study using the automated haematology
4
5
6
147 analyzer. About 50 µL of blood was aspirated in to the analyser for analysis to obtain the
7
8 148 result of the complete blood count (Sharma, 2019).
9
10
11 149 2.11 Effect of extract on Biochemical parameters
12
13
14 150 The serum was analyzed for biochemical markers such as total cholesterol (TC), triglycerides
15
16
17 151 (TG), low-density lipoprotein-Cholesterol (LDL-C), high-density lipoprotein-cholesterol
18
19 152 (HDL-C), Alanine transaminase (ALT), Aspartate transaminase (AST), urea, creatinine, and
20
21
22 153 bilirubin were estimated using commercial kits obtained from Randox Laboratories Ltd.
23
24 154 (Crumlin, UK) and following the guidelines described by the manufacturer. Also, total
25
26
155 cholesterol (TC), triglycerides (TG), low-density lipoprotein-Cholesterol (LDL-C) and high-
27
28
29 156 density lipoprotein-cholesterol (HDL-C) were calculated by Friedewald’s formula
30
31 157 (Friedewald et al., 1972).
32
33
34
158 2.12 Histopathological examination
35
36
37
38 159 Tissue samples of liver, kidney, brain, pancreas was prepared using the method described by
39
40 160 Baker and Silverston (1985). The tissue histology slides were viewed under the light
41
42
161 microscope at ×40 magnification. ( Baker and Silverton, 2014)
43
44
45
46 162 2.13 Statistical Analysis
47
48
49 163 Results obtained were expressed as mean ± SEM using GraphPad Prism 7 (GraphPad
50
51 164 Software Inc., La Jolla, CA, USA). The significance of the difference between the controls
52
53
54 165 and treated groups was determined using one-way and two analyses of variance (ANOVA. p<
55
56 166 0.05) followed by the Dunnett multiple comparison test.
57
58
59
167
60
61
62 7
63
64
65
 168 3.0. Results and Discussion
1
2 169 3.1. Phytochemical screening of HV
3
4
5
6
170 Phytochemical screening of the methanolic leaf extract of HV revealed the presence of
7
8 171 alkaloids (++), saponins (+++), cardiac glycosides (+++), tannins (+++), flavonoids (+++),
9
10 172 anthraquinones (+++) and terpenoids (+++).
11
12
13
14 173 3.2 Acute toxicity
15
16 174 No death or any behavourial changes were observed when 5, 50, 500, and 2000 mg/kg of
17
18 175 methanolic extract of HV were orally administered to mice. Also, there were no effects on the
19
20
21 176 skin, gastrointestinal, sensory and nervous systems of the animals. Similarly, no mortality or
22
23 177 acute toxicity signs were observed when the extract was administered intraperitoneally. This
24
25
26 178 indicated that the LD50 of the extract is greater than 2000 mg/kg and hence non toxic when
27
28 179 administered orally and intraperitoneally and that the doses used in the study were safe.
29
30
31 180
32
33 181
34
35
36
182
37
38 183
39
40 184
41
42
43 185
44
45 186
46
47
48 187
49
50 188
51
52
53 189
54
55 190
56
57
191
58
59
60 192
61
62 8
63
64
65
 193 Effect of HV treatment on blood glucose level in normoglycaemic rats.
1
2
3 NC H V 50 H V 150 H V 300 G LI 10
4
5
6
7
8 100
B l o o d g l u c o s e l e v e l ( m g /d L )

9 b c
c
10 a
11 80 a b
a
a
12
b
13
14 60 a
a
a a
15
a a
16
17 40
18
19
20 20
21
22
23 0
24 0 7 14 21
25
26 T im e ( D a y s )
27
28 194
29
30 195 Fig. 1: Dose related effect of HV leaf extract on normoglycaemic rats. Values are given as
31
32 196 mean ± SEM for each group of five rats NC: Negative control; GLI (10 mg/kg):
33
34
35 197 Glibenclamide (positive control), HV 50-300: 50, 150 and 300 mg/kg of Hippocratea
36
37 198 velutina extract, values with different superscripts are significantly different (p < 0.05) while
38
39
40 199 values with similar superscripts are comparable (p > 0.05).
41
42 200
43
44
45 201 The blood glucose levels of the negative control group of rats that were given distilled water
46
47
48 202 remained unchanged throughout the period of the experiment. The extract on day 7 gave a
49
50 203 comparable (p>0.05) hypoglycaemic effect to glibenclamide (10 mg/kg) at the tested doses.
51
52
53 204 However, 150 and 300 mg/kg of the extract gave a time dependent and comparable (p > 0.05)
54
55 205 hypoglycaemic effect to glibenclamide on days 14 and 21. This indicated higher
56
57
206 hypoglycaemic activity of the extract at elevated doses and suggested that the extract may not
58
59
60 207 be safe for human consumption at high concentrations (Fig 1). This observation therefore
61
62 9
63
64
65
 208 called for caution in the use of the plant by human subjects. Similarly, the possibility of
1
2 209 Eugenia uniflora leaf to cause hypoglycaemia at higher doses has been reported (Adebajo et
3
4
5 210 al., 2013).
6
7
8 211 Effect of the extract in streptozotocin-induced diabetic rats
9
10
11
12 NC DU H V 50 H V 150 H V 300 G li 1 0
13
14
500
B l o o d g l u c o s e l e v e l ( m g /d L )

15
16
17 400
18
19
c
20 300 b
b
21
22
23 200 c
ba
24 a a
b a
25 aa a a a
26 100 a a a

27
28 0
29
30 0 7 14 21 28
31
32 T im e ( D a y s )
33 212
34
35
36 213 Fig. 2: Dose related effect of the HV leaf extract in streptozotocin-induced diabetic rats.
37
38 214 Values are given as mean ± SEM for each group of five rats NC: Non diabetic rats (Negative
39
40
41 215 control); DU: Diabetic untreated rats; GLI (10 mg/kg): Glibenclamide (positive control),
42
43 216 HV 50-300: 50, 150 and 300 mg/kg of Hippocratea velutina extract, values with different
44
45
46 217 superscripts are significantly different (p < 0.05) while values with similar superscripts are
47
48 218 comparable (p > 0.05).
49
50
51 219 The blood glucose level of the non-diabetic negative control group of rats that received
52
53
54 220 distilled water remained normal throughout the period of the experiment. Also, the diabetic
55
56 221 untreated rats were constantly hyperglycaemic for the 28 days of treatment with distilled
57
58
59
222 water which indicated that the hyperglycaemia observed in this group was due to the injected
60
61
62 10
63
64
65
 223 streptozotocin and was permanent (Fig 2). The extract elicited a non-dose dependent
1
2 224 antihyperglycaemic effect on day 7 of the study at the tested doses of 50, 150 and 300 mg/kg
3
4
5 225 that was significantly higher than that of glibenclamide (10 mg/kg). On day 14 however, 50
6
7 226 and 150 mg/kg of the extract was significantly more active than the 300 mg/kg dose and the
8
9
10 227 positive control. On days 21 and 28, the activity of the extract at all the doses and
11
12 228 glibenclamide were comparable. This showed the effectiveness of the extract at lower doses
13
14
15 229 while increasing the dose did not lead to significant increase in activity (Fig 2). The extracts
16
17 230 of Senecio biafrae, Terminalia superba, Caesalpinia pulcherrima and Entandrophragma
18
19
231 cylindricum have been reported for their significant antidiabetic effects on drug-induced
20
21
22 232 diabetes in rats (Ayoola et al., 2019, Oluwarotimi et al., 2019, Faloye et al., 2020, Oladoja et
23
24 233 al., 2021).
25
26
27
234
28
29
30
31 235
32
33
34 236
35
36
37 237
38
39
40 238
41
42
43 239
44
45
46 240
47
48
49
241
50
51
52
53 242
54
55
56 243
57
58
59 244
60
61
62 11
63
64
65
 245 Table 1: Effect of HV leaf extract of on hematological parameters
1
2
3 Blood Control (DW) HV (200 mg/kg) HV (400 mg/kg) HV (800 mg/kg)
4
5
6 paramete
7
8 rs Day 0 Day 90 Day 0 Day 90 Day 0 Day 90 Day 0 Day 90
9
10
11 RBC 6.50 6.42 6.91 7.02 6.52 7.10 6.52 6.50
12 ± ±0.16a + ±0.51a
- 0.21 - 0.14 + +
13 (106/µL) + 0.12
- ±0.6a - 0.24 - 0.5a
14
15
16
WBC +
8.8 - 8.0 8.7 14.8 8.9 15.4 9.1 16.8
17 + + b + + b
18 (103/µL) 0.8 + 0.1a - 1.4 - 0.2 - 1.9 - 0.4 +
- 2.1 +
- 0.6
b

19
20 PCV (%)
21
+
42.2 - 42.6 42.6 48.2 42.4 46.4 42.4 43.1
3.0 ±2.0a 1-+ + b
22 - 2.0 ±1.2 + b +
- 3.6 +
- 3.4a
23 3.0 - 2.0
24 HGB 13.0 13.0
25
13.8 14.4 ±0.4a 14.1 14.2 14.4 14.0
26 +
- 0.2 +
- 0.3a +
- 0.8 ±0.8 ±0.8a ±0.4
(g/dL) ± 0.6a
27
28
29 MCV(fl) 67.8 69.6
30 66.9 69.6 69.1 78.4 69.2 79.8±4.2b
31 ± 1.3 ±2.0a ±1.8 ±3.1b ±0.2
+
- 0.2 ±1.4a
32
33 MCH (pg) 22.1 21.5 22.4 19.6 22.8 19.3 22.7 19.8
34
+ ±0.8a ±0.1 ±0.4a ±1.2a
35 +
- 0.4 +
- 0.4
a - 0.8 ±0.4
36 MCHC(g/
37 29.7 29.8 28.8 27.6 29.1 29.8 29.2 29.6
± 1.8 ±1.0a + +
- 0.4 ±08 a
38
dl) ± 1.1 ±0.2a - 1.6 ±1.4a
39
40
41 246 Data showed the mean ± SEM haematological parameters at the different doses, n=8. Results
42
43
44 247 having separate superscripts within row are significantly different (p < 0.05), while those that
45
46 248 are alike are comparable (p > 0.05). DW: Distilled Water; HV (200, 400, 800): Extract of
47
48
49 249 Hippocratea velutina; WBC: White Blood Corpuscles, RBC: Red Blood Corpuscles, HGB:
50
51 250 Haemoglobin, MCV: Mean Corpuscular Volume, MCH: Mean Corpuscular Haemoglobin,
52
53
251 MCHC: Mean Corpuscular Haemoglobin Concentration, PCV: Packed cell volume.
54
55
56
57 252 Hematological parameters are useful indices that can be employed to assess the toxic
58
59 253 potentials of plant extracts in living systems (Sunmonu and Oloyede 2010). They can also be
60
61
62 12
63
64
65
 254 used to explain blood relating functions of chemical compound/plant extract. Such laboratory
1
2 255 investigations have been reported to be highly sensitive, accurate, and reliable and it remains
3
4
5 256 the bedrock of ethical and rational research, disease diagnosis, prevention and treatmen
6
7 257 (Okokon et al., 2004) The various doses of HV extract in this study significantly increased
8
9
10 258 WBC, PVC and MCV levels of the rats’ blood. This result reflected leucopoetic and possible
11
12 259 immunomodulatory effects of the extract (Bashir et al., 2015). This will possibly enhance the
13
14
15 260 ability of the animals to produce antibodies in the process of phagocytocis with higher degree
16
17 261 of resistance to infections (Okunlola et al., 2015). Maintenance of Red Blood Cells,
18
19
262 Haemoglobin, Packed cell Volume, ` Mean Corpuscular Haemoglobin and Mean Corpuscular
20
21
22 263 Haemoglobin Concentration levels by the extract during the period of the experiment also
23
24 264 suggested that the extract had some positive effect on the haemopoietic system of the test rats
25
26
27 265 (Table 1). Extracts of Mangifera indica stem bark and Telfaria occidentalis had been reported
28
29 266 to show similar effects on the haematological components in rats (Nwinuka et al., 2008,
30
31
32 267 Shittu, 2015).
33
34
35 268
36
37
38 269
39
40
41 270
42
43
44 271
45
46
47 272
48
49
50
273
51
52
53
54 274
55
56
57 275
58
59
60 276
61
62 13
63
64
65
 277 Table 2: Effect of HV leaf extract of on differential blood count
1
2
3Blood Control HV (200 mg/kg) HV (400 mg/kg) HV (800 mg/kg)
4
5
6parameters Distilled water
7
8
9(103/mm3)
10
Day 0 Day 90 Day 0 Day 90 Day 0 Day 90 Day 0 Day 90
11
12
13Lymphocyts 60.2 + 0.3 61.0 60.1 62.6 60.6 66.1 60.1 66.4 ± 0.6b
14 ±1.0 a +0.1 + 1.9 a
15 - + 0.4 + 0.8b ±0.2
- -
16Neutrophils
17 20.6 + 0.4 22.0 22.2 26.9 22.8 28.1 + 28.0
23.6 -
18 ±0.4 a
± 1.4 ±0.1
b
±0.6b 0.4 b
± 0.9 +
- 0.2
19
20Eosinophils 3.0 + 0.6 + 0.5a
3.4 - + 0.2 4.8 -+ 0.4a + + 0.2a 4.8 - + + 0.4a
5.0 -
- 4.8 - 4.6 - 4.6 -
21
22 0.3 0.8
23Monocytes
24 + 0.4
3.0 - + ±0.00a 3.6 -
3.2- + 0.5 4.2- + 0.4a +
3.4 - + 0.2a 3.6 -
3.9- + + 0.8a
3.8-
25 0.6 0.2
26
27Basophils + 0.02 1.0 -
1.01 - + 0.0a + 0.0a
+ 0.1 1.0 -
1.00 - 1.00 -+ 1.0 - + 0.0a 1.00 - + 1.0 - + 0.1a
28 0.00
29 0.1
30 278 Data shows the mean ± SEM haematological parameters at the different doses, n=8. Results
31
32
279 having separate superscripts within row are significantly different (p < 0.05), while those that
33
34
35 280 are alike are comparable (p > 0.05).
36
37 281 In the differential blood count experiment, the various doses of the extract did not affect the
38
39
40 282 eosinophils, monocytes and basophils level similar to the negative control. However, 400 and 800
41
42 283 mg/kg of the extract significantly increased the lymphocyte level while all the tested doses
43
44
45 284 significantly caused an increase in the neutrophils level. This indicated that the extract had the
46
47 285 ability to cause lymphocytosis. Similar observation had been made with the aqueous extract of
48
49
50
286 Mangifera indica stem bark (Nwinuka et al., 2008).
51
52
53 287
54
55
56 288
57
58
59 289
60
61
62 14
63
64
65
 290 Table 3. Effect of HV leaf extract on biochemical parameters
1
2
3 Parameters Control (DW) HV 200 mg/kg HV 400 mg/kg HV 800 mg/kg
4
5 Creatinine + 4.00a
42.50 - 4.20a
43.10 - + 5.12a
43.80 - + 1.00a
44.10 -
6 kinase
7 Aspartate
8 + 3.06a
26.20 - + 1.02a
26.92 - + 5.42a
27.40 - + 2.62a
27.48 -
transaminase
9
10 Alanine + 4.42a
24.10 - + 3.04a
23.30 - + 9.56a
22.41 - + 2.62a
22.10 -
11 transaminase
12
13
Bilirubin + 2.20a
1.62 - + 2.40a
1.73 - + 0.94a
1.79 - + 0.49a
1.83 -
14
15 + 0.12a + 0.10a + 0.03a + 0.02a
16 Total protein 4.63 - 4.60 - 4.58 - 4.68 -
17
18 Urea + 5.42a
56.19 - + 4.88a
58.72 - + 3.20a
57.12 - + 1.00a
58.11 -
19
20 Creatinine + 0.01a
0.31 - + 0.02a
0.32 - + 0.04a
0.31 - + 0.03a
0.33 -
21 291 Data showed the mean ± SEM haematological parameters at the different doses, n=8. Results
22
23
24 292 having separate superscripts within row are significantly different (p < 0.05), while those that
25
26 293 are alike are comparable (p > 0.05). DW: Distilled Water; HV (200, 400, 800): Extract of
27
28
29 294 Hippocratea velutina.
30
31 295 The result of this study showed that there was no significant difference in all the important
32
33
34 296 biochemical components investigated compared to the negative control. The AST, ALP,
35
36 297 bilirubin, creatinine kinase, total protein, urea and creatinine levels were not affected by all
37
38
298 the tested doses of the extract (Table 3). This result indicated safety of the extract on the
39
40
41 299 heart, liver and kidney of the rats. High AST and ALT levels have been associated with liver
42
43 300 diseases or hepatotoxicity (Brautbar and Williams, 2002; Desai et al., 2012).
44
45
46 301
47
48 302
49
50
51 303
52
53 304
54
55
56
305
57
58 306
59
60
61
62 15
63
64
65
 307 Table 4: Effect of HV leaf extract on lipid profile
1
2
3 Biochemical Control HV 200 mg/kg HV 400 mg/kg HV 800 mg/kg
4 parameters (DW)
5 (mg/dl)
6
TC a 64.33 + 3.99a a a
7 66.10 +
- 4.17 - 64.00 +
- 3.68 63.96+- 1.28
8
9 TG (mg/dl) 16.10 - a
+ 1.21 10.23 - + 1.36b 10.17 -+ 1.32b + 2.05b
9.73 -
10 a
11 VLDL-C 3.00 +
- 0.15 + 0.13a
2.21 - 2.14 +
- 0.12
a 2.04 +
- 0.23
a
12
13
LDL-C - 4.17a 28.81 -
30.45 + + 4.44a + 4.18a
27.89 - 29.14 +
- 2.02
a
14 HDL-C
15 - 1.14a 24.13 +
23.15 + - 1.05
a 23.32 +
- 0.86
a
22.91 +
- 0.68
a
16
17
18 308 Data showed the mean ± SEM biochemical parameters at the different doses, n=8. Results
19
20 309 having separate superscripts within row are significantly different (p < 0.050), while those
21
22
23 310 that are alike are comparable (p > 0.05). DW: Distilled Water; HV (200, 400, 800): Extract of
24
25 311 Hippocratea velutina; TC: Total cholesterol, TG: Triglycerides, VLDL-C: Very Low-
26
27
28 312 density lipoprotein, LDL-C: Low density lipoprotein Cholesterol and HDL-C: High-density
29
30 313 lipoprotein – Cholesterol.
31
32
33 314 The extract gave a comparable (p>0.01) effect with the negative control on total cholesterol,
34
35 315 very low-density lipoprotein, low density lipoprotein – cholesterol and high-density
36
37 316 lipoprotein – cholesterol at all its tested concentrations. This showed that the extract lacked
38
39
40 317 appreciable hypolipidemic effect. However, it significantly reduced triglyceride level at 200-
41
42 318 800 mg/kg suggesting a possible triglyceride lowering effect of the extract (Table 4).
43
44
45 319
46
47
48 320
49
50
51 321
52
53
54 322
55
56
57 323
58
59
60
61
62 16
63
64
65
 324 Histopathological studies
1
2
3
4
5
6
7
8
9
10 A
B
11
12
13
14
15
16
17
18
19 325 C D
20
21
326 Plate 1: Photomicrograph of the histology of the liver. A: Control group, B-D: Treatment
22
23
24 327 groups with 200, 400 and 800 mg/kg of HV extract, respectively.
25
26
27 328 The liver of the control group of animals showed normal and well differentiated histological
28
29
329 structure of the central vein (black thick arrow), hepatocytes (black circle), sinusoids (black
30
31
32 330 thin arrow) and well-arranged hepatic plates. The liver histology of the rats that received 200
33
34 331 and 400 mg/kg HV extract showed normal preservation of hepatic architecture, slight
35
36
37 332 pyknotic cells (black arrow head), central vein (black thick arrow), hepatocytes (red circle )
38
39 333 and thin fibrotic septa. However, that of the rats that were treated with 800 mg/kg showed
40
41
42 334 enlarged central vein (blue thick arrow) with inflammatory cells, pyknotic cells (green arrow
43
44 335 head) and steatosis were observed around the hepatocytes (black circle). This suggested
45
46
47
336 possible inflammation of the liver at this dose (Plate 1).
48
49
50 337
51
52
53 338
54
55
56 339
57
58
59
60
61
62 17
63
64
65
 1
2
3
4
5
6
7 B
A
8 A
9
10
11
12
13
14
15
16
17 340 C D
18
19
20 341 Plate 2: Photomicrograph of the histology of the brain. A: Control group, B-D: Treatment
21
22
342 groups with 200, 400 and 800 mg/kg of HV extract, respectively.
23
24
25
26 343 The brain of the control group of rats had normal and well differentiated histological structure
27
28 344 of the granular layer (black thick arrow), the molecular layer (black arrow head) and the
29
30
345 neurons (black circle) are intact. The extract at 200 mg/kg preserved the brain archtitecture,
31
32
33 346 granulayer layer (black thick arrow), molecular layer (blue arrow head) and neurons but there
34
35 347 was slight distortion of the purkinje layer (black thin arrow) without any loss of functions. At
36
37
38 348 400 and 800 mg/kg however, there was degeneration of the brain structures (Plate 2). This
39
40 349 called for caution in the use of the plant at high doses.
41
42
43 350
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 18
63
64
65
 1 PCT
2
3
4
5
6
7 DCT
8 PCT
9
DCT
10 A B
11
12 DCT
13 DCT
14
15
16
17
18
19
20
PCT PCT
21
22 C D
23 351
24
25
26
352 Plate 3: Photomicrograph of the histology of the kidney. A: Control group, B-D: Treatment
27
28 353 groups with 200, 400 and 800 mg/kg of HV extract, respectively.
29
30
31 354 Renal histology of the control group of animals showed well differentiated and normal renal
32
33
34
355 architecture, the capsular space (black thin arrow), glomerular layer (black thick arrow),
35
36 356 proximal and distal tubules (PCT & DCT) were intact. All the renal structures were without
37
38 357 any loss of function in the group of rats treated with 200 mg/kg of HV. There were
39
40
41 358 constricted proximal and distal tubules, slight degeneration of the glomerular tuft (blue thick
42
43 359 arrow) with intact capsular spaces (black thin arrow) in the 400 mg/kg group of rats while
44
45
46 360 800 mg/kg of the extract caused severe degeneration of the glomerular tuft (red thick arrow),
47
48 361 constricted proximal and distal tubules with constricted capsular space(black thin arrow).
49
50
51 362 This also indicated possible toxicity of the extract to the kidney at high doses (Plate 3).
52
53
54
55
56
57
58
59
60
61
62 19
63
64
65
 1
2
3
4
5
6
7
8 A B

9
10
11
12
13
14
15
16
17
C D
18 363
19
20
21 364 Plate 4: Photomicrograph of the histology of the pancreas. A: Control group, B-D: Treatment
22
23 365 groups with 200, 400 and 800 mg/kg of HV extract, respectively.
24
25
26 366 Pancreatic histology of the controlled group of rats showed well differentiated and normal
27
28
29 367 pancreatic architecture, the pancreatic islets (black thick arrow), pancreatic acini (blue thin
30
31 368 arrow), and the interlobular connective tissue (black thin arrow) were all intact. The 200
32
33
34 369 mg/kg extract showed no significant histological changes in all the pancreatic features. While
35
36 370 400 mg/kg of the extract gave slight degeneration of the pancreatic islets (red thick arrow),
37
38
39
371 pancreatic acini (blue thin arrow) and the interlobular connective tissue (black thin arrow),
40
41 372 the effect of 800 mg/kg was severe (Plate 3). This also indicated lethal effect of the extract on
42
43 373 the pancreas at high doses.
44
45
46
47 374 4. Conclusion
48
49 375 The results obtained from this study revealed that the methanolic leaf extract of Hippocratea
50
51 376 velutina is effective as an antidiabetic agent without toxic effects on various haematological
52
53
54 377 and biochemical parameters of normal animal blood samples as well as on histology of
55
56 378 different body organs especially at low doses. It therefore justified its use in the management
57
58
59 379 of diabetes mellitus among Yoruba people in Nigeria.
60
61
62 20
63
64
65
 380 ACKNOWLEDGMENTS
1
2
3 381 The authors would sincerely like to appreciate the effort and guidance of Prof Ahmed
4
5
6
382 Adedeji for his unparalleled support towards the success of the research.
7
8
9 383 FUNDING
10
11
12 384 This research did not receive any specific grant from funding agencies in the public,
13
14 385 commercial or not-for-profit sectors.
15
16
17 386 AUTHORS CONTRIBUTIONS
18
19
20
21 387 FO, EI, MDA, OK, OS and OB planned the experimental procedure and collected the data
22
23 388 needed for the study. FO, MA, OE, and MDA performed the data analysis. Finally, FO, EI,
24
25 389 MDA and OK composed the article. All the contributors thoroughly read and approved the
26
27
28 390 final manuscript.
29
30
31 391 Conflict of interests
32
33
34 392 The authors declare that they have no known competing financial interests or personal
35
36
37 393 relationships that could have appeared to influence the work reported in this paper
38
39
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466 Oladoja, Farouk, Emmanuel Irokosu, Oluwafemi Kale, and Taiwo Olubodun-Obadun. 2021.
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Conflict of Interest

Conflict of interests

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper

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