Available online on 15.12.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                Research Article

Effect of Extract (Interface) from Stem Bark of Antidiabetic Anogeissus leiocarpus (African Birch Tree) on Random Blood Glucose Levels of Adult Female Wistar Rats: Optimisation for Therapeutic Hypoglycaemic Dose

Lauretta Oghenekevwe Esievo ^1,2, Dahiru Sani ³, Kingsley Oghenerukevwe Esievo ⁴, Edith Monica Esievo ^5,6, Emmanuel Oluwadare Balogun ^7,8, Ochuko Orakpoghenor ⁹, Peter Ibrahim Rekwot ^1,10, Lushaikyaa Allam ^1,11 King Akpofure Nelson Esievo ^12,13,14+

1. Department of Theriogenology and Production, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria.
2. Department of Theriogenology, College of Veterinary Medicine, Federal University of Agriculture, Zuru, Nigeria.
3. Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria.
4. Department of Agronomy, Faculty of Agriculture, Ahmadu Bello University, Zaria, Nigeria.
5. Department of Veterinary Surgery, College of Veterinary Medicine, Federal University of Agriculture, Zuru, Nigeria.
6. Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, United Kingdom.
7. Department of Biochemistry, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Nigeria.
8. African Centre of Excellence for Neglected Tropical Diseases and Forensic Biotechnology (ACENTDFB), Ahmadu Bello University, Zaria, Nigeria.
9. Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
10. Artificial Insemination Unit, National Animal Production Research Institute, Ahmadu Bello University, Shika, Zaria, Nigeria.
11. Veterinary Teaching Hospital, Ahmadu Bello University, Zaria, Nigeria.
12. Department of Veterinary Pathology, College of Veterinary Medicine, Federal University of Agriculture, Zuru, Nigeria.
13. Lecturer/Resource Person, College of Veterinary Surgeons, Nigeria, Pathology Faculty, Samaru, Zaria Study Centre, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria.
14. Consultant, Research and Diagnosis Unit, Kanesco Global Services Limited (RC 829505), Hayin Malam, Samaru, Zaria, Nigeria.

 

INTRODUCTION

Worldwide increases in type 2 diabetes mellitus (T2DM) without and with microvascular and macrovascular complications of nephropathy and retinopathy implied that DM may become a leading cause of death, by WHO, come 2030 but a worse situation was projected for 2035¹, thus creating challenging unresolved health concerns for the 21^st century². This has impacted on the global economy and the national budgets of numerous countries worldwide; the direct consequences of the risk factors induced by DM on the progression, prognosis and mortality of COVID-19, obesity, as a predisposing factor of DM and delayed wound healing or diabetic ulcers that led to amputation of limbs with their accompanying social menace had been highlighted earlier^3,4,5,6,7. 

Several reports^2,8,9,10,11 emphasized on the benefits of medicinal plants and their phytochemical constituents for the treatment of diabetes mellitus; this fits the situation in countries with reduced economic growth, which influenced traditional methods for the treatment of diabetes mellitus.

From numerous studies^{3,4,5,6,7,12,13} one medicinal plant, Anogeissus leiocarpus stand out. Crude ethanolic extracts of A. leiocarpus stem bark modulated sialic acids of plasma glycoproteins and red blood cells and revealed elevated serum sialic acids as a potent biomarker, predictive and prognostic in alloxan-induced diabetic dogs³. In addition, the crude ethanolic extracts ameliorated hyperglycaemia, hepato-renal damages, deranged electrolytes, acid-base balance and enhanced haematopoiesis in alloxan-induced diabetic dogs, further exhibiting a promise to prevent progression to type 2 diabetes mellitus since there was no reversal to hyperglycaemic state following withdrawal of administration of the extracts⁴.

The antioxidant activities coupled with the antidiabetic properties of the crude ethanolic extract of A. leiocarpus in alloxan-induced diabetic Wistar rats⁵ provided a boost to the plants efficacy in the treatment of DM. in addition, the dyslipidaemia produced in alloxan-induced diabetic dogs was attenuated by crude ethanolic extracts of A. leiocarpus stem bark⁶. The latter accelerated healing of surgically-induced deep skin wounds in alloxan-induced diabetic dogs⁷ showing landmarks of enhanced inflammatory responses of healing processes⁷. The guill and perr leaf of A. leiocarpus were effective on the hyperglycaemia and the associated dyslipidaemia in alloxan-induced diabetic rats¹² while total extract and fractions exhibited antihyperglycaemic activity in mice¹³. The attenuation of dyslipidaemia on alloxan-induced diabetic dogs⁶ and rats¹² by crude ethanolic extracts of A. leiocarpus is very important, laudable and requires more attention, since different classes of glucose lowering drugs, such as sulfonyl urea and meglitnides biguanides and thiazoliniediones^14,15 controlled the hyperglycaemia but did not effectively control the hyperlipidaemia associated with the DM, apart from the toxicity and resistance some patients experienced¹⁶ and the exorbitant costs^17,18 for developing countries.

Due to the above-listed endowed medicinal activities and properties of A. leiocarpus, under the context of a non-conventional treatment for DM, the ethanolic extract of its stem bark was purified to the point of crystallization and four components, an interface and three fractions were produced¹⁹ and the interface had a much higher proportion than the three other fractions¹⁹. Bioassay and toxicity studies with the interface in Wistar rats at a limit dose of 2000 mg/kg.bd.wt. caused no death within 24hours, no hepato-renal damages and pregnancy, gestation, parturition and reproductive performance were normal, with no teratogenic effects on pups¹⁹.

It became imperative to have an insight into the effect of the antidiabetic compound of extract (interface) of A. leiocarpus stem bark on the ovarian functions of adult female Wistar rats and this aspect is strategically designed to investigate an appropriate and hence the optimization for therapeutic hypoglycaemic dose.

MATERIALS AND METHODS

Anogeissus leiocarpus Stem Bark Harvest and All Purification Processes:

The harvest of stem bark from A. leiocarpus; its authentication; the fertility assessment of the tree grown soils, the ethanolic extractions, along with the qualitative and quantitative phytochemical screenings; partitioning of the ethanolic extracts into fractions and the final purification processes with column chromatography and thin layer chromatography were adequately described with details¹⁹. The bioassay and toxicity studies, using the interface component of the four different purified components were performed¹⁹. The yields of the interface and the other three fractions in grams were reported earlier. 

All purified extracts were stored in sample bottles, at room temperature (plate 1) and are reconstituted freshly when required. Storage of the purified A. leiocarpus stem bark extracts commenced on June, 2022.

 

 

 

Plate 1: Sample bottles that contained purified extracts of interface and fractions A, B and C

 

 

Constitution of purified extract

Five hundred (500) mg. of the extract, interface was dissolved in 1ml of distilled water to produce a concentration of 500 mg/ml.

Experimental Animals and experimental design

Twenty (20) adult female Wistar rats that weighed between 155 g and 235 g commenced the experiment on day 0. They were separated, at random, into 4 groups (cages) of 5 female rats each. Based on the major objective of the study, which is the effect of the antidiabetic compound of A. leiocarpus stem bark on ovarian functions, an adult male Wistar rat was placed into each of the 4 groups (cages), on day 0 and labelled groups A, B, C and D.

Group A (n=5): Served as control and received normal saline daily for 12 days.

Group B (n=5): Received oral administration of constituted extract, interface at a dose of 5 mg./kg.bd.wt. daily for 12 days.

Group C (n=5): Received oral administration of constituted extract, interface at a dose of 10 mg./kg.bd.wt. daily for 12 days.

Group D (n=5): This group received oral administration of constituted extract, interface at a dose of 20 mg/kg.bd.wt. daily for 12 days.

This design was aimed at investigating an appropriate, hence optimization for therapeutic hypoglycaemic dose for the antidiabetic A. leiocarpus and the choice of 12 days was due to the short oestrous cycle of the rats.

All the rats were fed commercial feeds (superstarter chow) and water was supplied ad. libitum.

Exclusion Criteria:

Exclusion criteria were applied to groups B (5 mg/kg.bd.wt.) and C (10 mg/kg.bd.wt.) which reduced the Wistar rats in groups B and C to 4 adult females each. (These are addressed under Results of this manuscript).

In group B, one female Wistar rat exhibited naturally acquired type 2 diabetes mellitus, noticeable on days 0 and 1. The exciting management of this naturally occurring type 2 diabetes mellitus in a female Wistar rat, as a case report, is part of another manuscript.

For group C, a female Wistar rat died from an unusual and unexpected cage related accident, three days after the commencement of the study.

Oral Administration of extract, interface of A. leiocarpus stem bark:

This was performed using 18G cannula oral gavage.

 

 

Samples Collections:

RBG:

On day 0, blood samples were collected from all female rats, through the ocular vein and baseline RBG levels were measured by placing a drop of blood directly onto an Accu chek® test strip inserted into the portable glucometer (Accu chek® Active, Roche, Roche Diabetes Care, Middle East, FZCO).

Thereafter all female rats were orally administered the extract, interface at the optimized dose corresponding with the designated group (as enumerated above) 5, 10 or 20 mg/kg.bd.wt. using 18G cannula oral gavage. Blood sample collections for RBG continued daily for the 12 days and to avoid traumatizing the ocular vein, blood samples were alternatively collected, that is, every other day, by cutting the rats tail tips.

Hormonal Assays:

Oestrogen and Progesterone:

Blood samples for the assay of oestrogen and progesterone were collected every other day post (daily) administration of the extract, by cutting the rats tail tips. Oestrogen and progesterone were measured using the human ELIZA kits.

Vaginal Cytology:

Vaginal swabs were collected every other day post (daily) administration of the extract, with the use of cotton balls.

Body Weights: The body weights of all female rats were measured in grams, at intervals, during the 12 day study.

Statistical analysis: The results obtained from this study were presented in charts and tables of means ± standard error of means (SEM). The data were subjected to ANOVA using the GraphPad Prism® statistical package with Tukey’s post-hoc tests to analyze the difference between groups. Values of P<0.05 were considered significant.

RESULTS

Random Blood Glucose (RBG)

On day 0 the mean ± SD of the RBG levels showed no significant (P>0.05) difference in all groups, but the values were nonsignificantly higher in groups B and D compared to groups A and C.

At day 1, the mean ± SD of the RBG values were significantly (P<0.05) increased in group B than in groups A, C and D.

From day 2 upto day 12, the mean ± SD values of RBG significantly (P<0.05) decreased in group B; non-significant fluctuations occurred in all other groups. (Table 1 and Fig. 1)

Random Blood Glucose (RBG) values (mg/dl.) of adult female Wistar rats of control and groups administered the optimized doses of 5, 10 and 20 mg/kg.bd.wt. of extract (Interface) of A. leiocarpus stem bark

 

 

 

Table 1: Mean ± SD glucose level

Dates (and Day)	Group A Control	Group B 5 mg/kg	Group C 10 mg/kg	Group D 20 mg/kg
11/09/23 (0)	68.8 ± 14.0	89.5 ± 5.21	77.8 ± 8.3	83.2 ± 7.6
12/09/23 (1)	99.0 ± 13.2a	129.5 ± 57.7b,2	101.8 ± 3.2a	102.0 ± 11.9a
13/09/23 (2)	76.8 ± 8.7	81.5 ± 5.81	84.0 ± 5.4	90.4 ± 15.0
14/09/23 (3)	87.2 ± 6.8	85.5 ± 6.11	91.8 ± 11.5	92.2 ± 8.3
15/09/23 (4)	98.6 ± 12.5	91.0 ± 14.31	76.3 ± 9.8	90.8 ± 23.8
16/09/23 (5)	90.4 ± 3.8	86.8 ± 5.11	90.3. ± 12.8	86.6 ± 11.7
17/09/23 (6)	91.8 ± 16.1	83.0 ± 2.51	84.5 ± 7.6	88.8 ± 17.0
18/09/23 (7)	93.8 ± 13.5	100.0 ± 5.41	94.0 ± 9.8	101.0 ± 7.7
19/09/23 (8)	90.4 ± 14.5	86.3 ± 11.21	111.3 ± 4.8	106.2 ± 18.7
20/09/23 (9)	103.6 ± 17.8	115.8 ± 10.81	107.3 ± 12.5	106.2 ± 13.0
21/09/23 (10)	95.8 ± 13.9	107.8 ± 11.91	100.8 ± 11.8	106.2 ± 20.5
22/09/23 (11)	93.2 ± 15.9	99.3 ± 8.31	99.0 ± 18.6	103.2 ± 11.3
23/09/23 (12)	86.6 ± 10.0	98.3 ± 15.61	73.3 ± 22.2	87.0 ± 10.2

Mean ± SD values with different superscript alphabets in the same row differ significantly at P < 0.05; values with different superscript numbers in the same column differ significantly at P < 0.05

Figure 1: Line graph showing the pattern of changes in glucose levels

The variations of the RBG values of all the adult female Wistar rats under the study are presented in Table 2.

 

Table 2: Variations of random blood glucose levels (mg/dl) of all adult female Wistar rats under study.

Table 2a Group A: Control (n=5)

Table 2b Group B: Administered 5mg/kg.bd.wt. (n=4)

 

Table 2c Group C: Administered 10mg/kg.bd.wt. (n=4)

 

Table 2d Group D: Administered 20mg/kg.bd.wt. (n=5)

 

Body Weight 

Body weights increased in groups A, B and C, but decreased in group D, from day 0 to day 2. Further increases in body weights occurred in groups A and C as against group B which experienced further decreases up to day 14. Group D experienced increased body weights at day 7 and then decreased at day 14. (Table 3 and Fig. 2). The variations in body weights of all adult female Wistar rats are in Table 4.

 

 

Table 3: Mean ± SD body weight in gram

Days	0	2	7	14
Group A - Control	209.6 ± 8.3	212.4 ± 15.1	217.2 ± 16.1	217.8 ± 12.9
Group B - 5 mg/kg	215.0 ± 14.7	223.8 ± 14.2	216.0 ± 21.1	210.8 ± 19.2
Group C - 10 mg/kg	178.5 ± 15.7	183.0 ± 12.7	193.0 ± 14.0	197.3 ± 17.2
Group D - 20 mg/kg	174.2 ± 15.8	164.8 ± 21.5	167.8 ± 10.9	165.2 ± 2.6

 

Figure 2: Chart showing the changes in body weights

 

Table 4: Body weight changes (gm) of adult female Wistar rats of control and groups administered the optimized doses of 5, 10 and 20mg/kg.bd.wt. of extract (interface) of A. leiocarpus stem bark

Table 4a = Group A: Control (n=5)

Dates (and Days)	11/9/23 (0)	13/9/23 (2)	18/9/23 (7)	25/9/23 (14)
S/No
1a	219	234	240	230
2a	206	200	208	212
3a	209	217	221	224
4a	198	196	197	198
5a	216	215	220	225

 

Table 4b = Group B: Administered 5mg./kg.bd.wt. (n=4)

Dates (and Days)	11/9/23 (0)	13/9/23 (2)	18/9/23 (7)	25/9/23 (14)
S/No
1b	235	245	246	238
3b	216	217	204	209
4b	208	215	215	202
5b	201	218	199	194

 

Table 4c = Group C: Administered 10mg/kg.bd.wt. (n=4)

Dates (and Days)	11/9/23 (0)	13/9/23 (2)	18/9/23 (7)	25/9/23 (14)
S/No
1c	185	189	204	210
3c	188	190	205	214
4c	186	189	187	185
5c	155	164	176	180

 

Table 4d = Group D: Administered 20mg/kg.bd.wt. (n=5)

Dates (and Days)	11/9/23 (0)	13/9/23 (2)	18/9/23 (7)	25/9/23 (14)
S/No
1d	163	154	160	162
2d	169	160	165	168
3d	171	154	169	167
4d	202	203	186	166
5d	166	153	159	163

 

 

Correlation between RBG levels and body weights at days 0, 2 and 7

There was a significant strong positive correlation between RBG levels and body weights at days 0, 2 and 7 in groups A and C. while in group B, a non-significant strong negative correlation existed between the RBG levels and body weights at days 0, 2 and 7. In group D, the correlation was non-significant and moderately negative (Table 5).

Correlation between RBG and body weight mean values of group B was non-significant and strongly negative on days 0, 2 and 7.

Table 5: Correlation between glucose level and body weight at days 0, 2 and 7

	r	P-value
Group A - Control	0.999	0.034*
Group B - 5 mg/kg	-0.770	0.440
Group C - 10 mg/kg	0.997	0.046*
Group D - 20 mg/kg	-0.566	0.617

* Levels of significance 

DISCUSSION

This aspect of the current study has shown that 5mg/kg.bd.wt. of the extract (interface) of A. leiocarpus stem bark significantly reduced mean RBG levels in adult normal female Wistar rats (group B) by day 12 post administration. In addition, it has provided reasons to suggest that the 5 mg/kg.bd.wt. can be therapeutically hypoglycaemic in adult female Wistar rats. Further observation revealed that 10 mg.bd.wt. of the extract (interface) of A. leiocarpus stem bark non-significantly reduced the mean RBG levels in the adult female Wistar rats (group C) by day 12 post administration, when compared to the mean RBG levels of day 1. While a dose dependent glucose reduction response was expected, it is paradoxical that 20 mg/kg.bd.wt. of the extract (interface) of A. leiocarpus stem bark equally produced a non-significant reduction of the mean RBG levels in the adult female Wistar rats (group D) by day 12 post administration; for the latter, (groups C and D) further observation to exceed 12 days did not occur due to the original design of the overall study on ovarian functions. 

Crude ethanolic extract at 1000 mg/kg.bd.wt. reduced blood glucose levels and enhanced haematopoiesis after a 14-day oral administration in adult Wistar rats²⁰; the crude ethanolic extracts contained all the fractions, prior to its purification. The limit dose of 2000 mg/kg.bd.wt. of the extract (interface) produced an overwhelming and a highly significant (P<0.0001) reduction of blood glucose levels in normal adult male and female Wistar rats combined over a 24 hour period¹⁹. In a related study to investigate the molecular basis of the activities of antidiabetic A. leiocarpus, 100 mg/kg.bd.wt. of the extract (interface) reduced blood glucose levels in alloxan-induced diabetic Wistar rats, 3 days after the development of marked hyperglycaemia (part of another manuscript).

In the current study, mean RBG levels of all the groups increased on day 1 post administration of the extract (interface) with the increase in the same group B (5 mg/kg.bd.wt.) being significant when compared with groups A, C and D. The observed increase that occurred in control group A (without extract administration) an increase that is similar to those of groups C and D [administered with the extract (interface)], albeit, non-significant, strongly suggest that the extract oral administration may not be implicated for the increases of the mean RBG values on day 1. It is being suggested that the increased mean RBG values of all groups on day 1 was in part, caused by the unusual and initial handling of these Wistar rats, accompanied by excitement, fright and secretion of epinephrine with expected increases of glucose in the peripheral circulation²¹ of these Wistar rats, until the rats got used to the daily routines of samples collections.

From days 2 to 14, group B rats experienced decreases in mean body weights; indeed a non-significant strong negative correlation existed between the mean RBG levels and body weights.

CONCLUSION

Daily oral administration of a minimal dose, 5 mg/kg.bd.wt of extract (interface) of A. leiocarpus stem bark is therapeutically hypoglycaemic, as it significantly reduced mean RBG levels by day 12. Clinical application of these lower doses is maintenance of normoglycaema, for a while, after “crashing” down the hyperglycaemia of DM, with a much higher therapeutic dose.

The shelf life/expiry date of the extract (interface) of A. leiocarpus stem bark is greater than seventeen (17) months when stored at room temperature.

Acknowledgement

The Authors acknowledge the monumental technical assistance of Dennis Otie and the contribution of Sunusi Musa, Department of Pharmacology and Toxicology, ABU, Zaria

Author’s contribution 

Esievo, KAN: Conceptualisation; Supervision; Investigation; Writing, Editing, 

Esievo, LO: Investigation; Ethanolic Extraction and Purification; Writing. 

Sani, D: Supervision; Investigation; Bioassay; Writing, Editing; 

Esievo, KO: Investigation; Soil composition; Ethanolic Extraction and Purification, Writing. 

Esievo, EM: Investigation; Ethanolic Extraction and   Purification; Writing.

Balogun, EO: Supervision; Investigation; Writing; Editing.

Rekwot, PI: Supervision; Investigation; Writing; Editing.

Allam, L: Supervision; Investigation; Writing; Editing.

Orakpoghenor, O: Investigation; Writing; Editing.

Funding Source

The funding was partly provided by the Research and Diagnosis Unit of Kanesco Global Services Limited (RC 829505) with additional funds from supervising Authors.

Conflict of Interest

The Authors declare that they have no conflict of interest.

Ethical Approval

All applicable international, national and institutional guidelines for the care and use of animals were followed. This article contains studies with animal subjects performed by the authors under an existing ethical approval by the committee on Animal use and care. (Ethical clearance No. ABUCAC/2019/16).

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