







































 

_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: drmichaeloku@gmail.com, michaeloku@unical.edu.ng; 
 
Cite as: M.E, Oku, Akpaso, M.I, Odey, P.A, Eru, E.M, Anani, S.E., and Umoh, N.M. 2024. “Stereological Studies on 
Ameliorative Role of Ethanolic Extracts of Vernonia Amygdalina and Gongronema Latifolium Against Streptozocin- Induced 
Diabetic Splenic Tissue Damage in Wistar Rats”. Asian Journal of Immunology 7 (1):131-48. 
https://journalaji.com/index.php/AJI/article/view/138. 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 131-148, 2024; Article no.AJI.122015 
 

 
 

 

 

Stereological Studies on Ameliorative 
Role of Ethanolic Extracts of Vernonia 

amygdalina and Gongronema latifolium 
against Streptozocin- Induced Diabetic 
Splenic Tissue Damage in Wistar Rats 

 
Oku, M.E a*, Akpaso, M.I a, Odey, P.A a, Eru, E.M a,  

Anani, S.E. a and Umoh, N.M a 

 
a Department of Anatomy, University of Calabar, Nigeria. 

 
Authors’ contributions  

 
This work was carried out in collaboration among all authors. All authors read and approved the final 

manuscript. 
 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2024/v7i1138  
 

Open Peer Review History: 
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,  

peer review comments, different versions of the manuscript, comments of the editors, etc are available here: 
https://www.sdiarticle5.com/review-history/122015  

 
 
 

Received: 18/06/2024 
Accepted: 20/08/2024 
Published: 29/08/2024 

 
 

ABSTRACT 
 

Background:  Diabetes mellitus is a chronic metabolic dysfunction marked with prolonged 
excessive glucose level, with disruptions in the metabolism of starch, lipids, and amino acids due to 
excessive production and/or inadequate utilization of glucose. 
Objective: To assess the protective impact of Vernonia amygdalina (VA) and Gongronema 
latifolium (GL) on splenic cytoarchitechure in STZ induced glycemic disorder rats using quantitative 
and histological methods. 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1138
https://www.sdiarticle5.com/review-history/122015


 
 
 
 

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Materials and Methods: A total of thirty Wistar rats were randomly divided into six groups of five 
rats each, weighing between 150-200g.  The groups were marked as A, B, C, D, E and F. Group A 
[Normal control] received distilled water, B [Diabetic control] received 45mg/kg body weight of 
Streptozocin, C received 100mg/kg of the extract VA only; D received 100mg/kg of GL extract only. 
E received 5mg/kg of Metformin and F received combined 100mg/kg of VA and GL for fourteen 
days. Twenty four hours after the last administration, the animals were sacrificed and the spleen 
was harvested, fixed, processed, stained with H&E and the spleen volume was estimate using 
Cavalieri method and data were analyzed using ANOVA at p<0.05.  
Results: The diabetic group treated with STZ only revealed reduced white pulp and the marginal 
zone and an apparent decrease in the cellular density of the lymphocyte component of the pulp, 
with positive Perl’s stain. The stereological analysis of the spleen showed a significant decrease of 
the splenic pulp volume density and a significant increase in the connective tissue volume density, 
which significantly improved after treatment with the combined extract of VA+GL-treated rats. The 
reduction of splenic pulp was mainly due to the decrease in the volume density of all structural 
components of the white pulp. 
Conclusion: it can be suggested that combined ethanolic extracts of VA+GL when used in 
combination can be used in management of diabetes. 

 

 
Keywords: Diabetes; spleen; Vernonia amygdalina; Gongronema latifolium; STZ. 
 

1. INTRODUCTION  
 

Diabetes is a chronic disorder associated with 
many debilitating multi-systemic complications. 
The spectrum of the diabetic pathology is 
complex and thus would require beyond single 
therapy for management of such complications. 
The more acceptable approach should embrace 
alternatives to orthodox care. Diabetes remains 
a public health concern with an estimated 
463 million cases worldwide [8.8% of the adult 
population] [1]. 
 

“Diabetic patients have significant defects of 
antioxidant protection which may increase their 
vulnerability to oxidative damage and the 
development of diabetic complications” [2]. 
“Reactive Oxygen Species [ROS] are chemically 
reactive molecules containing oxygen, such as, 
H2O2, HOCl, and free radicals such as 
Superoxide anion, hydroxyl radicals” [3]. 
“Antioxidants are molecules involved in 
scavenging of free radicals. This defense 
mechanism involves both enzymatic and non-
enzymatic strategies” [3]. “In 2017, diabetes 
resulted in approximately 4.2 million deaths. It is 
the seventh leading cause of death globally” [4]. 
The pooled Prevalence of 5.77% in Nigeria 
suggests that about 11.2 million people [1 out of 
every 17 adults] are living with the disease. 
 

Recent research has also shown a connection 
between diabetes and dementia, hearing loss, 
and some forms of cancer [5]. Diabetes 
increases the risk of early death, and diabetes-
related complications can lower quality of life [6]. 
The High cost of anti-diabetic drugs, its side 

effects and numerous complications associated 
with poor prognosis arising from orthodox drugs 
has led many to embrace plant-based therapy 
[7]. As several studies revealed amelioration of 
Alzheimer’s disease post-administration of 
Telfairia occidentalis and Talinium triangulare on 
rats induced with scopolamine [8,9,10,11,12], 
Musa paradisiaca stem juice limit the extent of 
status epilepticus, increased neuronal protein 
synthesis, reduced cytoarchitectural damage 
and astriogliosis as well as enhancing long term 
recognition memory in rats [13,14], star fruit 
ameliorates neurotoxicity in rats [15], other 
researchers also reported the antihyperglycemic 
and hypoglycemic action of Vernonia 
amygdalina [VA] and Gongronema latifolium 
[GL] in diabetic and non-diabetic rats [16]. The 
aqueous leaf extract has been shown to possess 
antihyperlipidemic and hypolipidemic effects on 
diabetic and non-diabetic rats [17].  According to 
[18], VA has continued to receive a lot of 
attention due to the numerous curative potentials 
that it has demonstrated; It is purported to 
possess antioxidant activity from radical 
scavengers [19]. Many people also believe in 
traditional ways of living, and this has influenced 
their health-seeking behavior [20], hence, the 
rationale to investigate the potentials of VA and 
GL on the cytoarchitecture of the spleen in 
streptozocin-induced diabetic Wistar rats in the 
present study. 
 

2. MATERIALS AND METHODS 
 

A total of 30 rats [150-200g] with ethical approval 
[FAREC/PA/[UC/049] were acquired from the 



 
 
 
 

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animal house, College of Medical Sciences, 
University of Calabar. The rats were kept under 
standard conditions of temperature [27oC-30oC] 
and fed with rat chow purchased from Agro Feed 
Mill Nigeria Limited, Calabar and provided with 
distilled water for drinking. They acclimatized for 
a week prior to commencement of the 
experiment. The animals were housed in 
properly ventilated plastic cages. 
 

2.1 Plant Extracts Preparation 
 
The fresh leaves of VA and GL were purchased 
from Marian market, Calabar, Cross River State-
Nigeria. These plants were identified by a 
botanist with reference number Bot/ 
Herb/UCC/0188 and Bot/Herb/UCC/0718 
respectively. The plants were cleaned and air 
dried, after which they were grounded into 
powered form. A measured amount of 120 
grams of powdered leaves was extracted using 
2.5 liters of absolute ethanol for 48hours with 
intermittent gyration of sample holder. After 
48hours of soaking, the extract solution was first 
double filtered with chess cloth, then with filter 
paper [Whatman/filter paper]. The filtrate 
[extract] was thereafter concentrated under 
reduced pressure at 45oc in rotary evaporator to 
10% volume and then to complete dryness using 
regulated temperature water bath, yielding 22 
grams [about 19.5g] of crude extract. The extract 
obtained was stored in a refrigerator until 
required. 
 

2.2 Induction of Diabetes to the 
Experimental Rats 

 
Diabetes was induced in overnight fasted 
experimental animals by a single dose of 
intraperitoneal injection of freshly prepared 
Streptozocin [STZ] at 45mg/kg body weight 
reconstituted in 0.1M sodium citrate buffer at pH 
4.5-5.0 as solvent using Lorke’s method. 
Diabetes was confirmed by use of glucometer to 
test for the fasting blood glucose concentration 
of experimental animals 48hrs after STZ 
administration, values above 180mg/dl were 
considered diabetic and found suitable for the 
study. 
 

2.3 Determination of LD50 

 
LD50 of ethanol leaf extracts of VA and GL was 
established to be >2000mg/kg according to 
Lorke’s method [1983].  The dosage was 
determined using 5% [100mg/kg] of 2000mg/kg 

body weight of ethanol extract of VA and GL 
leaves. 
 

2.4 Plant Extract and Metformin 
Administration 

 
Group A, the Normal control rats were given only 
distil water and feed, Group B was the diabetic 
control rats, induced with diabetes [received 
45mg/kg of STZ], Group C animals were induced 
with diabetes [received 45mg/kg of STZ] and 
were given Vernonia Amygdalina [100mg/kg] 
only. Group D were induced for diabetes 
[received 45mg/kg of STZ] and were given 
Gongronema latifolium [100mg/kg] only. Group E 
were induced with diabetes [received 45mg/kg of 
STZ] and were given Metformin [100mg/kg] and 
Group F was induced with diabetes        
[received 45mg/kg of STZ] and were given both 
extracts of Vernonia Amygdalina [100mg/kg] and 
Gongronema latifolium [100mg/kg]. Pre and post 
induction of STZ fasting blood sugar [FBS] and 
weight of animals were assessed. These 
procedures were carried out daily during the 
course of extracts administration. 
 

2.5 Tissue Processing Procedure 
 
Twenty four hours after the last administration, 
the experimental animals were sacrificed and the 
spleen was fixed with 10% formol saline. The 
tissues were dehydrated using ascending grades 
of alcohol followed by clearing with xylene, 
infiltrated, embedded with paraffin wax and 
sectioned [5µm] with rotary microtome. The 
ribbons of the sections were collected using 
clean slides and first straighten with 10% ethanol 
before being gently lowered into the surface of a 
warm water bath so as to straighten wrinkles on 
sectioned tissues. The floated sections were 
mounted on an albumenized slides and it was 
dried in the oven at 60 degrees Celsius, for one 
hour before staining. 
 

2.6 Hematoxylin and Eosin Staining 
Procedure [Cole, 1943] 

 
Tissue sections were dewaxed in xylene for 5-10 
minutes, transferred through descending grades 
of alcohol [100%, 95% and 70%] 5mins 
respectively. Sections were rinsed in running tap 
for 10mins and stained in hematoxylin for 10 
minutes. They were rinsed in water and Blueing 
in tap water for 5 minutes. Sections were 
differentiated in 1% acid alcohol so as to help 
nucleus absorbs the stain`. They were counter 



 
 
 
 

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stained in 1% Eosin for 30 seconds. Various 
sections were blued under a running tap water 
for 2-3mins. Finally, sections were passed 
through ascending grades of alcohol, cleared in 
xylene and mounted in distyrene, plasticizer and 
xylene [DPX]. 
 

2.7 Stereology 
 

The absolute volume of the spleen was 
estimated using the Cavalieri estimator of 
volume according to the method of West et al. 
[1999]. To achieve this, spleen of each Wistar rat 
per group was isolated, processed and 
sectioned serially using a microtome, as shown 
by [21] to provide a number of sections after pilot 
study on how many slices could be derived and 
how many slices will give the lowest coefficient 
of error, each section had a section of the 
spleen. Tissue sections of the spleen were 
selected using a systematic uniform random 
sampling method. The sections derived were 
stained using H and E stains. A transparent 
counting grid was placed randomly over the cut 
surface of every spleen slice. The number of 
points hitting the spleen was counted. The 
volume was then estimated using the Cavalieri’s 
principle [22] as follows: 
 

V total [mm3]: =𝑇 × 𝑎
𝑝⁄ × ∑𝑝𝑖 

 

where ‘‘: =’’ indicates that the result is the 
estimated value rather than the true value, “V” is 
the total volume of spleen, “T” = 100.  Slice 
thickness =5 µm x100 = 500 µm. Convert 500 

µm to mm, 500/1000 = 0.5 mm is the average 
slice thickness, “a/p” ¼ is the area per point 
associated with each point in the counting grid 
[4mm²], and “Ʃp” is the total number of points 
hitting the spleen. 
 

2.8 Data Analysis 
 
Results obtained at the end of the experiment 
were analyzed using the statistical software, 
statistical package for social science [IBM SPSS 
version 23.0] and Microsoft Office Excel 2019 
were used for charts. Results were expressed as 
mean ± S.E.M. One-way analysis of variance 
[ANOVA] was used to compare mean difference 
between groups followed by least significant 
difference [LSD] post hoc test. Paired t-test was 
employed for the comparisons of means as 
appropriate. Values were considered significant 
when p<0.05. 
 

3. RESULTS  
 
H&E stained Sections from the control group 
showed normal histology of the spleen with 
normal white pulp [WP] and numerous white 
blood cells, red pulp [RP], the marginal zone and 
the central vessels [BV] following H&E staining 
[Plate 1]. Group B revealed shrunken white pulp 
[WP] and enlarged red pulp [RP] with reduced 
germinal center using, the spleen also showed 
numerous lymphocytes in the shrunken white 
pulp area indicating the presence of 
inflammation and immune response and 

 

 
 

Plate 1. Photomicrograph of spleen of Wistar rats in normal control group treated with H20 
showing normal white pulp and red pulps with germinal center, H&E stain, [mag x 100] 



 
 
 
 

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distortion of the splenic cytoarchitecture, [Plate 
2]. Group C revealed signs of regenerating white 
pulp [WP] compared to the diabetic group, with 
lymphocytes infiltrating into the red pulp [RP] 
area [Plate 3]. Group D showed prominent white 
pulp (WP) with numerous white blood cells and 
reduced red pulp [RP] [Plate 4]. Group E showed 
normal appearing white pulp [WP] with white 
blood cells and reduced red pulp [RP] [Plate 5]. 
Group F revealed signs of regenerating normal 
white pulp, red pulp, increased lymphocytes in 
the white pulp with normal central vessel [Plate 
6].  

Stereological estimation of Spleen Volume of 
diabetic control group B [970±250.98] [CE = 
0.035] was significantly [p<0.05] lower when 
compared to normal control group A 
[2398.7±352.70] [CE = 0.018]. Spleen       
volume of all treated groups: group C 
[1630±321.23] [CE = 0.028], group D 
[2538±284.32] [CE = 0.015], group E 
[3458±515.54] [CE = 0.004] and group F 
[2548±871.34] were significantly [p<0.05] higher 
when compared to the volume of the        
diabetic control group B [970±250.98] [CE = 
0.023]. 

 

 
 

Plate 2. Photomicrograph of spleen of Wistar rats in Diabetic control group treated with 45 
mg/kg streptozocin only showing shrunken and distorted white pulp and reduced germinal 

center with blood vessel, H&E stain, [mag x100] 
 

 
 

Plate 3. Photomicrograph of spleen of Wistar rats in group C treated with 45 mg/kg 
streptozocin and 100 mg/kg of VA only showing regenerating white pulp [rWP] with 

lymphocytes [LYM] and reduced red pulp [RP] area, H&E stain, [mag x 100] 



 
 
 
 

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Plate 4. Photomicrograph of spleen of Wistar rats in group D inducedwith 45 mg/kg 
streptozocin and treated with 100 mg/kg GL only showing regenerating white pulp [WP] filled 

with increased white blood cells [WBC] and reduced red pulp [RP] area, H&E stain, [mag x100] 
 

 
 

Plate 5. Photomicrograph of Group E [Metformin solution 5mg/kg] showing regenerating 
splenic white pulp with white blood cells infiltrating the reduced red pulp [RP], H&E stain, 

[mag x 100] 
 



 
 
 
 

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Plate 6. Photomicrograph of the spleen of Wistar rats in Group F treated with 45 mg/kg 
streptozocin and combined extracts of 200 mg/kg VA + GL showing regenerating white pulp, 

with reduced red pulp with presence of numerous normal lymphocytes [LYM] in the white pulp 
and central vessel. H&E stain [mag x100)] 

 

 
 
Plate 7. Photomicrograph of spleen of Wistar rats in normal control group treated with distilled 
water showing weakly positive Perl’s stain with sparse hemosiderin deposits. Perl’s Prussian 

stain, [mag x100] 



 
 
 
 

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Plate 8. Photomicrograph of spleen of Wistar rats in group B treated with 45 mg/kg 
Streptozocin only showing intense Perl’s staining of hemosiderin deposits [arrows] within the 

red pulp.  Perl’s Prussian stain [mag x 100] 
 

 
 

Plate 9. Photomicrograph of Group C splenic tissue treated with 45 mg/kg streptozocin and 
100 mg/kg of VA only showing moderate staining of hemosiderin deposits [bluish colouration] 

within the red pulp [Perl’s Stain X 100] 



 
 
 
 

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Plate 10. Photomicrograph of spleen of Wistar rats ingroup D treated with 45 mg/kg 
streptozocin and 100 mg/kg of GL only showing moderate Perl’s stain with moderate 
hemosiderin deposits [arrow] within the red pulp. Perl’s Prussian stain, [mag x 100] 

 

 
 

Plate 11. Photomicrograph of the spleen of Wistar rats in group E treated with 45 mg/kg 
streptozocin and 5mg/kg metformin showing moderate Perl’s stain of hemosiderin deposits 

[arrow] within the red pulp. Perl’s Prussian stain, [mag x 100] 



 
 
 
 

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Plate 12. Photomicrograh of spleen of Wistar rats in group F treated with 45 mg/kg 
streptozocin and combined extract of 200 mg/kg VA and GL showing weak Perl’s stain with 

moderate hemosiderin deposits [bluish coloration] within the red pulp.  Perl’s Prussian stain, 
[mag x100] 

 

 
 

Plate 13. Stereological image of the spleen in normal control group of wistar rat showing 
normal morphology and splenic volume 

 



 
 
 
 

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Plate 14. Stereological image of splenic tissue of diabetic group that received 45mg/kg of STZ 
only, showing splenic tissue atrophy with distorted morphology and reduced spleen volume 

 

 
 

Plate 15. Stereological image of splenic tissue of animals that received 45mg/kg of STZ and 
treated with 100mg/kg of VA only showing increased splenic volume compared to the diabetic 

control group 



 
 
 
 

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Plate 16. Stereological image of splenic tissue of animals that received 45mg/kg of STZ and 
treated with 100mg/kg of GL only showed increased volume of the spleen when compared to 

the diabetic group 
 

 
 

Plate 17. Stereological image of splenic tissue of animals that received 45mg/kg of STZ and 
treated with Metformin at 5mg/kg daily, showed significantly increased volume of the spleen 

when compared to the diabetic group 



 
 
 
 

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Plate 18. Stereological image of splenic tissue of animals that received 45mg/kg of STZ and 
treated with 200mg/kg of combined extract of VA+GL showed significantly increased volume 

of the spleen compared to the diabetic control group 
 

4. DISCUSSION 
 
Diabetes decreases immune response capacity, 
including the suppression of immune cell 
function, atrophy of immune organs [22,23]. 
Studies have been done to ascertain the 
potential utility of natural products as 
immunomodulatory agents to enhance hosts 
responses to diseases [24]. This study focuses 
on the effect of diabetes on one of the important 
immunologic organs- the spleen and the 
possible ameliorative effect of combined extract 
of Vernonia amygdalina and Gongronema 
latifolium. Diabetes-related spleen damage 
increases immune dysfunction, which often 
results in the high risks of infection, morbidity 
and mortality in diabetic patients. 

 
“Herbs have been used in many capacities to 
improve human health and also serve as a 
source of natural templates for pharmaceutical 
synthesis of drugs. Many local communities 
across Afro-Asian regions of the world rely 
heavily on their ethno-botanical heritage to meet 
most of their primary healthcare needs” [25]. 
This study demonstrated that anti-diabetic effect 
of VA+GL in rats is mediated through complex 
multiple mechanisms. 
 

“Immunodeficiency is one of the major causes of 
diabetic complications.  The spleen, as a 
secondary lymphoid organ, acts as a site of 
initiation of most of the immune responses.  
Spleen harbors stem cells that act as precursors 
to insulin producing β cells of the pancreas. STZ 
selectively accumulates in the pancreatic β cells 
and induces hyperglycemic conditions           
through mitochondrial complication mediated 
glucotoxicity of the β cells” [26]. 
 
In this study, administration of the combined 
extracts post diabetes induction was found to 
reduce the increased blood glucose level and 
restore the normal level of serum insulin, spleen 
size and the spleen weight in the treated rats 
compared to the diabetic rats. 
 
In the diabetic control [Group B] induced with 
45mg/kg of streptozocin, the spleen showed 
signs of depleted white pulp compared to the 
enlarged size in the normal control group which 
is in line with the work of [27] who reported that 
the lymphocyte number were dramatically 
declined in both peripheral blood and the spleen 
following a distortion of the white pulp, dilation in 
the blood vessels, an increase of collagen 
deposition and a depletion in the iron particles 
detected in the red pulp. This indicates that the 



 
 
 
 

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lymphocytes are stressed by diabetic toxicity 
with high levels of free radicals which, increases 
the levels of pro-inflammatory cytokines leading 
to programmed cell death. The white pulp of the 
spleen is responsible for both adaptive and 
humoral immunity involving the B and T-
lymphocytes. Shrunken white pulp leads to a 
reduction in immune response, thereby making 
the body susceptible to diabetic complications. 
 
In groups C and D, which received 100mg/kg of 
Vernonia amygdalina [Bitter leaf] and 
Gongronema latifolium [Utazi] respectively 
showed significant reversible changes in the 
histological integrity of the spleen compared with 
the results in the diabetic group. This result 
suggests the extracts individually has effect on 
the diabetic alterations that occurred in the 
spleen after STZ induction to cause reversible 
changes in the splenic cytoarchitecture, with 
regenerating white pulp and presence of 
numerous normal lymphocytes in the spleen. In 
group E, treated with 5mg/kg of Metformin, 
changes in the histology of the spleen were 
observed. The shrunken white pulp seen in the 
diabetic group B was seen regenerating with 
increased size in this group. This shows that 
Metformin has anti-oxidant properties in line with 
[28]. There was a significant increase in the 
number of white blood cells which help to 
strengthen the body's immune response. 
 
In group F that received 100mg/kg of both V. 
amygdalina and G. latifolium showed similar 
histological profile to that of the normal control. 
This suggests a possible regeneration of the 
splenic cell functions and a significant reversal of 
diabetic insults on the splenic cytoarchitechure 
by the combined extract. This observation is in 
line with previous studies on the potentials of 
Vernonia Amygdalina, Gongronema Latifolium 
and Azadirachta Indica to cause a regeneration 
of pancreatic beta cells of STZ induced diabetic 
rats. [29,16,30]. The effects of the plant extracts 
were more pronounced when used in 
combination. This study observed that the 
improvement in the histology of the spleen was 
more remarkable in the group that received the 
combined extract [VA+GL] than in the groups 
that received individual extracts, [VA and GL]. In 
another study by [27], the administration of 
100mg/kg of Whey Proteins restored the 
histological integrity of the spleen following STZ 
induced diabetic degenerative changes were 
seen. Plant extracts can be useful in reversing 
diabetic insults on the histology of the spleen as 
was discovered from this study. 

The findings of this research are consistent with 
previous research on the regenerative potential 
of Vernonia Amygdalina, Gongronema latifolium, 
and Azadirachta indica in restoring pancreatic 
beta cells in STZ-induced diabetic rats [29]. 
Moreover, the synergistic impact of the mixed 
extract [VA+GL] was more pronounced in 
improving the histology of the spleen compared 
to the individual extracts [VA and GL] in this 
study. These results align with research by [27], 
which illustrated that the administration of whey 
proteins at a dosage of 100mg/kg restored the 
histological integrity of the spleen in the 
presence of STZ-induced diabetic degenerative 
changes. Therefore, the use of plant extracts 
shows promise in reversing diabetic-induced 
damage to the spleen's histology, as evidenced 
by the findings of this study. 
 
Staining intensity of the white pulp by Perl’s 
Prussian blue in the spleen sections of the 
normal control and the treated groups [C, D, E 
and F] groups were weakly positive, by contrast 
to the Diabetic group which showed higher 
intensity staining. The immunosuppressive 
states induced by hyperglycemia is largely 
responsible for the decline in lymphocyte 
population and subsequent increased 
hemosiderin deposition in the red pulp area as a 
consequence of impaired phagocytic activity 
[27].  The findings of [31] further support the 
current study's results, as they reported that the 
administration of whey protein to diabetic rats 
improved the lymphocyte population in the white 
pulp of the spleen. This improvement may be 
attributed to the inhibition cascade of the 
programmed cell death pathway, indicating a 
potential mechanism by which whey protein 
restores lymphocytic activity. Additionally, the 
restoration of iron deposition to approximately 
normal levels observed in the study suggests a 
further improvement in lymphocytic function. 
Therefore, the use of whey protein as a 
therapeutic agent holds promise for enhancing 
lymphocyte activity and restoring the normal 
functioning of the spleen in diabetic conditions. 
Changes observed in the cytoarchitecture of the 
spleen suggested that Whey Proteins could 
enhance immune response. This suggests that 
the ethanolic extract in single or combined doses 
administered reversed splenic cell destruction 
from STZ induced diabetes. 
 
The immunosuppressive states induced by 
hyperglycemia is largely responsible for the 
decline in lymphocyte population and 
subsequent increased hemosiderin deposition in 



 
 
 
 

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the red pulp area as a consequence of impaired 
phagocytic activity [27].  This agrees with the 
findings in another study by [31] who reported 
that the improvement of the lymphocyte 
population in the white pulp by Whey Protein 
treated diabetic rats may be due to the inhibition 
cascade of the programmed cell death pathway. 
Whey Proteins was found to restore the iron 
deposition to the approximately the normal level, 
indicating improvement of lymphocytic activity.  
Changes observed in the cytoarchitecture of the 
spleen suggested that Whey Proteins could 
enhance immune response.  This suggests that 
the ethanolic extract in single or combined doses 
administered reversed splenic cell destruction 
from STZ induced diabetes. 
 
The stereological analysis of the spleen showed 
significant decrease of the splenic pulp volume 
density in the diabetic group when compared to 
the control group and the treated group of Wistar 
rats. Reducing the presence of splenic pulp was 
mainly due to the decrease in the volume density 
of all structural components of the white pulp. 
This observation aligns with another study by 
[32], following administration of Dexamethasone 
at 150mg/kg body weight, which showed 
significant decrease of the splenic pulp volume 
density and significant increase of the 
connective tissue volume density. The 
cytoarchitecture of the spleen was restored 
following treatment with the combined extracts of 
VA+GL. 
 
Stereological studies also revealed 
that combined extracts of VA+GL treatment 
remarkably improved the volume of the splenic 
white pulp depleted by STZ induced diabetes. 
 
The point-counting method was utilized to study 
the volume densities of the following tissue 
compartments: red pulp; white pulp [this 
compartment was divided in two sub 
compartments: follicles and per arteriolar 
lymphocyte sheath]; marginal zone; and 
connective tissue. 
 
Stereology is a number of mathematical and 
statistical methods that permit the evaluation of 
3- dimensional structural information from 2-
dimensional sections [or histological slices]. 
Thus, researchers obtain important quantitative 
structural information, such as the volume, 
surface area or numbers of cells within 
described regional lines. The need for such 
quantitative information biological studies is of 
importance when evaluating the effect of various 

experimental treatments on any specific organs, 
tissues and cells in the body.  Spleen volumes 
were significantly increased in animals treated 
with VA+GL and individual extracts of VA and 
GL. Stereological studies also revealed 
that VA+GL treatment remarkably improved the 
volume of the splenic tissues and the numerical 
density depleted by STZ diabetes.  
 
Possible antidiabetic mechanism of VA+GL in 
STZ induced diabetes is through the 
regeneration of atrophic splenic structural 
integrity and its strong antioxidant potential. 
Diabetes is known to involve oxidative stress 
and changes in lipid metabolism. Gongronema 
latifolium has been shown to possess anti-
hyperglycemic, anti-lipidemic, antioxidant and 
anti-inflammatory properties. [33], showed that 
ethanolic extracts of GL leaves have anti-
hyperglycemic effects in Streptozocin induced 
diabetic rats, thought to be mediated through the 
activation of hexose and glucose -6-phosphate 
dehydrogenase, G6PDH. VA strengthens the 
immune system through its effect on cytokine 
regulation [34].  A study by [35], showed ferulic 
acid provides protection against hyperglycemia 
induced, oxidative stress mediated splenotoxicity 
by regulating the plasma insulin level, blood 
glucose level, intracellular inflammation and 
mitochondria dependent intrinsic pathway of 
apoptosis in the spleen.  
 
This study highlighted that the spleen volume of 
diabetic control group [970±250.98] [CE = 0.035] 
was significantly [p<0.05] lower when compared 
to normal control group [2398.7±352.70] [CE = 
0.018] and group F [2548±871.34] were 
significantly [p<0.05] higher when compared to 
the volume of the diabetic control group 
[970±250.98] [CE = 0.023] [36-41]. 
 

5. CONCLUSION 
 
From this study, it could be inferred that 
combined and individual ethanolic extracts of 
VA+GL possesses anti-diabetic and antioxidant 
properties that can mitigate the impact                       
of STZ induced diabetes on the spleen of Wistar 
rats. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 

Author(s) hereby declare that NO generative AI 
technologies such as Large Language Models 
(ChatGPT, COPILOT, etc) and text-to-image 
generators have been used during writing or 
editing of manuscripts. 



 
 
 
 

Oku et al.; Asian J. Immunol., vol. 7, no. 1, pp. 131-148, 2024; Article no.AJI.122015 
 
 

 
146 

 

CONSENT 
 
It is not applicable. 
 

ETHICAL APPROVAL 
 
Animal Ethic committee approval has been 
collected and preserved by the author(s) 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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