Biomedicine & Pharmacotherapy 149 (2022) 112833
Contents lists available at ScienceDirect 
Biomedicine & Pharmacotherapy 
journal homepage: www.elsevier.com/locate/biopha 
Hypoglycaemic activity of Oleanonic acid, a 3-oxotriterpenoid isolated 
from Aidia Genipiflora (DC.) Dandy, involves inhibition of carbohydrate 
metabolic enzymes and promotion of glucose uptake 
Benjamin Kingsley Harley a,*, Isaac Kingsley Amponsah b, Inemesit Okon Ben c, Nana 
Ama Mireku-Gyimah d, Daniel Anokwah e, David Neglo f, Cedric Dzidzor K. Amengor g, 
Theophilus Christian Fleischer a 
a Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana 
b Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, 
Kumasi, Ghana 
c Department of Pharmacology and Toxicology, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana 
d Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Ghana, Accra, Ghana 
e Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Cape-Coast, Cape-Coast, Ghana 
f Department of Basic Science, School of Basic and Biomedical Sciences, University of Health and Allied Sciences, Ho, Ghana 
g Department of Pharmaceutical Chemistry, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana   
A R T I C L E  I N F O   A B S T R A C T   
Keywords: The present study evaluated the antidiabetic activities of the 70% ethanol stem bark extract of Aidia genipiflora 
Streptozotocin-induced diabetes (AGB) and one of its constituents, oleanonic acid in streptozotocin (40 mg/kg)-induced diabetic rats. In vitro 
Glucose uptake assays of glucose uptake and inhibition of carbohydrate metabolizing enzymes were then used to investigate 
α-Glucosidase their mechanism(s) of hypoglycaemic action. In silico evaluation of the pharmacokinetic and toxicity properties 
α-Amylase 
Oleanonic acid of the compound was also carried out. Administration of AGB (100–400 mg/kg) and oleanonic acid (15 – 60 mg/ 
Aidia genipiflora kg) resulted in significant reductions (p < 0.001) in the blood glucose and considerable decrease (p < 0.05) in the 
elevated lipid parameters of the diabetic animals. AGB activity at 200 and 400 mg/kg; and oleanonic acid at 60 
mg/kg were comparable to glibenclamide (5 mg/kg). The extract and its isolate strongly inhibited α-glucosidase 
and α-amylase activity with IC50 values of (10.48 ± 1.39 µg/mL and 14.51 ± 1.26 µg/mL) and (36.52 ± 1.95 µM 
and 105.84 ± 1.08 µM) respectively. The glucose uptake assays showed that AGB and oleanonic acid exerted 
both insulin-dependent and independent promotional effect of glucose transport into the periphery by upregu-
lating the expression of PI3K and PPARγ transcripts with a concomitant increase in GLUT-4 transcripts. Although 
oleanonic acid was predicted to be teratogenic, it was found to be generally non-lethal with favourable phar-
macokinetics properties making it suitable for further studies. The study has shown that the stem bark of 
A. genipiflora is a source of new hypoglycaemic agents and that oleanonic acid possesses hypoglycaemic and anti- 
hyperlipidaemic activities.   
1. Introduction lifestyles, population ageing, urbanization, and economic development [2]. 
Nearly half a million people are at risk of the disease with 4.2 million 
The menace of diabetes mellitus and its complications is a major global people dying from it in the year 2019 [3]. The management of diabetes 
concern in healthcare. The International Diabetes Federation (IDF) esti- mellitus is a global health issue with a cure yet to be discovered. Currently 
mates 463 million diabetic patients worldwide with this number expected available medications for diabetes include insulin and various oral hypo-
to rise to 700 million by 2045 [1]. The increasing epidemic of diabetes has glycaemic drugs like the biguanides (metformin), sulfonylureas (gliben-
been attributed to factors such as unhealthy eating habits and sedentary clamide, glipizide and glimepiride), α-glucosidase inhibitors (miglitol and 
* Correspondence to: Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Health and Allied Sciences PMB 31, Ho, Volta Region, 
Ghana. 
E-mail address: bkharley@uhas.edu.gh (B.K. Harley).  
https://doi.org/10.1016/j.biopha.2022.112833 
Received 31 January 2022; Received in revised form 11 March 2022; Accepted 16 March 2022   
Available online 19 March 2022
0753-3322/© 2022 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
acarbose), meglitinides and the thiazolidinediones (pioglitazone and rosi- and radioactive [3H] 2-deoxyglucose were sourced from PerkinElmer 
glitazone) [4]. Though effective, the use of these agents is associated with (Waltham, MA, USA). 
serious adverse effects and therefore the search for more effective and safer All organic solvents used were of analytical grade and obtained from 
antidiabetic drugs is a pressing need. Almost 80% of the adult diabetic BDH, Laboratory Supplies (Merck Ltd, Lutterworth, UK). 
population and those that suffer the most debilitating complications of the 
disease reside in low- and middle-income countries because they do not 2.2. Plant collection, extraction and isolation of oleanonic acid 
have access to and/or cannot afford the oral hypoglycaemic medicines and 
therefore rely mostly on traditional medicines for their treatment [5]. The stem bark of A. genipiflora was collected from Kwahu Asakraka in 
The application of traditional practices in treating diseases has the Eastern Region, Ghana in April 2020 and authenticated at the 
existed since the beginning of time across all ethnic groups. It is esti- Institute of Traditional and Alternative Medicine (ITAM), University of 
mated that about 85% of the world’s population especially those Health and Allied Sciences (UHAS) where voucher specimen (Voucher 
dwelling in the rural areas of developing nations rely exclusively on number: UHAS/ITAM/2020/SB010) has been kept. The collected plant 
traditional medical practices for their primary healthcare [6]. Over the material was thoroughly washed under running water, chopped into 
past decades, herbal preparations and medicinal herbal products have pieces, air-dried for 7 days and ground into powder. Afterwards, 1.3 kg 
been shown to be effective in the management of diabetes and prevent of the powdered material was Soxhlet-extracted using 70% ethanol for 
the onset of secondary complications [7]. Whereas some medicinal 24 h. The extract obtained was evaporated under reduced pressure to 
plants have been proven to exert their activity through the stimulation of obtain a brown residue (AGB, 138 g) of which 125 g was reconstituted in 
regenerated and/or surviving β-cells of the pancreas to increase insulin distilled water and sequentially extracted with petroleum ether (pet 
release, others have been shown to help by stimulating the uptake of ether), chloroform (CHCl3), ethyl acetate (EtOAc) and n-butanol 
glucose into the peripheral tissues and inhibiting carbohydrate metab- (ButOH) and the solvents evaporated off to obtain five fractions: AGB- 
olizing enzymes [8]. Furthermore, some medicinal plants have also been Pet-ether, AGB-CHCl3, AGB-EtOAc, AGB-ButOH and aqueous portions 
shown to modulate the key effector molecules of the PI3K/Akt pathway [23]. 
to exert their antidiabetic actions [9] whiles others have been shown to The ethyl acetate portion (AGB-EtOAc, 20 g) was column chroma-
possess antioxidant and anti-hyperlipidaemic activities in addition to tographed (CC) over Silica gel 60 (70–230 mesh) using CHCl3 – meth-
their hypoglycaemic effects. anol (MeOH) mixtures (100:1, 90:1, 80:1, 70:1–1:1) to obtain 108 
Aidia genipiflora (DC) Dandy of the family Rubiaceae is a plant eluates (100 mL) which were pooled into 5 fractions (F1-F5). Purifica-
distributed across Central and West African tropical sub-regions. The tion of fraction F3 (5.6 g) on CC by gradient elution using pet ether:/ 
leaves and stem bark of A. genipiflora are used in managing various EtOAc mixtures followed by Sephadex LH-20 column eluting with 
communicable and non-communicable diseases in African Traditional CHCl3: MeOH (1:1) yielded oleanonic acid (1.8 g) as a white amorphous 
Medicine including drowsiness, diabetes, oedema, wounds, and gout powder. 
[10]. Anokwah et al. reported the antimicrobial activity of the stem bark The identity of the isolated compound as oleanonic acid was deter-
of the plant and further isolated some of its constituents responsible for mined using NMR spectroscopic methods and by comparing to reported 
its use as an anti-infective agent in traditional medicine [11]. However, literature. The NMR spectroscopic data are provided in the supple-
studies to validate the other traditional uses of the plant are yet to be mentary material. 
conducted. 
One of the bioactive constituents of A. genipiflora is oleanonic acid 2.3. Intestinal α-glucosidase inhibition 
which has also been identified in several plant species [12–14]. The 
compound and its derivatives have been extensively investigated for its Rat intestinal acetone powder (300 mg) was homogenized in 600 mL 
effects in the management of cancer and inflammation [15–18]. Similar of phosphate buffer (0.1 M, pH 6.9) and centrifuged at 12 000 g for 
studies have been conducted to establish its anti-infective activity which 30 min to obtain the enzyme solution used in the test. 100 μL of the 
includes antiparasitic [19] and antibacterial [20] activities. Through enzyme solution obtained were incubated with 50 μL of the test samples 
computational studies, Petersen et al. identified oleanonic acid as a (AGB [0.1 µg/mL – 100 µg/mL]; oleanonic acid and acarbose 
PPARγ agonist [21]. Elsewhere, the compound induced glucose uptake [1–1000 µM]) at 25 ◦C for 10 min in 96-well plates. Afterwards, 50 μL of 
in L6 skeletal myotubes [22]. Interestingly, although both studies sug- 5 mM p-nitrophenyl-α-D-glucopyranoside solution was added to each 
gest that oleanonic may be useful in the management of diabetes, no well and the plates incubated at 37 ◦C for 30 min. The absorbances of the 
studies have been conducted to identify its effect in an in vivo model. plates were then measured at 405 nm on a microplate reader (Thermo-
This study therefore seeks to investigate the antidiabetic potential of max, Molecular Device Corp., Sunnyvale, CA). Blank determination was 
A. genipiflora and evaluate the in vivo hypoglycaemic activity of ole- carried out using 50 μL of buffer solution without any test sample [24]. 
anonic acid in STZ-induced diabetic rats. The effect of oleanonic acid on 
glucose transport into the periphery as well as the effect of the com- 2.4. Inhibition of pancreatic α-amylase 
pound on the activities of α-amylase and α-glucosidase are also reported. 
Porcine pancreatic α-amylase powder (350 mg) was homogenized in 
2. Materials and methods 700 mL of 0.02 M phosphate buffer (pH 6.9) to get the enzyme solution 
employed in the assay. 500 μL of the test samples (AGB [0.1 µg/mL – 
2.1. Reagents and chemicals 100 µg/mL]; oleanonic acid and acarbose [1–1000 µM]) were mixed 
with 500 μL starch solution (1% w/v) and incubated at 25 ◦C for 10 min. 
C2C12 myoblasts (CRL-1772) and 3T3-L1 pre-adipocytes (CL-173) Afterwards 500 μL enzyme solution was added to each mixture and 
were products of American Type Culture Collection (ATCC) (Manassas, incubated for 10 min at room temperature. A mL each of dinitrosalicylic 
Virginia, USA) whereas newborn calf serum (NBCS), foetal calf serum acid colour reagent was then added to each mixture to end the reaction. 
(FCS), L-glutamine, Dulbecco’s modified Eagle’s Medium (DMEM), The mixtures were then heated for 10 min at 100 ◦C, diluted and their 
penicillin/streptomycin and horse serum were bought from Gibco absorbances measured at 540 nm [23]. 
(Berlin, Germany). Dinitrosalicylic acid (DNS), streptozotocin (STZ), 
cytochalasin B, 3-Isobutyl-1-methylxanthine (IBMX), glibenclamide, 2.5. Glucose transport assay 
acarbose, intestinal acetone powder, porcine pancreatic α-amylase, 
accutase, 2-deoxy-D-glucose and p-nitrophenyl-α-D-glucopyranoside 2.5.1. Glucose transport in 3T3-L1 adipocytes 
were obtained from Sigma Aldrich (St. Louis, MO, USA). Ultima Gold XR Fully differentiated and confluence (90%) 3T3-L1 adipocytes grown 
2
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 1% 2.6. Streptozotocin-induced diabetes assay 
antibiotics and 10% NBCS in 5% CO2 at 37 ◦C; and differentiated in 
DMEM augmented with 1 μmol/L dexamethasone, 1% antibiotics, 3-iso- 2.6.1. Experimental animals 
butyl-1-methylxanthine (IBMX, 0.5 mmol/L), 10 μmol/L human insulin Male Sprague-Dawley rats weighing between 180 and 205 g bought 
and 10% FCS were serum starved in 24- well plates for 4 h before and kept at the animal house of the Department of Pharmacology, 
treating with the test samples (AGB [0.1 − 100 µg/mL], oleanonic acid Kwame Nkrumah University of Science and Technology (KNUST) were 
[0.1 − 100 µM] and Insulin [0.01 – 10 nM]) for an hour. Afterwards, the housed in stainless steel cages lined with saw dust under environmental 
cells were washed with KRP-buffer (2 ×) and incubated with 400 μL conditions of temperature (25 ± 2 ◦C) and 12 h light: dark cycle. The 
glucose uptake-buffer (KRP-buffer supplemented with 10 μM 2-deoxy- animals were fed on standard pellet diet (R36, GHAFCO, Tema, Ghana) 
glucose and 1 μCi/mL radioactive [3H] 2-deoxyglucose) for 10 min. and freely allowed to drink water. 
Glucose uptake was then halted by the addition of 10 µM Cytochalasin B Male Sprague-Dawley rats were employed in the study because their 
in KRP buffer (CB-stop solution) and the cells iced for 10 min before pancreatic β-cells are susceptible to the necrotic actions of streptozoto-
rinsing thrice with cold phosphate buffered saline (PBS) solution, lysed cin and produces stable diabetes model easily [27]. 
0.2 M NaOH and mixed with liquid scintillation cocktail (4 mL). The The National Institute of Health (NIH) Guidelines for the care and use 
radioactivity present in the lysed cells were then measured in triplicate of laboratory animals (NIH, Department of Health and Human Services 
with a scintillation counter. The protein contents of the lysed cells were publication No 5, revised 1985) were adhered to in the treatment of 
measured using the BCA-Protein Assay Kit after which the glucose up- experimental rats. 
take was calculated as radioactive count per min (cpm)/mg of protein. Ethical clearance was obtained from the Department of Pharma-
Final data were expressed as the percentage of control. Cytochalasin B cology Ethics Committee, KNUST (CHRPE/AP/212/19). 
(10 µM) was used to determine the non-specific glucose uptake which 
was subtracted from each value [23]. 2.6.2. Acute toxicity studies 
The toxicity of A. genipiflora 70% ethanol stem bark extract (AGB) 
2.5.2. Glucose transport in C2C12 myotubes and oleanonic acid on male Sprague Dawley rats were evaluated as per 
Our previously published method was employed [25]. Fully differ- the Organization of Economic Cooperation and Development (OECD) 
entiated and confluence (90%) myotubes grown in DMEM containing Guidelines number 425. Overnight fasted (18 h) experimental animals 
1% penicillin/streptomycin, 10% FCS, 2% horse serum and 2 mM in groups of five were orally treated with a single dose of the test samples 
glutamine in 5% CO at 37 ◦2 C were incubated in serum-free DMEM for [AGB: 50, 500, 5 000 mg/kg; oleanonic acid: 10, 100 mg/kg or normal 
4 h in 24- well plates and treated with the test samples (AGB saline (0.9% 10 mL/kg)]. The rats were observed closely for toxicity 
[0.1 − 100 µg/mL], oleanonic acid [0.1 − 100 µM] and Insulin growth signs, behavioural changes, or death at 0, 15, 30, 60, 120 and 180 min, 
factor-1 (IGF-1) [0.1 – 100 nM]) for 60 min. The cells were then rinsed 24 h and 14 days after administration of the test samples. 
3 × with HBS-buffer and treated with 400 μL glucose uptake-buffer 
(HBS-buffer containing 2-deoxyglucose (10 μM) and radioactive [3H] 2.6.3. Induction of diabetes and experimental design 
2-deoxyglucose (2 μCi/mL)) for 5 min. The glucose transport reaction Diabetes was induced in the experimental rats by injection of STZ 
was then stopped by the addition of CB-stop solution (Cytochalasin B (40 mg/kg [i.p]) freshly dissolved in 0.1 M cold sodium citrate buffer 
(10 µM) in HBS buffer) and the cells kept on ice for 15 min before rinsing (pH 4.5). The diabetes induced rats were allowed to drink freely 5% 
with ice-cold PBS solution (3 ×). The myotubes were then lysed with glucose solution for the next 12 h to prevent initial drug-induced 
NaOH (0.2 M), added to 4 mL Liquid Scintillation Cocktail and the [3 H] hypoglycaemic mortality. Hyperglycaemia was confirmed by elevated 
2-deoxyglucose content measured in triplicate. The protein content of levels of blood glucose concentrations determined after 72 h using 
the lysed cells was also determined using the BCA-Protein Assay Kit after OneTouch Select glucometer (LifeScan, Inc. Milpitas, CA 95035 USA) 
which the glucose uptake was calculated as radioactive count per min [28]. Diabetic rats selected for the study had fasting blood glucose (FBG) 
(cpm)/mg of protein. Final data were expressed as the percentage of above 10 mmol/L with demonstratable signs of hyperglycaemia such as 
control. Cytochalasin B (10 µM) was used to determine the non-specific polyuria, polydipsia and hyperphagia [29]. 
glucose uptake which was subtracted from each value. The diabetic animals were randomly selected and put into groups of 
six animals - negative diabetic control group (vehicle: 2% tragacanth 
2.5.3. Reverse transcriptase-polymerase chain reaction (RT-PCR) solution), diabetic treatment groups (test samples: 100, 200 and 
A previously described reverse transcriptase-polymerase chain re- 400 mg/kg for AGB [p.o.]; 15, 30 and 60 mg/kg for oleanonic acid [p. 
action (RT-PCR) method with slight modifications was employed [26]. o.]) and diabetic positive control group (glibenclamide: 5 mg/kg [p.o.]). 
Briefly, differentiated C2C12 myotubes grown in 12-well plates were A negative normal control group (vehicle: 2% tragacanth solution 
treated with the EC50 of test samples (AGB, oleanonic acid, and IGF-1) [1 mL]) was also created with normoglycaemic rats. The treatment was 
obtained from the glucose transport assay in C2C12 myotubes and started a day after diabetes was confirmed, and this was considered day 
rosiglitazone (10 µM) for 24 h. After incubation, the total RNA from the one (1) of the study which continued for 3 weeks [30]. 
cells were isolated using TRIzol (Invitrogen) in accordance with the 
manufacturer’s instructions. Thereafter, cDNA was synthesized using 2.6.4. Determination of glucose level and lipid parameters of experimental 
SuperScript first-strand synthesis kit (Invitrogen) and analysed by PCR animals 
using an Applied Biosystems 7500 Fast PCR machine. The primers used The plasma glucose concentrations of the experimental rats were 
were Glucose transporter-4 (GLUT-4): sense, 5′-CGG GAC GTG GAG CTG measured prior to administering the first dose and 6 h into the study 
GCC GAG GAG-3′; anti-sense, 5′-CCC CCT CAG CAG CGA GTG A-3′; from blood obtained from their tail veins after the application of 
phosphoinositide-3-kinase (PI3K): sense, 5′-TGA CGC TTT CAA ACG lignocaine. The Fasting blood glucose (FBG) levels were determined 
CTA TC-3′; antisense, 5′-CAG AGA GTA CTC TTG CAT TC-3′; and weekly for 21 days. 
peroxisomal proliferator-activated receptor-gamma (PPARγ): sense, The experiment was concluded on day 22 and the rats sacrificed by 
5′-GGA TTC ATG ACC AGG GAG TTC CTC- 3′; anti-sense, 5′-GCG GTC cervical decapitation in the morning under anaesthesia with ketamine 
TCC ACT GAG AAT AAT GAC-3′. For PCR reaction, cDNA mixture (1 μL) (24 mg/kg [i.p]). Blood samples were collected by cardiac puncture for 
was mixed with PCR reaction solution consisting of 10X PCR buffer, 10 analysis of lipid parameters which include total cholesterol (TC), triglyc-
pM of paired primers, 2 mM dNTP and 2 units of Taq polymerase. eride (TG), and high-density lipoprotein cholesterol (HDL-C) using the BS- 
200 biochemistry analyser (www.mindray.com). Low density lipoprotein 
cholesterol (LDL-C) was then determined by the Friedewald equation [31]. 
3
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
2.7. In silico predictions of the pharmacokinetic and toxicity profile of Table 2 
oleanonic acid Effect of A. genipiflora stem bark extract on the lipid parameters of diabetic rats.  
Experimental Lipid parameters (mg/dL) 
The pharmacokinetic and toxicity properties of oleanonic acid was Groups 
Total Triglycerides LDL-C HDL-C 
investigated using QikProp, admetSAR and ADMETlab [32,33]. Molec- cholesterol 
ular descriptors for drug-likeness investigated include molecular weight, 
molecular polar surface area (PSA), number of hydrogen bond acceptors Normal control 76.08 ± 2.42 70.94 ± 2.54 17.48 39.91 
± 1.49 ± 1.48 
and donors and partition coefficient (cLogP), solubility and number of Diabetic control 125.50 ± 1.40 147.20 ± 0.92 77.84 18.29 
primary metabolites. The ADME properties estimated were Caco-2 ± 2.01 ± 0.25 
permeability, blood brain barrier (BBB) penetration, cytochrome P450 AGB (100 mg/kg) 112.80 136.30 ± 2.61 69.20 24.64 
a a 
metabolism and inhibition and human oral absorption. Toxicity pre- ± 0.81 b ± 1.16 ± 0.92
AGB (200 mg/kg) 104.20 128.10 62.38 27.58 
diction included reproductive toxicity, genotoxicity, carcinogenicity, ± 2.67c ± 0.73a ± 3.29a ± 1.64a 
hepatotoxicity, Ames mutagenicity, and toxicity dose levels. AGB (400 mg/kg) 86.80 ± 2.04c 107.80 54.32 29.95 
± 5.03c ± 2.88c ± 1.43c 
c c 
2.8. Statistical analyses Glibenclamide 76.70 ± 2.57 95.75 ± 2.52 35.21 31.62 
(5 mg/kg) ± 4.62c ± 1.45c  
Data from the in vivo assays are expressed as mean ± SEM whiles a p < 0.01 
data from in vitro assays are displayed as mean ± SD. Analyses were b p < 0.05 
c
carried out using one-way analysis of variance (ANOVA) with GraphPad p < 0.001 compared to diabetic control. AGB: 70% ethanol stem bark extract 
for Windows version 8 (GraphPad Prism Software, San Diego, USA). of A. genipiflora. 
Multiple comparisons between treatment groups for in vivo assays were 
done using Dunnet’s post hoc test whereas Tukey’s post hoc test was throughout the 21-day period (Fig. 2). 
employed comparison between groups for in vitro experiments. p < 0.05 Values are expressed as mean ± SEM (n = 6), *** p < 0.001 
was considered statistically significant. compared to diabetic control. AGB: 70% ethanol stem bark extract of 
A. genipiflora. 
3. Results 
3.2.2. Effect of AGB on the lipid profile of experimental rats 
3.1. Acute toxicity studies The 70% ethanol stem bark extract of A. genipiflora (AGB) exhibited a 
significant (p < 0.05) hypolipidaemic activity on the lipid parameters of 
Experimental animals treated with 70% ethanol stem bark extract of the STZ-induced diabetic rats. High plasma levels of triglycerides (TG), 
A. genipiflora (AGB) up to 5 000 mg/kg and oleanonic acid up to total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) 
100 mg/kg did not exhibit any difference in body weight and food experimental observed in the diabetic rats were considerably (p < 0.05) 
intake compared to saline solution-treated animals. They also did not reduced whereas the low high-density lipoprotein cholesterol (HDL-C) 
demonstrate any changes in behavioural patterns. Furthermore, no gross appreciated significantly (p < 0.01). 
pathological differences were observed between the treated groups and 
the control. None of the treated animals (extract and compound) 3.3. In vitro hypoglycaemic activity of A. genipiflora stem bark extract 
exhibited any sign of toxicity 14 days after administration (data not (AGB) and oleanonic acid 
shown). 
3.3.1. Effect of AGB and oleanonic acid on the activities of α-glucosidase 
3.2. Antidiabetic activity of A. genipiflora stem bark extract (AGB) in α-amylase 
STZ-induced diabetic rats The ethanol stem bark extract of A. genipiflora (AGB) and oleanonic 
acid inhibited considerably the activities of intestinal α-glucosidase and 
3.2.1. Effect of AGB on the blood glucose of the diabetic rats pancreatic α-amylase. The extract demonstrated strong inhibitory ac-
Treating the diabetic rats with the stem bark extract of A. genipiflora tivities against the enzymes with IC50 values of 10.48 ± 1.39 µg/mL and 
(AGB) and glibenclamide resulted in a significant (p < 0.001) hypo- 14.51 ± 1.26 µg/mL against α-glucosidase and α-amylase respectively 
glycaemic effect which was dose and duration dependent compared to (Table 3) while oleanonic acid exerted a higher inhibitory activity 
the diabetic control group. AGB reduced the blood glucose concentra- against α-glucosidase (~5-fold higher than acarbose) than α-amylase. 
tion of the experimental animals by 41.36%, 32.52% and 17.41% at 400, However, the α-amylase inhibitory activity of oleanonic acid was lower 
200 and 100 mg/kg respectively 6 h into the study whereas glibencla- than acarbose respectively. 
mide caused a reduction of 49.26% by the same period (Table 1). The 
daily oral supplementation of the diabetic animals with AGB (100 – 
400 mg/kg) and glibenclamide (5 mg/kg) reduced drastically the fast-
ing blood glucose (FBG) of the diabetic rats bringing them below the 10 
mmmol/L threshold at 200 and 400 mg/kg at the end of the study while 
the FBG of the untreated diabetic rats remained persistently high 
Table 1 
Effect of A. genipiflora stem bark extract on the blood glucose of diabetic rats 
within 6 h of the study.  
Experimental Groups 0 h 6th hour Variation (%) 
Normal control – – – 
Diabetic control 28.90 ± 0.53 30.98 ± 0.43 -7.20 
AGB (100 mg/kg) 29.30 ± 0.64 24.20 ± 0.75 *** 17.41 
AGB (200 mg/kg) 28.20 ± 0.97 19.03 ± 0.47 *** 32.52 
AGB (400 mg/kg) 29.28 ± 0.63 17.17 ± 0.55 *** 41.34 
Glibenclamide (5 mg/kg) 30.15 ± 0.54 15.40 ± 0.94 *** 49.26  Fig. 1. Structure of oleanonic acid.  
4
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
Fig. 2. Effect of A. genipiflora stem bark extract on fasting blood glucose of diabetic rats. Values are expressed as mean ± SEM (n = 6). ** p < 0.01, *** p < 0.001 
compared to diabetic untreated group. AGB: 70% ethanol stem bark extract of A. genipiflora. 
However, oleanonic acid stimulated a higher increase (2.5-fold above 
Table 3 control) of the PPARγ gene transcripts than AGB (~1.7-fold above the 
Effect of A. genipiflora stem bark extract and oleanonic acid on carbohydrate control). Rosiglitazone on the other hand demonstrated the highest 
metabolising enzymes.  
upregulation of PPARγ gene transcripts with an increase of 3.0-fold 
Test samples IC50 above the control (Fig. 3). 
α-Glucosidase α-Amylase 
AGB 10.48 ± 1.39 µg/mL 14.51 ± 1.26 µg/mL 3.4. In vivo antidiabetic activity of oleanonic acid 
Oleanonic acid 36.52 ± 1.95 µM 105.84 ± 1.08 µM 
Acarbose 183.68 ± 1.49 µM 98.36 ± 1.08 µM 3.4.1. Effect on blood glucose of diabetic rats 
Values displayed as mean ± SD (n = 3). AGB: 70% ethanol stem bark extract of The activity of oleanonic acid (15 – 60 mg/kg) on the blood glucose 
A. genipiflora. of the diabetic animals prior to and 6 h after administration of the initial 
doses are presented in Table 5 whereas its effect on the FBG of the 
3.3.2. Effect of AGB and oleanonic acid on the glucose transport in C2C12 diabetic animals recorded weekly over the 21 days study are shown in  
myotubes and 3T3-L1 adipocytes Fig. 4. The compound reduced the blood glucose level of the experi-
The activity of the stem bark extract of A. genipiflora (AGB) and mental animals with a considerable (p < 0.05) reduction at 60 mg/kg 
oleanonic acid on the enhancement of glucose transport were evaluated 6 h after treatment with the first dose and continued to significantly 
in C2C12 myotubes and 3T3-L1 adipocytes. AGB and oleanonic acid (p < 0.05) decrease the FBG over time till the end of the study compared 
exerted considerable stimulation of glucose uptake above the control to the diabetic control group. 
with EC50 of 32.05 ± 0.58 µg/mL and 15.57 ± 0.93 µg/mL for AGB and 
16.28 ± 0.30 µM and 7.26 ± 0.88 µg/mL for oleanonic acid in in C2C12 3.4.2. Effect on the lipid profile of the diabetic animals 
myotubes and 3T3-L1 adipocytes, respectively. Their activities were Oleanonic acid demonstrated considerable anti-hyperlipidaemic ac-
however lower than the positive controls (Table 4). tivity in the diabetic rats reducing significantly (p < 0.001) the elevated 
plasma levels of TC, TG and LDL-C and at the same time producing a 
3.3.3. Effect of AGB and oleanonic acid on GLUT-4, PI3K and PPARγ significant (p < 0.01) increase in the plasma HDL-C levels mostly at 30 
transcripts expression and 60 mg/kg (Table 6). 
A. genipiflora stem bark extract (AGB) and oleanonic acid signifi-
cantly (p < 0.001) increased the expression GLUT-4 and PI3K genes 3.5. Predicted ADMET profile of oleanonic acid 
above the control which was statistically like IGF-1 and rosiglitazone. 
Their increase of GLUT-4 transcripts was 3.20- and 3.50-folds respec- Computer-aided analysis showed that oleanonic acid fell within the 
tively above the control whereas they increased PI3K transcripts by stipulated ranges of Lipinski’s rule howbeit with one violation, lip-
2.55- and 2.83-folds respectively when compared to the control. ophilicity (cLogP ≤5) (Table 7). The compound was predicted to be 
devoid of blood-brain permeability (BBB-). Although oleanonic acid was 
predicted to exhibit poor solubility, it was shown to have > 90% oral 
Table 4 
Effect of A. genipiflora stem bark extract and oleanonic acid on glucose uptake in absorption and great permeability. It could also undergo passive ab-
C2C12 myotubes and 3T3-L1 adipocytes.  sorption via P-glycoprotein however it was neither a P-glycoprotein 
substrate nor inhibitor. Apart from CYP450 3A4, oleanonic was found to 
Test samples EC50 be neither a substrate nor an inhibitor of Cytochrome P450 family of 
C2C12 myotubes 3T3-L1 adipocytes isozymes. In terms of its toxicity, the compound was predicted to 
AGB 32.05 ± 0.59 µg/mL 15.57 ± 0.93 µg/mL generally non-toxic possessing no hERG inhibitory potential, non- 
Oleanonic acid 16.28 ± 0.30 µM 7.26 ± 0.88 µM carcinogenicity, non-hepatotoxicity, and non-genotoxicity. However, it 
IGF-1 2.85 ± 0.11 nM – was predicted to be teratogenic (Table 8). 
Insulin – 0.76 ± 0.04 nM 
Values displayed as mean ± SD (n = 3). AGB: 70% ethanol stem bark extract of 
A. genipiflora. 
5
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
Fig. 3. Effect of A. genipiflora stem bark extract and oleanonic acid on a) GLUT-4 b) PI3K and c) PPARγ gene expression in C2C12 myotubes. Values are expressed as 
mean ± SD (n = 3). * * p < 0.01, *** p < 0.001 compared to control (medium). Ros: Rosiglitazone. AGB: 70% ethanol stem bark extract of A. genipiflora. 
patients. It continued to lower the fasting blood glucose (FBG) of the 
Table 5 diabetic animals throughout the study and at 200 and 400 mg/kg 
Effect of oleanonic acid on the blood glucose of diabetic rats after 6 h of initial 
treatment.  brought the FBG to below 10 mmol/L by the 21st day which was com-
parable to glibenclamide. 
Experimental Groups 0 h 6th hour Variation (%) Inhibiting the activity of carbohydrate metabolizing enzymes, 
Normal control 5.32 ± 0.46 5.70 ± 0.09 – α-glucosidase and α-amylase, and thereby delaying the digestion of di-
Diabetic control 30.40 ± 1.13 29.55 ± 1.56 2.80 etary carbohydrate to glucose has been shown as an effective method to 
Oleanonic acid (15 mg/kg) 30.05 ± 1.35 26.30 ± 1.82 12.48 hinder the progression to diabetes in pre-diabetic individuals and high- 
Oleanonic acid (30 mg/kg) 31.20 ± 1.58 24.81 ± 1.60 20.50 
Oleanonic acid (60 mg/kg) 30.70 ± 1.02 23.20 ± 1.51 * 24.43 risk persons as well as a way to prevent the development of diabetic 
Glibenclamide (5 mg/kg) 29.92 ± 1.26 16.81 ± 1.34 *** 43.80 complications [34]. The extract considerably inhibited the activities of 
Values are expressed as mean SEM (n 6), * p 0.05, *** p 0.001 α-glucosidase and α-amylase with IC50 of 10.48 ± 1.39 µg/mL and ± = < <
compared to diabetic control. 14.51 ± 1.26 µg/mL respectively. The inhibition of these enzymes by 
AGB could account for the observed hypoglycaemic activity of the 
4. Discussion extract in the diabetic rats and indicate the presence of plant constitu-
ents with such mechanisms of hypoglycaemic action within the extract. 
In our continuous search for alternative treatments for diabetes, we The tendency of AGB to promote the transport of glucose into the 
evaluated the hypoglycaemic and hypolipidaemic activities of the peripheral tissues as another possible mechanism of hypoglycaemic 
hydroethanolic stem bark extract of A. genipiflora (AGB) in rat experi- activity was evaluated in mouse C2C12 skeletal muscles and 3T3-L1 
mental model of diabetes using streptozotocin (STZ, 40 mg/kg) as the adipocytes. The extract significantly promoted the uptake of radioac-3
inducing agent. The extract exhibited a fast onset of action reducing tive [ H] 2-deoxyglucose into the cell lines with EC50 of 32.05 
drastically (p < 0.001) the elevated blood glucose levels of the diabetic ± 0.58 µg/mL and 15.57 ± 0.93 µg/mL in C2C12 myotubes and 3T3-L1 
rats by 17 – 42% at 100–400 mg/kg within 6 h indicating its tendency to adipocytes respectively. The considerably enhanced glucose uptake 
manage sudden spikes in blood glucose concentration in diabetic observed in the skeletal muscle cell line correlated with significant 
(p < 0.001) increase in the expression of glucose transporter-4 (GLUT-4) 
Fig. 4. Effect of oleanonic on fasting blood glucose of diabetic rats. Values are depicted as mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to 
diabetic untreated group. 
6
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
Table 6 and phosphoinositide-3-kinase (PI3K) transcripts which is characteristic 
Effect of oleanonic acid on the lipid profile diabetic rats.  of the classical pathway for insulin-dependent glucose uptake mecha-
Experimental Lipid parameters (mg/dL) nism where there is the translocation of GLUT-4 from the cytosol to the 
Groups membranes of peripheral cells via the activation of PI3-kinase [35]. AGB 
Total Triglycerides LDL-C HDL-C 
cholesterol also significantly (p < 0.01) increased the peroxisomal 
proliferator-activated receptor-gamma (PPARγ) transcripts. The activa-
Normal control 76.72 67.75 ± 2.00 17.51 40.56 
3.44 0.80 2.13 tion of this enzyme also results in the increased utilisation of glucose in ± ± ±
Diabetic control 123.90 150.90 ± 2.66 86.97 17.16 the periphery by the expression and translocation of GLUT-4 through an 
± 1.39 ± 1.70 ± 1.16 insulin-independent glucose uptake process [36]. Thus, the observed 
Oleanonic acid 120.00 145.30 ± 1.72 78.08 21.19 glucose uptake effect of AGB is because of its tendency to stimulate both 
(15 mg/kg) ± 2.48 ± 4.25 ± 0.56 insulin-dependent and independent glucose uptake mechanisms. This 
Oleanonic acid 115.40 134.20 70.16 23.94 
(30 mg/kg) ± 3.14 ± 2.19 *** ± 2.84 *** ± 0.46 ** makes the extract an effective alternative in the management of both 
Oleanonic acid 94.30 121.39 64.31 25.44 type 1 and type 2 diabetes mellitus. 
(60 mg/kg) ± 2.23 *** ± 4.72 *** ± 3.39 *** ± 1.55 *** Hyperlipidaemia remains the major risk factor in the development of 
Glibenclamide 80.14 83.44 36.54 29.47 hypertension, atherosclerosis, and other cardiovascular diseases [37]. 
(5 mg/kg) ± 2.08 *** ± 1.77 *** ± 1.90 *** ± 1.31 *** Thus, the reduction in total cholesterol (TC), triglycerides (TG) and 
* * p < 0.01, * ** p < 0.001 compared to diabetic control. low-density lipoprotein-cholesterol (LDL-C) is considered a suitable 
approach in preventing such diabetic cardiovascular complications. 
Elevated levels of serum TC, TG and LDL-C of the diabetic animals were 
Table 7 significantly (p < 0.01) reduced with a concomitant increase (p < 0.01) 
Molecular properties of oleanonic acid.  in high-density lipoprotein-cholesterol (HDL-C) levels upon treatment 
Descriptor Calculated value Standard value/range with AGB which can be attributed to the presence of hypolipidaemic 
Molecular weight 454.69 ≤ 500 compounds in the extract and enhancement in glucose homeostasis 
clogP 6.18 ≤ 5 through improved insulin secretion and action due to the continuous 
H-bond donor 1 ≤ 5 supplementation of A. genipiflora stem bark extract. The quick onset and 
H-bond acceptor 3 ≤ 10 
LogS -7.325 5.7 long duration of hypoglycaemic activity coupled with the observed > −
Polar surface area 54.37 140 Ӑ2 hypolipidaemic activity of AGB in the diabetic animals indicate that the 
No. of Primary metabolites 3 < 7  stem bark extract of A. genipiflora is a good therapeutic adjunct in the 
management for the diabetes. 
Phytochemical studies led to the purification of oleanonic acid from 
Table 8 the ethanol stem bark extract of A. genipiflora. Its hypoglycaemic activity 
Predicted pharmacokinetic and toxicity properties of oleanonic acid.  was then established through in vitro and in vivo experiments. Oleanonic 
Model Result Probability/ acid inhibited α-glucosidase and α-amylase activities with IC50 values of 
Value 36.52 ± 1.95 µM and 105.84 ± 1.08 µM, respectively. Molecular dock-
ing studies have shown that triterpenoids form stable binding in-
Absorption   
Blood-Brain Barrier BBB - 0.9970 teractions such as hydrogen bonds, hydrophobic interactions with 
Human Intestinal Absorption HIA + 0.9919 amino acids and some H-Pi interactions in non-polar amino acids in the 
Caco-2 Permeability Caco-2 + 0.5282 binding sites of α-glucosidase and α-amylase to exert their inhibitory 
P-glycoprotein Substrate Non-Substrate 0.6903 activities [38,39]. Thus, the more and stronger interactions the tri-
P-glycoprotein Inhibitor Non-inhibitor 0.8289 
Renal Organic Cation Transporter Non-inhibitor 0.9985 terpene has with the enzymes, the higher the inhibitory effect. This may 
Bioavailability (F ) F - 0.157 therefore explain the higher inhibitory effect of oleanonic acid on 20% 20% 
Distribution   α-glucosidase (~3 ×) than α-amylase. The inhibition of these enzymes 
Plasma Protein Binding High protein-bound 93.35% also shows that oleanonic can reduce effectively postprandial hyper-
Volume of Distribution Optimal VD 0.846 glycaemia and prevent diabetes-related complications. 
Fraction unbound in plasma Low Fu 3.899% 
Subcellular localization Lysosomes 0.6599 Oleanonic acid exerted significant ability to promote glucose uptake 
Metabolism   into the periphery with EC50 of 16.28 ± 0.30 µM and 7.26 ± 0.88 µM in 
CYP450 3A4 Substrate Substrate 0.6063 C2C12 myotubes and 3T3-L1 adipocytes respectively corroborating its 
CYP450 2D6 Substrate Non-substrate 0.8683 previously reported ability to enhance glucose uptake in L6 muscle cells 
CYP450 2C9 Substrate Non-substrate 0.6106 
CYP450 1A2 Inhibitor Non-inhibitor 0.8772 [22]. It also produced a corresponding increase in GLUT-4, PI3K and 
CYP450 3A4 Inhibitor Non-inhibitor 0.8315 PPARγ gene transcripts indicating that oleanonic acid promotes glucose 
CYP450 2C9 Inhibitor Non-inhibitor 0.8584 uptake through GLUT-4 translocation via both insulin and non-insulin 
CYP450 2C19 Inhibitor Non-inhibitor 0.8273 signalling pathways. As previously stated, glucose transport through 
CYP450 2D6 Inhibitor Non-inhibitor 0.9514 the insulin signalling pathway involves the activation of PI3K and the 
CYP Inhibitory Promiscuity Non- CYP Inhibitory 0.9345 
Promiscuity subsequent translocation of GLUT-4 from the cytoplasm to the cell 
Excretion   membrane in peripheral cells. On the other hand, activating PPARγ 
Clearance (CL) Low 2.818 through its agonists, the thiazolidinediones, also enhance glucose 
Half-life short half-life (<3 h) 0.181 transport through GLUT-4 translocation. Oleanonic acid stimulated a 
Toxicity   
AMES Toxicity Non-AMES toxic 0.8500 comparable increase in PPARγ transcripts to rosiglitazone confirming 
Human Ether-a-go-go-Related Gene Non-inhibitor 0.5098 the earlier findings of Petersen et al. (2011) [21] that the compound is 
Inhibition also a PPARγ agonist. 
Carcinogens Non-carcinogen 0.9303 Following the in vitro assays, the hypoglycaemic and anti- 
Teratogenicity Teratogenic 0.9333 hyperlipidaemic activity of oleanonic acid was evaluated in STZ- 
Hepatotoxicity Non-hepatotoxic 0.7000 
Acute Oral Toxicity III 0.7802  induced diabetic rats. The compound at the doses (15 – 60 mg/kg) 
administered demonstrated significant (p < 0.001) glucose-lowering 
activity and corrected to some extent the dyslipidaemia present in the 
animals at the end of the study by lowering the high levels of TC, TG and 
7
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
LDL-C and at the same time increasing HDL-C levels. Generally, the Acknowledgments 
overall antidiabetic activity of the A. genipiflora extract was higher than 
oleanonic acid indicating the contribution of unidentified active com- The authors wish to thank the technical staff of the staff of the animal 
pounds such as flavonoids, glycosides, sterols and other triterpenoids to house of the Department of Pharmacology, KNUST and the technical 
the bioactivity of the plant extract. These compounds may be more staff of the School of Pharmacy, UHAS for their assistance. 
potent than and/or act synergistically or additively with oleanonic acid 
which was isolated in this study. CRediT authorship contribution statement 
Interestingly oleanolic acid, the reduced derivative of oleanonic acid 
has also been shown to exert antidiabetic activity in rats [40]. Loza-R- Benjamin Kingsley Harley: Conceptualization, Data curation, 
odríguez et al. (2020) [41] revealed that oleanolic acid enhances Formal analysis, Investigation, Methodology, Project administration, 
GLUT-4 translocation through PPARγ/α expression to exert its Supervision, Validation, Writing – original draft, Writing – review & 
anti-hyperglycaemic activity in C2C12 myoblasts. Several plant tri- editing. Isaac Kingsley Amponsah: Writing – original draft, Writing – 
terpenoids have also been shown to possess hypoglycaemic activities review & editing. Inemesit Okon Ben: Data curation, Formal analysis, 
with some exhibiting similar mechanisms of action as oleanonic acid and Investigation, Methodology. Nana Ama Mireku-Gyimah: Formal 
its reduced derivative, oleanolic acid [42–44]. analysis, Writing – original draft, Writing – review & editing. Daniel 
In silico pharmacokinetics prediction showed that oleanonic acid was Anokwah: Data curation, Formal analysis, Investigation, Methodology, 
within stipulated ranges except for one violation to Lipinski’s rule, Writing – original draft. David Neglo: Writing – original draft. Cedric 
falling short of lipophilicity (cLogP = 6.18). The compound was found to Dzidzor K. Amengor: Formal analysis, Investigation, Writing – review 
lack the tendency to cross the blood brain barrier and therefore devoid & editing. Theophilus Christian Fleischer: Conceptualization, Formal 
of any central nervous system (CNS) activity. Although the poor solu- analysis, Methodology, Supervision, Writing – review & editing. 
bility and non-polar nature could impair intestinal absorption, oleanonic 
acid was predicted to be highly orally absorbed and possess great Competing Interest 
permeability which could be because of its tendency undergo absorption 
via transporter carriers such as the P-glycoproteins [45]. The authors wish to declare no competing interest. 
Toxicity wise, oleanonic was predicted to be generally non-toxic and 
non-lethal possessing no carcinogenicity, no hepatotoxicity and no Supplementary Materials 
genotoxicity. However, it was found to be exhibit teratogenic potentials 
and should not be given to, including the plant extract from which it was The NMR spectra of oleanonic acid are included in the supplemen-
isolated from, pregnant and lactating women [46]. The compound was tary data. 
found not to exhibit any hERG inhibitory potential and therefore would 
not interfere with cardiac conduction and function. The inhibition of Appendix A. Supporting information 
hERG is a red flag in drug development which must be assessed at the 
early stages of the drug development process [47]. Oleanonic acid was Supplementary data associated with this article can be found in the 
identified as a category III drug for its acute oral toxicity with LD50 online version at doi:10.1016/j.biopha.2022.112833. 
between 500 and 5000 mg/kg, far above doses employed in this study 
(15 – 60 mg/kg). This was confirmed in the acute toxicity studies where References 
treated animals even at 100 mg/kg did not exhibit any signs of toxicity. 
The present study has demonstrated the hypoglycaemic activity of [1] M.G. Tinajero, V.S. Malik, An update on the epidemiology of Type 2 diabetes: A 
the 70% ethanol stem bark extract of A. genipiflora (AGB) and its isolate, global perspective, Endocrinol. Metab. Clin. North Am. 50 (2021) 337–355, https://doi.org/10.1016/j.ecl.2021.05.013. 
oleanonic acid using in vitro and in vivo assays and given scientific [2] N.G. Forouhi, N.J. Wareham, Epidemiology of diabetes, Medicine 47 (2019) 22–27, 
credence to the use of the plant as an antidiabetic agent in traditional https://doi.org/10.1016/j.mpmed.2018.10.004. 
medicine as well as a potential source of novel compounds for the dis- [3] M.A.B. Khan, M.J. Hashim, J.K. King, R.D. Govender, H. Mustafa, J. Al Kaabi, 
Epidemiology of type 2 diabetes–global burden of disease and forecasted trends, J 
covery of new hypoglycaemic drugs. Epidemiol, Glob. Health 10 (2020) 107–111, https://doi.org/10.2991/jegh. 
k.191028.001. 
Conclusion [4] J.M. Pappachan, C.J. Fernandez, E.C. Chacko, Diabesity and antidiabetic drugs, 
Mol. Asp. Med 66 (2019) 3–12, https://doi.org/10.1016/j.mam.2018.10.004. 
[5] P. Saeedi, I. Petersohn, P. Salpea, B. Malanda, S. Karuranga, N. Unwin, S. Colagiuri, 
In conclusion, the antidiabetic activity of the ethanol stem bark L. Guariguata, A.A. Motala, K. Ogurtsova, J. Shaw, D. Bright, Global and regional 
extract of A. genipiflora and oleanonic acid in STZ-induced diabetic rats diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results 
has been demonstrated in this study. The extract and its constituent have from the International Diabetes Federation Diabetes Atlas, Diabetes Res. Clin. Pract. 157 (2019), 107843, https://doi.org/10.1016/j.diabres.2019.107843. 
also been shown to exert their hypoglycaemic effects through the pro- [6] C. Smith-Hall, H.O. Larsen, M. Pouliot, People, plants and health: a conceptual 
motion of glucose uptake in C2C12 myotubes and 3T3-L1 adipocytes framework for assessing changes in medicinal plant consumption, J. Ethnobiol. 
through both insulin-dependent and independent pathways; as well as Ethnomed. 8 (2012) 1–11, https://doi.org/10.1186/1746-4269-8-43. [7] R. Vinayagam, J. Xiao, B. Xu, An insight into anti-diabetic properties of dietary 
the inhibition of α-glucosidase and α-amylase. Thus, A. genipiflora and phytochemicals, Phytochem Rev. 16 (2017) 535–553, https://doi.org/10.1007/ 
oleanonic acid may be useful in the management of diabetes. Further s11101-017-9496-2. 
phytochemical studies could be carried out to unearth the other anti- [8] B. Jacob, R.T. Narendhirakannan, Role of medicinal plants in the management of diabetes mellitus: a review, 3 Biotech 9 (2019) 1–17, https://doi.org/10.1007/ 
diabetic constituents of the plant. The antidiabetic activity coupled with s13205-018-1528-0. 
the in silico studies also showed that oleanonic acid could serve as [9] R. Vinayagam, M. Jayachandran, S.S.M. Chung, B. Xu, Guava leaf inhibits hepatic 
template for further hypoglycaemic drug development. gluconeogenesis and increases glycogen synthesis via AMPK/ACC signaling 
pathways in streptozotocin-induced diabetic rats, Biomed. Pharmacother. 103 
(2018) 1012–1017, https://doi.org/10.1016/j.biopha.2018.04.127. 
Funding [10] H. Burkill, The useful plant of west tropical Africa, in: Families MR, Second ed., vol. 
4, Royal Botanical Gardens,, Kew, United Kingdom, 1997. 
[11] D. Anokwah, E. Asante-Kwatia, A.Y. Mensah, C.A. Danquah, B.K. Harley, I. 
The authors received no specific funding for this work. K. Amponsah, L. Oberer, Bioactive constituents with antibacterial, resistance 
modulation, anti-biofilm formation and efflux pump inhibition properties from 
Data availability Aidia genipiflora stem bark, Clin. Phytosci 7 (2021) 28, https://doi.org/10.1186/ 
s40816-021-00266-4. 
[12] B. Yang, M. Jin, L. Tong, Y. Chen, Isolation and identification of oleanonic acid 
No data was used for the research described in the article. from Patrinia scabiosaefolia, Zhong Yao cai 22 (1999) 23–24. 
8
B.K. Harley et al.                                                                                                                                                                                 B  i o m   e d  i c  in  e   &   P  h  a  r m   a c  o  t h e  r a  p  y 149 (2022) 112833
[13] H. Gao, H. Liu, T. Tang, X. Huang, D. Wang, Y. Li, P. Huang, Y. Peng, Oleanonic hyperglycaemia in streptozotocin-induced diabetic rats possibly via glucose uptake 
acid ameliorates pressure overload-induced cardiac hypertrophy in rats: The role of enhancement and α-amylase inhibition, Biomed. Pharmacother. 132 (2020), 
PKCζ-NF-κB pathway, Mol. Cell. Endocrinol. 470 (2018) 259–268, https://doi.org/ 110847, https://doi.org/10.1016/j.biopha.2020.110847. 
10.1016/j.mce.2017.11.007. [31] W.T. Friedewald, R.I. Levy, D.S. Fredrickson, Estimation of the concentration of 
[14] S. Begum, S.M. Raza, B.S. Siddiqui, S. Siddiqui, Triterpenoids from the aerial parts low-density lipoprotein cholesterol in plasma, without use of the preparative 
of Lantana camara, J. Nat. Prod. 58 (1995) 1570–1574, https://doi.org/10.1021/ ultracentrifuge, Clin. Chem. 18 (6) (1972) 499–502. 
np50124a014. [32] J. Dong, N.-N. Wang, Z.-J. Yao, L. Zhang, Y. Cheng, D. Ouyang, A.-P. Lu, D.-S. Cao, 
[15] E.M. Giner-Larza, S. Máñez, M.C. Recio, R.M. Giner, J.M. Prieto, M. Cerdá-Nicolás, ADMETlab: a platform for systematic ADMET evaluation based on a 
J.L. Rıós, Oleanonic acid, a 3-oxotriterpene from Pistacia, inhibits leukotriene comprehensively collected ADMET database, J. Chemin.-. 10 (2018) 29, https:// 
synthesis and has anti-inflammatory activity, Eur. J. Pharm. 428 (2001) 137–143, doi.org/10.1186/s13321-018-0283-x. 
https://doi.org/10.1016/S0014-2999(01)01290-0. [33] H. Yang, C. Lou, L. Sun, J. Li, Y. Cai, Z. Wang, W. Li, G. Liu, Y. Tang, admetSAR 2.0: 
[16] S. Ghosh, M.D. Sarma, A. Patra, B. Hazra, Anti-inflammatory and anticancer web-service for prediction and optimization of chemical ADMET properties, 
compounds isolated from Ventilago madraspatana Gaertn., Rubia cordifolia Linn. Bioinformatics 35 (2019) 1067–1069, https://doi.org/10.1093/bioinformatics/ 
and Lantana camara Linn, J. Pharm. Pharm. 62 (2010) 1158–1166, https://doi. bty707. 
org/10.1111/j.2042-7158.2010.01151.x. [34] U. Hossain, A.K. Das, S. Ghosh, P.C. Sil, An overview on the role of bioactive 
[17] Y.-M. Chiang, J.-Y. Chang, C.-C. Kuo, C.-Y. Chang, Y.-H. Kuo, Cytotoxic triterpenes α-glucosidase inhibitors in ameliorating diabetic complications, Food Chem. 
from the aerial roots of Ficus microcarpa, Phytochemistry 66 (2005) 495–501, Toxicol. 145 (2020), 111738, https://doi.org/10.1016/j.fct.2020.111738. 
https://doi.org/10.1016/j.phytochem.2004.12.026. [35] K. Haghani, S. Pashaei, S. Vakili, G. Taheripak, S. Bakhtiyari, TNF-α knockdown 
[18] B.N. Irungu, J.A. Orwa, A. Gruhonjic, P.A. Fitzpatrick, G. Landberg, F. Kimani, alleviates palmitate-induced insulin resistance in C2C12 skeletal muscle cells, 
J. Midiwo, M. Erdélyi, A. Yenesew, Constituents of the roots and leaves of Biochem. Biophys. Res. Commun. 460 (4) (2015) 977–982, https://doi.org/ 
Ekebergia capensis and their potential antiplasmodial and cytotoxic activities, 10.1016/j.bbrc.2015.03.137. 
Molecules 19 (2014) 14235–14246, https://doi.org/10.3390/ [36] R. Anandharajan, S. Jaiganesh, N.P. Shankernarayanan, R.A. Viswakarma, 
molecules190914235. A. Balakrishnan, In vitro glucose uptake activity of Aegles marmelos and Syzygium 
[19] C.S. Funari, Ld Almeida, T.G. Passalacqua, I. Martinez, D.L. Ambrosio, R.M. cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes, 
B. Cicarelli, D.H.S. Silva, M.A. Graminha, Oleanonic acid from Lippia lupulina Phytomedicine 13 (6) (2006) 434–441, https://doi.org/10.1016/j. 
(Verbenaceae) shows strong in vitro antileishmanial and antitrypanosomal activity, phymed.2005.03.008. 
Acta Amaz. 46 (2016) 411–416, https://doi.org/10.1590/1809-4392201600204. [37] G.D. Flora, M.K. Nayak, A brief review of cardiovascular diseases, associated risk 
[20] S. Begum, A. Ayub, B.S. Siddiqui, S. Fayyaz, F. Kazi, Nematicidal triterpenoids from factors and current treatment regimes, Curr. Pharm. Des. 25 (38) (2019) 
Lantana camara, Chem. Biodivers. 12 (2015) 1435–1442, https://doi.org/ 4063–4084, https://doi.org/10.2174/1381612825666190925163827. 
10.1002/cbdv.201400460. [38] J. Wang, J. Zhao, Y. Yan, D. Liu, C. Wang, H. Wang, Inhibition of glycosidase by 
[21] R.K. Petersen, K.B. Christensen, A.N. Assimopoulou, X. Fretté, V.P. Papageorgiou, ursolic acid: In vitro, in vivo and in silico study, J. Sci. Food Agric. 100 (3) (2020) 
K. Kristiansen, I. Kouskoumvekaki, Pharmacophore-driven identification of PPARγ 986–994, https://doi.org/10.1002/jsfa.10098. 
agonists from natural sources, J. Comput. Aided Mol. Des. 25 (2011) 107–116, [39] M. Shah, S. Bashir, S. Jaan, H. Nawaz, U. Nishan, S.W. Abbasi, S.B. Jamal, A. Khan, 
https://doi.org/10.1007/s10822-010-9398-5. S.G. Afridi, A. Iqbal, Computational Analysis of Plant-Derived Terpenes as 
[22] K. Kawabata, K. Kitamura, K. Irie, S. Naruse, T. Matsuura, T. Uemae, S. Taira, α-Glucosidase Inhibitors for the Discovery of Therapeutic Agents against Type 2 
H. Ohigashi, S. Murakami, M. Takahashi, Y. Kaido, B. Kawakami, Triterpenoids Diabetes Mellitus, S. Afr. J. Bot. 143 (2021) 462–473, https://doi.org/10.1016/j. 
isolated from Ziziphus jujuba enhance glucose uptake activity in skeletal muscle sajb.2021.09.030. 
cells, J. Nutr. Sci. Vitaminol. 63 (2017) 193–199, https://doi.org/10.3177/ [40] S. Dash, G. Pattnaik, N. Sahoo, B. Kar, The promising role of oleanolic acid in the 
jnsv.63.193. management of diabetes mellitus: A review, J. Appl. Pharm. Sci. 11 (09) (2021) 
[23] B.K. Harley, R.A. Dickson, I.K. Amponsah, R.A. Ngala, D. Berkoh, T.C. Fleischer, 149–157, https://doi.org/10.7324/JAPS.2021.110918. 
Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition [41] H. Loza-Rodríguez, S. Estrada-Soto, F.J. Alarcón-Aguilar, F. Huang, G. Aquino- 
and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes, Jarquín, Á. Fortis-Barrera, A. Giacoman-Martínez, J.C. Almanza-Pérez, Oleanolic 
Asian Pac, J. Trop. Biomed. 10 (9) (2020) 387–396, https://doi.org/10.4103/ acid induces a dual agonist action on PPARγ/α and GLUT4 translocation: A 
2221-1691.290129. pentacyclic triterpene for dyslipidemia and type 2 diabetes, Eur. J. Pharmacol. 883 
[24] B.K. Harley, R.A. Dickson, T.C. Fleischer, Antioxidant, glucose uptake stimulatory, (2020), 173252, https://doi.org/10.1016/j.ejphar.2020.173252. 
α-glucosidase and α-amylase inhibitory effects of Myrianthus arboreus stem bark, [42] A. Giacoman-Martínez, F.J. Alarcón-Aguilar, A. Zamilpa, S. Hidalgo-Figueroa, 
Nat. Prod. Chem. Res. 5 (2017) 273–279, https://doi.org/10.4172/2329- G. Navarrete-Vázquez, R. García-Macedo, R. Román-Ramos, J.C. Almanza-Pérez, 
6836.1000273. Triterpenoids from Hibiscus sabdariffa L. with PPARδ/γ dual agonist action: In 
[25] B.K. Harley, I.K. Amponsah, I.O. Ben, D.W. Adongo, N.A. Mireku-Gyimah, M. Vivo, in vitro and in silico studies, Planta Med 85 (05) (2019) 412–423, https:// 
K. Baah, A.Y. Mensah, T.C. Fleischer, Myrianthus libericus: Possible mechanisms of doi.org/10.1055/a-0824-1316. 
hypoglycaemic action and in silico prediction of pharmacokinetics and toxicity [43] P.B. Kasangana, T. Stevanovic, Studies of pentacyclic triterpenoids structures and 
profile of its bioactive metabolite, friedelan-3-one, Biomed. Pharmacother. 137 antidiabetic properties of Myrianthus genus, Studies in Natural Products, 
(2021), 111379, https://doi.org/10.1016/j.biopha.2021.111379. Chemistry 68 (2021) 1–27. 
[26] S. Das, F. Morvan, B. Jourde, V. Meier, P. Kahle, P. Brebbia, G. Toussaint, D. [44] S. Indu, P. Vijayalakshmi, J. Selvaraj, M. Rajalakshmi, Novel Triterpenoids from 
J. Glass, M. Fornaro, ATP citrate lyase improves mitochondrial function in skeletal Cassia fistula Stem Bark Depreciates STZ-Induced Detrimental Changes in IRS-1/ 
muscle, Cell Metab. 21 (6) (2015) 868–876, https://doi.org/10.1016/j. Akt-Mediated Insulin Signaling Mechanisms in Type-1 Diabetic Rats, Molecules 26 
cmet.2015.05.006. (22) (2021) 6812, https://doi.org/10.3390/molecules26226812. 
[27] M. Kleinert, C. Clemmensen, S.M. Hofmann, M.C. Moore, S. Renner, S.C. Woods, [45] D. Smith, P. Artursson, A. Avdeef, L. Di, G.F. Ecker, B. Faller, J.B. Houston, 
P. Huypens, J. Beckers, M.H. De Angelis, A. Schürmann, Animal models of obesity M. Kansy, E.H. Kerns, S.D. Kr̈amer, H. Lennern̈as, H. van de Waterbeemd, 
and diabetes mellitus, Nat. Rev. Endocrinol. 14 (2018) 140–162, https://doi.org/ K. Sugano, B. Testa, Passive lipoidal diffusion and carrier-mediated cell uptake are 
10.1038/nrendo.2017.161. both important mechanisms of membrane permeation in drug disposition, Mol. 
[28] V. Ramachandran, R. Saravanan, P. Senthilraja, Antidiabetic and Pharm. 11 (2014) 1727–1738, https://doi.org/10.1021/mp400713v. 
antihyperlipidemic activity of asiatic acid in diabetic rats, role of HMG CoA: in vivo [46] R.R.D. Moreira, F.R. Camargo, A.M. Quílez, L. Salgueiro, C. Cavaleiro, Medicinal 
and in silico approaches, Phytomedicine 21 (3) (2014) 225–232, https://doi.org/ plants in pregnancy and lactation: perception of the health risk and practical 
10.1016/j.phymed.2013.08.027. educational group in Araraquara, São Paulo State, Brazil, J. Gen. Pract. 2 (6) 
[29] R.A. Dickson, B.K. Harley, D. Berkoh, R.A. Ngala, N.A. Titiloye, T.C. Fleischer, (2014) 190, https://doi.org/10.4172/2329-9126.1000190. 
Antidiabetic and haematological effect of Myrianthus arboreus P. Beauv. Stem bark [47] P. Saxena, E.-M. Zangerl-Plessl, T. Linder, A. Windisch, A. Hohaus, E. Timin, 
extract in streptozotocin-induced diabetic rats, Int. J. Pharm. Sci. Rev. Res 7 (2016) S. Hering, A. Stary-Weinzinger, New potential binding determinant for hERG 
4812–4826, https://doi.org/10.13040/IJPSR.0975-8232.7(12).4812-4826. channel inhibitors, Sci. Rep. 6 (2016) 24182, https://doi.org/10.1038/srep24182. 
[30] B.K. Harley, R.A. Dickson, I.K. Amponsah, I.O. Ben, D.W. Adongo, T.C. Fleischer, 
S. Habtemariam, Flavanols and triterpenoids from Myrianthus arboreus ameliorate 
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