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Original Article
ARTICLE IN PRESS
doi:
10.25259/JHS-2024-10-15-R1-(1623)

Fatty Acids Component of Essential Oil from Alchornea Cordifolia Modulated Activities of α-Amylase and α-Glucosidase Involved in Postprandial Hyperglycaemia

, , , , , , , , ,
1Department of Environmental Health Science, Faculty of Health Sciences, National Open University of Nigeria, Abuja, Nigeria
2Center of Excellence in Functional Foods and Gastronomy, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand
3Department of Biosciences, University of Technology Malaysia
4Department of Science Technology, School of Science and Technology, The Federal Polytechnic Offa, Offa
5Department of Biological Sciences, Thomas Adewumi University, Oko-Irese
6Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Kwara, Nigeria

* Corresponding author: Dr. Oluwafemi Ayodeji Idowu, Department of Environmental Health Science, Faculty of Health Sciences, National Open University of Nigeria, Abuja, Nigeria. id4phemy@gmail.com

Received: , Accepted: ,
© 2025 Published by Scientific Scholar on behalf of Journal of Health and Allied Sciences NU
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Idowu OA, Eya CP, Ebhodaghe F, Saliu OA, Tijjani H, Yupanqui CT, et al. Fatty Acids Component of Essential Oil from Alchornea cordifolia Modulated Activities of α-Amylase and α-Glucosidase Involved in Postprandial Hyperglycaemia. J Health Allied Sci NU. doi: 10.25259/JHS-2024-10-15-R1-(1623)

Abstract

Objectives

Essential oils are well-known important natural components in cosmetics and perfumery products because of their rich antioxidant sources. Recently, studies have focused more on their pharmacological activities, including antidiabetic activity. The present study focused on the antioxidant and modulatory activities of the fatty acids’ component of essential oil from Alchornea cordifolia seed on key enzymes involved in postprandial hyperglycaemia.

Material and Methods

Extraction was carried out via hydro-distillation, and chemical components of the oil were profiled by gas chromatography-spectroscopy. Antioxidant activity, which employs 1,1-diphenyl-2-picryhydazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic (ABTS) acid scavenging assays, as well as the modulatory potential of the oil for α-amylase and α-glucosidase activities, were investigated at concentrations of 10-50 mg/mL.

Results

An oil yield of 0.38% was obtained by the end of extraction, comprising twenty-six (26) chemical constituents, with over 40% primarily being fatty acids. 9,12-Octadecadienoic (49.35%) and n-Hexadecanoic (29.74%) acids constituted the most predominant fatty acids of the oil and facilitated the inhibition of α-amylase and α-glucosidase activities, with the highest enzyme inhibition at 50 mg/mL. The fatty acids also contributed significantly (p < 0.05) to the dose-dependent antioxidant activity of the oil against DPPH and ABTS radicals.

Conclusion

Essential oil from Alchornea cordifolia seed demonstrated antioxidant activity and modulated the activities of α-amylase and α-glucosidase enzymes, primarily involved in postprandial hyperglycaemia. The essential oil’s fatty acids component, particularly 9,12-Octadecadienoic acid, enabled these actions. Thus, this component of the essential oil could serve as an alternative to synthetic antioxidants and as a potential candidate in the development of new drugs to regulate blood sugar.

Keywords

α-glucosidase
α-amylase
Alchornea cordifolia
Essential oil
Fatty acids

INTRODUCTION

Diabetes mellitus (DM) is among the severe metabolic diseases confronting the globe and is characterised by hyperglycaemia. It occurs when the pancreas cannot secrete insulin either in a sufficient amount (called Type I DM) or when the produced insulin is not utilised by the body cells (called Type II DM), thus causing the blood sugar level to spike (hyperglycaemia). Insulin is a peptide hormone secreted by the β-pancreatic islet cells, mainly to regulate blood sugar.[1] Another type of DM may emerge only in women, especially during pregnancy, called gestational DM. DM is known to cause about 1.5 million deaths annually.[2] As of 2023, the global prevalence of diabetes among different adult age brackets was put at 537 million, with the African region having the highest prevalence (60%).[2] This global figure is predicted to skyrocket to 643 million and 783 million in 2030 and 2045, respectively.[2] An estimate of 19 million adults across the African continent have been reported to be diagnosed with diabetes in 2019.[3] In 2021, the number in this region increased to 24 million, with a predicted figure of reaching 55 million by 2045 if no urgent attention and interventions are given.[2] In Nigeria, there is no specific database that reflects the exact prevalence of DM. However, the prevalence of DM among adults aged 20-69 years old in 2021 was estimated to be 3.7% by the International Diabetes Federation.[2] Contrarily, it is widely presumed that this figure on DM prevalence by IDF grossly under-reflects the actual burden of DM in Nigeria, given that the data were extrapolated from other countries. Some reports have documented the prevalence of DM to be between 2% and 12% across the country in recent times.[4,5] Poor lifestyle, poor feeding habits, and side effects associated with synthetic drugs employed in the treatment of diabetes, among other factors, have contributed to the burden of DM globally. The use of medicinal plants by people to treat different diseases has been a common practice for many decades because of their presumed accessibility, affordability, pharmacological active ingredients, and safety. In recent times, studies on the aromatic extracts of medicinal plants called essential oils (EOs) have demonstrated multiple beneficial therapeutic activities for several metabolic disorders, including DM. EOs are important aromatic substances primarily produced in plants to protect them from predators, such as pests, to attract pollinators and also constitute an essential immune system of plants. They are secreted in varying amounts as highly volatile substances by certain parts, such as leaves, seeds, flowers, berries, rhizomes, roots, bark, resin, petals, etc., of different plants. These volatile substances can be extracted by distilling or pressing the secretory organs of the plants where they are found. The peel of citrus plants, for instance, is cold-pressed while the stem, leaves, flowers, root, and some other parts of the plant may be distilled to extract the oils. All these processes produce aromatic concentrates (EOs), which are a good source of chemically active substances, such as flavonoids, terpenoids, fatty acids, etc., with therapeutic value.[6] Hitherto, in the cosmetic industry, EOs have been extensively employed as important ingredients in perfumes and other beauty products as natural preservative agents and antioxidants for skin care. Recently, research on the pharmacological attributes of EOs has gained focus. EOs of different plants have been reported to demonstrate anti-diabetic effects. For instance, lime (Citrus aurantifolia) oil has been shown to have an antihyperglycemic effect by significantly reducing the fasting blood and hepatic glucose in an animal model.[7] Also, EOs of different Pinus species demonstrated antidiabetic activity by inhibiting α-glucosidase activity in vitro.[8] The combined administration of EOs from Carum carvi and Coriandrum sativum seeds has demonstrated better antidiabetic activity.[9] In addition to the antidiabetic effects, EOs have demonstrated other therapeutic effects. EOs from Citrus aurantifolia leaf have been shown to be an excellent antibacterial and anti-pathogenic agent against Staphylococcus aureus and Escherichia coli strains.[10] Citrus sinensis EOs exhibited antioxidant and antipathogenic activities against food spoilage bacteria.[11] According to Othman et al.,[12] four different species of Citrus plant (C. limon, C. hystrix, C. pyriformis, and C. microcarpa) leaves contained EOs with antioxidant, anti-proliferative, and anti-cancer activities. EO constituents of Tasmannia lanceolata also exhibit acaricidal and insecticidal activities.[13] Mahendran and Vimolmangkang[14] reported that EOs of Ocimum americanum and Ocinum basilicum possessed antimicrobial and mosquito larvicidal activities. A study on Mentha piperita showed that this plant contained EO with anti-Alzheimer’s potential.[15] Bialon et al.[16] recommended lemon EOs as an effective remedy against candidiasis caused by C. albicans. Posadzki et al.[17] also reported that Eucalyptus odorata oil demonstrated antibacterial effects and was strong cytotoxic against S. aureus, H. influenzae, S. pyogenes, and S. pneumonia. Alchornea cordifolia, commonly known as Christmas bush and belonging to the Euphorbiaceae family, is an evergreen shrub that perennially grows up to 4-8 m high. Alchornea cordifolia is widely distributed in sub-Saharan Africa, including Congo, Ivory Coast, Ghana, and Nigeria.[18] In Nigeria, A. cordifolia is indigenously called Ewe-eepa/Esinsin/Ipa by the Yoruba tribe, Ububo by the Igbo tribe, and Sambami by the Hausa tribe (the major three tribes of Nigeria). The plant parts (leaves, roots, and stem bark) made into different decoctions are commonly used in ethnomedicine throughout its areas of distribution to treat wounds, ulcers, diarrhoea, respiratory problems, venereal diseases, and female infertility.[19,20] These parts of A. cordifolia contain saponins, terpenoids, flavonoids, tannins, glycosides, carbohydrates, gallic acid, ellagic acid, gentisic acid, anthranilic acid, protocatechuic acid, imidazopyrimidine, and guanidine alkaloids like alchorneine and alchornidine.[19,21,22] Moreover, different extracts of the plant, particularly the leaf, root, and stem, are documented in literature by many studies to demonstrate different biological activities, including antidiabetic activity.[22-28] Conversely, the antidiabetic potential of the essential oil of A. cordifolia seed, also considered to be medicinal, is yet to be fully explored. Thus, this study focuses on the antioxidant and antidiabetic effects of EO from Alchornea cordifolia seeds as a potential candidate for the development of anti-diabetic drugs from plant origins.

MATERIAL AND METHODS

Sample collection

Fresh fruits of Alchornea cordifolia were harvested from the premises of Oduduwa University, Osun State, Nigeria, in the early hours (9 a.m.) on the 15th of March, 2024. Authentication of the plant was carried out at the Botany Department, University of Obafemi Awolowo, Ile-Ife, Osun State, Nigeria, where a specimen number (Ife-17942) was registered.

Extraction of oil

The seeds of the harvested fruits of A. cordifolia were removed and air-dried at room temperature (25°C) for 14 days and later oven-dried for 24 hours at 30°C to prevent the loss of active ingredients. The dried seeds were powdered mechanically using a blender. The resulting powder (200 g) was subjected to steam distillation (oil extraction) with n-hexane (500 mL) as the organic solvent for 7 hours using a Soxhlet apparatus. The oil extract was concentrated using a rotary evaporator at 60°C to remove the solvent. The resulting oil extract was weighed to know the yield and thereafter was transferred into a properly labelled dark amber glass bottle to prevent oxidative degradation and rancidity of the oil and was stored in a refrigerator at 4oC until required for further analysis.

Profiling of the essential oil of A. cordifolia by GC-MS

The extracted oil was analysed for its chemical components using a gas chromatography mass spectroscopic (GC-MS) analyser of a 7890A model Agilent gas chromatographic system with a 5975C mass selective detector (MSD) and 7693 series autosampler loaded with data system (Santa Clara, CA, United States). An HP-5ms fused silica capillary with 5% phenyl-methyl polysiloxane stationary phase of 30 m x 0.25 µm film thickness was employed as the GC column. The carrier gas was helium, having a column head pressure of 7.07 psi and a flow rate of 1 mL/min. Inlet and MSD temperatures of 200°C and 280°C, respectively, were employed. The GC oven temperature program initially used was 40°C for 10 minutes, and then it was increased by 3°C/min to 200°C and again by 2°C/min to 220°C. The oil was made to dissolve in chloroform, and 1 µL of the oil sample was placed into the machine via an injection technique. The EO components were profiled and identified according to the individual retention index with reference to a homologous series of normal alkanes and were compared to their mass spectrum using the Chem Station data system, the National Institute of Standards and Technology (NIST), and literature.

Antioxidant determination

DPPH scavenging activity assay

The antioxidant activity of the essential oil against 1,1-diphenyl-2-picryhydazyl (DPPH) was carried out in a 96-well microplate at concentrations of 10-50 mg/mL. The reagents for the DPPH assay were tested in a 96–well microplate, and the procedure is stated as follows: 20 µL of the oil at the different concentrations was mixed with 180 µL of DPPH solution (0.1 mM). It was shaken and incubated in the dark at room temperature for 30 minutes. Aluminium foil was used to protect the sample mixture from light photo-degradation. Butylated hydroxyl toluene (BHT) was used as the antioxidant reference compound at the same concentrations (10-50 mg/mL) with the oil sample. The absorbance was read at 517 nm. This process was repeated three times, and the inhibition of the DPPH radical by the oil sample was calculated in percentage using the formula: % &nbsp;inhibition&nbsp;of&nbsp;DPPH = [ Absorbance&nbsp;of&nbsp;control – Absorbance&nbsp;of&nbsp;sample / Absorbance&nbsp;of&nbsp;control ] × 1 00

ABTS (2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)) scavenging activity assay

The ABTS radical scavenging assay for the oil was carried out in a 96-well microplate by modifying the procedure of Re et al.[29] The ABTS radical was initially generated by oxidising the ABTS with potassium persulfate. Briefly, the ABTS (7 mM) solution was made to react with potassium persulfate solution (2.45 mM) and then incubated in the dark for 12 hours until a dark-coloured solution containing the ABTS radical was formed. Before the commencement of the assay, 50% of methanol was used to dilute the ABTS+ radical for an initial absorbance of about 0.70 (±0.02) at 745 nm, controlled at 30°C. ABTS radical scavenging activity was evaluated by the mixture of 3 mL of ABTS with 300 μL of the oil at varying concentration (10-50 mg/mL) in the well plate. Reduction in the absorbance was read at 60-second intervals following the mixture of the solution for 6 minutes. This procedure was replicated three times. BHT of the same concentrations (10-50 mg/mL) with the oil sample was used as the antioxidant reference compound. The inhibition of the ABTS radical was calculated in percentage by the expression: % &nbsp;inhibition&nbsp;of&nbsp;ABTS = [ Absorbance&nbsp;of&nbsp;control – Absorbance&nbsp;of&nbsp;sample / Absorbance&nbsp;of&nbsp;control ] × 1 00

Antidiabetic assay

α-Amylase (EC 3.2.1.1) assay

The α-amylase inhibitory activity of Alchornea cordifolia EO was determined according to the modified assay described by.[30] Different concentrations of the EO (10, 20, 30, 40, and 50 mg/ml) were prepared in dimethyl sulfoxide (DMSO). EO (250 μL) of each concentration and 250 μL α-amylase of the different concentrations were dispensed into test tubes and incubated (first incubation) at room temperature for 10 minutes. Following the initial incubation, 250 µL of 1% starch solution was mixed with 0.02 M sodium phosphate buffer (pH 6.9) in each tube interval, and the reaction was halted by adding 100 µL of dinitro salicylic acid reagent. The test tubes were incubated for the second time in a hot water bath for 5 minutes and cooled at 25°C room temperature. The reaction mixture was later diluted with 5 mL of distilled water, and the absorbance was taken at 540 nm. The absorbance taken was compared with the control containing DMSO and buffer only and not the oil extract. Acarbose at concentrations of 10, 20, 30, 40, and 50 mg/mL was used as a reference (positive) test to replace the oil extract. This assay was carried out in triplicates for accuracy. The α-amylase inhibitory activity of the EO was calculated using the formula below: Inhibition   ( % ) = [ ( A c + ) &nbsp; − &nbsp; ( A c − ) ] − [ A s − A b ] [ ( A c + ) − ( A c − ) ] × 100

Ac+ = enzyme activity absorbance at 100%

Ac- = enzyme activity absorbance (only solvent without enzyme) at 0%,

As = absorbance of oil sample with enzyme,

Ab = absorbance of blank (oil sample without enzyme)

α-Glucosidase (E.C. 3.2.1.20) assay

α-glucosidase inhibitory activity of EOs of A. cordifolia was determined in a 96-well microplate using the method described by Asghari et al.[31] with some modifications. The 96-well micro plate consisted of 250 µL phosphate buffer (0.02M; pH 6.9); 250 µl of (0.3 U/mL) α-glucosidase (Sigma Aldrich Chemical Co, USA), and 250 µL each of different concentrations (10-50 mg/mL) of the oil extract and acarbose (reference drug), both prepared in DMSO, as the reaction mixture was incubated for 15 minutes at 37oC. A 100 µL of 2.5 mM p-nitrophenyl-α-d-glucopyranoside (pNPG) as substrate was added to each well of the reaction mixture and again incubated at 37oC for ten minutes. The reaction was stopped with the addition of 100 µL sodium carbonate (0.1 M). DMSO was used as the blank control in the tubes together with the enzyme and substrate, while acarbose at concentrations of 10-50 mg/mL was used to replace the oil extract as the positive control. The activity of the enzyme was considered proportional to the absorbance of the resulting pNPG at 405 nm. The procedure assay was repeated three times, and % inhibition was calculated as expressed below: Inhibition   ( % ) = absorbance   of   the   control   blank − absorbance   of   the   oil   extract absorbance   of   the   control   blank × 100

Statistical analysis

All data were represented in triplicate and expressed as mean ± standard error of mean (SEM). Data were subjected to statistical analysis using SPSS version 20. Values with p < 0.05 were considered statistically different.

RESULTS

Percentage yield and gas chromatographic analysis of Alchornea cordifolia seed oil constituents

The percentage yield of the EO extracted from 200 g of powdered seed of Alchornea cordifolia was 0.38%. The chromatogram of the relative abundance of the EO constituents of Alchornea cordifolia seed and their respective retention times has been presented in Figure 1.

Gas chromatogram of essential oils from Alchornea codifolia seeds.
Figure 1:
Gas chromatogram of essential oils from Alchornea codifolia seeds.

Twenty-six chemical constituents were identified via GC-MS analysis in the extracted oil of A. cordifolia. The compounds present include groups of fatty acids, alcohols, esters, ketones, ethers, and hydrocarbons. However, only seven of the identified compounds were richly present and they include: 9,12-octadecadienoic (49.35%), n-hexadecenoic acid (29.74%), cyclopropane (6.08%), 2-dimethylsilyloxytetradecane (3.46%), 2-buten-1-ol (3.28%), thiirane (2.45%), and propane nitrile (2.21%), while phthalic acid (0.40%), 1-butanol (0.17%), 1-pentatonic acid (0.15%), octanoic acid (0.13%), oleic acid (0.10%), and others were present only in minute traceable amounts as shown in Table 1. Their chemical structures are given in Figure 2.

Table 1: Chemical constituents of Alchornea cordifolia seed oil
S/N Compound RT (min) Area %
1 L-Methioninol 2.400 0.03
2 Hydrazinecarbothiomide 2.543 0.01
3 Ethanone 2.669 0.01
4 2-Dimethylsilyloxytetradecane 3.601 3.46
5 2-Buten-1-ol 3.789 3.28
6 Thiirane 3.963 2.45
7 Cyclopropane 4.558 6.08
8 Pentanoic acid (valeric acid) 5.196 0.15
9 1-Butanol 5.346 0.17
10 Peroxide 5.677 0.55
11 Propane nitrile 5.990 2.21
12 Pentane 6.728 0.17
13 Hexanoic acid (caproic acid) 7.216 0.09
14 3-Buten-1-ol 7.498 0.01
15 Butanedioic acid (succinic acid) 8.286 0.10
16 4-amino-3-mercapto-1,2,4-triazole 8.567 0.06
17 Octanoic acid (caprylic acid) 9.850 0.13
18 1,4-Benzenediamine 10.488 0.03
19 2-Decenal 10.713 0.02
20 1,5-Hexadiene 11.082 0.01
21 2-Pentanone 11.295 0.03
22 Decanoic acid (capric acid) 11.601 0.03
23 Oleic acid 12.977 0.10
24 Phthalic acid 14.235 0.40
25 n-Hexadecanoic acid (palmitic acid) 16.080 29.74
26 9,12-Octadecadienoic (linolenic acid) 18.050 49.35

RT: Retention time

Chemical structures and formulae of the major oil extract constituents from A. cordifolia seed.
Figure 2:
Chemical structures and formulae of the major oil extract constituents from A. cordifolia seed.

Antioxidant and anti-diabetic activity

The antioxidant activity of seed EO from A. cordifolia against DPPH and ABTS radicals has been elucidated in Figures 3 and 4, respectively. The oil showed a concentration-dependent inhibitory effect on both DPPH and ABTS radicals like the expression noticed for BHT, a synthetic antioxidant employed as the reference antioxidant agent for this study, i.e., the higher the concentration, the higher the percentage inhibition and vice-versa. The oil extract demonstrated better activity in scavenging both radicals than BHT at all concentrations (10, 20, 30, 40, and 50 mg/mL) investigated with the highest inhibitory effects of 90% and 85%, respectively, at 50 mg/mL.

Inhibitory effect of oil extract from Alchornea codifolia seed on DPPH radical values are the mean of three replicates with significant difference at p<0.05. DPPH: 1,1-diphenyl-2-picryl-hydrazyl-hydrate, EOAC: Essential oil of Alchornea cordifolia, BHT: Butylated hydroxytoulene.
Figure 3:
Inhibitory effect of oil extract from Alchornea codifolia seed on DPPH radical values are the mean of three replicates with significant difference at p<0.05. DPPH: 1,1-diphenyl-2-picryl-hydrazyl-hydrate, EOAC: Essential oil of Alchornea cordifolia, BHT: Butylated hydroxytoulene.
Inhibitory effect of oil extract from Alchornea cordifolia seed on ABTS radical Values are mean of three replicates with significant difference at p<0.05. ABTS: 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), EOAC: Essential oil of Alchornea cordifolia, BHT: Butylated hydroxytoulene.
Figure 4:
Inhibitory effect of oil extract from Alchornea cordifolia seed on ABTS radical Values are mean of three replicates with significant difference at p<0.05. ABTS: 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), EOAC: Essential oil of Alchornea cordifolia, BHT: Butylated hydroxytoulene.

The effects of the seed EO of A. cordifolia on α-amylase and α-glucosidase activities are presented in Figures 5 and 6. The oil also showed concentration-dependent inhibitory effects on α-amylase and α-glucosidase activities in vitro, similar to acarbose employed as the reference drug. Though acarbose exhibited stronger inhibitory action on both enzymes’ activity than the oil extract, nevertheless, the seed EO of A. cordifolia was also able to express an active inhibitory effect on α-glucosidase than α-amylase at all concentrations investigated (10, 20, 30, 40, and 50 mg/mL) in this study. At 50 mg/mL, which was the highest investigated concentration, the percentage inhibitory effect of the seed oil on α-amylase and α-glucosidase activities was 65.14% and 72.30%, respectively, while that of acarbose was 92.11% and 98.64%, respectively.

Inhibitory effect of oil extract from Alchornea cordifolia seed on α-amylase activity Values are mean of three replicates with significant difference at p<0.05. EOAC: Essential oil of Alchornea cordifolia.
Figure 5:
Inhibitory effect of oil extract from Alchornea cordifolia seed on α-amylase activity Values are mean of three replicates with significant difference at p<0.05. EOAC: Essential oil of Alchornea cordifolia.
Inhibitory effect of oil extract from Alchornea cordifolia seed on α-glucosidase activity Values are mean of three replicates with significant difference at p<0.05. EOAC: Essential oil of Alchornea cordifolia seed.
Figure 6:
Inhibitory effect of oil extract from Alchornea cordifolia seed on α-glucosidase activity Values are mean of three replicates with significant difference at p<0.05. EOAC: Essential oil of Alchornea cordifolia seed.

DISCUSSION

The findings from this study showed 0.38% oil yield from A. cordifolia seed powder, indicating that 0.76 g of the EO was contained in a total volume of 500 ml of hexane extract of the seed plant, corresponding to a mass of 0.152 g oil extract from 100 mL, or 152 mg/100 mL. A total of 26 chemical constituents were identified in the EO, with 9,12-octadecadienoic acid present in relative abundance. The oil extract also demonstrated antioxidant activity against DPPH and ABTS radicals and modulated α-amylase and α-glucosidase enzymes involved in postprandial hyperglycaemia. The percentage yield of A. cordifolia seed oil (0.38%) from our study was higher than the 0.17% w/w oil yield earlier reported by Essien et al.[32] In addition, Essien et al.[32] identified 38 constituents in the 0.17% w/w oil yield of A. cordifolia seeds. This conversely is higher than the 26 chemical constituents identified in this current study despite the plant used in both studies were harvested within the same geographical (south-west) region of Nigeria. This variation may be attributed to the ecological factors, time of harvesting, type of solvent used, methods, and length of oil extraction employed, among others.[33,34] Nevertheless, our study showed similarity with Essien et al.[32] as the EO components from both studies belong to the same groups of terpenoids, fatty acids, and hydrocarbons. 9,12-octadecadienoic acid, which was more abundant than other chemical compounds in the seed oil of A. cordifolia from our study, belongs to the class of polyunsaturated fatty acids (PUFAs). 9,12-octadecadienoic acid is commonly called linoleic acid and is a PUFA with the double bonds located at the 9th and 12th carbon positions of the fatty acid, while n-hexadecenoic acid, the second most prominent chemical compound found in abundance, is commonly called palmitic acid and belongs to the class of saturated fatty acids. Previous studies have also reported an appreciable amount of PUFAs in the seed oil of A. cordifolia and other species of the plant.[26,32,35] This suggests that A. cordifolia is a rich source of PUFAs. The liver is a major site for metabolism; therefore, regulating key hepatocyte metabolic enzymes, including those involved in glucose uptake and oxidation, is crucial for the management of metabolic disorders such as DM. PUFAs such as 9,12-octadecadienoic acid, which is the major constituent of A. cordifolia seed EO, are known to regulate genes that encode glycolytic and lipogenic enzymes as well as other key metabolic enzymes involved in glucose and fatty acid oxidation.[36] In addition, PUFAs with multiple bonds in their structures assist in intracellular state stability, thus promoting the well-being of the body, especially the heart, and reducing the risk of cardiovascular diseases and their complications. Thus, PUFAs are capable of ameliorating hyperlipidaemic and hyperglycaemic conditions. For instance, the anti-hyperlipidaemic activity of the ethanolic leaf extract of A. cordifolia found to contain linoleic acid (9, 12-octadecadienoic acid) has been reported.[22] Free radicals or reactive oxygen species are usually unstable molecules that react with biological components to gain stability and subsequently induce oxidative stress, which leads to cell, protein, and Deoxyribonucleic acid (DNA) damage. Oxidative stress has been implicated in the pathogenesis of many diseases.[37] DM is linked to oxidative stress-mediated complications as well as hyperglycaemia, which are regarded as critical underlying factors in the pathogenesis of the disease.[38-40] Agents that can scavenge free radicals play a crucial role in the treatment and prevention of diabetes and its related complications. Antioxidant agents act to combat oxidative stress by reacting with the free radicals through electron donation, thus preventing their radical stability. It is evident from this study that the oil of A. cordifolia seed demonstrated antioxidant activity by its ability to scavenge DPPH and ABTS radicals higher than the reference antioxidant compound, BHT, at the different concentrations (10-50 mg/mL) investigated and in dose dependent. An earlier study by Ekeleme et al.[22] also reported dose-dependent antioxidant activity for both DPPH and ABTS radicals by A. cordifolia ethanol leaf extract. DPPH and ABTS assays are among the most common assays employed when investigating an agent for its antioxidant potential. DPPH radicals are usually characterised by a purple colouration, which, in the presence of an antioxidant, easily decolorises to yellow, appearing as (2,2-diphenyl-1-picrylhydrazine - reduced form of DPPH) in non-radical form.[41] It is worth noting that nearly 40% of the chemical constituents of A. cordifolia seed oil in this study were fatty acids. This corroborates earlier reports that fatty acids are among the abundant chemical constituents in A. cordifolia.[42] The fatty acid component of the oil, especially 9, 12-octadecadienoic acid, could have facilitated this activity, owing to its high relative abundance in the oil. The antioxidant activity of 9, 12-octadecadienoic acid has also been reported.[43-45] The antioxidant mechanism of this fatty acid is yet to be fully understood. However, one possible antioxidant mechanism of linolenic acid may be through its multiple bonds, capable of creating intracellular stability as a chemical compound by donating hydrogen atoms to acceptors like reactive oxygen species or free radicals to reduce their radical potentiality. The double bonds in the molecular structure of PUFAs, such as 9,12-octadecadienoic, are responsible for most of their biological activities.[46] Hexadecenoic acid (palmitic acid) has also been reported to demonstrate antioxidant activities.[47,48] The synergistic effect of 9, 12-octadecadienoic acid and hexadecenoic acid and may likely also drive the antioxidant activity exhibited by the oil. Other biological activities of 9, 12-octadecadienoic acid (linoleic acid) and hexadecenoic acid (palmitic acid) have also been documented.[47-51] One of the strategies adopted in the management of DM involves inhibiting α-amylase and α-glucosidase, which are carbohydrate-hydrolysing enzymes.[52] α-amylase, an enzyme located in the oral cavity and intestine, is the first major enzyme that initiates the hydrolysis or breakdown of carbohydrates, more specifically starch, into disaccharides, while α-glucosidase acts on the glycosidic bonds in disaccharides and hydrolyses them to monosaccharides. Both α-amylase and α-glucosidase are responsible for postprandial hyperglycaemia, and their inhibitions have been proven to lower postprandial glucose levels.[52,53] Therefore, inhibitors of α-amylase and α-glucosidase will help slow down the digestion of glucose, reduce glucose absorption, and assist in the management of hyperglycaemia and diabetes. It is possible that the fatty acids, particularly 9,12-octadecadienoic (linoleic acid), may be responsible for the anti-diabetic activity demonstrated by the seeds’ EO from A. cordifolia through the inhibition of α-amylase and α-glucosidase enzymes acting in synergy with other fatty acid components like n-hexadecenoic acid. Shettar et al.[54] have also identified 9,12-octadecadienoic and n-hexadecenoic acids as major chemical constituents in Ximenia americana chloroform and aqueous extracts profiled with GC-MS in an anti-diabetic study. Their findings revealed that the aqueous extract of X. americana, which contained 9,12-octadecadienoic as the only compound, demonstrated the strongest inhibitory activity on α-amylase and also led to delayed starch digestion with over 67% glucose uptake in yeast cells than the chloroform extract, which contained 9,12-octadecadienoic and n-hexadecenoic acid as part of the major constituents of the chloroform. It is interesting to note from this current study that the EO of A. cordifolia seed demonstrated a better inhibitory effect on α-glucosidase activity than α-amylase activity at all the concentrations investigated, with inhibitory activity of over 60% recorded at the lowest concentration (10 mg/mL) for α-glucosidase activity compared to α-amylase activity at the same concentration. The higher inhibitory activity exerted by the oil on alpha-glucosidase activity suggests a synergistic effect of the fatty acids’ components, majorly 9,12-octadecadienoic and n-hexadecanoic acids, resulting in higher inhibition of the enzyme. The low inhibitory activity on α-amylase by the oil extract suggests antagonistic effects of these constituents. This similar mechanism perhaps played out in the earlier report of Shettar et al.[54] where the chloroform extract of Ximenia americana containing both 9,12-octadecadienoic and n-hexadecenoic acids demonstrated lower α-amylase inhibitory activity than the aqueous extract, which contained only 9,12-octadecadienoic, although the authors did not determine the α-glucosidase inhibitory activity of the extracts in their study. Furthermore, in an in-silico study carried out by Ekeleme et al.[22] linoleic acid demonstrated a stronger binding affinity for the protein α-glycosidase (-4.5 kcal/mol) than α-amylase (-3.9 kcal/mol). It possibly means that the major target point of fatty acids in regulating blood sugar is likely at the point of conversion of disaccharide sugars to monosaccharides during carbohydrate digestion, which solely is the responsibility of α-glucosidase at this stage. This, therefore, suggests that fatty acids are better inhibitors of α-glucosidase than α-amylase, as evident in this study since the oil demonstrated a better inhibitory effect on α-glucosidase than α-amylase. Another possible mechanism corroborating the antidiabetic effect demonstrated by the essential oil of A. cordifolia seed is the antioxidant effect since the oil was also effective in scavenging free radicals capable of causing oxidative stress, which has been implicated in the pathogenesis of diabetes. More recently, studies have proven that PUFAs like linoleic acid in trace amounts protect against insulin resistance, atherosclerotic processes and regulate glucose metabolism.[55-57] In addition, some effects of PUFAs are mediated by preventing the reduction in palmitic acid-mediated adenosine monophosphate-activated protein kinase (AMPK) activity, resembling the action of metformin, a reference drug widely employed in diabetic studies.[58] Earlier studies by Eliakim-Ikechukwu and Obri[24] and Mohammed et al.[26] also accounted for the antidiabetic activity of the n-butanol fraction and ethanolic extract of Alchornea cordifolia leaves. The antimicrobial and cytotoxic activity of thiirane, which is one of the chemical constituents also identified in A. cordifolia seed oil in this present study, has been established.[59] Chai et al.[60] reported that plant extracts with cytotoxic actions are useful in the treatment of diabetes. Arguably, this is the first study to report the antidiabetic activity of the seed EO of A. cordifolia in modulating α-amylase and α-glucosidase activities involved in postprandial hyperglycaemia, thus suggesting the plant as a potential therapeutic natural source for the management of DM.

CONCLUSION

This study revealed that EO of the Alchornea cordifolia seed exerted inhibitory effects on α-glucosidase and α-amylase activities, which may be attributed to its high concentration of fatty acids, especially 9,12-octadecadienoic (linoleic acid), thus suggesting it as a potential candidate in developing novel drugs to control blood sugar in diabetic conditions. We recommend the active principles be isolated and an in vivo study be carried out to substantiate its antidiabetic candidature further.

Acknowledgment

The authors appreciate the Nigeria Tertiary Education Trust Fund (TETfund) board for providing the grant to carry out this research under the 2024 Institutional Based Research Grant.

Ethical approval

The research/study approved by the University Research Ethics Committee of the National Open University of Nigeria with an issuance number ETC/2024/NOUN/04/010, dated, 12th October 2024.

Declaration of patient consent

Patient’s consent not required as patients identity is not disclosed or compromised.

Financial support and sponsorship

The authors appreciate the Nigeria Tertiary Education Trust Fund (TETfund) board for providing the grant to carry out this research under the 2024 Institutional Based Research Grant.

Conflicts of interest

There are no conflicts of interest.

Use of artificial intelligence (AI)-assisted technology for manuscript preparation

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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