Inhibition of key enzymes linked to diabetes by Annona senegalensis Pers (Annonaceae) leaf in vitro

Accepted Manuscript Title: Inhibition of Key Enzymes Linked to Diabetes by Annona senegalensis Pers (Annonaceae) Leaf In vitro Authors: Auwal Ibrahim, Isma’ila Alhaji Umar, Idowu A. Aimola, Aminu Mohammed PII: DOI: Reference:

S2210-8033(18)30059-9 https://doi.org/10.1016/j.hermed.2018.11.004 HERMED 248

To appear in: Received date: Revised date: Accepted date:

14 November 2017 20 April 2018 19 November 2018

Please cite this article as: Ibrahim A, Umar IA, Aimola IA, Mohammed A, Inhibition of Key Enzymes Linked to Diabetes by Annona senegalensis Pers (Annonaceae) Leaf In vitro, Journal of Herbal Medicine (2018), https://doi.org/10.1016/j.hermed.2018.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of Key Enzymes Linked to Diabetes by Annona senegalensis Pers (Annonaceae) Leaf In vitro

Auwal Ibrahim, Isma’ila Alhaji Umar, Idowu A. Aimola, and Aminu Mohammed*

IP T

Department of Biochemistry, Faculty of Life Sciences, Ahmadu Bello University, Zaria,

SC R

Nigeria.

*Corresponding author Dr. Aminu Mohammed,

U

Department of Biochemistry, Faculty of Life Sciences,

N

Ahmadu Bello University, Zaria,

A

Nigeria.

M

[email protected]

ED

+2348061333502

PT

Abstract

Annona senegalensis Pers (Annonaceae) known as Wild Custard Apple is used locally in the

CC E

treatment of diabetes in Nigeria. This study was aimed at investigating the inhibitory potential of A. senegalensis leaf extracts and fractions on the activities of some enzymes (α-amylase and

A

α-glucosidase) linked to diabetes. Plant samples were extracted with n-hexane (HEX), ethyl cetate (EtOAc) and ethanol (EtOH) and the extracts were subjected to in vitro antidiabetic studies. The most active extract was further fractionated using column chromatography and the fractions obtained were screened for the inhibitory activities whilst the possible bioactive compounds were determined by Gas Chromatography Mass Spectroscopy (GC-MS). From the

1

results, ethanolic extract possessed lowest IC50 values (α-amylase: 204.04 ± 6.38 μg/ml, αglucosidase: 97.91 ± 2.40 μg/ml) compared to other extracts. The most active fraction (Ffraction) from the ethanolic extract showed lower IC50 values for α-amylase (237.14 ± 31.19 μg/ml) and α-glucosidase (88.25 ± 0.59 μg/ml). The data further showed that F-fraction is a competitive inhibitor (Vmax: 27.03 μmol/min, Km: 0.24%, ki value: 8.46 μg/ml) for α-amylase

IP T

and non-competitive inhibitor for α-glucosidase (Vmax: 1.10 μmol/min, Km: 3.7 mmol/l, ki value: 1.26 μg/ml). Possible compounds revealed by GC-MS from F-fraction were hexadecanoic acid,

SC R

methyl ester, 1,3-octadecenal, and 1,2-benzenedicarboxylic acid, and bis (2-methylpropyl)

ester. Therefore, our present data showed that A. senegalensis showed inhibitory potentials on

U

the activities of α-amylase and α-glucosidase, attributed to the possible presence of identified compounds.

M

A

N

Keywords: Annona senegalensis, diabetes, α-amylase, α-glucosidase, leaf

1.1 Introduction

ED

Diabetes mellitus (DM) is a metabolic disorder linked to defective insulin secretion, insulin insensitivity and/or both. It is characterized by chronic hyperglycemia with multiple

PT

etiological complications and is associated with alterations in the metabolisms of carbohydrate,

CC E

fat and protein. (ADA, 2014). Recent information showed the global prevalence of DM to be 415 million people and is estimated to double by 2040 (Nasli-Esfahani et al. 2017). This placed DM as a global public health challenge which thus, requires an holistic approach to fully

A

identify the best treatment. Presently, there are various treatment targets that are used for the management of DM

and its associated complications. One of such treatment targets is the control of postprandial hyperglycemia via inhibition of α-glucosidase and α-amylase actions in the early stage of diabetes. α-Amylase located in the pancreatic juice and saliva breaks down insoluble starch 2

molecules into soluble disaccharides and oligosaccharides (Norton et al. 2008). Similarly, αglucosidase found in the mucosal brush border of the small intestine catalyzes the digestion of dietary starch and disaccharides into absorbable monosaccharides (Sukumaran et al. 2016). Therefore, inhibitors of α-amylase and α-glucosidase delay carbohydrate digestion to absorbable monosaccharides in the small intestine and diminish the postprandial blood glucose

IP T

excursion (Zhang et al., 2017). However, the use of synthetic α-glucosidase and α-amylase inhibitors such as acarbose and miglitol have some adverse consequences, such as diarrhea and

SC R

abdominal pain (Naveen and Baskaran, 2017). For these reasons several studies are ongoing to discover plant-derived extracts or active ingredients with inhibitory potential on key enzymes

U

linked to diabetes, since fewer side effects were reported on the use of plant-derived products

N

for the treatment of various diseases (Atanasov et al. 2015).

A

Despite several existing approaches employed to select plant-derived products for

M

pharmacological screening, selection based on traditional usage has been widely utilized by most scientists and pharmaceutical companies (Cartwright and Matthews, 2016). Interestingly,

ED

Annona senegalensis Pers (Annonaceae) is among the most promising plants used locally for DM treatment in Nigeria and other African countries (Etuk and Mohammed, 2009; Karou et

PT

al. 2011; Ahombo et al. 2012; Diallo et al. 2012; Laleye et al. 2015; Shinkafi et al. 2015). In Nigeria, a polyherbal antidiabetic remedy (ADD-199) containing A. senegalensis leaf extract

CC E

as an active ingredient was reported to ameliorate hyperglycemia and diabetic-induced complications (Okine et al. 2005). More recently, Chedi et al (2017) have reported the

A

antidiabetic effect of A. senegalensis stembark ethanol extract in alloxan induced diabetic rats. In addition, other Annona species such as A. squamosa L. and A. muricata L. were reported to possess blood glucose lowering potential in diabetic rat models (Gupta et al. 2005; Kaleem et al. 2006; 2008; Florence et al. 2014). Furthermore, previous studies have shown that various A. senegalensis leaf extracts possessed anticonvulsant (Konate et al. 2012), antimicrobial (Jada

3

et al. 2014), antioxidant (Ajboye et al. 2010; Potchoo et al. 2008), anti-inflammatory and analgesic activities (Suleiman et al. 2014; Yeo et al. 2011). However, the scientific data to validate the traditional claim of A. senegalensis leaf in DM treatment is not yet available either in vitro, in vivo or in human subjects. Therefore, the present study aimed to investigate the inhibitory potential of A. senegalensis leaf on some key

IP T

enzymes linked to diabetes as an initial step to validate the ethnobotanical usage of the plant in

N

U

SC R

DM treatment.

A

2 Materials and Methods

M

2.1 Chemicals and Reagents

Saccharomyces cerevisiae α-glucosidase (E.C. 3.2.1.20), porcine pancreatic α-amylase 3.2.1.1),

p-nitrophenyl-α-d-glucopyranoside

ED

(E.C.

(pNPG),

p-nitrophenol,

starch,

dinitrosalicylic acid (DNS) and maltose were obtained from Sigma-Aldrich through Bristol

CC E

PT

Scientific Company Limited, Lagos, Nigeria.

2.2 Plant Material

A fresh sample of the A. senegalensis was obtained from Mando, Igabi, Kaduna State

A

in the month of June, 2016. The sample was identified at the herbarium unit of Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher specimen (1709) was deposited in the herbarium. The leaf of the A. senegalensis was plucked from the stem, washed and then dried under shade. The dried sample was then grounded into powder in a laboratory

4

using a mortar and pestle. The powdered sample was kept in a closed plastic container and used for the subsequent experiment. 2.3 Extraction and fractionation of the sample The dried powdered (25 g) leaf of A. senegalensis was extracted separately for 48 h in 500 ml of 3 different solvents (n-hexane (HEX), ethyl acetate (EtOAc) and ethanol (EtOH)),

IP T

after which were filtered using Whatmann filter paper. The HEX, EtOAc and EtOH extracts were concentrated using a rotary evaporator.

SC R

2.4 α-Amylase (E.C. 3.2.1.1) inhibitory effects

The α-amylase inhibitory effect of the extracts, fractions or acarbose (anti-diabetic drug

U

used to treat diabetes mellitus type 2) was carried out using a modified method of McCue and

N

Shetty (2004). Briefly, a 250 µl aliquot of the extracts, fractions or acarbose at different concentrations (30, 60, 120 and 240 µg/ml) was placed in a tube and 250 µl of 0.02 M sodium

M

A

phosphate buffer (pH 6.9) containing α-amylase (2.0 U/ml) solution was added. This solution was pre-incubated at 25°C for 10 min, after which 250 µl of 1% starch solution in 0.02 M

ED

sodium phosphate buffer (pH 6.9) was added at a time interval of 10 sec and then further incubated at 25°C for 10 min. The reaction was terminated after incubation by adding 1 ml of

PT

dinitrosalicylic acid (DNS) reagent. The tube was then boiled for 10 min and cooled to room temperature. The reaction mixture was diluted with 5 ml distilled water and the absorbance was

CC E

measured at 540 nm using a Shimadzu UV mini 1240 spectrophotometer. A control was prepared using the same procedure, replacing the extracts/fractions/acarbose with distilled

A

water.

The results of the α-amylase assay were expressed as a percentage of the control (blank)

according to the following formula: % Inhibition = [(Abs of control-Abs of compound)/Abs of control] x 100. 2.5 α-Glucosidase (E.C. 3.2.1.20) inhibitory effects

5

The inhibitory effect of the plant extracts, fractions or acarbose on α-glucosidase activity was determined according to the method described by Kim et al. (2005) using αglucosidase from Saccharomyces cerevisiae. The substrate solution 0.5 mM pnitrophenylglucopyranoside (pNPG) was prepared in 20 mM phosphate buffer, pH 6.9. An aliquot of 500 µl of α-glucosidase (1.0 U/ml) was then pre-incubated with 250 µl of the

IP T

different concentrations of the extracts, fractions or acarbose (30, 60, 120 and 240 µg/ml) for 10 min. Thereafter, a 250 µl of 0.5 mM pNPG was added as substrate to start the reaction. The

SC R

reaction mixture was incubated at 37°C for 30 min. The α-glucosidase activity was determined by measuring the yellow coloured p-nitrophenol released from pNPG at 405 nm.

U

The results of the α-glucosidase assay were expressed as a percentage of the control

N

(blank) according to the following formula:

M

2.6 Fractionation of crude ethanol extract

A

% Inhibition = [(Abs of control-Abs of sample)/Abs of control] x 100.

The extract with the highest inhibitory action (EtOH extract (20 g)) was subjected to

ED

column chromatography on a Merck silica gel 60 (0.040–0.063 mm) using solvent systems of increasing polarity HEX:EtOAc and EtOAc:MeOH with increments of 10% in each step to

PT

afford 85 fractions. After monitoring the TLC, fractions with similar profiles were pooled together to afford 7 major fractions (A: 11-13; B: 14-17; C: 20-25; D: 30-32; E: 33-34; F: 35-

CC E

47 and G: 48-55) that exhibited considerable inhibitory effect against α-amylase and αglucosidase actions.

A

2.7 Mechanism of α-glucosidase and α-amylase inhibitions The most active fraction was subjected to kinetic experiments to determine the type of

inhibition exerted on α-glucosidase and α-amylase. The experiment was conducted according to the protocols mentioned above at a constant concentration of the fractions (240 µg/ml) with a variable concentration of substrate. For the α-glucosidase inhibition assay, 0.313-5.0 mmol/l

6

of pNPG was used and 0.063-1.0% of starch was used for the α-amylase inhibition assay. The initial rates of reactions were determined from calibration curves constructed using varying concentrations of p-nitrophenol (0.313-5.0 mmol/l) and maltose (0.063-1.0%) for the αglucosidase and α-amylase inhibition assays, respectively. The initial velocity data obtained were used to construct Lineweaver-Burke’s plot to determine the KM (Michaelis constant) and

IP T

Vmax (maximum velocity) of the enzyme as well as the ki (inhibition binding constant as a

2.8 Gas Chromatography-Mass spectrometry analysis

SC R

measure of affinity of the inhibitor to the enzyme) and the type of inhibition for both enzymes.

The F-fractions that shows higher α-amylase and α-glucosidase inhibitory activity was

U

subjected to Gas Chromatography-Mass spectrometry (GCMS) analysis (Model; QP 2010

N

series, Shimadzu, Tokyo, Japan) equipped with a VF-5ms fused silica capillary column of 30

A

m length, 0.25 mm diameter, and 0.25 mm film thickness. The carrier gas was ultra-pure helium

M

at a flow rate of 0.7 ml/min and a linear velocity of 37 cm/s. The injector temperature was set at 250 °C. The initial oven temperature was 60 °C which was programmed to 280 °C at the rate

ED

of 10 °C/min with a hold time of 3 min. Injections of 2 µL were made in the split less mode with a manual split ratio of 20:1. The mass spectrometer was operated in the electron ionization

PT

mode at 70 eV and electron multiplier voltage at 1859 V. Other MS operating parameters were

CC E

as follows: ion source temperature 230 °C, quadrupole temperature 150 °C, solvent delay 4 min and scan range 50-700 amu. The spectrum of the compounds was compared with the

A

spectrum of National Institute of Standard and Technology (NIST) library database. 2.9 Statistical analysis The results of triplicate analysis are presented as mean ± standard deviation (SD). The analysis of variance (ANOVA) (SPSS version 20.0) was used to compare the means of the groups. Post-hoc test was performed by using Tukey’s HSD Multiple Range; a probability level of less than 5% (P˂ 0.05) was considered as significant. 7

3. Results and Discussion 3.1 Yield recovery The results for the yield of extracts recovered are presented in Table 1. According to the data, EtOH extract was found to have significantly (p< 0.05) higher percentage yield than

IP T

other extracts. However, HEX extract was observed to have the least recovery (Table 1). The

selection of the solvents for the extraction was based on the data obtained from the traditional

SC R

herbalist that A. senegalensis is administered in form of tincture or decoction, which has been

documented previously (Ahombo et al. 2012). Moreover, based on our recent search A.

U

senegalensis alcoholic extracts showed greater promising potentials compared to the water

N

extract (More et al. 2008; Yeo et al. 2011; Suleiman et al. 2014). This could be attributed to

A

the nature of alcohol polarity that allows the extraction of both polar and non-polar ingredients

M

compared to either strongly polar or non-polar solvents. Occasionally this increases the phytoconstituents and extraction yield as well, leading to higher pharmacological activity

ED

which is in line with our present data (Table 1).

3.2 Inhibition of α-amylase and α-glucosidase action by the extracts/fractions

PT

The data on the inhibition of α-amylase and α-glucosidase action by various extracts

CC E

derived from A. senegalenses leaf is presented in Figure 1 and Table 2. From the results, EtOH extract showed the lowest IC50 compared to other extracts though significantly (p< 0.05) higher compared to acarbose. This indicates greater inhibitory potential which apparently signifies the

A

higher availability of active ingredients with inhibitory potentials in the EtOH extract. Interestingly, the IC50 value depicted by the EtOH extract for α-glucosidase inhibitory effect was significantly (p< 0.05) lower compared to the acarbose indicating higher inhibitory activity than acarbose (Table 2). This is of pharmacological interest as greater inhibitory activity of αglucosidase action is preferred. This leads to the lower availability of the absorbable 8

monosaccharides which may limits the intestinal glucose absorption and thus, reduced postprandial hyperglycemia. Furthermore, HEX extract exhibited (p< 0.05) weak αglucosidase inhibitory action (Figure 1 and Table 2). Fractionation of the crude EtOH extract by column chromatography generated seven pooled fractions (A-G) which were investigated for their inhibitory potentials and the results

IP T

are presented in Figure 2 and Table 3. It was observed that fraction-F (217 mg) had shown the lowest IC50 values against the activities of α-amylase and α-glucosidase actions compared to

SC R

other solvent fractions (Figure 2 and Table 3). Fraction-F was obtained within HEX:EtOAc 2:8

ratio, implying that the possible inhibitory ingredients are likely volatile in nature. This

U

apparently indicates that the active ingredients with enzyme inhibitory potentials from the crude EtOH extract are more readily available in the fraction-F. Therefore, fraction-F was

A

N

selected to investigate the mode of action towards the α-amylase and α-glucosidase inhibition

3.3 Mode of action of fraction-F

M

and the possible compounds.

ED

The data on the mode of inhibition of fraction-F on α-amylase and α-glucosidase actions are presented in Figure 3 and Table 4. According to the data, fraction F exhibited a competitive

PT

mode of inhibition on α-amylase action with unchanged Vmax (27.03 min/μmol) and increased Km (0.24%) compared to the control Km value of 0.11% (Figure 3 and Table 4). This implied

CC E

that fraction-F compounds(s) competed with the active site of α-amylase and reduced the activity of the enzyme. However, fraction-F showed a non-competitive mode of inhibition on

A

α-glucosidase action (Figure 3 and Table 4). The Km value was unchanged (3.7%) when the Vmax increased from 1.10 to 2.30 (min/μmol) of the control and the inhibitory constant (ki) was found to be 1.26 (Table 4). Hence, it is suggested that possible compound(s) in fraction-F bind to other site(s), apart from the active site of α-glucosidase and induced conformational change in the three-dimensional structure of the enzyme and thus inhibits its activity. The equilibrium

9

constant for inhibitor binding (ki) of the fraction-F were lower for α-glucosidase than αamylase, indicating greater stability of the enzyme-substrate complex for α-glucosidase compared to α-amylase (Table 4). 3.4 Possible compounds present in fraction-F The GC-MS data of the most active fraction-F is presented in Figure 4 and Table 5.

IP T

From the results, several peaks were visible from the chromatogram and the possible compounds were detected and compared with data on the NIST library. Detected compounds

SC R

detected were volatile aromatics (1, 2-benzenedicarboxylic acid, buty l octyl ester and 1,2-

benzenedicarboxylic acid, bis (2-methylpropyl) este) and fatty esters (hexadecanoic acid,

U

methyl ester and methyl 9-methyltetradecanoate) which are components of essential oils (Table

N

5). Interestingly, essential oils or some of its active ingredients such as 1, 2-

A

benzenedicarboxylic acid, buty l octyl ester and hexadecanoic acid, methyl ester were shown

M

to reduce hyperglycemia and lessen diabetes-related complications (Jung et al. 2009; Tahir et al. 2016; Al-Hajj et al. 2016). Therefore, despite the limitation of the present study to

ED

specifically isolate the active compounds(s) responsible for the observed inhibitory effects of fraction-F, the authors postulate that this activity is attributed to either individual or combined

CC E

PT

action of the compounds detected.

4. Conclusion

The present data showed that A. senegalensis leaf extract possessed comparatively

A

moderate inhibitory potentials toward α-amylase and α-glucosidase actions, attributed to the detected compounds. Therefore, further work is recommended via other mechanisms to fully validate the traditional usage of A. senegalensis in the treatment of diabetes.

Declaration of interest 10

We declare that we have no conflict of interest.

Reference

IP T

Ahombo, G., Raoul, A.M.P.A., Diatewa, M., Mpati, J., Abena, A.A., Ouamba, J.M., 2012. Investigating on related diabetes therapeutic plants used in traditional medicine at Brazzaville. J. Med. Plant Res. 6, 5630-5639.

SC R

Ajaiyeoba, E., Falade, M., Ogbole, O., Okpako, O., Akinboye, D., 2006. In vivo antimalarial

and cytotoxic properties of Annona senegalensis extract. Afr. J. Trad. CAM. 3, 137-

U

141.

N

Ajboye, T., Yakubu, M., Salau, A., Oladiji, A., Akanji, M., Okogun, J., 2010. Antioxidant and

A

drug detoxification potential of aqueous extract of Annona senegalensis leaves in

M

carbon tetrachloride-induced hepatocellular damage. Pharm. Biol. 48, 136-170. Al-Hajj, N.Q.M., Algabr, M., Sharif, H.R., Aboshora, W., Wang, H., 2016. In vitro and in

ED

vivo evaluation of antidiabetic activity of leaf essential oil of Pulicaria inuloidesAsteraceae. J. Food Nutr Res. 4, 461-470.

PT

American Diabetes Association. 2014. Diagnosis and classification of diabetes mellitus.

CC E

Diabetes Care. 37, S81-S90. Atanasov, A.G., Waltenberger, B., Pferschy-Wenzig, E.M., Linder, T., Wawrosch, C., Uhrin,

A

P., Temml, V., Wang, L., Schwaiger, S., Heiss, E.H., Rollinger, J.M., Schuster, D., Breuss, J.M., Bochkov, V., Mihovilovic, M.D., Kopp, B., Bauer, R., Dirsch, V.M., Stuppner, H., 2015. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv. 33, 1582-1614.

Cartwright, A.C., Matthews, B.R., 2016. International Pharmaceutical Product Registration, 200, CRC Press. 11

Chedi, B.A., Abubakar, J.G., Adoum, O.A. 2017. Acute toxicity and antihyperglycaemic effect of ethanol stem-bark extract of Annona senegalensis Pers.(Annonaceae) on alloxan induced diabetic rats. Afr. J. Pharmacol. Ther. 6, 155-160. Cos, P., Vlietinck, A.J., Berghe, D.V., Maes, L., 2006. Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of-concept’. J. Ethnopharmacol.

IP T

106, 290-302.

Diallo, A.A., Traore, M.S., Keita, S.M., Balde, M.A., Keita, A., Camara, M., van Miert, S.,

SC R

Pieters, L., Balde, A.M., 2012. Management of diabetes in Guinean traditional

medicine: An ethnobotanical investigation in the coastal lowlands. J. Ethnopharmacol.

U

144, 353-361.

N

Etuk, E.U., Mohammed, B.J., 2009. Informant consensus selection method: A reliability

A

assessment on medicinal plants used in north western Nigeria for the treatment of

M

diabetes mellitus. Afr. J. Pharm. Pharmacol. 3, 496-500. Florence, N.T., Benoit, M.Z., Jonas, K., Alexandra, T., Désiré, D.D.P., Pierre, K., Théophile,

ED

D. 2014. Antidiabetic and antioxidant effects of Annona muricata (Annonaceae), aqueous extract on streptozotocin-induced diabetic rats. J. Ethnopharmacol, 151, 784-

PT

790.

Gertsch, J., 2009. How scientific is the science in ethnopharmacology? Historical perspectives

CC E

and epistemological problems. J. Ethnopharmacol. 122, 177-183.

Gupta, R.K., Kesari, A.N., Murthy, P.S., Chandra, R., Tandon, V., Watal, G. 2005.

A

Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. J. Ethnopharmacol, 99, 75-81.

Jada, M., Usman, W., Adamu, Y., 2014. In vitro antimicrobial effect of crude tannins isolated from the leaf of Annona senegalensis. Int. J. Biochem. Res. Rev. 4, 615-623.

12

Jung, C.S., Kim M.J, Jin H.H., Kim J.K., Jin Jun W., Kim H.K., Kim E.K., Ok K.M., Yon C.H., Hwang H.J., Jun K.Y., Shin D.H., 2009. Ameliorative effect of 1, 2benzenedicarboxylic acid dinonyl ester against amyloid beta peptide-induced neurotoxicity. Amyloid. 16, 15-24. Kaleem, M., Asif, M., Ahmed, Q.U., Bano, B., 2006. Antidiabetic and antioxidant activity of

IP T

Annona squamosa extract in streptozotocin-induced diabetic rats. Singapore Med. J. 47, 670-675

SC R

Kaleem, M., Medha, P., Ahmed, Q.U., Asif, M., Bano, B., 2008. Beneficial effects of Annona

squamosa extract in streptozotocin-induced diabetic rats. Singapore Med J. 49, 800-

U

804.

N

Karou, S.D., Tchacondo, T., Tchibozo, M.A.D., Abdoul-Rahaman, S., Anani, K., Koudouvo,

A

K., Batawila, K., Agbonon, A., Simpore, J., de Souza, C., 2011. Ethnobotanical study

M

of medicinal plants used in the management of diabetes mellitus and hypertension in the Central Region of Togo. Pharm. Biol. 49, 1286-1297.

ED

Kim, Y.M., Jeong, Y.K., Wang, M.H., Lee, W.Y., Rhee, H.I., 2005. Inhibitory effects of pine bark extract on alpha-glucosidase activity and postprandial hyperglycemia. Nutrition.

PT

21, 756-761.

Konate, A., Sawadogo, W., Dubruc, F., Caillard, O., Ouedraogo, M., Guissou, I., 2012.

CC E

Phytochemical and anticonvulsant properties of Annona senegalensis Pers. (Annonaceae), plant used in Burkina folk medicine to treat epilepsy and convulsions.

A

Br. J Pharmacol Tox. 3, 245-250.

Laleye, F. O. A., Mensah, S., Assogbadjo, A. E., Ahissou, H., 2015. Diversity, knowledge, and use of plants in traditional treatment of diabetes in the republic of Benin. Ethnobot. Res. Applic. 14, 231-257.

13

McCue, P.P., Shetty, K., 2004. Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase in vitro. Asia Pac. J. Clin. Nutr. 13, 101-106. More, G., Tshikalange, T., Lall, N., Botha, F., Meyer, J., 2008. Antimicrobial activity of medicinal plants against oral microorganisms. J. Ethnopharmacol. 119, 473-477. Nasli-Esfahani, E., Farzadfar, F., Kouhnavard, M., Ghodssi-Ghassemabadi, R., Khajavi, A.,

IP T

Peimani, M., Razmandeh, R., Vala, M., Shafiee, G., Rambod, C., Aalaa, M., Ghodsi, Razi, M.F., Bandarian F., Sanjari, M. 2017. Iran diabetes research roadmap (IDRR)

SC R

study: a preliminary study on diabetes research in the world and Iran. J. Diabetes Metab. Disord. 16, 9.

U

Naveen, J., Baskaran, V., 2017. Antidiabetic plant-derived nutraceuticals: a critical review.

N

Eur. J. Nutr. doi: 10.1007/s00394-017-1552-6. [Epub ahead of print].

A

Norton, J.E., Espinosa, Y.G., Watson, R.L., Spyropoulos, F., Norton, I.T., 2015. Functional

M

food microstructures for macronutrient release and delivery. Food Funct. 6, 663-678. Okine, L.K.N., Nyarko, A.K., Osei-Kwabena, N., Oppong, I.V., Barnes, F., Ofosuhene, M.,

ED

2005. The antidiabetic activity of the herbal preparation ADD-199 in mice: a comparative study with two oral hypoglycaemic drugs. J. Ethnopharmacol. 97, 31-38.

PT

Potchoo, Y., Guissou, I., Lompo, M., Sakie, E., Yaro, B., 2008. Antioxidant activity of aqueous methanol and ethyl acetate extract of leaves of Annona senegalensis Pers.

CC E

from Togo versus the one originates from Burkina Faso. Int. J. Pharmacol. 1, 1-5.

Shinkafi, T.S., Bello, L., Hassan, S.W., Ali, S., 2015. An ethnobotanical survey of antidiabetic

A

plants used by Hausa-Fulani tribes in Sokoto, Northwest Nigeria. J. Ethnopharmacol. 172, 91-99.

Stitt, A.W., Curtis, T.M., Chen, M., Medina, R.J., McKay, G.J., Jenkins, A., Gardner, T.A., Lyons, T.J., Hammes, H.P., Simó, R., Lois, N., 2016. The progress in understanding and treatment of diabetic retinopathy. Prog. Retin. Eye Res. 51, 156-186.

14

Sukumaran, S.K., Yee, K.K., Iwata, S., Kotha, R., Quezada-Calvillo, R., Nichols, B.L., Margolskee, R.F. 2016. Taste cell-expressed α-glucosidase enzymes contribute to gustatory responses to disaccharides. Proc. Natl. Acad. Sci. U.S.A., 113, 6035-6040. Suleiman, M., Mamman, M., Igomu, E., Muhammad, Y., Abdullahi, A., Talba, A., 2014. Evaluation of analgesic and anti-inflammatory effects of the crude methanol extract of

IP T

the stem-bark of Annona senegalensis Pers. Int. J. Med. Arom. Plants. 4, 88-96.

Tahir, H.U., Sarfraz, R.A., Ashraf, A., 2016. Chemical composition and antidiabetic activity

SC R

of essential oils obtained from two spices (Syzygium aromaticum and Cuminum cyminum). Int. J. Food Prop. 19, 2156-2164.

U

Yeo, D., Dinica, R., Yapi, H., Furdui, B., Praisler, M., Djaman, A., N’Guessan, D., 2011.

A

senegalensis leaves. Therapie. 66, 73-80.

N

Evaluation of the anti-inflammatory activity and phytochemical screening of Annona

Zhang, B.W., Li, X., Sun, W.L., Xing, Y., Xiu, Z.L., Zhuang, C.L., Dong, Y.S., 2017. Dietary

M

flavonoids and acarbose synergistically inhibit α-glucosidase and lower postprandial

A

CC E

PT

ED

blood glucose. J. Agric. Food Chem. 65, 8319-8330.

15

Figure captions

70

d

Ethylacetate c

A

40

c

Ethanol

Acarbose a,b b

b

a

b 30

a a,b

SC R

b

20 10 0

60 120 Concentration (µg/mL)

A N-Hexane

80

b

ED

B

Ethanol

Acarbose

PT

30

c

Ethyl acetete

70

40

c

M

90

CC E

α-glucosidase inhibitory activity (%)

100

50

240

N

30

60

b

IP T

50

U

α amylase inhibitory activity (%)

N-Hexane 60

b

20

b b a,b a

10

a a

a a

A

0

30

60 120 Concentration (µg/mL)

240

Figure 1: α-Amylase (A) and α-glucosidase (B) inhibition (%) of leaf extracts of A. senegalensis. Data are presented as mean ± SD of triplicate determinations. a-d Different letters presented over the bars for a given concentration of each extract are significantly different from each other (Tukey’s HSD multiple range post hoc test, p < 0.05). 16

A C E Acarbose

60 50

A

d

e d c,d

d

c

20

cc

c

b a

b

b

a

0 30

M

F

Acarbose

ED

40

240

U

D

G

50

a

N

C E

60

30 20 10

B

cc

A

70

A

PT

α-glucosidase inhibitory activity (%)

B

a

60 120 Concentration (µg/mL)

100 90

c

IP T

30

80

d

d

f

40

10

e

B D F

SC R

α -amylase inhibitory activity (%)

70

CC E

30

60 120 Concentration (µg/mL)

240

A

Figure 2: α-Amylase (A) and α-glucosidase (B) inhibition (%) of leaf column fractions (A-G) of A. senegalensis. Data are presented as mean ± SD of triplicate determinations. a-d Different letters presented over the bars for a given concentration of each extract are significantly different from each other (Tukey’s HSD multiple range post hoc test, p < 0.05). fraction A: HEX (100%), fraction B: HEX:EtOAc (8:2), fraction C: HEX:EtOAc (6:4), fraction D: HEX:EtOAc (1:1), fraction E: HEX:EtOAc (4:6),F: HEX:EtOAc (2:8 ratio), fraction G: EtOAc (100%).

17

IP T SC R U N A M ED PT

Figure. 3. Lineweaver-Burke plot for α-amylase (A) and α-glucosidase (B) in the absence and

A

CC E

presence of the inhibitors (Fraction-F).

18

IP T SC R U N A M

A

CC E

PT

ED

Figure 4: GC-MS Chromatogram of fraction-F from A. senegalensis leaf

19

Table

Table1: Percentage yield of A. senegalensis extracts Solvent

Yield (%) 1.90 1.88

Ethanol

18.67

A

CC E

PT

ED

M

A

N

U

SC R

Ethyl acetate

IP T

1.90

n-Hexane

20

Table 2: IC50 values for the inhibition of α-amylase and α- glucosidase activity by the leaf extracts of A. senegalensis IC50 (μg /ml) α-Amylase inhibition

α-Glucosidase inhibition

n-Hexane

359.96 ± 14.16c

>1000d

Ethyl acetate

617.72 ± 62.93d

253.57 ± 36.30c

Ethanol

204.04 ± 6.38b

97.91 ± 1.40a

Acarbose

151.31 ± 4.01a

121.29 ± 0.10b

IP T

Extract/standard

SC R

Data are presented as the mean ± SD of 3 replications. a-cValues with different letters along the

columns for a given parameter are significantly different from each other (Tukey's-HSD

A

CC E

PT

ED

M

A

N

U

multiple range post hoc test, p < 0.05).

21

Table 3: IC50 values for the inhibition of α-amylase and α-glucosidase activities by the fraction of the leaf of A. senegalensis IC50 (μg /ml) α-Amylase inhibition

α-Glucosidase inhibition

A

527.11 ± 34.13c

>1000e

B

>1000e

>1000e

C

>1000e

283.41 ± 47.64c

D

>1000e

>1000e

E

870.66 ± 16.18d

227.83 ± 2.71c

F

237.14 ± 31.19b

88.25 ± 0.59a

G

>1000e

552.39 ± 19.35d

Acarbose

151.31 ± 4.01a

U

SC R

IP T

Extract/standard

N

121.29 ± 0.10b

A

Data are presented as the mean ± SD of 3 replications. a-eValues with different letters along the

M

columns for a given parameter are significantly different from each other (Tukey's-HSD

ED

multiple range post hoc test, p < 0.05). A: HEX (100%); B: HEX:EtOAc (8:2); C: HEX:EtOAc

A

CC E

PT

(6:4); D: HEX:EtOAc (1:1); E: HEX:EtOAc (4:6); F: HEX:EtOAc (2:8); G: EtOAc (100%).

22

Table 4: Kinetic analysis of α-amylase and α-glucosidase inhibition by fraction-F from A. senegalensis leaf. α-Amylase inhibition

Fraction Km

Vmax

ki

Km

Vmax

ki

(%)

(µmol/min)

(µg/mL)

(mM)

(µmol/min)

(µg/mL)

0.11

27.03

3.70

2.30

0.24

27.03

8.46

3.70

A

CC E

PT

ED

M

A

N

U

Fraction-F

SC R

A. senegalensis

23

IP T

Control

α-Glucosidase inhibition

1.10

1.26

Table 5: Compounds identified in the fraction-F from leaf of A. senegalensis by GC-MS analysis Retention time (min)

Mass/amu

1,2-Benzenedicarboxylic acid, buty l octyl ester

33, 45

334 [M]+

Similarity index 90

1,2-Benzenedicarboxylic acid, bis (2-methylpropyl) ester

34

278 [M]+

90

Hexadecanoic acid, methyl ester

34

270 [M]+

81

Methyl 9-methyltetradecanoate

38

242 [M]+

89

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Compounds

24