Chemical composition and bioactivities of the polyphenolic-rich extract of Ormenis africana Jord. and Fourr

ABSTRACT

Ormenis africana is an endemic North African species used in folk medicine because of its hypoglycemic property. In this study, the α-amylase and α-glucosidase inhibition and antioxidant activities of the polyphenolic-rich extract from O. africana were determined. The chemical composition was made using liquid chromatography with photodiode array and electrospray ionization mass spectrometry method and the identification of phenolics was assessed by comparing their retention times and ultraviolet and mass spectra with those of the standards and/or reported in the literature. The total phenolic content was estimated by the Folin–Ciocalteu method. The antidiabetic potential was estimated by the determination of α-amylase and α-glucosidase inhibition in vitro. Four assays were used for the evaluation of antioxidant activity of the extracts. Seventeen phenolic compounds were detected. The major peaks are chlorogenic acid, 5-O-di-caffeoylquinic acid, and apigenin and luteolin derivatives. The polyphenolic-rich extract showed remarkable α-amylase and α-glucosidase inhibition activity in a concentration dependent manner. Furthermore, the extract also demonstrated high antioxidant activities. O. africana can serve as a potential natural source for the development of a novel α-amylase and α-glucosidase inhibitory agents against diabetic complications.

KEYWORDS:

• Ormenis Africana
• Enzyme inhibitors
• Antioxidants
• Diabetes
• LC-PDA-ESI(±)/MS)

Introduction

In recent years, a significant revival of interest in natural products as a potential source for new medicines has been observed. On the other hand, Mediterranean region is rich in medicinal plants with pharmacological and biological properties making them potential candidates for exploitation by the food and pharmaceutical industries. Several studies showed that they represent a valuable source for novel phytochemicals for treating numerous chronic diseases, among them non-insulin dependent diabetes mellitus (DM) called also type 2 diabetes. The later is one of the major public health problems worldwide. It is characterized by an abnormally high blood glucose level that has been reported to induce non-enzymatic glycosylation of various macromolecules, generation of reactive oxygen species (ROS), and alteration of endogenous antioxidants.^[1] The inhibition of carbohydrate hydrolyzing enzymes including α-amylase and α-glucosidase is one of the major conventional therapies for controlling hyperglycaemia. However, therapeutic drugs such as acarbose, voglibose are often associated with undesirable side effects.^[2] In the recent years many works have been made to approach enzyme inhibitors and antioxidant agents from natural sources for DM treatment in order to provide more candidates of drug (with lesser or no side effect) choices.^[3]

Tunisian flora includes about 2500 wild species growing on various bioclimatic zones ranging from lower humid to Saharan.^[4–6] The north-west region of Tunisia involves an extremely rich and varied flora, characterized by its originality on the systematic scheme and its wide use in folk medicine. These characteristics make the flora study of this region of great scientific interest in the field of plant food and nutrition. However, little is known about the chemical composition of these resources and the plant diversity is still under explored. From these resources we cite Ormenis africana (Jord. and Fourr.) Lit. et Maire, the Asteraceae species endemic of the North African region that grows naturally in the rocky slopes.^[4] This plant has been used since ancient times for medicinal, food and spice purposes, and known for its healing effects.^[7,8] Santolina species are a rich source of essential oil and polyphenolic compounds. Plant phenolic compounds are responsible for the health promoting properties of plant products. Dietary polyphenols have been reported to exert beneficial effects in a multitude of diseases, including cancer, cardiovascular diseases, and diabetes.^[9] Several assays have been frequently used to evaluate antioxidant ability and anti-diabetic properties of single phenolic compounds and/or complex mixtures. High-performance liquid chromatography (HPLC) coupled to diode array detector with mass spectrometry (MS) has proved to be the best tool in the separation and identification of phenolics in several plant extracts. It provides a rich amount of qualitative information from which compound identity.^[10,11]

Ormenis africana is an edible plant which has been used both as food and as medicine in North Africa for centuries. Despite the claimed health benefits attributed to this plant there is still a lack of scientific data to support this information. In this work, antioxidant and antidiabetic activities of polyphenolic-rich extract from O. africana were investigated. The chemical composition of extracts was assessed by LC-photodiode array (PDA)-electrospray ionization (ESI)/MS method.

Materials and methods

Plant material and sample preparation

Ormenis africana samples were collected in April 2013 from plants growing in the region of Jendouba under upper semi-arid bioclimatic zone. Voucher specimens were deposited at the herbarium of National Institute of Applied Science and Technology, Tunisia (Voucher number: Sant-012). Aerial part was dried in air shade at room temperature, and the dry plant was powdered. Ten grams of powder was defatted in a soxhlet system with 100 mL of hexane (6 h) and then extracted by 100 mL of methanol (6 h). The organic extract was concentrated by rotary evaporation under vacuum at 35°C. Finally, the solvent was evaporated under reduced pressure. The MeOH extracts were re-suspended in MeOH and filtered through a 0.22 μm filter paper discs (Glass Microfibre filters, Whatman; 6 mm in diameter), and stored in deep freeze (–20°C) until further treatment.

List of reagents

Folin–Ciocalteu, 2,2 0 –azinobis(3-ethylbenzothiazoline-6-sul-phonic acid diammonium salt) (ABTS), gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-S-triazine (TPTZ), and Trolox, were sourced from Sigma–Aldrich (St. Louis, MO). Standard chlorogenic acid, 3,5-O-di-caffeoylquinic acid, apigenin 7-O-rutinoside, apigenin 7-O-β-D-glucoside, and luteolin 7-O-β-D-glucoside and Hog pancreatic α-amylase and α-glucosidase were purchased from Sigma-Aldrich. All chemicals and reagent used were either analytical reagent or HPLC grade.

Antioxidant activities

Total reducing power (TRP)

The TRP was assayed using the Folin-Ciocalteu reagent and gallic acid as a standard.^[12] Folin-Ciocalteu reagent (1.5 mL) was added to a solution containing 0.5 mL of extract with a known concentration. The solution was mixed and after 5 min, 1.5 mL of 7.5% sodium carbonate solution was added. The mixture was left to incubate for 90 min, and the absorbance was measured at 760 nm. The total TRP was calculated by a standard gallic acid graph, and the results expressed in mg of gallic acid equivalents per g (mg GAE/g) of dry weight of extract. The assay was performed in triplicate for each extract.

DPPH radical scavenging assay

The antioxidant ability was assessed using the stable free radical DPPH.^[13] Extract solution (1 mL) was mixed with 2 mL DPPH in methanol (0.1 mM). The control contained all the components except extract. The changes in absorbance at 517 nm were monitored after 50 min of incubation. The inhibition percentage I(%) of radical-scavenging activity was calculated as:

where A₀ is the absorbance of the control and A_S is the absorbance of the sample. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in the 1.0–100 µm range was chosen as a standard antioxidant and activity was expressed as µm of Trolox equivalents (TE), referring to 1 mL of extract.

Free radical-scavenging ability by the use of a stable ABTS radical cation

A stable stock solution of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; ABTS^∙+) was produced by reacting a 7 mmol aqueous solution of ABTS with 2.45 mmol ammonium persulfate (final concentration), and allowing the mixture to stand in the dark at room temperature for 12–16 h before use.^[14] At the beginning of the analysis day, an ABTS^∙+ working solution was obtained by diluting the stock solution with ethanol, to an absorbance of 0.70 ± 0.02 at 734 nm, verified with a ultraviolet (UV)–vis spectrophotometer (Varian, Waldbronn, Germany). Extract (10 µL) was mixed with the working solution, to a final volume of 1 mL. The inhibition percentage I(%) of radical scavenging activity was calculated as:

where A₀ is the absorbance of the control and A_S is the absorbance of the sample after 4 min of incubation. Trolox (in the range from 1.0 to 50 µm) was used as a reference standard. TEAC were expressed as µm of TE, referring to 1 mL of extract.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed according to a literature procedure with minor modification.^[15] The fresh working FRAP reagent was prepared daily by mixing 25 mL acetate buffer (300 mmol, pH 3.6), TPTZ solution (10 mmol in 40 mmol HCl) and 2.5 mL of FeCl₃ 6H₂O solution (20 mmol). The reagent was warmed to 37°C, then 1500 µL were placed in a cuvette and the initial absorbance was read. A 50 µL of extract was added to the cuvette and the absorbance was measured after 4 min at 593 nm. Trolox was chosen as a standard antioxidant, and extracts’ activity were expressed as µm of TE referring to 1 mL of extract.

Pancreatic α-amylase inhibition assay

Appropriate dilution of the extract (100 μL) was added to 250 μL of Hog pancreatic α-amylase (EC 3.2.1.1) solution (0.5 mg/mL in 0.02 M sodium phosphate buffer pH 6.9) and the mixture was incubated at 37°C for 30 min. After pre-incubation, 250 μL of a starch solution (1% in 0.02 M sodium phosphate buffer at pH 6.9) was added and incubated at 37°C for 30 min. The reaction was terminated by the addition of 500 μL of dinitrosalicylic acid color reagent. The tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 5 mL H₂O, and absorbance was measured at 540 nm. To eliminate the absorbance produced by plant extract, appropriate extract controls without the enzyme were also included. Acarbose was used as positive control. The inhibition percentage is calculated according to the formula given below:

where A_control is the absorbance of sample without extract or acarbose, and As_ample is the absorbance of sample containing plant extract. IC₅₀ values (half-maximal inhibitory concentration) of sample and acarbose were obtained graphically by an inhibition curve.

α-Glucosidase inhibition assay

Appropriate dilution of the extracts (50 μL) was added to 100 μL of α-glucosidase solution (1.0 U/mL) in 0.1 mol/L phosphate buffer (pH 6.9) and the mixture was incubated at 25°C for 10 min. Thereafter, 50 μL of 5 mmol/L p-nitrophenyl-α-D-glucopyranoside solutions in 0.1 mol/L phosphate buffer (pH 6.9) was added. The reaction mixture was then incubated at 25°C for 5 min, and then absorbance was measured at 405 nm. Acarbose was used as positive control. The α-glucosidase inhibitory activity was expressed as percentage inhibition:

where A_control is the absorbance of sample without extract or acarbose, and As_ample is the absorbance of sample containing plant extract. IC₅₀ values (half-maximal inhibitory concentration) of sample and acarbose were obtained graphically by an inhibition curve.

LC-PDA-ESI/MS conditions

The phenolic compounds of O. africana were identified using a 3100 mass detector (Waters Co., Milford, MA) and an Alliance e2695 HPLC system (Waters Co.) equipped with a 2998 PDA, in addition to an XTerra MS C18 column (3.5 µm, 150 × 4.6 mm, Waters, Milford, MA, USA). The analysis was conducted at a flow rate of 0.5 mL/min at a detection wavelength of 200–600 nm and the oven temperature was 25°C. The mobile phases used were 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B) and the gradient varied from 14% to 26% B in 40 min, to 15% B at 60 min, to 0% B at 75 min and finally to 14% B at 75 min and held at 14% B from 75 to 80 min. The DAD was set at 280, 320 and 360 nm to provide real time chromatograms and the UV/Vis spectra from 200 to 600 nm were recorded for plant component identification. Mass spectra were acquired using ESI in the negative and positive ionization mode. The MS parameters were each set to a cone voltage of 70 V, source temperature of 120°C, desolvation temperature of 350°C, and a desolvation N₂ gas flow of 780 L/h. The range of molecular weights was m/z 100–1000 in full scan mode.

Statistical analysis

All assays were run in triplicate. The one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test were employed to study the differences between means. Values of p < 0.05 and p < 0.001 were considered statistically significant and highly significant, respectively.

Results and discussion

Chemical composition

Data from the LC-PDA–ESI/MS were used to identify the phenolic acids and flavonoids (Table 1). The structure of compounds was identified by comparison with literature when reference standard is unavailable.^[16–19] The typical LC–DAD–TIC profile of phenolic components can be observed in Fig. 1. The hydroxycinnamic derivatives detected in this work belong to mono- and di-caffeoylquinic acid compounds (compounds 1, 3, 4, 14, 15 and 17). These data agree with the results in the literature cited.^[20,21] Compound 1, 3 and 4 present the same UV spectra, with a maximum at 244, 326–328 nm and a shoulder at 290 nm. They give a [M-H]⁻ ion at m/z 353 and a fragment ion at m/z 191 which represents quinic acid, resulting from the neutral loss of caffeic acid [M-H-162]⁻. They showed also a [M+H]⁺ ion at m/z 355 and a fragment ions at m/z 163 [M+H-162]⁺ and m/z 731 [2M+Na]⁺. Therefore, peaks 1, 3 and 4 have been assigned as mono-caffeoylquinic acid isomers. Furthermore, the major compound, i.e., compound 4 was identified as chlorogenic acid by comparison of its retention time, UV spectrum and mass spectrometric data with reference standard.

Table 1. Peak assignments of the methanol extract of S. africana.

Peak No.	RT (min)	UV λmax (nm)	[M+H]^+ (m/z)	[M-H]^− (m/z)	Identification	Occurrence
1	8.35	244, 290sh, 326	355.23	353.15	mono-Caffeoylquinic acid
2	8.53	325	188/205	−	Unknown
3	8.9	244, 290sh, 326	355	353.15	mono-Caffeoylquinic acid
4	11.09	244, 290sh, 326	355.23	353.15	Chlorogenic acid*	++++
5	13.01	271, 328	595/433	593.13/431/269	Apigenin di-glucoside	+
6	15.8	322	−	179	Caffeic acid
7	18.2	−	−	305	Unknown	+
8	19.2	350	−	225	Unknown
9	20.05	308	803 ([M+Na]^+)	779.3	Unknown	+
10	24.48	264,328	595/433	593.13/269	Apigenin di-glucoside	+++
11	25.97	264, 348	465	463/285	kaempferol glucoside
12	27.23	246, 348	449/287	447.27/285	Luteolin 7-O-β-D-glucoside*	+++
13	31.4	268, 329	579	577.12/431/269	Apigenin 7-O-rutinoside*	+
14	33.5	244, 290sh, 326	517.27	515.06	3,5-O-di-Caffeoylquinic acid*	++++
15	34.5	244, 290sh, 326	517.27	515.06	di-Caffeoylquinic acid isomer
16	35.04	266, 334	433	431/269	Apigenin 7-O-β-D-glucoside
17	37.54	244, 290sh, 326	517.27	515.13	di-Caffeoylquinic acid isomer	+

*: identified by comparison with a standard reference

Figure 1. LC-DAD-TIC chromatogram of Tunisian S. africana extract. The peak assignments are listed in Table 1.

Compound 14, 15 and 17 give [M-H]⁻ ion at m/z 515 and [M+H]⁺ ion at m/z 517 with a fragment ions at m/z 353 [M-H-162]⁻ and m/z 355 [M+H-162]⁺. The UV spectra of these compounds showed the expected λ max at around 326–328 nm and a shoulder peak at 290 nm. Thus, compounds 15 and 17 were identified as isomers of dicaffeoylquinic acid whereas compound 14 was identified as 5-O-di-caffeoylquinic acid by comparison of its UV/MS data with those of standard. Peak 6 produced an intense ion [M-H]⁻ at m/z 179. It was identified based on the analysis of its UV, mass spectra and comparison with reference standard as caffeic acid.

Seven flavonoids were detected on the extract of O. africana. The main aglycones detected were flavones apigenin and luteolin. Peak 5 and 10 showed a pseudomolecular ion [M-H]⁻ at m/z 593 with a base peak at m/z 431 corresponding to the loss of a glucose unit and another fragment [M-H-162-162]⁻ at m/z 269. It produced a [M+H]⁺ ion at m/z 595 with a fragment ion at m/z 433 [M+H-162]⁺ in the positive ionization mode. Its UV spectrum is characteristic of an apigenin derivative. On the basis of these informations compound 5 and 10 were tentatively identified as two isomers of apigenin di-glucoside.

Peak 11 produced a molecular ion [M+H]⁺ at m/z 465 and [M-H]⁻ at m/z 463. In the negative ionization mode it showed a base peak at m/z 285 corresponding to the aglycon moiety [M-H-162]⁻. The aglycon of peak 11 presents a Band II peak at 264 nm. Then aglycon of peak 11 was attributed to kaempferol and the molecule was tentatively identified as kaempferol glucoside.^[22]

Peak 12 produced a quasimolecular ion at m/z 447 ([M-H]⁻) and at m/z 449 ([M+H]⁺) in the negative and positive ionization mode, respectively. It is characterised by the loss of 162 Da corresponding to the elimination of a glucose moiety to give a fragment ion at m/z 285 and 287 in the negative and positive mode, respectively. This compound was identified based on the analysis of its UV/MS spectra and by comparison with reference standard as luteolin 7-O-β-D-glucoside.

Peak 13 produced a molecular ion [M+H]⁺ at m/z 579 in the positive ionization mode. In the negative ionization mode it showed a pseudomolecular ion [M-H]⁻ at m/z 577 with two base peaks at m/z 431 corresponding to the loss of a rhamnose unit and m/z 269 corresponding to aglycon [M-H-146-162]⁻. By comparing its UV/MS spectra with available reference standards, compound 13 was identified as apigenin 7-O-rutinoside. Peak 16 showed in the negative mode a pseudomolecular ion [M-H]⁻ at m/z 431 with a base peak at m/z 269 corresponding to the loss of a hexose unit. In the positive mode it presented [M+H]⁺ ion at m/z 433. It was positively identified as apigenin 7-O-β-D-glucoside by comparison of its UV/MS data with standard.

Overall, the dominant phenolics are the phenolic acids chlorogenic acid and 3,5-O-di-caffeoylquinic acid and the flavonoids luteolin 7-O-β-D-glucoside, apigenin di-glucoside and apigenin 7-O-rutinoside. However, four components (2, 7, 8, and 9) with retention times of 8.5, 15.8, 18.2, and 19.2 min, respectively were not identified.

Enzyme assay

This study investigated the ability of phenolic extract of O. africana to inhibit α-amylase and α-glucosidase (key enzymes linked to type II diabetes) and the results are presented in Table 2. From the result, the polyphenolic-rich extract inhibited α-amylase in a concentration dependent manner. The inhibition (%) of extracts (60–80 mg/mL) showed significantly higher inhibitory activity compared with acarbose (p < 0.001), whereas there was no significant difference in extract at concentration of 40 µg /mL and acarbose (p > 0.05). However, the half-inhibitory concentration (IC₅₀) was estimated to be 28.35 ± 1.18 µg/mL, while the IC₅₀ of acarbose was 15.25 ± 1.42 µg /mL. Furthermore, the polyphenolic-rich extract inhibited α-glucosidase activity in a concentration dependent manner. The inhibition reached 98% for the concentration of 120 mg/mL). At concentration of 40 µg/mL extract of O. africana showed the same % of inhibition (61 ± 0.14) when compared to acarbose (69 ± 0.33%). The extracts (60–100 µg /mL) showed significantly higher inhibitory activity than acarbose (p < 0.01). The IC50 values of extract and acarbose are 34.21±1.2 and 17.22 ± 1.3 µg /mL, respectively.

Table 2. α-Glucosidase and α-amylase inhibition activity (%) of S. africana extract.

Sample	α-amylase inhibition	α-glucosidase inhibition
MeOH extract (10 µg/mL)	12 ± 0.22 ^d	8 ± 0.10 ^e
MeOH extract (20 µg /mL)	31 ± 0.54 ^c	37 ± 0.09 ^d
MeOH extract (40 µg /mL)	64 ± 0.32 ^b	61 ± 0.14 ^c
MeOH extract (60 µg /mL)	92 ± 0.41 ^a	81 ± 0.21 ^b
MeOH extract (80 µg /mL)	97 ± 0.11^a	90 ± 0.17 ^a
MeOH extract (100 µg /mL)	−	98 ± 0.20^a
Acarbose (20 µg /mL)	67 ± 0.19^b	69 ± 0.33 ^c

Results are reported as mean ± standard deviation of three replicates. Values in the same column followed by the same letter are not significantly different (Duncan’s multiple range test at p >0.05).

Antioxidant activity

The ability of S. afriana extract to act as a natural antioxidant was assessed by means of DPPH and ABTS radicals scavenging and FRAP. The results (Table 3) show that, in line with other flavonoid-rich Asteraceae extracts, O. africana displays high quenching activity toward DPPH. radicals, even superior to that measured toward ABTS.⁺ radical cations (46 ± 0.5 versus 36 ± 2 µm TE, respectively). Ferric reducing power was found to be in the same range as DPPH radical bleaching (52 ± 3.1 µm TE). The TRP, measured by Folin–Ciocalteu method, was high (50 ± 0.408 mg GAE/100 g DM).

Table 3. Antioxidant capacity of the MeOH extract of Santolina africana.

	Antioxidant Capacity
Sample	TRP^a	ABTS^b	DPPH^b	FRAP^b
Methanolic extract	50 ± 0.408	36 ± 2	46 ± 0.5	52 ± 3.1

Results are reported as mean ± standard deviation of three replicates

^a: mg gallic acid equivalents/g dry weight; ^b: µm Trolox equivalents (TE).

The results obtained in this study revealed a high antioxidant activity of O. africana extract. This may be explained by the high amount of phenolic compounds.^[23] The DPPH scavenging ability of Achillea distans subsp. alpina and A. distans subsp. distans (Asteraceae family) was assessed.^[24] The work showed similar results of radical scavenging activity with TEAC values of 42.14 and 46.72 µmol Trolox/mg, respectively. Moreover, there are no previously data about the phenolic profile of O. africana certainly because of its position as endemic species. In addition, there are few works on other Santolina species. S. chamaecyparissus was shown to accumulate 7-O-glycosides of apigenin, luteolin, chrysoeriol, patuletin, hispidulin, pectolinaringenin, nepetin, jaceosidin and axillarin, luteolin 7-methyl ether and 5,4’-dihydroxy-6,7-dimethoxyflavone.^[25–27] Bioactivity-guided fractionation of the methanol extract from the leaves of S. insularis had led to the isolation of six flavonoids: hispidulin, nepetin, cirsimaritin, rhamnocitrin, luteolin, and luteolin 7-O-β-D-glucopyranoside.^[28,29] The major peaks detected in the extract of the Iberian endemic Santolina semidentata are hydroxicinammic acids derivatives, flavones, simple phenolic acids, flavonols, and coumarins.^[30] With these results, also ferulic acid and its derivatives were the principal components identified in the hydroethanolic extract of S. impressa.^[31] The important constituents, considered as chemical markers of Santolina species were not detected in our work suggesting a high chemical differentiation of S. africana probably because of its specific position as an endemic plant which could play an important role in flavonoid diversity.^[32]

Today, there are increasing studies to search for novel as α-amylase and α-glucosidase inhibitors from natural sources expected to be effective and safe. In this work the evaluation of antidiabetic potential the extracts was done in vitro by inhibitory assays using enzymes α-amylase and α-glucosidase. The results of enzyme assays agree with other studies on Calendula officinalis.^[33] It was found that IC⁵⁰ value of C. officinalis extract was significantly higher (lower inhibitory activity) than acarbose, but when all purified compounds of the extract were tested, several phenolics, e.g., caffeic acid, isorhamnetin and quercetin have shown significantly higher inhibitory effect than acarbose. In fact, some constituents have been considered to display a variable biological activity due to their possible synergistic or antagonistic effects.^[34] In our work, the main phenolic compounds present in the extract are mono-caffeoyl quinic acid and di-caffeoyl quinic acid which have been previously reported as inhibitors of the key enzymes of carbohydrates digestion process.^[35] However, it was also considered that minor components, as well as a possible interaction between the substances could also affect the biological activities.

Several reports have shown close relationship between total phenolic contents and antioxidative activity of plant extracts.^[36,37] ROS play an important role in the pathogenesis of various serious diseases including diabetes. The production of ROS is increased particularly in diabetic patients who are highly predisposed to cancer and cardio-vascular diseases.^[38] The antioxidant ability of phenolic compounds could be attributed to their properties such as reducing agents, hydrogen donors, singlet hydrogen quenchers and/or metal ion-chelators. Therefore, natural antioxidants can also inhibit the key enzymes α-amylase, α-glucosidase and control the post-prandial hyperglycemic conditions which are a potential approach to cure the type 2 DM.^[39–41] In several works the mode of inhibition of the two enzymes was studied. The competitive, uncompetitive and mixed modes were proposed depending on the nature of molecules. Interestingly, chlorogenic acid and its derivatives as well as caffeic acid were mixed inhibitors, exhibiting both competitive and uncompetitive characteristics.^[42] In our study Ormenis africana extract was dominated by chlorogenic acid and its derivatives (e.g. 3,5-O-di-Caffeoylquinic acid, di-Caffeoylquinic acid isomer). The mixed mode of inhibition can be proposed but this assumption must be verified by the appropriate assays.

Conclusion

The present study represents a contribution to the use of O. africana as a source of natural antioxidants and antidiabetic agents. The results suggest that O. africana should be given special attention because of its good biological activity which might due to the presence of the phenolic compounds with mono and di-caffeoylquinic acids, luteolin, and apigenin derivatives being the main constituents. Further analysis of compound isolation as well as their in vivo studies must be investigated.

Funding

This work was supported by the Ministry of Higher Education and Scientific Research, Tunisia (Research grant LR15INRAP02).

Additional information

Funding

This work was supported by the Ministry of Higher Education and Scientific Research, Tunisia (Research grant LR15INRAP02).