Determination of The Radical Scavenging Activities and Identification of Anthocyanins from Hexalobus monopetalus Ripe

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Determination of The Radical Scavenging Activities and Identification of Anthocyanins from Hexalobus monopetalus Ripe

Fruits

Arrounan Noba,¹ Adama Hema,^1* Elie Kabré,² Bazoin Sylvain Raoul Bazié,² Paulin Ouôba,³ Constantin M.

Dabiré,^1,4Remy K. Bationo,^1,5Moumouni Koala,^1,6 Eloi Palé,¹ and Mouhoussine Nacro¹

1Laboratoire de Chimie Organique et de Physique Appliquées (L.C.O.P.A.), Université Joseph KI-ZERBO, UFR/SEA, Département de Chimie 03 BP 7021 Ouagadougou 03 Burkina Faso.

2Laboratoire National de Santé Publique (L.N.S.P) 09 BP 24 Ouagadougou 09, Burkina Faso.

3Laboratoire de Biologie et Écologie Végétales, Université de Ouagadougou, 03 BP 7021, Ouagadougou 03, Burkina Faso.

4Laboratoire de Chimie et Energies Renouvelables, Université Nazi Boni, 01 BP 1091 Bobo 01, Burkina Faso.

5CNRST/IRSAT/ Département Substances Naturelles, 03 BP 7047 Ouagadougou 03 Burkina Faso.

6Institut de Recherche en Sciences de la Santé, Centre National de la Recherche Scientifique et Technologique (IRSS/CNRST), 03 BP 7047 Ouagadougou 03, Burkina Faso.

*Corresponding e-mail: [email protected] Received 24 August 2021; Accepted 25 April 2022

ABSTRACT

A wild fruit from classified forest of Dindéresso was analyzed for total phenolics, flavonoids, anthocyanins compounds using the Folin-Ciocalteu reagent, spectrophotometric method of Zhishen and colleagues and by the differential pH method respectively. Free radical- scavenging activities of studied fruits extracts were estimated using diammonium 2,2'-azino- bis-(3-ethylbenzothiazoline-6-sulfonate) salt method. Three major anthocyanins were identified using high performance liquid chromatography coupled with spray ionization interface mass spectrometry. Three identified anthocyanins in fruit were reported to be cyanidin 3-O-(p-coumaroyl) glucoside, pelargonidin 3-O-glucoside and pelargonidin 3-O- rutinoside. In addition, H. monopetalus fruit contained of about 1165±3.1 mg of GAE per 100 g of fresh fruit, 4490±20.2 mg of QE per 100 g of fresh fruit, and 36±0.17 mg of cyanidin 3-O-glucoside equivalents per 100 g of fresh fruit. Total anthocyanin extract had an EC50 = 4.24 mg per mL and a TEC50 time of 21 minutes (intermediate reaction). This free radical- scavenging activity was very low compared to those of the references used (0.024 and 0.034 mg/mL respectively for ascorbic acid and Trolox). The low antiradical activity and reactivity of the H. mucronata extract could be explained by several factors. In any case, fruits of this species were potential sources of natural bioactive substances having beneficial effects on the health of consumers

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Keywords: Hexalobus monopetalus, ripe fruits, HPLC-MS/MS, anthocyanins, antioxidants

INTRODUCTION

In rural and urban areas, "wild" fruits occupy an important part of the diet and their contribution to the survival of populations is undeniable in times of crisis (lean season, flooding, drought, famine, political-military conflict) [1]. Wild fruit species contribute to the food security of rural populations in particular and are the subject of a flourishing trade in which women are the main actors [2]. However, many plant species have disappeared or

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become rarefied, mainly during the last century, due to anthropogenic action (logging, agricultural expansion, urbanization, etc.

Fruits and their derivatives are an important source of nutrients and other bioactive compounds with multifaceted potential [1]. Indeed, they are rich in antioxidants that help to reduce the incidence of degenerative diseases such as cancer, arthritis, arteriosclerosis, heart disease, inflammation, brain dysfunction and the acceleration of the aging process [3,4,5].

Antioxidants are defined as substances that can prevent or delay oxidative damage to lipids, proteins and nucleic acids by reactive oxygen species. These reactive oxygen species are reactive free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and non-radicals such as hydrogen peroxide, hypochlorous, etc. Antioxidants trap radicals by inhibiting initiation and breaking the spread of chains or suppressing free radical formation [6].

The most abundant antioxidants in fruits are polyphenolics and vitamin C ; vitamins A, B and E and carotenoids are present to a lesser extent in some fruits [6,7].

Phenolics in general are of great importance in the nutritional, organoleptic and commercial properties of fruits and their derivatives.

Among these phenolics, flavonoids represent the most important and most represented class.

Their biological properties on human health are well established. These properties include anti- inflammatory, antimicrobial and antioxidant properties. These compounds would be responsible for the prevention of cancer and cardiovascular diseases [8].

In particular anthocyanins are at the origin of various biological effects [9]. They represent a class of plant pigments found in many fruits such as grapes, blackcurrants, cherries and blueberries. They have antioxidant, anti-inflammatory properties and are compounds that also exhibit protective properties of DNA and are associated with health benefits [9,10].

Consumption of anthocyanins can reduce concentrations of total cholesterol (TC), low-density lipoproteins (LDL) and triglycerides (TG) in humans [11,12].

The major bioactive (beneficial) effects of phenolics in general and anthocyanins in particular are well established. Therefore, the discovery of new anthocyanins in plant materials will only increase the potential sources of these antioxidants.

In general, the data in the literature have referred to several works on the different organs of the species Hexalobus monopetalus with the exception of fruits.

Indeed, there is few works done on the fruit of this species in particular its composition of bioactive substances including phenolics in general but particularly anthocyanins, as well as its biological activity such as free radical-scavenging activity. Nevertheless, Hamisi M. Malebo in his article "Diprenylated Indole Alkaloids from Fruits of Hexalobus monopetalus" indicated that he had isolated an alkaloid family compound : 5-(2",3"-Epoxy-3'-methylbutyl)-3-(3'- hydroxy-3'-methyl-1'-acetyloxybut-2'-yl) indole [9].

The objective of this work is to carry out a quantitative study on micronutrients in general and in particular a qualitative study of anthocyanins of this fruit. Therefore, we studied the anthocyanin pigment profiles of this fruit as well as total phenolics, flavonoids and anthocyanins contents. For this purpose, the anthocyanins present in this studied fruit were identified using High Performance Liquid Chromatography (HPLC) coupled with mass spectrometry (MS). Free radical-scavenging activity of methanolic acidified (1% HCl) extract of the fruit was determined using the radical-cation (ABTS^•+) method.

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EXPERIMENT Sample

The ripe fruits of Hexalobus monopetalus were collected in the classified forest of Dindéresso (Bobo Dioulasso) at the geographical coordinates: 11°11'55,08'' North Latitude;

4°23'22,098'' West Longitude, Altitude 404 m.a.s.l. Before the harvest, an identification of the plant species was carried out by Dr. Paulin OUÔBA, Senior Lecturer and Researcher in Nazi Boni University. Harvested fruits were carefully washed and kept in the freezer. To obtain the different extracts, fruits were lightly crushed with a porcelain mortar. The crushed material obtained was used for the different extractions.

Chemicals

Distilled water, Folin-Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), quercetin, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Ethyl acetate, gallic acid, sodium carbonate, Amberlite XAD-7, aluminum chloride were purchased from Sigma-Aldrich, potassium chloride, sodium hydroxide, acetic acid, chloroform were purchased from AnalaR NORMAPUR, sodium nitrite was obtained from Labosi, methanol, formic acid, acetone, acetonitrile were obtained from Carlo Erba, hydrochloric acid was purchased from Fisher Chemical. The solvents used were of analytical quality or HPLC grade.

Extraction of total phenolics

Phenolics in general and flavonoids in particular were extracted by maceration of 15 g of fruit in approximately 60 mL of an acetone-water-acetic acid (70 : 29.5 : 0.5, v/v) solvent system for 72 h at 4°C. The operation was repeated in triplicate. The extract obtained was subjected to a liquid-liquid extraction with chloroform in order to remove any non-phenolic compound from the extract. The volume obtained from these different steps was carefully measured and stored at 4°C until the measurements.

Extraction of anthocyanins

The anthocyanins were extracted by maceration of 50 g of fruit in acidified methanol (1% HCl) for 72 hours at 4°C. The operation was repeated in triplicate. The extract was concentrated almost dry under reduced pressure (40 mbar) using a rotary evaporator (Buchi Switzerland, Rotavapor R-300) and stored at 4°C until the total anthocyanin contents were measured.

HPLC-MS/MS analysis of anthocyanins Purification on Amberlite XAD-7

After extraction with acidified methanol (1% HCl), the anthocyanin crude extract was purified. Indeed, the crude extract obtained was concentrated to dryness and recovered with a minimum of acidified distilled water and then filtered. Thus, the fixation on Amberlite XAD- 7 was performed in a column of 24 cm (Amberlite) length and 3 cm diameter. Amberlite was prepared in ethanol. The resulting mixture was poured into the column and allowed to stand.

Prior to the deposition of the aqueous extract, the column was washed with water to remove the alcohol. The concentrated extract was carefully loaded onto the Amberlite XAD-7 resin. A preliminary wash with distilled water until the eluent is neutral was performed, then an elution with ethanol containing HCl (1%) was performed. The collected eluate was concentrated to dryness under reduced pressure (40 mbar) using a rotary evaporator (Buchi Switzerland,

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Rotavapor R-300). The temperature of the bath was fixed at 35°C, with a rotation speed of 120 rpm.min⁻¹.

The identification and evaluation of free radical scavenging activity was performed on the total anthocyanin extracts.

Identification of anthocyanins using HPLC-MS/MS

HPLC-ESI-MS analysis was performed using an Agilent-1290 infinitty HPLC system equipped with a 6430 triple quad LC/MS mass detector (MS), fitted with an electrospray ionization (ESI) interface (Agilent Technologies, USA) and an Agilent MassHunter version B.06.00 workstation for data processing. The separation was performed in a Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm). The column was thermostatically controlled at 25°C, and the flow rate was set at 600 μL/min. The mobile phase consisted of two solvents: water-formic acid (A, 95 : 5, v/v) and acetonitrile-formic acid (B, 95 : 5, v/v). The gradient of solvents in volumetric ratios was as follows: 0-5 min, 95% A; 5-15 min, 90% A; 15-25 min, 90% A; 25- 35 min, 88% A; 35-50 min, 85% A; 50-60 min, 82% A; 60-80 min, 75% A; 80-90 min, 70%

A; 90-100 min, 95% A. There was a 10 min post run, which returns to baseline conditions.

Fifty microliters (50 µL) of sample, previously filtered through a cellulose acetate membrane (Millipore 25 mm, 0.45 µm,) was injected. The mass spectra of the ions were recorded in positive ionization mode in the range of m/z 100-1200. Nitrogen was used as the drying and nebulizing gas at a flow rate of 10 L/min and a pressure of 15 psi. The nebulizer temperature was set at 200°C, and a potential of 3500 V was used on the capillary.

Determination of total phenolics content

Total phenolic compounds (TPC) were determined using Folin-Ciocalteu’s reagent method, with some modifications. The principle of the assay is based on the conditions of the improved method developed by Singleton and colleagues [13]. To 60 µL of each properly diluted extract, 60 µL of Folin Ciocalteu’s reagent was added. After 8 min of incubation, 120 µL of a sodium carbonate solution Na2CO3 (7.5%, w/v) was added. The whole was incubated at 37°C for 30 min and the reading was done at 760 nm with a SAFAS MP96 spectrophotometer against a blank. The contents were obtained by relating the absorbances read on a standard curve (figure 1) established using gallic acid as standard. These contents are expressed in micrograms of gallic acid equivalents (GAE) per 100 g of fresh plant material (mg GAE/100 g).

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Figure 1. Gallic acid standard curve Evaluation of total flavonoid content

Total flavonoids contents were determined using a spectrophotometric method previously described by Zhishen and colleagues in 1999 [14], Dewanto and colleagues in 2002 [15], Sakanaka and colleagues in 2003 [16] and Khan in 2012 [17] but slightly modified. Then, to 50 µL of the convenient diluted extract were added 15 µL of a 5% sodium nitrite (NaNO2) solution. After 5 minutes, 15 µL of a 10% aluminum chloride (AlCl3) solution was added and the mixture was allowed to stand for an additional 6 minutes before adding 50 µL of 1 N sodium hydroxide (NaOH). The resulting mixture was immediately diluted with 150 µL of bi-distilled water. Absorbances of the pink solution obtained were read immediately at 510 nm using a SAFAS MP96 spectrophotometer. The results were obtained by relating the absorbances read to a standard curve (figure 2) established using a quercetin solution of concentration 0.001 to 0.5 mg/L. These results were expressed in micrograms of quercetin equivalents per 100 g of fresh material (mg QE/100 g).

Figure 2. Standard curve for quercetin

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Determination of total anthocyanin content

Total anthocyanins contents of the extracts were determined using the differential pH method described by Giusti and Wrolstad in 2001 [18], Giusti and colleagues in 2014 [19]. This method uses two buffer systems (potassium chloride solution, pH 1.0 (0.025 M) and acetate solution, pH 4.5 (0.4 M)).

Approximately, 0.5 mL of the extract was mixed with 3.5 mL of the corresponding buffers and the absorbance was read against a blank at 510 and 700 nm exactly 15 minutes later. The absorbance A was calculated as follows:

𝐴𝐴 = (𝐴𝐴𝜆𝜆𝜆𝜆𝜆𝜆𝜆𝜆−𝑚𝑚𝑚𝑚𝑚𝑚 − 𝐴𝐴700)𝑝𝑝𝑝𝑝=1.0−(𝐴𝐴𝜆𝜆𝜆𝜆𝜆𝜆𝜆𝜆−𝑚𝑚𝑚𝑚𝑚𝑚− 𝐴𝐴700)𝑝𝑝𝑝𝑝=4.5.

The monomeric concentration of anthocyanin dyes in the extract is calculated as cyanidin 3- glucoside:

[𝐴𝐴𝐴𝐴𝐴𝐴ℎ𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐴𝐴𝑜𝑜𝐴𝐴𝑜𝑜]�^{𝑚𝑚𝑚𝑚}_𝑚𝑚 �= 𝐴𝐴×𝑃𝑃𝑃𝑃×𝐹𝐹𝐹𝐹×𝑉𝑉×1000 𝜀𝜀×𝑙𝑙×𝑚𝑚 ²⁰

MW: Molecular Weight and ε molar extinction coefficient correspond to those of cyanidin 3- O-glucoside where MW = 449.2 g/mol and ε = 26900 [21]; m: the mass of the sample in g;

1000: for conversion from g to mg.

Total anthocyanin content is expressed as microgram cyanidin 3-O-glucoside per 100 g fresh material (mg C-3-G/ 100 g).

Determination of antioxidant activity by the ABTS method

The ABTS^•+ radical-cation solution was obtained by oxidation of ABTS with sodium persulfate in the dark for at least 16 hours. Volumes of 25 µL of the extract are mixed with 225 µL of the ABTS^•+ solution. The reaction mixture was shaken vigorously for 10 seconds. At regular time intervals (1 min), absorbances at 734 nm were read (against methanol) using a SAFAS MP96 [22] spectrophotometer. The kinetics of ABTS^•+ reduction at different concentrations of each sample tested were followed over time until a plateau was reached at the final time (Teq). The reduction of free radicals was evaluated by the relative ratio of the residual concentration [[ABTS^•+]t=Teq remaining at the end of the kinetics to its initial concentration:

% ABTS^•+= ^[ABTS_[ABTS^•+_•+^]^{𝐭𝐭=𝐓𝐓𝐓𝐓𝐓𝐓}_]

𝐭𝐭=𝟎𝟎 × 100

From the curve plotting the relationship between the percentage of reduction %(ABTS^•+)R

(figures 11, 13 and 15) and the concentration of the extract, the EC50 concentration and the TCE50 time were deduced by graphical interpolation (figures 12, 14 and 16).

The antioxidant activity was better when the EC50 value was small. A solution of Trolox and ascorbic acid were used as standard.

RESULT AND DISCUSSION

Identification of anthocyanins by HPLC-ESI-MS/MS

Positive ESI analysis showed two types of ions: the protonated molecular ion [M+H]⁺ and a fragment ion [M+H-X]⁺ resulting from the loss of the saccharide portion. However, since anthocyanins have a natural residual positive charge, a molecular ion [M]⁺ and a fragment ion

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[M-X]⁺ are observed in MS (Figures 4, 6 and 8). Aglycones frequently met in nature are cyanidin, pelargonidin, delphinidin, petunidin, paeonidin and malvidin. The value of X based on the difference between the molecular ion mass and the fragment ion one gives an idea on the nature of the saccharide. Although the position of the saccharide on the aglycone is not given by mass spectrometry, it should be noted that most of the known monoglycosylated anthocyanins have the glycosyl in position 3 [23]. Indeed, the glucosylation of the hydroxyl (OH) in position 3 has particular properties and is essential for the stability of anthocyanin pigments [23].

HPLC analysis of the purified extract from H. monopetalus fruits revealed three compounds 1, 2 and 3 with the last two being major according to the HPLC chromatogram (Figure 3). These three compounds are identified as peaks 1, 2 and 3, respectively, in the HPLC chromatogram (figure 3).

Moreover, the ESI-MS analysis of the extract revealed fragment ions that would correspond to two anthocyanidins : pelargonidin at m/z 271 (2 and 3) and cyanidin at m/z 287 (1) (Table 2).

Figure 3. HPLC chromatogram of the methanolic extract

Compound 1 (RT = 27.972 min) was identified with the molecular ion at m/z 595 in accordance with the mass calculated from the formula C30H27O13. By loss of a fragment ion at m/z 308 u (Figure 4) it gives the fragment ion at m/z 287 which would therefore be cyanidin.

Thus, compound 1 would be a derivative of cyanidin. Indeed, the loss of a mass at 308 u ([M- 146-162]⁺) could correspond on the one hand to rutinoside and on the other hand to a successive loss of 162 you and 146 u. The mass of 162 u corresponds to a hexosyl (galactosyl or glucosyl) and that of 146 corresponds to either a rhamnosyl or coumaric acid. Compound 1 could correspond to cyanidin 3-rutinoside, cyanidin 3-glucoside-5-rhamnoside or cyanidin 3-(p- coumaroyl) glucoside. However, the absence of a fragment ion at m/z 449 suggests the absence of a 146 u loss. Furthermore, Giusti and colleagues in their paper "Electrospray and Tandem Mass Spectroscopy As Tools for Anthocyanin Characterization" reported that fragmentation of C3-substituted diglycosylated anthocyanins produces only one fragment, corresponding to

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m/z of aglycone [24]. Thus, compound 1 would not correspond to cyanidin 3-O-glucoside-5- rhamnoside. In addition, according to the same authors, the C3-O-substituted pelargonidine derivatives were exceptions to this fragmentation pattern, for example pelargonidine 3- rutinoside. Indeed, the 1-6 bond between rhamnose and glucose, which form rutinose, allows a free rotation and a greater accessibility of the gas used to produce the fragmentation, thus giving rise to the loss of rhamnosyl (146 u). The absence of this loss in the case of compound 1 suggests that it would not correspond to the cyanidin 3-rutinoside. Furthermore, it is reported that C3-substituted acylated anthocyanins produce only one fragment corresponding to the aglycone in their fragmentation [25], as observed in the MS-MS spectrum of peak 1. In sum, compound 1 could correspond to cyanidin 3-O-(p-coumaroyl) glucoside (Figure 5). This could be reinforce by the presence of a shoulder around 330 nm on recorded UV-visible spectrum which justifies this acylation. But the HPLC device used for the analysis didn’t have a Diiode strip detector to record the UV-visible spectra of the detected anthocyanin compounds.

The MS-MS analysis performed on compound 2 (RT= 32.3023 min) shows an intense peak at m/z 270.9000 (≈ 271) (Figure 6) which would correspond to the mass calculated using the formula C15H11O5 and a less intense peak at m/z 432.9000 (≈ 433) (Figure 6) corresponding to the mass calculated using the formula C21H21O10. Indeed, the formula C21H21O10 at m/z 433 would correspond to a monosaccharide derivative of pelargonidin. The fragment ion at m/z 270.9000 (≈ 271) was obtained after a loss of 162 u corresponding to a hexoxyl. Compound 2 would be pelargonidin 3-O-glucoside or pelargonidin 3-O-galactoside. Furthermore, the hexose commonly found in fruits is glucose [26,27]. Thus, compound 2 would correspond to pelargonidin 3-O-glucoside (Figure 7)

MS-MS analysis of compound 3 indicated an intense peak at m/z 270.9000 (≈ 271) (Figure 8) which corresponds to the mass calculated using the formula C15H11O5, a less intense peak at m/z 432.9000 (≈ 433) corresponding to the mass calculated using the formula C21H21O10, and a middle peak at m/z 578.9000 (≈ 579) corresponding to the mass calculated using the formula C27H31O14. The fragment ion at m / z 433 is obtained by loss of a unit of 146 which would be either a rhamnoside or coumaric acid. Moreover, the fragment ion at m/z 271 is derived from the loss of a mass of 162 u by the fragment ion at m / z 433. This loss would correspond to a glucosyl or galactosyl. Compound 3 could be pelargonidin 3-O-(p coumaroyl) glucoside, pelargonidin 3-O-rutinoside or pelargonidin 3-O-glucoside-5-rhamnoside.

However, the C3-substituted acylated anthocyanins produce only one fragment in MS-MS corresponding to the aglycone in their fragmentation [25] which is not the case in MS-MS of compound 3. The presence of a fragment ion at m/z 433 suggested that compound 3 cannot be pelargonidin 3-O-(p-coumaroyl) glucoside. In addition, reverse phase high performance liquid chromatography has the characteristic of predicting the order of elution of anthocyanins based on their polarity. Indeed, triglycosides are eluted before diglycosides which, in turn, are eluted before monoglycosides. An exception to this generalization is that 3-rutinosides have a longer retention time than their 3-glucoside counterparts because of the apolar methyl group of rhamnose [28]. This observation was made between compound 2 (RT= 32.3023 min) which would be a monoglucosylated pelargonidin derivative and compound 3 (R^T = 35.5933 min) which would be diglycosylated pelargonidin. All in all, compound 3 would be pelargonidin 3- O-rutinoside (Figure 9).

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Figure 4. Mass spectrum of compound 1

O OH

HO O

OH m/z 287 OH

O HO

OH OH

O

O HO

m/z 308

m/z 449 m/z 146

1 2

3 5 4

6

7 8

9

10 1'

2' 3' 4'

5' 6'

Figure 5. Structure of compound 1

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O OH

HO O

OH

HO O

HO

OH OH

m/z 271

m/z 162

1 2

3 5 4

6

7 8

10

9

1' 2'

3' 4' 5' 6'

OH

HO O

m/z 271 OH

O O

HO OH

OH

O

HO OH HO

H₃C m/z 146 m/z 449

m/z 308

1 2

3 5 4

6

7 8 1'

2' 3' 4' 5' 6'

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Table 1. Identification of anthocyanins in Hexalobus monopetalus fruit Pics Retention

time (min) Compound [M]⁺ [M- 162/146]⁺

162-[M- 146]⁺

Molecules identified

1 27.972 1 595 - 287 Cyanidin 3-(p-

coumaroyl) glucoside

2 32.3023 2 433 271 - Pelargonidin 3-

glucoside

3 35.5933 3 579 433 271 Pelargonidin 3-

rutinoside Total phenolic content

The method using the Folin-Ciocalteu reagent is a quantification technique that allows rapid assessment of total phenolic compounds in foods [29]. Table 3 shows the results from the quantitative analysis. These results were expressed as mg gallic acid equivalent (GAE) per 100 g fresh fruit from the equation of the curve (y = 24.941x + 0.1225; R² = 0.9995) (figure 1).

In general, the data in the literature have referred to several works on the different organs of this species with the exception of fruits. Indeed, there is little work done on fruits in particular their composition in bioactive substances. However, after the evaluation of the phenolic compounds content, it appears that the fruit of Hexalobus monopetalus contains on average 1165±3.1 mg of GAE per 100 g of fresh fruit (Table 2). The content of total phenolic compounds of this fruit studied is higher than those of some fruits consumed in Burkina Faso such as Diospyros mespiliformis, Ficus sycomorus, Gardenia erubescens, Parkia biglobosa, Vitellaria. paradoxa [30]. Thus, polyphenolics contents could make this fruit a potential source of antioxidants and nutrients for consumers.

Total flavonoid content (TFC)

Flavonoids, which are one of the most diverse and widespread groups of natural compounds, are probably the most important natural phenolic compounds [29]. In addition to their antioxidant activities, flavonoids exhibit a wide range of biological and pharmacological activities, including anti-inflammatory, antiviral, anti-allergenic, anti-carcinogenic, anti-aging [29]. The TFTs of the extract were expressed as mg Quercetin Equivalents (mg QE) per 100 g fresh fruit from the equation of the curve (y = 1.0409x + 0.3078; R² = 0.9974) (Figure 2). Table 3 shows the TFT values. Fruits of this species contains a non-negligible content of phenolic compounds (1165±3.1 mg GAE/100 g fresh fruit). Moreover, after the evaluation of total flavonoid contents, it is found that this fruit contains an average content of 4490±20.2 mg of QE/100 g of fresh fruit. Compared to some wild fruits consumed in Burkina Faso, such as Ximenia americana, Vitellaria paradoxa, Tamarindus indica, Saba senegalensis, Lannea microcarpa, Adansonia digitata, the total flavonoids contents of this fruit are not outdone [30].

Phenolic compounds in general and flavonoids in particular have been reported to contribute to antioxidant activity by inhibiting free radical oxidation through an electron or hydrogen atom transfer mechanism. Diets high in phenolic compounds such as flavonoids have been associated with lower mortality rates from coronary heart disease [31]. Thus, regular consumption of fruit may be beneficial to human health, such as reducing the risk of cardiovascular disease and cancer [32].

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Total anthocyanin content (TAC)

After determining the total anthocyanin content, the fruit of this species contains about 36±0.17 mg cyanidin 3-O-glucoside equivalents per 100 g of fresh material (Table 2). The content of anthocyanins is high in this fruit compared to that of red cabbage, red onion, red currant, plum and strawberry [27]. In addition, the anthocyanin content is high in this fruit compared to some temperate and tropical fruits [33]. Anthocyanins are characterized by their antioxidant properties, which are beneficial to health and particularly against cellular aging.

Thus, this remarkable anthocyanin content would make this fruit a potential source of anthocyanins for the benefit of consumers.

Radical-scavenging activities

The ABTS^•+radical cation implemented in this assay is not commercially available and must be chemically generated. Its excellent spectral characteristics, solubility in organic and aqueous solvents and stability over a wide range of pH values show particular interest for this radical in the estimation of the antioxidant activity of total extracts [33].

Using this method, the concentration of the radical-cation reduced by the total anthocyanin extracts for 35 minutes was measured. Thus, the reactivity was estimated by the effective concentration EC50 (or the inverse 1/EC50) of the extract, which corresponds to a 50% reduction in the activity (absorbance) of the radical cation ABTS^•+ in the reaction mixture. The antioxidant capacity of a compound is all the higher the smaller its EC50. In addition, to better characterize the anti-radical power of the extract, the monitoring of the kinetics of the reduction was carried out by the determination the TEC50 time necessary to reach the equilibrium at EC50

(Figure 11-16). The anthocyanin extract studied has an EC50 of 4.24 mg/mL against 0.024 and 0.034 mg/mL respectively for ascorbic acid and Trolox and a TEC50 time of 21 minutes (intermediate reaction) (Table 3). Indeed, the TEC50 time allows the classification of the reactivity of an extract. The reaction is fast when TEC50 is less than 5 minutes, intermediate when TEC50 is between 5 and 30 minutes and slow if TEC50 is greater than 30 minutes [22]. The low antiradical activity and reactivity of this extract could be explained on the one hand by the mechanism of action of trapping the ABTS^•+ radical-cation and on the other hand by the structure of the molecules.

Comparative studies carried out on anthocyanin aglycones reveal that hydroxyl and methoxyl groups influence the antioxidant activity. Indeed, anthocyanins with a single OH group in 4'-position on the B-ring (figure 10) such as pelargonidin, malvidin and peonidin have low antioxidant activity compared to cyanidin hydroxylated at the 3'- and 4'-positions [34].

Bors and colleagues [35] showed that hydroxyls at the 3' and 4' positions of the B-ring of anthocyanins are very crucial in the free radical scavenging activity. HPLC-ESI-MS/MS analysis reveals that our extract had three anthocyanic molecules (cyanidin 3-O-(p-coumaroyl) glucoside, pelargonidin 3-O-glucoside, pelargonidin 3-O-rutinoside), of which pelargonidin 3- O-rutinoside is the major compound, which would explain the low activity of H. monopetalus extract. It should also be noted that the different constituents of the extract can act in synergy, each one influencing more or less the global antioxidant capacity. In addition, glycosylation is a factor that influences the antioxidant activity of anthocyanins. It either increases, decreases or has no significant effect on the free radical-scavenging activity [34]. For the same substituent, the high number of sugars tends to decrease the antiradical activity.

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A C

B 7

8

6

5 9 4 3

1

2 1' 2'

3' 4' 5' 6' 10

Figure 10. Aglycone of anthocyanins

It should be noted that total anthocyanin extracts are complex mixtures whose antioxidant activities cannot be explained by structural considerations of anthocyanin constituents alone.

There could be non-anthocyanic compounds that affect the free radical-scavenging activity in one way or another. The activity of an antioxidant depends on its capacity to form (during the inhibition process of a free radical) a more stable radical [35]. The number of hydroxyl groups and sugars contributes to increase or decrease the stability, thus affecting the antioxidant potential. It also depends on the free radical chosen to assess antioxidant capacity. In addition to the anti-free radical activity of anthocyanins, they have enormous biological properties such as anti-inflammatory and anti-cancer activity and have a major impact on the prevention and control of cardiovascular diseases [36].

Figure 11. The curve showing the reduction of the radical by ascorbic acid as a function of time

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Figure 12. Determination of the effective concentration EC50 of the methanolic solution of ascorbic acid

Figure 13. The curve showing the reduction of the radical by Trolox as a function of time

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Figure 14. Determination of the effective EC50 concentration of the methanolic solution of Trolox

Figure 15. The curve showing the reduction of the radical by the extract as a function of time

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Figure 16. Determination of the effective concentration EC50 of the methanolic solution of the extract

Table 2. Total phenolic compounds (TPC), flavonoids (TFT) and anthocyanins content (TAC)

TPC TFC TAC

Content in mg/100 g 1165±3.1 4490±20.2 36±0.17

Table 3. Antioxidant activities of total anthocyanin extract determined by the ABTS method Total extracts Equation of the

curve Correlation coefficient

Antioxidant capacity IC50

(mg/mL) TEC50

(min)

Ascorbic acid 0.0577x^-1.809 0.9759 0.024 4

Trolox 0.3659x^-1.459 0.989 0.034 10

H. monopetalus 70.227x^-0.238 0.9901 4.24 21

CONCLUSION

Using HPLC-MS/MS method, three anthocyanins were identified in ripe fruits of H.

monopetalus : namely cyanidin 3-O-(p-coumaroyl) glucoside, pelargonidin 3-O-glucoside and pelargonidin 3-O-rutinoside. Pelargonidin 3-O-rutinoside was thought to be the major compound in the fruit of this species. In addition, antiradical activity of the anthocyanin-rich extract from ripe fruits of H. monopetalus was evaluated and ABTS^•+ radical cation reduction kinetics were monitored. The extract manifested a weaker anti-radical activity than that of the two standards used (Ascorbic acid, Trolox). This could be explained by several factors such as the trapping mechanism of the ABTS^•+ radical-cation and and also by the structure of the

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molecules. The evaluation of the micronutrient content showed that this fruit is a potential source of bioactive compounds (phenolic compounds, flavonoids and anthocyanins) very interesting for the prevention and the treatment of a great number of pathologies.

ACKNOWLEDGMENT

The authors would like to thank the Joseph KI ZERBO University and the National Public Health Laboratory (LNSP) (Burkina Faso) for all the efforts made to carry out this study.

CONFLICT OF INTEREST

Authors declare the submitted manuscript have no any conflict of interest.

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