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Chemical composition and antioxidant activity of Borago officinalis L. leaf extract growing in Algeria
⁎Corresponding author at: Laboratoire de biochimie et de toxicologie environnementale, Faculté des sciences, Université Badji Mokhtar, Annaba, Algeria.
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
The aqueous and hydroalcoholic extracts of borage (Borago officinalis) leaves from Annaba region (Algeria) were preliminary analyzed for their phenolic profile (total phenolics, total flavonoids, total flavonols, total tannins and total anthocyanins). These extracts were evaluated for their antioxidant properties by different methods such as DPPH radical scavenging, test NBT and total antioxidant activity. The two extracts have exhibited a high antiradical capacity. Indeed, the ethanolic extract showed the lower IC50 values and the highest amount of phenolics (94.09 ± 1.72 mg gallic acid/g dry extract). Using LC-MS/MS analysis, it was possible to identify phenolic acids, flavonoids, sterol and for the first time oleuropein was identified in the aqueous extract of the plant. The obtained results have demonstrated that phenolic compounds are the major contributor to the antioxidant activity of plants.
Keywords
Borago officinalis
Total phenol
Phenolic profile
DPPH
Antioxidant activity
- BHA
-
butylated hydroxy anisole
- BHT
-
butylated hydroxy toluene
- BO
-
Borago officinalis
- C
-
catechin
- C-3-G
-
cyanidin-3-glucoside
- DE
-
dry extract
- DPPH
-
1,1-diphenyl-2-picrylhydrazyl
- EDTA
-
ethylene diamine tetra acetic acid
- ESI-MS/MS
-
electrospray Ionization Mass Spectrometry
- GA
-
gallic acid
- HPLC
-
high performance liquid chromatography
- IC50
-
half maximal inhibitory concentration
- IP
-
inhibition percentage
- LC-MS/MS
-
liquid chromatography coupled to tandem mass spectrometry
- Mo
-
molybdate
- MS
-
mass spectrum
- NBT
-
nitroblue tetrazolium
- OD
-
optical density
- Q
-
quercitin
- R
-
rutin
- ROS
-
reactive oxygen species
- TBHQ
-
tert-butyl hydroquinone
Abbreviations
1 Introduction
Recently, the biological and medical sciences are invaded by a new concept called “oxidative stress”, in which the cell cannot control the excessive presence of reactive oxygen species (ROS). ROS such as superoxide anions (O2•−), hydroxyl radical (OH•) and nitric oxide (NO) are produced by biological combustion in the respiration process.
Currently, it is well known that although oxidative stress is not a disease in itself, it is potentially involved in many diseases as a trigger or associated with complications during their evolution as in cancer, cardiovascular diseases, inflammatory lung diseases, immune dysfunctions and neurodegenerative disorders. ROS can also cause a change in the organoleptic properties of foods by the oxidative degradation of their constituent lipids (Yamaguchi et al., 2004).
Hence, the balance between antioxidation and oxidation is believed to be a critical concept for maintaining a healthy biological system (Tiwari, 2001). So, to prevent or reduce the oxidative stress, sufficient amounts of antioxidants need to be consumed or added to foods.
The antioxidants can be of synthetic or natural origin. Fact, synthetic antioxidants, such as butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and tert-butyl hydroquinone (TBHQ), are widely used in the food industry. Although, they are effective and less expensive than natural antioxidants, some toxic effects have been reported (Choi et al., 2000). Thus, research for a safer and effective natural antioxidant is in progress. Among these natural antioxidants, phenolic antioxidants can be mentioned. Actually, various methods have been used to evaluate the antioxidant properties of phenolic compounds in vitro (Antolovich et al., 2002). These compounds are proved to be more potent antioxidants than Vitamins E and C and carotenoids. Besides, they are reported to quench the oxygen-derived and substrate-derived free radicals by donating a hydrogen atom or an electron to the free radical (Wettasinghe and Shahidi, 1999). Phenolic compounds are present in a wide range of vegetables and plants, playing an important role in defense mechanisms (Arici et al., 2014).
Borage (Borago officinalis L.) is a herbaceous plant of the Boraginaceae family native to North Africa and widely spread in many Mediterranean countries. In Algeria, this plant is used not only for preparing beverages and salads but also for different medicinal purposes. Over the last years, in spite of being the subject of increasing agricultural interest thanks to its high content of γ- linolenic acid in seed (Mhamdi et al., 2009), to the best of our knowledge, few research studies have been devoted to the investigation of its antioxidant activity. For example, Conforti et al. (2008) have demonstrated the strong antioxidant activity of borage leaves extract. Other research works have reported that the ethanolic extract of defatted seeds possess phenolic acids, antioxidant and free radical scavenging activities (Mhamdi et al., 2010a). However, the information pertaining to the active components of borage leaves is very scarce. It is worthy to mention that the presence of fatty acids (Ciriano et al., 2009), phenolic acids (Mhamdi et al., 2010b), pyrrolizidines alkaloids (El-Shazly and Wink, 2014), and sterols (Conforti et al., 2008) was previously noticed.
In folk medication, the most of medicinal plants are used by making their aqueous extracts as raw materials but it is important to note that same biomass is more beneficiary if extracted in ethanol rather than water. Therefore, the Algerian borage was preliminarily characterised by comparing the aqueous extract and the ethanol extract through their chemical compositions by using colorimetric methods and mass spectrometry. Furthermore, the antioxidant activity of both extracts of borage was carried out using three methods: 2,2- diphenyl-picrylhydrazyl radical-scavenging assay (DPPH test), total antioxidant activity by phosphomolybdenum method, and scavenging of superoxide radical (NBT test).
2 Materials and methods
2.1 Plants materials
Fresh entire leaves of Borago officinalis (BO) were collected in March 2012 from his natural habitat at the East flank of Mount Edough (facing the Mediterranean Sea) in Sidi Aissa. This semi-urban area, located to the north of the conurbation of Annaba (Latitude: 36° 55′30.84″N/Longitude: 7° 44′52.56″ E) is weakly urbanized and with very low traffic. Harvesting was carried out according to good harvesting practices for medicinal plants established by OMS (2003).
2.2 Chemicals
Ammonium molybdate, gallic acid and vanillin were obtained from Biochem, Chemopharma (Cosne Sur Loire, France). All other chemical products used in this study were purchased from Sigma Chemical Co (St Louis, France).
2.3 Extraction procedure
The leaves were washed thoroughly under tap water, air-dried and powdered using electric grinder to obtain a powder.
2.3.1 Aqueous extract
Ten grams of BO leaves powder were boiled with 200 mL of distilled water for 20 min with an occasional stirring. The decoction preparation was then filtered through a muslin cloth followed by filtration with filter paper. The extract was evaporated to one-fifth of its original volume and kept at 4 °C until its use.
2.3.2 Ethanol extract
Dried and powder materials were extracted with 80% EtOH (1:10, w/v) for 24 h, and concentrated under reduced pressure at a temperature of 60 °C in a rotary evaporator.
2.4 Determination of the total phenols
The determination of the total phenolic compounds was performed by means of the Folin–Ciocalteau reagent and the method described by Gargouri et al. (2013). The total phenolic content was expressed as milligrams of gallic acid (GA) equivalent per gram of extract. The total phenolics were carried out by using standard gallic acid curve with least square regression (y = 0.011x, R2 = 0.990). The optical density was measured at λ = 765 nm and carried out using a spectrophotometer (Shimadzu UV-1800 PC, Japan).
2.5 Total flavonoids contents
The total flavonoids were measured by a colorimetric assay adopted by Bouaziz et al. (2010). One mL aliquot of appropriately diluted sample or standard solution of quercitin was added to a 10 mL volumetric flask containing 4 mL double distillate water (ddH2O). At zero time, 0.3 mL 5% NaNO2 was added to the flask. After 5 min, 0.3 mL 10% AlCl3 was added. At 6 min, 2 mL of 1 M NaOH was added to the mixture. Immediately, the reaction flask was diluted with the addition of 2.4 mL of ddH2O and thoroughly mixed. The absorbance of the mixture –pink color– was determined at 510 nm compared to control water. The total flavonoid contents are expressed as mg quercitin (Q) equivalents/g of extract (Y = 0.008, R2 = 0.997).
2.6 Total flavonols content
The content of flavonols was determined by the method of Yermakov et al. (1987). A standard curve of rutin was performed by mixing 2 mL of different concentrations of methanolic solutions of rutin with 2 mL of AlCl3 (20 mg/mL) and 6 mL of sodium acetate (50 mg/mL). The absorbance was measured after 2.5 h at 440 nm. The same procedure was followed for 2 mL of plant extracts (3.5 mg/mL). The total flavonols were expressed as dry extract mg/g rutin equivalents (R). For rutin, the curve absorbance against concentration was described by the equation (y = 0.085x, R2 = 1).
2.7 Total tannins content
The total tannins content in the plant extracts were determined according the method of Bouaziz et al. (2008). 50 μl of the extract was added to 3 ml vanillin/methanol (4%). After stirring, 1,5 mL concentrated HCl was added. The absorbance was read at 500 nm after 15 min. The total tannin contents are expressed as mg catechin equivalent (C)/g of dry extract (y = 0.005x, R2 = 0.988).
2.8 Total anthocyanins content
The total anthocyanins are water-soluble pigments. The total anthocyanins were estimated by a pH differential method (Lee et al., 2005). The absorbance was measured in spectrophotometer at 520 nm and at 700 nm in buffers at pH 1 and 4.5, using a molar extinction coefficient of cyanidin-3-glucoside of 29,600.
The results of the equation were expressed as milligrams of cyanidin-3-glucoside equivalents per g of extract after the calculation of the following formula: where A is the absorbance calculated by the way: (Aλ520 − Aλ700)pH=1 − (Aλ520 − Aλ700)pH=4.5; MM is molecular mass of cyanidin-3-glucoside; DF is the dilution factor; ɛ is the extinction coefficient; d is the thickness of a curve.
2.9 LC-MS/MS analysis
LC-UV-MS/MS analyses were performed as described by Kite et al. (2007) on a Thermo Scientific System consisting of an Accela U-HPLC unit with a photodiode array detector and an LTQ Orbitrap XL mass spectrometer fitted with an electrospray source. The following parameters were employed throughout all MS experiments: for electrospray ionization with positive ion polarity the capillary voltage was set to 3.5 kV, the drying temperature to 350 °C, the nebulizer pressure to 40 psi, and the drying gas flow to 10 l min−1. The maximum accumulation time was 50 ms, the scan speed was 26,000 mz−1 s−1 (ultra scan mode) and the fragmentation time was 30 ms.
Chromatography was performed on 5 μL sample injections on to a 150 × 3 mm i.d., 3 μm, Luna C18 (2) column (Phenomenex). A 400 μL/min linear mobile phase gradient of methanol/water/acetonitrile +1% formic acid was used, changing from 0:90:10 to 90:0:10 over 20 min, followed by an isocratic phase for 5 min and then a column wash phase and equilibrium of column for 3 min before the next injection.
The ESI source of the mass spectrometer was operated in both positive and negative modes under the recommended manufacturers conditions for the mobile phase parameters. The orbitrap mass analyser was set to scan in the range of m/z 200–2000 at 30,000 resolutions in one polarity while the linear ion-trap analyser performed MSn analyses on the most abundant ions in both polarities using an ion-isolation window of +2 m/z and relative collision energy of 35%. For the accurate mass analyses of product ions generated by MS2 in the ion trap, the ions were scanned by the orbitrap at 7000 resolutions. Negative mode ESI was utilized since the phenolic compounds in question ionize better in this mode. The acidification of the LC mobile phase allows the best separation as the hydroxyl groups on compounds are kept in their acidic form, thereby increasing their retention on the column and decreasing peak broadening.
2.10 Determination of antioxidant activity
2.10.1 DPPH radical scavenging assay
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging effect was evaluated following the procedure described in a previous study (Bouaziz et al., 2008). In succinct terms, the aliquots (50 mL) of various concentrations of the compound tested were added to 5 mL of a 0.004% methanol solution of DPPH. After 30 min of incubation at room temperature, the absorbance was read against a blank at 517 nm. The inhibition of free radicals DPPH in percentage (IP%) was calculated in the following way:
IP% = [(Ablank − Asample)/Ablank] × 100, where IP is the inhibition percentage; Ablank is the absorbance of the control reaction (containing all reagents except the test extract), and Asample is the absorbance of the test compound. The results are expressed as IC50, the amount of antioxidant necessary to decrease the initial concentration of DPPH by 50%. The lower IC50 values indicate a higher antioxidant activity. The synthetic antioxidants butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls.
2.10.2 Scavenging of superoxide radical (NBT test)
The scavenging activity toward the superoxide radical (O2•−) was measured in terms of inhibition of O2•− generation. The nitroblue tetrazolium (NBT) reacts with the superoxide anion to give the oxidized NBT (tetrazolyl) which becomes formazan, water insoluble and purple (Yagi et al., 2002).
The reaction mixture consisted of 100 μL of samples, phosphate buffer, riboflavin, EDTA and NBT. The absorbance was read at 580 nm after illumination under UV lamp for 10 min against blank. The blank contained all the components except NBT. The percentage of inhibition was calculated using the following formula: IP% = [1 − (ODsample/OD100%)] × 100. Where IP is the inhibition percentage, OD sample is the absorbance of the test compound and OD 100% is the absorbance of the control reaction. The tested compound concentration, which provided 50% inhibition (IC50, expressed in μg/mL), was calculated from the graph plotted inhibition percentage against the extract concentration.
2.10.3 Total antioxidant activity by phosphomolybdenum method
The total antioxidant capacity was based on the reduction of ammonium molybdate (IV) to ammonium molybdate (V) by the sample and the subsequent formation of green phosphate/Mo (V) compounds with a maximum absorption at 695 nm (Prieto et al., 1999).
0.1 mL of each sample (10 mg/mL) was mixed with the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate in 100 mL). The tubes were incubated in boiling water bath at 95 °C for 90 min. Then the solution was cooled to room temperature and absorbance was read at 695 nm with spectrophotometer against blank. The total antioxidant activity results were the ratio between the positive control and the extracts under study. The results are expressed in μg positive control (BHT or Vit C)/μg extract, after the calculation of the following relation: x positive control (μg/mL)/x extract (μg/mL).
2.11 Statistical analysis
Three replicates of each sample were used for statistical analysis. Data were expressed as mean ± standard error (SE). In all experiments, data were subjected to correlation coefficient in Excel 2007.
3 Results
3.1 Extracts yields and polyphenolic contents
The aqueous and ethanolic extracts from Borago officinalis leaves yielded approximately 45% and 4.83% respectively.
The results for the quantitative determination of the total phenols, flavonoids, flavonols, tannins and anthocyanins contents of the aqueous and ethanolic extracts of Borago officinalis leaves are showed in Table 1. The Statistic analysis of phenolic content revealed a high significant difference (P ⩽ 0.001) between the aqueous (35.48 ± 2.70 mg GAE/g DE) and ethanolic extract (94.09 ± 1.72 mg GAE/g DE).
| Samples | Total polyphenols (mg GAE/g DE) | Flavonoids (mg QE/g DE) | Flavonols (mg R/g DE) | Tannins (mg C/g DE) | Anthocyanins (mg C-3-G/g DE) |
|---|---|---|---|---|---|
| Borago officinalis | |||||
| Ethanol extract | 94.09 ± 1.72 | 37.65 ± 3.93 | 11.39 ± 1.56 | 6.67 ± 0.69 | 0.27 ± 0.09 |
| Water extract | 35.48 ± 2.70 | 20.79 ± 2.95 | 10.88 ± 0.18 | 3.14 ± 0.60 | 0.09 ± 0.01 |
Values are mean ± SE of three replicates for each estimation.
The statistical differences between the ethanolic and aqueous extracts are shown in table as significant (∗) or very high significant (∗∗∗).
The total flavonoid content in the ethanolic extract (37.65 ± 3.93 mg QE/g DE) was higher than that in the aqueous extract (20.79 ± 2.95 mg QE/g DE). It varied significantly (P ⩽ 0.05) from one another. Moreover, no significant differences (P ⩾ 0.05) were found in the total flavonols, representing 11.39 ± 1.56 mg rutin/g DE for the ethanolic extract and 10.88 ± 0.18 mg/g DE for the aqueous extract.
Tannins and anthocyanins were also estimated in extracts. According to Table 1, the tannins and anthocyanins are present in low amounts in both extracts. In fact, while the variation of the tannins amount was significant (P ⩽ 0.05) between both extracts, that of anthocyanins amount was of no significance (P ⩾ 0.05).
3.2 Tentative identification of the chemical compounds by LC MS/MS analysis
Using the LC MS/MS apparatus, it was possible to identify the majority of the compounds of the analyzed plant. All the detected molecules were characterized by studying their fragmentation obtained by ESI-MS/MS in positive and negative mode, as well as by using the literature data and optionally by injection standards under the same chromatographic conditions. The identities, retention times, UV characteristics, and observed molecular and fragmented ions for individual components are presented in Table 2.
| No peak | RT (min) | UV (nm) | Formula | Extract | [M−H]−/[M+H]+ | MS/MS | Tentative identification |
|---|---|---|---|---|---|---|---|
| 1 | 1.33 | 256 | C17H22O3 | a,b | 273 | 175, 123 | m-Geranyl-p-hydroxybenzoic acid |
| 2 | 2.86 | 325 | C16H16O8 | b | 335 | 317, 179, 161 | Caffeoyl shikimate acid |
| 3 | 5.39 | 362 | C9H10O4 | a,b | 181 | 166,153, 139 | Syringaldehyde |
| 4 | 6.37/7.11 | 280, 340 | C8H8O3 | a | 181/183 | 137, 113, 109 | p-Hydroxyphenyl lactic acid |
| 5 | 7.53 | 222, 298 | C17H21O10 | a | /387 | 225, 207 | Sinapic acid hexoside |
| 6 | 10.25 | 254, 286, 308sh, | C36H30O16 | a | 717 | 537, 519, 493 | Lithospermic acid B |
| 7 | 10.38 | 260, 355 | C21H20O12 | a | /465 | 303, 273, 257 | Quercetin-3-O-glucoside (Isoquercetin) |
| 8 | 10.54 | 280 | C10H12O4 | b | /197 | 179, 153, 138 | Dihydroferulic acid |
| 9 | 10.58 | 210 | C29H50O | a | 413/ | Nd | β-Sitosterol |
| 10 | 11.65/11.40 | 253, 268, 345 | C21H19O11 | a | 447/449 | 285, 257, 243, 213, 199 | Luteolin 7-O-glucoside |
| 11 | 12.25/11.41 | 256, 352 | C21H19O11 | a | 447/449 | 301, 283, 257, 179, 151 | Quercetin-3-O-rhamnoside (Quercetin) |
| 12 | 12.55 | 278 | C21H24O11 | a | 451 | 289, 179, 165, 137 | Catechin-7-O-glucoside |
| 13 | 11.79 | 254 | C25H32O13 | b | 539 | 377, 307, 275 | Oleuropein |
| 14 | 12.96 | 268, 337 | C21H20O10 | a | 431 | 311, 269, 207 | Apigenin 8-C-glucoside (Vitexin) |
| 15 | 13.03 | 270, 334 | C21H20O10 | a | 431 | 311, 269, 251, 207 | Apigenin 6-C-glucoside (isovitexin) |
| 16 | 13.67 | 278 | C11H12O4 | b | 207 | 164, 134, 104 | 3,4-Dimethoxycinnamic acid |
| 17 | 14.96 | 322 | C9H8O4 | a | /181 | 178, 134 | Caffeic acid |
| 18 | 16.25 | 242, 270sh, 343 | C18H16O6 | b | 327 | 285 | Luteolin 7,3′,4′-trimethyl ether |
| 19 | 16.26 | 266, 353 | C18H16O6 | b | 327 | 285 | Kaempferol 3,7,4′-trimethyl ether |
| 20 | 19.83 | 280 | C14H21N4O3 | b | 291 | 273, 231, 145 | Coumaroyl hydroxyagmatine |
| 21 | 20.07 | 283, 340 | C21H22O10 | a,b | 433/435 | 271 | Naringenin O-hexosides |
| 22 | 20.48 | 256 | C13H16O8 | a,b | /301 | 139 | 4-Hydroxybenzoic acid glucoside |
(a): Identified in aqueous extract; (b) identified in ethanolic extract; sh: should; nd: not defined.
As regards the aqueous extract, 11 constituents were identified (Table 2). For example, compound 4 with a [M−H]− ion at m/z 181 was identified as a p-hydroxyphenyl lactic acid. Its spectrum revealed the typical fragmentation of its constituents groups, with fragmentations at m/z 137 and 109, due to the loss of –COO and –CO groups, respectively, as previously described (Santos et al., 2010). The identification of compound 13 as Oleuropein was performed by LC-MS as well as by the analysis of UV spectra. The mass spectrum ESI-MS showed a pseudomolecular ion at m/z 539 of the fragments tuned with the image of the fragmentation ion at m/z 377, emanating from the cleavage of the glycosyl link. Besides, the ion at m/z 307 is explained by the loss of a C4H6O fragment (Caruso et al., 2000), while the fragment at m/z 275 can be estimated from the rearranged fragments (De la Torre-Carbot et al., 2005) (Fig. 1).
(a) Chromatogram of ion at m/z 539 in the negative ion mode full MS spectra and MS2 spectra. (b) Chromatogram of ion at m/z 447 in the negative ion mode full MS spectra and MS2 spectra. (c) Chromatogram of ion at m/z 451 in the negative ion mode full MS spectra and MS2 spectra.
In the ethanol extract of Borago officinalis, 15 compounds were detected (Table 2). For example, the mass spectrum in the negative mode of compound 10 showed a base peak [M−H]− m/z 447 and an aglycone ion at m/z 285. The loss of 162 amu from the intermediate ion is due to the loss of glucose. The λmax of the UV spectrum at 253, 268, 345 nm suggests that is luteolin 7-O-glucoside. The compound 11 gives ion [M−H]− m/z 447 corresponding to the base peak in the negative mode spectrum. In MS2, the fragment ion m/z 301 [M−H-146]− brought about the indication of a rhamnoside and its O-glycosidic linkage. The UV spectrum gave birth to maxima at 256 and 352 nm pointing to a quercetin-type flavonol (Michel, 2011). Other peaks were observed in the same spectrum at m/z 283, 257, 179 and 151. The loss of a water molecule gave an ion at m/z 283. The retrocyclization of ion m/z 301 generated an ion at m/z 179, which lost a molecule of carbon monoxide (CO) to give ion at m/z 151. In addition, m/z 301 lost 28 amu and an ion corresponding to m/z 257 was generated. This suggests that it could be a quercetin 3-O-rhamnoside (quercetin) (Fig. 1).
A negative ion mass spectrum of compound 12 showed [M−H]− m/z 451 and an aglycone ion at m/z 289 in MS2 (Fig. 1). Besides, the MS2 spectrum exhibited fragments at m/z 179, 165 and 137. According to (Chua et al., 2008) catechin (m/z 289) was detected in the negative mode and a peak with low intensity m/z 137 [M−H-152]− resulted from a retrocyclization; type Retro Diels–Alder (RDA). The loss of 3,4 dihydroxy-phenyl and catechol generated fragments at m/z 165 and m/z 179, respectively (Fig. 2). This suggests that compound 12 with 278 maxima UV spectrum is catechin-7-O-glucoside.![Proposed scheme for fragmentation of [M−H]− of compound 12.](/content/184/2019/12/8/img/10.1016_j.arabjc.2014.11.059-fig2.png)
Proposed scheme for fragmentation of [M−H]− of compound 12.
The mass spectrum shows a monopic MS1 [M−H]− at m/z 431 in negative mode. The MS2 spectrum also showed a lower intensity peak at m/z 413, corresponding to the loss of a water molecule (H2O) and another at m/z 862 corresponding to [2M−H]−. The MS2 spectrum had a peak at m/z 311 obtained by the loss of 120 amu (C-glycoside) and another corresponding to the loss of a hexosyl (162 amu) at m/z 269. The ion peak m/z 269 was fragmented into a peak at m/z 251 (loss of 18 amu) and another at m/z 207 (loss of 44 amu). These suggestions along with the maxima UV spectrum show that compound 14 is vitexin (apigenin 8-C-glucoside).
The elected compound 15 gave a mass spectrum identical to that reported for vitexin with the presence of an intense ion m/z 431 and several fragments (m/z 311, 269 and 207), suggesting that it could be identified as isovitexin.
Equally, the compounds in Table 2 were identified by comparing HPLC retention times, UV spectra and mass spectra with the data obtained from standard in-house libraries.
3.3 Antioxidant activity
To screen the antioxidant properties of the samples, several chemical and biochemical assays were performed: scavenging activity on DPPH radicals, total antioxidant activity and scavenging of superoxide radical (NBT test).
The antioxidant activities of the investigated plant extracts were compared with two antioxidant, BHA and ascorbic acid, which were used as reference standards.
3.3.1 Free radical scavenging activity
The DPPH antioxidant activity of the extracts indicates their ability to dispose of hydrogen atoms. The free radical scavenging potency of the samples is presented in Table 3. As illustrated, the ethanolic extract of Borago officinalis was found to exhibit the highest radical scavenging (92.85 ± 3.07 μg/mL). The IC50 of BHT and ascorbic acid was 22.5 ± 0.62 μg/mL and 3.14 ± 0.36 μg/mL, respectively. The DPPH radical scavenging activity of the tested samples was in the order: ascorbic acid > BHT > ethanolic extract > aqueous extract.
| Samples | DPPH IC50 (μg/ml) | NBT IC50 (μg/ml) | Total antioxidant activity | |
|---|---|---|---|---|
| BHT (mg BHT/mg extract) | Vit C (mg vit C/mg extract) | |||
| Borago officinalis | ||||
| Ethanol extract | 92.85 ± 3.07 | 175.73 ± 0.60 | 1.374 | 0.235 |
| Water extract | 150.37 ± 0.99 | 346.25 ± 3.52 | 0.887 | 0.151 |
| BHT | 22.5 ± 0.62 | 189.27 ± 6.23 | – | – |
| VIT C | 3.14 ± 0.36 | 167.47 ± 1.40 | – | – |
Values are mean ± SE of three replicates for each estimation.
Only the statistical differences between the ethanolic and aqueous extracts are shown in table as significant (∗) or high significant (∗∗).
The Statistic results have revealed a high significant difference (P ⩽ 0.01) of the DPPH antioxidant activity between ethanolic and aqueous extracts. Therefore, there is a high difference (P ⩽ 0.01) between the standards and the ethanolic extract and a very high difference (P ⩽ 0.001) between the standards and the aqueous extract.
3.3.2 Scavenging of superoxide radical (NBT test)
The antioxidant activity was also determined by the inhibition of the oxidation of NBT, i.e., by inhibiting the formation of superoxide anion O2•−. The scavenging of superoxide radical estimated by NBT test of the extracts is shown in Table 3. The ethanolic extract has shown higher antioxidant activity (175.73 ± 0.60 μg/mL) than BHT (189.27 ± 6.23 μg/mL) and aqueous extract (346.25 ± 3.52 μg/mL), but lower than that of ascorbic acid (167.47 ± 1.40 μg/mL).
Statistically, a high significant difference (P ⩽ 0.01) of the antioxidant activity between the ethanolic and aqueous extracts is detected. Moreover, the antioxidative capacity of BHT reveals no significant differences (P ⩾ 0.05) with that of ethanolic extracts. However, it shows a high significant difference (P ⩽ 0.01) with that of aqueous extracts. Besides, ascorbic acid shows a very high difference (P ⩽ 0.001) in both extracts.
3.3.3 Total antioxidant activity
Table 3 shows that the total antioxidant activity of ethanolic extract of Borago officinalis is higher than BHT with a ratio of 1.37. While the aqueous extract has a lower activity compared with two positive controls, the magnitude of values for the successive extracts shows a similar trend in NBT test and total antioxidant activity.
4 Discussion
The increasing interest in the search for natural alternatives for synthetic antioxidants has led to the antioxidant evaluation of a number of plant sources. In the present study, Borago officinalis extracts are used as a potential source of bioactive compounds. To achieve this purpose, the chemical composition and antioxidant activity of the aqueous and ethanolic extracts of borage were investigated.
The data presented in this study demonstrate that the extracts of Borago officinalis possess antioxidant and free radical scavenging activities. This suggests that the investigated leaf extracts of borage could also exert protective effects in vivo against oxidative and free radical injuries occurring in different pathological conditions.
The antioxidant activity of plant extracts is usually linked to their phenolic content. For that reason several research studies have evaluated the relationships between the antioxidant activity of plant products and their phenolic content. In some studies, a correlation between them was found (Velioglu et al., 1998). In this study, the findings have shown a relationship between the antioxidant activity and total phenolic content. This agrees well with the idea that the phenolic compounds have a key role in free radical scavenging and/or reducing systems. Nevertheless, these results must be interpreted with caution as the method used for estimating the total phenolic content has weak selectivity because the Folin–Ciocalteu reagent reacts positively with different antioxidant compounds (phenolic and no phenolic substances) (Que et al., 2006).
The ethanolic extract has been shown to have higher total polyphenolic contents and antioxidant capacity than aqueous extract, probably due to the polarity and good solubility for phenolic components in ethanol (Siddhuraju and Becker, 2003). The amount of total phenol in the ethanolic extract (94.09 ± 1.72 mg GAE/g DE) was in accordance with the findings of Conforti et al. (2008). But the studied extract exhibited higher flavonoids content and lower antioxidant activity compared to the results reported for Italian Borage by Conforti et al. (2008). Furthermore, the results of DPPH antioxidant activity of ethanolic leaf extract have shown higher activity than those reported by Mhamdi et al. (2010a) for the methanolic leaf extract of Tunisian borage. Such differences could be due to the environmental conditions and geographical distribution, which can modify the constituents of the plant (Hossain and Shah, 2015). Moreover, the antioxidant activities of the plant extracts could be due to the different qualitative and quantitative compositions of their phenolic constituents, from phenolic acids to flavonoids and their derivatives. Consequently, a chemical analysis for the determination of the extract compounds is necessary. Thus, LC-MS was utilized to separate and evaluate the chemical constituents in borage leaves extracts and to assess their antioxidant activity. As shown in Table 2, the phytochemical investigation of borage leaves has led to the identification of 9 flavonoids, 7 phenolic acid derivates, three phenolic acids, a secoiridoid, a phenolic aldehyde and a sterol.
Among the phenolic acids identified, caffeic acid, p-hydroxyphenyl lactic acid and p-hydroxybenzoic acid were reported in methanolic seeds extract of borage by Zadernowskia et al. (2002). In fact, the phenolic compounds were already identified in methanolic leaf extract of borage by Mhamdi et al. (2010a) who have shown the presence of ferulic acid, cinnamic acid, syringic acid, sinapic acid and coumaric acid, which is in agreement with the presence of this phenolic acid derivates in our extracts. The identification of β-Sitosterol in ethanolic extract is in accordance with the findings by Conforti et al. (2008). Therefore, the oleuropein, known for its presence in olive leaves (Jemai et al., 2008), was reported in the aqueous extract of borage for the first time. Besides, all flavonoids and lithospermic acid were identified for the first time in Borago officinalis in the present study, though already reported in Boraginaceae family (Wollenwebera et al., 2002; Yamamoto et al., 2002).
The differences in the antioxidant capacity of the extracts could be attributed to the qualitative variability in compounds between them. For instance, the antioxidant activities of phenolic acids and their derivatives, such as esters, depend on the number of hydroxy groups in the molecules (Soobrattee et al., 2005). In addition, it has been reported that the antioxidant potency of flavonoids is roughly proportional to the total number of –OH groups and is positively affected by the presence of an o-dihydroxy moiety in the B-ring (Apak et al., 2008). In this regard, the presence of 7 flavonoids (quercetin, isoquercetin, catechin-7-O-glucoside, naringenin O-hexoside, luteolin 7-O-glucoside, vitexin and isovitexin) was reported for the ethanolic extract and three flavonoids (luteolin 7,3′,4′-trimethyl ether, kaempferol 3,7,4′-trimethyl ether and naringenin O-hexoside) for the aqueous extract. This partially explains the high antioxidant capacity of the ethanolic extract.
Moreover, a strong antioxidant capacity is also reported for the compounds 6 (Damašius et al., 2014), 9 (Conforti et al., 2008) and 17 (Gülçin, 2006) identified in the ethanolic extract and compound 13 (Jemai et al., 2008) present in the aqueous extract.
It is generally assumed that the ability to act as a hydrogen donor and the inhibition of oxidation are due to the synergism between the antioxidants in the samples, which makes the antioxidant capacity dependant not only on the concentration of phenols, but also on their structure and the interaction between them (Hmid et al., 2013). Thus it is possible to deduce that the antioxidant power of the aqueous extract is lower than the ethanolic extract due to its low content of phenols compared to ethanol extract.
5 Conclusion
In the present study, the high phenolic content and antioxidant potential of Algerian Borago officinalis have been demonstrated. The obtained results have shown that the ethanolic extract of borage was highly active against reactive oxygen species. In addition, the LC MS/MS analysis of leaf extracts of Borago officinalis showed the presence of 10 flavonoids and a secoiridoid (oleuropein), which are isolated for the first time in borage. The antioxidant activity of the extracts could be attributed to these compounds. Moreover, this research work has revealed that the Algerian borage can be an interesting source of antioxidants, with a potential use in the food and/or pharmaceutical industries. However, further research would be required.
Acknowledgments
The authors would like to thank the Algerian and Tunisian Ministries of Higher Education and Scientific Research for the support of this research work via the Algero-Tunisian project (2012-2014). They also wish to extend their thanks to Mrs. Leila MAHFOUDHI, an English teacher at the Sfax Faculty of Science, for having proofread this paper.
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