Saliha Dassamiourab,
Selsabil Meguellatib,
Hdouda Lamraouib,
Mohamed Sabri Bensaadac,
Rokayya Sami*d,
Garsa Alshehryd,
Eman Hillal Althubaitie and
Areej Suliman Al-Meshalf
aLaboratory of Biotechnology of Bioactive Molecules and Cellular Physiopathology (LBMBPC), Department of Microbiology and Biochemistry, Faculty of Natural and Life Sciences, University Batna 2, Fesdis, Batna 05078, Algeria. E-mail: s.dassamiour@univ-batna2.dz; m.bensaad@univ-batna2.dz
bDepartment of Microbiology and Biochemistry, Faculty of Natural and Life Sciences, University Batna 2, Fesdis, Batna 05078, Algeria. E-mail: katrykorina@gmail.com; onzukaeikich87@yahoo.com
cLaboratory of Cellular and Molecular Physio-Toxicology-Pathology and Biomolecules (LPTPCMB), Faculty of Natural and Life Sciences, University Batna 2, Fesdis, Batna 05078, Algeria
dDepartment of Food Science and Nutrition, College of Sciences, Taif University, P.O. 11099, Taif, 21944, Saudi Arabia. E-mail: rokayya.d@tu.edu.sa; garsa.a@tu.edu.sa
eDepartment of Biotechnology, Faculty of Science, Taif University, P.O. 11099, Taif, 21944, Saudi Arabia. E-mail: i.althubaiti@tu.edu.sa
fDepartment of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia. E-mail: a.almashal@psau.edu.sa
First published on 4th May 2022
The date palm (Phoenix dactylifera L.) is one of the most important crops in arid and semi-arid zones. Date fruit occupies a good place in traditional medicine among the Saharan residents, due to its therapeutic virtues; although there may be several therapeutic virtues yet to be discovered. The aim of this study was to investigate the phytochemical and pharmacological properties of the hexanic (EHx), chloroformic (ECh), ethyl acetate (EAc) and aqueous (EAq) extracts of Tanteboucht pulp. The phytochemical characterization and estimation of phenolic compounds were done based on an HPLC-DAD approach. The antioxidant activity was evaluated by a DPPH scavenging effect test. The sensitivity of 7 bacterial strains and Candida albicans to Tanteboucht extracts was tested using the diffusion disc on agar medium method. The membrane stabilization test was used to determine the in vitro anti-inflammatory effect of the fruit extracts. Fourteen phenolic compounds were detected in organic extracts and EAc was the richest followed by ECh and finally EHx which was very poor in these molecules. All extracts showed antioxidant, anti-inflammatory and antimicrobial properties which differ in rate. Indeed, ECh had the greatest scavenging effect on DPPH, followed by EAc and then EAq. EAc was the most potent inhibitor of microbial strains. EAc and ECh were more efficient at membrane stabilization followed by EAq and the three extracts had more anti-inflammatory capacity than the positive control acetyl salicylic acid. The obtained considerable activities were significantly correlated with flavonoid and tannin contents in the extracts.
The date palm (Phoenix dactylifera L.) is one of the most important crops in arid and semi-arid zones, and plays an important role in the economic and social life of the populations of these regions.2 It is one of the fruit species whose cultivation has existed since ancient times.3
The date is a fruit obtained from the date palm, has significant pharmaceutical, nutritional and commercial value. In addition to its high energy value4 due to its high carbohydrate content, date pulp is rich in phytochemicals such as phenolic compounds, especially anthocyanins, procyanidins, flavonoids and phenolic acids. These compounds are the subject of increasing interest5,6 besides their pharmacological properties. Indeed, several activities such as antioxidant, antimicrobial, anti-inflammatory and anti-tumour activities are attributed to these metabolites.7–9 The latter remain less defined in the case of dates in general and that of its common varieties in particular. Tanteboucht, which is one of these varieties, is the subject of this study where we (the authors) targeted its phytochemical composition and its related biological activities.
The microbial strains were obtained from the microbiology laboratory of the University of Batna 1-Algeria.
Detection was performed at three different wavelengths, 250 nm, 280 nm and 340 nm.10
In addition, the anti-free radical efficacy is calculated as follows: EA = 1/IC50
Where the IC50 is the concentration of extract required to obtain 50% of the reduced form of the DPPH radical.
Phenolic compounds | Content (μg g−1) of EHx | Content (μg g−1) of ECh | Content (μg g−1) of EAc | En μg/100 g of FFw |
---|---|---|---|---|
a ‘—’: absent; FFW: fruit fresh weight. | ||||
Gallic acid | — | 11.27 ± 0.35 | 8.91 ± 1.33 | 60.38 ± 5.72 |
Caffeic acid | — | 12.89 ± 0.16 | 5.94 ± 0.79 | 53.58 ± 3.27 |
Chlorogenic acid | — | 40.28 ± 2.79 | 32.47 ± 3.75 | 218.08 ± 20.55 |
4-Hydroxybenzoic acid | — | 26.87 ± 1.77 | 4051.10 ± 71.98 | 14812.63 ± 266.4 |
Coumaric acid | — | — | — | |
Trans-ferulic acid | — | — | ||
Salicylic acid | — | — | — | |
Vanillin | 1.83 ± 0.17 | 29.68 ± 1.39 | — | 95.16 ± 3.60 |
Coumarin | 50.59 ± 5.74 | 22.58 ± 2.02 | — | 107.64 ± 10.42 |
Rutin | — | 21.02 ± 1.60 | — | 52.13 ± 3.96 |
Quercetin | — | — | 207.05 ± 25.55 | 753.66 ± 93.02 |
Catechin | — | 19.69 ± 2.91 | 17.01 ± 2.62 | 110.75 ± 16.75 |
Epicatechin | — | 14.65 ± 2.02 | — | 36.33 ± 5.02 |
Luteolin | — | — | 12.69 ± 1.85 | 46.19 ± 6.74 |
Cyanidine chloride | — | 20.02 ± 4.48 | 4.26 ± 1.75 | 65.16 ± 17.50 |
Tannic acid | — | 7.25 ± 2.15 | 8.11 ± 1.61 | 47.5 ± 11.20 |
Procyanidine B2 | — | 6.85 ± 0.78 | 4.20 ± 0.49 | 32.28 ± 3.73 |
These results are in agreement with those earlier found by Mansouri et al.11 for the same date variety; where they highlighted the presence of flavonoids and phenolic acids as caffeic acid and derivatives of cinnamic acid. It has been observed that Gallic acid was present, in large amount in several date varieties.14,15 Caffeic, ferulic, 4-hydroxybenzoic and chlorogenic acids are some of the compounds detected in many date varieties, besides different flavonoids and derivatives like: rutin, catechin, quercetin, luteolin, vanillin and coumarin,14,16 and these results are in agreement with ours.
Extracts/standards | IC50 (mg mL−1) | AE |
---|---|---|
a 1st letter: comparison (p <0.001) of all samples to quercetin standard.b 2nd letter: comparison (p <0.001) of all samples to ascorbic acid standard. | ||
EAc | 1.19 ± 0.02ab | 0.83 ± 0.02ab |
ECh | 0.86 ± 0.08ab | 1.16 ± 0.11ab |
EAq | 2.84 ± 0.90ab | 0.37 ± 0.12ab |
EHx | — | — |
Quercetin | 0.07 ± 0.01 | 14.48 ± 0.32 |
Ascorbic acid | 0.25 ± 0.02 | 4.01 ± 2.09 |
These results revealed that all the extracts tested had an anti-free radical activity at the maximum concentration (10 mg mL−1); ECh exhibited the highest anti-free radical activity (92.22%), followed by EAc (85.25%) and then EAq (70.5%). The IC50 of DPPH of EHx could not be really quantified because at the maximum concentrations tested, only a fraction of 21% of DPPH was captured, indicating a low concentration of antioxidants in this extract. Moreover, ECh was found to be the most active extract with an IC50 of 0.86 mg mL−1 and an AE estimated at 1.16 followed by EAc with an IC50 of around 1.19 mg mL−1 and an AE of 0.83. The EAq gave the lowest anti-free radical activity with an IC50 of 2.84 mg mL−1 and an AE of around 0.37.
For comparative purposes, ascorbic acid and quercetin were used as antioxidant standards; they showed an interesting anti-free radical activity with an IC50 of the order of 0.25 and 0.07 mg mL−1, therefore an AE of the order of 4.01 and 14.48 respectively.
In comparison with ascorbic acid and quercetin all the extracts tested were found to be less active. Noting that, these standards are pure while the extracts are of crude composition.
Ghiaba et al.17 found IC50 equal to: 10.83, 13.20, 16.77 mg L−1 for methanolic extracts of the varieties Deglet Nour, Ghars, and Degla Baidha, these values are higher than those of the variety Tanteboucht; which indicates an antiradicalaire efficiency lower than that of Tanteboucht extracts. Noting that, these are not only different species but also the combined action of the different compounds with anti-free radical activity that they may contain.
Antioxidant molecules such as ascorbic acid, tocopherol, flavonoids and tannins have been shown to reduce and discolor DPPH, due to their ability to transfer the hydrogen atom and single electron.18 The polyphenols in the date extracts are probably responsible for the antioxidant activity of these extracts. Indeed the phytochemical investigation on Tanteboucht revealed its considerable contents of several flavonoids and phenolic acids besides tannins which may contribute to the pharmacological properties of this fruit. Substantial linear coefficients of determination (R2) of 0.71, 0.68 and 0.73, were obtained between the anti-free radical efficacy (EA) and the total identified phenolics, phenolic acids and tannins contents respectively. Furthermore, a very significant coefficient of determination of 0.96 existed between AE and flavonoid content. It is evident therefore, that the high activity of the ECh and EAc extracts is attributed in large part to phenolic acids, flavonoids and tannins detected in Tanteboucht extracts.
Noting that, Alam et al.19 conclude that the antioxidant activity depends on the polymerization degree as well as the hydroxylation of phenolic compounds, when studying this activity in several date varieties. Phenolic acids such gallic and chlorogenic acids play an important role in antioxidant reactions; these compounds can increase levels of superoxide dismutase, glutathione and catalase, which enhance considerably antioxidant defense mechanisms.20,21 On the other hand, Sato et al.22 reported that antioxidant activity of caffeic acid is stronger than that of its precursor chlorogenic acid.
In the same way, Shim et al.23 found that p-hydroxybenzoic acid had strong scavenging activity of DPPH radical. It was mentioned that vanillin had potent neuroprotective effect against oxidative brain damage.24 Coumarins are aromatic compounds, having good antioxidant capacity; which was reported in several studies.
Khalil and Mustafa25 found that isolated coumarins from Granny Smith apple seeds had considerable free radicals scavenging capacity. They were able to enhance antioxidant defence and thus improve tomato plant tolerance to salinity in the study of Antonijević et al. (2021).26 Indeed, they were found to be good inhibitors, with flavonoids, of peroxyl radicals.27
Rutin and quercetin are considered from the most potent antioxidant agents and several studies28,29 reported that these flavonoids may considerably reduce oxidative stress and lipid peroxidation in brain, kidney and liver by enhancing the expression of key genes involved in antioxidant processes like glutathione S transferase α (GSTα), paraoxonase-1(PON-1) and glutamate-cysteine ligase (GCL). This process will considerably decrease the plasma level of malondialdehyde (MDA) and Glutathione (GSH) considered as the principal markers of oxidative stress.30
Otherwise, quercetin and catechin have protective effect against collagen fragmentation, premature skin cancer and skin photo-aging processes31,32 by significantly reducing and neutralizing free radical and oxygen-mediated damage in cells and also in extracellular matrix.33 The pre-treatment with cyanidin chloride provided a neuroprotective effect againt ROS in a study on the nematode Caenorhabditis elegans (C. elegans).34 Otherwise, a strong correlation was obtained between antioxidant properties and cyanidin and tannins content of blackberry.35 Luteolin was found to ameliorate the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase36 and thus showed a high activity in antioxidant process.
A research study highlighted antioxidant properties of this molecule against pro-oxidant effect at higher doses.37 These activities occurred by interacting with H2O2 and metal ions.
Fig. 3 Major effects of Tanteboucht extracts on the sensitivity of the microbial studied strains; 1: EAc, 2: ECh, 3: EHx, 4: EAq, 5: DMSO, 6: Sterile distilled water. |
Extract/strain | EAc | EHx | ECh | EAq | Standards |
---|---|---|---|---|---|
Enterobacter Cancerogenus | 10.55 ± 0.20 | — | 6.60 ± 0.35 | 6.55 ± 0.41 | — |
MRSA | 7.75 ± 0.37 | — | 6.70 ± 0.43 | — | — |
Staphylococcus Mu50 | 10.90 ± 0.33 | 8.00 ± 0.05 | 9.50 ± 0.11 | — | — |
Salmonella typhimurium | 7.30 ± 0.21 | — | 9.20 ± 0.26 | 16.75 ± 0.30 | 19 (cefoxitine) |
22 (imipenem) | |||||
Acinetobacter Sp | 19.75 ± 0.45 | — | 8.00 ± 0.15 | — | — |
E. coli | 20.00 ± 0.32 | — | 8.50 ± 0.45 | — | 20 (amoxicilline) |
25(Gentamycine) | |||||
Staphylococcus aureus | 22.00 ± 0.38 | 8.00 ± 0.5 | 9.00 ± 0.16 | — | 19 (oxacilline) |
19 (vancomycine) | |||||
Bacillus polyma | 10.60 ± 0.15 | — | — | — | — |
P. aeruginosa | 22.00 ± 0.37 | — | 9.00 ± 0.35 | — | 10 (tetracycline) |
20 (Gentamycine) | |||||
C. albicans | 10.00 ± 0.18 | 8.50 ± 0.13 | 8.50 ± 0.18 | — | — |
ECh gave a lower antibacterial activity compared to EAc and whose most sensitive strains were Staphylococcus Mu50 and S. typhimurium with inhibition diameters of 9.5 and 9.2 mm respectively; followed by P. aeruginosa and Staphylococcus aureus which gave 9.00 mm and finally E. coli and Candida albicans with 8.5 mm of inhibition diameter. However, it did not give any activity against Bacillus polyma. Daas amiour et al.12 found that alcoholic extracts from three varieties of dates had minimal inhibitory effect against the Escherichia coli strain which agree with this result.
EHx has been shown to be active only against Candida albicans (8.5 mm), Staphylococcus aureus and Staphylococcus Mu50 both with 8 mm as the diameter of inhibition zone. This activity may be due to the richness of this extract in lipid compounds which can hinder microbial growth. Indeed according to Branen et al.38 long chain fatty acids have interesting antibacterial effects.
The aqueous extract gave a good zone of inhibition against Gram (−) strains mainly S. typhimurium (16.75 mm), however, it gave minimal effect with Enterobacter. This activity can be explained by the richness of this extract in tannins which are capable of combining with microbial enzymes and of chelating certain metals such as iron,39 thus stopping microbial growth.
The fungal strain C. albicans appears to be sensitive to all organic extracts with diameters of 10 mm, 8.5 mm, 8.5 mm for EAc, ECh and EHx respectively, it may be due to the presence of phenol acids and flavonoids in these extracts. Indeed, Branen et al.38 confirmed that phenolics have both antibacterial and antifungal activity. In contrast, no inhibition of Candida albicans was observed with the aqueous extract.
To ensure that the activity is intrinsic to the extracts, the activities of the solvents used for the extraction and that used for the dissolution (DMSO) of the organic extracts were tested and gave negative results, one concentration initially. To determine the minimum inhibitory concentration (MIC) of EAc extract which had the most potent antimicrobial effect with maximum diameter of inhibitory zone of 22 mm (Table 3), dilutions (50%, 25%, 12.5%, 6.25%, 3.12%) were tested and the results are mentioned in Table 4.
Strain/dilution | 50 | 25 | 12.5 | 6.25 | 3.12 |
---|---|---|---|---|---|
S. aureus | 18.2 ± 0.1 | 12.9 ± 0.16 | 10.75 ± 0.08 | 10.00 ± 0.01 | 6.5 ± 0.01 |
E. coli | 17.78 ± 0.47 | 14.22 ± 0.02 | 11.93 ± 0.10 | 10.53 ± 0.20 | 6.75 ± 0.02 |
C. albicans | 15.55 ± 0.04 | 11.02 ± 0.02 | 06.73 ± 0.09 | — | — |
According to the results obtained by HPLC, the ECh and EAc extracts from Tanteboucht contain several flavonoids and phenolic acids, which have significant antibacterial activity.40 Indeed, this author signalled that these molecules could exert antibacterial effects since they are powerful in vitro as inhibitors of DNA gyrase. He added that they have antimicrobial activity attributed to their phenolic function, this activity is supposed to increase with number of hydroxyl, methoxyl or glucosyl substituents and according to Mansouri et al.11 these types of flavonoids are present in dates.
The mechanism of the antimicrobial effects of polyphenols is undoubtedly very complex, among the hypotheses put forward, we can cite: Inhibition of extracellular microbial enzymes, the sequestration of the substrate necessary for microbial growth, the chelation of metals such as iron and the inhibition of microbial metabolism.40
The correlation results between the content of phenolic compounds and the antimicrobial effect of the extracts showed that the inhibitory effect exerted on S. Mu50, C. albicans and Enterobacter cancerogenus by the hexane, chloroform and ethyl acetate extracts seems to be due to flavonoids, given that there is a strong correlation between the content of flavonoids and the inhibitory effect whose correlation coefficients are (R2 = 0.98), (R2 = 0.89), (R2 = 0.85), respectively. Tannic acid was found as inhibitor of both Gram-positive and Gram-negative bacteria, such as: Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Listeria innocua.41
Coumarin was found active on Escherichia coli 81nr.149 SKN541, Enterobacter aerogenes CIP 104 725, Salmonella typhimurium SKN533 and Salmonella infantis SKN 557 in the study carried out by Nitiema et al.,42 where minimum inhibitory concentration of this molecule was from 0.625 to 5.0 mg mL; however, the minimum bactericidal concentration was ≤5 mg mL−1. Additionally, Rutin from Peumus boldus extract had highest antibacterial activity, especially against E. coli and Bacillus cereus.43
On the other hand, it was signalled in the study of Gutiérrez-Venegas et al.44 that luteolin, quercetin and rutin were actives against Escherichia coli, staphylococcus aureus and Candida albicans while catechin inhibited only staphylococcus aureus. It was found also in the study of Tyagi et al.45 that gallic, caffeic and tannic acids besides catechin and quercetin have considerable inhibition of strains of Escherichia coli and Pseudomonas aeruginosa.
Furthermore, it was reported by Paz et al.46 that, identified phenolics in extracts of Hamelia patens were able to inhibit Escherichia coli, Staphylococcus aureus and Salmonella typhi growth. This is because, these phenolic compounds contain hydroxycinnamic acid, quercetin, procyanidin B2, catechin besides epicatechin; therefore, they concluded that the last compound was more implicated in this activity.
The significant membrane stabilizing activity of extracts from the Tanteboucht date can be attributed to the identified flavonoids, in particular flavonols and flavanols, as well as tannins; indeed, examination of these results revealed a significant linear coefficient of determination (R2 = 0.64) between membrane stabilizing effect and flavonoid content and a very significant coefficient of determination (R2 = 0.99) between this activity and tannin content. According to Lavanya et al.47 all these compounds are endowed with anti-inflammatory power.
The obtained results allow us to conclude that Tanteboucht phenolics are involved in the membrane stabilization capacity of its extracts which decreases the possibility of installation of inflammation as it was indicated in several studies that inhibition of erythrocyte membrane damage is considered to be an index of anti-inflammatory activity.48,49
In the study of Yin et al.,50 Procyanidin B2 from grape seed showed an anti-inflammatory activity and thus provided protective effect against the damage of the diabetic pancreas. Lesjak and his collaborators51 carried out a study on antioxidant and anti-inflammatory activities of quercetin and its derivatives and concluded that, these compounds may act as effective anti-inflammatory and antioxidant agents. Soyocak et al.52 signalled that tannic acid may contribute to the treatment of inflammation by decreasing myeloperoxidase activity in rats against formalin-induced paw oedema. Hence, the p-hydroxybenzoic acid found abundantly in Tanteboucht extracts has potent biological activities, as previously confirmed in several studies. Indeed, this molecule reduced oxidative stress induced by hydrogen peroxide and contributed in inhibition of neurodegeneration process, which confirm its anti-inflammatory effect in the study conducted by Winter et al.53 On the other hand, Maleki et al.54 mentioned that flavonoids have beneficial properties in vitro in inflammatory diseases and they can inhibit enzymes or transcription factors involved in inflammation process.
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