|Year : 2015 | Volume
| Issue : 2 | Page : 47-55
Improving the production of unsaturated fatty acid esters and flavonoids from date palm pollen and their effects as anti-breast-cancer and antiviral agents: An in-vitro study
Kadry Z Ghanem1, Manal M Ramadan PhD 2, Hassan Z Ghanem3, Mohamed Fadel4
1 Department of Food Science and Nutrition, National Research Centre, Giza, Egypt; Department of Clinical Nutrition, Faculty of Applied Medical Sciences, Jizan University, KSA
2 Department of Chemistry of Flavour and Aroma, National Research Centre, Giza, Egypt
3 Department of Therapeutical Chemistry, National Research Centre, Giza, Egypt
4 Department of Microbial Chemistry, National Research Centre, Giza, Egypt
|Date of Submission||05-May-2015|
|Date of Acceptance||12-Aug-2015|
|Date of Web Publication||8-Feb-2016|
Manal M Ramadan
Department of Chemistry of Flavour and Aroma, National Research Centre, Dokki, 12622, Giza
Source of Support: None, Conflict of Interest: None
Pollens from different plants contain unsaturated fatty acid esters (USFAEs) and flavonoids that play a very important role as bioactive compounds. Therefore, the present study was designed to improve the production of volatile USFAEs and flavonoids from date palm pollen (DPP) in a culture of Trichoderma koningii and test its activities as an anti-breast-cancer and antiviral agent.
Materials and methods
The volatile esters of fermented and nonfermented date palm pollens (FDPPs) were identified using gas chromatographic-mass spectrometric (GC-MS) analysis. Antioxidant activities were determined using three different methods: the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, the ferric reducing antioxidant power assay, and the 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assay. Polyphenols (phenolics and flavonoids) were also determined. Anti-breast-cancer and antiviral activities were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
GC-MS analysis showed an improvement in the level of USFAE in FDPP (47.99%) almost double that of the DPP results (24.11%) extract concentration. Flavonoids content of the FDPP extract (93.4 ± 6.3 mg/ml) was higher than that obtained by the DPP extract (45.4 ± 2.1 mg/ml), which was more than double the value. Antioxidant activity of the FDPP extract increased 3.16, 3.42, and 2.14 times that of the DPP extract as determined by the ABTS, ferric reducing antioxidant power (FRAP), and DPPH assays, respectively. The extract of FDPP showed strong anticancer activity against the MCF-7 cell line (IC 50 : 9.52 μg/ml) compared with the DPP extract (IC 50 : 96.22 μg/ml). Also, the FDPP extract had strong antiviral activity (CC 50 : 16.5 μg/ml) compared with DPP (CC 50 : 38.8 μg/ml). This is the first report in which the FDPP extract is used in biological studies as anti-breast-cancer and antiviral agents.
Fermentation of DPP by T. koningii improves many bioactive volatile USFAE and flavonoid contents; these have anti-breast-cancer and antiviral activity.
Keywords: anticancer, antioxidants, antiviral, flavonoids, pollen, volatile esters
|How to cite this article:|
Ghanem KZ, Ramadan MM, Ghanem HZ, Fadel M. Improving the production of unsaturated fatty acid esters and flavonoids from date palm pollen and their effects as anti-breast-cancer and antiviral agents: An in-vitro study. J Arab Soc Med Res 2015;10:47-55
|How to cite this URL:|
Ghanem KZ, Ramadan MM, Ghanem HZ, Fadel M. Improving the production of unsaturated fatty acid esters and flavonoids from date palm pollen and their effects as anti-breast-cancer and antiviral agents: An in-vitro study. J Arab Soc Med Res [serial online] 2015 [cited 2017 Dec 18];10:47-55. Available from: http://www.new.asmr.eg.net/text.asp?2015/10/2/47/175555
| Introduction|| |
Cancer is a major health problem and one of the main causes of mortality worldwide. At present, breast cancer is the most common cancer in women worldwide. Currently, breast cancer (23% of all new cancer cases) is the second leading cause of death in women worldwide including Egypt as well. Despite the progress achieved in the use of anticancer drugs in the treatment of malignant diseases, they are frequently associated with unfavorable side effects. Recently, many natural products from Egyptian plants have been reported to have anticancer activity , . The therapeutic effects of dietary fatty acids on cancer cell progression have been verified by both in-vitro and in-vivo studies  . Saturated fatty acids are nonessential fatty acids and are harmful if excessively ingested in food. However, polyunsaturated fatty acids (PUFAs) are designated as 'essential' for good health as their metabolic precursors cannot be synthesized in the body and must be ingested by food intake. PUFAs have important effects on the structure and physical properties of localized membrane domains. They modulate enzyme activities, carriers and membrane receptors (low-density lipoprotein receptors, insulin, antibodies neurotransmitters, drugs receptors, etc.)  . Some esters of fatty acids show antitumor activity against Ehrlich ascites tumor in mice  . Flavonoids are naturally occurring polyphenolic compounds with low molecular weight. These compounds are ubiquitous, found in almost all plant kingdoms. In humans, the vesicular stomatitis virus (VSV) causes an acute influenza-like illness with symptoms such as fever, muscle aches, headache, malaise, and blisters in the mouth  . Flavonoids were reported to have antiviral, antioxidant, antiproliferative, antitumor, anti-inflammatory, antiallergic, and anticarcinogenic properties that have been proven through in-vitro and in-vivo studies. Flavonoid consumption through plant-derived foods and from medicinal plants has beneficial health effects in humans ,, . Pollens are the male reproductive cells of flowers. Most noncultivated plants produce pollens. The early Egyptians and ancient Chinese used pollen as a rejuvenating medicinal agent. Date palm (Phoenix dactylifera L.) pollens are commonly used in the Middle East, especially in Egypt  . Phytochemicals screening showed that dried date palm pollens (DPPs) contain sterols, triterpenes, saponins, and glycosides  . A significant change in bioactive volatile and nonvolatile components of the fermentation process by Trichoderma spp. has been reported recently  .
The present study was designed to improve the production of unsaturated fatty acid ester (USFAE) and flavonoids from DPP in a culture of Trichoderma koningii as well as to examine DPP and fermented date palm pollen (FDPP) extracts as anti-breast-cancer and antivirus agents (VSV).
| Materials and methods|| |
Raw materials and chemicals
Date palm pollen
Egyptian dry DPP (P. dactylifera L.) was obtained from the Department of Medicinal and Aromatic Plants, Ministry of Agriculture (Cairo, Egypt).
Microorganisms and culture conditions
T. koningii was obtained from Microbial Chemistry Lab., National Research Center (Dokki, Cairo, Egypt) and maintained on potato dextrose agar slants at 30°C for 72 h. The spore suspensions were prepared by adding 10 ml of sterilized water to slant cultures and the surface was gently rubbed with a sterilized wire loop. The fermentation was carried out in 250 ml Erlenmeyer flasks containing 5 g of DPP moistened to 50% (v/w) with distilled water. One milliliter of spore suspension (106 spores) was used as an inoculum. The cultures were incubated at 30°C for 3 days by solid-state fermentation.
Date palm pollen extraction
Fatty acid alkyl esters were obtained by the trans-esterification of triglycerides (in most cases, vegetable oils and animal fats) with methanol or ethanol  . Volatile (esters) and nonvolatile (phenolic and flavonoid) compounds were extracted from DPP grain (DPP and FDPP) samples with ethanol. Ethanol is a better extracting solvent compared with methanol  . The extraction was performed on the basis of the modification reported by Rajeswari et al.  . Five grams of each of DPP and FDPP was weighed into a 250 ml conical flask and extracted using 150 ml 95% ethanol. The flask was shaken every hour for the first 6 h, then kept aside and shaken again after 24 h. The solvent layer was separated from the solid residue by centrifuging at 2000g for 10 min. The clear supernatant was transferred to a clean rounded flask and evaporated using a vacuum evaporator at less than 50°C. The extract was concentrated to 2 ml and stored at −20°C until further use.
Gas chromatographic-mass spectrometric analysis
About 2 μl of each extract was used. The analysis was carried out using a coupled gas chromatography (Hewlett-Packard Model 5890; Hewlett-Packard, Perkin Elmer Co., USA)/mass spectrometry (Hewlett-Packard-MS 5970; Hewlett-Packard). The ionization voltage was 70 eV and the mass range was m/z 39-400 amu. The oven temperature was maintained initially at 50°C for 5 min and then programmed from 50 to 250°C at a rate of 4°C/min. Helium was used as the carrier gas at a flow rate of 1.1 ml/min. The injector and detector temperatures were 220 and 250°C, respectively. The isolated peaks were identified by matching with data from the library of mass spectra (National Institute of Standard and Technology). Quantitative determination was carried out on the basis of peak area integration. Interpretation on mass spectrum gas chromatographic-mass spectrometric (GC-MS) was carried out using the database of the NIST library.
Determination of phenolic content
The phenolic content was determined according to the Folin-Ciocalteu procedure. It was determined by means of a calibration curve prepared with gallic acid and expressed as milligrams of gallic acid equivalent per milliliter of sample  .
Determination of flavonoid content
The total flavonoid content was determined as reported by Thaipong et al.  . It was expressed as milligram of catechin equivalent per milliliter of sample  .
Determination of antioxidant activity
The 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay was used as reported by Thaipong et al.  . The antioxidant activity was determined by a calibration curve prepared with ascorbic acid and expressed as milligram of ascorbic acid equivalent per milliliters of sample  .
For the 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assay, the procedure followed the method of Arnao et al.  . Results are expressed in millimoles per liter Trolox equivalents per milliliter extract. Additional dilution was needed if the ABTS value measured was beyond the linear range of the standard curve  .
Ferric reducing antioxidant power assay
The ferric reducing antioxidant power assay was performed according to the Benzie and Strain's  method. Results are expressed in millimoles per liter Trolox equivalents per milliliter extract. Additional dilution was needed if the ferric reducing antioxidant power value measured was beyond the linear range of the standard curve  .
Cell cultures and treatments
The human breast cancer cell line (MCF-7) was obtained from the American Type Culture Collection (Rockville, Maryland, USA). Cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% MEM nonessential amino acid solution, and 1% penicillin streptomycin solution (10 000 U of penicillin and 10 mg of streptomycin in 0.9% NaCl) in a humidified atmosphere of 5% CO 2 and 95% air at 35°C. The passage number range for both cell lines was maintained between 20 and 25. The cells were cultured in 75 cm 2 cell culture flasks. For experimental purposes, cells were cultured in 96-well plates (0.2 ml of cell solution/well). The optimum cell concentration as determined by the growth profile of the cell line was 2 × 10 5 cells/ml (cells were allowed to attach for 24 h before treatment with tested extracts). The stock solution was filtered with Minisart filters Merck (Darmstadt, Germany) (0.22 μm). The cells were exposed to 11 dilutions (0.16, 0.32, 0.64, 1.28, 2.56, 5.12, 10.24, 20.48, 40.96, 81.92, and 163.84 μg/ml) of test extracts. Cell monolayers were washed with PBS and the additional serially diluted materials were dispensed to the precultured plates to determine the toxicity of test materials  .
The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay is based on the protocol described for the first time by Mossmann  . The assay was optimized for the cell lines used in the experiments. Briefly, for the purposes of the experiments at the end of the incubation period, cells were incubated for 4 h with 0.8 mg/ml of MTT and dissolved in serum-free medium. Washing with PBS (1 ml) was followed by the addition of DMSO (1 ml) and gentle shaking for 10 min so that complete dissolution was achieved. Aliquots (200 μl) of the resulting solutions were transferred to 96-well plates and absorbance was recorded at 560 nm using the microplate spectrophotometer system (Spectra Max190; Molecular Devices). The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated  .
Cells and viruses
Vero and BHK-21 cells were grown in Eagle's minimal essential medium (MEM; GIBCO) containing 5% inactivated calf serum and 50 g/ml gentamicin. Maintenance medium (pH 7.5) consisted of MEM supplemented with 1.5% inactivated calf serum and gentamicin. The VSV was propagated on Vero cells. Virus stocks were plaque assayed on Vero cells  .
Vero cells grown in 96-well tissue culture plates were incubated with 12 concentrations (0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, 102.4, 204.8, and 409.6 μg/ml) of each extract for 24 h at 37°C. Cell viability was measured using the MTT procedure. The cytotoxicity of each extract was expressed as the 50% cytotoxic concentration (CC 50 ), which is the concentration required to reduce cell viability to 50% of the control  .
The results are reported as mean ± SD for at least three experiments. Statistical differences were analyzed using the one-way ANOVA test.
| Results|| |
Gas chromatographic-mass spectrometric analysis of date palm pollen and fermented date palm pollen
Extract of DPP and FDPP were analyzed by gas chromatography coupled with mass spectrometry. Twenty-three volatile compounds were identified. GC-MS analysis showed that the extract of DPP included four unsaturated fatty acids, six saturated fatty acid esters, six USFAEs, and a small number of benzene derivatives [Table 1].
|Table 1 Gas chromatographic-mass spectrometric analysis of date palm pollen and fermented date palm pollen by Trichoderma koningii|
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Palmitoleic and oleic acids, as monounsaturated fatty acids, comprised 2.12 and 2.73% of total volatiles. The saturated fatty acids ester (SFAE) identified in DPP included dodecanoic acid-ethyl ester, tetradecanoic acid-ethyl ester, hexadecanoic acid-methyl ester, hexadecanoic acid-ethyl ester, octadecanoic acid-ethyl ester, and docosanoic acid-ethyl ester. In general, the predominant SFAE of DPP was hexadecanoic acid-ethyl ester (35.32%) of the total volatiles. The USFAEs identified in DPP included ethyl-9-hexadecenoate, 9, 12, 15-octadeca trienoic acid-ethyl ester, ethyl oleate, linoleic acid-ethyl ester, 11-eicosenoic acid-methyl ester, and 9-hexadecenoic acid-9-octadecenyl ester. In general, the predominant USFAE of DPP were ethyl-9-hexadecenoate, 9, 12, 15-octadeca trienoic acid-ethyl ester, and ethyl oleate. Ethyl-9-hexadecenoate and ethyl oleate, as monounsaturated fatty acids, comprised 8.42 and 1.35% of the total volatiles of DPP. 9, 12, 15-Octadeca trienoic acid-ethyl ester, as PUFAs, comprised 9.68% of the total volatiles of DPP. However, GC-MS analysis showed that the extract of FDPP included two unsaturated fatty acids, seven saturated fatty acid esters, six USFAEs, and a small number of benzene derivatives [Table 1].
9,12-Octadecadienoic and oleic acids, as monounsaturated fatty acids, comprised 1.03 and 1.37% of the total volatiles of the FDPP extract. The SFAE identified by the GC-MS in FDPP extract included tetradecanoic acid-ethyl ester, pentadecanoic acid-ethyl ester, pentadecanoic acid-14-methyl-methylester, hexadecanoic acid-ethyl ester, heptadecanoic acid-ethyl ester, octadecanoic acid-ethyl ester, and docosanoic acid-ethyl ester. In general, the predominant SFAE of FDPP was hexadecanoic acid-ethyl ester, comprising 36.58% of the total volatiles. The USFAE identified in FDPP included ethyl-9-hexadecenoate, 16-octadecenoic acid-methyl ester, linoleic acid-ethyl ester, 9, 12, 15-octadecatri-enoic acid-ethyl ester, ethyl oleate, and 6, 9, 12, 15-docosatetraenoic acid-methyl ester. In general, the predominant USFAE of FDPP extract were ethyl-9-hexadecenoate, linoleic acid-ethyl ester, and 9, 12, 15-octadeca trienoic acid-ethyl ester. Ethyl-9-hexadecenoate and ethyl oleate, as monounsaturated fatty acid esters, comprised 9.83 and 2.0% of the total volatiles of the FDPP extract. Linoleic acid-ethyl ester and 9, 12, 15-octadeca trienoic acid-ethyl ester, as polyunsaturated fatty acid esters, comprised 21.06 and 13.93% of the total volatiles of the FDPP extract. Results showed that fermented palm pollen extract of T. koningii increased the level of linoleic acid-ethyl ester 4.8 times that in DPP extract; it was 4.39% of the total volatile esters of DPP and increased to 21.06% in FDPP. In general, the level of total USFAEs in FDPP (47.99%) is almost twice that in DPP (24.11%) as shown in [Figure 1].
|Figure 1: Total percentage of unsaturated fatty acid esters (USFAE) in date palm pollen (DPP) and fermented date palm pollen (FDPP)|
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Polyphenols (phenolics and flavonoids)
The results indicated that the flavonoid content of the FDPP extract was 93.4 ± 6.3 mg/ml. This value was higher than the value of 45.4 ± 2.1 mg/ml obtained by the DPP extract. It was observed that the levels of phenolic compounds of DPP and FDPP were 38.1 ± 1.0 and 39.3 ± 1.4 mg/ml [Table 2].
|Table 2 Phenolic and flavonoid content of date palm pollen and fermented date palm pollen|
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[Table 3] shows that the means of the antioxidant activity levels in FDPP were 1.664 ± 0.03 and 1.656 ± 0.02 mmol/l Trolox equivalent/ml extract as determined by ABTS and FRAB assays and 0.240 ± 0.01 mg ascorbic acid equivalent/ml extract by DPPH assay, whereas the means of the antioxidant activity levels in DPP were 0.525 ± 0.02 and 0.484 ± 0.01 mmol/l Trolox equivalent/ml extract by ABTS and FRAB assays and 0.112 ± 0.00 mg ascorbic acid equivalent/ml extract by the DPPH assay.
|Table 3 Antioxidant activity of date palm pollen and fermented date palm pollen extracts by ABTS, FRAB, and|
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The data obtained in [Figure 2] show the cytotoxic effect of DPP and FDPP extracts against the MCF-7 breast cancer cell line. The cytotoxicity of each extract was expressed as the 50% cytotoxic concentration (IC 50 ), which is the concentration required to reduce cell viability to 50% of the control. Both DPP and FDPP extracts showed powerful activity as anti-breast-cancer agents. The FDPP extract showed strong anticancer activity against MCF-7 cells (IC 50 : 9.52 μg/ml) compared with the DPP extract (IC 50 : 96.22 μg/ml) [Figure 2].
|Figure 2: Cytotoxicity (IC50) of date palm pollen (DPP) and fermented date palm pollen (FDPP) extracts against MCF-7 breast cancer cell lines|
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The data obtained in [Figure 3] show the cytotoxic effect of both DPP and FDPP extracts against VSV. [Figure 3] represents the CC 50 values, which are expressed in μg/ml. The FDPP extract showed strong antiviral activity (CC 50 : 16.5 μg/ml) compared with the DPP extract (CC 50 : 38.8 μg/ml).
|Figure 3: Cytotoxicity (CC50) of date palm pollen (DPP) and fermented date palm pollen (FDPP) extracts against ve sicular stomatitis virus|
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| Discussion|| |
In recent years, the use of microbial lipids, which can be obtained by fermenting non-food feedstocks, has attracted increasing attention  . A number of oleaginous microorganisms such as microalgae  , yeasts  , and fungi  have been tested for their capability for lipid accumulation. In addition to variations in lipid content, pollen also varies in the relative proportions of fatty acids as well as in their diversity  . Our results were agreement with those of Boatose et al.  , who reported that oleic and palmitoleic acids were identified as unsaturated fatty acids in palm pollen, whereas Hazem  reported that the predominant fatty acids of palm pollen grains were palmitic, linoleic, and myristic acid. Al-Shahib and Marshall  showed that the major fatty acid composition in date pollen extracts may differ within the varieties and different climatic conditions of growing. Monounsaturated fatty acids and PUFAs contribute toward the characteristics of the aroma  .
Our GC-MS results showed that palmitoleic and oleic acids, the main monounsaturated fatty acids, in the DPP extract showed a good flavor as reported by many authors ,, .
Volatile esters are responsible for the fruity nature of fermented extracts and thus constitute a vital group of aromatic compounds. Many fermentation parameters are known to affect volatile ester production, especially ethyl esters, where ethyl esters were the predominant esters in DPP and FDPP extracts, as shown in our results. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after the addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that the addition of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes an increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability plays an important role in ethyl ester production  .
Aroma-active esters are formed intracellularly by fermenting yeast cells. As they are lipid soluble, ethyl esters can diffuse through the cellular membrane into the fermenting medium. Unlike acetate ester excretion, which is rapid and complete, the transfer of ethyl esters to the fermenting medium decreases markedly with increasing chain length. The rate of ethyl ester formation is dependent on three factors: the concentrations of the two cosubstrates (the acyl coenzyme A component and ethanol) and the activity of the enzymes involved in their synthesis and hydrolysis. Hence, all parameters that influence substrate concentrations or enzyme activity may affect ethyl ester production  . In strain selection, specific attention to the levels of expression of other genes (e.g. fatty acid synthesis genes) appears to be more relevant than the levels of expression of the EEB1 and EHT1 genes for the final ethyl ester levels  .
Many phytochemical studies of DPP report results that are in agreement with our results, which indicated the presence of many flavonoids compounds in DPP extracts ,, . This experiment was designed to improve the production of flavonoids from DPP by culture of T. koningii. The present results showed an improvement in the flavonoid content in FDPP (93.4 ± 6.3 mg/ml); it was more than twice that of the DPP results (45.4 ± 2.1 mg/ml). Considerable effort has been directed toward elucidating the flavonoid biosynthetic pathway from a molecular genetics point of view by identifying and analyzing genes or cDNAs for flavonoid biosynthetic enzymes and regulatory factors. Moreover, analysis of enzyme structures and functions involved in flavonoid biosynthesis, generation of transgenic plants, development of protein engineering tools, and heterologous production of flavonoids in microbial systems has led to considerable progress in the characterization of the flavonoid biosynthesis pathway. This pathway produces a large number of secondary metabolites, such as lignins, stilbenes, phenolic acids, and other polyphenols  .
Pollen may be considered an effective natural and functional dietary food supplement because of its remarkable content of polyphenol substances and significant radical scavenging capacity, in addition to their nutritional-physiological implications and their health-promoting effect  . Antioxidants are chemicals that interact and deactivate the free radicals, therefore preventing them from causing harm  . Studies indicated that the aqueous extracts of dates have potent antioxidant activity  . The antioxidant activity is attributed to the wide range of phenolic compounds in dates including p-coumaric, ferulic, sinapic acids, flavonoids, and procyanidins  .
Abdulaziz  indicated that the Egyptian dates were rich in phenolic compounds and flavonoids with high antioxidant activity. The excellent antioxidant activity can be attributed not only to the presence of the polyphenols but also that of volatile constituents. These components could transform free radicals such as DPPH into nonradical DPPH-H , .
This experiment was designed to evaluate the role of DPP as an antioxidant in vitro using three different methods: ABTS, FRAB, and DPPH. Data indicated that the FDPP extract showed stronger antioxidant activity than the DPP extract. In general, data showed that the fermentation process of data palm pollen (FDPP) by T. koningii increased the antioxidant activity levels 3.16, 3.42, and 2.14 times that of nonfermented (row) DPP as determined by ABTS, FRAB, and DPPH assays, respectively. This may be attributed to the high levels of unsaturated fatty acids and polyphenol content as well as the presence of butylated hydroxyl toluene, a strong natural antioxidant, which represents 2.11% of the total volatiles in FDPP extract [Table 1].
Our results are in agreement with those of many previous studies on date pale pollen. Rahmani et al.  reported that palm pollen extracts contained some antioxidants that play an important role in reactive oxygen species scavenging. Al-Farsi and Lee  reported that date extracts have the potential to be used as a supplement for antioxidants in nutraceutical, pharmaceutical, and medicinal products. Date extracts are rich sources for nutritive substances, polyphenols, and bioactive compounds. Date extracts have been shown to impair the cytotoxicity of azoxymethane-induced cancer in colonic tissue in rats  .
It is important to explore the extraction of health-promoting bioactive compounds, such as antimicrobial, antioxidant, anti-inflammation, and anticancer activities.
Pollens from different plants were reported to have antibacterial  , antifungal  , antioxidant  , and anti-inflammatory activity  . To our knowledge, no data have been published on FDPP and its activity against breast cancer. We designed the current work to study whether DPP and FDPP extracts show cytotoxicity in MCF-7 (human breast cancer cell lines). In general, the results indicated that both DPP and FDPP extracts have anti-breast-cancer activity. The results indicated a significant difference between DPP and FDPP extracts (P < 0.05). Increasing the dose decreased the cell viability significantly. This bioactivity may be attributed to the presence of polyphenolic compounds (phenolics and flavonoids). Flavonoids are known for their immunomodulatory and anti-inflammatory activities and inhibition of proinflammatory cytokine production and their receptors  . They also inhibit histamine release from different tissues  and downregulate transcription factor NF-kB/IkB involved in immune and inflammatory responses  . Metwaly et al.  reported that palm pollen has an anti-inflammatory effect. Different studies have reported that USFAEs can modulate the activity of the components of intracellular signaling ,, .
Particular attention has been paid to the role of Ca 2+ entry through store-operated channels  . Thus, store-operated Ca 2+ entry (SOCE) has been involved in cell signaling that occurs in nonexcitable cells to induce different cell processes including gene regulation and cell growth  . It has been observed recently that the addition of PUFA to cells cultured in vitro modifies the extent of SOCE  . Thus, oleic acid can inhibit SOCE in Ehrlich tumor cells, possibly because they intercalate into the plasma membrane and directly affect the activity of the channels involved  .
In addition, a SOCE-inhibitory effect induced by oleic acid in a colon adenocarcinoma cell line has been reported recently  . Studies on the activity of other esters by Kato et al.  showed that esters of fatty acids and the propylene glycol ester of myristic acid were the most effective against the tumor. You et al.  showed that USFAEs of 4′′-methyl-deoxypodophyllotoxin increased in-vivo antitumor activity. David  showed that the protection of olive oil against breast cancer may be because of oleic acid components.
The following study aims to investigate the antiviral potential of DPP and FDPP extracts against the VSV. Because of the alarming emergence of resistance to antivirus drugs, there is a need to identify new naturally occurring antiviral molecules  .
We tested the FDPP extract for antiviral properties. The MTT assay is probably one of the most widely used colorimetric indicators of cell viability, and it assesses mitochondrial cellular function on the basis of the enzymatic reduction of the tetrazolium salt by the mitochondrial dehydrogenases in viable cells  .
In this study, the cytotoxicity of the DPP and FDPP extracts was evaluated by this assay. Our results showed that FDPP is rich in polyphenols and alkyl esters. Tillekeratne et al.  have reported that alkyl esters have antivirus activity. Some studies have shown the effectiveness of plant extracts as in-vitro inactivators of several enteric viruses; this was attributed to phenolic compounds. Several commercially available phenolic compounds of plant origin were effective; these natural antiviral agents seem to depend on the stability of these complexes, such as tannins and related compounds, during passage through the digestion system  . This is the first report in which fermented palm pollen extracts have been used in biological studies as anti-breast-cancer and antiviral agents.
In conclusion, fermentation of Egyptian DPP by T. koningii improves many bioactive volatile USFAEs and flavonoid contents. FDPP extract has strong antioxidant activity and anti-breast-cancer activity. It has also antiviral activity. Further investigations are needed to be applied as additives, in food and pharmaceutical industries.
The authors are grateful to the Department of Medicinal and Aromatic Plants, Ministry of Agriculture (Cairo, Egypt) for providing the Egyptian dry date palm pollen (Phoenix dactylifera L.).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ramadan MM, Ali MM, Ghanem KZ, El-Ghorab AH. Essential oils from Egyptian aromatic plants as antioxidant and novel anticancer agents in human cancer cell lines. Grasas Ceites 2015; 66:1-9.
El-Ghorab AH, Ramadan MM, Abd El-Moez SI. Essential oil, antioxidant, antimicrobial and anticancer activities of Egyptian Pluchea dioscoridis
extract. Res J Pharm Bio Chem Sci 2015; 6:1255-1265.
Amer M, Alanazi AM, Ali Khan A. Anticancer role of novel saturated fatty acid ester conjugates on cultured cancer cells. J Cancer Sci Ther 2013; 5:118-125.
Zamaria N. Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev 2004; 44:273-282.
Kato A, Ando K, Tamura G, Arima K. Effects of some fatty acid esters on the viability and transplantability of Ehrlich ascites tumor cells. Cancer Res 1971; 31:501-504.
Haidari M, Ali M, Ward Casscells S III, Madjid M. Pomegranate (Punica granatum
) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir. Phytomedicine 2009; 16:1127-1136.
Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-like compounds - nature, occurrence, dietary intake and effects on nutrition and health. J Sci Food Agric 2000; 80:1094-1117.
Corsinovi L, Biasi F, Poli G, Leonarduzzi G, Isaia G. Dietary lipids and their oxidized products in Alzheimer's disease. Mol Nutr Food Res 2011; 2:161-172.
Gutierrez-Merino C, Lopez-Sanchez C, Lagoa R, Samhan-Arias AK, Bueno C, Garcia-Martinez V. Neuroprotective actions of flavonoids. Curr Med Chem 2011; 18:1195-1212.
Kroyer G, Hegedus N. Evaluation of bioactive properties of pollen extracts as functional dietary food supplement. Innov Food Sci Emerg Technol 2001; 2:171-174.
Mahran GH, Abdel-Wahab SM, Attia AM. A phytochemical study of date palm pollen. Planta Med 1976; 29:171-175.
Ramadan MM, Elbandy MA, Fadel M, Ghanem KZ. Biotechnological production of volatile and non volatile antioxidant compounds from fermented soy bean meal with Trichoderma
sp. Res J Pharm Bio Chem Sci 2014; 5:537-547.
Knothe G. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol 2005; 86:1059-1070.
Ramadan MM, Yehia HA, Shaheen MS, Abed EL-Fattah SM. Aroma volatiles, antibacterial, antifungal and antioxidant properties of essential oils obtained from some spices widely consumed in Egypt. Am Euras J Agric Environ Sci 2014; 14:486-494.
Rajeswari G, Murugan M, Mohan VR. GC-MS analysis of bioactive components of Hugonia mystax
L. (Linaceae). Res J Pharm Biol Chem Sci 2012; 3:301-308.
Thaipong K, Boonprakoba U, Crosby K, Cisneros L. Distributions of phenolic compounds, yellow pigments and oxidative enzymes in wheat grains and their relation to antioxidant capacity of bran and debranned flour. J Cereal Sci 2012; 56:652-658.
Thaipong K, Boonprakoba U, Crosby K, Cisneros L, Byrnec DH. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J Food Comp Anal 2006; 19:669-675.
Arnao MB, Cano A, Acosta M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem 2001; 73:239-244.
Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of 'antioxidant power': the FRAP assay. Anal Biochem 1996; 239:70-76.
Romero D, Gomez-Zapata M, Luna A, Garcia-Fernandez JA. Morphological characterization of BGM (Buffalo Green Monkey) cell line exposed to low doses of cadmium chloride. Toxicol In Vitro 2003; 17:293-299.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55-63.
Wachsman MB, López EM, Ramirez JA, Galagovsky LR, Coto CE. Antiviral effect of brassinosteroids against herpes virus and arenaviruses. Antivir Chem Chemother 2000; 11:71-77.
Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007; 25:294-306.
Saenge C, Cheirsilp B, Suksaroge TT, Bourtoom T. Potential use of oleaginous red yeast Rhodotorula glutinis
for the bioconversion of crude glycerol from biodiesel plant to lipids and carotenoids. Process Biochem 2011; 46:210-218.
Vicente G, Bautista LF, Rodríguez R, Gutiérrez FJ, Sádaba I, Ruiz-Vázquez RM, et al
. Biodiesel production from biomass of an oleaginous fungus. Biochem Eng J 2009; 48:22-27.
Boatose DH, Barth MO, Rocha CI, Cunha IB, Carvalho PO, Torres EA, Michelan M. Fatty acids composition and palynological analysis of bee (Apis) pollen loads in the states of Sao Paulo and Minas Gerais, Brazil. J Apicultural Res 2004; 43:35-39.
Hazem MH. Chemical composition and nutritional value of palm pollen grains. Global J Biotechnol Biochem 2011; 6:01-07.
Al-Shahib W, Marshall RJ. Fatty acid content of the date seeds from 14 varieties of date palm Phoenix dactylifera
L. Int J Food Sci Technol 2003; 38:709-712.
Ramadan MM, Abd Algader NN, El-Kamali HH, Ghanem KZ, Farrag AH. Chemopreventive effect of Coriandrum sativum
fruits on hepatic toxicity in male rats. World J Med Sci. 2013; 8:322-333.
Besbes S, Blecker C, Deroanne C, Drira N, Attia H. Date seeds: chemical composition and characteristic profiles of the lipid fraction. Food Chem 2004; 84:577-584.
Nehdi I, Omri S, Khalil MI, Al-Resayes SI. Characteristics and chemical composition of date palm (Phoenix canariensis
) seeds and seed oil. Ind Crop Prod 2010; 32:360-365.
Habib HM, Kamal H, Ibrahim WH, Al Dhaheri AS. Carotenoids, fat soluble vitamins and fatty acid profiles of 18 varieties of date seed oil. Ind Crop Prod 2013; 42:567-572.
Saerens SM, Delvaux F, Verstrepen KJ, Van Dijck P, Thevelein JM, Delvaux FR. Parameters affecting ethyl ester production by Saccharomyces cerevisiae
during fermentation. Appl Environ Microbiol 2008; 74:454-461.
Nykanen L, Nykanen I. Production of esters by different yeast strains in sugar fermentations. J Inst Brew 1977; 83:30-31.
Furukawa K, Yamada T, Mizoguchi H, Hara S. Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae
. J Biosci Bioeng 2003; 95:448-454.
Anderson OM, Markham KR. Flavonoids chemistry, biochemistry and applications
. New York: CRC Press Taylor and Francis Group; 2006. 219-262.
Abbas FA, Ateya AM. Estradiol, esteriol, estrone and novel flavonoids from date palm pollen. Aust J Basic Appl Sci 2011; 5:606-614.
Suresh S, Guizani N, Al-Ruzeiki M, Al-Hadhrami A, Al-Dohani H, Al-Kindi I, Rahman MS. Thermal characteristics, chemical composition and polyphenol contents of date-pits powder. J Food Eng 2013; 119:668-679.
Krover G, Hegedus N. Evaluation of bioactive properties of pollen extracts as functional dietary food supplement. Innov Food Sci Emerg Technol 2001; 2:171-174.
Gulcin I. Fe3+Fe2+ transformation method: an important antioxidant assay advanced protocols in oxidative stress III
. Springer; 2015;1208:233-246.
Mansouri A, Embarek G, Kokkalou E, Kefalas P. Phenolic profile and antioxidant activity of the Algerian ripe date palm fruit (Phoenix dactylifera
) Food Chem 2005; 89:121-127.
Gu L, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D, Prior RL. Screening of foods containing proanthocyanidins and their structural characterization using LCMS/MS and thiolytic degradation. J Agric Food Chem 2003; 51:7513-7521.
Abdulaziz EA. Study of anti-nutrients and antioxidant in date palm fruits (Phoenix dactylifera
L.) from Saudi Arabia and Egypt. J Am Sci 2014; 10:231-238.
Ramadan MM, Abd-Algader NN, El-Kamali HH, Ghanem KZ, Farrag AH. Volatile compounds and antioxidant activity of the aromatic herb Anethum graveolens.
J Arab Soc Med Res 2013; 8:79-88.
Abd-Algader NN, El-Kamali HH, Ramadan MM, Ghanem KZ, Farrag AH. Xylopia aethiopica
volatile compounds protect against panadol-induced hepatic and renal toxicity in male rats. World Appl Sci J 2013; 27:10-22.
Rahmani AH, Aly SM, Ali H, Babiker AY, Srikar S, Khan AA. Therapeutic effects of date fruits (Phoenix dactylifera
) in the prevention of diseases via modulation of anti-inflammatory, anti-oxidant and anti-tumour activity. Int J Clin Exp Med 2014; 7:483-491.
Al-Farsi MA, Lee CY. Optimization of phenolics and dietary fibre extraction from date seeds. Food Chem 2008; 108:977-985.
Waly MI, Al-Ghafri BR, Guizani N, Rahman MS. Phytonutrients effect of date pit extract against azoxymethane-induced oxidative stress in rat colon. Asian Pac J Cancer Prev 2013; 20:321-329.
Baltrusaityte V, Venskutonis PR, Ceksteryte V. Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chem 2007; 101:502-514.
Ozcan M. Inhibition of Aspergillus parasiticus
NRRL 2999 by pollen and propolis extracts. J Med Food 2004; 7:114-116.
Leja M, Mareczek A, Wyzgolik G, Klepacz BJ, Czekonska K. Antioxidative properties of bee pollen in selected plant species. Food Chem 2007; 100:237-240.
Choi EM. Antinociceptive and antiinflammatory activities of pine (Pinus densiflora
) pollen extract. Phytother Res 2007; 21:471-475.
Kempuraj D, Madhappan B, Christodoulou S, Boucher W, Cao J, Papadopoulou N, et al
. Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells. Br J Pharmacol 2005; 145:934-944.
Pearce F, Befus AD, Bienenstock J. Mucosal mast cells: III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells. J Allergy Clin Immunol 1984; 73:819-823.
Tsai PJ, Wu SC, Cheng YK. Role of polyphenols in antioxidant capacity of napier grass from different growing seasons. Food Chem 2008; 106:27-32.
Metwaly MS, Dkhil MA, Al-Quraishy S. Anti-coccidial and anti-apoptotic activities of palm pollen grains on Eimeria papillata
-induced infection in mice. Biologia (Bratisl) 2014; 69:254-259.
Rao CV, Reddy BS. Modulating effect of amount and types of dietary fat on ornithine decarboxylase, tyrosine protein kinase and prostaglandins production during colon carcinogenesis in male F344 rats. Carcinogenesis 1993; 14:1327-1333.
Marignani PA, Sebaldt RJ. Formation of second messenger diradylglycerol in murine peritoneal macrophages is altered after in vivo (n-3) polyunsaturated fatty acid supplementation. J Nutr 1995; 125:3030-3040.
de Jonge HW, Dekkers DH, Lamers JM. Polyunsaturated fatty acids and signalling via phospholipase C-beta and A2 in myocardium. Mol Cell Biochem 1996; 157:199-210.
Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev 2005; 85:757-810.
Parekh AB, Penner R. Store depletion and calcium influx. Physiol Rev 1997; 77:901-930.
Gamberucci A, Fulceri R, Benedetti A. Inhibition of store-dependent capacitative Ca 2+
influx by unsaturated fatty acids. Cell Calcium. 1997; 21:375-385.
Carrillo C, Cavia Mdel M, Alonso-Torre SR. Oleic acid inhibits store-operated calcium entry in human colorectal adenocarcinoma cells. Eur J Nutr 2012; 51:677-684.
Kato A, Ando K, Suzuki S, Jamura G, Arima K. Antitumor activity of fatty acids and their esters. II. Activity of monoglycerides and other esters. In: H Umezawa, editor. Progress in antimicrobial and anticancer chemotherapy. Proceedings of the Sixth International Congress of Chemotherapy. Tokyo, Japan: University of Tokyo Press; 1970. Vol. 2:142.
You YJ, Kim Y, Nam NH, Ahn BZ. Antitumor activity of unsaturated fatty acid esters of 4¢-demethyldeoxypodophyllotoxin. Bioorg Med Chem Lett 2003; 13:2629-2632.
David TW. Oleic acid - the anti-breast cancer component in olive oil. AU JT 2005; 9:75-78.
Tillekeratne LM, Sherette A, Fulmer JA, Hupe L, Hupe D, Gabbara S, et al
. Differential inhibition of polymerase and strand-transfer activities of HIV-1 reverse transcriptase. Bioorg Med Chem Lett 2002; 12:525-528.
Chiang LC, Chiang W, Chang MY, Ng LT, Lin CC. Antiviral activity of Plantago major extracts and related compounds in vitro
. Antiviral Res 2002; 55:53-62.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]