Natural Predominance of Abscisic Acid in Pongammia pinnata

May 23, 2017 - In test conditions, test compound (honey-abscisic acid) and EMS (133 mM) ... In test, abscisic acid solution (500 μL) was mixed with d...
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Natural Predominance of Abscisic Acid in Pongammia pinnata (“Karanj”) Honey Contributed to its Strong Antimutagenicity Sudhanshu Saxena, Jyoti Tripathi, Suchandra Chatterjee, and Satyendra Gautam* Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400094, India ABSTRACT: Various samples of raw (unprocessed) floral honey collected from different geographical locations of India were assayed for its antimutagenicity against ethyl methanesulfonate in E. coli MG1655 cells through rifampicin resistance assay. A monofloral honey (“Pongammia pinnata”, local name “Karanj”) displayed maximum antimutagenicity (78.0 ± 1.7; P ≤ 0.05). Solid phase extraction (using Amberlite XAD-2 resin) followed by HPLC resulted into different peaks displaying varying antimutagenicity. Peak at retention time (Rt) 27.9 min (henceforth called P28) displayed maximum antimutagenicity and was further characterized to be abscisic acid (ABA) using ESI−MS and NMR. Its antimutagenicity was reconfirmed through human lymphoblast cell line (TK6) mutation assay using thymidine kinase (tk+/−) cell line. Although ABA from this honey displayed strong antimutagenicity, it lacked any in vitro antioxidant capacity indicating noninvolvement of any radical scavenging in the observed antimutagenicity. KEYWORDS: honey, mutagen, rifampicin assay, human lymphoblast mutation assay, antioxidant capacity

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INTRODUCTION Honey primarily consists of sugars, enzymes, amino acids, organic acids, carotenoids, vitamins, minerals, aromatic substances, and phenolics including flavonoids.1 Floral sources and honey bee species could affect the physicochemical attributes of honey. Quality characteristics could also be affected by geographical conditions, processing, and storage conditions.2 Health protective properties of honey are primarily contributed by their phytoconstituents. These bioactive phytochemicals are mostly contributed through flower nectar or its metabolic byproducts formed in the digestive tract of the honey bee. Diverse prophylactic properties of honey include antioxidant, antimicrobial, immunomodulatory, wound healing potential, antimutagenicity, and many others.3−6 Some of these bioactivities directly impact on molecular events leading to diseased state whereas others work indirectly involving complex regulatory pathways such as immunomodulation and antimutagenicity. Increasing mutagenic burden in the environment from various sources such as industrial (toxic effluents), vehicular air pollutants, and biocides from agricultural applications is a matter of health concern. Food contaminants, additives, and adulterants also contribute to the etiology of human cancer, where some of these carcinogenic contaminants include heterocyclic amines, nitrates, pesticides, dioxins, and other organochlorines.7,8 Mutation associated diseases could possibly be prevented by nutritional chemoprevention through dietary bioactive components.9 This concept has been endorsed by various organizations of high repute including WHO, American Cancer Society, and American Institute of Cancer Research.10−13 In current study, antimutagenic potential of raw honey collected from different geographical regions of India was compared and potent bioactive from honey possessing maximum antimutagenic potential was further identified and characterized. Antimutagenicity assay involved both bacterial as well as human cell line model systems. Findings will help in © 2017 American Chemical Society

understanding the nutraceutical efficacy of honey in terms of its antimutagenic potency.

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MATERIALS AND METHODS

Chemicals. Fetal bovine serum (FBS), Luria−Bertani (LB) medium, rifampicin, and RPMI (Roswell Park Memorial Institute) 1640 medium were procured from Hi Media laboratories Pvt. Ltd., India. cis,trans-Abscisic acid, aminopterin, 5-azacytidine (5-AZ), biotin, histidine, cytidine, thymidine, trifluorothymidine (TFT), hypoxanthine, L-glutamine, and ethyl methanesulfonate (EMS) were obtained from Sigma-Aldrich (St. Louis, Mo., U.S.A.). Pen-Strep (Penicillin and streptomycin) was procured from Gibco Life Technologies, U.S.A. Acetic anhydride and glacial acetic acid were procured from Thomas Baker (Chemicals) Limited, Mumbai. Sulfuric acid was obtained from SD-Fine Chem Ltd., Mumbai. Toluene, ethyl acetate, formic acid, and methanol were procured from Chemco Fine Chemicals, Mumbai. Honey Samples. A total of 17 raw and unprocessed floral honey samples were procured from all the four geographical regions of India from reliable and authentic suppliers. Bacterial Strains and Cell Lines. Ames strains Salmonella typhimurium TA100 (hisG46 gal bio ch1005 rfa1004 uvrB pKM101) and TA102 (hisΔ(G)8476 galE503 rfa1027/pAQ1/pKM101), were procured from Xenometrix, Inc. U.S.A. E. coli MG1655 (F-λ-ilvG-rfb50 rph-1) was gifted by Prof. M.Z. Humayun, UMDNJ, U.S.A. Thymidine kinase (TK6) human lymphoblast cell line (genotype: tk+/−) was purchased from NCCS, Pune, India. Cells were cultured in standard RPMI 1640 medium supplemented with penicillin (200 units/mL; 1 unit = 0.6 μg), streptomycin (50 μg/mL), L-glutamine (0.3 mg/mL), and fetal bovine serum (10%) at 37 °C in humidified atmosphere containing 5% CO2. Pollen Analysis of Honey. Pollen analysis was performed as per the method of International Commission for Bee Botany as described earlier.14 Permanent pollen slides were prepared and examined under microscope. Pollen identification was performed using reference pollen Received: Revised: Accepted: Published: 4624

April 3, 2017 May 22, 2017 May 23, 2017 May 23, 2017 DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

Article

Journal of Agricultural and Food Chemistry slides at Birbal Sahni Institute of Paleobotany Herbarium, Lucknow, India and also the published literature.15 Upon the basis of pollen frequency (P.F.), honey could be monofloral i.e., frequency of single pollen type >45%: secondary types (P.F. 16−45%); important minor types (P.F. 3−15%); and minor types (P.F. < 3%). Antimutagenicity Assay. rpoB/RifR Rifampicin Assay. Assay was performed as per the method described earlier.14−16 Overnight grown E. coli MG1655 was subcultured to mid log phase and placed on ice (15 min) to arrest the growth. Cell suspension was centrifuged (8000g, 10 min), and pellet was washed twice using LB broth (50 mL) and resuspended in the same (25 mL). Mutagenesis was induced by adding EMS (70 μL; effective concentration: 130 mM) into cell suspension (5 mL) and incubating (37 °C) in a rotary shaker (125 rpm) with or without honey solution for 45 min. Cells were centrifuged, and the pellet was washed twice with LB and resuspended in LB (5 mL). An aliquot (500 μL) of this was inoculated in LB broth (in replicates) and cultures were grown overnight in shaking condition (125 rpm) at 37 °C. Respective dilutions were spread plated on LB-rifampicin (100 μg/ mL) as well as LB plates and incubated (37 °C) for 24 h. Colony forming units were counted and mutation frequency was enumerated as the ration of total no. of RifR (rifampicin resistant) mutants to the total no. of viable cells in a unit volume (mL). Antimutagenicity (mutation lowering frequency) was equated by enumerating the extent of reduction in the mutation frequency conferred by the test antimutagenic agent and calculated in comparison to the positive control (EMS) as follows: Antimutagenicity (%) = [1 − (mutation frequency in the presence of test antimutagenic compound/mutation frequency in the presence of mutagen alone) × 100]. Ames Test. Ames test was performed as per the procedure described earlier.18,19 Salmonella typhimurium tester strains TA100 and TA102 were cultured in nutrient broth (16 h at 37 °C), and its aliquot (1 mL) was centrifuged and suspended in nutrient broth (500 μL, 2X). In control set EMS (133 mM) was added, gently mixed and incubated (37 °C for 20 min). In test conditions, test compound (honey-abscisic acid) and EMS (133 mM) were added to the cell suspension and coincubated as above. A 2 mL aliquot of molten agar was mixed, and the suspension was overlaid on minimal agar plates. The plates were incubated (37 °C) for 48 h, and the number of his+ revertant colonies was counted at the end of incubation both in control as well as test conditions. Thymidine Kinase Mutation Assay. Thymidine kinase mutation assay was performed as per the modified OECD procedure described earlier.17−21 Exponential phase cells (density ≈ 2 × 105 cells/mL) were subjected to EMS (0.4 mM) and further grown for 4 h at 37 °C in a humidified CO2 incubator (CO2 5%). Selection of mutagen concentration was based on the earlier studies.17 Cells were washed twice and counted using a hemocytometer. Cultures were adjusted at a density of ∼2 × 105 cells/mL and grown for further 2 days in nonselective conditions to allow expression of tk−/− phenotype. Cells (∼2 × 105) from each treatment were grown in selective medium containing TFT (5 μg/mL) for 3 days at 37 °C in CO2 incubator and counting of cell no. was performed as described below. Similar experiments were also performed in the presence of HPLC purified bioactive (antimutagenic) compound from most antimutagenic honey. The result was expressed as the relative number of mutants (tk−/− phenotype) per ∼105 seeded cells, where, relative number of mutants =

Hamburg, Germany). This was subsequently dissolved in Milli-Q water (4 mL) and used for further analyses. High Performance Liquid Chromatography (HPLC). HPLC was performed using a reverse-phase system (UltiMate 3000 Dionex Corporation, CA, U.S.A.; VWD-3000 detector; C-18 column: Acclaim 120, size: 4.6 × 250 mm2, particle size: 5 μm) and Chromeleon Version 6.8 SR8 software. Mobile phase comprised of A (methanol) and B (2% glacial acetic acid). Injection volume was 30 μL. Following linear solvent gradient was used: start 10% A in B, gradient: until 28 min 60% A in B; until 30 min 90% A in B at the flow rate of 1 mL/ min. Chromatogram was monitored at 280 nm. Electrospray Ionization−Mass Spectrometry (ESI−MS). HPLC purified peak fraction displaying highest antimutagenicity was vacuum-dried, dissolved in methanol and analyzed by mass spectrometry (6550 iFunnel Q-TOF LC-MS; Agilent Technologies, U.S.A.) equipped with a jet stream technology dual spray ESI source. Source parameters were: gas temperature 250 °C, flow rate 13 L/min, VCap 1660 V, fragmentor 175 V, skimmer 165 V, octopole RF Peak 750 V, ion source HPLC-Chip (ChipCube, 75 μm × 43 mm × 5 μm; C-18 ZX-SB80, Model G4240A), and injection volume 15 μL. QTOF parameters were as follows: acquisition mode, targeted MS/MS (range: 100−1500 m/z) and scan rate: 1 spectrum/s. Data acquisition and processing was done using MassHunter workstation data acquisition software (Version B.05.01, Agilent Technologies). Assessment of Antioxidant Capacity. Radical Scavenging Activity. The in vitro antioxidant capacity of HPLC purified antimutagenic compound (HPAC) was assayed using different assays. For DPPH radical scavenging, HPAC was diluted with 70% ethanol (to 600 μL), mixed with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) solution (400 μL, 0.2 mM), and incubated (26 ± 2 °C) for 15 min. Absorbance was measured at 517 nm.23 For 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate (ABTS) scavenging assay HPAC was mixed with 1 mL of diluted ABTS+ solution (dilution 1:132), incubated (6 min), followed by measurement of absorbance at 734 nm.24 Gallic acid (antioxidant) was used as a positive control in both the assays. Radioprotective Capacity of HPAC. E. coli Survival Study. Extent of protection (if any) offered to E. coli cells subjected to γ radiation induced oxidative stress was evaluated in terms of decimal reduction dose.17 Single colony of E. coli MG1655 was grown overnight in LB (10 mL) broth in a rotary shaker (125 rpm, 37 °C). Culture was diluted to ∼105 cells/mL using saline. In test, abscisic acid solution (500 μL) was mixed with diluted cell suspension, whereas, in control set instead of abscisic acid saline (500 μL) was added. Cell suspensions were exposed to γ radiation (0.1−0.5 kGy) and centrifuged (20 000g for 15 min at 4 °C). Cell pellet was washed using saline and resuspended in the same (1 mL). For the enumeration of total viable count, cell suspensions were serially diluted, spread plated on LB agar plates and incubated (37 °C for 24 h) in an incubator. The D10 value was determined by dose-survival curve by taking the negative reciprocal of the survival curve slope. In Vitro DNA Protective Activity of HPAC. In-vitro DNA protection offered to the naked plasmid DNA was performed as described earlier.17 An aliquot of HPAC (15 μL) was mixed with ultrapurified (cesium chloride density gradient) pBR322 plasmid DNA (15 μL, ∼400 ng) and irradiated (0.15 kGy). DNA was subjected to agarose (1%) gel electrophoresis along with control samples, and the band intensity was quantified using a gel documentation system (Syngene gene genius bioimaging system, Syngene, Frederick, MD). DCFDA Assay. 2′,7′-Dichlorofluorescin diacetate (DCFDA) assay was performed as per the method described earlier.25 Approximately 2 × 105 cells/mL were seeded in 24-well plate and incubated for 24 h. Media were replaced with serum free RPMI media, and the purified antimutagenic compound was added. After 30 min incubation, hydrogen peroxide (50 μM) was added to the cell suspension to generate oxidative stress. Cells were washed after 1 h and resuspended in RPMI medium containing DCFDA dye (5 μM). Cells were examined under fluorescence microscope with an excitation wavelength of 485 nm (Carl Zeiss AxioVert.A1, Germany) and imaged using equipped Tucsen ISH500 camera.

number of mutants in test number of spontaneous mutants

Partial Purification of Honey Phenolics by Removing Sugars and Other Macromolecules. Honey phenolics were extracted as described earlier.22 Honey (∼100 g) was mixed with acidified water (500 mL, pH 2 adjusted with HCL) and filtered. Activated Amberlite XAD-2 resin was added to the filtrate followed by stirring the mixture for 1 h at ambient temperature. Amberlite resin was packed in a column, washed with acidified water (pH 2; 400 mL) and further with deionized water (400 mL) for removal of sugar and other compounds. Adsorbed phenolics were eluted with methanol (300 mL) and elute was vacuum-dried (Eppendorf concentrator 5301; Eppendorf, 4625

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Table 1. Comparative Antimutagenic Activity of Honey against Ethyl Methanesulfonate Induced Mutagenesis in E. coli MG1655 Cells Evaluated Using rpoB/RifR Assay honey NER-1 IGR-1 IGR-2 IGR-3 IGR-4 IGR-5 IGR-6 SPR-1 SPR-2 SPR-3 SPR-4 SPR-5 SPR-6 SPR-7 SPR-8 SPR-9 SPR-10

collection site Imphal, Manipur East Champaran, Bihar Muzaffarpur, Bihar Samastipur, Bihar Begusarai, Bihar Bharatpur, Rajasthan Neemuch, Madhya Pradesh Baroda, Gujrat Sahyadri, Maharashtra Bhimashankar, Maharashtra Kastalav, Maharashtra Mahabaleshwar, Maharashtra Kolhapur, Maharashtra Nagpur, Maharashtra Nanded, Maharashtra Akola, Maharashtra Warangal, Andhra Pradesh

latitude, longitude 24.8170° 26.6098° 26.1209° 25.8630° 25.5184° 27.2170° 24.4764° 22.3072° 18.9838° 19.0720° 17.6805° 17.9307° 16.7050° 21.1458° 19.1383° 20.7059° 17.9689°

N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N,

93.9368° 84.8568° 85.3647° 85.7810° 86.1752° 77.4895° 74.8624° 73.1812° 73.7695° 73.5357° 74.0183° 73.6477° 74.2433° 79.0882° 77.3210° 77.0219° 79.5941°

antimutagenicity (%) 3.0 34.0 14.0 50.0 78.0 10.0 10.0 3.0 26.0 4.0 30.0 72.0 2.0 40.0 2.0 2.0 7.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

k

0.7 0.8e 0.5h 2.8c 1.7a 0.5i 0.5i 0.5k 1.5g 0.5k 0.5f 2.4b 0.5l 0.5d 0.5d 0.5l 0.5j

number of RifR mutants/108 cells 2134 1452 1892 1100 484 1980 1980 2134 1628 2112 1540 616 2156 1320 2156 2156 2046

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

47a 38g 27d 28i 19k 39c 42c 44a 32e 30a 23f 22j 45a 38h 33a 40a 41b

Letters depict the significance (p ≤ 0.05) of differences in mean values when analyzed by Two-way analysis of variance (ANOVA). Same letter across the column indicate insignificant differences, whereas, different letters indicate significant differences. EMS (positive control) induced mutation frequency was 2200 RifR/108 cells. Spontaneous mutation frequency (negative control) was ∼1.0/108 million cells. a−l

of South Eastern Asia and Australia.26 Beekeeping products including propolis, bee bread, honey mixtures, and royal jelly were also reported to possess antimutagenicity against the mutagenic effect of various chemical and physical mutagens.27 Mixture of honey bee products has been reported to minimize the genotoxic side effects of the anticancer drug cyclophosphamide and thus possesses chemopreventive potential.28 The rpoB gene encodes the β-subunit of DNA dependent RNA polymerase that binds with rifampicin when E. coli is exposed to rifampicin, thus blocking the elongation of nascent RNA.29 Mutation(s) in the rpoB decrease the binding affinity of rifampicin to RNA polymerase thus making it insensitive to rifampicin conferring RifR mutator phenotype to E. coli. As rpoB gene contains many hot spots of mutation, it provides an excellent tool to assess the antimutagenic potential of compounds of interest. Other associated advantage of rpoB/ Rif R system is its low (∼1.0/108 million cells) spontaneous mutation frequency.14,16,17 HPLC Based Purification of Bioactive from P. pinnata Honey. P. pinnata honey was purified using Amberlite XAD-2 resin employing solid phase extraction.30 SPE purified fraction was further subjected to HPLC analysis that resulted into several peaks (Figure 1A). Peak fractions were collected, vacuum-dried and evaluated for antimutagenicity using rpoB/ RifR assay. Peak fraction resolved at a retention time of 27.9 min displayed >75% antimutagenicity at 100 μg/mL concentration, whereas other three peak fractions displayed significantly lesser antimutagenicity at same concentration. Peak fraction at 27.9 min (P28) also displayed antimutagenicity (∼56%) against other chemical mutagen like 5-azacytidine (data not shown). P28 also displayed antimutagenicity in Ames test with TA100 and TA102 strains (Table 2B). Antimutagenicity of P28 in HLM Assay. To assess the wide spectrum antimutagenic potential, TK6 human lymphoblast cell line based HLM assay was also used to investigate the antimutagenicity of P28 compound (Figure 1D,E). The method described is the modified OECD procedure for testing antimutagenicity of potent compounds, where the mutant

Thin Layer Chromatography. Equimolar (2.35 mM) concentrations of analytes catechin (CA), gallic acid (GA) and abscisic acid (ABA) were subjected to TLC on a fluorescent silica TLC plate (TLC Silica gel 60 F254; Merck, Germany) using a mobile phase toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2). DPPH° radical scavenging potential was evaluated by spraying DPPH° solution (2.54 mM) on the TLC plate where the development of a yellow color on a purple background is indicative of antioxidant action of the analyte. Similary, ABTS+ radical scavenging activity was assessed by spraying ABTS+ solution (7 mM) on the TLC plate. NMR Analysis. The NMR spectra were recorded with a Bruker AC5000 MHz FT NMR spectrometer (Bruker, Fallanden, Switzerland) using CDCl3, and TMS was used as an internal standard. The usual abbreviations employed are s = singlet and d = doublet.

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STATISTICAL ANALYSIS Statistical analysis was performed using BioStat 2009 Version Professional 5.8.0.0 (AnalystSoft Inc., Canada). All of the analyses were performed in three independent sets. In each set samples were used in triplicates. Means and standard deviations were calculated taking all the readings in consideration. The mean values were further compared using two-way ANOVA (analysis of variance) test for assessing the significance of their difference. The analyses were performed at the level of significance where P ≤ 0.05.

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RESULTS AND DISCUSSION

Different Collections of Floral Honey Displayed Varying Antimutagenicity. Comparative antimutagenicity of different unprocessed floral honey against ethyl methanesulfonate indicated a wide variation (Table 1). In presence of directly acting mutagen EMS the no. of RifR mutants/100 million cells was ∼2200.14,16,17 “Pongammia pinnata” (“Karanj” or “Indian beech”) honey displayed maximum (∼78%; No. of RifR mutants/108 cells: 484) antimutagenicity followed by a honey SPR-5 that displayed ∼72% (no. of RifR mutants/108 cells: 616) antimutagenicity. Pongamia pinnata (L.) belongs to family Leguminosae and subfamily Papilionaceae. It is a mediumsized glabrous, perennial tree that grows in the littoral regions 4626

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Journal of Agricultural and Food Chemistry

Figure 1. (A) HPLC profile of SPE purified fraction from “Pongammia pinnata” honey; (B) antimutagenicity evaluation of HPLC purified peak fractions in E. coli through rpoB/RifR assay; (C) antimutagenic potential of P28 peak fraction on rifampicin selective plate; (D) antimutagenicity of HPLC purified P28 in Human lymphoblast cell line: (i) cells without treatment (negative control), (ii) cells treated with EMS (positive control), mutant (tk−/−) cells seen as cellular aggregates (marked), (iii) cells treated with EMS in the presence of P28, and (E) relative number of mutants against EMS in the presence of HPLC purified P28. a−dLetters depict the significance (p ≤ 0.05) of differences in mean values when analyzed by Twoway analysis of variance (ANOVA). Different letters on the bar indicate significant differences. 4627

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Table 2. Antimutagenicity (A) of P28 under Different Treatment Conditions Using E. coli Based rpoB/Rif R Assay System; (B) P28 Using Ames Test (A) conc. (μg/mL)

pretreatment

post-treatment

100 200

27 ± 2a 65 ± 4a (B)

3 ± 1b 17 ± 2b

strain

his− revertants (EMS)

his− revertants (EMS + P28)

S. Typhimurium TA100 S. Typhimurium TA102

1936 ± 8x 1900 ± 12x

1264 ± 6y 1214 ± 14y

a,b,x,y

letters depict the significance (p < 0.05) of differences in mean values when analyzed by Two-way analysis of variance (ANOVA). Same letter across the row or column indicates insignificant differences, whereas different letters indicate significant differences.

Figure 2. P28 fraction displaying lack of in vitro radical scavenging activities (A) ABTS; (B) DPPH; (C) cell survival in terms of decimal reduction dose (D10); (D) plasmid DNA protection assay [lane 1: pBR322 plasmid DNA; lane 2: DNA subjected to γ radiation (160 Gy); lane 3−5: DNA subjected to γ radiation (160 Gy) in the presence of P28 (100, 250, and 500 μg/mL, respectively)].

(tk−/− phenotype) cells in selective media are enumerated and expressed as the relative number of mutants per ∼105 seeded cells. This is compared with that of the positive control to calculate the percentage reduction in mutagenicity. Similar methodology has been reported earlier for assessing the antimutagenic potential of an ethoxy-substituted phylloquinone derivative from spinach.18 The number of induced mutant cells (tk−/−) upon exposure to EMS (0.5 mM) was found to be ∼8 × 104 (±3 × 103) cells per ∼105 seeded cells. Mutagenicity reduced by ∼55% in the presence of P28 (50 μg/mL) and by ∼75% at its higher (200 μg/mL) concentration. This clearly indicated a dose dependent increase in antimutagenic potential. However, no induced mutation and cytotoxicity were observed in TK6 cell line when P28 was tested alone at this concentration. Observed antimutagenic effect of honey extract in human cell line signifies its therapeutic potential. TK6 cells are mammalian mismatch repair proficient and O6-alkyl guanine DNA alkyltransferase deficient cells which are sensitive

to cytotoxic and mutagenic effects of alkylating agents causing the formation of O6-methylguanine and O6-ethylguanine DNA adducts, with GC→ AT transitions. The TK6 gene mutation assay employs the heterozygous thymidine kinase gene locus which undergoes forward mutation (tk+/− → tk−/− phenotype) upon mutagen exposure. Mutant tk−/− cells lack thymidine kinase enzyme and thus can survive the cytotoxic effect of pyrimidine analogue trifluorothymidine (TFT) and can be detected. Gene mutation studies using TK6 human lymphoblast cells have been performed earlier for assessing the antimutagenic potential of different compounds screened through Ames test.17 Possible Mechanism(s) of Antimutagenicity. To elucidate the possible underlying mechanism, E. coli cells were treated with P28 1 h prior to mutagen exposure (pretreatment) or cotreated along with mutagen (EMS) simultaneously or the cells were first treated with EMS followed by addition of P28 (post-treatment). In pre- and 4628

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Figure 3. DCFDA assay indicating no antioxidant capacity of P28 in human lymphoblast TK6 cell line subjected to oxidative stress: (A) Control; (B) H2O2; (C) H2O2 + BHT; and (D) H2O2 + P28.

Figure 4. TLC profile of abscisic acid (ABA) along with standard antioxidants [Catechin (CA); gallic acid (GA); and (A) UV 254 nm; (B) DPPH spraying; and (C) ABTS spraying.

shown strong bioantimutagenic potency. As P28 provided protection against the mutagenic effect of EMS in all three treatment conditions, therefore it is considered as both desmutagen as well as bioantimutagen. Certain antimutagens act both as desmutagens as well as bioantimutagens such as docosahexaenoic acid, eicosapentaenoic acid, and PUFA. To understand whether P28 fraction is exerting antimutagenicity through a mechanism involving antioxidant capacity (radical scavenging), different radical scavenging assays were employed. P28 fraction lacked DPPH and ABTS radical scavenging activities (Figure 2A,B). Standard antioxidant gallic acid displayed prominent DPPH (concentration range: 0−5.5 μM; y = 15.76x + 1.4474; R2 = 0.9932) as well as ABTS (concentration range: 0−3.5 μM; y = 27.005x + 4.4983; R2 =

cotreatment conditions a statistically significant decrease in the mutation frequency was observed with respect to positive control (Table 2 A). The antimutagenic effect was found to be 27% and 3% in case of pretreatment and post-treatment, respectively (Table 2A). Antimutagens that are active in pre- or cotreatment are considered as desmutagens, whereas those active in post-treatment condition are called bioantimutagens.31 Desmutagens exert their antimutagenic action by mediating biochemical modification of mutagen at the extracellular level. Some of the reported desmutagenic compounds are Burdock, water extract of guava, refined corn bran, coriander juice, galangin, and quercitin. However “bioantimutagens” interfere with cellular damage fixation processes. S-methylmethanethiosulfonate and cinnamaldehyde have been reported to have 4629

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Journal of Agricultural and Food Chemistry

Figure 5. Interaction of ABA with mutagens as indicated by HPLC profile (a) ABA only, (b) ABA plus EMS, (c) ABA plus Mitomycin C, and (d) ABA plus 5-AZ.

Interaction Studies with Mutagen. HPLC purified P28 fraction was incubated with mutagens EMS, 5-AZ, MMC, and their interaction was examined by HPLC analysis (Figure 5). Interestingly, incubation of P28 fraction with mutagens did not lead to any change in the HPLC profile of P28 fraction. Upon the basis of these observations it could be inferred that the P28 fraction is exerting antimutagenicity likely through indirect inactivation of mutagen but not by direct interaction leading to a complex formation. Characterization of the P 28 Fraction by Mass Spectrometric Analyses. HPLC purified P28 fraction was subjected to ESI−MS/MS analyses in the negative ion mode. A molecular ion of m/z 263 was detected that resulted in three characteristic daughter ions with m/z 219.1, 203, and 201.1 upon fragmentation (Figure 6). These corresponded to product ions from losses of CO2, CO2 H2O. In positive ion mode, dehydration ions were observed with m/z 247.1 as one of the predominant fragmentation products. Upon the basis of the m/ z value of the molecular ion and the characteristic fragmentation pattern, the prospective identity of P28 resembles abscisic acid.32 HPLC analysis of cis,trans-abscisic acid (Sigma, St. Louis, MO) showed resemblance with the P28 profile (retention time 27.9 min). Spiking of standard abscisic acid with honey (IGR-6) displaying weak antimutagenicity also corroborated the above findings. NMR Analysis of Pab. Compound P28 was an off-white powder, exhibiting the characteristic NMR spectral features of

0.9941) scavenging activity. P28 fraction offered no protection to cells and DNA against γ radiation mediated oxidative damage (Figure 2C,D). Antioxidant capacity of the P28 fraction was further assessed in human lymphoblast TK6 cell line by DCFDA assay that evaluates the total reactive oxygen species (ROS) level in the cells. Cells under oxidative stress (induced by hydrogen peroxide treatment) displayed prominent fluorescence due to ROS generation (Figure 3B). Treatment with standard antioxidant (BHT, Butylated hydroxytoluene) fluorescence reduced to background due to ROS scavenging (Figure 3C). Prominent fluorescence was also noticed in cells subjected to P28 fraction in the presence of hydrogen peroxide (Figure 3D). These observations confirmed that P28 does not possess in vitro antioxidant capacity. The above observations were further corroborated by the TLC profiling. Prominent dark bands of gallic acid (GA, standard antioxidant), catechin (CA, standard antioxidant), and abscisic acid (ABA) were observed on a fluorescent background under UV 254 nm exposure (Figure 4A). The Rf of CA, GA, and ABA were 0.44, 0.48, and 0.64, respectively. TLC plate was further subjected to DPPH° as well as ABTS+ spraying. Under both conditions standard antioxidants catechin and gallic acid developed clear yellow and white spots in the case of DPPH° and ABTS+, respectively, owing to their antioxidant activity but ABA showed no scavenging (Figure 4B,C). In TLC profiling too, abscisic acid was not found to display any antioxidant capacity. 4630

DOI: 10.1021/acs.jafc.7b01529 J. Agric. Food Chem. 2017, 65, 4624−4633

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Figure 6. Electrospray ionization mass spectrum of HPLC P28 peak fraction.

abscisic acid (Table 3). Two singlet protons at δ 5.76 and 5.94 were assigned as H2 and H8, respectively. Two doublets at δ

different season of harvest, occurrence of abscisic acid was quite prominent, thus indicating it as a biochemical marker for “Karanj” honey. Abscisic Acid and Its Diverse Regulatory Role. Although abscisic acid does not structurally resemble a phenolic acid, it however exhibits similar chromatographic resolution as phenolic acids in a suitable solvent system.33 Abscisic acid has been biochemically characterized as a hormone and is conserved from cyanobacteria to higher plants.34,35 Endogenous ABA synthesis has been demonstrated in lower Metazoa, Porifera, and Hydrozoa. ABA is a sesquiterpenoid derived from C40 carotenoids and is reported to have diverse and important physiological roles in higher plants particularly during abiotic stresses, seed dormancy and germination, stomatal closure, and certain gene transcription. Under stress conditions when soil begins to dry, abscisic acid is accumulated in root tissues, released to the xylem vessels and transported to the shoot to help the plant cope with the stress situation.36 These stress conditions increase the ABA concentration in the nectar, and therefore, in honey in varying amounts. Natural occurrence of abscisic acid has been reported in different floral honey including Acacia, Heather, Manuka, Rape, Sage, and Strawberry.34,37−39 The role of ABA in plant adaptive response has been proposed where UV-B led to a NOS-like mechanism, which helped in maintaining cell homeostasis and attenuated UV-B-induced cell damage.40 (+)-cis,trans-Abscisic

Table 3. 1H NMR Data of the Compound P28 proton position

chemical shift

2 4 5 8 10a 10b 12 13 14 15

5.76 7.82 6.28 5.94 2.55 2.20 1.08 1.05 1.95 2.06

peak multiplicity s d d s d d s s s s

(J = 16.1) (J = 16.1) (J = 20.0) (J = 20.0)

7.82 (J = 16.1) and 6.28 (J = 16.1) were due to the protons of 4 and 5 positions. The other two doublets at δ 2.55 (J = 20.0) and 2.20 (J = 20.0) represented two different protons (a and b) of H10. Four singlet protons of four methyl groups were noted at 1.08, 1.05, 1.95, and 2.06 were assigned as protons of 12, 13, 14, and 15 positions, respectively. The 1H NMR (Table 2) analysis clearly corroborated the above findings and further confirmed P28 as abscisic acid. Besides, in Pongammia pinnata (“Karanj”) honey procured in 4631

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Journal of Agricultural and Food Chemistry

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acid is a natural and active isomer of the abscission accelerating plant hormone. cis−trans abscisic acid in honey has been reported to come mainly from nectar, honeydew, and pollen, but not from bees, although bees may be responsible for converting some cis−trans isomer into trans−trans.41 ABA also plays a role in mammalian physiology as an endogenous immune regulator and has multiple bioactivities.42 Recent evidence indicates that ABA plays a fundamental role in regulating many human cell responses to diverse stimuli. ABA production has been demonstrated in human granulocytes, β pancreatic cells, and mesenchymal stem cells, where ABA stimulates cell-specific functional effects through signaling.42 Recently it has been reported that naturally occurring ABA enhances immune defense in Apis mellifera and contributes to the overall colony fitness.43−45 However, there is limited information on the mechanistic basis of beneficial health effects of ABA in humans. ABA was reported as an anticancer compound (US patent no. 838902432). As compared to control mice, ABA treatment substantially increased survival rates in mice transplanted with tumor and displayed no toxic side effects. Unlike some drugs that are cytotoxic, ABA when functioning as a growth regulator does not have significant toxic side effects on animal cells. ABA could be proposed as a potential therapeutic agent and experimental tool to modulate human cellular activities in vivo as this possesses potential for future development into clinical applications. In this context availability of ABA through natural dietary routes such as honey could serve as an important health promoting supplement. Findings in the current study provide credible evidence establishing a functional (antimutagenic) role of honey through ABA, a compound of diverse biological significance across the living systems.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-022-25595379; fax: 91-22-25505151/25519613; email: [email protected] (S.G.). ORCID

Satyendra Gautam: 0000-0002-9312-2183 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

Authors thank the technical assistance provided by Mr. Rahul Jain and Ms. Kanchan Deshmukh during various assays.

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