Mass spectrometric identification and toxicity assessment of degraded products of aflatoxin B1 and B2 by Corymbia citriodora aqueous extracts

This study explores the detoxification potential of Corymbia citriodora plant extracts against aflatoxin B1 and B2 (AFB1; 100 μg L−1 and AFB2; 50 μg L−1) in In vitro and In vivo assays. Detoxification was qualitatively and quantitatively analyzed by TLC and HPLC, respectively. The study was carried out by using different parameters of optimal temperature, pH and incubation time period. Results indicated that C. citriodora leaf extract(s) more effectively degrade AFB1 and AFB2 i.e. 95.21% and 92.95% respectively than C. citriodora branch extract, under optimized conditions. The structural elucidation of degraded toxin products was done by LCMS/MS analysis. Ten degraded products of AFB1 and AFB2 and their fragmentation pathways were proposed based on molecular formulas and MS/MS spectra. Toxicity of these degraded products was significantly reduced as compared to that of parent compounds because of the removal of double bond in the terminal furan ring. The biological toxicity of degraded toxin was further analyzed by brine shrimps bioassay, which showed that only 17.5% mortality in larvae was recorded as compared to untreated toxin where 92.5% mortality was observed after 96hr of incubation. Therefore, our finding suggests that C. citriodora leaf extract can be used as an effective tool for the detoxification of aflatoxins.

much emphasis has been focused on the control or elimination of these toxic metabolites in food grains and livestock feeds 8 .
Detoxification of aflatoxins appears to be a more attractive approach. Detoxification strategies have been arbitrarily divided into physical, chemical, or microbiological processes to detoxify by destroying, modifying, or absorbing the mycotoxin so as to reduce or eliminate the toxic effects 9 . However, each treatment has its own limitations. During physical methods of aflatoxin detoxification like cooking and roasting various nutrients are destroyed from treated food commodity 9,10 . While chemicals methods like ammoniation, treatment with formaldehyde and sodium bisulphite have been found to be effective in detoxification of aflatoxins but their use in food industry is restricted because of food safety issues 11 . Biological detoxification of aflatoxins by employing microorganisms have been demonstrated by several researchers with major drawback of utilizing nutrients from food for their own growth and multiplication and release of undesirable compounds 12,13 . So there is a need to identify biologically safe and cost effective aflatoxin detoxifying compounds for use in food and feed industries. Since the treated product should be safe and unaffected by the chemicals used and the nutritive values of the treated product should not be altered.
Natural plant products may provide alternative way to prevent food or feed from fungal or mycotoxin contamination. Powder and extract of many medicinal herbs and higher plants have been reported to inhibit the growth of toxigenic fungi and production of toxins 14,15 . Reddy et al. 16 investigated the potential of certain plant extracts and biocontrol agents for the reduction of aflatoxin B1 (AFB1) in stored rice. Among the plant extracts tested, Syzigium aromaticum (L.) Merr. Et Perry, Curcuma longa (L.), Allium sativum L. and Ocimum sanctum (Linn.) effectively inhibited the A. flavus growth and AFB1 production. Similarly, Velazhahan et al. 17 evaluated various medicinal plants extracts for their ability to detoxify aflatoxin G1 (AFG1) by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Of the various plant extracts, the seeds extract of Trachyspermum ammi showed maximum degradation of AFG1. Another study by Vijayanandraj et al. 18 also demonstrated the effect of different parameters on aqueous extracts of various medicinal plants for detoxification of aflatoxin B1 (AFB1). They found that leaf extracts of Adhatoda vasica Nees showed 98% degradation of AFB1 after incubation for 24h at 37 °C. Correspondingly, Kannan and Velazhahan 19 explored the potential of some indigenous medicinal plants extracts for detoxification of aflatoxins. Their study showed that among various tested plants, Barleria lupulina Lindl. leaf extract(s) exhibited maximum detoxification of aflatoxin B1, B2, G1 and G2 at pH 10 whereas detoxification percentage decreased at pH 7 and 3. Time course study of aflatoxin detoxification by B. lupulina extract showed that degeneration of aflatoxin occurred within 10 min and this percentage was increased with increase in incubation period. In this study Corymbia citriodora aqueous extract is used which has been reported to possess known antibacterial, antifungal, anti-tumor, antioxidant, analgesic and anti-inflammatory effects by various researchers. So we use this plant to explore its potential to detoxify aflatoxins by developing a cost effective and an eco-friendly strategy for detoxification.

Results and Discussion
In the present study the aqueous extracts of Corymbia citriodora leaf and branch were evaluated for their ability to detoxify AFB1 and AFB2. Degradation capability of plant extracts were qualitatively analyzed by TLC, which exhibited that in the presence of C. citriodora leaf extract(s), as compared to C. citriodora branch extract, fluorescence of recovered AFB1 and AFB2 was very weak and more incubation lead to more distinct decline in fluorescence. While no loss of fluorescence was observed in toxin recovered from untreated control samples. Detoxification was quantitatively analyzed by HPLC. Results showed that maximum degradation of AFB1 and AFB2 was observed in C. citriodora leaf extract(s) i.e. 95.21% and 92.95% respectively. Vitro). Toxin detoxification was conducted at different temperatures for 3, 6, 12, 24, 48 and 72 hr of incubation ( Table 1). The extent of detoxification was compared with control under same conditions. Time course study of toxin degradation showed that detoxification of AFB1 and AFB2 occurred within 3 hrs and percentage of degradation gradually increased with increase in incubation time. Maximum degradation was observed after 72 hrs of incubation. Similar findings were also observed by earlier workers (Hajare et al. 20 ; Velazhahan et al. 17 ; Vijayanandraj et al. 18 ; Kannan and Velazhahan 19 ).

Effect of temperature and incubation period on toxin detoxification by plant extracts (In
In case of temperature, highest inactivation was observed at 60 °C. At this temperature, respective control (water) showed 13.36% and 8.64% detoxification of AFB1 and AFB2 after 72 hrs of incubation, respectively. However, toxin treated with C. citriodora leaf and branch extracts showed 99.56% and 85.21% detoxification of aflatoxin B1 while detoxification of aflatoxin B2 was 92.20% and 69.89% respectively, under same conditions. This detoxification may be due to synergistic effect of heat and moisture 15,16 . Similarly, Hajare et al. 20 worked on aflatoxin inactivation by using Ajwain seeds extract under optimized conditions. According to his findings, highest inactivation was observed at 60 °C but further studies were conducted on 45 °C to eliminate the effect of heat and moisture on toxin inactivation.
In the present study, only 4.19% and 3.41% of AFB1 and AFB2 was found to be inactivated in control samples at 30 °C. While in treated samples at 30 °C, C. citriodora leaf extract(s) exhibited higher degree of AFB1 and AFB2 detoxification i.e., 82.55% and 83.27% than C. citriodora branch extract which showed 73.50% and 60.96% detoxification of AFB1 and AFB2, respectively (Table 1). Hence, further studies were conducted only at 30 °C after 72 hrs of incubation in order to eliminate the effects of external factors on toxin detoxification.

Effect of pH on toxin detoxification by plant extracts (In Vitro).
Studies on the effect of pH on detoxification of aflatoxins by Corymbia plant extracts revealed that maximum detoxification was observed at pH 10 followed by pH 8. Results showed that C. citriodora leaf extract(s) significantly detoxify AFB1 and AFB2 than C. citriodora branch extract (Table 2 & 3). After treatment with C. citriodora leaf extract(s), degradation of AFB1 and AFB2 was 99.59% and 96.77% respectively at pH 10 while 95.21% and 92.95% degradation of AFB1 and AFB2 was observed at pH 8. However, C. citriodora branch extract showed 83.96% and 69.15% degradation of AFB1 and AFB2 at pH 10. As compared to C. citriodora leaf extract(s) at pH 8, degradation of AFB1 and AFB2 by C. citriodora branch extract was 78.29% and 66.91% respectively. Distilled water with pH adjusted to 2, 4, 6, 8, and 10 was used as a control. Control  data showed that at pH 10, 19.94% of AFB1 and 17.45% of AFB2 was degraded after 72 hr of incubation at 30 °C while 15.88% and 13.05% degradation of AFB1 and AFB2 was observed at pH 8 under same conditions. The percentage of degradation decreases as the pH decreases to neutral or acidic range (Tables 2 & 3). Subsequently, Mendez-Albores et al. 21 also found that aflatoxin florescence, attributed to the coumarin moiety, diminish or even disappear in alkaline treatment. In addition, the similar results were in accordance with the findings of Kannan and Velazhahan 19 who explored the potential of Barleria lupulina leaf extract on detoxification of aflatoxins. Similarly, Hajare et al. 20 found that aflatoxin inactivation using aqueous Trachyspermum ammi seeds extract was maximum at pH 10. In the present study, pH 8 was selected for further studies because at this pH C. citriodora leaf extracts showed significant (P < 0.05) detoxification of AFB1 and AFB2 and these results were closely comparable with the results obtained at pH 10. Moreover, at high basic pH conditions aflatoxins are known to become unstable and sensitive, therefore to omit this possibility pH 8 was selected which is 100 times less alkaline than pH 10.
In Vivo Detoxification of Aflatoxins in maize samples. In Vivo analysis followed a similar trend as that has been recorded in In Vitro studies. Data obtained from In Vivo studies showed that maximum detoxification of AFB1 and AFB2 in spiked maize samples was carried out by C. citriodora leaf extract after 72 hrs of incubation i.e., 91.71% and 88.77% respectively. As compared to C. citriodora leaf extract, in C. citriodora branch extract 70.26% and 58.84% detoxification of AFB1 and AFB2 was observed in spiked samples (Table 4). Similarly in a study, conducted by Hajare et al. 20 , decontamination of spiked corn samples was carried out using ajowan extract. Their findings showed that aflatoxin-contaminated agricultural commodities could be decontaminated using appropriate conditions of temperature and pH along with ajowan inactivation factor. HPLC chromatograms confirmed that after C. citriodora leaf extract treatment, trace amount of aflatoxin was present along with other peaks whose footprints were not found in chromatogram of parent compounds which may be attributed to toxins degradation products ( Supplementary Figures 1 & 2  Structural characterization of AFB1, AFB2 and their degradation products. To predict molecular formulae as well as elemental composition of degraded products of AFB1 and AFB2 after treatment with C. citriodora leaf extract(s), samples were analyzed by mass spectrometer with electrospray ionization (ESI).
Aflatoxin B1 and B2 exhibited good ESI ionization efficiency in the positive ion mode with molecular base ion at m/z 313.17 and m/z 315.17 for protonated adduct [M+ H] + while m/z 335 and m/z 337 for sodium adduct [M+ Na] + respectively. To validate the identity of the parent, these ions were fragmented into daughter ions. Because the sodium adduct did not exhibit specific fragmentation for any compound, the protonated molecule was chosen as the precursor ion for aflatoxins in the product ion scan mode.

MS/MS analysis of AFB1 and AFB2. MS/MS spectrum of AFB1
showed that continuous loss of carbon monoxide (CO) was the main fragmentation pathway. Methyl and methanol losses occurred on methoxy group located on side chain of benzene. The double bond equivalence (DBE) of AFB1 was 12 (Fig. 1a). However, MS/MS fragmentation pathway of AFB2 revealed that daughter ions were formed by loss of carbon monoxide, oxygen, hydrogen and methyl group (Fig. 1b). The DBE of AFB2 was 11.
The degradation products were identified on the basis of accurate mass measurement of ions and similar fragmentation pathways with AFB1 and AFB2.
Results indicated that after treatment with C. citriodora leaf extract(s) ten possible degraded products of AFB1 and AFB2 were formed with structural alteration in parent compound. Structural formulas of possible degraded products of AFB1 and AFB2 are shown in Fig. 2a,b.

MS/MS analysis for confirmation of degraded products of AFB1.
The degradation product C 17 H 10 O 6 (with 311.17 m/z) formed by the loss of two hydrogen atoms from AFB1. The DBE of C 17 H 10 O 6 was 13 which was one more than AFB1. The fragmentation pathway was different from that of AFB1.  (Fig. 3).
Degradation product C 16 H 6 O 5 with ion peak at m/z 279.25 had one less CH 6 O molecule than AFB1 whereas the DBE of C 16 H 6 O 5 was more than AFB1 i.e., 14. Loss of carbon monoxide (CO), carbon dioxide (CO 2 ) and oxygen (O) was the main fragmentation pathway. Removal of double bond occurred due to the addition of hydrogen (Fig. 4).
The degradation products C 16 H 22 O 5 (with m/z 295.17) and C 16 H 20 O 5 (with m/z 293.17) were formed due to the loss of carbon monoxide (CO) by the opening of lactone ring and addition of hydrogen atoms to AFB1 molecule. Difference between these two degraded products is only of two hydrogen atoms. The DBE content of both products was same i.e., 6 which was less than AFB1. The fragmentation pathway of both products was different from that of AFB1. The precursor ion C 16 (Fig. 7).
The degradation products C 16 H 20 O 6 (with 309.33 m/z) and C 16 H 18 O 6 (with 307.25) were formed by the loss of carbon monoxide from the side chain of benzene ring and addition of OH group on the double bond of terminal furan ring on left side. While addition of hydrogen atoms occurred on both furan and lactone rings. Difference between them was only of two hydrogen atoms. The DBE of C 16 H 20 O 6 and C 16 H 18 O 6 was lower than AFB1 i.e., 7 and 8 respectively. The more detail on fragmentation pathway of these two degraded products are shown in Figs 8 & 9.
The degradation product C 16 H 16 O 7 (with m/z 321.25) had one less carbon atom, as well as four more hydrogen atoms and one more oxygen atom than AFB1. The DBE of C 17 H 20 O 6 was less than AFB1 i.e., 9. Loss of water, oxygen, and carbon monoxide was the main fragmentation pathway (Fig. 10).

MS/MS analysis for confirmation of degraded products of AFB2.
The degradation product C 16 H 12 O 6 (with m/z 301.08) was formed by the replacement of methoxy group with hydroxyl group on the side chain of benzene ring. The DBE of C 16 (Fig. 11).     The degradation product C 17 H 16 O 6 at m/z 317.25 had two more hydrogen atoms and one less DBE than AFB2. Thus, by implication an additional reaction occurred. More detail on fragmentation pathway is shown in Fig. 12.
Several studies have been conducted with the micro-organisms, physical and chemical agents, ultraviolet (UV) rays, Gamma rays and plant products for the aflatoxin detoxification, have shown change of structure of the aflatoxin molecule after detoxification [22][23][24][25][26][27][28][29]17,18 . Aflatoxins have been widely researched for their toxicity by various scientists 1,30,31 . Their toxicity data showed that aflatoxins have cyclopentene ring and furan moiety in their chemical structure. In AFB1 presence of double bond in the terminal furan ring is key factor for its toxic and carcinogenic activities 29 . In contrast, aflatoxin B2 which have saturated furan ring is hundreds times less carcinogenic 32 . The degraded products of AFB2 may be active but were less potent than that of parent compound. In this present study, data showed that eight possible degradation products of AFB1 were obtained after treatment with C. citriodora leaf extract. Among them, 25% products (with m/z 293, 295) were formed after modification of lactone ring and 12% products (with m/z 327) were formed after removal of double bond in terminal furan ring. While in 37% products (with m/z 307, 309, 321) removal of double bond in the terminal furan ring occurred along with modification of lactone ring. Other studies in literature also supported the similar findings. Lee et al. 33 observed that lactone ring plays an important role in fluorescence of aflatoxin molecule. On its cleavage, the molecule becomes non florescent with subsequent significant reduction in toxicity. Velazhahan et al. 17 reported detoxification of aflatoxin G1 by seed extract of Ajowan (T. ammi) and suggested the modification of lactone ring structure of AFG1 as mechanism of detoxification. Similar findings were observed by Vijayanandraj et al. 18 after detoxification of aflatoxin B1 by an aqueous extract from leaves of  Adhatoda vasica Nees. A study conducted by Wang et al. 29 on structure elucidation of radiolytic products of aflatoxin B1 in methanol water solution revealed that in most of radiolytic products addition reaction occurred on the double bond of terminal furan ring, resulting significantly reduced toxicity as compared to that of AFB1. Experiments by Luo et al. 34 showed that aflatoxin B1 can be effectively degraded by using ozone in aqueous system. According to them, due to conjugate addition reaction on the double bond of terminal furan ring for AFB1, the toxicity of degradation products was significantly decreased compared with that of AFB1.
Assessment of biological toxicity of degraded products. Furthermore, biological toxicity of degraded toxin products were tested by brine shrimps (Artemia salina) bioassay. The brine shrimps assay proved to be a convenient system for monitoring biological activity 35 . In this study, the degraded toxin products were incubated with brine shrimp larvae at 26 °C for 24 to 96 hrs. The percentage of mortality was compared with that of control (Table 5). Results indicated that only 12.5-17.5% mortality in brine shrimps larvae was observed after treatment with degraded toxin products. However, mortality level was 82.5-92.5% when larvae were incubated with untreated toxin AFB1 (100 μ g L −1 ) and AFB2 (50 μ g L −1 ), under same conditions. Percentage of mortality was increased with increase in incubation period. The outcome of study clearly implies that the degraded compounds showed much lower toxicity towards brine shrimps larvae than AFB1 and AFB2 at the tested concentration. Similar findings were also observed by Samuel et al. 23 , who worked on detoxification of aflatoxin B1 by Pseudomonas putida. He compared the toxicity of treated and untreated AFB1 towards HeLa cells and concluded that degraded products are nontoxic (D1) or much less toxic (D2 and D3) than AFB1 to the cells at the tested concentrations.

Conclusion
To our knowledge, this is the first report to demonstrate the ability of Corymbia citriodora plant extract to detoxify AFB1 and AFB2 in both In Vitro and In Vivo studies. Maximum detoxification was shown by C. citriodora leaf extract(s) at pH 8 and 30 °C temperature after 72 hrs of incubation. Data obtained after structural characterization and biological toxicity assessment of degraded toxin products revealed that their toxicity level is much more less than parent compound. The outcome of study clearly implies the efficient detoxification of aflatoxins by using C. citriodora leaf extract(s). These extracts are not only cost effective and environmental safe but also are available to the consumers ubiquitously. The use of these toxin degrading natural products can pave the path for the availability of high quality food products for the end users.

Methods
Chemical and reagents. Aflatoxin B1 and B2 purified from A. flavus were prepared in laboratory under optimized conditions and compared through HPLC with standard AFB1 and AFB2 purchased from (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions of AFB1 (1000 μ g L −1 ) and AFB2 (500 μ g L −1 ) were prepared in methanol and stored at 4 °C. The working solutions of AFB1 (100 μ g L −1 ) and AFB2 (50 μ g L −1 ) were prepared by diluting the stock solution.
Safety Information. Workers should always wear a face mask, disposable surgical gloves and lab coat when working with the above chemicals and mould cultures.

Preparation of plant extract. Corymbia citriodora leaves and branches were collected from
Quaid-e-Azam campus, University of the Punjab, Lahore. Samples were surface-sterilized using 1% sodium hypochlorite for 10 min and washed several times with sterile distilled water. After surface sterilization, aqueous plant extract was prepared by homogenizing 10 g of leaves/branches with 10 mL of sterile distilled water. Homogenate was filtered through muslin cloth and centrifuged at 14,000 rpm for 20 min. Supernatant was sterilized using syringe filter assembly and used for further detoxification studies.
In Vitro Toxin inactivation assay. For detoxification studies, 50 μ L of working solution containing (100 μ g L −1 ) AFB1 and (50 μ g L −1 ) AFB2 was mixed with 250 μ L of C. citriodora plant extracts and incubated for various intervals of time. After incubation period, reaction was terminated by addition   of equal volume of chloroform. The residual toxin was extracted by vortexing the mixture thoroughly. Chloroform fraction was separated by low speed centrifugation. After centrifugation, organic phase was withdrawn, evaporated to dryness under gentle stream of nitrogen and re-dissolved in methanol. Control consisted of 50 μ L of toxin in 250 μ L of water and was incubated under same conditions.

Determination of optimal pH for toxin detoxification by plants extract (In Vitro). The optimal
pH was determined by modifying the original pH of the C. citriodora aqueous extracts in the range of 2.0 to 10.0 (adjusted using either 1 N HCl or 1 N NaOH) and then assayed for toxin detoxification activity. Distilled water with same pH range as well as untreated extract was used as control.
Determination of optimal temperature and incubation time period for toxin detoxification by plants extract (In Vitro). For  Quantitative analysis of treated and untreated toxin was done by using High Performance Liquid Chromatography (HPLC) after derivatization. A HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (Merck, Darmstadt, Germany) and a fluorescence detector was used for quantification. Mobile phase consisting of water: methanol: acetonitrile in the volume ratio 60:20:20 at a flow rate of 1 mL/min was applied and aflatoxin was detected at excitation and emission wavelengths of 360 nm and 440 nm respectively. For HPLC method validation, calibration curves were drawn using a series of calibration solutions in methanol. Each standard solution was chromatographed in duplicate. Further, identification of degraded toxin metabolites was carried out by mass spectral studies. LCMS analysis of degraded toxin. Degraded toxin products were analyzed by using surveyor LC system equipped with mass spectrophotometer and PDA plus detectors (Thermo Fisher Scientific). System was validated with known standards individually and in mixture form. All analysis were performed in triplicate using luna phenomenex C 18 column (150 × 4.6 mm, 3 μ m), in isocratic mode. Following are the LC-MS conditions for Aflatoxins. Injection volume was 10 μ L. Mobile phase consisted of Methanol: Acetonitrile: Water (22.5: 22.5: 55.0 v/v). Column temperature was maintained at 30 °C. The total operation time was 25 min with the flow rate of 0.5 mL min −1 . MS conditions were as follows: capillary temperature was 335 °C, sheath gas flow and Auxillary gas flow was 20 L min −1 and 4 L min −1 respectively. Source voltage, capillary voltage and tube lens voltage was 5 KV, 49 V and 120 V respectively.

ESI-MS/MS Conditions for Aflatoxins through direct insertion pump. Mass spectrometery/
Mass spectrometery was performed on a Thermo Scientific LTQ XL System fitted with electrospray ionization (ESI) source operating in positive ionization mode with optimum conditions set as follows: capillary voltage to 49.0 V, source voltage to 5.0 KV, Tube lens voltage to 110 V, and capillary temperature to 275 °C. Sheath and auxillary gas flow were adjusted to get stable spray i.e., 3 L min −1 and 0.4 L min −1 respectively. Data was collected in positive mode within the range of 100 m/z to 500 m/z. The final identification of an unknown compound was then performed based on the accurate measurement of mass of parent ions and fragments, as well as other useful MS/MS spectrum information.
Testing biological toxicity of degraded products. The biological toxicity of degraded toxin products was tested by brine shrimps (Artemia salina) bioassay. The procedure for bioassay generally followed the method developed by Solis 36 , with some modifications. Brine shrimps dry eggs were procured from local market. 100-200 mg of shrimps eggs were hatched in artificial sea water (34 g sea salt/ L of deionized water) by incubation under 60 W lamp, providing direct light and warmth (26 °C). Throughout hatching period, the same conditions of light sensitivity and temperature were maintained. After an incubation period, the hatched nauplii were separated from shells and transferred to fresh sea water.
The volume of 300 μ l of treated and untreated AFB1 (100 μ g L −1 ) & AFB2 (50 μ g L −1 ) solution was added to 96 well plate separately and dried overnight. After complete evaporation of solvent, toxins were redissolved in 200 μ L of sea water. After that, 200 μ L of sea water containing 40-45 organisms were pipetted into each well, resulting in a final volume of 400 μ L and incubated for 24-96 hrs at 26 °C. Mortality was determined by counting the immobile (dead) larvae under stereoscope microscope. Toxicity of each solution was evaluated in triplicate.

Statistical analysis.
Results obtained in various experiments were subjected to statistical analysis by using DSSTAT software. Data were analyzed by analysis of Variance (ANOVA) and differences among the means were determined for significance at P ≤ 0.05 using Tukey's multiple range test.