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Biotransformation of newly synthesized coumarin derivatives by Candida albicans as potential antibacterial, antioxidant and cytotoxic agents
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biotransformation of newly synthesized coumarin derivatives by Candida albicans as potential antibacterial, antioxidant and cytotoxic agents ⁎
Ambreena,1, Shafiul Haqueb,1, Vineeta Singha, , Diksha Katiyarc, Mohd Tariq Ali Khand, ⁎ Vikash Tripathie, Hesham El Enshasyf,g, Mukesh Pasupuletie, Bhartendu Nath Mishraa, a
Department of Biotechnology, Institute of Engineering and Technology, Dr. A.P.J. Abdul Kalam Technical University, Lucknow, 226021, Uttar Pradesh, India Research & Scientific Studies Unit, College of Nursing & Allied Health Sciences, Jazan University, Jazan, 45142, Saudi Arabia c Department of Chemistry MMV, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India d Endocrinology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India e Microbiology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India f Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Skudai, 81310, Johor Bahru, Johor, Malaysia g City of Scientific Research and Technological Applications, New Burg Al Arab, Alexandria, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacteria Biotransformation Candida albicans Mass spectrometry Coumarin
In this study, one bioactive coumarin analog was obtained as a result of biotransformation of three inactive coumarin derivatives by free cells of Candida albicans. The bioactive analog was purified by Column chromatography and HPLC. The presence of coumarin moiety in the biotrasformed product was confirmed by λmax at 350–400 nm and FT-IR spectrum. The structure of the purified compound established by LC–MS and 1H NMR suggests the chances of biotransformation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2Hchromen-2-one (MW 289 Da) into 7-(3-Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethyl-chromen-2-one as a main product (MW 318 Da). The extra peak of 332 Da in LC–MS further confirms the presence of small proportion of 7-(3Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2-one apart from the main product. Oxidation followed by methylation reaction might be responsible for this conversion. The biotransformed product showed antimicrobial activity against Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Salmonella typhi followed by decent antioxidant activity (6.756 μg IC50). The efficacy of coumarin-analog on cellular proliferation was found at 40 μM concentration against human breast cancer MDA-MB-231 cells in MTT assay, which is insignificant against normal breast tissue MCF-10A cells at the same concentration. These findings suggest the potential use of C. albicans for achieving pharmacologically active coumarin analogs showing antibacterial, antioxidant and cytotoxic activity.
1. Introduction Biotransformation is an emerging tool of green chemistry and has increasing recognition in the chemical and pharmaceutical sector for the conversion of existing ineffective or less effective compounds into new or useful products [1,2]. The term biotransformation can be defined as a specific modification of a defined compound to a defined product with structural similarity by using biological catalysts [3]. A biological catalyst can be an enzyme, free cells or system. Microorganisms are capable of producing various enzymes in a short period of time because of their natural character to multiply and they can also survive under extreme environments of temperature and pH conditions.
Therefore, microbial transformation can make reactions feasible, which are not likely to be carried out by traditional synthetic procedures. Microbial modifications of chemical compounds can be done by oxidation, reduction, hydrolysis, condensation, isomerization reactions, formation of the new C]C double bonds etc.[4]. Enzymes produced during microbial metabolic activities are generally involved in the bioconversion of organic compounds. As these biocatalysts are highly specific for their activity, hence regio-specific or enantiomer-specific bioconversion leads to the formation of a specific product. Bioconversion process facilitated by microbial cells and enzymes has proven to be superior compared to chemical synthesis and other conventional methodologies as it can incorporate stereospecificity
⁎
Corresponding authors. E-mail addresses: [email protected] (V. Singh), [email protected] (B.N. Mishra). 1 Ambreen and Shafiul Haque contributed equally to this work. https://doi.org/10.1016/j.procbio.2019.08.024 Received 19 February 2019; Received in revised form 24 July 2019; Accepted 30 August 2019 1359-5113/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Ambreen, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.08.024
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or reduce the complexity of the purification steps [5]. Besides this, the superiority of microbial transformation over other accounts is basically due to greater surface-volume ratio, high growth rate, sterility maintenance, which is comparatively easier than plant or animal cell culture and above all they possess a high rate of metabolism for efficient transformation of substrate added [6]. In the present work, coumarins have been selected as a parent molecule, which are non-toxic and non-inhibitory in nature against the biotransforming organism. Coumarins are chemically known as benzopyran-2-one or chromen-2-one or simply benzo-α-pyrones ring system. Coumarins can be synthesized via.chemical route or are widely available in Umbelliferae, Rutaceae and other plant families [7]. These coumarins have been reported to possess anti-inflammatory, anti-allergic, antioxidant, antiviral, hepatoprotective, antithrombotic, and anti-carcinogenic activities. There are reports of profound use of coumarins in photochemotherapy, anti-HIV therapy and stimulants of CNS, and apart from this they are also active as anti-coagulant, estrogenic, vasodilator, molluscacidal, anti-helminthic, sedative, analgesic, dermal photosensitizing, antimicrobials and possess hypnotic and hypothermic activity [1,7]. In the present study, the practicability of biotransformation of newly synthesized inactive coumarin derivatives into biologically active coumarin-analogs via.free cells of C. albicans was evaluated followed by purification, characterization and prospection of the transformed product for antibacterial, antioxidant and cytotoxic activity.
synthesized coumarin derivatives were: C1: 7-(3-(cyclopropylamino)-2hydroxypropoxy)-2H-chromen-2-one; C2: 7-(3-(butylamino)-2-hydroxypropoxy)-2H-chromen-2-one; C3: 7-(3-(cyclohexylamino)-2-hydroxypropoxy)-2H-chromen-2-one; C4: 7-(2-hydroxy-3-morpholinopropoxy)-2H-chromen-2-one; C5: 7-(3-(cyclopropylamino)-2hydroxypropoxy)-4-methyl-2H-chromen-2-one; C6: 4-(2-hydroxy-3morpholinopropoxy)-2H-chromen-2-one.
2. Materials and methods
2.3. Biotransformation by free cells method
2.1. Synthesis of coumarin derivatives
The microbial culture of C. albicans was prepared in potato dextrose broth (PDB) after 48 h of growth and used for biotransformation studies. Biotransformation was carried out by the free cells of C. albicans with the starting reaction mixture of 1 mg coumarin compounds, 1 ml of free fungal cells (5 × 105 CFU/ml) and 10 ml of trisodium citrate buffer. The reaction mixture was incubated for at 32 °C for 48 h at 120 rpm. After 48 h, the reaction mixture was withdrawn and centrifuged at 8000 rpm at 4 °C for 10 min to obtain the supernatant. The aqueous supernatant was extracted with ethyl acetate and the organic solvent was concentrated to dryness under vacuum condition in a rotary evaporator (Buchi labs, Switzerland) to obtained the biotransformed product. The activity of the biotransformed product against the test strains was checked by the disc diffusion method. Bioconversion was further evaluated by thin layer chromatography (TLC) using hexane: methanol (8:2, v/v) as a solvent system and developed it in iodine chamber. Rf value of the substrate and the product was calculated as the ratio of distance traveled by the compound to the distance traveled by the solvent.
2.2. Activity profiling of coumarins The antibacterial activity of coumarins was examined against Grampositive Bacillus pumilus MTCC 1604, Bacillus subtilis MTCC 441, and Staphylococcus aureus MTCC 537 and Gram-negative Escherichia coli MTCC 1304, Klebsiella pneumonia MTCC 3384, and Salmonella typhi MTCC 537 bacteria. The bacterial cultures were maintained on nutrient agar (NA) at 4 °C. The working cultures were prepared by inoculating the sterile NB medium with the overnight grown test strains until the solution turbidity reached 0.5 McFarland standards. The stock solutions of the synthesized coumarin derivatives were prepared in methanol. The activity profile of 200 μg of coumarin compounds against bacterial and fungal strains was determined by agar plate method [10]. The zone of inhibition (zoi; measured in mm) was recorded after 24 h for all the bacterial strains. Drugs, like novobiocin and vancomycin were used as antibacterial drug standards, whereas, amphotericin B was used as an antifungal drug standard.
The chemicals used in this work were of analytical grade and purity. Coumarinyl amino alcohols C1–C6 were synthesized following the method reported earlier [8,9]. The reaction of 7-hydroxycoumarin with epichlorhydrin in the presence of K2CO3 led to the formation of oxirane derivative, which on regioselective nucleophilic ring opening with cyclopropyl amine, butyl amine, cyclohexyl amine and morpholine in the presence of ethanol at room temperature afforded coumarinyl amino alcohols 1–4, respectively, with good yield (Fig. 1). The coumarin compound 5 was synthesized via.nucleophilic opening of oxirane ring by cyclopropyl amine. Oxirane was synthesized by the reaction of 7hydroxy-4-methyl-coumarin with epichlorohydrin. Whereas, the synthesis of oxirane intermediate was performed by the reaction between 4-hydroxy coumarin and epichlorohydrin followed by its nucleophilic ring opening by morpholine and yielded coumarin compound C6 in good amount. The spectral data of all the synthesized compounds were in good agreement with the previously reported data. The
Fig. 1. Chemical synthesis scheme used for coumarinyl amino alcohols showing the reaction conditions required for the formation of the reaction intermediates. 2
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The biotransformed product was purified by column chromatography using silica gel (mesh size 230–400) column (1.8 × 70 cm) as a packing material. The column was eluted under an isocratic condition with ethyl acetate and 5 ml fractions were collected. The collected fractions were dried and checked for antimicrobial activity against Bacillus pumilus MTCC1607, Staphylococcus aureus MTCC 902, Bacillus subtilis MTC441, Escherichia coli MTCC 1304, Salmonella typhi MTCC 537 and Klebsiella pneumonia MTCC3384 by the disc diffusion method. The purity of the active fractions was tested by using HPLC system (Waters) equipped with 515 HPLC binary pump, 2998 Photodiode array (PDA) detector, and 2707 autosampler. HPLC separation was achieved on a reverse phase, reverse phase silica column (RP18) with a mobile phase consisting of the water-acetonitrile gradient at a flow rate of 1 ml/min. Further, the detailed structure of the pure product was established by UV–vis spectroscopy, LC–MS, IR, and NMR.
cells (200 μL) were seeded in a 96-well plate and different concentrations (1, 2, 5, 10, 20, 40 and 80 μM) of coumarin derivatives were added in triplicate. DMSO (0.1%) and centchroman (10 μM) were used as negative and positive control, respectively. All the group cells were treated with 0.2% DMSO in triplicate and incubated for 24 and 48 h. Following the incubation, the experiment was terminated by adding 20 μL of MTT (stock: 5 mg/ml of MTT in PBS, Life technologies catalog M6469) in each of the treatment as well as the control well. Further, the plate (96-well) was incubated in 5% CO2 incubator at 37 °C for 2 h. Afterwards the plate was observed under the microscope to observe the formation of formazan crystals. The medium from the wells was discarded and 200 μL of DMSO was added in each well. The 96-well plate was kept on shaker for 10 min for dissolving the crystals in DMSO. The absorbance was recorded at 595 nm for each well of the plate using iMARK microplate reader (Bio-rad, Hercules, CA, USA). The data obtained were expressed as percent proliferation compared to the control and graphs were plotted.
2.5. Biological activity of transformed product
2.7. Statistical analysis
The antimicrobial activity of the biotransformed coumarins expressed in terms of minimum inhibitory concentration (MIC) values was determined by micro-broth dilution method as described by Priyanka et al. [9]. Stock solutions were prepared as 1 mg/ml concentration of the biotransformed coumarin derivatives and standard antibiotics, i.e., Novobiocin and Vancomycin. The antioxidant activity of the biotransformed compound was analyzed using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging assay with minor modifications [11]. Different concentrations of the coumarin compounds in ethanol (222.5–2225 μg in 100 μl) were taken in test tubes and 2.9 ml of DPPH solution (2.5 mg/100 ml of ethanol) was added. The reaction mixture was vortexed and incubated for 30 min in dark and absorbance was measured at 520 nm by using spectrophotometer (UV–vis Shimadzu). The reaction mix without compound was considered as a control standard. Ascorbic acid was used as a reference compound and the experiment was performed in triplicate. The percentage scavenging effect of DPPH of the tested sample was calculated using the following formula:
Student t-test was used to calculate the significance of the data obtained from MTT assay. In t-test, the control was compared with the treated values and also among the pairs. All the significant analysis was done for triplicate values. Where * shows significant (p < 0.05), ** very significant (p < 0.001) and *** highly significant (p < 0.0001).
2.4. Purification and characterization
3. Results 3.1. Antimicrobial activity of the substrate/product Six coumarin derivatives were evaluated for their antimicrobial activity against the bacterial test strains by measuring the zone of inhibition with a concentration of 200 μg. The results of antibacterial testing indicate that the synthesized coumarin compounds possessed little antimicrobial potential, with the highest zoi of 12 mm only in the case of compound C1 against S. typhi (data not shown). From antimicrobial activity profile of the synthesized coumarin series, the most inactive derivatives C4–C6 were selected for the biotransformation purpose. Biotransformation studies were performed for the above mentioned three compounds using free cells of C. albicans, however, only compound number C5 was found active. The biotransformed product was tested against the same group of bacteria and the results of MIC values expressed in μg/ml of the biotransformed compound C5-P (7-(3-(cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2one) and the standard drugs are summarized in Table 1. The results indicated that the biotransformed coumarin derivative is capable of inhibiting both Gram-positive and Gram-negative bacteria.
Percentage scavenging effect of DPPH or % inhibition = (A0 ̶ A1/ A0) × 100 Where, A0 is the absorbance of control reaction and A1 is the absorbance of the test of the standard sample. 2.6. Cytotoxicity assay MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay was performed against human breast carcinoma cell lines (MDA-MB-231) following the protocol as reported by Srisawat et al., with minor modifications [12]. MDA-MB-231 cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and incubated in a CO2 incubator for 24 h at 37 °C. When the cells reached a confluency of 40–50%, the complete media was replaced with fresh medium. Around 5 × 103 MDA-MB-231
3.2. Antioxidant activity The results of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging experiment suggest that the biotransformed product possess good antioxidant properties (Table 2). The experimental regression value 0.98 further emphasized the modest antioxidant activity
Table 1 Antimicrobial activity profile in terms of MIC values (μg/ml) of coumarin derivatives. Test strains (MTCC)
C1
C2
C3
C4
C5
C6
C5-P
N
V
Bacillus subtilis MTCC 441 Bacillus pumilus MTCC 1604 Staphylococcus aureus MTCC 537 Salmonella typhi MTCC 537 Klebsiella pneumonia MTCC 3384 Escherichia coli MTCC 1304
200 > 200 200 100 > 200 100
> 200 > 200 200 100 > 200 200
> 200 > 200 > 200 200 200 200
> 200 > 200 > 200 200 > 200 > 200
> 200 > 200 > 200 > 200 200 > 200
> 200 100 > 200 > 200 > 200 > 200
100 25 50 100 100 50
6.25 6.25 6.25 25 25 25
1.56 1.56 1.56 0.35 0.35 R
C1-C6: Coumarin derivatives; C5-P = biotransformed product; N = Novobiocin; V = Vancomycin; R = Resistant. 3
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Table 2 Percentage DPPH scavenging effect against varying concentrations of the biotransformed coumarin derivative. S. No.
Concentration of biotransformed coumarin (μg)
DPPH scavenging effect (% inhibition)
Ascorbic acid DPPH scavenging effect (%)
1 2 3 4 5
250 500 750 1000 1250
27.5 43.5 63.5 70..5 89.5
30 51 72 78 91
of the biotransformed coumarin derivative. Effective concentration EC50 value demonstrated the concentration of coumarin-derivative required to scavenge 50% of DPPH free radical. In the performed experiment, the EC50 value was 6756.76 μg, means 6.7 mg of the biotransformed product of coumarin derivative is effective in showing antioxidant activity.
3.3. Cytotoxicity Fig. 3. Effect of biotransformed coumarin derivative on cellular proliferation against normal human breast MCF10A cells at different time points (a) 24 h and at (b) 48 h. Note: ns: non-significant.
The efficacy of the biotransformed coumarin derivative was studied in a dose-dependent (1, 2, 5, 10, 20, 40 and 80 μM) and time-dependent manner (24 and 48 h) against cellular proliferation of human breast MDA-MB-231 cancer cells using MTT assay (Fig. 2). The inhibition of cellular proliferation by the purified biotransformed compound was not very significant at the lower concentration, i.e.,up to 10 μM. However, from 20 μM onwards it was found significant, when compared with the control under t-test analysis. With the increase in the compound concentration, the inhibition of cellular proliferation increased and at 80 μM concentration showed maximum inhibition (p < 0.0001). IC50 value, which depicts 50% inhibition of the cell proliferation, was found to be at 54 and 40.08 μM of the biotransformed coumarin derivative at 24 and 48 h time points, respectively. The substrate coumarin at a concentration of 80 μM was able to inhibit only 35 and 40% cellular proliferation at 24 and 48 h, respectively. Centchroman (CC) was used as a positive control and the IC50 value of CC was 15 and 10 μM at 24 and 48 h, respectively in MDA-MB-231 cells. The results of 24 and 48 h treatment suggest that with the increase in time interaction, the effectiveness of the compound increases. The above experiment was compared by studying the efficacy of the biotransformed coumarin derivative on cellular proliferation against human normal breast MCF10A cells using MTT assay for the cytotoxicity under the same experimental conditions of dose and time dependency. The compound had no or insignificant influence (p > 0.05) over cellular proliferation from 10 to 40 μM as they increased cellular proliferation by 1–9% in 24 h and 9–14% in 48 h. The maximum
experimental dose, i.e.,80 μM was found to show 18% cellular inhibition. At the concentration of 40 μM, the biotransformed product inhibited cellular proliferation by 9 and 14% at 24 and 48 h incubation, respectively; and 18% inhibition at 80 μM concentration; out of which, only 48 h incubation was found significant. Whereas, the original product (i.e., substrate) inhibited 6 and 2% cells at 80 μM concentration. It is clear from MTT cytotoxicity assay that biotransformed product has no cytotoxic effect on human normal MCF10A cells between 10–40 μM concentration at 24 h and 48 h, respectively, and can be further explored as anti-cancer compound (Fig. 3). 3.4. Chemical characterization of the substrate/biotransformed product The absorption bands observed at 1716 and 3422 cm1 in IR spectrum indicated the presence of δ lactone and hydroxyl functions, respectively. The ESI MS spectrum of the coumarin compound showed a peak at m/z 290 corresponding to its [M+H]+. In IH NMR spectrum signals corresponding to H-5 of benzopyrone ring appeared as doublet at δ 7.25 with J value of 8.8 Hz, while H-6 and H-8 were observed as multiplet at δ 6.81. H-3 was observed as singlet at δ 6.12. However, signals for propan-2-ol linker appeared as multiplets at δ 4.10 (C(1′)H2,
Fig. 2. Effect of biotransformed coumarin derivative on cellular proliferation against human breast cancer MDA-MB-231 cells at different time points (a) 24 h and at (b) 48 h. Note: ns: non-significant; * less than control; ** equal to control; *** greater than control, i.e., centchroman. 4
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of benzene moiety in the structure; the signals at 0.30 marks the presence of cyclopropane ring; at 1.93 (t, 3H) shows CH region, 7.58 (d, 4H) denotes CH2 bonding; whereas at 2.7 multiple peaks are observed confirming the presence of eNH and eCH; signals at 3.9 confirms the presence of eOH and eCH bonding; signal at 4.04 confirms CH2 bonding; at 3.24 it is CH3 bonding and OH shares peak at 2.0.J coupling region and chemical shift (δ) indicates that either modifying methyl group or hydroxyl group in the original structure might results in the final product. Therefore, oxidation at CH3 followed by methylation or oxidation at CH3 followed by methylation at eOH lead to the molecular weight of 318 Da, whereas oxidation of CH3 followed by methylation of CH3 and eOH both results in a molecular weight of 332 Da (Fig. 6).
NH, C(2′)H) and 3.12 (C(3′)H2). The methylene protons of cyclopropyl ring appeared as multiplets at δ 1.26 and 0.83. However, methine proton of cyclopropyl ring was observed as multiplet at δ 2.42 merged with signal for methyl protons of the 4-methyl substitutent. The 13C NMR spectrum of 5 exhibited signals at δ 102.8, 127.7, 111.7 and 107.6 corresponding to C-3, C-5, C-6 and C-8 of benzopyrone ring. The signals for quaternary carbons C-2, C-4, C-7, C-9 and C-10 were observed at δ 161.8, 153.8, 157.6, 151.2 and 116.8, respectively, while signals corresponding to carbons of propan-2-ol linker appeared at δ 72.3, 62.8 and 49.8 for C-1′, C-2′, and C-3′, respectively. The methyl carbon of 4methyl substituent was observed at δ 18.7. The signal at δ 35.2 was attributed to methine and at δ 9.3 and 7.8 were attributed to methylene carbons of cyclopropyl ring. The efficiency of biotransformation reaction was found to be 64%, i.e., biotransformation of 100 mg of coumarin-derivative resulted 64 mg of the pure product. The product was oily in nature and showed solubility in DMSO. The process of bioconversion held was confirmed by thin layer chromatography showing spots with different Rf value than the substrate. The complete structure identification of the biotransformed compound was done on the basis of spectral studies. The absorbance in the UV region for the substrate and product was 350 nm and 400 nm, respectively (Fig. 4a) falling in the range (between 300–400 nm) of coumarins [8]. IR spectra: peaks at 1072, 1283 in the product and 1071, 1286 in the crude corresponds to aryl-alkyl-ether (AryleOeR). The peaks, 3425 and 3013 in the product belongs to eOH, eNH bonding, respectively. These similarities suggest that the side chain present in the product is intact as same as in the crude; a peak at 2925 in the product corresponds to eCH stretching region; The absorption bands observed at 1716 and 3422 cm−1 in the substrate and product indicate the presence of δ lactone and hydroxyl functions, respectively, which further confirm the intact lactam ring. As, benzene is a stable structure in comparison to lactam ring, hence the chances of group modification at the side chain or at pyrone ring are speculated (Fig. 4b). In ESI-MS the major compounds corresponding to the retention time 8.41, 9.64 and 9.99 possess molecular weight 290.3, 318.3 and 332 Da, respectively (Fig. 5). Mass distribution from LC–MS predicts the product mass to be 318 or 332 at a retention time of 9.64 and 9.99 respectively (Fig. 5). ESI-MS also establishes the distinct molecular weight of 318; it is certain that the main product corresponds to the molecular weight 318 with the smaller fraction being converted to the molecular weight 332. While, FT-IR suggests that the major functional groups and ring remained intact and provides maximum structural similarity in the substrate and product; it is likely that there is a modification of either eOH, eCH3, eNH group (Fig. 4b). The presence of signals between J = 7.7–7.0 confirm the presence
4. Discussion The ability of biocatalysts to selectively produce useful products under relatively mild conditions compared to its chemical catalyst counterpart make biocatalysts an interesting and powerful tool. Biocatalyst can be a free enzyme or a whole cell catalyzing a chemical reaction and producing highly enantioselective, low toxic product [4]. Recent technology advancements have increased the focus of industry to discover new biocatalysts and optimize their performance. In order to create an eco-friendly system, certain studies on the modification of benzopyrone ring system employing different fungi and bacteria, as functional group transferring agents, have been undertaken [1]. Coumarins are common example of benzopyrone ring system, and popularly named as benzopyran-2-one or chromen-2-one or simply benzo-αpyrones ring system. Coumarins are one of the most common phytochemicals present in various natural products. Due to its diverse bioactivity [13] it is also synthesized chemically [14]. In this direction, through chemical synthesis a series of coumarins derivatives (coumarinyl amino alcohols) have been synthesized; unfortunately, the compounds of this series are not very effective in bioactivity, therefore, biotransformation method has been used to make an effort to convert the inactive derivative into an active one. The modifications at the chemical level of inactive coumarins generally lead the conversion into active structure which is mainly useful for pharmaceutical industries and opens the door for novel drug development. In this direction, microorganisms play an important role for structural modifications [15]. Biotransformation of coumarin-derivatives have been reported in various literatures using a variety of microorganisms, like Streptomyces griseus [16], Candida tropicalis [1], Arthrobacter species, Aspergillus niger [17], Fusarium solani [18] and Cunninghamella elegans [19]. The structure of any molecule plays an important role in order to reflect its biological activity. Kayser and Kolodziej [20] reported that the addition of eOH moiety to coumarin core structure significantly
Fig. 4. (a) UV absorption pattern of the biotransformed product C5-P: showing absorbance near 400 nm; (b) FTIR of the biotransformed product C5-P: showing the presence of basic functional groups of coumarin derivatives. 5
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Fig. 5. Mass spectra of the substrate/product reflecting variations in the mass of the substrate and the product endorsing biotransformation process.
Fig. 6. 1H NMR of the biotransformed product C5-P confirms the presence of benzopyrone and cyclopropyl ring.
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Acknowledgements
reduced the antibacterial activity against all the tested bacteria. Their findings suggest that the antibacterial activity of oxygenated coumarins apparently depends on the position of polar (eOH) and less polar (Me/ OMe) functional group at the aromatic nucleus of the coumarin structure. This assumption match with the present study proving the last three compounds (C4–C6) from the series were most inactive against a group of tested bacteria. The addition of methyl group further depicted fairly low activity of the compound C5 against all the tested strains. The active compounds favor its permeation more efficiently through the lipid layer of the fungi [21]. Nigam et al. [1] reported the application of Saccharomyces griseus and Candida tropicalis for microbial biotransformation purposes for the modification of coumarin substrates. They reported the biotransformations of coumarins derivatives 7-ethoxy-4-methyl coumarin into 7-dihydroxy-4-methyl coumarin by Saccharomyces griseus and 7hydroxy-4-methyl coumarin into 7-methoxy-4-methyl coumarin by Candida tropicalis. The report of Nigam et al. [1] provides the prime reason to examine the ability of Candida albicans for bioconversion process. The results of bioconversion of inactive coumarin derivative (C5) into active product suggest the efficiency of C. albicans towards biotransformation. The results regarding the structure elucidation of the product suggest the most possible reaction of the conversion is the oxidation at CH3 moiety present on chromone ring followed by methylation at specific positions, which ultimately offer main and side products. Methylation and oxidation are the common chemical reactions involve in the mechanism of bioconversion using microbes. Beside antimicrobial activity, the modified coumarin possessed ROS activity and toxic activity against the breast cancer cell line MDA-MB231. Previously, the toxic activity has been diligently mentioned in the work of Musa et al. [7] regarding coumarin and its active metabolite 7hydroxy coumarins particularly against breast cancer, which is a leading cause of death. Musa et al. [7], have proved that bioactive analog of coumarin works as a potent selective estrogen receptor modulators (SERMs) as well as strong chemotherapeutic agent in breast cancer. Few naturally occurring coumarin derivatives including umbelliferone, warfarin, aesculetin (6,7 dihydroxycoumarin), 7-methoxy coumarin, psoralen, and imperatorin have shown their potential in the past. In addition, coumarins work as lipid-lowering agents and hydroxycoumarins are powerful chain-breaking antioxidants [22], and can prevent free radical injury by scavenging reactive oxygen [7].
The authors are grateful to IET, Lucknow for providing the laboratory facility and partial financial support under World Bank assisted TEQIP-Government of India program-phase II for this research work. The author, Shafiul Haque, is grateful to Jazan University, Saudi Arabia for providing the access to the Saudi Digital library. The author, Hesham El Enshasy would like to acknowledge the support of MOE and UTM-RMC through HICOE grant no. R.J130000.7846.4J262. References [1] S. Nigam, J.V. Rao, B.S. Jayashree, Microbial biotransformation—a novel approach for modification on coumarin substrates, Ind. J. Biotechnol. 12 (2013) 379–385. [2] M.E. Hegazy, T.A. Mohamed, A.I. ElShamy, H.M. Abou-El-Hamd, U.A. Mahalel, E.H. Reda, A.M. Shaheen, W.A. Tawfik, A.A. Shahat, K.A. Shams, N.S. Abdel-Azim, Microbial biotransformation as a tool for drug development based on natural products from mevalonic acid pathway: a review, J. Adv. Res. 6 (2015) 17–33. [3] M.D. Lilly, Eighth P. V. Danckwerts memorial lecture presented at Glaziers’ Hall, London, U.K. 13 May 1993: advances in biotransformation processes, Chem. Engg. Sci. 49 (1994) 151–159. [4] F. Garzón-Posse, L. Becerra-Figueroa, J. Hernández-Arias, D. Gamba-Sánchez, Whole cells as biocatalysts in organic transformations, Mol. 23 (2018) 1265. [5] E.M. Sales, T.F. Barros, E.D.S. Velozo, Biotransformation of coumarins by Saccharomyces cerevisiae, World J. Pharm. Pharm. Sci. 3 (2014) 209–216. [6] M.S. Smitha, S. Singh, R. Singh, Microbial Biotransformation: a process for chemical alterations, J. Bacteriol. Mycol. 4 (2017) 85. [7] M.A. Musa, J.S. Cooperwood, M.O.F. Khan, A review of coumarin derivatives in pharmacotherapy of breast cancer, Curr. Med. Chem. 15 (2008) 2664–2679. [8] S. Misra, L.K. Singh, Priyanka, J. Gupta, S. Misra-Bhattacharya, D. Katiyar, Synthesis and biological evaluation of 4-oxycoumarin derivatives as a new class of antifilarial agents, Eur. J. Med. Chem. 94 (2015) 211–217. [9] Priyanka, V. Singh, Ekta, D. Katiyar, Synthesis, antimicrobial, cytotoxic and E. coli DNA gyrase inhibitory activities of coumarinyl amino alcohols, Bioorg. Chem. 71 (2017) 120–127. [10] S.I.G.H.M. Montalvão, V. Singh, S. Haque, Bioassays for bioactivity screening. Analysis of Marine samples in search of bioactive compounds, in: T. Rocha-Santos, A. Duarte (Eds.), Comprehensive Analytical Chemistry Series, Elsevier B.V., 2014ISBN9780444633590. [11] Shekhar, C. Tailor, A. Goyal, Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. leaves, Am. J. Ethnomed. 1 (2014) 244–249. [12] T. Srisawat, Y. Sukpondma, P. Graidist, S. Chimplee, K. Kanokwiroon, The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of vaticadiospyroides symington type SS fruit include effects on mode of cell death, Pharmacogn. Mag. 11 (Suppl. 1) (2015) S148–S155. [13] S. Tavakoli, M.R. Delnavazi, R. Hadjiaghaee, S. Jafari-Nodooshan, F. KhalighiSigaroodi, M. Akhbari, A. Hadjiakhoondi, N. Yassa, Bioactive coumarins from the roots and fruits of Ferulagotrifida Boiss. an endemic species to Iran, Nat. Prod. Res. 32 (2018) 2724–2728. [14] Priyanka, R.K. Sharma, R.J. Butcher, D. Katiyar, Facile construction of 4H-chromenes via Michael addition of phenols to benzylideneoxobutanoates and their successful conversion into pyranocoumarins, Tetrahedron Lett. 59 (2018) 2347–2351. [15] J.S. doNascimento, J.C. Conceição, E. de Oliveira Silva, Biotransformation of coumarins by filamentous fungi: an alternative way for achievement of bioactive analogs, Mini-Rev. Org. Chem. 16 (2019) 1–10. [16] F.S. Sariaslani, J.P. Rosazza, Novel biotransformations of 7-ethoxycoumarin by Streptomyces griseus, Appl. Environ. Microbiol. 46 (1983) 468–474. [17] C.B. Aguirre-Pranzoni, G.I. Furque, C.E. Ardanaz, A. Pacciaroni, V. Sosa, C.E. Tonn, M. Kurina-Sanz, ARKIVOC: Online J. Org. Chem. (2011) 170–181 (vii). [18] H.S. Shieh, A.C. Blackwood, Use of coumarin by soil fungi, Can. J. Microbiol. 15 (1969) 647–648. [19] G.A. Attia, K.A. Abou-El-Seoud, A.R. Ibrahim, Biotransformation of coumarins by cunninghamellaelegans, Afr. J. Pharm. Pharmacol. 10 (2016) 411–418. [20] O. Kayser, H. Kolodziej, Antibacterial activity of simple coumarins: structural requirements for biological activity, Z. Naturforsch. C 54 (1999) 169–174. [21] S.U. Rehman, Z.H. Chohan, F. Gulnaz, C.T. Supuran, In-vitro antibacterial, antifungal and cytotoxic activities of some coumarins and their metal complexes, J. Enzyme Inhib. Med. Chem. 20 (2005) 333–340. [22] A.A. Kadhum, A.A. Al-Amiery, A.Y. Musa, A.B. Mohamad, The antioxidant activity of new coumarin derivatives, Int. J. Mol. Sci. 12 (2011) 5747–5761.
5. Conclusion In the present study, biotransformation was done by oxidation followed by methylation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)4-methyl-2H-chromen-2-one by Candida albicans to 7-(3Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethylchromen-2-one as main product with molecular weight 318 Da. In addition to the main peak, a small peak of molecular weight 332 Da was also observed in the LC–MS region that indicates the presence of 7-(3Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2one in addition to the main product; and this was confirmed by the spectral analysis. Besides this, attempts were made to evaluate the success of bioconversion of the substrate into the final product. The biotransformed product showed significant results for its biological activity assessed in terms of decent antibacterial, antioxidant cytotoxicity properties, and reflects its therapeutic use in the near future after several in vivo trials. Declaration of Competing Interest The authors declare no conflicts of interest.
7
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biotransformation of newly synthesized coumarin derivatives by Candida albicans as potential antibacterial, antioxidant and cytotoxic agents ⁎
Ambreena,1, Shafiul Haqueb,1, Vineeta Singha, , Diksha Katiyarc, Mohd Tariq Ali Khand, ⁎ Vikash Tripathie, Hesham El Enshasyf,g, Mukesh Pasupuletie, Bhartendu Nath Mishraa, a
Department of Biotechnology, Institute of Engineering and Technology, Dr. A.P.J. Abdul Kalam Technical University, Lucknow, 226021, Uttar Pradesh, India Research & Scientific Studies Unit, College of Nursing & Allied Health Sciences, Jazan University, Jazan, 45142, Saudi Arabia c Department of Chemistry MMV, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India d Endocrinology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India e Microbiology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India f Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Skudai, 81310, Johor Bahru, Johor, Malaysia g City of Scientific Research and Technological Applications, New Burg Al Arab, Alexandria, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacteria Biotransformation Candida albicans Mass spectrometry Coumarin
In this study, one bioactive coumarin analog was obtained as a result of biotransformation of three inactive coumarin derivatives by free cells of Candida albicans. The bioactive analog was purified by Column chromatography and HPLC. The presence of coumarin moiety in the biotrasformed product was confirmed by λmax at 350–400 nm and FT-IR spectrum. The structure of the purified compound established by LC–MS and 1H NMR suggests the chances of biotransformation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2Hchromen-2-one (MW 289 Da) into 7-(3-Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethyl-chromen-2-one as a main product (MW 318 Da). The extra peak of 332 Da in LC–MS further confirms the presence of small proportion of 7-(3Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2-one apart from the main product. Oxidation followed by methylation reaction might be responsible for this conversion. The biotransformed product showed antimicrobial activity against Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Salmonella typhi followed by decent antioxidant activity (6.756 μg IC50). The efficacy of coumarin-analog on cellular proliferation was found at 40 μM concentration against human breast cancer MDA-MB-231 cells in MTT assay, which is insignificant against normal breast tissue MCF-10A cells at the same concentration. These findings suggest the potential use of C. albicans for achieving pharmacologically active coumarin analogs showing antibacterial, antioxidant and cytotoxic activity.
1. Introduction Biotransformation is an emerging tool of green chemistry and has increasing recognition in the chemical and pharmaceutical sector for the conversion of existing ineffective or less effective compounds into new or useful products [1,2]. The term biotransformation can be defined as a specific modification of a defined compound to a defined product with structural similarity by using biological catalysts [3]. A biological catalyst can be an enzyme, free cells or system. Microorganisms are capable of producing various enzymes in a short period of time because of their natural character to multiply and they can also survive under extreme environments of temperature and pH conditions.
Therefore, microbial transformation can make reactions feasible, which are not likely to be carried out by traditional synthetic procedures. Microbial modifications of chemical compounds can be done by oxidation, reduction, hydrolysis, condensation, isomerization reactions, formation of the new C]C double bonds etc.[4]. Enzymes produced during microbial metabolic activities are generally involved in the bioconversion of organic compounds. As these biocatalysts are highly specific for their activity, hence regio-specific or enantiomer-specific bioconversion leads to the formation of a specific product. Bioconversion process facilitated by microbial cells and enzymes has proven to be superior compared to chemical synthesis and other conventional methodologies as it can incorporate stereospecificity
⁎
Corresponding authors. E-mail addresses: [email protected] (V. Singh), [email protected] (B.N. Mishra). 1 Ambreen and Shafiul Haque contributed equally to this work. https://doi.org/10.1016/j.procbio.2019.08.024 Received 19 February 2019; Received in revised form 24 July 2019; Accepted 30 August 2019 1359-5113/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Ambreen, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.08.024
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or reduce the complexity of the purification steps [5]. Besides this, the superiority of microbial transformation over other accounts is basically due to greater surface-volume ratio, high growth rate, sterility maintenance, which is comparatively easier than plant or animal cell culture and above all they possess a high rate of metabolism for efficient transformation of substrate added [6]. In the present work, coumarins have been selected as a parent molecule, which are non-toxic and non-inhibitory in nature against the biotransforming organism. Coumarins are chemically known as benzopyran-2-one or chromen-2-one or simply benzo-α-pyrones ring system. Coumarins can be synthesized via.chemical route or are widely available in Umbelliferae, Rutaceae and other plant families [7]. These coumarins have been reported to possess anti-inflammatory, anti-allergic, antioxidant, antiviral, hepatoprotective, antithrombotic, and anti-carcinogenic activities. There are reports of profound use of coumarins in photochemotherapy, anti-HIV therapy and stimulants of CNS, and apart from this they are also active as anti-coagulant, estrogenic, vasodilator, molluscacidal, anti-helminthic, sedative, analgesic, dermal photosensitizing, antimicrobials and possess hypnotic and hypothermic activity [1,7]. In the present study, the practicability of biotransformation of newly synthesized inactive coumarin derivatives into biologically active coumarin-analogs via.free cells of C. albicans was evaluated followed by purification, characterization and prospection of the transformed product for antibacterial, antioxidant and cytotoxic activity.
synthesized coumarin derivatives were: C1: 7-(3-(cyclopropylamino)-2hydroxypropoxy)-2H-chromen-2-one; C2: 7-(3-(butylamino)-2-hydroxypropoxy)-2H-chromen-2-one; C3: 7-(3-(cyclohexylamino)-2-hydroxypropoxy)-2H-chromen-2-one; C4: 7-(2-hydroxy-3-morpholinopropoxy)-2H-chromen-2-one; C5: 7-(3-(cyclopropylamino)-2hydroxypropoxy)-4-methyl-2H-chromen-2-one; C6: 4-(2-hydroxy-3morpholinopropoxy)-2H-chromen-2-one.
2. Materials and methods
2.3. Biotransformation by free cells method
2.1. Synthesis of coumarin derivatives
The microbial culture of C. albicans was prepared in potato dextrose broth (PDB) after 48 h of growth and used for biotransformation studies. Biotransformation was carried out by the free cells of C. albicans with the starting reaction mixture of 1 mg coumarin compounds, 1 ml of free fungal cells (5 × 105 CFU/ml) and 10 ml of trisodium citrate buffer. The reaction mixture was incubated for at 32 °C for 48 h at 120 rpm. After 48 h, the reaction mixture was withdrawn and centrifuged at 8000 rpm at 4 °C for 10 min to obtain the supernatant. The aqueous supernatant was extracted with ethyl acetate and the organic solvent was concentrated to dryness under vacuum condition in a rotary evaporator (Buchi labs, Switzerland) to obtained the biotransformed product. The activity of the biotransformed product against the test strains was checked by the disc diffusion method. Bioconversion was further evaluated by thin layer chromatography (TLC) using hexane: methanol (8:2, v/v) as a solvent system and developed it in iodine chamber. Rf value of the substrate and the product was calculated as the ratio of distance traveled by the compound to the distance traveled by the solvent.
2.2. Activity profiling of coumarins The antibacterial activity of coumarins was examined against Grampositive Bacillus pumilus MTCC 1604, Bacillus subtilis MTCC 441, and Staphylococcus aureus MTCC 537 and Gram-negative Escherichia coli MTCC 1304, Klebsiella pneumonia MTCC 3384, and Salmonella typhi MTCC 537 bacteria. The bacterial cultures were maintained on nutrient agar (NA) at 4 °C. The working cultures were prepared by inoculating the sterile NB medium with the overnight grown test strains until the solution turbidity reached 0.5 McFarland standards. The stock solutions of the synthesized coumarin derivatives were prepared in methanol. The activity profile of 200 μg of coumarin compounds against bacterial and fungal strains was determined by agar plate method [10]. The zone of inhibition (zoi; measured in mm) was recorded after 24 h for all the bacterial strains. Drugs, like novobiocin and vancomycin were used as antibacterial drug standards, whereas, amphotericin B was used as an antifungal drug standard.
The chemicals used in this work were of analytical grade and purity. Coumarinyl amino alcohols C1–C6 were synthesized following the method reported earlier [8,9]. The reaction of 7-hydroxycoumarin with epichlorhydrin in the presence of K2CO3 led to the formation of oxirane derivative, which on regioselective nucleophilic ring opening with cyclopropyl amine, butyl amine, cyclohexyl amine and morpholine in the presence of ethanol at room temperature afforded coumarinyl amino alcohols 1–4, respectively, with good yield (Fig. 1). The coumarin compound 5 was synthesized via.nucleophilic opening of oxirane ring by cyclopropyl amine. Oxirane was synthesized by the reaction of 7hydroxy-4-methyl-coumarin with epichlorohydrin. Whereas, the synthesis of oxirane intermediate was performed by the reaction between 4-hydroxy coumarin and epichlorohydrin followed by its nucleophilic ring opening by morpholine and yielded coumarin compound C6 in good amount. The spectral data of all the synthesized compounds were in good agreement with the previously reported data. The
Fig. 1. Chemical synthesis scheme used for coumarinyl amino alcohols showing the reaction conditions required for the formation of the reaction intermediates. 2
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The biotransformed product was purified by column chromatography using silica gel (mesh size 230–400) column (1.8 × 70 cm) as a packing material. The column was eluted under an isocratic condition with ethyl acetate and 5 ml fractions were collected. The collected fractions were dried and checked for antimicrobial activity against Bacillus pumilus MTCC1607, Staphylococcus aureus MTCC 902, Bacillus subtilis MTC441, Escherichia coli MTCC 1304, Salmonella typhi MTCC 537 and Klebsiella pneumonia MTCC3384 by the disc diffusion method. The purity of the active fractions was tested by using HPLC system (Waters) equipped with 515 HPLC binary pump, 2998 Photodiode array (PDA) detector, and 2707 autosampler. HPLC separation was achieved on a reverse phase, reverse phase silica column (RP18) with a mobile phase consisting of the water-acetonitrile gradient at a flow rate of 1 ml/min. Further, the detailed structure of the pure product was established by UV–vis spectroscopy, LC–MS, IR, and NMR.
cells (200 μL) were seeded in a 96-well plate and different concentrations (1, 2, 5, 10, 20, 40 and 80 μM) of coumarin derivatives were added in triplicate. DMSO (0.1%) and centchroman (10 μM) were used as negative and positive control, respectively. All the group cells were treated with 0.2% DMSO in triplicate and incubated for 24 and 48 h. Following the incubation, the experiment was terminated by adding 20 μL of MTT (stock: 5 mg/ml of MTT in PBS, Life technologies catalog M6469) in each of the treatment as well as the control well. Further, the plate (96-well) was incubated in 5% CO2 incubator at 37 °C for 2 h. Afterwards the plate was observed under the microscope to observe the formation of formazan crystals. The medium from the wells was discarded and 200 μL of DMSO was added in each well. The 96-well plate was kept on shaker for 10 min for dissolving the crystals in DMSO. The absorbance was recorded at 595 nm for each well of the plate using iMARK microplate reader (Bio-rad, Hercules, CA, USA). The data obtained were expressed as percent proliferation compared to the control and graphs were plotted.
2.5. Biological activity of transformed product
2.7. Statistical analysis
The antimicrobial activity of the biotransformed coumarins expressed in terms of minimum inhibitory concentration (MIC) values was determined by micro-broth dilution method as described by Priyanka et al. [9]. Stock solutions were prepared as 1 mg/ml concentration of the biotransformed coumarin derivatives and standard antibiotics, i.e., Novobiocin and Vancomycin. The antioxidant activity of the biotransformed compound was analyzed using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging assay with minor modifications [11]. Different concentrations of the coumarin compounds in ethanol (222.5–2225 μg in 100 μl) were taken in test tubes and 2.9 ml of DPPH solution (2.5 mg/100 ml of ethanol) was added. The reaction mixture was vortexed and incubated for 30 min in dark and absorbance was measured at 520 nm by using spectrophotometer (UV–vis Shimadzu). The reaction mix without compound was considered as a control standard. Ascorbic acid was used as a reference compound and the experiment was performed in triplicate. The percentage scavenging effect of DPPH of the tested sample was calculated using the following formula:
Student t-test was used to calculate the significance of the data obtained from MTT assay. In t-test, the control was compared with the treated values and also among the pairs. All the significant analysis was done for triplicate values. Where * shows significant (p < 0.05), ** very significant (p < 0.001) and *** highly significant (p < 0.0001).
2.4. Purification and characterization
3. Results 3.1. Antimicrobial activity of the substrate/product Six coumarin derivatives were evaluated for their antimicrobial activity against the bacterial test strains by measuring the zone of inhibition with a concentration of 200 μg. The results of antibacterial testing indicate that the synthesized coumarin compounds possessed little antimicrobial potential, with the highest zoi of 12 mm only in the case of compound C1 against S. typhi (data not shown). From antimicrobial activity profile of the synthesized coumarin series, the most inactive derivatives C4–C6 were selected for the biotransformation purpose. Biotransformation studies were performed for the above mentioned three compounds using free cells of C. albicans, however, only compound number C5 was found active. The biotransformed product was tested against the same group of bacteria and the results of MIC values expressed in μg/ml of the biotransformed compound C5-P (7-(3-(cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2one) and the standard drugs are summarized in Table 1. The results indicated that the biotransformed coumarin derivative is capable of inhibiting both Gram-positive and Gram-negative bacteria.
Percentage scavenging effect of DPPH or % inhibition = (A0 ̶ A1/ A0) × 100 Where, A0 is the absorbance of control reaction and A1 is the absorbance of the test of the standard sample. 2.6. Cytotoxicity assay MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay was performed against human breast carcinoma cell lines (MDA-MB-231) following the protocol as reported by Srisawat et al., with minor modifications [12]. MDA-MB-231 cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and incubated in a CO2 incubator for 24 h at 37 °C. When the cells reached a confluency of 40–50%, the complete media was replaced with fresh medium. Around 5 × 103 MDA-MB-231
3.2. Antioxidant activity The results of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging experiment suggest that the biotransformed product possess good antioxidant properties (Table 2). The experimental regression value 0.98 further emphasized the modest antioxidant activity
Table 1 Antimicrobial activity profile in terms of MIC values (μg/ml) of coumarin derivatives. Test strains (MTCC)
C1
C2
C3
C4
C5
C6
C5-P
N
V
Bacillus subtilis MTCC 441 Bacillus pumilus MTCC 1604 Staphylococcus aureus MTCC 537 Salmonella typhi MTCC 537 Klebsiella pneumonia MTCC 3384 Escherichia coli MTCC 1304
200 > 200 200 100 > 200 100
> 200 > 200 200 100 > 200 200
> 200 > 200 > 200 200 200 200
> 200 > 200 > 200 200 > 200 > 200
> 200 > 200 > 200 > 200 200 > 200
> 200 100 > 200 > 200 > 200 > 200
100 25 50 100 100 50
6.25 6.25 6.25 25 25 25
1.56 1.56 1.56 0.35 0.35 R
C1-C6: Coumarin derivatives; C5-P = biotransformed product; N = Novobiocin; V = Vancomycin; R = Resistant. 3
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Table 2 Percentage DPPH scavenging effect against varying concentrations of the biotransformed coumarin derivative. S. No.
Concentration of biotransformed coumarin (μg)
DPPH scavenging effect (% inhibition)
Ascorbic acid DPPH scavenging effect (%)
1 2 3 4 5
250 500 750 1000 1250
27.5 43.5 63.5 70..5 89.5
30 51 72 78 91
of the biotransformed coumarin derivative. Effective concentration EC50 value demonstrated the concentration of coumarin-derivative required to scavenge 50% of DPPH free radical. In the performed experiment, the EC50 value was 6756.76 μg, means 6.7 mg of the biotransformed product of coumarin derivative is effective in showing antioxidant activity.
3.3. Cytotoxicity Fig. 3. Effect of biotransformed coumarin derivative on cellular proliferation against normal human breast MCF10A cells at different time points (a) 24 h and at (b) 48 h. Note: ns: non-significant.
The efficacy of the biotransformed coumarin derivative was studied in a dose-dependent (1, 2, 5, 10, 20, 40 and 80 μM) and time-dependent manner (24 and 48 h) against cellular proliferation of human breast MDA-MB-231 cancer cells using MTT assay (Fig. 2). The inhibition of cellular proliferation by the purified biotransformed compound was not very significant at the lower concentration, i.e.,up to 10 μM. However, from 20 μM onwards it was found significant, when compared with the control under t-test analysis. With the increase in the compound concentration, the inhibition of cellular proliferation increased and at 80 μM concentration showed maximum inhibition (p < 0.0001). IC50 value, which depicts 50% inhibition of the cell proliferation, was found to be at 54 and 40.08 μM of the biotransformed coumarin derivative at 24 and 48 h time points, respectively. The substrate coumarin at a concentration of 80 μM was able to inhibit only 35 and 40% cellular proliferation at 24 and 48 h, respectively. Centchroman (CC) was used as a positive control and the IC50 value of CC was 15 and 10 μM at 24 and 48 h, respectively in MDA-MB-231 cells. The results of 24 and 48 h treatment suggest that with the increase in time interaction, the effectiveness of the compound increases. The above experiment was compared by studying the efficacy of the biotransformed coumarin derivative on cellular proliferation against human normal breast MCF10A cells using MTT assay for the cytotoxicity under the same experimental conditions of dose and time dependency. The compound had no or insignificant influence (p > 0.05) over cellular proliferation from 10 to 40 μM as they increased cellular proliferation by 1–9% in 24 h and 9–14% in 48 h. The maximum
experimental dose, i.e.,80 μM was found to show 18% cellular inhibition. At the concentration of 40 μM, the biotransformed product inhibited cellular proliferation by 9 and 14% at 24 and 48 h incubation, respectively; and 18% inhibition at 80 μM concentration; out of which, only 48 h incubation was found significant. Whereas, the original product (i.e., substrate) inhibited 6 and 2% cells at 80 μM concentration. It is clear from MTT cytotoxicity assay that biotransformed product has no cytotoxic effect on human normal MCF10A cells between 10–40 μM concentration at 24 h and 48 h, respectively, and can be further explored as anti-cancer compound (Fig. 3). 3.4. Chemical characterization of the substrate/biotransformed product The absorption bands observed at 1716 and 3422 cm1 in IR spectrum indicated the presence of δ lactone and hydroxyl functions, respectively. The ESI MS spectrum of the coumarin compound showed a peak at m/z 290 corresponding to its [M+H]+. In IH NMR spectrum signals corresponding to H-5 of benzopyrone ring appeared as doublet at δ 7.25 with J value of 8.8 Hz, while H-6 and H-8 were observed as multiplet at δ 6.81. H-3 was observed as singlet at δ 6.12. However, signals for propan-2-ol linker appeared as multiplets at δ 4.10 (C(1′)H2,
Fig. 2. Effect of biotransformed coumarin derivative on cellular proliferation against human breast cancer MDA-MB-231 cells at different time points (a) 24 h and at (b) 48 h. Note: ns: non-significant; * less than control; ** equal to control; *** greater than control, i.e., centchroman. 4
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of benzene moiety in the structure; the signals at 0.30 marks the presence of cyclopropane ring; at 1.93 (t, 3H) shows CH region, 7.58 (d, 4H) denotes CH2 bonding; whereas at 2.7 multiple peaks are observed confirming the presence of eNH and eCH; signals at 3.9 confirms the presence of eOH and eCH bonding; signal at 4.04 confirms CH2 bonding; at 3.24 it is CH3 bonding and OH shares peak at 2.0.J coupling region and chemical shift (δ) indicates that either modifying methyl group or hydroxyl group in the original structure might results in the final product. Therefore, oxidation at CH3 followed by methylation or oxidation at CH3 followed by methylation at eOH lead to the molecular weight of 318 Da, whereas oxidation of CH3 followed by methylation of CH3 and eOH both results in a molecular weight of 332 Da (Fig. 6).
NH, C(2′)H) and 3.12 (C(3′)H2). The methylene protons of cyclopropyl ring appeared as multiplets at δ 1.26 and 0.83. However, methine proton of cyclopropyl ring was observed as multiplet at δ 2.42 merged with signal for methyl protons of the 4-methyl substitutent. The 13C NMR spectrum of 5 exhibited signals at δ 102.8, 127.7, 111.7 and 107.6 corresponding to C-3, C-5, C-6 and C-8 of benzopyrone ring. The signals for quaternary carbons C-2, C-4, C-7, C-9 and C-10 were observed at δ 161.8, 153.8, 157.6, 151.2 and 116.8, respectively, while signals corresponding to carbons of propan-2-ol linker appeared at δ 72.3, 62.8 and 49.8 for C-1′, C-2′, and C-3′, respectively. The methyl carbon of 4methyl substituent was observed at δ 18.7. The signal at δ 35.2 was attributed to methine and at δ 9.3 and 7.8 were attributed to methylene carbons of cyclopropyl ring. The efficiency of biotransformation reaction was found to be 64%, i.e., biotransformation of 100 mg of coumarin-derivative resulted 64 mg of the pure product. The product was oily in nature and showed solubility in DMSO. The process of bioconversion held was confirmed by thin layer chromatography showing spots with different Rf value than the substrate. The complete structure identification of the biotransformed compound was done on the basis of spectral studies. The absorbance in the UV region for the substrate and product was 350 nm and 400 nm, respectively (Fig. 4a) falling in the range (between 300–400 nm) of coumarins [8]. IR spectra: peaks at 1072, 1283 in the product and 1071, 1286 in the crude corresponds to aryl-alkyl-ether (AryleOeR). The peaks, 3425 and 3013 in the product belongs to eOH, eNH bonding, respectively. These similarities suggest that the side chain present in the product is intact as same as in the crude; a peak at 2925 in the product corresponds to eCH stretching region; The absorption bands observed at 1716 and 3422 cm−1 in the substrate and product indicate the presence of δ lactone and hydroxyl functions, respectively, which further confirm the intact lactam ring. As, benzene is a stable structure in comparison to lactam ring, hence the chances of group modification at the side chain or at pyrone ring are speculated (Fig. 4b). In ESI-MS the major compounds corresponding to the retention time 8.41, 9.64 and 9.99 possess molecular weight 290.3, 318.3 and 332 Da, respectively (Fig. 5). Mass distribution from LC–MS predicts the product mass to be 318 or 332 at a retention time of 9.64 and 9.99 respectively (Fig. 5). ESI-MS also establishes the distinct molecular weight of 318; it is certain that the main product corresponds to the molecular weight 318 with the smaller fraction being converted to the molecular weight 332. While, FT-IR suggests that the major functional groups and ring remained intact and provides maximum structural similarity in the substrate and product; it is likely that there is a modification of either eOH, eCH3, eNH group (Fig. 4b). The presence of signals between J = 7.7–7.0 confirm the presence
4. Discussion The ability of biocatalysts to selectively produce useful products under relatively mild conditions compared to its chemical catalyst counterpart make biocatalysts an interesting and powerful tool. Biocatalyst can be a free enzyme or a whole cell catalyzing a chemical reaction and producing highly enantioselective, low toxic product [4]. Recent technology advancements have increased the focus of industry to discover new biocatalysts and optimize their performance. In order to create an eco-friendly system, certain studies on the modification of benzopyrone ring system employing different fungi and bacteria, as functional group transferring agents, have been undertaken [1]. Coumarins are common example of benzopyrone ring system, and popularly named as benzopyran-2-one or chromen-2-one or simply benzo-αpyrones ring system. Coumarins are one of the most common phytochemicals present in various natural products. Due to its diverse bioactivity [13] it is also synthesized chemically [14]. In this direction, through chemical synthesis a series of coumarins derivatives (coumarinyl amino alcohols) have been synthesized; unfortunately, the compounds of this series are not very effective in bioactivity, therefore, biotransformation method has been used to make an effort to convert the inactive derivative into an active one. The modifications at the chemical level of inactive coumarins generally lead the conversion into active structure which is mainly useful for pharmaceutical industries and opens the door for novel drug development. In this direction, microorganisms play an important role for structural modifications [15]. Biotransformation of coumarin-derivatives have been reported in various literatures using a variety of microorganisms, like Streptomyces griseus [16], Candida tropicalis [1], Arthrobacter species, Aspergillus niger [17], Fusarium solani [18] and Cunninghamella elegans [19]. The structure of any molecule plays an important role in order to reflect its biological activity. Kayser and Kolodziej [20] reported that the addition of eOH moiety to coumarin core structure significantly
Fig. 4. (a) UV absorption pattern of the biotransformed product C5-P: showing absorbance near 400 nm; (b) FTIR of the biotransformed product C5-P: showing the presence of basic functional groups of coumarin derivatives. 5
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Fig. 5. Mass spectra of the substrate/product reflecting variations in the mass of the substrate and the product endorsing biotransformation process.
Fig. 6. 1H NMR of the biotransformed product C5-P confirms the presence of benzopyrone and cyclopropyl ring.
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Acknowledgements
reduced the antibacterial activity against all the tested bacteria. Their findings suggest that the antibacterial activity of oxygenated coumarins apparently depends on the position of polar (eOH) and less polar (Me/ OMe) functional group at the aromatic nucleus of the coumarin structure. This assumption match with the present study proving the last three compounds (C4–C6) from the series were most inactive against a group of tested bacteria. The addition of methyl group further depicted fairly low activity of the compound C5 against all the tested strains. The active compounds favor its permeation more efficiently through the lipid layer of the fungi [21]. Nigam et al. [1] reported the application of Saccharomyces griseus and Candida tropicalis for microbial biotransformation purposes for the modification of coumarin substrates. They reported the biotransformations of coumarins derivatives 7-ethoxy-4-methyl coumarin into 7-dihydroxy-4-methyl coumarin by Saccharomyces griseus and 7hydroxy-4-methyl coumarin into 7-methoxy-4-methyl coumarin by Candida tropicalis. The report of Nigam et al. [1] provides the prime reason to examine the ability of Candida albicans for bioconversion process. The results of bioconversion of inactive coumarin derivative (C5) into active product suggest the efficiency of C. albicans towards biotransformation. The results regarding the structure elucidation of the product suggest the most possible reaction of the conversion is the oxidation at CH3 moiety present on chromone ring followed by methylation at specific positions, which ultimately offer main and side products. Methylation and oxidation are the common chemical reactions involve in the mechanism of bioconversion using microbes. Beside antimicrobial activity, the modified coumarin possessed ROS activity and toxic activity against the breast cancer cell line MDA-MB231. Previously, the toxic activity has been diligently mentioned in the work of Musa et al. [7] regarding coumarin and its active metabolite 7hydroxy coumarins particularly against breast cancer, which is a leading cause of death. Musa et al. [7], have proved that bioactive analog of coumarin works as a potent selective estrogen receptor modulators (SERMs) as well as strong chemotherapeutic agent in breast cancer. Few naturally occurring coumarin derivatives including umbelliferone, warfarin, aesculetin (6,7 dihydroxycoumarin), 7-methoxy coumarin, psoralen, and imperatorin have shown their potential in the past. In addition, coumarins work as lipid-lowering agents and hydroxycoumarins are powerful chain-breaking antioxidants [22], and can prevent free radical injury by scavenging reactive oxygen [7].
The authors are grateful to IET, Lucknow for providing the laboratory facility and partial financial support under World Bank assisted TEQIP-Government of India program-phase II for this research work. The author, Shafiul Haque, is grateful to Jazan University, Saudi Arabia for providing the access to the Saudi Digital library. The author, Hesham El Enshasy would like to acknowledge the support of MOE and UTM-RMC through HICOE grant no. R.J130000.7846.4J262. References [1] S. Nigam, J.V. Rao, B.S. Jayashree, Microbial biotransformation—a novel approach for modification on coumarin substrates, Ind. J. Biotechnol. 12 (2013) 379–385. [2] M.E. Hegazy, T.A. Mohamed, A.I. ElShamy, H.M. Abou-El-Hamd, U.A. Mahalel, E.H. Reda, A.M. Shaheen, W.A. Tawfik, A.A. Shahat, K.A. Shams, N.S. Abdel-Azim, Microbial biotransformation as a tool for drug development based on natural products from mevalonic acid pathway: a review, J. Adv. Res. 6 (2015) 17–33. [3] M.D. Lilly, Eighth P. V. Danckwerts memorial lecture presented at Glaziers’ Hall, London, U.K. 13 May 1993: advances in biotransformation processes, Chem. Engg. Sci. 49 (1994) 151–159. [4] F. Garzón-Posse, L. Becerra-Figueroa, J. Hernández-Arias, D. Gamba-Sánchez, Whole cells as biocatalysts in organic transformations, Mol. 23 (2018) 1265. [5] E.M. Sales, T.F. Barros, E.D.S. Velozo, Biotransformation of coumarins by Saccharomyces cerevisiae, World J. Pharm. Pharm. Sci. 3 (2014) 209–216. [6] M.S. Smitha, S. Singh, R. Singh, Microbial Biotransformation: a process for chemical alterations, J. Bacteriol. Mycol. 4 (2017) 85. [7] M.A. Musa, J.S. Cooperwood, M.O.F. Khan, A review of coumarin derivatives in pharmacotherapy of breast cancer, Curr. Med. Chem. 15 (2008) 2664–2679. [8] S. Misra, L.K. Singh, Priyanka, J. Gupta, S. Misra-Bhattacharya, D. Katiyar, Synthesis and biological evaluation of 4-oxycoumarin derivatives as a new class of antifilarial agents, Eur. J. Med. Chem. 94 (2015) 211–217. [9] Priyanka, V. Singh, Ekta, D. Katiyar, Synthesis, antimicrobial, cytotoxic and E. coli DNA gyrase inhibitory activities of coumarinyl amino alcohols, Bioorg. Chem. 71 (2017) 120–127. [10] S.I.G.H.M. Montalvão, V. Singh, S. Haque, Bioassays for bioactivity screening. Analysis of Marine samples in search of bioactive compounds, in: T. Rocha-Santos, A. Duarte (Eds.), Comprehensive Analytical Chemistry Series, Elsevier B.V., 2014ISBN9780444633590. [11] Shekhar, C. Tailor, A. Goyal, Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. leaves, Am. J. Ethnomed. 1 (2014) 244–249. [12] T. Srisawat, Y. Sukpondma, P. Graidist, S. Chimplee, K. Kanokwiroon, The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of vaticadiospyroides symington type SS fruit include effects on mode of cell death, Pharmacogn. Mag. 11 (Suppl. 1) (2015) S148–S155. [13] S. Tavakoli, M.R. Delnavazi, R. Hadjiaghaee, S. Jafari-Nodooshan, F. KhalighiSigaroodi, M. Akhbari, A. Hadjiakhoondi, N. Yassa, Bioactive coumarins from the roots and fruits of Ferulagotrifida Boiss. an endemic species to Iran, Nat. Prod. Res. 32 (2018) 2724–2728. [14] Priyanka, R.K. Sharma, R.J. Butcher, D. Katiyar, Facile construction of 4H-chromenes via Michael addition of phenols to benzylideneoxobutanoates and their successful conversion into pyranocoumarins, Tetrahedron Lett. 59 (2018) 2347–2351. [15] J.S. doNascimento, J.C. Conceição, E. de Oliveira Silva, Biotransformation of coumarins by filamentous fungi: an alternative way for achievement of bioactive analogs, Mini-Rev. Org. Chem. 16 (2019) 1–10. [16] F.S. Sariaslani, J.P. Rosazza, Novel biotransformations of 7-ethoxycoumarin by Streptomyces griseus, Appl. Environ. Microbiol. 46 (1983) 468–474. [17] C.B. Aguirre-Pranzoni, G.I. Furque, C.E. Ardanaz, A. Pacciaroni, V. Sosa, C.E. Tonn, M. Kurina-Sanz, ARKIVOC: Online J. Org. Chem. (2011) 170–181 (vii). [18] H.S. Shieh, A.C. Blackwood, Use of coumarin by soil fungi, Can. J. Microbiol. 15 (1969) 647–648. [19] G.A. Attia, K.A. Abou-El-Seoud, A.R. Ibrahim, Biotransformation of coumarins by cunninghamellaelegans, Afr. J. Pharm. Pharmacol. 10 (2016) 411–418. [20] O. Kayser, H. Kolodziej, Antibacterial activity of simple coumarins: structural requirements for biological activity, Z. Naturforsch. C 54 (1999) 169–174. [21] S.U. Rehman, Z.H. Chohan, F. Gulnaz, C.T. Supuran, In-vitro antibacterial, antifungal and cytotoxic activities of some coumarins and their metal complexes, J. Enzyme Inhib. Med. Chem. 20 (2005) 333–340. [22] A.A. Kadhum, A.A. Al-Amiery, A.Y. Musa, A.B. Mohamad, The antioxidant activity of new coumarin derivatives, Int. J. Mol. Sci. 12 (2011) 5747–5761.
5. Conclusion In the present study, biotransformation was done by oxidation followed by methylation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)4-methyl-2H-chromen-2-one by Candida albicans to 7-(3Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethylchromen-2-one as main product with molecular weight 318 Da. In addition to the main peak, a small peak of molecular weight 332 Da was also observed in the LC–MS region that indicates the presence of 7-(3Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2one in addition to the main product; and this was confirmed by the spectral analysis. Besides this, attempts were made to evaluate the success of bioconversion of the substrate into the final product. The biotransformed product showed significant results for its biological activity assessed in terms of decent antibacterial, antioxidant cytotoxicity properties, and reflects its therapeutic use in the near future after several in vivo trials. Declaration of Competing Interest The authors declare no conflicts of interest.
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