European Journal of Medicinal Chemistry 94 (2015) 1e7
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Original article
Clerodane type diterpene as a novel antifungal agent from Polyalthia longifolia var. pendula Asish K. Bhattacharya a, *, Hemender R. Chand a, Jyothis John b, Mukund V. Deshpande b a b
Division of Organic Chemistry, CSIR-National Chemical Laboratory (CSIR-NCL), Dr. Homi Bhabha Road, Pune 411 008, India Division of Biochemical Sciences, CSIR-National Chemical Laboratory (CSIR-NCL), Dr. Homi Bhabha Road, Pune 411 008, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 26 February 2015 Accepted 27 February 2015 Available online 28 February 2015
Bioactivity-guided chemical examination of methanolic extract of leaves of Polyalthia longifolia var. pendula led to the isolation of the active constituent, a diterpene 1 which was identified as 16ahydroxycleroda-3,13(14)Z-dien-15,16-olide on the basis of its spectral data. Among the tested strains, diterpene 1 was found to exhibit antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 mM against Candida albicans NCIM3557, Cryptococcus neoformans NCIM3542 (human pathogens) and Neurospora crassa NCIM870 (saprophyte), respectively. Initial, structureeactivity-relationship (SAR) data generated by synthesizing some derivatives revealed that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. The mode of action of 1 in C. albicans is due to compromised cell membrane permeability and also probably due to disruption of cell wall structures. The red blood cell haemolysis of all the compounds (1e4) did not show any significant haemolysis and was found to be less than 15% for all the compounds when tested at highest concentration, i.e. 1200 mM. Interestingly, all the tested compounds inhibited YeH transition in dimorphic C. albicans NCIM3557 at much lower concentration than their MIC90 values. Determination of ROS generation by diterpene 1 using DCFH-DA and DHR123 (dihydrorhodamine) staining of C. albicans NCIM3557 indicated production of intracellular ROS as a mechanism of antifungal activity. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Diterpene Polyalthia longifolia Isolation Flash chromatography Natural products Antifungal activity
1. Introduction Susceptibility to fungal pathogens is a common occurrence in both humans and plants; however, in recent years, the problem is aggravated due to development of resistance in several species of fungi to available drugs/fungicides [1]. It is widely believed that more than 75% of all the fungal infections in humans are caused by the Candida species viz. Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis. Research on development of new antifungal agents over the past few decades has resulted in only a few antifungal drugs, which are being clinically used. At present, the drugs being used to treat fungal infections could be broadly classified into five classes of compounds, which include azoles, polyenes, fluoro pyrimidines, echinocandins and allyl amines [2,3a]. However, most of these are of synthetic origin except a few e.g. polyenes [3b and c] and echinocandins [3dej] classes of compounds, which are of natural origin. Furthermore, the resistance
* Corresponding author. E-mail address: [email protected] (A.K. Bhattacharya). http://dx.doi.org/10.1016/j.ejmech.2015.02.054 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
developed by pathogens against these drugs as well as complications due to drug interactions with the host cells, side effects, their poor bioavailability etc. further compromise their wide spread utility. In recent times, several classes of compounds have been isolated from diverse plants, which are reported to have antifungal activities [4,5]. However, these could not be developed into useful antifungal drugs, as some or other drawbacks were associated with these molecules. Hence, there is an urgent need to develop new naturally occurring antifungal agents having target specificity, broadspectrum activity and different mechanism of action than the existing drugs.
2. Results and discussion The genus Polyalthia (Annonaceae) has been credited with seventy species out of which only seven are indigenous to India. Polyalthia longifolia var. pendula Linn. is popularly known as “ulta Ashok” in India and widely grown in the gardens of tropical and subtropical Asia as an evergreen ornamental tree [5]. This plant has
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been reported to be widely used in traditional system of medicine for the treatment of hypertension, fever, diabetes, helminthiasis and skin diseases [6]. The chemical examination of P. longifolia var. pendula has resulted in the isolation of several classes of compounds such as diterpenes, including clerodane and triterpenes and aporphine alkaloids and these have been investigated for various biological activities [7]. In continuation of our work [8] on naturally occurring bioactive secondary metabolites, we initiated systematic chemical examination of P. longifolia var. pendula for its antifungal constituents. The activity-guided fractionation of the crude methanolic extract over flash SiO2 column furnished 6 sub-fractions (AeF). The antifungal assays of these six major fractions against a screen of fungal strains led us to an active fraction (fraction B), which showed promising activities. Further, efforts in isolation of the active secondary metabolite from fraction B by automated flash chromatography using RediSep® column (SiO2, 2 12 g, stacked) resulted in the isolation of a pure compound 1, which was identified as 16ahydroxycleroda-3,13(14)Z-dien-15,16-olide (Fig. 1) on the basis of its spectral data [7d]. Total synthesis of diterpene 1 has been reported in the literature [9]. The isolated pure compound 1 was assayed against five human pathogens such as C. albicans NCIM3557, C. glabrata NCIM3237, Cryptococus neoformans NCIM3542, Aspergilus fumigatus NCIM902, Aspergillus niger NCIM628, C. albicans NCIM3471 (non-pathogenic), phytopathogen, Fusarium oxysporum NCIM1043 and a saprophyte, Neurospora crassa NCIM870 (Table 1). The diterpene 1 showed MIC90 values of 50.3, 100.6 and 201.2 mM against C. albicans NCIM3557, C. neoformans NCIM3542 and N. crassa NCIM870, respectively. The standard antifungal drugs fluconazole and amphotericin B exhibited MIC90 values of 32 and 2 mg/mL against C. albicans NCIM3557, respectively [10]. In order to shed some light on the structureeactivity relationship (SAR) and to deduce information on pharmacophores responsible for the remarkable antifungal activities of the secondary metabolite 1, we undertook some analogue synthesis of the same (Scheme 1). Compound 1 on treatment with Ac2O/pyridine afforded the acetate 2 as a colourless solid, m.p. 134e136 C. The 1H and 13C NMR of this acetate clearly revealed it to be a mixture of acetate isomers due to epimerization [11] at C-16. The epoxidation of diterpene 1 with m-CPBA in DCM afforded the epoxide 3 as a viscous oil, [a]27 D 24.43 (c 0.95, CHCl3). On treatment of compound 1 with Boc2O in presence of TEA and catalytic amount of DMAP in DCM led to formation of Boc derivative 4 which was also found to
Fig. 1. Structure of isolated diterpene 1 from P. longifolia var. pendula.
Table 1 MIC90 values of compound 1. Strains
1 (mM)
Candida albicans NCIM3557 Candida albicans NCIM3471 Candida glabrata NCIM3237 Cryptococcus neoformans NCIM3542 Aspergillus fumigatus NCIM902 Aspergillus niger NCIM628 Fusarium oxysporum NCIM1043 Neurospora crassa NCIM870
50.3 805.0 805.0 100.6 805.0 805.0 805.0 201.2
be a mixture of isomers due to the epimerization [11] at C-16 by the analysis of its 1H and 13C NMR spectra. When we tried to reduce the double bond between C3eC4 (or eventually C13eC14 also) with 10% PdeC/H2 in EtOAc, even after 5d we could get only a complex mixture which could not be separated using flash chromatography. The three semi-synthetic derivatives 2e4 of the diterpene 1 were assayed against all the test organisms. However, the MIC90 values of synthesized derivatives (2e4) were found to be >600 mM against all the strains. The preliminary structureeactivity-relationship (SAR) studies from the assay of synthesized derivatives (2e4) indicate that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. However, the complete SAR remains to be concluded with the synthesis of some more derivatives/analogues of 1. Further, it was necessary to evaluate the natural product 1 and its semi-synthetic derivatives (2e4) for their haemolytic potential on red blood cells (RBCs) [12]. The compounds (1e4) were incubated with RBCs and release of haemoglobin due to the RBC lysis was measured (Fig. 2). It is pertinent to mention here that at the tested concentrations closer to the MIC of C. albicans (NCIM3557), none of the compounds exhibited any significant haemolysis. The red blood cell haemolysis was found to be less than 15% for all the compounds (1e4) when tested at highest concentration, i.e. 1200 mM. In several human and plant pathogenic fungi, direct correlation between the ability of fungus to switch between yeast (Y) and hypha (H) forms and pathogenicity has been reported [13]. Hence, all the compounds (1e4) were tested for their Y to H transition inhibition. All the tested compounds inhibited YeH transition in C. albicans NCIM3557 at much lower concentration than their MIC90 values (Table 2). Propidium iodide (PI) has been found to be an excellent membrane impermeable nucleic acid staining fluorescent dye [14a]. PI enters the cells with compromised permeability only and it binds to the double stranded nucleic acids thus producing a red fluorescence when excited at 480 nm. We studied the uptake of PI by Candida cells in the presence of the natural diterpene 1 at two concentrations, 50.3 mM and 100.6 mM by epifluorescence microscopy (Fig. 3) and at three concentrations, 25.2 mM, 50.3 mM (MIC90) and 100.6 mM by Confocal microscopy (Fig. 4) [14b]. In order to deduce antifungal mechanism of our natural diterpene 1, we studied the generation of reactive oxygen species (ROS) by compound 1 since the generation of reactive oxygen species (ROS) is considered to be associated with apoptosis (a programmed cell death) and necrosis. Hence, we studied ROS generation by diterpene 1 using DHR123 (dihydrorhodamine) [15] (Fig. 5) and DCFH-DA staining [16] (Fig. 6). C. albicans NCIM3557 cells were incubated with different concentrations (50.3, 100.6 and 201.2 mM) of diterpene 1, and then treated with DHR123 for 30 min. Fluorescence resulting from oxidation of the dye DHR123 were observed at 525 nm. It was found that with increasing concentration of compound 1, the relative fluorescence increased (Fig. 5). This
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Scheme 1. Preparation of derivatives of 1. Reagents and conditions: (i) Py, Ac2O, rt, 24 h; (ii) mCPBA, DCM, 1.5 h; (iii) Boc2O, TEA, DMAP (cat), DCM, 0 C, 2 h; (iv) 10% Pd/C, H2 (1 atm), EtOAc, rt, 5d.
Fig. 2. Haemolytic activity of compounds (1e4).
Table 2 Effect of compounds on yeastehypha transition in Candida albicans NCIM3557. Compound
Inhibition of 50% germ tube formation in C. albicans (mM)
1 2 3 4
25.14 177.80 191.60 153.10
clearly indicated generation of intracellular ROS. Cells without the compound served as negative control whereas cells with H2O2 served as positive control. Similarly, C. albicans NCIM3557 cells were incubated at different concentrations (25.2 mM, 50.3 mM and 100.6 mM) of diterpene 1 and subsequently incubated with DCFH-DA. Fluorescent cells resulting from oxidation of dye DCFH-DA were observed under epifluorescence microscope using I3 filter. Cells treated with diterpene 1 expressed fluorescence at all the three concentrations (25.2 mM, 50.3 mM and 100.6 mM) with an increase in relative fluorescence at increasing concentration thereby indicating production of
Fig. 3. Fluorescence microscope images of membrane permeabilization by propidium iodide (PI) uptake: (a) Untreated control (without compound 1); (b) positive control (heat-killed); (c) C. albicans (NCIM3557) cells with compound 1 (50.3 mM); (d) C. albicans (NCIM3557) cells with compound 1 (100.6 mM).
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Fig. 4. Confocal laser scanning microscopy (CLSM) images of membrane permeabilization by propidium iodide (PI) uptake: (a) Untreated control (without compound 1); (b) positive control (heat-killed); (c) C. albicans (NCIM3557) cells with compound 1 (25.2 mM); (d) C. albicans (NCIM3557) cells with compound 1 (50.3 mM); (e) C. albicans (NCIM3557) cells with compound 1 (100.6 mM).
intracellular ROS. Cells without the compound served as the negative control (Fig. 6). 3. Conclusions We have isolated a diterpene 1 identified as 16a-hydroxycleroda-3,13(14)Z-dien-15,16-olide using automated flash chromatography without following solventesolvent extraction protocols from the methanolic extract of the leaves of P. longifolia var. pendula. The isolated diterpene 1 was assayed against five human pathogens (C. albicans NCIM3557, C. glabrata NCIM3237, C. neoformans NCIM3542, A. fumigatus NCIM902 and A. niger NCIM628), C. albicans NCIM3471 (non-pathogenic), phytopathogen, F. oxysporum NCIM1043 and N. crassa NCIM870 (saprophyte). The diterpene 1 exhibited considerable antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 mM against C. albicans NCIM3557, C. neoformans NCIM3542 and N. crassa NCIM870, respectively. Our initial structureeactivity-relationship (SAR) studies from the assay of synthesized derivatives (2e4) indicate that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. Further, we have demonstrated that the natural product 1 has a broad spectrum fungicidal activity with its mode of action in C. albicans due to compromised cell membrane permeability (thereby enabling enhanced entry into the cells) and also probably due to disruption of cell wall structures. In addition, all the compounds (1e4) exhibit less than 15% haemolysis of red blood cells even when tested at highest concentration, i.e. 1200 mM. Moreover, all the tested compounds inhibited YeH transition in a dimorphic C. albicans at much lower concentration than their MIC90 values. Further, intracellular ROS generation by diterpene 1, confirmed by using DCFH-DA and DHR123 staining of C. albicans NCIM3557 cells, suggests mechanism of antifungal activity of diterpene 1. It is presumed that our studies on this molecule could be further utilized for target-based approach to explore its therapeutic potentials.
Fig. 5. Determination of ROS levels by DHR123 (dihydrorhodamine) staining in the presence of different concentrations of the diterpene 1 in C. albicans (NCIM3557) cells by epifluorescence microscopy: (a) Control (without compound 1); (b) positive control with H2O2; (c) with compound 1 (50.3 mM); (d) with compound 1 (100.6 mM); (e) with compound 1 (201.2 mM).
4. Experimental section 4.1. General The FT-IR spectra were recorded on an FT-IR-8300 Shimadzu spectrometer. NMR spectra were recorded on Bruker ACF 200 and AV200 (200 MHz for 1H NMR and 50 MHz for 13C NMR) and AV400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometers, using CDCl3 as solvent. Tetramethylsilane (0.00 ppm) served as an internal standard in 1H NMR and CDCl3 (77.0 ppm) in 13C NMR, respectively. Chemical shifts are expressed in parts per million (ppm). Mass spectra were recorded on LC-MS/MS-TOF API QSTAR PULSAR spectrometer, samples introduced by infusion method using the Electrospray Ionization Technique (ESI). HRMS (ESI) of samples was taken on an Orbitrap (quadrupole plus ion trap) and TOF mass analyser. Flash chromatography was performed with CombiFlash Rf 200i with UV/VIS and ELSD, Isco Teledyne Inc., USA using RediSep® column (SiO2). All other chemicals were of analytical grade. Epifluorescence microscopy was done using Epifluorescence Microscope, Leitz Laborlux, Germany. The laser scanning
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3H), 0.79 (s, 3H); dC (100 MHz, CDCl3) 171.7, 170.6, 144.3, 120.4, 117.1, 99.9, 46.5, 38.7, 38.2, 36.7, 36.3, 34.8, 27.4, 26.8, 21.4, 20.0, 18.3, 18.2, 18.0, 16.0; ESI-MS m/z 341.1985 (M þ Na)þ; HRMS (ESI) calcd for C20H30O3Na 341.2087, found 341.2089. 4.1.4. Preparation of acetate derivative (2) To a solution of compound 1 (21 mg, 0.066 mmol) in pyridine (0.3 mL), Ac2O (0.6 mL) was added and left at ambient temp for overnight. Toluene (3 5 mL) was then added to remove pyridine and Ac2O by co-distillation on rotary evaporator under reduced pressure. Crude product on complete dryness followed by flash chromatography on RediSep® column (SiO2, 12 g) eluting with DCM (isocratic) afforded compound 2 as a colourless solid (20 mg, 84%), which was a mixture of acetate isomers due to epimerization at C16 [11]. M.p. 134e136 C; Rf 0.39 (MeOH-DCM, 1:9); [a]27 D 19.56 (c 0.9, CHCl3); ymax (CHCl3)/cm1 3752, 2967, 2927, 1757, 1649, 1454, 1379, 1212, 1053, 982, 756; dH (200 MHz, CDCl3) 6.84 (s, 1H), 5.94 (s, 1H), 5.19 (brs, 1H), 2.38e2.22 (m, 1H), 2.18e2.14 (m, 4H), 2.11e1.93 (m, 2H), 1.77e1.64 (m, 2H), 1.60 (m, 5H), 1.53e1.39 (m, 6H), 1.01 (s, 3H), 0.82 (t, J ¼ 3.0 Hz, 3H), 0.77 (s, 3H); dC (100 MHz, CDCl3) 170.0, 169.2, 168.2, 168.1, 144.5, 144.5, 120.3, 118.2, 118.1, 93.9, 93.9, 46.6, 46.6, 38.7, 38.2, 36.7, 36.4, 35.0, 34.9, 27.4, 26.9, 26.9, 21.3, 21.2, 20.8, 20.0, 18.4, 18.3, 18.1, 16.1, 16.0; ESI-MS m/z 383.01 (M þ Na)þ; HRMS (ESI) calcd for C22H32O4Na 383.2193, found 383.2191.
Fig. 6. Determination of ROS levels by DCFH-DA staining in the presence of different concentrations of the diterpene 1 in C. albicans (NCIM3557) cells by epifluorescence microscopy: (a) Control (without compound 1); (b) with compound 1 (25.2 mM); (c) with compound 1 (50.3 mM); (d) with compound 1 (100.6 mM).
confocal microscopy was performed on a Zeiss LSM 710 Microscope. 4.1.1. Plant material Plant leaves were collected from the garden maintained at CSIRNCL, Pune during the month of June, 2010. 4.1.2. Extraction and isolation Air-dried and grounded leaves (100 g) of P. longifolia var. pendula were extracted with MeOH (5 1.0 L) at room temperature for five days. After completion of the extraction, the solvent was evaporated under reduced pressure to afford the MeOH extract (27.2 g). A portion of the MeOH extract (5.2 g) was fractionated on SiO2 (200 g, 230e400 mesh) column eluting with DCM: MeOH (0 / 20%) to furnish 6 sub-fractions (AeF). The compound 1 was present in DCM: MeOH (99:1) fraction (fraction B). The fraction B (1.250 g) was flash chromatographed on CombiFlash Companion, Isco Teledyne Inc., USA using RediSep® column (SiO2, 2 12 g, stacked together) and isocratic elution was done with DCM to furnish the pure compound 1 (270 mg) with 1.4% overall yield. 4.1.3. Spectral data [7d] of 16a-hydroxycleroda-3,13(14)Z-dien15,16-olide (1) Rf 0.36 (MeOH-DCM, 9:1); [a]27 D 42.57 (c 1.49, MeOH); ymax (CHCl3)/cm1 3376, 2930, 1748, 1650, 1459, 1386, 1130, 948, 755; dH (400 MHz, CDCl3) 6.07 (s, 1H), 5.85 (s, 1H), 5.20 (brs, 1H), 2.37e2.20 (m, 2H), 2.10e2.01 (m, 2H), 1.76e1.65 (m, 2H), 1.60 (s, 3H), 1.57e1.43 (m, 6H), 1.37e1.19 (m, 5H), 1.02 (s, 3H), 0.83 (d, J ¼ 6.5 Hz,
4.1.5. Preparation of epoxide (3) To a stirred solution of compound 1 (43 mg, 0.135 mmol) in DCM (4 mL) cooled in ice-water bath, was added m-CPBA (33 mg, 0.189 mmol, 1.4 eq.). After stirring for 1.5 h, DCM (10 mL) was added and the organic layer was separated, washed with aq. 10% KI solution (2 20 mL), followed by 10% Na2S2O3 solution (2 20 mL), 10% NaHCO3, (2 20 mL) solution and then finally with H2O (2 20 mL). The organic layer was then dried (anhydrous Na2SO4), evaporated in vaccuo, which on flash chromatography on (RediSep® SiO2 column, 12 g) eluting with 2% MeOH-DCM (isocratic) furnished compound 3 as a viscous oil (25 mg, 45%). Rf 0.57 1 (MeOH-DCM, 1:9); [a]27 D 24.43 (c 0.95, CHCl3); ymax (CHCl3)/cm 3369, 2926, 2857, 1752, 1648, 1577, 1560, 1458, 1141, 951, 757; dH (200 MHz, CDCl3) 6.00 (s, 1H), 5.82 (s, 1H), 2.94 (m, 1H), 2.41e2.18 (m, 2H), 1.94e1.86 (m, 2H), 1.74e1.57 (m, 4H), 1.46e1.37 (m, 5H), 1.53e1.37 (m, 5H), 1.01 (s, 3H), 0.80 (3H), 0.73 (s, 3H); dC (100 MHz, CDCl3) 171.8, 170.8, 117.0, 99.4, 67.0, 61.4, 47.9, 39.2, 38.5, 37.9, 36.1, 34.6, 26.8, 21.5, 21.4, 21.4, 18.3, 17.7, 16.3, 16.0; ESI-MS m/z 357.08 (M þ Na)þ; HRMS (ESI) calcd for C20H30O4Na 357.2036, found 357.2034. 4.1.6. Preparation of Boc derivative (4) Compound 1 (31 mg, 0.097 mmol) was dissolved in DCM (4 mL) and cooled in ice-water bath, Boc2O (80 mg, 0.37 mmol, 3.7 eq.) was then added followed by TEA (10 mL, 0.074 mmol) and catalytic amount of DMAP (2 mg, 0.016 mmol) and reaction mixture was stirred for 2 h. After completion of the reaction (TLC), the reaction mixture was then evaporated in vaccuo and directly subjected to flash chromatography (RediSep® SiO2 column, 12 g) eluting with EtOAc:petroleum ether (1:19) to furnish the pure compound 4 which was obtained as a viscous oil (33 mg, 80%) and found to be an inseparable mixture of C-16 epimers [11]. Rf 0.62 (DCM); 1 [a]27 3683, 3618, 3440, D 27.15 (c 1.0, CHCl3); ymax (CHCl3)/cm 3020, 2928, 2856, 2400, 1799, 1765, 1649, 1458, 1373, 1256, 1216, 757, 669; dH (500 MHz, CDCl3) 6.66 (d, J ¼ 8.2 Hz, 1H), 5.93 (s, 1H), 5.20 (s, 1H), 2.37e2.27 (m, 2H), 2.25e2.21 (m, 1H), 2.12e1.98 (m, 5H), 1.75e1.64 (m, 4H), 1.59 (m, 4H), 1.54e1.53 (m, 9H), 1.50e1.44 (m, 9H), 1.01 (s, 3H), 0.81 (m, 3H), 0.77 (s, 3H); dC (125 MHz, CDCl3) 169.8, 167.6, 167.5, 151.5, 144.5, 144.4, 120.4, 120.3, 118.2, 118.1, 104.4, 96.4, 84.8, 84.7, 46.5, 46.5, 38.7, 38.7, 38.2, 36.7, 36.4, 36.3, 34.9,
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34.8, 31.9, 29.6, 29.4, 29.3, 27.6, 27.3, 26.8, 26.8, 21.3, 21.2, 19.9, 18.3, 18.2, 18.0, 16.0, 15.9; ESI-MS m/z 441.08 (M þ Na)þ; HRMS (ESI) calcd for C25H38O5Na 441.2611, found 441.2612. 4.1.7. Reduction of diterpene (1) Compound 1 (18 mg, 0.056 mmol) was dissolved in EtOAc (3 mL) and PdeC (10%, 9 mg) was added to the reaction mixture. Stirring was continued at rt under an atmosphere of hydrogen for 5d. After 5d, the catalyst was filtered off over a celite bed and the reaction mixture was then evaporated in vaccuo. The reaction mixture showed a complex TLC pattern (30% EtOAc-PE), which could not be further purified using flash chromatography (RediSep® SiO2 column, 4 g) eluting with petroleum ether: EtOAc (0 / 30, gradient). 4.1.8. Antifungal assays Fungi growth conditions: Human and plant pathogenic fungal strains, C. albicans NCIM3557, C. albicans NCIM3471, C. glabrata NCIM3237, C. neoformans NCIM3542, A. niger NCIM628, A. fumigatus NCIM902, F. oxysporum NCIM1043 and a saprophyte model, N. crassa NCIM870 were obtained from National Collection of Industrial Microorganisms (NCIM), CSIR-National Chemical Laboratory, Pune, India. The human pathogenic fungal strains were maintained on slants of YPG agar (yeast extract, 0.3%; peptone, 0.5%; glucose, 1.0%; agar, 2.0%) and the plant pathogenic fungi were maintained on potato dextrose agar (2% PDA) slants at 28 C and sub-cultured every 15 days. During experimentation, the fungal strains were grown in YPG broth. 4.1.9. Minimum inhibitory concentration (MIC) determination The purified final compounds were evaluated for antifungal susceptibility testing by microbroth dilution method according to the recommendations of the CLSI [17]. Appropriate amount of test compounds were dissolved in DMSO to get 100 final strength. The stock was then diluted 1:40 in YPG medium and 200 mL from this was added to the first row of a 96-well microtiter plate. The compound was serially diluted two fold in successive wells to get a range of 4e512 mg/mL. Fungal yeast cells (~2 104 cfu/mL, spores for phytopathogens), freshly grown in YPG broth in logarithmic phase, were suspended in the medium and inoculated (100 mL) in the wells of the plate. The microtiter plates were incubated for 24e48 h, and the absorbance was measured at 600 nm by using microtiter plate reader to measure the cell growth. The MIC was defined as the lowest concentration required for >90% inhibition of growth with respect to the growth in control and IC50 was the concentration at which 50% growth inhibition was observed. 4.1.10. Membrane integrity assay Propidium iodide (PI) staining was used for checking integrity of fungal plasma membrane following treatment with diterpene 1. C. albicans NCIM3557 cells were harvested at the logarithmic phase and 1 106 cells/mL were added in phosphate buffer saline (PBS, 0.1 mM, pH 7.2), containing inhibitor. The tubes were incubated at 37 C for 2 h. Cells were separated by centrifugation and washed with PBS. Cells were then incubated with 3 mM of PI for 10 min, harvested by centrifugation, washed (using PBS) and suspended in PBS. PI stained cells were counted using epifluorescence microscope (Leitz Laborlux, Germany). A filter set, N 2.1 filter block with excitation filter BP 515-560 and emission filter LP 580 were used. 4.1.11. Confocal microscopy The effect of compound 1 on membrane integrity of fungal cell was confirmed by confocal microscopy using PI [14b]. The C. albicans (NCIM3557) cells (~1 106 CFU/ml) growing in log phase were suspended in an RPMI-1640 medium containing diterpene 1 at its MICs (50.3 mM) and PI (3 mM). The mixture was
incubated at 37 C for 2 h at 180 rpm. The cells were harvested by centrifugation and resuspended in PBS (pH 7.4). The cells were observed under confocal microscope with a wavelength N560 nm for PI. All images were captured at 400 magnification. 4.1.12. Cellular toxicity assay The cellular toxicity of compounds was determined by red blood cells (RBC) lysis assay [12]. In brief, the RBCs of sheep blood were washed with 2e3 times with PBS (pH 7.0) and finally RBCs were resuspended in PBS so as to obtain the 4% solution. Then, 1000 mL of PBS containing the appropriate concentration of test compound was mixed with 1000 mL of 4% RBC suspension and incubated at 37 C for 2 h. No haemolysis and 100% haemolysis were observed in PBS and 0.1% Triton-X 100, respectively. The reaction mixture was centrifuged at 2000 rpm for 5 min and the absorbances of supernatant were read at 545 nm. Percent haemolysis was calculated as: ¼ [(A540 in the test A540 in PBS)/(A540 in 0.1% Triton-X 100 A540 in PBS) 100. All experiments were done in triplicate and the average values were given as percent haemolysis. 4.1.13. Measurement of reactive oxygen species (ROS) production Fluorescence based assays such as 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine123 (DHR123) staining were used to monitor the generation of reactive oxygen species (ROS) in C. albicans cells after incubation with the diterpene 1. The cell-permeant dye DCFH-DA and DHR123 are oxidized by ascorbic acid, peroxinitrite and hydroxyl radicals (OH ) to yield the fluorescent molecule 20 ,70 -dichlorofluorescein and rhodamine123. 4.1.13.1. DHR123 (dihydrorhodamine) staining [15]. Dihydrorhodamine123 is the reduced form of rhodamine123, commonly used as a fluorescent mitochondrial dye. Dihydrorhodamine123 itself is nonfluorescent, but it readily enters cells and gets oxidized by reactive oxygen species (ROS) to fluorescent rhodamine123 that accumulates in mitochondrial membranes. The DHR123 staining was carried out according to the reported procedure [15]. 1 106 cells of C. albicans NCIM3557 were inoculated in YPG broth containing different concentrations of inhibitor and incubated at 37 C for 200 min. After completion of incubation, cells were harvested by centrifugation and washed with PBS. 5 mg/mL DHR123 was added (from a 2.5 mg/mL stock solution in ethanol) to the cells, suspended in PBS and tubes were further incubated for 30 min. Cells were separated by centrifugation. Cell pellet was washed and resuspended in PBS. Cells were observed for fluorescence with excitation and emission wavelengths of 480 nm and 525 nm respectively. 4.1.13.2. DCFH-DA staining [16]. Amount of ROS generated was measured by fluorometric assay with DCFH-DA. 1107 cells of C. albicans NCIM3557 were inoculated in PBS containing compound and incubated at 37 C for 60 min. After completion of incubation, 10 mM of DCFH-DA was added and incubated for 2 h. Cells were separated by centrifugation. Cell pellet was washed with PBS and resuspended in PBS. The fluorescence intensities (excitation 485 nm and emission 538 nm, respectively) of the resuspended cells were measured with a spectrofluorometer. Conflict of interest Authors declare no conflict of interest. Acknowledgements This work was supported by Council of Scientific and Industrial Research (CSIR), New Delhi sponsored network project (NaPAHA,
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CSC0130). HRC is grateful to CSIR, New Delhi for the award of a Senior Research Fellowship (SRF). Authors are grateful to Dr. A. Sen, Entomology Unit, Division of Organic Chemistry and Dr. S. Tupe, Biochemical Sciences Division, CSIR-NCL, Pune for fruitful discussions and suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.02.054. References [1] (a) M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta, S. Gadhwe, Bioorg. Med. Chem. 20 (2012) 5678; (b) D. Barrett, Biochim. Biophys. Acta 1587 (2002) 224; (c) F.E. Dayan, C.L. Cantrell, S.O. Duke, Bioorg. Med. Chem. 17 (2009) 4022. [2] (a) R.E. Lewis, Mayo Clin. Proc. 86 (2011) 805; (b) Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang, Y. Liu, J. Yao, Z. Miao, W. Zhang, C. Sheng, Eur. J. Med. Chem. 82 (2014) 490; (c) Y. Zou, S. Yu, R. Li, Q. Zhao, X. Li, M. Wu, T. Huang, X. Chai, H. Hu, Q. Wu, Eur. J. Med. Chem. 74 (2014) 366; (d) S. Mert, R. Kasımogulları, T. Iça, F. Çolak, A. Altun, S. Ok, Eur. J. Med. Chem. 78 (2014) 86. [3] (a) A.C. Pasqualotto, D.W. Denning, J. Antimicrob. Chemother. 61 (2008) 119; (b) A.W.K. Ng, K.M. Wasan, G. Llopez-Berestein, J. Pharm. Pharm. Sci. 6 (2003) 67; (c) S.B. Zotchev, Curr. Med. Chem. 10 (2003) 211; (d) R. Nyfeler, W. Keller-Schierlein, Helv. Chim. Acta 57 (1974) 2459; (e) R.E. Schwartz, D.F. Sesin, H. Joshua, K.E. Wilson, A.J. Kempf, K.A. Goklen, D. Kuehner, P. Gailliot, C. Gleason, R. White, E. Inamine, G. Bills, P. Salmon, L. Zitano, J. Antibiot. 45 (1992) 1853; (f) G.F. Bills, G. Platas, F. Palaez, P. Masurekar, Mycol. Res. 103 (1999) 179; (g) G. Keating, D. Figgiti, Drugs 63 (2003) 2235; (h) R.A. Fromtling, Drugs Today 38 (2002) 245; valo, A.-J. Carrillo-Mun ~ oz, J. Salgado, D. Cardenes, S. Brio , (i) M.P. Are s, A. Espinel-Ingroff, J. Antimicrob. Chemother. 51 (2003) 163; G. Quindo (j) G. Maschmeyer, A. Glasmacher, Mycoses 48 (2005) 227. [4] R.E. Schultes, in: W.A.R. Thomson (Ed.), The Kingdom of Plants, in Medicines from the Earth, McGraw-Hill Book Co., New York, 1978, p. 208. [5] (a) T. Arif, J.D. Bhosale, N. Kumar, T.K. Mandal, R.S. Bendre, G.S. Lavekar, R. Dabur, J. Asian Nat. Prod. Res. 11 (2009) 621; (b) C.Y. Chen, F.R. Chang, Y.C. Shih, T.J. Hsieh, Y.C. Chia, H.Y. Tseng, H.C. Chen, S.J. Chen, M.C. Hsu, Y.C. Wu, J. Nat. Prod. 63 (2000) 1475. [6] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, International Book Distributors, Dehradun, India, 1995, p. 562. [7] (a) A.P. Phadnis, S.A. Patwardhan, N.N. Dhaneshwar, S.S. Tavale, T.N. Guru Row, Phytochemistry 27 (1988) 2899; (b) S. Faizi, R.A. Khan, N.R. Mughal, M.S. Malik, K.S. Sajjadi, A. Ahmad, Phytother. Res. 22 (2008) 907; (c) G. Zhao, J.H. Jung, D.I. Smith, K.V. Wood, J.L. McLaughlin, Planta Med. 57 (1991) 380; (d) P. Misra, K.V. Sashidhara, S.P. Singh, A. Kumar, R. Gupta, S.S. Chaudhaery, S. Sen Gupta, H.K. Majumder, A.K. Saxena, A. Dube, Br. J. Pharmacol. 159 (2010) 1143; (e) S. Faizi, R.A. Khan, S. Azher, S.A. Khan, S. Tauseef, A. Ahmad, Planta Med. 69
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Clerodane type diterpene as a novel antifungal agent from Polyalthia longifolia var. pendula Asish K. Bhattacharya a, *, Hemender R. Chand a, Jyothis John b, Mukund V. Deshpande b a b
Division of Organic Chemistry, CSIR-National Chemical Laboratory (CSIR-NCL), Dr. Homi Bhabha Road, Pune 411 008, India Division of Biochemical Sciences, CSIR-National Chemical Laboratory (CSIR-NCL), Dr. Homi Bhabha Road, Pune 411 008, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2014 Received in revised form 26 February 2015 Accepted 27 February 2015 Available online 28 February 2015
Bioactivity-guided chemical examination of methanolic extract of leaves of Polyalthia longifolia var. pendula led to the isolation of the active constituent, a diterpene 1 which was identified as 16ahydroxycleroda-3,13(14)Z-dien-15,16-olide on the basis of its spectral data. Among the tested strains, diterpene 1 was found to exhibit antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 mM against Candida albicans NCIM3557, Cryptococcus neoformans NCIM3542 (human pathogens) and Neurospora crassa NCIM870 (saprophyte), respectively. Initial, structureeactivity-relationship (SAR) data generated by synthesizing some derivatives revealed that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. The mode of action of 1 in C. albicans is due to compromised cell membrane permeability and also probably due to disruption of cell wall structures. The red blood cell haemolysis of all the compounds (1e4) did not show any significant haemolysis and was found to be less than 15% for all the compounds when tested at highest concentration, i.e. 1200 mM. Interestingly, all the tested compounds inhibited YeH transition in dimorphic C. albicans NCIM3557 at much lower concentration than their MIC90 values. Determination of ROS generation by diterpene 1 using DCFH-DA and DHR123 (dihydrorhodamine) staining of C. albicans NCIM3557 indicated production of intracellular ROS as a mechanism of antifungal activity. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Diterpene Polyalthia longifolia Isolation Flash chromatography Natural products Antifungal activity
1. Introduction Susceptibility to fungal pathogens is a common occurrence in both humans and plants; however, in recent years, the problem is aggravated due to development of resistance in several species of fungi to available drugs/fungicides [1]. It is widely believed that more than 75% of all the fungal infections in humans are caused by the Candida species viz. Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis. Research on development of new antifungal agents over the past few decades has resulted in only a few antifungal drugs, which are being clinically used. At present, the drugs being used to treat fungal infections could be broadly classified into five classes of compounds, which include azoles, polyenes, fluoro pyrimidines, echinocandins and allyl amines [2,3a]. However, most of these are of synthetic origin except a few e.g. polyenes [3b and c] and echinocandins [3dej] classes of compounds, which are of natural origin. Furthermore, the resistance
* Corresponding author. E-mail address: [email protected] (A.K. Bhattacharya). http://dx.doi.org/10.1016/j.ejmech.2015.02.054 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
developed by pathogens against these drugs as well as complications due to drug interactions with the host cells, side effects, their poor bioavailability etc. further compromise their wide spread utility. In recent times, several classes of compounds have been isolated from diverse plants, which are reported to have antifungal activities [4,5]. However, these could not be developed into useful antifungal drugs, as some or other drawbacks were associated with these molecules. Hence, there is an urgent need to develop new naturally occurring antifungal agents having target specificity, broadspectrum activity and different mechanism of action than the existing drugs.
2. Results and discussion The genus Polyalthia (Annonaceae) has been credited with seventy species out of which only seven are indigenous to India. Polyalthia longifolia var. pendula Linn. is popularly known as “ulta Ashok” in India and widely grown in the gardens of tropical and subtropical Asia as an evergreen ornamental tree [5]. This plant has
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been reported to be widely used in traditional system of medicine for the treatment of hypertension, fever, diabetes, helminthiasis and skin diseases [6]. The chemical examination of P. longifolia var. pendula has resulted in the isolation of several classes of compounds such as diterpenes, including clerodane and triterpenes and aporphine alkaloids and these have been investigated for various biological activities [7]. In continuation of our work [8] on naturally occurring bioactive secondary metabolites, we initiated systematic chemical examination of P. longifolia var. pendula for its antifungal constituents. The activity-guided fractionation of the crude methanolic extract over flash SiO2 column furnished 6 sub-fractions (AeF). The antifungal assays of these six major fractions against a screen of fungal strains led us to an active fraction (fraction B), which showed promising activities. Further, efforts in isolation of the active secondary metabolite from fraction B by automated flash chromatography using RediSep® column (SiO2, 2 12 g, stacked) resulted in the isolation of a pure compound 1, which was identified as 16ahydroxycleroda-3,13(14)Z-dien-15,16-olide (Fig. 1) on the basis of its spectral data [7d]. Total synthesis of diterpene 1 has been reported in the literature [9]. The isolated pure compound 1 was assayed against five human pathogens such as C. albicans NCIM3557, C. glabrata NCIM3237, Cryptococus neoformans NCIM3542, Aspergilus fumigatus NCIM902, Aspergillus niger NCIM628, C. albicans NCIM3471 (non-pathogenic), phytopathogen, Fusarium oxysporum NCIM1043 and a saprophyte, Neurospora crassa NCIM870 (Table 1). The diterpene 1 showed MIC90 values of 50.3, 100.6 and 201.2 mM against C. albicans NCIM3557, C. neoformans NCIM3542 and N. crassa NCIM870, respectively. The standard antifungal drugs fluconazole and amphotericin B exhibited MIC90 values of 32 and 2 mg/mL against C. albicans NCIM3557, respectively [10]. In order to shed some light on the structureeactivity relationship (SAR) and to deduce information on pharmacophores responsible for the remarkable antifungal activities of the secondary metabolite 1, we undertook some analogue synthesis of the same (Scheme 1). Compound 1 on treatment with Ac2O/pyridine afforded the acetate 2 as a colourless solid, m.p. 134e136 C. The 1H and 13C NMR of this acetate clearly revealed it to be a mixture of acetate isomers due to epimerization [11] at C-16. The epoxidation of diterpene 1 with m-CPBA in DCM afforded the epoxide 3 as a viscous oil, [a]27 D 24.43 (c 0.95, CHCl3). On treatment of compound 1 with Boc2O in presence of TEA and catalytic amount of DMAP in DCM led to formation of Boc derivative 4 which was also found to
Fig. 1. Structure of isolated diterpene 1 from P. longifolia var. pendula.
Table 1 MIC90 values of compound 1. Strains
1 (mM)
Candida albicans NCIM3557 Candida albicans NCIM3471 Candida glabrata NCIM3237 Cryptococcus neoformans NCIM3542 Aspergillus fumigatus NCIM902 Aspergillus niger NCIM628 Fusarium oxysporum NCIM1043 Neurospora crassa NCIM870
50.3 805.0 805.0 100.6 805.0 805.0 805.0 201.2
be a mixture of isomers due to the epimerization [11] at C-16 by the analysis of its 1H and 13C NMR spectra. When we tried to reduce the double bond between C3eC4 (or eventually C13eC14 also) with 10% PdeC/H2 in EtOAc, even after 5d we could get only a complex mixture which could not be separated using flash chromatography. The three semi-synthetic derivatives 2e4 of the diterpene 1 were assayed against all the test organisms. However, the MIC90 values of synthesized derivatives (2e4) were found to be >600 mM against all the strains. The preliminary structureeactivity-relationship (SAR) studies from the assay of synthesized derivatives (2e4) indicate that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. However, the complete SAR remains to be concluded with the synthesis of some more derivatives/analogues of 1. Further, it was necessary to evaluate the natural product 1 and its semi-synthetic derivatives (2e4) for their haemolytic potential on red blood cells (RBCs) [12]. The compounds (1e4) were incubated with RBCs and release of haemoglobin due to the RBC lysis was measured (Fig. 2). It is pertinent to mention here that at the tested concentrations closer to the MIC of C. albicans (NCIM3557), none of the compounds exhibited any significant haemolysis. The red blood cell haemolysis was found to be less than 15% for all the compounds (1e4) when tested at highest concentration, i.e. 1200 mM. In several human and plant pathogenic fungi, direct correlation between the ability of fungus to switch between yeast (Y) and hypha (H) forms and pathogenicity has been reported [13]. Hence, all the compounds (1e4) were tested for their Y to H transition inhibition. All the tested compounds inhibited YeH transition in C. albicans NCIM3557 at much lower concentration than their MIC90 values (Table 2). Propidium iodide (PI) has been found to be an excellent membrane impermeable nucleic acid staining fluorescent dye [14a]. PI enters the cells with compromised permeability only and it binds to the double stranded nucleic acids thus producing a red fluorescence when excited at 480 nm. We studied the uptake of PI by Candida cells in the presence of the natural diterpene 1 at two concentrations, 50.3 mM and 100.6 mM by epifluorescence microscopy (Fig. 3) and at three concentrations, 25.2 mM, 50.3 mM (MIC90) and 100.6 mM by Confocal microscopy (Fig. 4) [14b]. In order to deduce antifungal mechanism of our natural diterpene 1, we studied the generation of reactive oxygen species (ROS) by compound 1 since the generation of reactive oxygen species (ROS) is considered to be associated with apoptosis (a programmed cell death) and necrosis. Hence, we studied ROS generation by diterpene 1 using DHR123 (dihydrorhodamine) [15] (Fig. 5) and DCFH-DA staining [16] (Fig. 6). C. albicans NCIM3557 cells were incubated with different concentrations (50.3, 100.6 and 201.2 mM) of diterpene 1, and then treated with DHR123 for 30 min. Fluorescence resulting from oxidation of the dye DHR123 were observed at 525 nm. It was found that with increasing concentration of compound 1, the relative fluorescence increased (Fig. 5). This
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3
Scheme 1. Preparation of derivatives of 1. Reagents and conditions: (i) Py, Ac2O, rt, 24 h; (ii) mCPBA, DCM, 1.5 h; (iii) Boc2O, TEA, DMAP (cat), DCM, 0 C, 2 h; (iv) 10% Pd/C, H2 (1 atm), EtOAc, rt, 5d.
Fig. 2. Haemolytic activity of compounds (1e4).
Table 2 Effect of compounds on yeastehypha transition in Candida albicans NCIM3557. Compound
Inhibition of 50% germ tube formation in C. albicans (mM)
1 2 3 4
25.14 177.80 191.60 153.10
clearly indicated generation of intracellular ROS. Cells without the compound served as negative control whereas cells with H2O2 served as positive control. Similarly, C. albicans NCIM3557 cells were incubated at different concentrations (25.2 mM, 50.3 mM and 100.6 mM) of diterpene 1 and subsequently incubated with DCFH-DA. Fluorescent cells resulting from oxidation of dye DCFH-DA were observed under epifluorescence microscope using I3 filter. Cells treated with diterpene 1 expressed fluorescence at all the three concentrations (25.2 mM, 50.3 mM and 100.6 mM) with an increase in relative fluorescence at increasing concentration thereby indicating production of
Fig. 3. Fluorescence microscope images of membrane permeabilization by propidium iodide (PI) uptake: (a) Untreated control (without compound 1); (b) positive control (heat-killed); (c) C. albicans (NCIM3557) cells with compound 1 (50.3 mM); (d) C. albicans (NCIM3557) cells with compound 1 (100.6 mM).
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Fig. 4. Confocal laser scanning microscopy (CLSM) images of membrane permeabilization by propidium iodide (PI) uptake: (a) Untreated control (without compound 1); (b) positive control (heat-killed); (c) C. albicans (NCIM3557) cells with compound 1 (25.2 mM); (d) C. albicans (NCIM3557) cells with compound 1 (50.3 mM); (e) C. albicans (NCIM3557) cells with compound 1 (100.6 mM).
intracellular ROS. Cells without the compound served as the negative control (Fig. 6). 3. Conclusions We have isolated a diterpene 1 identified as 16a-hydroxycleroda-3,13(14)Z-dien-15,16-olide using automated flash chromatography without following solventesolvent extraction protocols from the methanolic extract of the leaves of P. longifolia var. pendula. The isolated diterpene 1 was assayed against five human pathogens (C. albicans NCIM3557, C. glabrata NCIM3237, C. neoformans NCIM3542, A. fumigatus NCIM902 and A. niger NCIM628), C. albicans NCIM3471 (non-pathogenic), phytopathogen, F. oxysporum NCIM1043 and N. crassa NCIM870 (saprophyte). The diterpene 1 exhibited considerable antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 mM against C. albicans NCIM3557, C. neoformans NCIM3542 and N. crassa NCIM870, respectively. Our initial structureeactivity-relationship (SAR) studies from the assay of synthesized derivatives (2e4) indicate that the double bond between C3eC4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. Further, we have demonstrated that the natural product 1 has a broad spectrum fungicidal activity with its mode of action in C. albicans due to compromised cell membrane permeability (thereby enabling enhanced entry into the cells) and also probably due to disruption of cell wall structures. In addition, all the compounds (1e4) exhibit less than 15% haemolysis of red blood cells even when tested at highest concentration, i.e. 1200 mM. Moreover, all the tested compounds inhibited YeH transition in a dimorphic C. albicans at much lower concentration than their MIC90 values. Further, intracellular ROS generation by diterpene 1, confirmed by using DCFH-DA and DHR123 staining of C. albicans NCIM3557 cells, suggests mechanism of antifungal activity of diterpene 1. It is presumed that our studies on this molecule could be further utilized for target-based approach to explore its therapeutic potentials.
Fig. 5. Determination of ROS levels by DHR123 (dihydrorhodamine) staining in the presence of different concentrations of the diterpene 1 in C. albicans (NCIM3557) cells by epifluorescence microscopy: (a) Control (without compound 1); (b) positive control with H2O2; (c) with compound 1 (50.3 mM); (d) with compound 1 (100.6 mM); (e) with compound 1 (201.2 mM).
4. Experimental section 4.1. General The FT-IR spectra were recorded on an FT-IR-8300 Shimadzu spectrometer. NMR spectra were recorded on Bruker ACF 200 and AV200 (200 MHz for 1H NMR and 50 MHz for 13C NMR) and AV400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometers, using CDCl3 as solvent. Tetramethylsilane (0.00 ppm) served as an internal standard in 1H NMR and CDCl3 (77.0 ppm) in 13C NMR, respectively. Chemical shifts are expressed in parts per million (ppm). Mass spectra were recorded on LC-MS/MS-TOF API QSTAR PULSAR spectrometer, samples introduced by infusion method using the Electrospray Ionization Technique (ESI). HRMS (ESI) of samples was taken on an Orbitrap (quadrupole plus ion trap) and TOF mass analyser. Flash chromatography was performed with CombiFlash Rf 200i with UV/VIS and ELSD, Isco Teledyne Inc., USA using RediSep® column (SiO2). All other chemicals were of analytical grade. Epifluorescence microscopy was done using Epifluorescence Microscope, Leitz Laborlux, Germany. The laser scanning
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5
3H), 0.79 (s, 3H); dC (100 MHz, CDCl3) 171.7, 170.6, 144.3, 120.4, 117.1, 99.9, 46.5, 38.7, 38.2, 36.7, 36.3, 34.8, 27.4, 26.8, 21.4, 20.0, 18.3, 18.2, 18.0, 16.0; ESI-MS m/z 341.1985 (M þ Na)þ; HRMS (ESI) calcd for C20H30O3Na 341.2087, found 341.2089. 4.1.4. Preparation of acetate derivative (2) To a solution of compound 1 (21 mg, 0.066 mmol) in pyridine (0.3 mL), Ac2O (0.6 mL) was added and left at ambient temp for overnight. Toluene (3 5 mL) was then added to remove pyridine and Ac2O by co-distillation on rotary evaporator under reduced pressure. Crude product on complete dryness followed by flash chromatography on RediSep® column (SiO2, 12 g) eluting with DCM (isocratic) afforded compound 2 as a colourless solid (20 mg, 84%), which was a mixture of acetate isomers due to epimerization at C16 [11]. M.p. 134e136 C; Rf 0.39 (MeOH-DCM, 1:9); [a]27 D 19.56 (c 0.9, CHCl3); ymax (CHCl3)/cm1 3752, 2967, 2927, 1757, 1649, 1454, 1379, 1212, 1053, 982, 756; dH (200 MHz, CDCl3) 6.84 (s, 1H), 5.94 (s, 1H), 5.19 (brs, 1H), 2.38e2.22 (m, 1H), 2.18e2.14 (m, 4H), 2.11e1.93 (m, 2H), 1.77e1.64 (m, 2H), 1.60 (m, 5H), 1.53e1.39 (m, 6H), 1.01 (s, 3H), 0.82 (t, J ¼ 3.0 Hz, 3H), 0.77 (s, 3H); dC (100 MHz, CDCl3) 170.0, 169.2, 168.2, 168.1, 144.5, 144.5, 120.3, 118.2, 118.1, 93.9, 93.9, 46.6, 46.6, 38.7, 38.2, 36.7, 36.4, 35.0, 34.9, 27.4, 26.9, 26.9, 21.3, 21.2, 20.8, 20.0, 18.4, 18.3, 18.1, 16.1, 16.0; ESI-MS m/z 383.01 (M þ Na)þ; HRMS (ESI) calcd for C22H32O4Na 383.2193, found 383.2191.
Fig. 6. Determination of ROS levels by DCFH-DA staining in the presence of different concentrations of the diterpene 1 in C. albicans (NCIM3557) cells by epifluorescence microscopy: (a) Control (without compound 1); (b) with compound 1 (25.2 mM); (c) with compound 1 (50.3 mM); (d) with compound 1 (100.6 mM).
confocal microscopy was performed on a Zeiss LSM 710 Microscope. 4.1.1. Plant material Plant leaves were collected from the garden maintained at CSIRNCL, Pune during the month of June, 2010. 4.1.2. Extraction and isolation Air-dried and grounded leaves (100 g) of P. longifolia var. pendula were extracted with MeOH (5 1.0 L) at room temperature for five days. After completion of the extraction, the solvent was evaporated under reduced pressure to afford the MeOH extract (27.2 g). A portion of the MeOH extract (5.2 g) was fractionated on SiO2 (200 g, 230e400 mesh) column eluting with DCM: MeOH (0 / 20%) to furnish 6 sub-fractions (AeF). The compound 1 was present in DCM: MeOH (99:1) fraction (fraction B). The fraction B (1.250 g) was flash chromatographed on CombiFlash Companion, Isco Teledyne Inc., USA using RediSep® column (SiO2, 2 12 g, stacked together) and isocratic elution was done with DCM to furnish the pure compound 1 (270 mg) with 1.4% overall yield. 4.1.3. Spectral data [7d] of 16a-hydroxycleroda-3,13(14)Z-dien15,16-olide (1) Rf 0.36 (MeOH-DCM, 9:1); [a]27 D 42.57 (c 1.49, MeOH); ymax (CHCl3)/cm1 3376, 2930, 1748, 1650, 1459, 1386, 1130, 948, 755; dH (400 MHz, CDCl3) 6.07 (s, 1H), 5.85 (s, 1H), 5.20 (brs, 1H), 2.37e2.20 (m, 2H), 2.10e2.01 (m, 2H), 1.76e1.65 (m, 2H), 1.60 (s, 3H), 1.57e1.43 (m, 6H), 1.37e1.19 (m, 5H), 1.02 (s, 3H), 0.83 (d, J ¼ 6.5 Hz,
4.1.5. Preparation of epoxide (3) To a stirred solution of compound 1 (43 mg, 0.135 mmol) in DCM (4 mL) cooled in ice-water bath, was added m-CPBA (33 mg, 0.189 mmol, 1.4 eq.). After stirring for 1.5 h, DCM (10 mL) was added and the organic layer was separated, washed with aq. 10% KI solution (2 20 mL), followed by 10% Na2S2O3 solution (2 20 mL), 10% NaHCO3, (2 20 mL) solution and then finally with H2O (2 20 mL). The organic layer was then dried (anhydrous Na2SO4), evaporated in vaccuo, which on flash chromatography on (RediSep® SiO2 column, 12 g) eluting with 2% MeOH-DCM (isocratic) furnished compound 3 as a viscous oil (25 mg, 45%). Rf 0.57 1 (MeOH-DCM, 1:9); [a]27 D 24.43 (c 0.95, CHCl3); ymax (CHCl3)/cm 3369, 2926, 2857, 1752, 1648, 1577, 1560, 1458, 1141, 951, 757; dH (200 MHz, CDCl3) 6.00 (s, 1H), 5.82 (s, 1H), 2.94 (m, 1H), 2.41e2.18 (m, 2H), 1.94e1.86 (m, 2H), 1.74e1.57 (m, 4H), 1.46e1.37 (m, 5H), 1.53e1.37 (m, 5H), 1.01 (s, 3H), 0.80 (3H), 0.73 (s, 3H); dC (100 MHz, CDCl3) 171.8, 170.8, 117.0, 99.4, 67.0, 61.4, 47.9, 39.2, 38.5, 37.9, 36.1, 34.6, 26.8, 21.5, 21.4, 21.4, 18.3, 17.7, 16.3, 16.0; ESI-MS m/z 357.08 (M þ Na)þ; HRMS (ESI) calcd for C20H30O4Na 357.2036, found 357.2034. 4.1.6. Preparation of Boc derivative (4) Compound 1 (31 mg, 0.097 mmol) was dissolved in DCM (4 mL) and cooled in ice-water bath, Boc2O (80 mg, 0.37 mmol, 3.7 eq.) was then added followed by TEA (10 mL, 0.074 mmol) and catalytic amount of DMAP (2 mg, 0.016 mmol) and reaction mixture was stirred for 2 h. After completion of the reaction (TLC), the reaction mixture was then evaporated in vaccuo and directly subjected to flash chromatography (RediSep® SiO2 column, 12 g) eluting with EtOAc:petroleum ether (1:19) to furnish the pure compound 4 which was obtained as a viscous oil (33 mg, 80%) and found to be an inseparable mixture of C-16 epimers [11]. Rf 0.62 (DCM); 1 [a]27 3683, 3618, 3440, D 27.15 (c 1.0, CHCl3); ymax (CHCl3)/cm 3020, 2928, 2856, 2400, 1799, 1765, 1649, 1458, 1373, 1256, 1216, 757, 669; dH (500 MHz, CDCl3) 6.66 (d, J ¼ 8.2 Hz, 1H), 5.93 (s, 1H), 5.20 (s, 1H), 2.37e2.27 (m, 2H), 2.25e2.21 (m, 1H), 2.12e1.98 (m, 5H), 1.75e1.64 (m, 4H), 1.59 (m, 4H), 1.54e1.53 (m, 9H), 1.50e1.44 (m, 9H), 1.01 (s, 3H), 0.81 (m, 3H), 0.77 (s, 3H); dC (125 MHz, CDCl3) 169.8, 167.6, 167.5, 151.5, 144.5, 144.4, 120.4, 120.3, 118.2, 118.1, 104.4, 96.4, 84.8, 84.7, 46.5, 46.5, 38.7, 38.7, 38.2, 36.7, 36.4, 36.3, 34.9,
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34.8, 31.9, 29.6, 29.4, 29.3, 27.6, 27.3, 26.8, 26.8, 21.3, 21.2, 19.9, 18.3, 18.2, 18.0, 16.0, 15.9; ESI-MS m/z 441.08 (M þ Na)þ; HRMS (ESI) calcd for C25H38O5Na 441.2611, found 441.2612. 4.1.7. Reduction of diterpene (1) Compound 1 (18 mg, 0.056 mmol) was dissolved in EtOAc (3 mL) and PdeC (10%, 9 mg) was added to the reaction mixture. Stirring was continued at rt under an atmosphere of hydrogen for 5d. After 5d, the catalyst was filtered off over a celite bed and the reaction mixture was then evaporated in vaccuo. The reaction mixture showed a complex TLC pattern (30% EtOAc-PE), which could not be further purified using flash chromatography (RediSep® SiO2 column, 4 g) eluting with petroleum ether: EtOAc (0 / 30, gradient). 4.1.8. Antifungal assays Fungi growth conditions: Human and plant pathogenic fungal strains, C. albicans NCIM3557, C. albicans NCIM3471, C. glabrata NCIM3237, C. neoformans NCIM3542, A. niger NCIM628, A. fumigatus NCIM902, F. oxysporum NCIM1043 and a saprophyte model, N. crassa NCIM870 were obtained from National Collection of Industrial Microorganisms (NCIM), CSIR-National Chemical Laboratory, Pune, India. The human pathogenic fungal strains were maintained on slants of YPG agar (yeast extract, 0.3%; peptone, 0.5%; glucose, 1.0%; agar, 2.0%) and the plant pathogenic fungi were maintained on potato dextrose agar (2% PDA) slants at 28 C and sub-cultured every 15 days. During experimentation, the fungal strains were grown in YPG broth. 4.1.9. Minimum inhibitory concentration (MIC) determination The purified final compounds were evaluated for antifungal susceptibility testing by microbroth dilution method according to the recommendations of the CLSI [17]. Appropriate amount of test compounds were dissolved in DMSO to get 100 final strength. The stock was then diluted 1:40 in YPG medium and 200 mL from this was added to the first row of a 96-well microtiter plate. The compound was serially diluted two fold in successive wells to get a range of 4e512 mg/mL. Fungal yeast cells (~2 104 cfu/mL, spores for phytopathogens), freshly grown in YPG broth in logarithmic phase, were suspended in the medium and inoculated (100 mL) in the wells of the plate. The microtiter plates were incubated for 24e48 h, and the absorbance was measured at 600 nm by using microtiter plate reader to measure the cell growth. The MIC was defined as the lowest concentration required for >90% inhibition of growth with respect to the growth in control and IC50 was the concentration at which 50% growth inhibition was observed. 4.1.10. Membrane integrity assay Propidium iodide (PI) staining was used for checking integrity of fungal plasma membrane following treatment with diterpene 1. C. albicans NCIM3557 cells were harvested at the logarithmic phase and 1 106 cells/mL were added in phosphate buffer saline (PBS, 0.1 mM, pH 7.2), containing inhibitor. The tubes were incubated at 37 C for 2 h. Cells were separated by centrifugation and washed with PBS. Cells were then incubated with 3 mM of PI for 10 min, harvested by centrifugation, washed (using PBS) and suspended in PBS. PI stained cells were counted using epifluorescence microscope (Leitz Laborlux, Germany). A filter set, N 2.1 filter block with excitation filter BP 515-560 and emission filter LP 580 were used. 4.1.11. Confocal microscopy The effect of compound 1 on membrane integrity of fungal cell was confirmed by confocal microscopy using PI [14b]. The C. albicans (NCIM3557) cells (~1 106 CFU/ml) growing in log phase were suspended in an RPMI-1640 medium containing diterpene 1 at its MICs (50.3 mM) and PI (3 mM). The mixture was
incubated at 37 C for 2 h at 180 rpm. The cells were harvested by centrifugation and resuspended in PBS (pH 7.4). The cells were observed under confocal microscope with a wavelength N560 nm for PI. All images were captured at 400 magnification. 4.1.12. Cellular toxicity assay The cellular toxicity of compounds was determined by red blood cells (RBC) lysis assay [12]. In brief, the RBCs of sheep blood were washed with 2e3 times with PBS (pH 7.0) and finally RBCs were resuspended in PBS so as to obtain the 4% solution. Then, 1000 mL of PBS containing the appropriate concentration of test compound was mixed with 1000 mL of 4% RBC suspension and incubated at 37 C for 2 h. No haemolysis and 100% haemolysis were observed in PBS and 0.1% Triton-X 100, respectively. The reaction mixture was centrifuged at 2000 rpm for 5 min and the absorbances of supernatant were read at 545 nm. Percent haemolysis was calculated as: ¼ [(A540 in the test A540 in PBS)/(A540 in 0.1% Triton-X 100 A540 in PBS) 100. All experiments were done in triplicate and the average values were given as percent haemolysis. 4.1.13. Measurement of reactive oxygen species (ROS) production Fluorescence based assays such as 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine123 (DHR123) staining were used to monitor the generation of reactive oxygen species (ROS) in C. albicans cells after incubation with the diterpene 1. The cell-permeant dye DCFH-DA and DHR123 are oxidized by ascorbic acid, peroxinitrite and hydroxyl radicals (OH ) to yield the fluorescent molecule 20 ,70 -dichlorofluorescein and rhodamine123. 4.1.13.1. DHR123 (dihydrorhodamine) staining [15]. Dihydrorhodamine123 is the reduced form of rhodamine123, commonly used as a fluorescent mitochondrial dye. Dihydrorhodamine123 itself is nonfluorescent, but it readily enters cells and gets oxidized by reactive oxygen species (ROS) to fluorescent rhodamine123 that accumulates in mitochondrial membranes. The DHR123 staining was carried out according to the reported procedure [15]. 1 106 cells of C. albicans NCIM3557 were inoculated in YPG broth containing different concentrations of inhibitor and incubated at 37 C for 200 min. After completion of incubation, cells were harvested by centrifugation and washed with PBS. 5 mg/mL DHR123 was added (from a 2.5 mg/mL stock solution in ethanol) to the cells, suspended in PBS and tubes were further incubated for 30 min. Cells were separated by centrifugation. Cell pellet was washed and resuspended in PBS. Cells were observed for fluorescence with excitation and emission wavelengths of 480 nm and 525 nm respectively. 4.1.13.2. DCFH-DA staining [16]. Amount of ROS generated was measured by fluorometric assay with DCFH-DA. 1107 cells of C. albicans NCIM3557 were inoculated in PBS containing compound and incubated at 37 C for 60 min. After completion of incubation, 10 mM of DCFH-DA was added and incubated for 2 h. Cells were separated by centrifugation. Cell pellet was washed with PBS and resuspended in PBS. The fluorescence intensities (excitation 485 nm and emission 538 nm, respectively) of the resuspended cells were measured with a spectrofluorometer. Conflict of interest Authors declare no conflict of interest. Acknowledgements This work was supported by Council of Scientific and Industrial Research (CSIR), New Delhi sponsored network project (NaPAHA,
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CSC0130). HRC is grateful to CSIR, New Delhi for the award of a Senior Research Fellowship (SRF). Authors are grateful to Dr. A. Sen, Entomology Unit, Division of Organic Chemistry and Dr. S. Tupe, Biochemical Sciences Division, CSIR-NCL, Pune for fruitful discussions and suggestions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.02.054. References [1] (a) M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta, S. Gadhwe, Bioorg. Med. Chem. 20 (2012) 5678; (b) D. Barrett, Biochim. Biophys. Acta 1587 (2002) 224; (c) F.E. Dayan, C.L. Cantrell, S.O. Duke, Bioorg. Med. Chem. 17 (2009) 4022. [2] (a) R.E. Lewis, Mayo Clin. Proc. 86 (2011) 805; (b) Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang, Y. Liu, J. Yao, Z. Miao, W. Zhang, C. Sheng, Eur. J. Med. Chem. 82 (2014) 490; (c) Y. Zou, S. Yu, R. Li, Q. Zhao, X. Li, M. Wu, T. Huang, X. Chai, H. Hu, Q. Wu, Eur. J. Med. Chem. 74 (2014) 366; (d) S. Mert, R. Kasımogulları, T. Iça, F. Çolak, A. Altun, S. Ok, Eur. J. Med. Chem. 78 (2014) 86. [3] (a) A.C. Pasqualotto, D.W. Denning, J. Antimicrob. Chemother. 61 (2008) 119; (b) A.W.K. Ng, K.M. Wasan, G. Llopez-Berestein, J. Pharm. Pharm. Sci. 6 (2003) 67; (c) S.B. Zotchev, Curr. Med. Chem. 10 (2003) 211; (d) R. Nyfeler, W. Keller-Schierlein, Helv. Chim. Acta 57 (1974) 2459; (e) R.E. Schwartz, D.F. Sesin, H. Joshua, K.E. Wilson, A.J. Kempf, K.A. Goklen, D. Kuehner, P. Gailliot, C. Gleason, R. White, E. Inamine, G. Bills, P. Salmon, L. Zitano, J. Antibiot. 45 (1992) 1853; (f) G.F. Bills, G. Platas, F. Palaez, P. Masurekar, Mycol. Res. 103 (1999) 179; (g) G. Keating, D. Figgiti, Drugs 63 (2003) 2235; (h) R.A. Fromtling, Drugs Today 38 (2002) 245; valo, A.-J. Carrillo-Mun ~ oz, J. Salgado, D. Cardenes, S. Brio , (i) M.P. Are s, A. Espinel-Ingroff, J. Antimicrob. Chemother. 51 (2003) 163; G. Quindo (j) G. Maschmeyer, A. Glasmacher, Mycoses 48 (2005) 227. [4] R.E. Schultes, in: W.A.R. Thomson (Ed.), The Kingdom of Plants, in Medicines from the Earth, McGraw-Hill Book Co., New York, 1978, p. 208. [5] (a) T. Arif, J.D. Bhosale, N. Kumar, T.K. Mandal, R.S. Bendre, G.S. Lavekar, R. Dabur, J. Asian Nat. Prod. Res. 11 (2009) 621; (b) C.Y. Chen, F.R. Chang, Y.C. Shih, T.J. Hsieh, Y.C. Chia, H.Y. Tseng, H.C. Chen, S.J. Chen, M.C. Hsu, Y.C. Wu, J. Nat. Prod. 63 (2000) 1475. [6] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, International Book Distributors, Dehradun, India, 1995, p. 562. [7] (a) A.P. Phadnis, S.A. Patwardhan, N.N. Dhaneshwar, S.S. Tavale, T.N. Guru Row, Phytochemistry 27 (1988) 2899; (b) S. Faizi, R.A. Khan, N.R. Mughal, M.S. Malik, K.S. Sajjadi, A. Ahmad, Phytother. Res. 22 (2008) 907; (c) G. Zhao, J.H. Jung, D.I. Smith, K.V. Wood, J.L. McLaughlin, Planta Med. 57 (1991) 380; (d) P. Misra, K.V. Sashidhara, S.P. Singh, A. Kumar, R. Gupta, S.S. Chaudhaery, S. Sen Gupta, H.K. Majumder, A.K. Saxena, A. Dube, Br. J. Pharmacol. 159 (2010) 1143; (e) S. Faizi, R.A. Khan, S. Azher, S.A. Khan, S. Tauseef, A. Ahmad, Planta Med. 69
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