Govanoside A, a new steroidal saponin from rhizomes of Trillium govanianum

Steroids xxx (2015) xxx–xxx

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Govanoside A, a new steroidal saponin from rhizomes of Trillium govanianum Shafiq-ur-Rahman a,b,c,⇑, Muhammad Ismail a,⇑, Muhammad Raza Shah c, Achyut Adhikari c, Itrat Anis d, Malik Shoaib Ahmad c, Muhammad Khurram b a

Department of Pharmacy, University of Peshawar, Peshawar 25120, Pakistan Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal, Dir (U) 18000, Pakistan H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan d Department of Chemistry, University of Karachi, Karachi 75270, Pakistan b c

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 8 October 2015 Accepted 20 October 2015 Available online xxxx Keywords: Trillium govanianum Trilliaceae Spirostane glycosides Govanoside A Antifungal

a b s t r a c t A new spirostane steroidal saponin, govanoside A (1) along with three known compounds borassoside E (2) pennogenin (3) and diosgenin (4) were isolated from rhizomes of Trillium govanianum. Their structures were elucidated through 1D, 2D-NMR spectroscopic data analysis and acid hydrolysis. Compound (2) in genus Trillium and all compounds (1–4) in T. govanianum are reported herein for the first time. Furthermore, compounds 1 & 2 exhibited good to moderate activities against Aspergillus niger ATCC 16888, Aspergillus flavus ATCC 9643, Candida albicans ATCC 18804, and Candida glabrata ATCC 90030. This is a significant finding keeping in view the limited antifungal drugs for aspergillosis and candidiasis. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Trillium govanianum Wall, belongs to family Trilliaceae and is primarily distributed in South Asia from Pakistan to Bhutan between the altitudinal ranges of 2500–3800 m [1,2]. The rhizomes of T. govanianum are called nag chatri (Hindi), matar zela (pushtu), and are used traditionally in folk medicine for dysentery, healing of wounds, inflammation, antiseptic, boils, menstrual and sexual disorders [3–5]. The genus Trillium is comprised of long lived herbaceous flowering plants. Their different species are widely distributed throughout the world, and species reported from Pakistan is T. govanianum [6]. The North American species of Trillium are known to have uterine stimulant, antimicrobial, antifungal, and antibacterial properties [7–9]. So far a number of steroids, steroidal glycosides and steroidal saponins have been isolated from the different species of Trillium [10,11]. However, literature survey concerning the secondary metabolites of T. govanianum showed that no systematic chemical work has been carried out on this plant specie, therefore our attention was ⇑ Corresponding authors at: Department of Pharmacy, University of Peshawar, Peshawar 25120, Pakistan (Shafiq-ur-Rahman). E-mail addresses: [email protected] ( Shafiq-ur-Rahman), m_ismail@ upesh.edu.pk (M. Ismail).

directed to the chemical constituents of the underground parts of T. govanianum, on which a detailed phytochemical investigation was carried out. This paper presents the isolation and characterization of a new spirostane saponin (1), along with three (2–4) known compounds. Structural determinations of these compounds were carried out on the basis of modern spectroscopic analysis, including HRFAB-MS, 1D and 2D-NMR. The antifungal activity of isolated compounds is also discussed.

2. Experimental 2.1. General experimental procedures The 1H-, 13C- and 2D-NMR spectra were recorded on Bruker Avance 600 MHz NMR spectrometers. Specific rotations were measured on a JASCO P-2000 polarimeter. The FAB spectrum was recorded on a JEOL TMS-HX110 (Japan) Mass Spectrometer. Column Silica gel (E. Merck, 70–230 mesh) and flash silica gel (E. Merck, Germany, 230–400 mesh) was used for column chromatography. TLC was carried out on pre-coated silica gel F254 aluminum sheets (0.25 mm thickness). TLCs spots were visualized by heating after spraying with 1% Ce(SO4)2 – 10% aqueous H2SO4.

http://dx.doi.org/10.1016/j.steroids.2015.10.013 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Shafiq-ur-Rahman et al., Govanoside A, a new steroidal saponin from rhizomes of Trillium govanianum, Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.10.013

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Shafiq-ur-Rahman et al. / Steroids xxx (2015) xxx–xxx Table 1 H-NMR and

1

13

C-NMR (MeOH, 600 & 150 MHz) chemical shift assignments in compound 1.

C. No.

dC

dH (J, Hz)

C. No. 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

84.5 37.5 69.2 43.3 139.5 126.1 32.6 34.2 51.0 43.4 24.8 40.9 41.7 57.9 33.2 84.4 58.8 17.2 15.4 46.6 62.9 112.1 72.1 83.5 144.5 62.1

27

114.0

3.51 overlap 1.71, 2.09 m 3.39 m 2.22, 2.24 m – 5.55 br d (5.4) 1.55, 1.96 1.54 m 1.38 m – 1.41, 2.46 1.19, 1.69 m – 1.22 m 1.54, 1.98 m 4.56 q (7.2) 1.82 dd (7.8, 6.6) 0.91 s 1.08 s 2.71 dd (7.2, 6.6) 3.52, 3.63 overlap – 3.74 d (4.2) 4.26 d (4.2) – 3.71 d (12.0), 4.46 d (12.0) 4.98 br s 5.08 br s

2.2. Plant material Rhizomes of T. govanianum were collected from Khyber Pakhtunkhwa, Dir Upper, Kohistan valley (34° 540 and 35° 520 North latitudes and 72° 430 and 73° 570 East longitudes), in August, 2013. The plant was identified by Mr. Ghulam Jelani (Curator), Department of Botany, University of Peshawar. A voucher specimen [No. Bot. 20092 (PUP)] has been deposited in the herbarium, Department of Botany, University of Peshawar, Pakistan for reference. 2.3. Extraction and isolation The shade-dried rhizomes of T. govanianum (07 kg) was ground and extracted with MeOH (40 L) at room temperature, three times for a period of seven days (3  40 L). The combined methanolic extract was evaporated to dryness to yield brownish gummy residue (512 g), which was further fractionated (solid–liquid partition) into hexane (HF, 81 g), chloroform (CF, 94 g), ethyl acetate (EF, 85 g) and n-butanol (BF, 115 g). The chloroform soluble fraction (74 g) was subjected to column chromatography over silica gel and the elution was carried out with mixtures of hexane, chloroform, EtOAc and MeOH in increasing order of polarity to yield seventeen fractions (CFA–CFR). The sub fraction (CFB, 728 mg) obtained from 20% EtOAc/chloroform were re-chromatographed over silica gel eluting with mixture of EtOAc and hexane in increasing order of polarity yielded compound 4 (diosgenin, 94 mg, 20% EtOAc/ hexane) and compound 3 (pennogenin, 21 mg, 25% EtOAc/hexane). The n-butanol soluble fraction (BF, 30 g) was subjected to column chromatography over silica gel and the elution was carried out with mixtures of EtOAc and MeOH in increasing order of polarity to yield five fractions (BFA–BFE). The sub fraction, BFA (322 mg) which was obtained with 10% MeOH/EtOAc was re-chromatographed over silica gel eluted with mixture of MeOH and EtOAc in increasing order of polarity afforded compound 2

1-O-b-D-Glc. 1 20 30 40 50 60 30 -O-b-D-Glc. 100 200 300 400 500 600 600 -O-b-Api. 1000 2000 3000 4000 5000 24-O-6-deoxy-b-D-Gul 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 4000 0 -O-a-L-Rha 1000 00 2000 00 3000 00 4000 00 5000 00 6000 00

dC

dH (J, Hz)

100.2 77.3 88.6 69.8 78.1 63.6 105.3 74.8 78.0 71.8 77.5 67.1 112.6 77.5 65.4 80.0 75.0

4.40 d (7.2) 3.50 m 3.66 m 3.37 m 3.25 m 3.60, 3.69 4.38 d (7.2) 3.51 m 3.67 m 3.37 m 3.27 m 3.60, 3.73 5.18 d (2.4) 3.99 d (3.0) 3.35s – 3.76 s, 3.78 d

103.4 70.2 70.9 80.3 70.7 16.1

4.72 3.64 3.90 3.45 4.10 1.10

d (8.4) m m m m d (6.6)

101.6 72.8 72.1 73.4 69.9 18.7

5.37 3.90 3.67 3.41 4.13 1.24

br s m m m (9.6, 6) d (6.6)

(borassoside E, 48 mg, 5% MeOH/EtOAc). The sub fraction (BFB, 492 mg) was re-chromatographed over silica gel, eluted with mixture of MeOH and EtOAc in increasing order of polarity yielded sub fractions (BFBa–BFBe). The sub fraction, BFBc (198 mg) which was obtained with 30% MeOH/EtOAc was re-chromatographed over silica gel eluted with mixture of MeOH and EtOAc in increasing order of polarity yielded compound 1 (govanoside A, 32 mg, 20% MeOH/EtOAc). 2.3.1. Govanoside A (1) White powder; (32 mg); [a]25 D = 139° (c = 0.50, MeOH); HR-FAB-MS (Positive): 1225.54 (calcd 1224.54, C56H88O29); IR (KBr): 3420 cm1 (OH). M.P = 276–281 °C; 1H-NMR (MeOD, 600 MHz); Table 1; 13C-NMR (MeOD, 150 MHz); Table 1. 2.3.2. Borassoside E (2) White powder; (48 mg); [a]25 D = 47.2° (c = 0.50, MeOH); HR-FAB-MS (Negative): 867.46 (calcd 868.48, C45H72O16); IR (KBr): 3409 cm1 (OH); M.P = 263–266 °C; 1H-NMR (MeOD, 600 MHz); Table 2; 13C-NMR (MeOD, 150 MHz); Table 2. 2.3.3. Pennogenin (3) Colorless crystalline solid; (21 mg); [a]25 D = 99.8° (c = 0.70, MeOH); FAB-MS (Positive): 431 (calcd 430.31, C27H42O4); IR (KBr): 3490 cm1 (OH); M.P = 206–210 °C. 2.3.4. Diosgenin (4) Colorless crystalline solid; (94 mg); [a]25 D = 124° (c = 1.3, MeOH); FAB-MS (Positive): 415 (calcd 414.31, C27H42O3); IR (KBr) cm1: 3415 (OH) 1601 (C@C); M.P = 204–207 °C. 2.3.5. Acid hydrolysis Compounds 1 and 2 (10 mg) were dissolved in 5 mL MeOH, mixed with 5 mL of 5% HCl (v/v), and then refluxed for 2 h on boiling water bath. Methanol was distilled off and the hydrolysate was

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Shafiq-ur-Rahman et al. / Steroids xxx (2015) xxx–xxx Table 2 H-NMR and

1

13

C-NMR (MeOH, 600 & 150 MHz) chemical shift assignments in compound 2.

C. No.

dC

dH (J, Hz)

1 2 3 4

38.0 30.7 78.0 39.5

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

141.8 122.6 32.4 31.4 51.7 38.5 21.9 41.4 40.9 57.8 33.2 80.0 61.9 16.7 19.8 42.9 17.4 110.6 29.8 28.2 32.8 69.7 17.8

1.10, 168 m 1.92, 2.10 m 3.68 m 2.68 dd (11.2, 12.9) 2.78 dd (2.2, 13.6) – 5.63 br 1.52, 1.90 m 1.42 m 1.10 m – 1.44 m 1.10, 1.88 m – 1.12 m 1.56, 1.90 m 4.02 m 1.80 dd (6.0, 8.8) 1.02 s 1.08 s 2.52 m 1.08 d (7.6) – 1.60 m 1.24 m 1.44 m 3.80 br 1.01 d (6.2)

shaken with CH2Cl2 to separate the aglycone of glycoside. The CH2Cl2 extract was dried over anhydrous sodium sulfate and the solvent was distilled off. The residue was dissolved in methanol and subjected to determination of aglycone moiety. The acidic mother liquor was neutralized with silver oxide and the precipitate formed, was filtered off and washed several times with distilled water. The combined filtrate and washings were evaporated to dryness under reduced pressure. The residue was then dissolved in few drops of methanol and used for identification of the sugar moieties by comparison with standard sugar samples [12,13]. 2.4. Antifungal assay Test strains were from the American Type Culture Collection (ATCC) (Manassas, VA) and included Candida albicans ATCC 18804, Candida glabrata ATCC 90030, Aspergillus fumigatus ATCC 1022, Aspergillus flavus ATCC 9643, and Aspergillus niger ATCC 16888. Susceptibility testing was performed with some modification of previously reported method [14]. Briefly, samples (dissolved in dimethyl sulfoxide) were serially diluted using 20% dimethyl sulfoxide in 0.9% saline and transferred in duplicate to 96-well flat-bottom micro titer plates. Candida spp. inocula were prepared by picking 1 to 3 colonies from agar plates and resuspending in 4 ml 0.9% sterile saline. The optical density at 630 nm of the saline suspensions was compared to the 0.5 McFarland standard. The microorganisms were diluted in broth (RPMI 1640 at pH 4.5) to afford final target inocula of 5.0  103 for Candida spp. The Aspergillus spp. inocula were made by carefully removing spores from agar slants, transferring to 4 ml 0.9% saline, and filtering through Miracloth (Merck Millipore, USA). The filtrate was diluted appropriately in 5% Alamar blue (Life technologies, USA)-RPMI 1640 broth (at pH 7.3) to afford a final target inoculum of 4.0  104 CFU/ml. The fungal inocula were added to the samples to achieve a final volume of 200 ll. Negative control (media only) and positive control (amphotericin B) were included on each test plate. All organisms were read at 630 nm

C. No. 3-O-D-Glc 10 20 30 40 50 60 20 -O-Rha 100 200 300 400 500 600 40 -O-Rha 1000 2000 3000 4000 5000 6000

dC

dH (J, Hz)

100.4 77.7 76.8 79.3 78.0 62.10

4.87 d (6.9) 4.21 m 3.58 m 3.92 m 4.10 m 3.90, 4.10

102.2 72.5 73.0 74.2 70.4 19.2

6.27 4.66 4.35 4.72 4.70 1.52

br s m dd (1.9, 9.4) m m d (6.4)

102.6 73.0 72.8 74.6 70.2 18.8

5.60 3.98 4.26 4.10 4.62 1.82

br s m dd (2.2, 9.0) m m d (6.3)

using BioTek reader (Bio-Tek, USA) prior to and after incubation (Candida spp. at 25 °C for 18 to 24 h; Aspergillus spp. at 25 °C for 72 h). The MIC was defined as the lowest test concentration that allowed no detectable growth in comparison to controls. 3. Results and discussion Compound 1 was obtained as white amorphous powder. HRFAB-MS showed pseudo molecular ion [M+H]+ at m/z 1225.5426 (calcd 1224.5411), which was consistent with the molecular composition C56H88O29. 1H-NMR and 13C-NMR data of compound 1 was largely similar to diosgenin, the difference in compound 1 was the number of sugar moieties. The 1H-NMR spectrum showed the presence of three olefinic protons signals at d 5.55 as broad doublet (J = 5.4 Hz), 5.08 as br s and 4.98 br s, which showed the presence of two C@C in compound 1. Similarly, 1 H-NMR also showed the signals for five anomeric protons at d 5.41 br s, 5.18 d (J = 2.4 Hz), 4.72 d (J = 8.4 Hz), 4.39 d (J = 7.2 Hz), 4.38 d (J = 7.2 Hz), which suggested the presence of five monosaccharides including one apiose. In addition to this the 1H-NMR spectrum showed signals for two methyl at d 0.91 s and 1.08 s attached to quaternary carbon, and two methyl signals at d 1.10 d (J = 6.6 Hz), and 1.24 d (J = 6.6 Hz), attached to tertiary carbon. The 13C-NMR spectrum showed signals for sugar moieties and olefinic functionalities. The attachment of carbon in steroidal skeleton of saponin and sugar moieties was assigned on the basis of HSQC, HMBC and COSY correlations (Figs. 2 and 3). The olefinic proton at d 5.55 (H-6) showed HMBC correlations to two quaternary carbons at d 139.5 and 43.4 attributed to C-5, and C-10, respectively. HSQC spectrum showed the correlations of protons at d 3.51 and 3.39 with carbon at d 84.5 and 69.2, suggesting the presence of two OH groups in ring A of steroidal skeleton. The C–C bond connectivity in ring A and B was deduced through COSY correlations. H-1 (d 3.51) showed COSY cross peaks with C-2 methylene protons (d 2.09, 1.71), which further showed cross peaks with H-3 (d 3.39). Similarly, H-3 showed COSY cross peaks with methylene protons at d 2.24 and 2.22. This suggested the connectivity of C-1

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Shafiq-ur-Rahman et al. / Steroids xxx (2015) xxx–xxx

1

2

3

4 Fig. 1. Structures of compounds 1–4.

to C-4. H-6 (d 5.55) showed COSY correlations with H2-7 (d 1.96, 1.55), which further showed connectivity with C-8 through COSY cross peaks with H-8 (d 1.54). The COSY correlation of H-20 with H-17 (d 1.82) and H2-21 (d 3.63, 3.2), along with HMBC correlations of H-21 (d 3.63, 3.63) with C-20 (d 46.6) and C-22 (d 112.1) inferred an OH at C-21. Similarly, the HMBC correlations of H-24 (d 4.26), H2-26 (d 4.46, 3.71) with C-22 suggested oxygenated nature of ring-F. The stereochemistry of steroidal skeleton was deduced on the basis of NOESY correlations (Fig. 3). H-1 showed NOESY crosspeaks with H-3, this could be only possible if both these protons are axially oriented. Therefore, H-1 and H-3 are a-oriented. H-8 showed NOESY correlations with H3-18 and H3-19, suggesting there same orientation i.e., b-oriented. Similarly, H-9 and H-14

were found to be a-oriented, as H-9 showed NOESY cross-peaks with H-1 (axially oriented). The HMBC correlations of H-24 (d 4.26) with C-23 (d 72.1), C-25, (d 144.5), and C-27 (d 114.0), along with COSY cross peaks of H-24 with proton at d 3.74 suggested the position of OH groups at C-23 and C-24, and an exocyclic C@C bond between C-25 and C-27. The C-24 signal appeared at d 83.3, which indicated the glycoside linkage with this position. This connectivity was further confirmed through the HMBC correlation of anomeric proton at d 4.72 (H-10000 ) with C-24. H-23 showed NOESY cross peaks with H-20 and H-24, which suggested the b-orientation of these protons. The coupling constant value of H-23 and H-24 was consistent with literature reported value of similar structure i.e. 4.2 Hz, which further confirmed an a-OH at C-23 and C-24.

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Shafiq-ur-Rahman et al. / Steroids xxx (2015) xxx–xxx

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Fig. 2. Key HMBC correlations in compound 1.

Fig. 3. Key NOESY correlations in compound 1.

The connectivity between sugar molecules was inferred on the basis of HMBC correlations. H0 -1 (d 4.40) showed HMBC correlations with C-1 (d 84.5), C-20 (d 77.3), C-30 (d 88.6), and C-50 (d 78.1), suggesting the connectivity of glucose moiety with C-1. Furthermore, the HMBC correlations of H-100 (d 4.38), with C-30 (d 88.6) indicated the C-30 -O-C-100 connectivity, i.e. the second glucose molecule attached with C0 -3. The connectivity of an apiose molecule was inferred through the HMBC correlations of H-1000 (d 5.18) with C-600 (d 67.1), C-2000 (d 77.5), and C-5000 (d 75.0). The coupling constant (J = 5.18) and 13C-NMR data was similar to the literature reported [15], which indicated the presence of an apiose unit connected to glucose. The presence of a deoxy b-D-gulose in connection with C-24 was inferred through HMBC correlation of H-10000 with C-24. Similarly, the presence of an a-L-rhamnose was deduced through the HMBC correlations of H-100000 with C-40000 (d 80.3) and NMR data reported by Ono et al. [11]. Compound 1 was structurally characterized as (1b,3b,23S,24S)-1-[O-b-D-glucopyranosyl (1?3)-O-b-D-glucopyranosyl (1?6)-O-b-D-apiofuranosyl]-3,23 dihydroxyspirosta-5,25-dienyl-24-[O-a-L-rhamnopyranosyl (1?4)b-D-6-deoxygulopyranoside] (Fig. 1). To the best of our knowledge this compound is not reported previously and is a new compound. A common name was proposed for compound 1 as govanoside A. Compound 2 was isolated in the form of white powder, and was identified by comparing observed spectroscopic data with already

reported in literature [16]. FAB-MS showed [MH] at m/z 867.46 which was in agreement with the molecular formula C45H72O16. The 1H-NMR and 13C-NMR data showed the presence of steroidal skeleton and sugars units in compound 2. Compound 3 (pennogenin) was identified by comparing the spectroscopic data with that of reported in literature. FAB-MS showed [M+H]+ at m/z 331 which was in agreement with the molecular formula C27H43O4. The 1H-NMR and 13C-NMR data of compound 3 was in agreement with previous reports [17,18]. Compound 4 (diosgenin) was obtained in the form of white crystalline powder and identified by comparing the observed data with that of reported in literature. FAB-MS showed [M+H]+ at m/z 415 which was in agreement with the molecular formula C27H43O3. The 1H-NMR and 13C-NMR data of compound was in agreement with previously reported literature [19,20]. The antifungal susceptibility results indicated that steroidal saponins 1 and 2 were active against C. albicans, and C. glabrata with effects more prominent against C. albicans. In case of filamentous fungi A. niger and A. flavus responded while A. fumigates remained unaffected. However, compounds 3 and 4 were inactive at the highest test concentration of 20 lg/ml. The antifungal activities of the test compounds are shown in Table 3. In comparison compound 2 exhibited good activities against Candida spp. while compound 1 had moderate activities. In case of A. niger compound 1 exhibited good control while compound 2 had moderate activity

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Table 3 Antifungal activity of isolated compounds (1–4). Compounds

1 2 3 4 Amphotericin B *

References

Antifungal activity of compounds (1–4) (MIC lg/ml) C. glabrata

C. albicans

A. niger

A. fumigatus

A. flavus

20* 10 >20 >20 2.5

5.0 2.5 >20 >20 0.625

5.0 10 >20 >20 5.0

>20 >20 >20 >20 5.0

20 10 >20 >20 5.0

Concentration range 0.312–20 lg/ml; MIC (minimum inhibitory concentration).

against it. As far as A. flavus is concerned compound 2 had a better control in comparison to compound 1. Compounds 3 and 4 had no activity on test fungi as far as this study is concerned. To our best knowledge, the in vitro antifungal activity of compounds 1 and 2 has not been reported so far. Especially compound 1 is reported herein for the first time and has good activity against yeast as well as filamentous fungi. As steroidal saponins have shown to possess antifungal potentials [21,22] which endorse the findings of this study with respect to compound 1 and 2. 4. Concluding remarks T. govanianum is an indigenous medicinal herb of Pakistan. To our best knowledge, this is the first report of its steroidal chemical constituents and their antifungal activities. Govanoside A and borassoside E, both steroidal saponins, exhibit considerable antifungal activities. The sugar moieties attached to aglycone steroidal nuclei probably effects the antifungal activity. Borassoside E has three sugar units and shows better antifungal activity in comparison to govanoside A which has five sugar units. This can be due to the high polar nature of govanoside A, which may hinder its fungal membrane permeability, and thus possess less antifungal activity as compared to borassoside E [23]. As fungal infections are a major cause of morbidity and mortality and there is need for the discovery of new antifungal drugs, therefore further detail studies with respect to structure activity relationship and derivatization on both compounds are required to develop effective antifungal drugs. Acknowledgements The authors are thankful to Higher Education Commission (HEC) of Pakistan for financial support and H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Pakistan for providing laboratory facilities for isolation and characterization of these compounds.

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Please cite this article in press as: Shafiq-ur-Rahman et al., Govanoside A, a new steroidal saponin from rhizomes of Trillium govanianum, Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.10.013