Tuesday, 15 May 2018
2016 Antimycobacterial triterpenes from the Canadian medicinal plant Sarracenia purpurea
y Volume 188, 21 July 2016, Pages 200-203 Journal of Ethnopharmacology Ethnopharmacological communication Author links open overlay panelSteven A.MorrisonaHaoxinLiaDuncanWebsterbJohn A.JohnsonaChristopher A.Grayac a Department of Biological Sciences, University of New Brunswick, Saint John, New Brunswick, Canada E2L 4L5 b Division of Infectious Diseases, Department of Medicine, Saint John Regional Hospital, Saint John, New Brunswick, Canada E2L 4L2 c Department of Chemistry, University of New Brunswick, Saint John, New Brunswick, Canada E2L 4L5 Received 4 February 2016, Revised 21 April 2016, Accepted 28 April 2016, Available online 9 May 2016. crossmark-logo https://doi.org/10.1016/j.jep.2016.04.052 Get rights and content Abstract Ethnopharmacological relevance The purple pitcher plant, Sarracenia purpurea, is a medicinal plant used by the Canadian First Nations to treat a wide variety of illnesses. The Mi’kmaq and Wolastoqiyik (Maliseet) peoples of Eastern Canada have traditionally used infusions of S. purpurea for the treatment of tuberculosis-like symptoms. Previous investigations have shown methanolic extracts of S. purpurea to possess antimycobacterial activity. Aim of the study To isolate and identify antimycobacterial constituents from S. purpurea. Materials and Methods Methanolic extracts of S. purpurea were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The antimycobacterial constituents were identified by NMR, MS and polarimetry. Results The triterpenes betulinaldehyde, β-sitosterol, betulinic acid, and ursolic acid were isolated from S. purpurea. Betulinaldehyde, betulinic acid, and ursolic acid exhibited MICs of 450, 950, and 450 μM and IC50s of 98, 169, and 93 μM against M. tuberculosis H37Ra respectively whilst β-sitosterol was inactive (MIC and IC50 of >1000 μM). Conclusions Betulinaldehyde, betulinic acid, and ursolic acid were identified as the principal constituents responsible for the antimycobacterial activity of S. purpurea. This work is consistent with the ethnopharmacological use of S. purpurea by Canadian First Nations as a treatment against infectious diseases. Graphical abstract fx1 Download high-res image (290KB)Download full-size image Previous article in issue Next article in issue Abbreviations [α]Dspecific rotation measured at 589 nm and the temperature indicated 1Done dimensional 2Dtwo dimensional ACSAmerican Chemical Society cconcentration in g/100 mL CH2Cl2dichloromethane EtOAcethyl acetate HPLChigh performance liquid chromatography HRESIMShigh resolution electrospray ionization mass spectrometry IC50median inhibitory concentration IRinfra-red LC-MSliquid chromatography – mass spectrometry MeOHmethanol MICminimum inhibitory concentration MRAmicroplate resazurin assay MSmass spectroscopy nBuOH1-butanol NMRnuclear magnetic resonance SDstandard deviation TLCthin layer chromatography Keywords Antimycobacterial Betulinaldehyde β-Sitosterol Betulinic acid Ursolic acid Sarracenia purpurea Mycobacterium tuberculosis 1. Introduction Tuberculosis is one of the leading causes of global morbidity and mortality associated with an infectious disease and it is estimated that approximately one third of the global population is currently infected with Mycobacterium tuberculosis (WHO, 2014). Due to the emergence of drug resistant strains of M. tuberculosis, there is a need for the development of new drugs with unique mechanisms of action (Koul et al., 2011). Sarracenia purpurea L. (Sarraceniaceae), or the purple pitcher plant, is a carnivorous herbaceous perennial that has been used by Canadian First Nations for the treatment of a wide variety of illnesses (Moerman, 1998). The Mi’kmaq and Wolastoqiyik (Maliseet) peoples of Eastern Canada have long used S. purpurea as a remedy for tuberculosis-like symptoms either by infusing the plant as a tea or through direct consumption of the herb (Moerman, 1998). Previous bioassays have shown that methanolic extracts of S. purpurea inhibited the growth of M. tuberculosis H37Ra (O’Neill et al., 2014) and these observations, in conjunction with the plants historical use by Canadian First Nations, have prompted our current research to identify and isolate the antimycobacterial constituents from S. purpurea. 2. Materials and methods 2.1. General experimental procedures All solvents for extraction and isolation were ACS certified or HPLC grade. NMR spectra were recorded on an Agilent 400-MR DD2 instrument at 400 MHz for 1H and 100 MHz for 13C using standard 1D and 2D pulse programs. HRESIMS data were recorded on Thermo LTQ Exactive Orbitrap LC-MS. Optical rotations were measured with a Rudolph Autopol III polarimeter. Flash chromatography was performed using a Biotage Flash+ chromatography system with KP-Sil 25+S and C18 25+S silica cartridges (40–63 µm, 60 Å). Normal phase semi-preparative HPLC was performed using a Waters 510 pump, a Phenomenex silica column (10 µm, 100 Å, 250×10 mm) and a Waters R401 refractive index detector at a flow rate of 4 mL/min. Antimycobacterial testing was performed using the culture broth supplied in Mycobacteria Growth Indicator Tubes (BBL™ MGIT™) in non-tissue culture treated, low-binding, black 96-well microlitre plates sealed with polyester films (50 µm). Fluorometric readings (in relative fluorescence units, RFU) were recorded using a Molecular Devices Gemini EM dual-scanning microplate spectrofluorometer (530 nm excitation filter and a 590 nm emission filter operating in top-scan mode). 2.2. Plant material and extraction S. purpurea was collected by hand in July 2011 from the woods of Prince of Wales, New Brunswick, Canada (45° 11.988′ N; 66° 13.814′ W). Plant tissue was cleaned by hand, rinsed with deionized water, freeze-dried and stored at –20 °C. Plants were identified by Dr. Stephen Clayden of the New Brunswick Museum and a voucher specimen has been deposited in the New Brunswick Museum Herbarium (Number: NBM VP-39665). The freeze dried plants (40.0 g) were ground in a domestic blender, exhaustively extracted in methanol (2×200 mL; 7 h per extraction) using a Soxhlet extractor and the resulting solution concentrated in vacuo to give a crude methanolic extract (12.9 g). 2.3. Mycobacterial strains and growth conditions M. tuberculosis strain H37Ra (ATCC 25,177) was grown as described by O’Neill et al. (2014) and diluted to a turbidity equivalent to a 1.0 McFarland standard (107 CFU). Suspensions were cryogenically preserved for up to four weeks, thawed, and diluted prior to use. 2.4. Microplate resazurin assay (MRA) The MRA was carried out as described by O’Neill et al. (2014) using rifampin (0.1 μg/mL) and 2% DMSO as the positive and negative controls, respectively. The percentage inhibition of mycobacterial growth was then defined as 1−(test or positive control well fluorescence/mean negative control well fluorescence)×100 (Collins and Franzblau, 1997). Fractions that caused more than 50% inhibition were considered to have significant activity against M. tuberculosis H37Ra. 2.5. Extraction, isolation and identification The S. purpurea extract exhibited antimycobacterial activity against M. tuberculosis H37Ra in our screening bioassay (mean inhibition ± standard deviation =24.0±0.1%; tested at 100 μg/mL) and fractionation of the extract was bioasay guided using the MRA. Initially, the crude extract was fractionated by a modified Kupchan solvent–solvent partition protocol to give five fractions as follows: the organic extract (7.0 g) was dissolved in 9:1 MeOH/H2O (600 mL) and extracted with hexanes (3×200 mL), then diluted with H2O (300 mL) and extracted with CH2Cl2 (3×200 mL). The aqueous fraction was then concentrated, dissolved in H2O (600 mL) and extracted with EtOAc (3×200 mL) and n-BuOH (3×200 mL). The five partition fractions were concentrated in vacuo to give the following fractions: hexanes (290 mg), CH2Cl2 (766 mg), EtOAc (366 mg), n-BuOH (1.31 g), and aqueous (3.08 g). The hexanes fraction (290 mg) was subjected to silica gel flash chromatography using a stepwise gradient of hexanes to EtOAc (10% increments of EtOAc, 200 mL per eluent) to afford 11 fractions. Fraction 2 (220 mg) was further purified by silica gel flash chromatography using a stepwise gradient of 100% hexanes to 9:1 hexanes/EtOAc (2% increments of EtOAc, 130 mL per eluent) followed by washes of 17:3 hexanes/EtOAc and 4:1 hexanes/EtOAc. The eluents were combined according to their respective TLC profiles to yield nine fractions. Of these nine fractions, fraction 5 (18 mg) exhibited antimycobacterial activity and was further purified using normal phase HPLC (eluted with 9:1 hexanes/EtOAc) to give compounds 1 (2 mg) and 2 (1 mg). The third column fraction obtained from the hexanes partition (19 mg) also exhibited antimycobacterial activity and was further purified by normal phase HPLC directly (eluted using 17:3 hexanes/EtOAc) to give compound 3 (6 mg) and a mixture of fatty acid glycerides. The CH2Cl2 liquid-liquid partition fraction (766 mg) was subjected to silica gel flash chromatography using a stepwise gradient of hexanes to EtOAc (10% increments of EtOAc, 200 mL per eluent) to afford 11 fractions. Fraction 4 (78 mg) exhibited antimycobacterial activity and was further purified by normal phase HPLC (4:1 hexanes/EtOAc) to give compound 4 (6 mg). 2.6. Spectroscopic and spectrometric data Betulinaldehyde (1). White solid; [α]25D=−4° (c 6×10−4, CH2Cl2); IR (thin film) υmax 3438, 2936, 2868, 1723, 1456, 1373, 1247, 1038, 887 cm−1; HRESIMS m/z 441.3728 [M + H+] (calculated for C30H49O2, 441.3727). 1H and 13C NMR data were consistent with literature values (Barthel et al., 2008). β-Sitosterol (2). White solid; [α]25D=−29° (c 4×10−3, CH2Cl2); IR (thin film) υmax 3429, 2937, 2869, 1665, 1455, 1377, 1051, 958, 801 cm−1; HRESIMS m/z 397.3807 [M – H2O + H+] (calculated for C29H49, 397.3829). 1H and 13C NMR data were consistent with literature values (Chang et al., 2000). Betulinic acid (3). White solid; [α]25D=−8° (c 3×10−3, CH2Cl2); IR (thin film) υmax 3448, 2938, 1686, 1455, 1373, 1232, 1034, 882, 803 cm−1; HRESIMS m/z 457.3676 [M + H+] (calculated for C30H49O3, 457.3676). 1H and 13C NMR data were consistent with literature values (Peng et al., 1998). Ursolic acid (4). White solid; [α]25D=0° (c 6×10−4, CHCl3); IR (thin film) υmax 3438, 2933, 2638, 1687, 1456, 1373, 1237, 1033, 881, 803 cm−1; HRESIMS m/z 457.3677 [M + H+] (calculated for C30H49O3, 457.3676). 1H and 13C NMR data were consistent with literature values (Seebacher et al., 2003). 2.7. Determination of minimum inhibitory concentrations (MIC) and median inhibitory concentrations (IC50) MICs and IC50 values against M. tuberculosis H37Ra were determined as previously described (O’Neill et al., 2014) on dilution series comprising 12 concentrations (400–0.20 μg/mL) in triplicate. The MIC of a compound was considered to be the lowest concentration at which it inhibited mycobacterial growth by more than a mean value of 90% (Collins and Franzblau, 1997), and the corresponding IC50 was estimated by fitting a four parameter logistic curve (Sebaugh, 2011) to the mycobacterial growth data using GraphPad Prism version 6 (GraphPad Software, California, USA). 3. Results and discussion The genus Sarracenia is comprised of 11 species found mainly on the south eastern coast of the United States with the exception of S. purpurea which is widely distributed up the east coast of North America into the Canadian Maritime provinces and across Canada into eastern British Columbia (MacDaniel, 1971; Ne’eman, et al., 2006; Schnell, 2002). The First Nations peoples of eastern Canada have traditionally used S. purpurea as a treatment for a multitude of illnesses, with the Mi’kmaq and Wolastoqiyik using infusions of the plant to treat respiratory illnesses, including tuberculosis (Moerman, 1998). Methanolic extracts of S. purpurea were therefore screened for antimycobacterial activity against M. tuberculosis H37Ra using the microplate resazurin assay. Bioassay guided fractionation involving solvent partition, flash chromatography, and normal phase HPLC led to the isolation of betulinaldehyde (1, 0.03% dry weight), betulinic acid (3, 0.08% dry weight), and ursolic acid (4, 0.02% dry weight) as the principal antimycobacterial constituents. β-Sitosterol (2, 0.02% dry weight) was also isolated, but was found to be significantly less active than the other triterpenes. The molecular formulae of 1, 2, 3, and 4 were determined from the pseudomolecular ions observed from HRESIMS and all structures (see Fig. 1) were confirmed through comparison of the NMR data obtained for these compounds with literature values (1: Barthel et al., 2008; 2: Chang et al., 20003: Peng et al., 1998; 4: Seebacher et al., 2003). Fig. 1. Download high-res image (140KB)Download full-size image Fig. 1. The triterpenes betulinaldehyde (1), β-sitosterol (2), betulinic acid (3), and ursolic acid (4) isolated from Sarracenia purpurea. All compounds exhibited inhibitory activity against M. tuberculosis H37Ra with betulinaldehyde and ursolic acid being the most active (97.8 and 92.7 μM respectively), and β-sitosterol possessing only weak antimycobacterial activity (>1000 μM; Table 1). Betulinic acid and ursolic acid have been previously isolated from S. purpurea (Muhammad et al., 2013) and betulinaldehyde and β-sitosterol have been isolated from Sarracenia flava (Bhattacharyya et al., 1976). All of the compounds have been reported to exhibit antimycobacterial activity at levels similar to those observed for the S. purpurea natural products (Hongmanee, 2011; Jesus et al., 2015; Jiménez et al., 2005; Wächter et al., 1999). However, this is the first report identifying these compounds as antimycobacterial constituents of S. purpurea and is consistent with the ethnopharmacological use of this plant by Canadian First Nations as a treatment against tuberculosis. Table 1. Biological activities (MICs and IC50s in μM) of the antimycobacterial constituents of Sarracenia purpurea. Compound Mycobacterium tuberculosis H37Ra MIC IC50 (95% CI)a Betulinaldehyde (1) 450 97.8 (93.9–101.8) β-Sitosterol (2) >1000b >1000b Betulinic acid (3) 875 168.8 (161.5–176.4) Ursolic acid (4) 450 92.7 (87.7–98.0) Rifampin (positive control) 6.0×10−3 1.06×10−3 (0.96×10−3–1.18×10−3) a 95% confidence interval (n =3). b Compound not sufficiently active to calculate data. Acknowledgements The authors would like to thank Stephen Clayden (New Brunswick Museum), Larry Calhoun (University of New Brunswick), and Fabrice Berrué and Patricia Boland (University of Prince Edward Island) for their assistance with plant identification, recording 2D NMR data, and obtaining HRMS data respectively. Financial support for this research was provided by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant 350764-2009 to CAG and Undergraduate Student Research Assistantships to SM), the New Brunswick Innovation Foundation Foundation (Research Assistantship Initiative grants 2012-23 and 2013-66 to CAG), and Horizon Health Network (Health Promotion Research Fund Tier II grant 73118 to DW, CAG and JAJ) and is gratefully acknowledged. References Barthel et al., 2008 A. Barthel, S. Stark, R. Csuk Oxidative transformations of betulinol Tetrahedron, 64 (39) (2008), pp. 9225-9229 ArticleDownload PDFView Record in Scopus Bhattacharyya et al., 1976 J. Bhattacharyya, U. Kokpol, D.H. Miles The isolation from Sarracenia flava and partial synthesis of betulinaldehyde Phytochemistry, 15 (3) (1976), pp. 432-433 ArticleDownload PDFView Record in Scopus Chang et al., 2000 Y.C. Chang, F.R. Chang, Y.C. Wu The constituents of Lindera glauca J. Chin. Chem. Soc., 47 (2) (2000), pp. 373-380 CrossRefView Record in Scopus Collins and Franzblau, 1997 L. Collins, S.G. Franzblau Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium Antimicrob. Agents Chemother., 41 (5) (1997), pp. 1004-1009 View Record in Scopus Hongmanee, 2011 P. Hongmanee The data on MIC determination of Mycobacterium tuberculosis against different compounds Siriraj Med. J., 61 (1) (2011), pp. 56-59 View Record in Scopus Jesus et al., 2015 J.A. Jesus, J.H.G. Lago, M.D. Laurenti, E.S. Yamamoto, L.F.D. Passero Antimicrobial activity of oleanolic and ursolic acids: an update Evid.-Based Complement. Altern. Med. (2015) Article ID: 620472 Jiménez et al., 2005 A. Jiménez, M. Meckes, V. Alvarez, J. Torres, R. Parra Secondary metabolites from Chamaedora tepejilote (Palmae) are active against Mycobacterium tuberculosis Phytother. Res., 19 (2005), pp. 320-322 CrossRefView Record in Scopus Koul et al., 2011 A. Koul, E. Arnoult, N. Lounis, J. Guillemont, K. Andries The challenge of new drug discovery for tuberculosis Nature, 469 (7331) (2011), pp. 483-490 CrossRefView Record in Scopus MacDaniel, 1971 MacDaniel, S., 1971. The genus Sarracenia (Sarraceniaceae). Bulletin of Tall Timbers Research Station, 9. Moerman, 1998 D.E. Moerman Native American Ethnobotany Timber Press, Portland, OR (1998) Muhammad et al., 2013 A. Muhammad, P.S. Haddad, T. Durst, J.T. Arnason Phytochemical constituents of Sarracenia purpurea L. (pitcher plant) Phytochemistry, 94 (2013), pp. 238-242 ArticleDownload PDFView Record in Scopus Ne'eman et al., 2006 G. Ne'eman, R. Ne'eman, A.M. Ellison Limits to reproductive success of Sarracenia purpurea (Sarraceniaceae) Am. J. Bot., 93 (11) (2006), pp. 1660-1666 CrossRefView Record in Scopus O’Neill et al., 2014 T.E. O’Neill, H. Li, C.D. Colquhoun, J.A. Johnson, D. Webster, C.A. Gray Optimisation of the microplate resazurin assay for screening and bioassay-guided fractionation of phytochemical extracts against Mycobacterium tuberculosis Phytochem. Anal., 25 (5) (2014), pp. 461-467 CrossRefView Record in Scopus Peng et al., 1998 C. Peng, G. Bodenhausen, S. Qiu, H.H.S. Fong, N.R. Farnsworth, S. Yuan, C. Zheng Computer-assisted structure elucidation: application of CISOC–SES to the resonance assignment and structure generation of betulinic acid Magn. Reson. Chem., 36 (4) (1998), pp. 267-278 CrossRefView Record in Scopus Schnell, 2002 D.E. Schnell Carnivorous Plants of the United States and Canada Timber Press, Portland, OR (2002) Sebaugh, 2011 J. Sebaugh Guidelines for accurate EC50/IC50 estimation Pharm. Stat., 10 (2) (2011), pp. 128-134 CrossRefView Record in Scopus Seebacher et al., 2003 W. Seebacher, N. Simic, R. Weis, R. Saf, O. Kunert Complete assignments of 1H and 13C NMR resonances of oleanolic acid, 18α-oleanolic acid, ursolic acid and their 11-oxo derivatives Magn. Reson. Chem., 41 (8) (2003), pp. 636-638 CrossRefView Record in Scopus Wächter et al., 1999 G.A. Wächter, S. Valcic, M.L. Flagg, S.G. Franzblau, G. Montenegro, E. Suarez, B.N. Timmermann Antitubercular activity of pentacyclic triterpenoids from plants of Argentina and Chile Phytomedicine, 6 (5) (1999), pp. 341-345 ArticleDownload PDFView Record in Scopus World Health Organization, 2014 World Health Organization, 2014. Global Tuberculosis Report 2014. Geneva, Switzerland. © 2016 Elsevier Ireland Ltd. All rights reserved.