Isolation, characterisation and antibacterial activity studies of coumarins from Rhododendron lepidotum Wall. ex G. Don, Ericaceae

Estudos de isolamento, caracterização e atividade antibacteriana de cumarinas de Rhododendron lepidotum Wall. ex G. Don, Ericaceae

Isolation, characterisation and antibacterial activity studies of coumarins from Rhododendron lepidotum Wall. ex G. Don, Ericaceae

Estudos de isolamento, caracterização e atividade antibacteriana de cumarinas de Rhododendron lepidotum Wall. ex G. Don, Ericaceae

Shakeel-U-Rehman^I; Reehana Khan^I; Khursheed A. BhatI, ^*; Alsaba F. Raja^I; Abdul S. Shawl^I; Mohd S. Alam^II

^IIndian Institute of Integrative Medicine Sanatnagar, Srinagar 190005, India

^IIDepartment of Chemistry Jamia Hamdard, New Delhi 110062, India

RESUMO

Seis cumarinas dafinina (1), dafinetina (2), dafinetina glicosídeo (3), rodonetina (4), rodonina (5) e umbeliferona (6) foram isoladas do extrato metanólico das partes aéreas de Rhododendron lepidotum Wall. ex G. Don, Ericaceae. Os compostos e seus derivados acetilados foram testados para verificar sua atividade antibacteriana contra Staphylococcus aureus ATCC-29213, Escherichia coli resistente à meticilina, Staphylococcus aureus ATCC-15187, ATCC-8739, Pseudomonas aeruginosa ATCC-9027, pelo método de microdiluição, usando ciprofloxacina como referência. A substância 2 apresentou a melhor atividade antibacteriana com o MIC 125 μg/mL contra S. aureus ATCC-29213 e MRSA ATCC-15187 seguido pela substância 4, que apresentou o valor de CIM de 250 μg/mL contra as quatro cepas testadas. Todas as moléculas apresentaram melhor atividade antibacteriana do que seus derivados acetilados.

Unitermos:Rhododendron lepidotum, glicosídeos cumarínicos, HMBC, antibacteriano, daphnin, daphnetin.

ABSTRACT

Six coumarins daphnin (1), daphnetin (2), daphnetin glucoside (3), rhodonetin (4), rhodonin (5) and umbelliferone (6) were isolated from the methanolic extract of Rhododendron lepidotum Wall. ex G. Don, Ericaceae (aerial part). The compounds and their acetyl derivatives were screened for antibacterial activity against Staphylococcus aureus ATCC-29213, methicillin resistant Staphylococcus aureus ATCC-15187, Escherichia coli ATCC-8739, Pseudomonas aeruginosa ATCC-9027 by microdilution method as compared to the reference ciprofloxacin. Compound 2 displayed the best antibacterial activity with MIC 125 μg/mL against S. aureus ATCC-29213 and MRSA ATCC-15187 followed by 4 which exhibited the MIC value of 250 μg/mL against all the four tested strains. All molecules showed better antibacterial activity than their acyl derivatives.

Keywords:Rhododendron lepidotum, coumarin glycosides, HMBC, antibacterial, daphnin, daphnetin.

INTRODUCTION

Infectious diseases are the world's leading cause of premature deaths, killing almost fifty thousand people every day (Nelson et al., 2001). In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world (Nikaido, 2009; Andersson, 2003). Among multidrug resistant bacteria gram positive MRSA is of particular concern. The ability of S. aureus species to develop resistance to virtually all antibiotics needs to be addressed. So new antibacterial agents need to be explored to overcome this concern. Plants are known to produce certain chemicals which are naturally toxic to bacteria (Cowan, 1999). Some coumarin derivatives possess very promising antimicrobial activity and the most potent among them is novobiocin (Kawase et al., 2001). In 1945, Goth conducted the first antibacterial activity studies of coumarins and found that dicoumarol inhibited the growth of several bacterial strains (Goth, 1945). Melliou et al. (2005) found that pyranocoumarin derivatives have a broad spectrum of activity. Dadak & Hodak (1966) suggested that the coumarins with antibacterial activity act selectively against gram positive bacteria. Recently T. Smyth et al. (2009) studied a number of coumarin compounds for antimicrobial activity and came up with some interesting observations like he discovered that 8-iodo-5,7-dihydroxy coumarin displayed similar activity as that of vancomycin which is the current drug of choice for the treatment of MRSA infections. Natural products and their derivatives represent a major breakthrough for the treatment of infectious diseases (Fischbach & Walsh, 2009). Herein, we report the isolation, identification and antibacterial activity of six natural coumarin molecules (and their derivatives) from Rhododendron lepidotum Wall. ex G. Don, Ericaceae. The compounds were screened for antibacterial activity against Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa using microdilution method against the reference compound ciprofloxacin. The antibacterial activity of these compounds was expressed as minimum inhibitory concentration.

MATERIAL AND METHODS

General

Melting points were determined on a Buchi 570 melting point apparatus. UV and IR spectra were measured, using Shimadzu UV-1650PC UV-Vis and Perkin-Elmer FT-IR Paragon 500 spectrometers, respectively. The ¹H and ¹³C NMR spectra were recorded on a Bruker 200MHz spectrometer, using TMS as internal standard. NMR spectra of 1, 3, 4 and 5 were recorded in deuterated DMSO. MS spectra were recorded on Jeol-MSD 300 and Bruker Esquire 3000. Column chromatography was performed using silica gel (60-120 mesh, E Merck).

Plant material

The aerial parts of Rhododendron lepidotum Wall. ex G. Don, Ericaceae, were collected from upper areas of Sonamarg (4000-6000 m), Kashmir, India. Voucher specimen is deposited in the herbarium of the institute (No.1454/93). The plant material was dried in shade and milled to powder form, which till its actual use, was kept in airtight amber bottle.

Extraction and isolation of 1, 2, 3, 4, 5 and 6

The shade dried stem part of Rhododendron lepidotum (500 g) was powdered and subjected to extraction with petroleum ether 60-80 ºC (3x 3.5 L) for 24 h at room temperature. The defatted plant material was extracted with methanol (3x 4.0 L) at room temperature. The combined extracts were concentrated under reduced pressure to yield a crude gum type extract (70 g). The methanolic extract thus obtained was dissolved in minimum amount of methanol and slurry was prepared. The slurry thus prepared was subjected to column chromatography over silica gel. The column was eluted with CHCl₃-MeOH (95:5-20:80) to afford 1-6 (Khan et al., 2008; Zhang et al., 2007).

8-Acetoxy-[2,3,4,6-tetraacetyl-]-7-O- β-D-gluco pyranonsyl benzopyranone (1a): white amorphous powder, mp 197.3 ºC; [α] D ²⁵ -53.3 º (c 0.60, MeOH), ESIMS: m/z at 550[M⁺], (Calcd for C₂₅H₂₆O₁₄), ¹H NMR (Table 1).

7,8-Diacetyl benzopyranone (2a): mp 122 ºC, ESIMS: m/z at 262 [M⁺], (Calcd. for C₁₃H₁₀O₆), ¹H NMR (Table 1).

7-Acetoxy[2,3,4,6-tetraacetyl-]-8-O-β-D-gluco pyranonsyl benzopyranone (3a): white amorphous powder, mp 88.8 ºC; [α]D ²⁵ -25.2º(c 0.50, MeOH), ESIMS: m/z at 550 [M⁺], (Calcd for C₂₅H₂₆O₁₄), ¹H NMR (Table 1).

Acetylation of compounds 1, 2, 3 and 6

In a general experiment compound (0.5 mmoL), pyridine (500 μL) and acetic anhydride (500 μL) were stirred at 40 ºC for 24 h. The reaction mixture was poured into ice-cold water and extracted with EtOAc. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo to get the desired acyl derivative in almost quantitative yield.

Medium used

Muller Hinton Agar and Muller Hinton Broth (Becton-Dickinson, Cockeysville, MD, USA; DIFCO laboratories).

Bacterial strains, culture conditions and antibiotics

The compounds were tested against following bacteria: Gram positive Staphylococcus aureus ATCC 29213, methicillin resistant Staphylococcus aureus ATCC 15187; Gram negative Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 obtained from American Type Cultures Collection (Manassas, VA, USA). The cultures were maintained on Tryptone soya agar and stored at -70 °C containing 50% glycerol (Himedia, Mumbia, India). Antibiotic was obtained from sigma Aldrich, Ciprofloxacin was used as a standard antibacterial agent for this study. Stock solution was prepared at 1 mg/mL.

Minimum inhibitory concentration assay

MIC was determined as per the guidelines of Clinical and Laboratory Standards Institute (Formerly the National Committee for Clinical Laboratory Standards) (Clinical and Laboratory Standards Institute, 2006). Bacterial suspensions were prepared by suspending 18 h grown bacterial culture in sterile normal saline. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standards (equivalent to 1.5x10⁸ CFU/mL) at wavelength 625 nm. The 2-fold serial of compounds (1, 2 and 3, stock solution prepared in dimethylsulphoxide) were prepared in Mueller Hinton Broth (MHB; DIFCO laboratories) in 100 μL volume in 96-well U bottom microtitre plates (Tarson, Mumbai, India). The above mentioned bacterial suspension was further diluted in the MHB and 100 μL volume of this diluted inoculum was added to each well of the plate resulting in the final inoculum of 5x10⁵ CFU/mL in the well and the final concentrations of compounds ranged from 2000 to 3.90 μg/mL till 10^th column. Column 11 and column 12, containing 100 μL and 200 μL of medium without drug, served as growth and medium control respectively. The plates were incubated at 37 °C for 18 h. The plates were visually read and the minimum concentration of the compound showing no turbidity was recorded as MIC.

Results and discussions

In the course of bioprospection of natural products from plants, the shade dried stem of Rhododendron lepidotum Wall. ex G. Don, Ericaceae, was finely powdered and defatted with petroleum ether. The defatted plant material was subjected to methanol extraction. The methanolic extract was subjected to column chromatography on silica gel. The mobile phase used consisted of a mixture of MeOH and CHCl₃ (05:95-80:20). Upon increasing the polarity of mobile phase gradually compounds 1-6 were furnished one after another. To the best of our knowledge, the acetyl derivatives of 1, 2, 3 and 4 are being reported for the first time. The spectral data (¹H NMR, ¹³C NMR and mass) of compounds 1 and 3 were similar to each other. However their different melting points and specific optical rotation (223 ºC and [α]D ²⁵ -77.8º for 1; 194.3 ºC and [α]D 25+21º for 2) suggested 1 and 3 to be two different compounds. The presence of a typical pair of doublets in the ¹H NMR spectrum of 1 at ´6.29 and 7.94 and that of 3 at ´6.28 and 7.9 for 32 -H and 42 -H protons suggest the compounds to be coumarins (Murry et al., 1982). Further the ¹³C NMR and DEPT spectra of 1 showing nine signals at ´160.5, 148.6, 145.2, 143.1, 134.4, 118.8, 114.9,113.9, 112.7 (aromatic carbons) and six resonances at ´ 102.3, 77.8, 76.2, 73.7, 70.3, 61.2 (glycosidic region) and that of 3 showing nine aromatic carbons with ´ value as 160.0, 153.4, 147.7, 144.7, 131.0, 124.0, 113.4, 112.0, 111.5 and six glycosidic carbons with ´ 103.7, 77.2, 76.2, 73.8, 69.6 and 60.7 account for their coumarin glycosidic skeleton. The spectral data of 1 is in agreement with that of daphnin (Zhang et al., 2007). Compound 2 was identified as daphnetin using IR, mass and NMR spectra (Ma et al., 2007). However physicochemical properties, mp, [α] D ²⁵, and ¹³C NMR of 3 were slightly different from daphnetin-8-O-glucoside (3) (Ullah et al., 1999; Kayser & Kolodzie, 1999 ). So an HMBC experiment was done to determine the linkage of glucose moiety with the coumarin skeleton. The HMBC experiment showed a long range correlation between H-1' at 4.8 (1H, d, J = 8.0) and C-8 (´131.0) for 3 (Table 2). Thus the structure of compound 3 was identified as daphnetin-8-O-glucoside and hence mp, [α]D²⁵ and ¹³C NMR for 3 have been corrected. In earlier paper we have reported the isolation and characterization of compounds 4 and 5 (Khan et al., 2008). 6 was identified by comparison of its spectral data with that of reported in literature (Razdan et al., 1987).

The antibacterial activity of ten coumarin compounds expressed as minimum inhibitory concentration, are shown in Table 3. Coumarins usually show a broad range of antibacterial activity against tested pathogens. The antibacterial properties of coumarins were first recognised in 1945 when Goth (1945) conducted an investigation with dicoumarol and found it to inhibit the growth of several bacterial strains. Kayser et al. (1999) evaluated the antimicrobial activity of fourteen oxygenated coumarins using a microtitre method against eight different microorganisms. Among these compounds, the highly oxygenated 7-hydroxy-5,6-dimethoxycoumarin and 6,8-dihydroxy-5,7-dimethoxycoumarin were reported to be the most potent antibacterial coumarins. None of the authors has studied coumarin glycosides for antibacterial structure-activity relationship. We have screened coumarin isolates which include coumarin glycosides, umbelliferone, daphnetin and their acyl derivatives for antibacterial activity. The compounds were screened for antibacterial activity against Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa using microdilution method against the reference compound ciprofloxacin. The antibacterial activities of these compounds are expressed as minimum inhibitory concentration.

Compound 2 is the most active compound out of all the coumarins tested, which exhibited MIC ranged from 125-500 μg/mL against the tested pathogens. From Table 3, it is clear that 1 and 4 show better activity than 3 and 5. All the four compounds 1, 3, 4 and 5 are coumarin monoglycosides and differ in the position of free hydroxyl and glycoside. Compounds 1 and 4 possess free hydroxyl at C8 unlike 3 and 5 in which the free hydroxyl lies at C7 in 3 and is protected as methoxyl at C8 in 5. Compound 6 shows antibacterial activity with MIC 1000 μg/mL; 1 and 4 exhibited better antibacterial activity against all the strains with an MIC value of 250 μg/mL compared to 3 and 5 showing an MIC value 1000 μg/mL. Compound 6, which has a free hydroxyl at C7, does not possess good antibacterial activity. From the above data, it can be concluded that coumarins with free hydroxyl at C8 possess better antibacterial activity and the acetyl derivatives are in general less active than their parent compounds.

CONCLUSIONS

The coumarins under study showed a broad range of antibacterial activity. Some molecules were selectively active against gram positive bacteria only. In particular, it has been demonstrated that the nature and position of substituent can give rise to increased or decreased antibacterial activity for these compounds. The most active coumarin 2, which displayed activity with MIC 125 μg/mL against S. aureus ATCC-29213 and MRSA ATCC -15187, needs to be modified for SAR studies.

• Andersson DI 2003. Persistence of antibiotic resistant bacteria. Curr Opin Microbiol 6: 452-456.
• Clinical and Laboratory Standards Institute 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 7. ed. Approved standard. M7-A7. Wayne PA: CLSI.
• Cowan MM 1999. Plant products as antimicrobial agents. Clin Microbiol Rev 12: 564-582.
• Dadak K, Hodak K 1966. Some relations between the structure and the antibacterial activity of natural coumarins. Experientia 22: 38-39.
• Fischbach MA, Walsh CT 2009. Antibiotics for emerging pathogens. Science 325: 1089-1093.
• Goth A 1945. The antibacterial properties of dicumarol. Science 101: 383.
• Kawase M, Varu B, Shah A, Motohashi M, Tani S, Saito S 2001. Antimicrobial activity of new coumarin derivatives. Arzneimittel-Forsch 51: 67-71.
• Kayser O, Kolodziej H 1999. Antibacterial activity of simple coumarins: structural requirements for biological activity. Z Naturforsch 54: 169-174.
• Khan R, Shawl AS, Tantray M, Alam MS 2008. New coumarin glycosides from Rhododendron lepidotum. Fitoterapia 79: 232-233.
• Ma B, Guo HF, LouHX 2007. A new lignan and two eudesmanes from Lepidozia vitrea. Helv Chim Acta 90: 58-62.
• Melliou E, Magiatis P, Mitaku S, Skaltosounis AL, Chinou E, Chinou I 2005. Natural and synthetic 2,2-dimethylpyranocoumarins with antibacterial activity. J Nat Prod 68: 78-82.
• Murry HRD, Medez J, Brown AS 1982. The natural coumarins, occurrence, chemistry and biochemistry. John Wiley & Sons: New York.
• Nelson KE, Williams CM, Graham NMH 2001. Infectious disease epidemiology: theory and practice. Gaithersburg: Aspen Publishers.
• Nikaido H 2009. Multidrug resistance in bacteria. Annu Rev Biochem 78: 119-146.
• Razdan TK, Qadri B, Harkar S, Waight ES 1987. Chromones and coumarins from Skimmia laureola. Phytochemistry 26: 2063-2069.
• Smyth T, Ramachandran VN, Smyth WF 2009. A study of the antimicrobial activity of selected naturally occurring and synthetic coumarins. Int J Antimicrob Ag 33: 421-426.
• Ullah N, Ahmed S, Mohammad P, Rabnawaz H, Malik A 1999. Chemical constituents of Daphne oleoides. Fitoterapia 70: 214-215.
• Zhang W, Shen YH, Liu RH, Zhang C, Chen HS, Fu P, Shan L, Zhang WD 2007. Coumarins from the Stem bark of Daphne marginata. Chem Nat Compd 43: 317-318.

Publication Dates

• Publication in this collection
  12 Nov 2010
• Date of issue
  Dec 2010