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Antimicrobial compounds from Alpinia conchigera
Journal of Ethnopharmacology 145 (2013) 798–802
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antimicrobial compounds from Alpinia conchigera Ahmad Nazif Aziz a, Halijah Ibrahim b, Devi Rosmy Syamsir b, Mastura Mohtar c, Jaya Vejayan d, Khalijah Awang e,f,n a
Department of Chemical Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu (UMT), 21030 Kuala Terengganu, Terengganu, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia c Medicinal Plants Programme, Biotechnology Division, Forest Research Institute Malaysia (FRIM) 52109, Kepong, Selangor, Malaysia d School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor, Malaysia e Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia f Centre for Natural Products and Drug Discovery (CENAR), Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2012 Received in revised form 10 October 2012 Accepted 14 December 2012 Available online 21 December 2012
Ethnopharmacological relevance: The rhizome of Alpinia conchigerahas been used as a condiment in the northern states of Peninsular Malaysia and occasionally in folk medicine in the east coast to treat fungal infections. In some states of Peninsular Malaysia, the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish. This study aimed to investigate the chemical constituents of the pseudostems and rhizomes of Malaysian Alpinia conchigera and to evaluate the antimicrobial activity of the dichloromethane (DCM) extracts of the pseudostems, rhizomes and the isolated compounds against three selected fungi and five strains of Staphylococcus aureus. Materials and methods: The dried and ground pseudostems (0.8 kg) and rhizomes (1.0 kg) were successively extracted in Soxhlet extractor using n-hexane, dichloromethane (DCM) and methanol. The n-hexane and DCM extracts of the pseudostem and rhizome were subjected to isolation and purification using column chromatography on silica gel using a stepwise gradient system (n-hexane to methanol). Briefly, a serial two fold dilutions of the test materials dissolved in DMSO were prepared prior to addition of 100 ml overnight microbial suspension (108 cfu/ml) followed by incubation at 37 1C (bacteria) or 26 1C (dermatophytes and candida) for 24 h. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibition to bacterial/candida/dermatophytes’ growth. Antibiotic cycloheximide was used as reference for anticandidal and antidermatophyte comparison while oxacilin was used as reference for antibacterial testing. DMSO served as negative control. Turbidity was taken as indication of growth, thus the lowest concentration which remains clear after macroscopic evaluation was taken as the minimum inhibitory concentration (MIC). Results: The isolation of n-hexane and DCM extracts of the rhizomes and pseudostems of Alpinia conchigera via column chromatography yielded two triterpenes isolated as a mixture of stigmasterol and b-sitosterol: caryophyllene oxide, chavicol acetate 1, p-hydroxy cinnamaldehyde 2, 10 S-10 acetoxychavicol acetate 3, trans-p-coumaryl diacetate 4, 10 S-10 -acetoxyeugenol acetate 5, 10 -hydroxychavicol acetate 6, p-hydroxycinnamyl acetate 7 and 4-hydroxybenzaldehyde. The DCM extract of the rhizome of Alpinia conchigera indicated potent antifungal activity against Candida albicans, Microsporum canis and Trycophyton rubrum with MIC values of 625 mg/ml, 156 mg/ml and 156 mg/ml, respectively. It also showed significant inhibitory activity with MIC values between 17.88 and 35.75 mg/ml against the mutant Staphylococci isolates MSSA, MRSA and Sa7. Amongst the isolated compounds, the lowest inhibition observed were of 10 S-10 -acetoxyeugenol against the dermatophytes (MIC 313 mg/ml) followed by trans-p-coumaryl diacetate against both dermatophytes and candida (MIC 625 mg/ml). The compound p-hydroxycinnamyl acetate strongly inhibited Staphylococcus aureus strain VISA (MIC 39 mg/ml) followed by trans-p-coumaryl diacetate and 10 -hydroxychavicol acetate with MIC value of 156 mg/ml. Conclusion: In conclusion, the observed antibacterial, anticandidal and antidermatophyte activity of the extracts and compounds obtained from the rhizome confirm the traditional use of Alpinia cochigera rhizome in the treatment of skin infection. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alpinia conchigera Zingiberaceae Phenylpropanoids Antimicrobial Antifungal
n Corresponding author at: Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: þ 603 796 740 64; fax: þ 603 796 741 93. E-mail address: [email protected] (K. Awang).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.024
A.N. Aziz et al. / Journal of Ethnopharmacology 145 (2013) 798–802
1. Introduction Alpinia conchigera Griff. is a herbaceous perennial, 2 to 5 ft. tall, found in eastern Bengal and southwards to Peninsular Malaysia and Sumatera. In Malaysia, it is also locally known as lengkuas ranting, lengkuas kecil, lengkuas padang, lengkuas genting or chengkenam (Burkill, 1966). This species is semi-wild, common in open wet grounds such as edges of rice fields, streams as well as under the shade of palm oil and rubber trees. The rhizome is used as a condiment in the northern states of Peninsular Malaysia and occasionally in folk medicine in the east coast to treat fungal infections (Ibrahim et al., 2000). In some states of Peninsular Malaysia the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish (Ibrahim et al., 2009). There were few studies on the chemical constituents of Alpinia conchigera reported to date. Yu et al. (1988) reported the presence of nonacosane, b-sitosterol, 10 -acetoxychavicol acetate and 10 -acetoxyeugenol acetate in the fruit, the two phenylpropanoid derivatives showing anti-inflammatory activity. Later, Athamaprasangsa et al. (1994) communicated the detection of four known phenylpropanoids: chavicol acetate, 10 -hydroxychavicol acetate, 4-acetoxycinnamyl alcohol and 4-acetoxycinnamyl acetate from the aqueous layer obtained from the hydrodistillation of the fresh rhizomes of Alpinia conchigera Griff. from Thailand. They also reported five diarylheptanoids: 1,7-diphenyl-3,5-heptanedione, 1,7-diphenyl-5-hydroxy-3-heptanone, 5-hydroxy-7-(40 -hydroxy-30 -methoxyphenyl)-1-phenyl-3 heptanone, 1,7-diphenylhept-4-en-3-one and 7-(40 -hydroxy-30 methoxyphenyl)-1-phenylhept-4-en-3-one and two flavonoids: 3,5,7-trihydroxyflavone and 3,5,7-trihydroxy-40 -methoxyflavone from the n-hexane and DCM extracts of the dried rhizomes. Recent studies on chemical constituents of Alpinia conchigera by Phan et al. (2005) reported b-sitosterol, stigmasterol and three flavonoids: cardamomin, alpinetin and naringenin 5-Me ether isolated from the rhizomes of Alpinia conchigera Griff. from Vietnam. Further study on the methanolic extract of the rhizomes resulted with b-sitosterol, stigmasterol, cardamomin, chalconaringenin 20 -O-Me ether, alpinetin, and naringenin 5-O-Me ether (Le et al., 2007). In addition, Giang et al. (2007) isolated flavokawin B, b-sitosterol, stigmasterol, alpinetin, (3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene, b-D-fructopyranose, b-D-fructofuranose, and 2-O-Me b-D-fructofuranose from the fruits of Alpinia conchigera Griff. (Zingiberaceae) from Vietnam. 10 S-10 acetoxychavicol acetate and 10 S-10 -acetoxyeugenol acetate obtained from Alpinia conchigera were found to induce apoptosis in oral squamous carcinoma cells (HSC-4) and human breast cancer cells (MCF-7), respectively (Awang et al., 2010; Hasima et al., 2010). Ibrahim et al. (2009) communicated the essential oil constituents of Alpinia conchigera. All of the above mentioned studies involved the constituents from the rhizomes and fruits of Alpinia conchigera from China, Thailand, Vietnam and Malaysia and fruits of ract of the rhizomes. In this study, the chemical constituents from the pseudostems and rhizomes of Malaysian Alpinia conchigera will be investigated and discussed. In addition, investigation on the antimicrobial activity of the DCM extracts of the pseudostems, rhizomes and the isolated compounds against three selected fungi and five strains of Staphylococcus aureus will be carried out.
799
herbarium series number KL 5049. The samples were authenticated by Professor Dr. Halijah Ibrahim of the Institute of Biological Sciences, University of Malaya and the voucher specimens were deposited in the herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur. 2.2. Extraction and isolation The dried and ground pseudostems (0.8 kg) and rhizomes (1.0 kg) were extracted in Soxhlet extractor using n-hexane, dichloromethane (DCM) and methanol (MeOH) successively. The pseudostem and rhizome extracts were dried in vacuo. The n-hexane and DCM extracts were further subjected to chemical compound isolation and antimicrobial activity (antibacterial and antifungal activity) studies. The n-hexane and DCM extracts of the pseudostems and rhizomes were subjected to column chromatography (CC) on silica gel using a stepwise gradient system (n-hexane to MeOH). All compounds were successfully isolated using CC with a combination of n-hexane-ethyl acetate (EtOAc) as the solvent system. 2.3. General experimental procedure All NMR spectra were recorded on a JEOL LA-400 spectrometer (400 MHz), in CDCl3; The mass of the compounds were obtained from GC–MS (Hewlett Packard 5989 A) on HP-5 column. 2.4. Antimicrobial activity 2.4.1. Test isolates Microbes selected for this study are those implicated in skin infections (Williams et al., 1999; Pereira Gonzales and Maisch, 2012). The crude extracts and the isolated compounds were individually tested against five strains of Staphylococcus aureus (each differing in their antibiotic resistance profile) namely Methicillin sensitive Staphylococcus aureus ATCC 29213 (MSSA), Methicillin resistant Staphylococcus aureus ATCC 33591 (MRSA), Vancomycin intermediate resistant Staphylococcus aureus ATCC 700699 (Sa7), MRSA with intermediate resistance to vancomycin (VISA24) and MRSA with complete resistance to vancomycin (VRSA156), a candida: Candida albicans ATCC 10231, two dermatophytes: Microsporum canis (ATCC 36299) and Trichophyton rubrum (ATCC 28188). All isolates were purchased from American Type Culture Collection (ATCC) except two mutant isolates namely VISA24 and VRSA156 that were obtained through a step wise lab-passage procedure as described previously (Mohtar et al., 2009). 2.4.2. Inoculum preparation Media was sterilized by autoclaving at 120 1C for 15 min and all subsequent manipulations were carried out in a class 2 biohazard cabinet. The Staphylococcus aureus strains (coded as Sa 2, Sa 3, Sa 7) were cultured in Mueller Hinton Broth (MHB), VISA and VRSA in Tryptic soy broth (TSB) overnight (24 h) at 37 1C while the candida and dermatophytes were cultured in Potato dextrose broth (PDB) overnight at 26 1C. The resulting inoculum was further adjusted to obtain a turbidity comparable to that of McFarland standard tube No. 0.5 (Vandepitte et al., 1991) prior to use.
2. Material and methods 2.1. Plant material The pseudostems and rhizomes of wild Alpinia conchigera Griff. were collected from Jeli province of Kelantan, Malaysia with
2.4.3. Minimum inhibitory concentration (MIC) The minimum inhibitory concentration (MIC) value determination assay was carried out to evaluate the potential compounds as inhibitory agent against test isolate using double-broth microdilution method involving 96-wells microtitre-plates as described
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A.N. Aziz et al. / Journal of Ethnopharmacology 145 (2013) 798–802
previously (Saiful et al., 2008). Briefly, a serial two fold dilutions of the test compounds dissolved in DMSO were prepared prior to addition of 100 ml overnight microbial suspension (108 cfu/ml) followed by incubation at 37 1C (bacteria) or 26 1C (dermatophytes and Candida) for 24 h. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibition to bacterial/candida/dermatophytes’ growth. DMSO served as negative control. Antibiotic cycloheximide was used as reference for anticandidal and antidermatophyte comparison while oxacilin was used as reference for antibacterial testing. Turbidity was taken as indication of growth, thus the lowest concentration which remains clear after macroscopic evaluation was taken as the minimum inhibitory concentration (MIC). For further reconfirmation, 20 ml of MTT reagent (1 mg/ml) was added to the suspension in the selected wells, followed by 20 min incubation at 37 1C. The reagent-suspension colour will remain clear or yellowish indicating complete inhibition (cidal) activity as opposed to dark blue for growth (Eloff, 1998). The MIC was recorded as the most repeatable minimum concentration of triplicate.
Table 1 List of isolated compounds from the n-hexane and DCM extracts of pseudostems and rhizomes of Alpinia conchigera and their percentage yield (%). Compound
Pseudostem (% yield)
Rhizome (% yield) Extract
Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 Caryophyllene oxide p-Hydroxy cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Ni: not isolated.
n-hexane
DCM
n-hexane
DCM
0.31 Ni Ni 0.07 0.15 0.68 Ni Ni 0.13 0.20
Ni Ni Ni 0.18 9.08 0.95 Ni 0.37 0.48 1.10
0.25 0.08 0.05 0.59 40.07 0.19 0.12 0.15 0.61 0.12
0.14 Ni Ni 0.20 25.25 0.80 0.03 0.19 0.12 0.92
3. Results and discussion Soxhlet extraction of the pseudostems yielded 5.89 g (0.74% w/w) of n-hexane extracts, 14.70 g (1.84% w/w) of DCM extracts and 72.30 g (9.03% w/w) of MeOH extracts while the rhizomes produced 22.68 g (2.27% w/w) of n-hexane extracts, 15.87 g (1.59% w/w) of DCM extracts and 150.36 g (15.04% w/w) of MeOH extracts. The isolation of n-hexane extracts of the rhizomes of Alpinia conchigera via column chromatography yielded two triterpenes ¨ ´ r, 2004; Xu isolated as a mixture of stigmasterol (Forgo and Kove et al., 2005) and b-sitosterol (De-Eknamkul and Potduang, 2003); one sesquiterpene; caryophyllene oxide (Silverstein and Webster, 1998), seven phenyl propanoids; chavicol acetate 1 (Yu et al., 1988; Mitsui et al., 1976), p-hydroxy cinnamaldehyde 2 (Leem et al., 1999), 10 S-10 -acetoxychavicol acetate 3 (Janssen and Scheffer, 1985; Noro et al., 1988; Yang and Eilerman, 1999), trans-p-coumaryl diacetate 4, 10 S-10 -acetoxyeugenol acetate 5 (Noro et al., 1988; Yang and Eilerman, 1999), 10 -hydroxychavicol acetate 6 (Janssen and Scheffer, 1985; Yang and Eilerman, 1999), p-hydroxycinnamyl acetate 7 (Kiuchi et al., 2002), and one phenolic compound; 4-hydroxybenzaldehyde (Noro et al., 1988). The DCM extract of the rhizome afforded nine compounds which were similar to that of the compounds found in the n-hexane extract, but deprived of chavicol acetate 1 and caryophyllene oxide. As for the purification of the extracts from the pseudostem, seven compounds were found in the n-hexane crude extract including a mixture of stigmasterol and b-sitosterol, 2, 3, 4, 7 and 4-hydroxybenzaldehyde while 2, 3, 4, 6, 7 and 4-hydroxybenzaldehyde were successfully isolated from the DCM extract (Table 1, Fig. 1). The dicloromethane (DCM) crude extract of Alpinia conchigera and the nine isolated compounds obtained were evaluated for their antimicrobial activity against three fungi (Table 2) and five strains of Staphylococcus aureus (Table 3) using minimum inhibitory concentration (MIC) assay. Cycloheximide and oxacillin were included as comparison for the antifungal and antibacterial potential, respectively. The results from the antimicrobial assay exhibited a distinct inhibitory pattern in the major targeted groups namely the candida, dermatophytes and staphylococci, hence will be discussed accordingly. The DCM extract of the rhizome of Alpinia conchigera indicated potent antifungal activity against Candida albicans, Microsporum canis
Fig. 1. The structures of compound 1–7 isolated from the pseudostems and rhizomes of Alpinia conchigera.
A.N. Aziz et al. / Journal of Ethnopharmacology 145 (2013) 798–802
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Table 2 Activity of rhizome DCM crude extract and isolated compounds of Alpinia conchigera against selected fungi. No.
1 2 3 4 5 6 7 8 9 10 11
MIC (mg/mL)
Isolated compounds
DCM crude extract Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 p-Hydroxy cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Cycloheximide
Candida albicans (ATCC 10231)
Microsporum canis (ATCC 36299)
Trycophyton rubrum (ATCC 28188)
625 42500 42500 2500 42500 625 1250 42500 1250 1000 1250
156 42500 42500 2500 42500 625 313 42500 1250 2000 2180
156 4 2500 4 2500 2500 4 2500 625 313 4 2500 1250 2000 2180
All the samples were run in triplicate (n¼ 3).
Table 3 Activity of rhizome DCM crude extract and isolated compounds of Alpinia conchigera against five strains of Staphylococcus aureus. No.
1 2 3 4 5 6 7 8 9 10 11
Isolated compound
DCM crude extract Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 p-hydroxy Cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Oxacillin
MIC (mg/ml) Sa 2 (ATCC 25923)
Sa 3 (ATCC 33591)
Sa 7 (ATTC 700699)
VISA 24
VRSA 156
1250 Nt 35.75 625 625 625 Nt 1250 1250 1250 o 19.5
1250 Nt 17.88 313 625 625 Nt 625 1250 1250 156
1250 Nt 35.75 156 625 625 Nt 1250 1250 2500 625
42500 Nt 471.5 2500 313 156 Nt 156 41250 39 313
42500 Nt 471.5 2500 313 1250 Nt 625 41250 625 o 19.5
All the samples were run in triplicate (n¼ 3), Nt: not traced.
and Trycophyton rubrum with MIC values: 625 mg/ml, 156 mg/ml and 156 mg/ml, respectively which were lower than the control antibiotic, cycloheximide; an antifungal agent which is active against many pathogenic fungi (MIC: 1250, 2180, 2180 mg/ml) (Whiffen, 1948, 1950; Barbary et al., 2010). Amongst the isolated compounds, the lowest inhibition observed were of 10 S-10 -acetoxyeugenol 5 against the dermatophytes (MIC: 313 mg/ml) followed by trans-p-coumaryl diacetate 4 against both dermatophytes and candida (MIC: 625 mg/ ml). At a concentration of as high as 2500 mg/ml, neither anticandidal nor anti-dermatophyte activity could be detected from 10 S-10 -acetoxychavicol acetate 3, the major compound (25.25%) present in the DCM extracts. Although differing in assay technique (broth dilution vs agar diffusion) the observation rhymes very well with earlier report by Janssen and Scheffer (1985) where 5 ml of a very high stock concentration (14 mg/ml) was required to inhibit the dermatophytes. The inhibitory potential of each individual compounds are lower than DCM extract itself. This could be due to the synergism of individual compounds in mixture form. The five staphylococci isolates used in this study differ in their antibiotics’ resistance profile. In contrast to the anti-fungal activity, the DCM extract exhibited weak inhibitory activity (1250 mg/ml) against the non mutant Staphylococci (Table 3). However, significant inhibitory activity with MIC values between 17.88 and 35.75 mg/ml was observed against the MSSA, MRSA and Sa7 isolates. The compound p-hydroxycinnamyl acetate 7 strongly inhibited VISA (MIC 39 mg/ml) followed by trans-pcoumaryl diacetate 4 and 10 -hydroxychavicol acetate 6 with MIC value of 156 mg/ml as compared to the antibiotic, oxacillin (313 mg/ml). The former two compounds are similar in structure
differing only in one of the functional group. In this regard, replacement of a hydroxyl group (7) with an acetoxyl group (4) had resulted in a four fold reduction in the inhibitory activity. VRSA on the other hand was most affected by 10 S-10 -acetoxychavicol acetate 3, the major compound present in the DCM rhizome extract with inhibitory activity value of 313 mg/ml. In conclusion, the observed antibacterial, anticandidal and antidermatophyte activity of the DCM extract and compounds obtained from the rhizome support the traditional use of Alpinia cochigera rhizome in the treatment of skin infection. In addition, our previous study has shown that the essential oils were devoid of this activity which is not surprising as the oils did not contain the active compounds; 10 S-10 -acetoxychavicol acetate and 10 S-10 acetoxyeugenol acetate, discovered in this study.
Acknowledgements This project was funded by a grant under the Chair of Dr. Siti Hasmah Mohd. Ali. The authors wish to thank Mr. Din Mohd. Nor, Mrs. Noryati Jamil (UM), Mr. Saiful Azmi and Mrs. Mazurah Mohd. Isa (FRIM) for their technical assistance.
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Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antimicrobial compounds from Alpinia conchigera Ahmad Nazif Aziz a, Halijah Ibrahim b, Devi Rosmy Syamsir b, Mastura Mohtar c, Jaya Vejayan d, Khalijah Awang e,f,n a
Department of Chemical Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu (UMT), 21030 Kuala Terengganu, Terengganu, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia c Medicinal Plants Programme, Biotechnology Division, Forest Research Institute Malaysia (FRIM) 52109, Kepong, Selangor, Malaysia d School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor, Malaysia e Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia f Centre for Natural Products and Drug Discovery (CENAR), Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2012 Received in revised form 10 October 2012 Accepted 14 December 2012 Available online 21 December 2012
Ethnopharmacological relevance: The rhizome of Alpinia conchigerahas been used as a condiment in the northern states of Peninsular Malaysia and occasionally in folk medicine in the east coast to treat fungal infections. In some states of Peninsular Malaysia, the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish. This study aimed to investigate the chemical constituents of the pseudostems and rhizomes of Malaysian Alpinia conchigera and to evaluate the antimicrobial activity of the dichloromethane (DCM) extracts of the pseudostems, rhizomes and the isolated compounds against three selected fungi and five strains of Staphylococcus aureus. Materials and methods: The dried and ground pseudostems (0.8 kg) and rhizomes (1.0 kg) were successively extracted in Soxhlet extractor using n-hexane, dichloromethane (DCM) and methanol. The n-hexane and DCM extracts of the pseudostem and rhizome were subjected to isolation and purification using column chromatography on silica gel using a stepwise gradient system (n-hexane to methanol). Briefly, a serial two fold dilutions of the test materials dissolved in DMSO were prepared prior to addition of 100 ml overnight microbial suspension (108 cfu/ml) followed by incubation at 37 1C (bacteria) or 26 1C (dermatophytes and candida) for 24 h. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibition to bacterial/candida/dermatophytes’ growth. Antibiotic cycloheximide was used as reference for anticandidal and antidermatophyte comparison while oxacilin was used as reference for antibacterial testing. DMSO served as negative control. Turbidity was taken as indication of growth, thus the lowest concentration which remains clear after macroscopic evaluation was taken as the minimum inhibitory concentration (MIC). Results: The isolation of n-hexane and DCM extracts of the rhizomes and pseudostems of Alpinia conchigera via column chromatography yielded two triterpenes isolated as a mixture of stigmasterol and b-sitosterol: caryophyllene oxide, chavicol acetate 1, p-hydroxy cinnamaldehyde 2, 10 S-10 acetoxychavicol acetate 3, trans-p-coumaryl diacetate 4, 10 S-10 -acetoxyeugenol acetate 5, 10 -hydroxychavicol acetate 6, p-hydroxycinnamyl acetate 7 and 4-hydroxybenzaldehyde. The DCM extract of the rhizome of Alpinia conchigera indicated potent antifungal activity against Candida albicans, Microsporum canis and Trycophyton rubrum with MIC values of 625 mg/ml, 156 mg/ml and 156 mg/ml, respectively. It also showed significant inhibitory activity with MIC values between 17.88 and 35.75 mg/ml against the mutant Staphylococci isolates MSSA, MRSA and Sa7. Amongst the isolated compounds, the lowest inhibition observed were of 10 S-10 -acetoxyeugenol against the dermatophytes (MIC 313 mg/ml) followed by trans-p-coumaryl diacetate against both dermatophytes and candida (MIC 625 mg/ml). The compound p-hydroxycinnamyl acetate strongly inhibited Staphylococcus aureus strain VISA (MIC 39 mg/ml) followed by trans-p-coumaryl diacetate and 10 -hydroxychavicol acetate with MIC value of 156 mg/ml. Conclusion: In conclusion, the observed antibacterial, anticandidal and antidermatophyte activity of the extracts and compounds obtained from the rhizome confirm the traditional use of Alpinia cochigera rhizome in the treatment of skin infection. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alpinia conchigera Zingiberaceae Phenylpropanoids Antimicrobial Antifungal
n Corresponding author at: Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: þ 603 796 740 64; fax: þ 603 796 741 93. E-mail address: [email protected] (K. Awang).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.024
A.N. Aziz et al. / Journal of Ethnopharmacology 145 (2013) 798–802
1. Introduction Alpinia conchigera Griff. is a herbaceous perennial, 2 to 5 ft. tall, found in eastern Bengal and southwards to Peninsular Malaysia and Sumatera. In Malaysia, it is also locally known as lengkuas ranting, lengkuas kecil, lengkuas padang, lengkuas genting or chengkenam (Burkill, 1966). This species is semi-wild, common in open wet grounds such as edges of rice fields, streams as well as under the shade of palm oil and rubber trees. The rhizome is used as a condiment in the northern states of Peninsular Malaysia and occasionally in folk medicine in the east coast to treat fungal infections (Ibrahim et al., 2000). In some states of Peninsular Malaysia the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish (Ibrahim et al., 2009). There were few studies on the chemical constituents of Alpinia conchigera reported to date. Yu et al. (1988) reported the presence of nonacosane, b-sitosterol, 10 -acetoxychavicol acetate and 10 -acetoxyeugenol acetate in the fruit, the two phenylpropanoid derivatives showing anti-inflammatory activity. Later, Athamaprasangsa et al. (1994) communicated the detection of four known phenylpropanoids: chavicol acetate, 10 -hydroxychavicol acetate, 4-acetoxycinnamyl alcohol and 4-acetoxycinnamyl acetate from the aqueous layer obtained from the hydrodistillation of the fresh rhizomes of Alpinia conchigera Griff. from Thailand. They also reported five diarylheptanoids: 1,7-diphenyl-3,5-heptanedione, 1,7-diphenyl-5-hydroxy-3-heptanone, 5-hydroxy-7-(40 -hydroxy-30 -methoxyphenyl)-1-phenyl-3 heptanone, 1,7-diphenylhept-4-en-3-one and 7-(40 -hydroxy-30 methoxyphenyl)-1-phenylhept-4-en-3-one and two flavonoids: 3,5,7-trihydroxyflavone and 3,5,7-trihydroxy-40 -methoxyflavone from the n-hexane and DCM extracts of the dried rhizomes. Recent studies on chemical constituents of Alpinia conchigera by Phan et al. (2005) reported b-sitosterol, stigmasterol and three flavonoids: cardamomin, alpinetin and naringenin 5-Me ether isolated from the rhizomes of Alpinia conchigera Griff. from Vietnam. Further study on the methanolic extract of the rhizomes resulted with b-sitosterol, stigmasterol, cardamomin, chalconaringenin 20 -O-Me ether, alpinetin, and naringenin 5-O-Me ether (Le et al., 2007). In addition, Giang et al. (2007) isolated flavokawin B, b-sitosterol, stigmasterol, alpinetin, (3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene, b-D-fructopyranose, b-D-fructofuranose, and 2-O-Me b-D-fructofuranose from the fruits of Alpinia conchigera Griff. (Zingiberaceae) from Vietnam. 10 S-10 acetoxychavicol acetate and 10 S-10 -acetoxyeugenol acetate obtained from Alpinia conchigera were found to induce apoptosis in oral squamous carcinoma cells (HSC-4) and human breast cancer cells (MCF-7), respectively (Awang et al., 2010; Hasima et al., 2010). Ibrahim et al. (2009) communicated the essential oil constituents of Alpinia conchigera. All of the above mentioned studies involved the constituents from the rhizomes and fruits of Alpinia conchigera from China, Thailand, Vietnam and Malaysia and fruits of ract of the rhizomes. In this study, the chemical constituents from the pseudostems and rhizomes of Malaysian Alpinia conchigera will be investigated and discussed. In addition, investigation on the antimicrobial activity of the DCM extracts of the pseudostems, rhizomes and the isolated compounds against three selected fungi and five strains of Staphylococcus aureus will be carried out.
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herbarium series number KL 5049. The samples were authenticated by Professor Dr. Halijah Ibrahim of the Institute of Biological Sciences, University of Malaya and the voucher specimens were deposited in the herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur. 2.2. Extraction and isolation The dried and ground pseudostems (0.8 kg) and rhizomes (1.0 kg) were extracted in Soxhlet extractor using n-hexane, dichloromethane (DCM) and methanol (MeOH) successively. The pseudostem and rhizome extracts were dried in vacuo. The n-hexane and DCM extracts were further subjected to chemical compound isolation and antimicrobial activity (antibacterial and antifungal activity) studies. The n-hexane and DCM extracts of the pseudostems and rhizomes were subjected to column chromatography (CC) on silica gel using a stepwise gradient system (n-hexane to MeOH). All compounds were successfully isolated using CC with a combination of n-hexane-ethyl acetate (EtOAc) as the solvent system. 2.3. General experimental procedure All NMR spectra were recorded on a JEOL LA-400 spectrometer (400 MHz), in CDCl3; The mass of the compounds were obtained from GC–MS (Hewlett Packard 5989 A) on HP-5 column. 2.4. Antimicrobial activity 2.4.1. Test isolates Microbes selected for this study are those implicated in skin infections (Williams et al., 1999; Pereira Gonzales and Maisch, 2012). The crude extracts and the isolated compounds were individually tested against five strains of Staphylococcus aureus (each differing in their antibiotic resistance profile) namely Methicillin sensitive Staphylococcus aureus ATCC 29213 (MSSA), Methicillin resistant Staphylococcus aureus ATCC 33591 (MRSA), Vancomycin intermediate resistant Staphylococcus aureus ATCC 700699 (Sa7), MRSA with intermediate resistance to vancomycin (VISA24) and MRSA with complete resistance to vancomycin (VRSA156), a candida: Candida albicans ATCC 10231, two dermatophytes: Microsporum canis (ATCC 36299) and Trichophyton rubrum (ATCC 28188). All isolates were purchased from American Type Culture Collection (ATCC) except two mutant isolates namely VISA24 and VRSA156 that were obtained through a step wise lab-passage procedure as described previously (Mohtar et al., 2009). 2.4.2. Inoculum preparation Media was sterilized by autoclaving at 120 1C for 15 min and all subsequent manipulations were carried out in a class 2 biohazard cabinet. The Staphylococcus aureus strains (coded as Sa 2, Sa 3, Sa 7) were cultured in Mueller Hinton Broth (MHB), VISA and VRSA in Tryptic soy broth (TSB) overnight (24 h) at 37 1C while the candida and dermatophytes were cultured in Potato dextrose broth (PDB) overnight at 26 1C. The resulting inoculum was further adjusted to obtain a turbidity comparable to that of McFarland standard tube No. 0.5 (Vandepitte et al., 1991) prior to use.
2. Material and methods 2.1. Plant material The pseudostems and rhizomes of wild Alpinia conchigera Griff. were collected from Jeli province of Kelantan, Malaysia with
2.4.3. Minimum inhibitory concentration (MIC) The minimum inhibitory concentration (MIC) value determination assay was carried out to evaluate the potential compounds as inhibitory agent against test isolate using double-broth microdilution method involving 96-wells microtitre-plates as described
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previously (Saiful et al., 2008). Briefly, a serial two fold dilutions of the test compounds dissolved in DMSO were prepared prior to addition of 100 ml overnight microbial suspension (108 cfu/ml) followed by incubation at 37 1C (bacteria) or 26 1C (dermatophytes and Candida) for 24 h. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibition to bacterial/candida/dermatophytes’ growth. DMSO served as negative control. Antibiotic cycloheximide was used as reference for anticandidal and antidermatophyte comparison while oxacilin was used as reference for antibacterial testing. Turbidity was taken as indication of growth, thus the lowest concentration which remains clear after macroscopic evaluation was taken as the minimum inhibitory concentration (MIC). For further reconfirmation, 20 ml of MTT reagent (1 mg/ml) was added to the suspension in the selected wells, followed by 20 min incubation at 37 1C. The reagent-suspension colour will remain clear or yellowish indicating complete inhibition (cidal) activity as opposed to dark blue for growth (Eloff, 1998). The MIC was recorded as the most repeatable minimum concentration of triplicate.
Table 1 List of isolated compounds from the n-hexane and DCM extracts of pseudostems and rhizomes of Alpinia conchigera and their percentage yield (%). Compound
Pseudostem (% yield)
Rhizome (% yield) Extract
Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 Caryophyllene oxide p-Hydroxy cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Ni: not isolated.
n-hexane
DCM
n-hexane
DCM
0.31 Ni Ni 0.07 0.15 0.68 Ni Ni 0.13 0.20
Ni Ni Ni 0.18 9.08 0.95 Ni 0.37 0.48 1.10
0.25 0.08 0.05 0.59 40.07 0.19 0.12 0.15 0.61 0.12
0.14 Ni Ni 0.20 25.25 0.80 0.03 0.19 0.12 0.92
3. Results and discussion Soxhlet extraction of the pseudostems yielded 5.89 g (0.74% w/w) of n-hexane extracts, 14.70 g (1.84% w/w) of DCM extracts and 72.30 g (9.03% w/w) of MeOH extracts while the rhizomes produced 22.68 g (2.27% w/w) of n-hexane extracts, 15.87 g (1.59% w/w) of DCM extracts and 150.36 g (15.04% w/w) of MeOH extracts. The isolation of n-hexane extracts of the rhizomes of Alpinia conchigera via column chromatography yielded two triterpenes ¨ ´ r, 2004; Xu isolated as a mixture of stigmasterol (Forgo and Kove et al., 2005) and b-sitosterol (De-Eknamkul and Potduang, 2003); one sesquiterpene; caryophyllene oxide (Silverstein and Webster, 1998), seven phenyl propanoids; chavicol acetate 1 (Yu et al., 1988; Mitsui et al., 1976), p-hydroxy cinnamaldehyde 2 (Leem et al., 1999), 10 S-10 -acetoxychavicol acetate 3 (Janssen and Scheffer, 1985; Noro et al., 1988; Yang and Eilerman, 1999), trans-p-coumaryl diacetate 4, 10 S-10 -acetoxyeugenol acetate 5 (Noro et al., 1988; Yang and Eilerman, 1999), 10 -hydroxychavicol acetate 6 (Janssen and Scheffer, 1985; Yang and Eilerman, 1999), p-hydroxycinnamyl acetate 7 (Kiuchi et al., 2002), and one phenolic compound; 4-hydroxybenzaldehyde (Noro et al., 1988). The DCM extract of the rhizome afforded nine compounds which were similar to that of the compounds found in the n-hexane extract, but deprived of chavicol acetate 1 and caryophyllene oxide. As for the purification of the extracts from the pseudostem, seven compounds were found in the n-hexane crude extract including a mixture of stigmasterol and b-sitosterol, 2, 3, 4, 7 and 4-hydroxybenzaldehyde while 2, 3, 4, 6, 7 and 4-hydroxybenzaldehyde were successfully isolated from the DCM extract (Table 1, Fig. 1). The dicloromethane (DCM) crude extract of Alpinia conchigera and the nine isolated compounds obtained were evaluated for their antimicrobial activity against three fungi (Table 2) and five strains of Staphylococcus aureus (Table 3) using minimum inhibitory concentration (MIC) assay. Cycloheximide and oxacillin were included as comparison for the antifungal and antibacterial potential, respectively. The results from the antimicrobial assay exhibited a distinct inhibitory pattern in the major targeted groups namely the candida, dermatophytes and staphylococci, hence will be discussed accordingly. The DCM extract of the rhizome of Alpinia conchigera indicated potent antifungal activity against Candida albicans, Microsporum canis
Fig. 1. The structures of compound 1–7 isolated from the pseudostems and rhizomes of Alpinia conchigera.
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Table 2 Activity of rhizome DCM crude extract and isolated compounds of Alpinia conchigera against selected fungi. No.
1 2 3 4 5 6 7 8 9 10 11
MIC (mg/mL)
Isolated compounds
DCM crude extract Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 p-Hydroxy cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Cycloheximide
Candida albicans (ATCC 10231)
Microsporum canis (ATCC 36299)
Trycophyton rubrum (ATCC 28188)
625 42500 42500 2500 42500 625 1250 42500 1250 1000 1250
156 42500 42500 2500 42500 625 313 42500 1250 2000 2180
156 4 2500 4 2500 2500 4 2500 625 313 4 2500 1250 2000 2180
All the samples were run in triplicate (n¼ 3).
Table 3 Activity of rhizome DCM crude extract and isolated compounds of Alpinia conchigera against five strains of Staphylococcus aureus. No.
1 2 3 4 5 6 7 8 9 10 11
Isolated compound
DCM crude extract Stigmasterol and b-sitosterol (mixture) Chavicol acetate 1 p-hydroxy Cinnamaldehyde 2 10 S-10 -Acetoxychavicol acetate 3 Trans-p-coumaryl diacetate 4 10 S-10 -Acetoxyeugenol acetate 5 10 -Hydroxychavicol acetate 6 4-Hydroxybenzaldehyde p-Hydroxycinnamyl acetate 7 Oxacillin
MIC (mg/ml) Sa 2 (ATCC 25923)
Sa 3 (ATCC 33591)
Sa 7 (ATTC 700699)
VISA 24
VRSA 156
1250 Nt 35.75 625 625 625 Nt 1250 1250 1250 o 19.5
1250 Nt 17.88 313 625 625 Nt 625 1250 1250 156
1250 Nt 35.75 156 625 625 Nt 1250 1250 2500 625
42500 Nt 471.5 2500 313 156 Nt 156 41250 39 313
42500 Nt 471.5 2500 313 1250 Nt 625 41250 625 o 19.5
All the samples were run in triplicate (n¼ 3), Nt: not traced.
and Trycophyton rubrum with MIC values: 625 mg/ml, 156 mg/ml and 156 mg/ml, respectively which were lower than the control antibiotic, cycloheximide; an antifungal agent which is active against many pathogenic fungi (MIC: 1250, 2180, 2180 mg/ml) (Whiffen, 1948, 1950; Barbary et al., 2010). Amongst the isolated compounds, the lowest inhibition observed were of 10 S-10 -acetoxyeugenol 5 against the dermatophytes (MIC: 313 mg/ml) followed by trans-p-coumaryl diacetate 4 against both dermatophytes and candida (MIC: 625 mg/ ml). At a concentration of as high as 2500 mg/ml, neither anticandidal nor anti-dermatophyte activity could be detected from 10 S-10 -acetoxychavicol acetate 3, the major compound (25.25%) present in the DCM extracts. Although differing in assay technique (broth dilution vs agar diffusion) the observation rhymes very well with earlier report by Janssen and Scheffer (1985) where 5 ml of a very high stock concentration (14 mg/ml) was required to inhibit the dermatophytes. The inhibitory potential of each individual compounds are lower than DCM extract itself. This could be due to the synergism of individual compounds in mixture form. The five staphylococci isolates used in this study differ in their antibiotics’ resistance profile. In contrast to the anti-fungal activity, the DCM extract exhibited weak inhibitory activity (1250 mg/ml) against the non mutant Staphylococci (Table 3). However, significant inhibitory activity with MIC values between 17.88 and 35.75 mg/ml was observed against the MSSA, MRSA and Sa7 isolates. The compound p-hydroxycinnamyl acetate 7 strongly inhibited VISA (MIC 39 mg/ml) followed by trans-pcoumaryl diacetate 4 and 10 -hydroxychavicol acetate 6 with MIC value of 156 mg/ml as compared to the antibiotic, oxacillin (313 mg/ml). The former two compounds are similar in structure
differing only in one of the functional group. In this regard, replacement of a hydroxyl group (7) with an acetoxyl group (4) had resulted in a four fold reduction in the inhibitory activity. VRSA on the other hand was most affected by 10 S-10 -acetoxychavicol acetate 3, the major compound present in the DCM rhizome extract with inhibitory activity value of 313 mg/ml. In conclusion, the observed antibacterial, anticandidal and antidermatophyte activity of the DCM extract and compounds obtained from the rhizome support the traditional use of Alpinia cochigera rhizome in the treatment of skin infection. In addition, our previous study has shown that the essential oils were devoid of this activity which is not surprising as the oils did not contain the active compounds; 10 S-10 -acetoxychavicol acetate and 10 S-10 acetoxyeugenol acetate, discovered in this study.
Acknowledgements This project was funded by a grant under the Chair of Dr. Siti Hasmah Mohd. Ali. The authors wish to thank Mr. Din Mohd. Nor, Mrs. Noryati Jamil (UM), Mr. Saiful Azmi and Mrs. Mazurah Mohd. Isa (FRIM) for their technical assistance.
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