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Research ArticlePhytochemicals from Kaempferia angustifolia Rosc and TheirCytotoxic and Antimicrobial Activities

Sook Wah Tang1 Mohd Aspollah Sukari1 Bee Keat Neoh2 Yunie Soon Yu Yeap1

Ahmad Bustamam Abdul3 Nurolaini Kifli4 and Gwendoline Cheng Lian Ee1

1 Department of Chemistry Faculty of Science Universiti Putra Malaysia 43400 Serdang Selangor Malaysia2 Sime Darby Technology Center 2 Jalan Tandang 46050 Petaling Jaya Selangor Malaysia3 UPM-MAKNA Cancer Research Laboratory Institute of Bioscience Universiti Putra Malaysia 43400 Serdang Selangor Malaysia4 Pangiran Anak Puteri Rashidah Sarsquoadatul Bolkiah Institute of Health Science Universiti Brunei Darussalam Jalan Tungku LinkGadong BE1410 Brunei Darussalam

Correspondence should be addressed to Mohd Aspollah Sukari aspollahupmedumy

Received 30 April 2014 Accepted 4 June 2014 Published 25 June 2014

Academic Editor Jose Domingos Fontana

Copyright copy 2014 Sook Wah Tang et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Phytochemical investigation on rhizomes ofKaempferia angustifolia has afforded a new abietene diterpene kaempfolienol (1) alongwith crotepoxide (2) boesenboxide (3) 21015840-hydroxy-44101584061015840-trimethoxychalcone (4) zeylenol (5) 6-methylzeylenol (6) (24S)-24-methyl-5120572-lanosta-9(11) 25-dien-3120573-ol (7) sucrose 120573-sitosterol and its glycoside (8) The structures of the compounds wereelucidated on the basis of spectroscopic methods (IR MS and NMR) Isolation of 6-methylzeylenol (6) (24S)-24-methyl-5120572-lanosta-9(11) 25-dien-3120573-ol (7) and 120573-sitosterol-3-O-120573-D-glucopyranoside (8) from this plant species has never been reportedpreviously The spectroscopic data of (7) is firstly described in this paper Cytotoxic screening indicated that most of the purecompounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocyticleukemia) and MCF-7 (human breast cancer) cell lines However all extracts and most of the pure compounds tested were foundto be inactive against HT-29 (human colon cancer) and HeLa (human cervical cancer) cell lines Similarly none of the extracts orcompounds showed activity in the antimicrobial testing

1 Introduction

Kaempferia is a genus of herbs of over 50 species natives ofeast tropical Asia belonging to the Zingiberaceae family It issmall rhizomatous herb with usually thick aromatic tuberousroots and short rhizomes Kaempferia angustifolia is one ofthe Kaempferia species which is less well known in Malaysiaif compared with K galanga and K rotunda Itis locallyknown asKunci pepet Kuncimenir orKunci kunotThis plantis tuberous and stemless has small leaves and could be foundgrowing wildly in the forests of west and center of Java Ithas nice smell and is usually used as medicine to treat coldstomach-ache and dysentery while its rhizome is used forcoughs and as a masticatory [1]

In recent years many researchers have indicated thehigh potential of edible Southeast Asia plants for cancer

chemoprevention and as antimicrobial agents Zingiberaceaehave been found to be one of the desirable sources of effectivecancer-preventive agents since Zingiberaceous plantsdemonstrated promising inhibitory effect on the growth ofhuman breast cancer (MCF-7) colon cancer (HT-29 andCol2) lung cancer (A549) stomach cancer (SNU-638) andcervical cancer (CaSki) cell lines [2ndash4] Besides antimicrobialactivities of ginger were also a topic of interest Reports onthe screening of extracts or essential oils of members fromZingiberaceae family against bacterial strains fungi andyeast have been published [5ndash7] Alpinia Curcuma andZingiber species were frequently studied but literature onantimicrobial properties of Kaempferia species was not stud-ied as much Essential oil of Kaempferia angustifolia has beenreported [8] to have moderate antimicrobial activity towardsinhibition of Staphylococcus aureus and Pseudomonas

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 417674 6 pageshttpdxdoiorg1011552014417674

2 BioMed Research International

aeruginosa (1063 plusmn 023 1003 plusmn 022mm resp) butnot against Escherichia coli Bacillus subtilis Streptococcusfaecalis Candida albicans andMicrosporum gypseum

Phytochemical investigations of Kaempferia species haveyielded several compounds including cyclohexane diepoxidederivatives flavonoids and diterpenes [9ndash11] Most of theconsumable ginger rhizomes have long been used as folkmedicine for centuries but have not been thoroughly inves-tigated for their bioactive properties yet Thus here we wishto report the isolation and cytotoxic and antimicrobial prop-erties of chemical constituents from rhizomes of Kaempferiaangustifolia

2 Materials and Methods

21 General Experimental Procedures Melting points (uncor-rected) were determined using Barnstead Electrothermal IA9100 Series melting points equipment Optical rotations weremeasured on a Jasco P-2000 polarimeter equipped with asodium lamp (589 nm) The IR spectra were recorded usingKBr discs on Perkin Elmer FTIR spectrophotometer 1650 1Hand 13CNMR spectra were obtained on JEOL spectrometer at500 and 125MHz andor 400 and 100MHz respectively withtetramethylsilane (TMS) as internal standard Mass spectrawere recorded on Shimadzu model QP 5050A spectrometerUltraviolet spectra were recorded on a Shimadzu UV-1601spectrophotometer Separation by column chromatographywas carried out by using silica gel (Merck 7749 7734 and9385) while silica gel 60 PF

254aluminium sheets were used

for TLC analysis

22 Plant Material and Cytotoxic Cell Lines Kaempferiaangustifolia was collected from Java Indonesia in 2001 Theplant was identified by Dr Sugeng Riyanto Faculty of Phar-macy Gadjah Mada University and the voucher specimen(number 16SR0601-01) was deposited at the herbarium ofthe institutionTheHL-60 (human promyelocytic leukemia)MCF-7 (human breast cancer) HT-29 (human colon cancer)and HeLa (human cervical cancer) cell lines were obtainedfrom theNational Cancer InstituteMaryland USA Bacterialstrains were originally obtained from American Type CellCulture Collection (ATCC) USA

23 Extraction and Isolation The finely ground air-driedrhizomes (10 kg) were extracted repeatedly with petroleumether chloroform and methanol thrice each for 72 hoursThe extracts were filtered and concentrated under reducedpressure to obtain petroleum ether (840 g) chloroform(750 g) and methanol (670 g) extracts The petroleum etherextract was triturated with methanol at room temperature togive a solid product whichwas then filtered and recrystallizedin methanol to yield crotepoxide (2) (16mg) Brown oilextract (580 g) was obtained upon evaporation of the filtrateThe extract was subjected to vacuum column chromatog-raphy and eluted stepwise with 100 hexane hexane-ethylacetate ethyl acetate-methanol and 100 methanol Thisafforded boesenboxide (3) (40mg) 21015840-hydroxy-4 41015840 61015840-trimethoxychalcone (4) (26mg) and 120573-sitosterol (18mg)

Spectroscopic data of compounds 2ndash4 have been reportedpreviously [12] Chloroform extract was subjected to columnchromatography separation and eluted with mixture of hex-ane and ethyl acetate to give 23 fractions (200mL each)Fraction three which was eluted from hexane-ethyl acetate(5 5) was purified to afford zeylenol (5) (0238 g) whilefraction four obtained from elution with mixture of hexane-ethyl acetate (3 7) was rechromatographed over silica gel togive subfractions 17ndash19 which were then recrystallized withmethanol to afford 6-methylzeylenol (6) (42mg) Methanolextract was fractionated using column chromatography ina similar manner Eighteen fractions of 200mL each werecollected Combination and purification of fractions 14 and15 from elution with ethyl acetate-methanol (7 3) gavesucrose (11mg) A reinvestigation of the sample (7650 g)from the same source was conducted by extraction withmethanol (3 times 72 hours) to obtain a dark brown viscousliquid extract which was then partitioned with hexaneand water with 10 methanol (1 1) to give hexane-solublefraction (1060 g) as brown yellowish viscous liquid Thehexane fraction (800 g) was subjected to column chromatog-raphy eluted with mixtures of hexane ethyl acetate andmethanol of increasing polarity to give 86 fractions (200mLeach) Upon washing and recrystallizing with methanolfractions 13-4 which were eluted from hexane ethyl acetate(97 3) afforded (24S)-24-methyl-5120572-lanosta-9(11) and 25-dien-3120573-ol (7) (40mg) Fraction 84 which was eluted fromethyl acetate methanol (50 50) was further washed withmethanol to give 120573-sitosterol-3-O-120573-D-glucopyranoside (8)(8mg) Similarly compound 2 (10mg) compound 4 (7mg)and 120573-sitosterol (35mg) were also obtained from this extractChloroform-soluble fraction (40000 g) was fractionated ina similar manner to obtain 100 fractions (200mL each)which were combined into major fractions based on theirTLC profiles Compounds 1 (20mg) 2 (079 g) 3 (16mg) 4(10mg) and 5 (097 g) were subsequently obtained Isolationprocedures and spectroscopic data of compounds 1 and 5have been reported previously [13]

24 Spectral Data (24119878)-Methyl-5120572-lanosta-9(11)25-dien-3120573-ol (7) C

31H52O white needle-shaped crystal Melt-

ing point 180-181∘C [120572]119863 +675(c 004 CHCl

3) 119877119891 060

(Hexane-EtOAc 4 1) IR (KBr) 3684 3402 2940 1644 1098888 cmminus1 UVVis 120582max (CHCl

3) nm (log 120576) 2392 (3837) 1H

NMR (400MHz CDCl3) and 13C NMR (100MHz CDCl

3)

Table 1 EI-MS 119898119911() = 440 (M+ 27) 425 (56) 407 (31)313 (100) 69 (72) 55(81)

25 Cytotoxic Assay The cytotoxic assay was carried outaccording to the methods described previously [14] Cyto-toxic and proliferation profiles were determined by MTT(3-[45-dimethylthioazol-2-yl]-25-diphenyltetrazolium bro-mide) assay whereby cell viability of treated cell populationwas compared to the control cell population The cell via-bility was determined by the formation of blue formazancrystals of MTT in which the crystals were dissolved inDMSO and the absorbance was read with Elisa reader testwavelength of 570 nm and references wavelength of 630 nm

BioMed Research International 3

Table 1 1H (400MHz) and 13C-NMR (100MHz) spectral data ofcompound 7 (CDCl3)

Position 1205751H (ppm)

(multiplicity 119869 in Hz)12057513C

(ppm)1 135 (m) 145 (m) 3612 167 (m)a 172 (m) 2783 322 (dd 119 46) 7894 mdash 3915 087 (d 64)b 525

6 147 (dd 129 27)168 (m)a 214

7 130 (m) 2818 216 (m) 4189 mdash 148510 mdash 39411 522 (d 64) 115012 191 (br d 64) 371

208 (br d 64)13 mdash 44314 mdash 47015 133 (m)c 33916 187 (br d 46) 27917 157 (m) 50918 064 (s) 14419 104 (s) 22320 180 (m) 36021 087 (d 64)b 18422 133 (m)c 34023 115 (m) 142 (m) 31424 210 (m) 41625 mdash 150226 466 (s) 109427 164 (s) 18628 099 (s) 28229 082 (s) 15730 073 (s) 18531 100 (d 74) 202abcOverlapped signals

Four human cancer cell lines which included HL-60 (humanpromyelocytic leukemia) MCF-7 (human breast cancer)HT-29 (human colon cancer) and HeLa (human cervicalcancer) cell lines were tested against crude extracts andpure compounds isolated A graph of percentage of cellviability versus the concentration of crude extracts or purecompounds tested was plotted and the IC

50values were

determined The cytotoxic index used was IC50 which is

the concentration that yields 50 inhibition of the treatedcell compared with untreated control Extracts that showIC50lt 30 120583gmL are considered to have significant cytotoxic

activity whilst pure compounds that show cytotoxicity IC50lt

20 120583gmL are qualified for further cytotoxicity investigations[15]

26 Antimicrobial Assay The extracts and isolated com-pounds were subjected to antimicrobial testing against sev-eral microbes including methicillin resistant Staphylococcusaureus Pseudomonas aeruginosa (ATCC 60690) Salmonellacholeraesuis and Bacillus subtilis (wild) (B

29) for antibac-

terial screening and Candida albicans Saccharomyces cere-visiae and Aspergillus ochraceus (ATCC 398) for antifun-gal screening using disc diffusion method as described in[16] Ampicillin (Gram-negative bacteria) and streptomycin(Gram-positive bacteria) standards were used for each ofthe bacteria whilst nystatin was used for fungi and yeastas positive control The antimicrobial activity was evaluatedqualitatively bymeasuring the diameter of the clear inhibitionzones around the impregnated discs The activity index wasdetermined by dividing its zone of inhibition by the standardantibacterial agent The samples that show activity of gt05were considered to exhibit significant antibacterial activity[17]

3 Results and Discussion

Extraction and separation on the isolates of various extractsof K angustifolia led to the isolation and characterizationof kaempfolienol (1) crotepoxide (2) boesenboxide (3)21015840-hydroxy-44101584061015840-trimethoxychalcone (4) zeylenol (5) 6-methylzeylenol (6) (24S)-24-methyl-5120572-lanosta-9(11) and25-dien-3120573-ol (7) together with sucrose 120573-sitosterol and itsglycoside (8) (Figure 1) To the best of our knowledge com-pounds 6 7 and 8 were isolated for the first time from thisplant species In this report we describe the spectroscopicdata of compound 7 as well as cytotoxic and antimicrobialactivities of the plant constituentsThe details of the isolationand characterization of new compound kaempfolienol (1)zeylenol (5) and its derivatives have been described in ourprevious report [13] The compounds were characterizedusing spectroscopic methods and by comparison with theliterature [12 18ndash21]

Compound 6 (0042 g) with molecular formula ofC22H22O7was obtained as colourless needle-shaped crystals

with melting point of 206-207∘C The FAB-MS spectrumshowed the [M+H]+ peak at mz 399 which corresponds tothe molecular formula mentioned All spectral data were inaccordance with the previously reported data of compound 6which is isolated from Uvaria purpurea [14]

Compound 7 was isolated as white needle-shaped crystalwithmelting point of 180-181∘CTheEI-MS spectrum showeda molecular ion at mz 440 corresponding to molecularformula of C

31H52O indicative of six degrees of unsaturation

The IR spectrum of (7) indicated the presence of hydroxylgroup at 3684 and 3402 cmminus1 and olefinic functional group at1644 cmminus1 The 1H NMR spectrum demonstrated signals forsix tertiary methyl groups and two secondary methyl groupsranging from 120575 064 to 164 three olefinic protons at 120575 466(2H) and 522 (1H) and a deshieldedmethine proton at 120575 322These spectral data (Table 1) suggested that compound 7 is


O

OOCOR

O

R

(4)(1)

HO

HO

OH

OHOH

OH

OH

OH

(2) R = CH3

MeO OMe

OMeCH2OCOC6H5

OCOCH3

OCOC6H5

OCOC6H5

(5) R = OH

HO

HOHO

HOOH

H

123

4 56

7

8910

1112

1314

15

16

1718

19

20

21 22

2324

2526

27

2829

30

31

O

H

O

(7) (8)

(3) R = C6H5 (6) R = OCH3

Figure 1 Structures of chemical constituents fromKaempferia angustifolia kaempfolienol (1) crotepoxide (2) boesenboxide (3) 21015840-hydroxy-44101584061015840-trimethoxychalcone (4) zeylenol (5) 6-methylzeylenol (6) (24S)-24-methyl-lanosta-9(11) 25-dien-3120573-ol (7) and 120573-sitosterol-3-O-120573-D-glucopyranoside (8)

an unsaturated triterpene alcoholThe 13CNMR experimentsincluding DEPT sorted 31 signals into eight methyls tenmethylenes six methines and seven quaternary carbonsThe carbon signals for (7) were almost identical to thecorresponding carbon signals of reported data for its acetylderivative [20] except for C-2 to C-4 and the signals for 4120573-methyl (C-29) The chemical shifts values of C-3 C-2 andC-4 revealed an upfield shift of 20 ppm and a downfieldshift of 36 and 11 ppm respectively compared to those ofcorresponding data of (24S)-24-methyl-5120572-lanosta-9(11) 25-dien-3120573-acetate [20] It was thus clear that a hydroxyl groupwas located at C-3The precise NMR signals connectivities of(7) were deduced by interpretation of the significant HMBCspectrum and are shown in Figure 2 The olefinic proton at 120575522 (d 64Hz) was correlated with C-10 C-13 and C-8 via 3Jcorrelation and hence the double bond was placed at C-9C-11 Two exomethylene protons which resonated as singlet at 120575466 exhibited aw-coupling to the deshieldedmethyl group at120575 164 (H-27) in COSY spectrum andwere correlated to C-24C-25 andC-27 inHMBC spectrumOn the basis of the aboveresults compound 7 was characterized as (24S)-24-methyl-5120572-lanosta-9(11) 25-dien-3120573-ol This is the first isolation ofthe compound as triterpene alcohol since previous literaturereported it as its acetyl derivatives isolated from Glycosmisarborea and Neolitsea aciculata [20 21]

Compound 8 was obtained as white amorphous powderfrom the last fraction of hexane extract The molecular ionpeak for compound 8 which is suggested to be at mz 576

HO

H

12

34

29 28

H

19

67

830

1415

16

H

910

1113 17

18

2122

H23

2425

26

27

31

Figure 2 Selected HMBC correlations of (7)

could not be traced in the EI-MS spectrum However thefragment ions at mz 414 [M-C

6H10O5]+ 396 [M-C

6H10O5-

H2O]+ 255 [M-C

6H10O5-H2O-C10H21(side chain)]+ and

mz 127 73 and 57 which were obtained from the sugarmoiety suggested that compound 8 may be monoglycosidewith a C

29-sterol (mz 414) aglycone moiety Further analysis

of FAB-MS spectrum demonstrated the molecular ion peakat 599 [M+Na]+ which supported the molecular formula ofC35H60O6 From the evidences in spectral data and com-

parison with reported data [19] compound 8 was thereforeestablished as 120573-sitosterol-3-O-120573-D-glucopyranoside whichwas isolated previously from Prunella vulgaris


Table 2 IC50 values for pure compounds of K angustifolia against HL-60 MCF-7 HT-29 and HeLa cell lines

SampleslowastIC50 (120583gmL)

HL-60 MCF-7 HT-29 HeLa1 2422 plusmn 030 2350 plusmn 170 gt30 gt302 gt30 1809 plusmn 020 gt30 gt303 777 plusmn 048 1923 plusmn 069 gt30 gt304 749 plusmn 124 624 plusmn 057 gt30 gt305 1165 plusmn 052 gt30 gt30 gt306 1963 plusmn 051 1763 plusmn 084 mdash 1945 plusmn 0507 610 plusmn 022 575 plusmn 154 gt30 142 plusmn 0168 2309 plusmn 339 1983 plusmn 115 gt30 2395 plusmn 036Standard goniothalamin tamoxifen 5-fluorouracil 14 28 42 12lowastValues were means plusmn standard deviation of triplicate analyses

The cytotoxic screening results (Table 2) suggested theantagonist effect shown by the isolated compounds ofKaempferia angustifolia against HL-60 (human promyelo-cytic leukemia) MCF-7 (human breast cancer) and HeLa(human cervical cancer) cell lines All crude extracts testedwere inactive against all cell lines tested with IC

50gt

30 120583gmL Among the cell lines examined HL-60 and MCF-7 cells exhibited high sensitivity for all tested compounds(1ndash8) except for the weak activity shown by compound 2against HL-60 cell and compound 5 towards MCF-7 Most ofthe isolated compounds were inactive against HT-29 (humancolon cancer) and HeLa cell lines with the exception ofcompound 7 which showed strong activity with IC

50of

14 120583gmL which is comparable to standard (5-fluorouracil)Compounds 6 and 8 also showedmoderate inhibition againstHeLa cell line Extracts or isolated compounds which giveIC50lt 10 120583gmL were considered to have significant

cytotoxic activity [19] Cytotoxic agents that have been usedas standard (positive control) in the screening test weretamoxifen goniothalamin and 5-fluorouracil

Interestingly 21015840-hydroxy-44101584061015840-trimethoxychalcone(also known as flavokawain A) (4) appeared to be the mostpotent compound in cytotoxic screening against both HL-60and MCF-7 cell lines A recent publication [22] investigatedthe antiproliferative effects of this compound against a panelof cancerous cell lines The results revealed that flavokawainA (4) exhibited similar cytotoxic property against MCF-7cell line but demonstrated better anticancer effect againstHT-29 cell line as compared with our findings with GI

50

values of 175 120583M (55 120583gmL) and 453 120583M (142 120583gmL)respectively The presence of methoxy substituents and120572-methylation of the enone moiety and the presenceof 21015840 oxygenated substituents are favourable structuralrequirements for cytotoxic activity due to antimitotic activitywhile several ring-A methoxylated chalcones exhibitedcytotoxic activity by inhibiting tubulin polymerizationagainst a variety of tumor cell lines in the low micromolarrange (IC

50lt 50 120583M) [23] Kaempfolienol (1) showed mod-

erate inhibition against HL-60 and MCF-7 cell lines withIC50lt 30 120583gmL while zeylenol (2) demonstrated moderate

inhibition towards HL-60 only with IC50

of 116 120583gmLas reported previously [13]

As for antimicrobial testing crude extracts and con-stituents of Kaempferia angustifolia were assayed againstGram-positive Bacillus subtilis MRSA Gram-negative Pseu-domonas aeruginosa Salmonella choleraesuis Candida albi-cans (yeast) and fungi Aspergillus ochraceus and Saccha-romyces cerevisiae However no activity was detected for allthe samples against all microbes tested Compounds (4) and(6) were not tested towards C albicans and fungi due toinsufficient amount of samples The antifungal properties offlavokawain A (4) against Saccharomyces cerevisiae Candidaalbicans and some other fungi were reported to be inactive[24] and these results complement our findings Compound4 did not show any inhibition towards Gram-positive andGram-negative bacteria tested in this study

The isolation of triterpene from Kaempferia species isunprecedented besides reports on common plant sterolssuch as 120573-sitosterol and stigmasterol The physical proper-ties and spectral data of (24S)-24-methyl-5120572-lanosta-9(11)25-dien-3120573-ol (7) are firstly described in this paper Thebiotaxonomic distribution of lanostanes is quite restricted[25] They are abundant in Polyporaceae and other fungibut also appear in some Spermatophyta like OrchidaceaePinaceae and AnacardiaceaeThe isolation of lanostane-typetriterpene (compound 7) from Zingiberaceae has never beenreported before and hence this finding has contributed to thechemotaxonomic significance

4 Conclusion

The rhizome extracts of Kaempferia angustifolia Roscafforded eight compounds (terpene triterpene cyclohexanederivatives chalcone and glycoside) which showed cytotox-icity against some human cancer cell lines The structuresof the isolates were identified and elucidated based onspectroscopic method and comparison of literature reviews

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors wish to express their thanks to Professor DrHiromitsu Takayama of Chiba University (Japan) and DrMohd Azlan Nafiah of Universiti Pendidikan Sultan Idris(Malaysia) for obtaining the MS and NMR data Financialassistance from PASCA fellowship of the Malaysian Govern-ment and GRF fellowship from Universiti Putra Malaysia isgratefully acknowledged

References

[1] K Heyne Tumbuhan Berguna Indonesia I Badan Penelitianamp Pengembangan Department Kehutanan Jakarta Indonesia1987

[2] L R LingNAbdulWahab andN Zainal Abidin ldquoCytotoxicityactivity of selected ZingiberaceaerdquoMalaysian Journal of Sciencevol 24 no 1 pp 207ndash212 2005

[3] C Kirana I R Record G H McIntosh and G P JonesldquoScreening for antitumor activity of 11 species of Indonesianzingiberaceae using human MCF-7 and HT-29 cancer cellsrdquoPharmaceutical Biology vol 41 no 4 pp 271ndash276 2003

[4] G Park E J Lee H Y Min et al ldquoEvaluation of cytotoxicpotential of Indonesia medicinal plants in cultured humancancer cellsrdquoNatural Product Sciences vol 8 no 4 pp 165ndash1692002

[5] M Habsah M Amran M M Mackeen et al ldquoScreeningof Zingiberaceae extracts for antimicrobial and antioxidantactivitiesrdquo Journal of Ethnopharmacology vol 72 no 3 pp 403ndash410 2000

[6] C E Ficker M L Smith S Susiarti D J Leaman C Irawatiand J T Arnason ldquoInhibition of human pathogenic fungiby members of Zingiberaceae used by the Kenyah (IndonesiaBorneo)rdquo Journal of Ethnopharmacology vol 8521 no 4 pp289ndash293 2003

[7] I N Chen C C Chang C C Ng C Y Wang Y TShyu and T L Chang ldquoAntioxidant and antimicrobial activityof Zingiberaceae plants in Taiwanrdquo Plant Foods for HumanNutrition vol 6321 no 4 pp 15ndash20 2008

[8] N Vipunngeun C Palanuvej and N Ruangrungsi ldquoEssentialoil from Kaempferia angustifolia rhizome chemical composi-tions and antimicrobial activitiesrdquo Journal of Health Researchvol 21 no 4 pp 275ndash278 2007

[9] O Pancharoen P Tuntiwachwuttikul andW C Taylor ldquoCyclo-hexane oxide derivatives from Kaempferia angustifolia andKaempferia speciesrdquo Phytochemistry vol 28 no 4 pp 1143ndash1148 1989

[10] C Yenjai K Prasanphen S Daodee V Wongpanich and PKittakoop ldquoBioactive flavonoids from Kaempferia parviflorardquoFitoterapia vol 75 pp 89ndash92 2004

[11] U Prawat P TuntiwachwuttikulW C Taylor LM EngelhardtB W Skelton and A H White ldquoDiterpenes from Kaempferiaspeciesrdquo Phytochemistry vol 32 pp 991ndash997 1993

[12] M A Sukari B K Neoh N H Lajis et al ldquoChemical Con-stituents of Kaempferia angustifolia (Zingiberaceae)rdquo OrientalJ Chemistry vol 20 no 3 pp 451ndash456 2004

[13] S W Tang M A Sukari M Rahmani N H Lajis and AM Ali ldquoA new abietene diterpene and other constituents fromKaempferia angustifoliaRoscrdquoMolecules vol 16 no 4 pp 3018ndash3028 2011

[14] M A Sukari A Y L Ching G E C Lian M Rahmani and KKhalid ldquoCytotoxic constituents from Boesenbergia pandurata(Roxb) Schltrrdquo Natural Product Sciences vol 13 no 2 pp 110ndash113 2007

[15] A Lovy B Knowles R Labbe and L Nolan ldquoActivity of ediblemushrooms against the growth of human T4 leukemic cancercells HeLa cervical cancer cells and Plasmodium falciparumrdquoJournal of Herbs Spices and Medicinal Plants vol 6 no 4 pp49ndash57 1999

[16] M M Mackeen A M Ali S H El-Sharkawy et al ldquoAntimi-crobial and cytotoxic properties of some Malaysian traditionalvegetables (ULAM)rdquo Pharmaceutical Biology vol 35 no 3 pp174ndash178 1997

[17] E P Padla L T Solis R M Levida C-C Shen and CY Ragasa ldquoAntimicrobial isothiocyanates from the seeds ofMoringa oleifera Lamrdquo Zeitschrift fur Naturforschung C Journalof Biosciences vol 67 no 11-12 pp 557ndash564 2012

[18] Y Takeuchi Q Cheng Q W Shi T Sugiyama and T OritanildquoFour polyoxygenated cyclohexenes from the Chinese treeUvaria purpureardquo Bioscience Biotechnology and Biochemistryvol 65 no 6 pp 1395ndash1398 2001

[19] H Kojima N Sato A Hatano andH Ogura ldquoSterol glucosidesfrom Prunella vulgarisrdquo Phytochemistry vol 29 no 7 pp 2351ndash2355 1990

[20] A K Chakravarty B Das K Masuda and H Ageta ldquoTetra-cyclic Triterpenoids from Glycosmis arboreardquo Phytochemistryvol 42 no 4 pp 1109ndash1113 1996

[21] K Yano T Akihisa T Tamura and T Matsumoto ldquoFour 4120572-methylsterols and triterpene alcohols from Neolitsea aciculatardquoPhytochemistry vol 31 no 6 pp 2093ndash2098 1992

[22] J C Aponte M Verastegui E Malaga et al ldquoSynthesis cyto-toxicity and anti-Trypanosoma cruzi activity of new chalconesrdquoJournal of Medicinal Chemistry vol 51 no 19 pp 6230ndash62342008

[23] M L Go X Wu and X L Liu ldquoChalcones an update oncytotoxic and chemoprotective propertiesrdquo Current MedicinalChemistry vol 12 no 4 pp 483ndash499 2005

[24] P Boeck P C Leal R A Yunes et al ldquoAntifungal activityand studies on mode of action of novel xanthoxyline-derivedchalconesrdquo Archiv der Pharmazie vol 338 no 2-3 pp 87ndash952005

[25] E M Giner-Larza S Manez R M Giner-Pons M CarmenRecio and J-L Rıos ldquoOn the anti-inflammatory and anti-phospholipase A2 activity of extracts from lanostane-richspeciesrdquo Journal of Ethnopharmacology vol 73 no 1-2 pp 61ndash69 2000

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Microbiology


aeruginosa (1063 plusmn 023 1003 plusmn 022mm resp) butnot against Escherichia coli Bacillus subtilis Streptococcusfaecalis Candida albicans andMicrosporum gypseum

Phytochemical investigations of Kaempferia species haveyielded several compounds including cyclohexane diepoxidederivatives flavonoids and diterpenes [9ndash11] Most of theconsumable ginger rhizomes have long been used as folkmedicine for centuries but have not been thoroughly inves-tigated for their bioactive properties yet Thus here we wishto report the isolation and cytotoxic and antimicrobial prop-erties of chemical constituents from rhizomes of Kaempferiaangustifolia

2 Materials and Methods

21 General Experimental Procedures Melting points (uncor-rected) were determined using Barnstead Electrothermal IA9100 Series melting points equipment Optical rotations weremeasured on a Jasco P-2000 polarimeter equipped with asodium lamp (589 nm) The IR spectra were recorded usingKBr discs on Perkin Elmer FTIR spectrophotometer 1650 1Hand 13CNMR spectra were obtained on JEOL spectrometer at500 and 125MHz andor 400 and 100MHz respectively withtetramethylsilane (TMS) as internal standard Mass spectrawere recorded on Shimadzu model QP 5050A spectrometerUltraviolet spectra were recorded on a Shimadzu UV-1601spectrophotometer Separation by column chromatographywas carried out by using silica gel (Merck 7749 7734 and9385) while silica gel 60 PF

254aluminium sheets were used

for TLC analysis

22 Plant Material and Cytotoxic Cell Lines Kaempferiaangustifolia was collected from Java Indonesia in 2001 Theplant was identified by Dr Sugeng Riyanto Faculty of Phar-macy Gadjah Mada University and the voucher specimen(number 16SR0601-01) was deposited at the herbarium ofthe institutionTheHL-60 (human promyelocytic leukemia)MCF-7 (human breast cancer) HT-29 (human colon cancer)and HeLa (human cervical cancer) cell lines were obtainedfrom theNational Cancer InstituteMaryland USA Bacterialstrains were originally obtained from American Type CellCulture Collection (ATCC) USA

23 Extraction and Isolation The finely ground air-driedrhizomes (10 kg) were extracted repeatedly with petroleumether chloroform and methanol thrice each for 72 hoursThe extracts were filtered and concentrated under reducedpressure to obtain petroleum ether (840 g) chloroform(750 g) and methanol (670 g) extracts The petroleum etherextract was triturated with methanol at room temperature togive a solid product whichwas then filtered and recrystallizedin methanol to yield crotepoxide (2) (16mg) Brown oilextract (580 g) was obtained upon evaporation of the filtrateThe extract was subjected to vacuum column chromatog-raphy and eluted stepwise with 100 hexane hexane-ethylacetate ethyl acetate-methanol and 100 methanol Thisafforded boesenboxide (3) (40mg) 21015840-hydroxy-4 41015840 61015840-trimethoxychalcone (4) (26mg) and 120573-sitosterol (18mg)

Spectroscopic data of compounds 2ndash4 have been reportedpreviously [12] Chloroform extract was subjected to columnchromatography separation and eluted with mixture of hex-ane and ethyl acetate to give 23 fractions (200mL each)Fraction three which was eluted from hexane-ethyl acetate(5 5) was purified to afford zeylenol (5) (0238 g) whilefraction four obtained from elution with mixture of hexane-ethyl acetate (3 7) was rechromatographed over silica gel togive subfractions 17ndash19 which were then recrystallized withmethanol to afford 6-methylzeylenol (6) (42mg) Methanolextract was fractionated using column chromatography ina similar manner Eighteen fractions of 200mL each werecollected Combination and purification of fractions 14 and15 from elution with ethyl acetate-methanol (7 3) gavesucrose (11mg) A reinvestigation of the sample (7650 g)from the same source was conducted by extraction withmethanol (3 times 72 hours) to obtain a dark brown viscousliquid extract which was then partitioned with hexaneand water with 10 methanol (1 1) to give hexane-solublefraction (1060 g) as brown yellowish viscous liquid Thehexane fraction (800 g) was subjected to column chromatog-raphy eluted with mixtures of hexane ethyl acetate andmethanol of increasing polarity to give 86 fractions (200mLeach) Upon washing and recrystallizing with methanolfractions 13-4 which were eluted from hexane ethyl acetate(97 3) afforded (24S)-24-methyl-5120572-lanosta-9(11) and 25-dien-3120573-ol (7) (40mg) Fraction 84 which was eluted fromethyl acetate methanol (50 50) was further washed withmethanol to give 120573-sitosterol-3-O-120573-D-glucopyranoside (8)(8mg) Similarly compound 2 (10mg) compound 4 (7mg)and 120573-sitosterol (35mg) were also obtained from this extractChloroform-soluble fraction (40000 g) was fractionated ina similar manner to obtain 100 fractions (200mL each)which were combined into major fractions based on theirTLC profiles Compounds 1 (20mg) 2 (079 g) 3 (16mg) 4(10mg) and 5 (097 g) were subsequently obtained Isolationprocedures and spectroscopic data of compounds 1 and 5have been reported previously [13]

24 Spectral Data (24119878)-Methyl-5120572-lanosta-9(11)25-dien-3120573-ol (7) C

31H52O white needle-shaped crystal Melt-

ing point 180-181∘C [120572]119863 +675(c 004 CHCl

3) 119877119891 060

(Hexane-EtOAc 4 1) IR (KBr) 3684 3402 2940 1644 1098888 cmminus1 UVVis 120582max (CHCl

3) nm (log 120576) 2392 (3837) 1H

NMR (400MHz CDCl3) and 13C NMR (100MHz CDCl

3)

Table 1 EI-MS 119898119911() = 440 (M+ 27) 425 (56) 407 (31)313 (100) 69 (72) 55(81)

25 Cytotoxic Assay The cytotoxic assay was carried outaccording to the methods described previously [14] Cyto-toxic and proliferation profiles were determined by MTT(3-[45-dimethylthioazol-2-yl]-25-diphenyltetrazolium bro-mide) assay whereby cell viability of treated cell populationwas compared to the control cell population The cell via-bility was determined by the formation of blue formazancrystals of MTT in which the crystals were dissolved inDMSO and the absorbance was read with Elisa reader testwavelength of 570 nm and references wavelength of 630 nm






6 147 (dd 129 27)168 (m)a 214




50values were






29) for antibac-










O

OOCOR

O

R

(4)(1)

HO

HO

OH

OHOH

OH

OH

OH

(2) R = CH3

MeO OMe

OMeCH2OCOC6H5

OCOCH3

OCOC6H5

OCOC6H5

(5) R = OH

HO

HOHO

HOOH

H

123

4 56

7

8910

1112

1314

15

16

1718

19

20

21 22

2324

2526

27

2829

30

31

O

H

O

(7) (8)

(3) R = C6H5 (6) R = OCH3




HO

H

12

34

29 28

H

19

67

830

1415

16

H

910

1113 17

18

2122

H23

2425

26

27

31



6H10O5]+ 396 [M-C

6H10O5-

H2O]+ 255 [M-C











50gt


50of




50








4 Conclusion





Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






6 147 (dd 129 27)168 (m)a 214




50values were






29) for antibac-










O

OOCOR

O

R

(4)(1)

HO

HO

OH

OHOH

OH

OH

OH

(2) R = CH3

MeO OMe

OMeCH2OCOC6H5

OCOCH3

OCOC6H5

OCOC6H5

(5) R = OH

HO

HOHO

HOOH

H

123

4 56

7

8910

1112

1314

15

16

1718

19

20

21 22

2324

2526

27

2829

30

31

O

H

O

(7) (8)

(3) R = C6H5 (6) R = OCH3




HO

H

12

34

29 28

H

19

67

830

1415

16

H

910

1113 17

18

2122

H23

2425

26

27

31



6H10O5]+ 396 [M-C

6H10O5-

H2O]+ 255 [M-C











50gt


50of




50








4 Conclusion





Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O

OOCOR

O

R

(4)(1)

HO

HO

OH

OHOH

OH

OH

OH

(2) R = CH3

MeO OMe

OMeCH2OCOC6H5

OCOCH3

OCOC6H5

OCOC6H5

(5) R = OH

HO

HOHO

HOOH

H

123

4 56

7

8910

1112

1314

15

16

1718

19

20

21 22

2324

2526

27

2829

30

31

O

H

O

(7) (8)

(3) R = C6H5 (6) R = OCH3




HO

H

12

34

29 28

H

19

67

830

1415

16

H

910

1113 17

18

2122

H23

2425

26

27

31



6H10O5]+ 396 [M-C

6H10O5-

H2O]+ 255 [M-C











50gt


50of




50








4 Conclusion





Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






50gt


50of




50








4 Conclusion





Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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