Pharmacognosy

Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the

Leaves Extract of Dillenia indica Linn.
Apu AS, Muhit MA, Tareq SM, Pathan AH, Jamaluddin ATM, Ahmed M
Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka,
Dhaka 1000, Bangladesh

Address for correspondence: Mr. Apurba Sarker Apu; E-mail: apurba2sarker@yahoo.com

ABSTRACT

The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation
of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions
of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc
diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina)
lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions
showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average
zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 ␮g/disc. But the aqueous fraction was
found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC50 of 0.52 ␮g/ml), n-hexane
DQGFKORURIRUPIUDFWLRQVGHPRQVWUDWHGDVLJQL¿FDQWF\WRWR[LFDFWLYLW\KDYLQJ/&50 of 1.94 ␮g/ml and 2.13 ␮g/ml,
respectively). The LC50 values of the carbon tetrachloride and aqueous fraction were 4.46 ␮g/ml and 5.13 ␮g/ml,
UHVSHFWLYHO\7KH VWXG\ FRQ¿UPV WKH PRGHUDWH DQWLPLFURELDO DQG SRWHQW F\WRWR[LF DFWLYLWLHV RI Dillenia indica
leaves extract and therefore demands the isolation of active principles and thorough bioassay.

Key words: Antimicrobial activity, Artemia salina, brine shrimp lethality bioassay, Dillenia indica

DOI: 10.4103/0975-1483.62213

INTRODUCTION of cancer and diarrhea.[2]7KHIUXLWMXLFHRI WKLVSODQWKDV

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The genus Dillenia has 60 species, of which Dillenia indica DOVRHPSOR\HGLQFRXJKPL[WXUH[3]7KHVROYHQWH[WUDFWVRI 
/LQQDHXV)DPLO\'LOOHQLDFHDHLVWKHPRVWFRPPRQHGLEOH fruits of D. indica are reported to have antioxidant activity.[4]
VSHFLHV2ULJLQDOO\IURP,QGRQHVLDWKLVHYHUJUHHQWURSLFDO CNS depressant activities in mice were found from the
tree is now found from India to China. The common DOFRKROLFH[WUDFWRI WKHOHDYHVRI D. indica.[5] Considering
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of D. indicaOHDYHVEDUNDQGIUXLWVDUHPL[HGDQGJLYHQ
the source of many modern medicines.
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number of different methods used for the assessment of
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50 J Young Pharm Vol 2 / No 1

 Leaves extract of Dillenia indica

which one is the best method for in vitro assay.[6]0DMRULW\
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was then concentrated and dried by a rotary evaporator
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resistant strains of microorganisms.[10,11] It is important to
note that the disc diffusion method demonstrated activity
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were tested by the paper disc diffusion method.[7] Thirteen
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MATERIALS AND METHODS paper discs were impregnated with 400 ␮g of each of the
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for further reference.
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SUHLQRFXODWHGZLWKWKHWHVWEDFWHULDDQGIXQJL7KHSODWHV
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the test agents was determined by measuring the diameter
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 Apu, et al. J Young Pharm. 2010;2(1): 50-53
%ULQHVKULPSOHWKDOLW\ELRDVVD\ RESULT AND DISCUSSION
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cytotoxic activity[15,19] of the nKH[DQHFDUERQWHWUDFKORULGH of D. indica OHDYHV ZHUH DFWLYH DJDLQVW PRVW RI  WKH WHVWHG
FKORURIRUPDQGDTXHRXVIUDFWLRQVIURPPHWKDQROLFFUXGH RUJDQLVPV>7DEOH@7KHDYHUDJH]RQHRI LQKLELWLRQSURGXFHG
extracts. For the experiment, 4 mg of each of the extracts
by the nKH[DQH FDUERQ WHWUDFKORULGH DQG FKORURIRUP
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fraction was ranged from 6-8 mm, 7-8 mm, and 6-7 mm,
of varying concentrations (400, 200, 100, 50, 25, 12.5,
UHVSHFWLYHO\DWDFRQFHQWUDWLRQRI ␮g/disc. Against the
6.25, 3.13, 1.56, 0.78 ␮JPOZHUHREWDLQHGE\WKHVHULDO
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Table 1: Antimicrobial activity of chloroform, carbon
FDOFXODWHG$QDSSUR[LPDWHOLQHDUFRUUHODWLRQZDVREVHUYHG tetrachloride, n-hexane, and aqueous fraction of
ZKHQ ORJDULWKP RI  FRQFHQWUDWLRQ YHUVXV SHUFHQWDJH RI  methanolic extract of Dillenia indica leaves and positive
PRUWDOLW\[21]ZDVSORWWHGRQWKHJUDSKSDSHUDQGWKHYDOXHVRI  control kanamycin
LC50ZHUHFDOFXODWHGXVLQJ0LFURVRIW([FHO>)LJXUH@ Test microorganisms Diameter of zone of inhibition
(mm ⴞ SD) (n ⴝ 3)
9LQFULVWLQHVXOSKDWHZDVXVHGDVSRVLWLYHFRQWURO
CF CT HX AQ KM
Gram-positive bacteria
Bacillus cereus 7 ⫾ 0.3 8 ⫾ 0.2 7 ⫾ 0.3 - 36 ⫾ 0.2
Bacillus megaterium 7 ⫾ 0.2 8 ⫾ 0.4 7 ⫾ 0.1 - 37 ⫾ 0.3
Bacillus subtilis 7 ⫾ 0.3 8 ⫾ 0.2 6 ⫾ 0.3 - 40 ⫾ 0.3
Staphylococcus aureus - 8 ⫾ 0.3 7 ⫾ 0.2 - 28 ⫾ 0.2
Sarcina lutea 7 ⫾ 0.2 8 ⫾ 0.3 7 ⫾ 0.3 - 31 ⫾ 0.5
Gram-negative bacteria
Escherichia coli 7 ⫾ 0.2 - - - 32 ⫾ 0.5
Pseudomonus aeruginosa 6 ⫾ 0.5 7 ⫾ 0.2 7 ⫾ 0.3 - 30 ⫾ 0.4
Salmonella paratyphi 7 ⫾ 0.4 8 ⫾ 0.3 7 ⫾ 0.1 - 33 ⫾ 0.3
Salmonella typhi 7 ⫾ 0.1 8 ⫾ 0.3 7 ⫾ 0.5 - 35 ⫾ 0.1
Shigella boydii 7 ⫾ 0.3 8 ⫾ 0.1 6 ⫾ 0.4 - 31 ⫾ 0.5
Shigella dysenteriae 6 ⫾ 0.3 7 ⫾ 0.3 7 ⫾ 0.2 - 35 ⫾ 0.3
Vibrio mimicus 7 ⫾ 0.2 8 ⫾ 0.4 8 ⫾ 0.1 - 34 ⫾ 0.2
Vibrio parahemolyticus 7 ⫾ 0.3 8 ⫾ 0.3 8 ⫾ 0.5 - 35 ⫾ 0.3
Fungi
Candida albicans 7 ⫾ 0.3 8 ⫾ 0.3 7 ⫾ 0.4 - 32 ⫾ 0.3
Aspergillus niger 7 ⫾ 0.1 8 ⫾ 0.3 7 ⫾ 0.1 - 34 ⫾ 0.1
Figure 1: Plot of log concentration of n-hexane (— ̆ ō ECTDQP
Sachoromyces cerevacae 6 ⫾ 0.4 8 ⫾ 0.2 7 ⫾ 0.3 - 31 ⫾ 0.3
VGVTCEJNQTKFG
ō1ōEJNQTQHQTO
ōţōCPFCSWGQWU
ō⫻ —) fraction
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Table 2: Effect of n-hexane, carbon tetrachloride, chloroform and aqueous fraction of methanolic extract and positive
control vincristine sulphate on brine shrimp
Conc. (␮g/ml) Log C % mortality LC50 (␮g/ml) Vincristine sulphate
HX CT CF AQ HX CT CF AQ Conc. (␮g/ml) Log C % mortality LC50 (␮g/ml)
400 2.602 100 100 100 100 1.94 4.46 2.13 5.13 40 1.602 100 0.52
200 2.301 100 100 100 100 20 1.301 100
100 2.000 100 100 100 80 10 1.000 100
50 1.698 90 80 80 70 5 0.699 90
25 1.398 80 80 70 70 2.50 0.398 80
12.5 1.097 80 60 70 60 1.25 0.097 60
6.25 0.796 70 50 60 50 0.63 ⫺0.201 50
3.13 0.495 50 40 50 40 0.31 ⫺0.509 40
1.56 0.194 50 40 50 40 0.16 ⫺0.796 30
0.78 ⫺0.108 30 30 40 30 0.078 ⫺1.108 20

52 J Young Pharm Vol 2 / No 1

 Leaves extract of Dillenia indica

Table 3: The result of cytotoxic activity of n-hexane (HX), 0L]RUDP,QGLD)LWRWHUDSLD

carbon tetrachloride (CT), chloroform (CF), and aqueous  6KRPH8.KDQQD5.6KDUPD+33KDUPDFRJQRVWLFVWXGLHVRQDillenia indica
(AQ) fraction of methanolic extract and positive control /LQQ,,)UXLWDQG6HHG3URF,QGLDQ$FDG6FL3ODQW6FL
vincristine sulphate (VS) on brine shrimp  $EGLOOH0+6LQJK53-D\DSUDNDVKD*.-HQD%6$QWLR[LGDQWDFWLYLW\RI 
the extracts from Dillenia indica fruits. Food Chem 2005;90:891-6.
Sample LC50 (µg/ml) Regression equation R2
 %KDNXQL'6'KDU0/'KDU01'KDZDQ%10HKURWUD%16FUHHQLQJ
VS 0.52 y = 33.256x + 58.740 0.9580
RI ,QGLDQSODQWVIRUELRORJLFDODFWLYLW\,,,QGLDQ-([S%LRO
HX 1.94 y = 25.972x + 42.602 0.9253
 :LONLQVRQ-00HWKRGVIRUWHVWLQJWKHDQWLPLFURELDODFWLYLW\RI H[WUDFWV
CT 4.46 y = 29.192x + 30.585 0.8837 ,Q$KPDG,$TLO)2ZDLV0HGLWRUV0RGHUQSK\WRPHGLFLQH7XUQLQJ
CF 2.13 y = 24.159x + 41.863 0.9569 PHGLFLQDOSODQWVLQWRGUXJV*HUPDQ\:LOH\9&+S
AQ 5.13 y = 26.575x + 30.849 0.9706  %DXHU$:.LUE\:06KHUULV-&7XUFN0$QWLELRWLFVXVFHSWLELOLW\WHVWLQJ
E\DVWDQGDUGL]HGVLQJOHGLVFPHWKRG$P-&OLQ3DWKRO
 %HOERXNKDUL1&KHULWL$$QWLEDFWHULDODQGDQWLIXQJDODFWLYLWLHVRI FUXGH
5.13 ␮JPOIRUnKH[DQH+;FDUERQWHWUDFKORULGH&7 extracts from Launeae arborescens3DN-%LRO6FL
FKORURIRUP&)DQGDTXHRXV$4IUDFWLRQUHVSHFWLYHO\  .KDQ $ 5DKPDQ 0 ,VODP 06 $QWLEDFWHULDO DQWLIXQJDO DQG F\WRWR[LF
&RPSDUHG WR SRVLWLYH FRQWURO YLQFULVWLQH VXOSKDWH 96 DFWLYLWLHVRI DPEO\RQHLVRODWHGIURPAmorphophallus campanulatus. Indian J
3KDUPDFRO
LC50 0.52 ␮JPO DOO WKH IUDFWLRQV WHVWHG VKRZHG JRRG
EULQHVKULPSODUYLFLGDODFWLYLW\$JDLQWKHFUXGHH[WUDFWV
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further investigation.[22] The cytotoxic activity exhibited
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indicates the presence of potent bioactive compounds.[15]
CONCLUSION
7KH DQWLPLFURELDO DQG F\WRWR[LF DFWLYLWLHV RI  YDULRXV
fractions of D. indica OHDYHV IRXQG LQ WKLV VWXG\ PD\
H[SODLQVRPHRI WKHWUDGLWLRQDOPHGLFLQDOXVHVRI WKLVSODQW
7KHVH FRXOG EH RI  SDUWLFXODU LQWHUHVW LQ UHODWLRQ WR ÀQG
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RI FKHPLFDOO\LQWHUHVWLQJDQGELRORJLFDOO\LPSRUWDQWGUXJ
candidates.
ACKNOWLEDGEMENTS
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 0F/DXJKOLQ-/5RJHUV//$QGHUVRQ-(7KHXVHRI ELRORJLFDODVVD\VWR
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 0F/DXJKOLQ -/ 5RJHUV // 7KH XVH RI  ELRORJLFDO DVVD\V WR HYDOXDWH
ERWDQLFDOV'UXJ,QI -
 0LGGOHWRQ36WHZDUW)$O4DKWDQL6(JDQ32·5RXUNH&6DUNHU6'et al.
$QWLR[LGDQWDQWLEDFWHULDODFWLYLWLHVDQGJHQHUDOWR[LFLW\RI Alnus glutinosa,
Fraxinus excelsior and Papaver rhoeas. Iranian J Pharma Res 2005;2:81-6.
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J Nat Prod 1996;59:100-8.
Source of Support: Nil, &RQÀLFWRI,QWHUHVW None declared.
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