Antifungal Activity of Rose-Scented Geranium ( Pelargonium Species) Essential Oil and its Six Constituents

Abstract

Rose scented Geranium (Pelargonium species) leaf oil and six of its major constituents namely citronellol, linalool, geraniol, isomenthone, geranyl formate and citronellyl formate showed fungicidal property against Candida albicans and Crytococcus neoformans causing common fungal infections like candidiasis and cryptococcosis in human beings. In terms of antifungal activity citronellol was the most effective followed by geraniol, isomenthone, geranyl formate and citronellyl formate. The activity of pure oil seemed to differ from the constituents. The effect was observed both at 28 and 4 oC indicating temperature independent activity. Probably the antifungal activity is mediated through some mechanism other than membrane disruption and inhibition of cell division. The fungicidal activity .compared well with standard antifungal drugs. Citronellol isolated from rose-scented geranium oil, therfore holds promise for pharmaceutical uses.

Full text

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Jeobp 8 (2) ?005 PP I I 8-222
tssN 0972-060x
Antifungal Activity of Rose-Scentcd Ger:rnium ( Pchrgonium Species)
Chandi C. Rath*, S' K' Dash and B' R Rajesrvar Rao
*p.G. Department of Botany, North.ori:t.: -u]1,:^.t:i,il .srirr rr chatrdra vilrar
Takatptlr' Bariplda- /5 /UUJ lrlola
Central lnstitute otl,4eaici'rat rttcl Arotlratic Phnrs (CIMAP)
Field station Uppal Road' I Iyderabad' lnd ia
Atlstract: Rose scented Geran iurr (Pelctrgonitrnt species) ieaf oil and six of its major cott-
stituents namely citroneltol, tinaloot'*ge"'iif '-it"it*ft"'"' 9e1n1lliyate ana citronellyl formate
showed fungicidal p'optn'"' ugu'n"?' nd d ttll'it'rn\ ?rtl Ct)'tt'co(L tt.\ 4'''firmint cirusrngcom-
mon fungal inlecliols like *"0'o'"ti'ijllJ"*' o"'.""."''t-; ' ]iuttran b'iIg' ln lcrnrs oI antifungil
acrivitv. citronetlol *u, t'r't *ou ""ei''1i":roir'o't' ti r'5 gt"nior' i'o*tnLf,one' geranyl formate rnd
cirronellyl formate The *'i'ity "tpuf"ii "t'"'i to Alrif' lront lhe c''nstituents The cffect rrrs
observed both at l8 and 4'C i"dit'i;; 't*"'"'"t i"ltotn9t".':l'lllJ,'l;"ro"toolil[1,'';;:"XTl
:lilyJily*[l[:::11,lil"#::il;:]::',1'J;Illilii:llff,:i:l:lliilJjil,i,*' r "",,.r,",
isolated from rose-scentcd geraniunioil,,i,.r.r",", r,ou. nrorltise for phirrmaceutical uses'
Key wortls: Rose-scented geranitltn oil' aronratic cotr stilLtcnts' antifungal activitl"
Candida albicans, Cryptococcus neofornrturs'
Introduction: Rose-scented geranitltn 1l e ktrg.,ln iuttt ,sp l,is a Perennial herb, culti.
vated in semi-arid and tropical ;l*""i;. TLe ieaf a,id s'oot yield 0.28 - 0.38% and 0 15 -
0.24%o essential oit' "tptttiu"ry:' iii"-untiuottt'iur' ontifungal ancl nematocidal activities of
rose-scented geratriutrr olt tlaue Uetn reported tt"l'er 'll lllYl]"" Though' reports are
available on the antimicrobiar activity of rose-scerrtcd geraniLrn esscntial Qil, there have been
very few reports ,tgu'Oit'g tll" u"iittiitroUi't "'luity of tlte cottsliruelrrs of tltis essential oil
asainst human rungut puttlog"nt il;;;;;i;; " io undertake this work to studv against
F"ri iii, al b ic an s artd C ryp t ococ cus n L ofo r u' t t t s
6* r-pona i n g e u t hor : ( chandi. C Rath)
F--rrail: <chandi rath@yrhoo co'ttt>

Chandi C. Rath et allJeobp 8 (2) 2005 pp 2l 8-222
Materials and Methods
Fungi: Candida albicans MTCC-183 and C4ptococcus neoformqns MTCC-1437
were procured from microbial Type Culture Collection Centre (MTCC), IMTECH,
Chandigarh, India and rnaintained in our laboratory on Sabouraud dextrose agar slants.
Oil: Rose-scented Ceranium (Pelargoniunt sps. farnily geraniaceae) slroot oil was
obtained from Central [nstitute of Medicinal and Arornatic Plants (CIMAP) field station,
Hyderabad. The oil contains citronellol(29.3%), gerzniol (21 .8%), linalool (9.502), isomenthone
(8.6%), citronellyl fomate (5.4%) and geranyl forrnate (4.1%) as its rnain corlstituentsr.
Compounds: Six majoraromatic chem ical components ofthe oilviz. citronellol, lina-
lool, geraniol, geranyl formate, citronellyl forrnate and isomenthone were procured from
M/s S.H. Kelkar & Co. Ltd. Mumbai, India. Also a mixture of these six compounds at 1 :l
ratio was prepared in our laboratory and used in this study.
Media: Sabouraud dextrose broth (SDB) and Sabouraud dextrose agar (SDA) were
procured from Hi-media Mumbai Ltd. and prepared as per rlanufactlrrer's iustruction. So-
d iu:r taurocholate 0.75% (wiv) in the medium was used as an emulsifying agent to facilitate
the miscibility ofthe oil and the constituents in the mediurn. Mediurn with tauroclrolate served
as control sets through out the study.
Antifungal fl clivity studies
Determination of Minimrrfn Inhibitory Concentration (MlC) of the oil and the
aromatic constituents. This was determined follorving the tube dilLrtion method repofted
earliere. Briefly diflerent volurnes of the oil were mixed with sterile , cooled molten SDA
supplemented with 0.75% (Vv) ofsod iurn taurocholate to give a concentration of I .95- 1000
pl/ml by two fold serial dilution method. The statistical analysis of the MIC values from 3
experiments was carried out by using students t-test.
Determination of nature of toxicity of the oils and aromatic constituents: Tlris
(fungistatic or fungicidal toxicity) was determ ined reinocu Iating the frrrrgal discs (frorn M IC
dilution tubes) on to SDA plates, incubated at 28 * 7'C for 48 hrs. Frrngal growth after the
incubation period indicated tlie fungistatic activity, whereas, its absence derronstrated the
fungicidal nature.
Effect of temperatur€ dependant activity: An experiment rvas deJigned for it by
tube dilution methodr0, witlrtlight modifications. Two sets ofsabouraLrd dextrose taurocho-
late agar slants (SDTA) were prepared (supplenenting 0.7502 w/v socliurn taurocholate into
SDA medium) contaiu ing MIC level ofoil and constituents separately lor studying the effect
of neat oil and conrpounds respectively. The slants were inoculated with freshly grorvn
fungal rrats of 5mrr dianreter. One set was incubated at 4"C and the otlrer 28 + 20C. l'he
effect was studied by subculturing the fungal mats onto S DA plates and checked for viability
after 18-24 hrs of incLrbatiorr at 280C.
219

Chandi C Rath cl ai.iJeobp 8 (2) 1005 pP 218-122 220
Comparisonofactivityoftheoil|rnditsConstitucntSwithstnndardnntifungal
drugs: The antibiogram pattern ofthe patltogens rvas studied by disc diffusion rnethod of
Baulf et.ul.rt on SDA plates against Clotrimazole. Fluconazole, ltraconazole, Griseofulvin,
Amphotericin-B and Nystatin (disc procured l'r'our Hi-media N{unrbai Ltd., except Griseoful-
vin and Itraconazole. wlrich were pr.epared in oLrr laboratory). Sterile filter paper discs con-
taining MIC levels ofoil and aromatic constituents rvere also trsed for colnparison'
Dctermination of nature of action pirttcrn of the oil nnd its compountls: lt was
deterrrirred by disc diffirsion metllod of Bauer et el \t artd Rath el a/ ls'
ResultsantlDiscussion:FronrthelnininrLlllinhibitoryconcentratiollstudiesitwas
observedthatthepuleoilandallthecomponel]tSincludingthelnixtureshowedanit-fungal
activities against both Candido albictuts artd C\)Ptococctts neoformans' However' the
activity of ti;e compottnds in tertns of M lC seemecl to d iffer (Tabe l ) The pure oilinhibited '
C. albican.s artd C. ncofornnns at 7 8 and 31 25 ptl/nll respectively Auong the compo-
nents citronellol showed minimum MIC valuc against both test pathogerls. Wlrereas, ge-
raniol, isomenthone, and geranyl fortrate shorved siniilar MIC values agaillst both patho-
g"n., i.. 7.8,7.8 arid lS.eZ ptAnt respectively' Tlre nlixtr'rre ofthe componerts showed a
i,ttc'rutu" oi t .9 pl/ml against botlt C. albican.s and C. neoJbrntans in con:parisou to neat
geraniurn oil, wliich was observed to be 7.8 and 31 25 pl/m I against both the test pathogens
Iespectively. The lowest MIC valtre of the comPonent tllixtttre could be attribLrted to the
svrrersistic effect of the constituents. Kurita c1. o/. 't reportecl the simitar synergistic activi-
iL. *Itt, ,t," nrixtures of B-erifaidehyde, cinnamaldehyde, citral a,d citronellal against 18
irngi in .orrporiton to iitronellol oil. Further', it is pertillent to mentioll here that the MIC
uuti". oftl. ion.tponeuts correlated to its percellt value in tlrc oil, i'e higher percentage with
lowerMlCvaluesexceptlinaloolwlrichshowedsinilarMlCagainstC'a/bicarsasthatof
citronellol.
As no growth was reported on subculturing onto SDA plates from MIC dilution tubes
theactivityw"asreportedtobefUngicidatattheseconcentrations.Thisftlngicidaleffectwas
independent ofternperature occuring both at 28 and 4"C l-hc lirngicidal activity occr:red at
4,,C indicates the eriergy independerrt nature of activity of geranium oiland its constituerts.
Fronr the antibiograrl pattern it was observed thal C' alhicans was sensitive to
citracorazole. flttconarole and nystatin wlicrcas' C neofctt mans was resistallt to all the
drugsusedexcept itraconazole ani nystatin(Table2 ) FLtmhcr, geranium oil and its cpnstitu-
ents showed beite, zone of inhibitions, in cotllparison to the antifungal courpounds used
(Tabel 2.).
It is atr iurportant point to mentioll that the geralliuln oil an its coustitueuts' showed
better zores at very low concentration itt conrparisorl to arltifungal drugs ln general' there
seemedtobeoverallagreementbetweerrtlresizeofinhibitionzonesobtainedandMlC
uulues, i.e. Iarger zones if inhibition correlated rvith lorvcr M IC values' However' variations
did occur ur; illo*n in Table I and 2. An ilrportaut featur.e observed that, the cock-tail
mi>:ture ol'tlre six cotnporlellts shorved better zones of inhibition inconlparision to the neat
Ceran iLrrrr oil. Th is further inclicatcs tltat thc ncat o il colt.tpt ises soue acrolllatic conlpoLlnds

that represerts antr-::::': Rath er arlJeobp 8 ( 2) 2t)0 5 pp 2 1 8'2)'t
of inh ibition *"." oa...r.aTljl!.lr is}1 eif ctive const ir,er rs. Furrrrernorc. ,;,n itr, .or*,
osrnoprolectarr, ar',d borl,rh.::ll.' "' sDA arrd SDSA plates contrir:irg n t, ",.r".. ".'"li
e'nptiotericin-s, iil ;;,;X1#:rrcewere rcsisrarrt to Arnphorcricii-8. ,,,n cr;j"l'rr",ljl
brane pemeabitity u,a "u,.ing r*",r,#;:"li:::lillllo,il,,: o,'.,r,,,* rungar celr me,,,_
ages mitotic spindte and inh;u;r'."iii;:;,::.';il,:'l"tlluettts where as, criseofutvin danr-
, iurn oit. ,,. .l,,,.,i-,.,*'"iij,oJl:t l'.u';t;.." 'his irrrplics tlrrr proh:rbly rtre r. i,",,rlif i.rl-
n* y1:ltltf i*Tai:i:lt,#:n:i" :,ll;;lll;;:ll:Hif ,l:;fl Hr
u.."r1".,.r"',,ilili;,:i:Tr"fl;f,::l:.jl]'r":_-u,::l;,i,1,,,,::,,i,oi^,",,,,*;;fi';:l,ll:
Uur results, as descrihe
c",u,;iil..","lfi ,:,ffi ::l::ff ij,ll:::.,,,"fl j:#;,: m,f;n:i;:iiil Il[ ffji:[i:,
Acknowledgemen t: Th
v. v*", a;,=.i".il;. K:;#"jl,ly c.c. Rath hisht-v acknowtedge the hetp of Shri SLrresh
ents and director, Ciiln,;;.;:":I;,:l*,Y,1]lXll,; il",,pprvi,ig th" a,o',,;,i".ilfil
Referen ces
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Organisms
Candida albicans
MTCC I83
Cyplococcus
neoformans
MTCC 1437
HG
C
B
AMIC*
DE
7.8 + 3.9+ 3.9+ 7.8+
0.33 0.13 0.13 0.16
31.25 + 3.9'r 15.62+ 7 .8 +
0.46 0.13 0.14 0.19
7.8 + 15.62+ 61.5r 1.9a
0.12 0.09 0.14 0.03
7 .8 + 15.62+ 125 + I .9 +
o)2 0.13 0.22 0.05
Chandi C, Rath e/a/./Jeobp 8 (2) 2005 pp 218-l2l 222
Table 1. Determinaiton of MIC valucs of rose-sccntcd geranium oil (neat)
and six of its major constituents
*Oil and aromatic constituents are loaded at MIC level per disc
- Resistant (no zolres ofinhibition)
* MIC value ofneat oil is represented in pl/ml and arornatic constituents in pgllnl.
A - Gernaniurn neat oil: B-Citronellol; C-Linalool; D-Geranioli
E - Isomenthone; F - Geranyl forlnate; C-Citronellyl lbnnate;
H - cock tail Inixtur oftlle six constituents at I :1 ratio'
Table 2, Cornparison of activity of common antifungal agents and geranium oil
and its major chemical constituents (at MIC level) by disc diffusion
method on SDA and SDSA Platcs'
Compounds ,,rPotency disc Zone sizes in mm
C' alhicans C' nertfornutrs
. SDA SDSA SDA SDSA
Clotrimazole 25rtg
Itraconazole 30Pg 14 14 19 19
Griseofulvin 35Pg
Amphotericin-B ' l00U
Nystatin l00U 18 I 8 10 9
Fiuconazole l0Pg 22 22
Oil and its comPounds*
Geranium oil (neag 22 20 22 22
Cihonellol 22 24 24 24
Linalool 22 24 12 12
Geraniol 28 28 '11 27
lsomenthone 20 22 17 17
Geranyl formate ll ll 13 ]l
Citrorrellyal formate 14 ll 8 12
Cock taiimixtur ofsix compound at l:1 ratio >30 >30 >32 >32