Studies on aqueous solutions of essential oils

STUDlIES

ON

AQi-‘EOUS

SOLUTlUNS

OF

ESSENTIAL

OfLS

Various hiokgical effects Ilaw hen described after accidental or espcrimcntat intake of essential oils, both in mart and animals l-8. A stuciv of the tosic effects of variuus cssentiul oils on preparations in tissue culture ww therefore plnnncd. Aqueous solutions are readily incorporated into the medium, and are distributed uniformly to the cells. ‘T?tis eliminates the use of organic solvents or carriers for the preparation of test solutions which might interfere with processes of growth and maturation of tbc tissues in culture. However, a careful searclr of the literature did not produce any definite values nn the degree of water solubility of these substances, and were therefore cstahlishcd in this laboratory.

The essential oils used wcrc prcpruxzd for research (see Tumble I I ).

eitlicr

obtained

from

commercial

sources

or

Oil and distilled water were mixed by shaking in a separatury funnel. Oil was in cxccss and separated grossly from the uc~ucous phase on standing for XG 11or more. The aqueous phase was centrifuged for 30 min at 2500 rpm at rotm temperature in an IEC centrifuge. Ffoating oil droplets were removed and the supcrnatant solution was transkrred to clean tubes and recentrifuged under the same conditions twice more, For oils lighter than water (anethole and estragole) an additional r-lt funnel separation was done before the last ccntrifugation. Experiments were conducted in which hot distilled water and preheated scparatory funnels were used. Since no clifferences in results were found, this procedure was discontinued. Preparatians made by the above-mentioned method had an oil content above the stable concentration IcvcI. The following methods were tried in order to eliminate the excess. I. Adsorption on glass beads. Three passages over 6.5 g of glass beads of z.smm diameter. This was done routinely for all solutions except eugenol and isoeugenof. 2. Centrifugation. (a) Spinco Centrifuge, not refrigerated, at 40,000 rpm for

I IO Concentration

t4ml I

1 -’ \ ‘. -p,_ 600 500%

I 1:

ooyz ITig.

10 1

201

30 1

r . C.oncclltr;ttion

:;ji?,.p)

-x 2

lb-__._-.

cstmgolc;

40

50

-0------------a

60

70

80

R,

ICI __._ -_.

..-

ICStf%l~t>ll!

\lIctllolc

. . . ..-

cthcr

It:,

--

.

II’ 4

t2a

XICO

.. _-

“.,_...

Al pg.

._... ___

.-_.-.

MC0

AI

_ ..._

XlCO

n 1c’0 NV0 MC0 iiIC0


Jl’a

socLlgcllc~l nctllyl

ctllcr

l’r

.---

hTc0

Al

--

\saro1w

l’r

MC0

MC0 ---

hIro1

Al

_-__

-c-KI-r.~o

Pr

-

eIyristicinc so-illyristicitlc

Al 1%

-.--

-oc:I-I&-

.;:Llgl!ILOl

hl

-

-OCl-LSD” --0c1-1.~0 hlc0 0l.r.

.socllgcllol

E’r

-

MC0


SOSCL fro1

100

110

r;rngc of cssciiti;d oil ;ic~~icc~Lis (_I) isouugc1101, diluicxl 1 : 2 ; (5)

, Rr? __

90

-

--

OH.

hIf_!O itIC

-

120 A

solutir~tw. iscwugcnctl,

(I) I
A(?UEOUS

SOLUTIONS

OF

ESSEXTI~I,

OILS

III

I 11, in polypropylene or polyallorner tubes. (6) IEC Centrifuge, Model UV, at 1500 rpm for 30 min in Filterfuge stainless steel tubes with 2 Millipore filters of the MI? type wit11 pore size of 0.22 pm, diameter 25 mm. 3. Filtration. (a) Millipore filter as above, in Micro Syringe liolder ; the volume filtered was 2 ml. (b) Frittcd Glass, 1; (fine) and Ul; (ultrafine) filters, 2-s cm diameter ; the volumes filtered were 3-5 ml. Gravimetric solutions were prepared as follows. Oil in weighed amounts was mixed in glass-stoppered flasks with the necessary volume of distilled water to make solutions of the desired concentration, and then shaken on a wrist-action mech~mical shaker for 30 min. Absorl>ance curves tverc recorded for each oil, cm CL ~~3eclm~~r~ Model IIf< z at tile beginning of tllc espcriments and after various intervals Sl~cctrc~yliotomcter, of standing, wit11 and witflout access of air.

Equal voluiiies of tlie xclueous soluticm. of oil and carbon tctracl~loricle were shaken in separatory funnels wit11 teflon stopcocks. After separation, tile carbon tetrachloride fraction containing tile heavy oil was collected. Tllc absorbilnce was read on a I3CXl;klli~ll Spectrt,pllotonieter in ultraviolet liglit at the wavelength of peak ;ibsorb;Lnce for cac.11 oil, cw~cl tilt: concentrations were calculated from standard (:urves.

mdd YIU

Tllc solutions l~reparccl by tllc ~Ll.)ovc-dcscril~ccl metllocl still contained oil in esccss. Tllc escess separated from tile solution eitller by scclimcntation of oil drolActs or by volatilization and escape tllrough tllc rubber stolqlers covering the tubes, or ‘I‘lle process was greatly prolonged by both during relatively long periods of standing. 1 shows tlie decrease in minimizing volatilization. in glass stolq~erccl tubes. Figure concentration (,.ug ml-l) of tile solutions stored in rubber-stol,l,cred glass tubes as a function of time (days). Tile lligh values rcl>rescnt the average concentrations after preparation (see ~Wctlrods). Elcmicin, eugenol, isocugenol, estragolc, safrol and mpristicine reaclled a concentration level whicll rernainccl stable for zo days or more. For the other oils, the experiments \vere interrupted before the solutions reaclled a stable level, and the concentrations continuecl to decrease at a slow rate. average concenTable I contains the concentration range, ,i.e. the liigliest and the lowest average values trations (pug ml-l) after preparation of the solution reached, either at the stable concentration level or at the termination of experiments. The average period of time (days) to reacll the stable concentration level or the lowest values mentioned is also indicated. Standard deviations and coefficients of variance were calculated for tile oils which reached a stable concentration level. Concentrations maintained for IOO days or more in glass-stoppered tubes are mentioned in order to indicate the effect of volatilization. Table II includes the expected and actually obtained concentrations of gravimetric solutions. The solutions of asarone were unsatisfactorily cloudy and unstable altllough the solutions made by the usual procedure were clear at above 50 ,~g ml-l, Changes in aljsorbatice curves were observed in G markedly higher concentrations. APtal. Clriwa. Acla,

51 (1970)

IO+IXG

II2

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f/4hJ ?tli -‘)

I’ccck

C;hUl4&‘S it1

wuve-

UhWiJWlCC

-...... ___

--.__-

CtO’VCS

It~llgtlb

(Jlill)

__- -._.

9.1.

r>

3X.S ‘J-i. 5 fJl.‘J

g; c

(Se) (Fi) (19

-~-_1

(5)

I:)

_-_..__--~

____.__..__ __.___ __.___

-_ _.---- __..____--..--- _... _I_._. __--- .._- .~_____

Dr. A. ‘I’. S~ru~,c;rz*‘, I)ow Chc~ii. Lb.; (SC) I>f. T. 1\. %ro, l~ritzsclic 13ros.; (I<) I< & Ii IditlJS. Illc. ; (C) Cal. Lziochcm. CO.

nrCJuklyl1

St:ltC!

IdCaSpitd:

(vi)

I’kchCX

sci

of the x2 oils used after various periods ctf standing. Safrol and estrngolc prcscntcd appearance from the first day of occasionally a changed “aged” (spcctrograI>fticnIly) preparation. Also mentioned in Table II are tllc sources of the oils used and the peak wave length at which the concentrntion readings were made. Adsorption on glass beads reduced the concentration by IO to 4o%,. The adsorption was great mainly with the low solubility oils, when tile concentrations were far almve the solubility level. En all the cases the excess was reduced only by a fraction, leaving the conccntratiorls above the level of stable concentration (high values of tile concentration range in Tslble I and Fig. I). Centrifugation in the Spinco centrifuge reducccl tile concentration to a certain estent. The reduction was inconsistent and did not reach the stable concentration level. Visible sediment was never found. The effect of the procedure also depended upon the type of tubes used. Polypropylene tubes grossly changed their appearance

SOLUTIONS

AQUEOUS

OF ESSISSTIAL

OILS

113

a.fter use with elemicin solution; the reduction in concentration was striking and reached a level much below the stable concentration level (from 075 to 275 ,ug ml-l). The tubes could not be cleaned and had to be discarded. A reduction below the stable concentration level occurred also for myristicine (Go to 26 ,ug ml-l) and estragole (255 to 175 rug ml-‘), but to a lesser degree. Tl~e use of polypropylene tubes was then discontinued. With polyallomer tubes, the concentrations were reduced inconsistently but reached only to or above the values of the stable concentration, never below. Centrifugation in Vilterfuge tubes was made in relatively few instances, since it was found to parallel the results obtained by filtration through Millipore filters (see below) without presenting additional advantages. I;iltration thrctugh Millipore nlembrane, iMF tyl>e, was effective in reducing the concentration to or below the stable concentration level, independent of the level of saturation. Serious doubts that the reduction in concentration was clue to tile filtration process itself were raised by the fact that a@tation in a test tube containing the membrane and oil solution was sufficient to lower the concentration to the same values as filtration, or even more.

I

--I

Recovery

IOOL

%

’ 67.0

I

97.0

64.4

1

100

I

02.0

66.0

Pig. 2. Pcrwntagc of reduction in conccntrntion by 11s~ of Milliporc filters hll? close to the stnblc concwitration level (filtration or shaking), ant1 uf rccovcry from the Milliporc filters. Shaclcd column, filtration ; clottcci colun~n, shaking. * From lcvcl of 2qo pg WI-1.

Figure z shows the percentage reduction in concentration by filtration and by shaking for I 11. At the beginning: of the experiments tlte solutions were close to the stable concentration level and the process reduced the concentration much below that level. The reduction was usually &q-eater by shaking (62 to So%) than by filtration (g to 64%) ; only in the case of elemicin was the reduction identical with both processes. The Millipore membranes, which were shaken for I h with the oil solutions,

were blotted and shaken for f 11with carbon tetracl~loride. Tlx2 oil thus recovered from the ~IilIi~)ore membrane amounted to tjz-sooo$ of the oil “lost” by the previous contact of the solution with the filter (Fig. 2). ‘Filtration through frittcd glass filters reduced tllc concentrations inconsistcntly at I~igh concentration. At the stable concentration level, filtrrrtion and/or shaking with tile filter did not reduce tile concentrations below that level in any of the xz oils tested. The solutions filtered through the Ul: fritted glass filters are sterile, and tltus can be incorporated into media for tissue culture.

‘flue aim r)f tllis work was to c,btain and study saturated aclue~3s solutictns of essential oils, Twelve oils were studied; for clemicin, cugenol, isocugenol, estragole, safrol and myristicinc, the study was pursuccl to completion. For as;Lrffne, cugenol anetholc and isosafrol, only metllyl ether, isocugenol methyl ctlier, isomyristicine, partial results were obtained. It ~8s found tlliLt illteltllc initi8.1 prepar:Ltion of tile solutions, iL prolonged process of cc~uilil~rntion takes place, in wfiich scpar;~tion of fine oil droplets or volatilization, ffr hotlt, xc effective, An indication of tlte r&dive itn~wrtanco of citfwr process was ohtninccl by using h’lass-s~oppcrccl tulxs which n~initnizecl volatilixatirm ; tile concentrations rcmnined unc11niq:ecl for cstcnsivc periods of time in tire case in

wllicll volntilixation was tlw mnin means of escape (myristicinc, elcmicin, snfrol, etc.) and reduction to a wrtain ostent ~;LS cjfxxrved wlwre droplet formation w;i?i cffcctivc: (c:.g. warone, cugenol mctltyl ctlwr, cugcnctl, clc.) (Table L). The time ncccssary tc) reach ;I Icvel of stable concentrntion wts CLL.30 clays for clemicin, estrtrgolo, myristicinc ; considerably slwrter for safrol (,7 days), and longer for cugenol and isoeugcnol (over 100 days, if undiluted) (Fig. I). Scvcrul metliods of rcclucing tlw csccss of oil ut 211xccleratcclratc were tried. Ccntrifugatioti and filtrntion ;~s suclt wcrc htli utrsuccossful. From high crtncentmtions, thcrc was a rcducticltt to rclativcly lower vnluer; by either mctllocl, but tlw rate was unlxcclict~~l~lc, unreliai~lc ;uicl it proved to be clue to contact adsorption by the tub2 (&LSS, Ix3lyallc~mcr, l~olyprolg&2ne) or the filter (fritted glass, RIilliIwrc). At stable concentrntion lcvcls, ghss and l~o1~~allomctrtulws, as well as frittcd glass Filters were ineffective in further lo\vering tllo coI1cetttration, cvcn by prolonged contact, shaking or filtration. In contrast, wntacA xvitl3 tlw ~Iillil~or~~ mcmtx-nnc of Ml; type (mixecl ccllulosc esters) was cffcctivc in further reducing the conccntr;ttirm below the stable level by removing the oil from tlrc solution. As demonstrated, the rcmovnl was not due to tlw filtration l~rocess (l;iK. 2). l;or clcmicin, filtration (sllort contact) was sufficient nncl as efficient as one llour of contact by shaking. Par cugenol, filtration reduced the ctmcentrntion I,_y wily q’%, and shaking IowerctLit by G3’;/ol~elow solubility level. l;or eugertol methyl ether, cstragolc, snfrol and myristicine, filtration reduced the concentration by 40-50’!& and shaking lowered it by 70-80~& Recovery of the nil from the Millipore rncmbrnnc wllicll cstracted it, was possible by shaking the filter for I 11 with carbon tetracllloridc with a recovery rate of Sz-xoo~~~~,.No further studies wcrc made to determine tlic nature of this process. There arc indications that polypropylene might also 11avc a similar effect, l?ut its use was discontinued and not studied.

During long periods of standing, changes occurred in several of the oils in solution; these were detected by changes in their spectrographiti absorption curves (Table II). A similar phenomenon was also observed by other methods by TRUITT et al.10. et al.D and ZACKSO-SZASZ Correlating structure and volubility in tlie I2 oils studied, it appears that in any pair of isomers of the allyl-progeny1 structure, tile prolxnyl cc~n~pouncl has a lower solubility than the ally1 isomer. \\?e wish to tllank Dr. T. A. supply of elemicin ancl isomyristicinc. for supplying myristicinc.

SE-IV We

for his \~tluahlc suggestions arlcl fol- the are also indebted to Dr. -4. T. Sr-rrrtc;r~

Tile acclivous solutions of 12 essential oils \vcrc studied. A level of stable concentration, unchanged for at lcnst 20 days, was obtained for elemicin, cugenol, isoeugcnol, estragole, safrol and myristicine. i\nctliolc, isomJ*risticine, cugenol metllyl ctller, isocugenol methyl ether, asaronc and isosnfrol \vcrc only partially studied. Stable aqueous solutions filterccl tllrougll Ul; frittccl glass filters are sterile and can 1~ tested in tissue culture. l’ropcnyl isomers liavc lo\vcx- solubility tllilll tllc ally1 counterparts. Clianges cxcurrctl in scvcral oils after various pcriocls of standing, as ohervccl by sl~~ctrol>l~c.~tonzctric alxorption spectra. Millipore mcmlxxnes of the iV I; type, and 13ossitAy l~olyl~rc~l~ylcnc tubes \vc’rc found to r~niovc tlic oils Iron1 solution at concentrations \vllicli arc otllcrwisc stnblc.

t_Jn niveau stable clc concentration qui ii’x pas cllangi: pcnclant ail mains 20 jours fut obtenu avec I’~lhicinc, I’eug&iole, l’isoeugchole, l’cstragole, lc safrole et la myristicinc. L’an~tliolc, l’isomyristicinc, l’cugholc mdtl~yle 6tlicr, l’isocug&x~le m&hyle dtller, l’asaronc et l’isosafrole rmt dtc? Ctucli&x sculcincnt lxirtiellcment. Les solutions aclucuses stables, filtr&s SLh-avers lc filtrc clc vcrrc frittc Uli sent sthilcs et peuvent Ctre cssaydes dans la culture dc tissus. Les isrmi&res propdnylicIucs sont mains solubles clue lcs isom~rcs allyliqucs. On ;L put obscrvcr dcs cliangemcnts clans plusieurs liuiles, aprc5s clcs ph-iocles de tcmps cliffhentes, comme le prouvent les spcctrcs cl’absorption spcctrc~l~1iotc~iiidtriclucs. Les membranes Millipores clu type Ml; et probablcment les tubes de lx~lyprol~yl&n~: pcuvcnt retircr les lluilcs d’une solution, m6mc si lcs solutions sent ri dcs concentrations stables.

Die w5ssrigen I,Osungen van 12 iitllerischcn Olen sind untersucht worclen. Piit Elemizin, Eugenol, Is<)-cugenol, Hstragol, Safrol urzd. Myristizin wurdc einc HOcllstkonzentration ermittelt, die fiir wcnigstens 20 Tagc stabil blicb. Anetllol, Iso-myristizin, Eugenol methyllither, Iso-cugenol nietliyliitlier, Asaron und Iso-safrol wurclen nur teilweise untersucht. Stabile wiissrige LBsungen ,die durcli UF Glasfritten filtriert wcrden, sind sterii uncl kiinnen in Gcwebekulturen zuTestzweclcen vcrwenclet

1x6

31.ShMLiELL\-,

W.

KEUP

werden. Propenyl-isomcre haben eine nicdrigere LOslichkeit als ihrc Ally1 Analoge. Naclt verschiedener Zeitdauer des unbcriihrtcn Ahstehcns fandcn in mehreren Olen VerSinderungen statt, die sic11 in spektrophotometriscflen Absorptionsiindcrungen ausdriickten. MiIlipore Membrane (MF Typ) und. m6glicherweise Polypropylen%cntrifugenriihren fiihrten xur Entfernung clcr tiitherischcn Ole aus der Liisung in Konzentrationen, die unter underen Umstiinden stabil waren.