Research reports

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Research R e p o r t s B

O B

H

A R R I S

and 50% oil concentrations delayed

PAPILLOMAS AND SAGE

appearance by 1 and 3 weeks, respec-

Salvia libanotica (synonymous with

tively. At week 16, application of 5%,

S. triloba and S. fruticosa) is widespread

50% and 100% essential oil inhibited

in Lebanon and traditionally used by

tumour incidence by 11%, 30% and

Middle Eastern herbalists to treat

33% respectively and tumour yield by

headaches, gastric and respiratory

42%, 41% and 78%, respectively. The

problems. Although the antimicrobial

chronic topical applications of sage oil

properties of the essential oil have

did not affect skin morphology, weight,

been investigated, its effect on skin car-

survival or behaviour of treated ani-

cinogenesis has not been evaluated up

mals, implying that the applications

until now.

were not toxic.

As mutations caused by tumour

Sage oil was also applied to a

initiation are irreversible, it is impor-

receiving either sage oil intraperi-

tant in skin cancer prevention to iden-

toneally (4% and 8%), by gavage

tify compounds that act against the

(0.3 ml of 100%) or by topical applica-

reversible

of

tion (5%, 50% and 100%) 20 minutes

tumorigenesis. Lebanese researchers

before each TPA treatment. The inci-

(Gali-Muhtasib and Affara, 2001)

dence and yield of skin tumours were

therefore examined the effect of Salvia

recorded weekly for 20 weeks and

libanotica essential oil on the tumour-

tumour weight and volume were

promoting activities of 12-O-tetrade-

recorded at weeks 17 and 20.

propagation

phase

(SP-1) that was originally induced by treatment with TPA, i.e. the same as the in-vivo study. At doses higher than 50 ␮g/ml, the essential oil significantly inhibited cell proliferation in a dosedependent manner without being toxic. This result was consistent with the in-vivo topical application tests.

canoylphorbol-13-acetate (TPA) on

The gavage and i.p. applications

tumours initiated with 7,12-dimethyl-

of sage essential oil did not reduce

benz[a]anthracine (DMBA).

tumour incidence or yield, but signifi-

The essential oil was distilled

cantly reduced the weight of papillo-

from the fresh leaves (yield 1.5–1.9%)

mas by 80% and 55% (using the 8%

and found to contain 1,8-cineole

dose), respectively. These results were

(57.4%) and camphor (8.4%) as the

explained in terms of problems with

main components. The LD50 of the

pharmacodynamics and bioavailability.

essential oil in mice was determined as

In contrast, the topical applica-

a 12% concentration.

mouse papilloma-derived cell line

tion of sage essential oil significantly

The authors concluded that S. libanotica essential oil was a potent antitumour-promoting agent against carcinogenesis in mice and that further study could develop the oil as an effective chemopreventive agent against skin cancer.

TEA TREE AND INFLAMMATION

Mice were initiated with a single

reduced the tumour-promoting effects

The essential oil of Melaleuca alternifolia

topical application of DMBA and 3

of TPA both by inhibiting new papillo-

has demonstrated anti-inflammatory

weeks later, promotion was started

mas and the growth of existing ones.

activity in animal models and its main

twice weekly by topical application of

The

highest

component, terpinen-4-ol, has been

TPA to shaved dorsal skin. Test mice

inhibitory activity, causing a 4-week

found to suppress the production of

were divided into three groups, each

delay in tumour appearance, whilst 5%

pro-inflammatory cytokines in human

100%

T h e I n t e r n a t i o n a l J o u r n a l o f A r o m a t h e r a p y 2002

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monocytes. To further investigate the

acted as a selective barrier, only allowing

through the skin and their effect upon

mechanism of anti-inflammatory activ-

penetration of the more water-soluble

inflammatory cells in-vivo are required.

ity, an Australian group (Brand et al.,

components into the dermis. Used at concentrations found in

2001) focussed their attention on

the whole oil (and equivalent to

COLON CANCER

0.031% essential oil), terpinen-4-ol

For more than 10 years, epidemiological

demonstrated

dose-

studies have shown that the dietary

peroxide, superoxide, singlet oxygen,

dependent suppression of superoxide

consumption of plant products may

hydroxyl radicals, hypochlorous acid

production in monocytes stimulated

reduce the incidence of colorectal can-

and various chloramines. They are

with fMLP and LPS, but not PMA. The

cer. The identification of phytochemi-

formed when macrophages and neu-

two former compounds bind to cell

cals with antitumour activity is hoped

trophils are stimulated, assembling

receptors and act on intracellular pro-

to lead to significant advances in the

(NADPH) oxidase on their plasma

tein kinase C. As terpinen-4-ol did not

prevention of human cancer. Some

membranes,

oxygen-derived

reactive

species

(ODRS). These species include hydrogen

a

significant

subsequently

exert an effect before the addition of

monoterpenoids that are found in

promote the reduction of oxygen in

the agonist, it must act at a point after

many essential oils have already been

the extracellular fluid. The ODRS are

the receptor phase. At an equivalent

found to possess antitumour activity.

important for host defences, being

concentration of between 0.00025% and

For example, limonene has demon-

both antitumoral and antimicrobial,

0.001% essential oil, ␣-terpineol sup-

strated antitumour activity against

but their production during acute and

pressed the superoxide production

breast, lung, stomach, liver and skin

chronic inflammation is cytotoxic and

induced by all three agonists, suggest-

cancers in animals and is undergoing

may cause tissue and DNA damage.

ing that it worked on protein kinase

clinical trials in humans. Geraniol, a

which

Human neutrophils and mono-

C or a precursor. The two components

monoterpene alcohol, has also demon-

cytes were isolated from healthy volun-

therefore had a different inhibitory

strated antitumour activity against

teers and their superoxide production

mode of action upon superoxide

a range of animal cancer cells both

determined by means of chemilumi-

production.

in-vitro and in-vivo. Thus a French team

nescence resulting from oxidation of a

The water-soluble components

(Carnesecchi et al., 2001) decided to

fluorescent compound. Measurements

had no significant effect on stimulated

investigate its activity with regard to

were taken in the presence of one of

superoxide production by neutrophils.

colonic cancer cells.

three superoxide agonists; N-formyl-

1,8-cineole was inactive with regard to

They examined the in-vitro effect

methionyl-leucyl-phenylalanine

both cell types. Analysis of the uptake

of geraniol on the growth of a human

(fMLP),

from

of both components by monocytes and

colonic cancer cell line, Caco-2.

Escherichia coli (LPS) or phorbol 12-

neutrophils revealed no significant

Geraniol was added to cell cultures at

myristate 13-acetate (PMA).

differences.

concentrations between 100 ␮M and

lipopolysaccharide

The effects of tea tree essential oil

The study implied that M. alterni-

500 ␮M and was found to inhibit cell

and its water-soluble components (ter-

folia essential oil has potential as an

proliferation in a dose-dependent

pinen-4-ol, 1,8-cineole and ␣-terpineol)

anti-inflammatory agent since it con-

manner. At 400 ␮M, it caused a 70%

were investigated upon superoxide

tains water-soluble components that

inhibition of cell growth and this con-

production in the absence and pres-

may selectively regulate cell function

centration was therefore used in fur-

ence of agonists. It was found that

during inflammation. This means that

ther experiments.

whole tea tree oil contained compo-

neutrophils could be fully active in

Apoptosis and cytotoxicity induced

nents that activated cell superoxide

eliminating foreign antigens whilst

by geraniol were investigated as possi-

production. However, it was not

monocyte superoxide production was

ble causes of the growth inhibition,

thought likely that the blood cells

suppressed and oxidative tissue dam-

using DNA fragmentation and the

would be exposed to whole essential oil

age prevented. Further research into

release of lactate dehydrogenase (into

in-vivo, since the stratum corneum

the penetration of tea tree components

the culture medium), respectively.

© 2002 Elsevier Science Ltd.

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It was found that geraniol did not

effect on the growth of human colon

indicator. The minimum agar concen-

induce apoptosis or exhibit cytotoxicity

cancer cells, was cytostatic in effect,

tration (MAC) of essential oil was

and so cell cycle analysis was then per-

had no cytotoxicity and inhibited

defined as the concentration of oil

formed over a 6-day period. In the

DNA synthesis. The low toxicity of

absorbed in agar after incubation for

presence of geraniol, cells accumulated

geraniol will enable future in-vivo stud-

3 days on an MID plate.

in the S phase of growth (where DNA is

ies in colon cancer prevention to be

replicated) whilst numbers in the G1

performed.

A third test used a standard agar dilution assay where the essential oils or their major components were incor-

phase (the phase before S, where the

porated into the agar medium before

cells become metabolically active)

ANTIFUNGAL VAPOUR

gradually decreased. Interestingly, this

inoculation with the fungi. The mini-

result differed from the antitumour

The determination of the antimycotic

mum inhibitory concentration (MIC)

effects of another monoterpenoid, per-

activity of essential oils is usually per-

was defined as the lowest concentra-

illyl alcohol, which decreased the num-

formed in aqueous media (e.g. agar)

tion of essential oil or component

ber of human breast cancer cells in the

but there have been no reports of the

inhibiting visible colony formation

S phase whilst increasing cells in the

effect of essential oil vapour against

after 3 days. The essential oil/compo-

G1 phase.

dermatophytes. A Japanese study

nent content of the MIC agar was

It was thus shown that geraniol

(Inouye et al., 2001) developed a novel

determined as before; the MAC here

inhibited DNA synthesis within the

method of evaluating the effect of

defined as the concentration of essen-

cell. This was further proven by the

essential oil vapour on fungi and at the

tial oil/component remaining in the

addition of radiolabelled thymidine

same time, the concentration of essen-

agar of an MIC plate.

and geraniol to cell cultures; the incor-

tial oil absorbed into the agar medium.

The results for both fungal species

Seven essential oils were used in

were almost identical with antifungal

poration of thymidine into cellular the

bark,

activity in the order: cinnamon bark9

To investigate the mechanism

lemongrass, thyme CT carvacrol, per-

lemongrass9thyme9perilla9laven-

whereby geraniol inhibited DNA syn-

illa, lavender, tea tree and lemon)

der9tea tree9lemon. The MID values

thesis, the effects of geraniol on cellular

against

dermatophytes

were less than the MIC values in all the

polyamine metabolism were examined.

Trichophyton rubrum and T. mentagro-

oils examined, e.g. for cinnamon bark

The activities of two key enzymes were

phytes. The agar medium was inocu-

an MID of 0.25 and an MIC of 12.5.

monitored. In the presence of geraniol,

lated with either fungi and placed in

The MAC values by vapour contact

ornithine decarboxylase activity (an

an airtight box. A filter paper impreg-

were 1.4–4.7 times less than the MACs

activity that is enhanced during cancer

nated with 100 ␮l of essential oil (two-

by agar dilution. The results demon-

growth) was reduced by 50% whilst

fold dilutions in ethyl alcohol) was

strated that fungal growth was inhib-

S-adenosylmethionine decarboxylase

placed in each box. Then 1 ml of air

ited by lesser amounts of essential oils

activity increased by 30%. This was

was periodically extracted from each

by vapour contact than by solution con-

accompanied by a 40% decrease in the

box and the essential oil content veri-

tact, i.e. vapour contact was more effec-

cellular putrescine pool and a concomi-

fied. The minimum inhibitory dose

tive. The testing of the major essential

DNA was reduced by 55%.

experiment

the

⫺1

(cinnamon

two

tant 60% increase in the N-acetylsper-

(MID ␮g/ml

air space) was defined

oil components showed that these

midine pool. Overall, these results

as the minimum quantity of essential

components were responsible for the

demonstrated that geraniol activated

oil added to inhibit growth of the fungi

antimycotic activity of their parent oils.

intracellular polyamine catabolism,

after 3 days (at which time the controls

Using four selected essential oils,

although it was not certain if polyamine

had developed a visible colony).

their effect by vapour contact was

The agar from each MID plate

examined in more detail. Perilla,

was removed and the absorbed essen-

thyme and lemongrass killed fungal

research

tial oil extracted and estimated using

conidia (spores), inhibited germination

showed that geraniol had a potent

the predominant component as an

and hyphal elongation at 1–4 ␮g/ml⫺1,

depletion was solely responsible for the antiproliferative effect. In

conclusion,

the

© 2002 Elsevier Science Ltd.

T h e I n t e r n a t i o n a l J o u r n a l o f A r o m a t h e r a p y 2002

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IJAR-75Res.QXD

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whilst

lavender

was

Page 66

effective

at

⫺1

40–60 ␮g/ml . The in-vivo effectiveness of thyme and perilla essential oils was then

and

The effect of the oil and individ-

anisaldehyde (2.6%). The whole oil

ual components were also examined

and the three individual components

with regard to enhancement of human

were used in the experiments.

immune cell growth by adding them to

(87.8%),

limonene

(3.9%)

tested in experimental dermatophyto-

As antimutagenicity is one of the

cultures of T and B lymphocytes. At a

sis in guinea pigs. Their feet were inoc-

first methods to be employed when

concentration of 1 g/L the essential oil

ulated with T. mentagrophytes and after

screening new compounds for anticar-

enhanced the growth of cell lines by up

infection was established, the feet were

cinogenic activity, the test solutions

to 200%, whereas individual compo-

wrapped in gauze. A disc impregnated

were individually added to a trans-

nents had far fewer effects. Kinetic

with either 20 mg of thyme oil or 20 mg

formed Chinese hamster ovary cell line

analysis using the B lymphocyte cell

of perilla oil was then attached to the

(CHO AS-52), a line more efficient in

line revealed that the whole oil

gauze and occluded. This procedure

detecting deletion-type mutations than

enhanced growth by 180% within the

was repeated once a day for 3 days and

bacteria. In the presence of a strong

first 10 minutes of contact, whereas the

the level of infection evaluated. There

mutagenic substance, 1.0 g/L of essen-

individual components started to

was no significant difference in efficacy

tial oil improved antimutagenicity by

slowly

between the two oils. Neither resolved

75% as compared to the control. The

minutes.

the infection completely but the sever-

addition of the three main compo-

ity was significantly reduced in com-

nents in the same concentrations did

that the essential oil of A. rugosa could

parison to the controls.

not provide the same level of protec-

selectively inhibit the proliferation of

It was concluded that the use of

tion as the whole oil. Both anisalde-

human cancer cells and promote

essential oils in the vapour state for

hyde and limonene had greater activity

human immune cell growth. The

antimycotic purposes was promising.

than methyl chavicol.

essential oil was more effective than

enhance

growth

after

20

The series of experiments showed

There was homogenous absorption of

Cytotoxicity testing was carried

individual components, indicating a

vapour over a wide skin surface (effi-

out on a normal human liver cell line.

synergistic effect between them and/or

cient percutaneous absorption of

Both the whole oil and its three com-

other minor components within the

vaporized thyme and perilla was shown

ponents were shown to possess low

oil. It was stressed that as a preliminary

in mice but the data was not given) and

cytotoxicity by inhibiting less than 12%

report, more investigations using

there was little chemical irritation due

of normal growth at a concentration of

in-vivo methods were necessary to fully

to the slow rate of absorption. It was

0.8 g/L. The cytotoxicity of the three

elucidate the effects of the oil on

noted that most antifungal agents cur-

individual components was less than

human cancer cells.

rently in clinical use are non-volatile.

that of the whole oil, with anisaldehyde possessing the least. The antimutagenic effect of the

REFERENCES Brand, C., Ferrant, A., Prager, R.H.,

ANTICANCER AGASTACHE

whole oil and the three individual

Riley, T.V., Carson, C.F., Finlay-

Agastache rugosa (Korean mint or wrin-

components was then tested using

Jones, J.J., Hart, P.H. (2001) The

kled giant hyssop) is a plant widely dis-

human lung, liver, gastric and breast

water-soluble components of the

tributed throughout Asia and has been

carcinoma cell lines. The oil at 1 g/L

essential oil of Melaleuca alternifolia

used extensively as a source of flavour-

inhibited the proliferation of all cancer

(tea tree oil) suppress the production

ing and oil due to its unique taste char-

cell lines, having the greatest effect on

acteristics. A Korean team (Kim et al.,

lung cancer cells with an inhibition of

2001) investigated its effects on human

82%. Gastric carcinoma cells were the

cancer cell lines and lymphocytes.

least affected. The components had

of superoxide by human monocytes, but

not

neutrophils,

activated

in-vitro. Inflamm. Res. 50: 213–219. Carneseecchi,

S.,

Schneider,

J.,

Ceraline, J., Duranton, B., Gosse, F.,

The flower essential oil was

lower activities than the whole oil, with

Seiler, N., Raul, F. (2001) Geraniol, a

obtained by steam distillation and

methyl chavicol demonstrating the

component of plant essential oils,

found to contain mainly methyl chavicol

highest anticancer activity.

inhibits

growth

and

polyamine

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biosynthesis in human colon cancer

Inouye, S., Uchida, K., Yamaguchi, H.

Park, Y.S., Hwang, S.J., Kim, J.H.

cells. J. Pharm. Exper. Ther. 298:

(2001) In-vitro and in-vivo anti-

(2001) The effect of the oil of

197–200.

Trichophyton activity of essential oils

Agastache rugosa O. Kuntze and three

by vapour contact. Mycoses 44:

of its components on human cancer

99–107.

cell lines. J. Essent. Oil Res. 13:

Gali-Muhtasib, H.U., Affara, N.I. (2001) Chemopreventive effects of sage oil on skin papillomas in mice. Phytomedicine 7: 129–136.

Kim, M.H., Chung, W.T., Kim, Y.K.,

214–218.

Lee, J.H., Lee, H.Y., Hwang, B.,

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