842

J. Agric. Food Chem. 2010, 58, 842849

DOI:10.1021/jf9027206

Anti-Arthritic Effects and Toxicity of the Essential Oils of

Turmeric (Curcuma longa L.)
JANET L. FUNK,*, JENNIFER B. FRYE, JANICE N. OYARZO, HUAPING ZHANG, AND
BARBARA N. TIMMERMANN

Department of Medicine, University of Arizona, Tucson, Arizona 85724 and Department of Medicinal
Chemistry, School of Pharmacy, University of Kansas, Lawrence, Kansas 66045

Turmeric (Curcuma longa L., Zingiberaceae) rhizomes contain two classes of secondary metabolites, curcuminoids and the less well-studied essential oils. Having previously identified potent
anti-arthritic effects of the curcuminoids in turmeric extracts in an animal model of rheumatoid
arthritis (RA), studies were undertaken to determine whether the turmeric essential oils (TEO) were
also joint protective using the same experimental model. Crude or refined TEO extracts dramatically
inhibited joint swelling (90-100% inhibition) in female rats with streptococcal cell wall (SCW)induced arthritis when extracts were administered via intraperitoneal injection to maximize uniform
delivery. However, this anti-arthritic effect was accompanied by significant morbidity and mortality.
Oral administration of a 20-fold higher dose TEO was nontoxic, but only mildly joint-protective (20%
inhibition). These results do not support the isolated use of TEO for arthritis treatment but, instead,
identify potential safety concerns in vertebrates exposed to TEO.
KEYWORDS: Curcuma longa L.; turmeric; essential oil; arthritis; rheumatoid arthritis; liver; anemia

INTRODUCTION

Essential oils are complex mixtures of volatile terpenes selected

throughout evolution to protect plants from external threats by
various means including mimicking endogenous substrates in
herbivores (1). While biologic and potentially medicinal effects
of these essential oils are thus to be anticipated, these compounds
are often less well studied than other classes of secondary plant
metabolites, with polyphenols being a prominent example. Such is
the case for turmeric (Curcuma longa L., Zingiberaceae), a
medicinal botanical whose rhizome contains two major classes
of secondary metabolites, the phenolic curcuminoids and the
hydrophobic essential oils (2). Many laboratories, including our
own (3, 4), have demonstrated potent and physiologically important effects of the curcuminoids (5). For example, we have
elucidated a profound anti-arthritic effect of curcuminoid-containing turmeric extracts due to their ability to inhibit nuclear
factor-B (NF-B) activation in experimental rheumatoid arthritis, thus blocking multiple downstream signaling pathways critical
to joint inflammation, including cyclo-oxygenase (COX)-stimulated prostaglandin-E2 (PGE2) production (3). Far less studied
and indeed often discarded in the preparation of turmeric dietary
supplements (AR) are turmerics essential oils.
Reported biologic properties of the multicomponent essential
oils of turmeric include antifungal (6), mosquitocidal (7), antivenom (8), antibacterial, and antioxidant properties (9, 10). Medicinal effects of TEO in vertebrates have also been reported for
stroke and diabetes: a single acute dose of TEO (250-500 mg/kg
*To whom correspondence should be addressed. Tel: (520) 6263242. Fax: (520) 626-3644. E-mail: [email protected].

pubs.acs.org/JAFC

Published on Web 12/21/2009

oral or ip) was neuroprotective in rats subjected to occlusive

or embolic strokes (11-13), while chronic TEO dietary supplementation (g620 mg/kg/d) normalized serum glucose in diabetic
mice (14, 15).
Major compounds present in turmeric essential oils (TEO)
include ar-turmerone, R-turmerone, and -turmerone. The relative proportions of these terpenoids in TEO extracts vary depending on the rhizomes geographic origin and method of extraction (9, 16, 17). In isolation, these compounds have demonstrated
bioactivity in vitro, including cytotoxic and apoptotic effects in
various cells (18, 19); binding to the nuclear receptor, peroxisome
proliferator-activated receptor (PPAR-) (14); inhibition of
inducible PGE2 and NO production (2, 20); and inhibition of
platelet aggregation (21).
Emerging in vitro evidence from our laboratories and others
suggests that crude, multicomponent hexane extracts of TEO may
be more bioactive than any of their individual fractions or
components (2, 9, 11). For example, when our laboratories
isolated and compared the efficacy of 8 distinct fractions of a
crude TEO extract vs the extract itself in blocking COX-mediated
PGE2 production in vitro, the potency of the crude TEO extract
exceeded that of each of its fractions (2). Moreover, the crude
essential oils had the same potency as that of turmerics other
secondary metabolites, the curcuminoids, in this in vitro screening
assay. Thus, we have postulated that the essential oils of turmeric
may also have significant medicinal anti-arthritic effects in vivo,
independent of those attributed to turmerics polyphenols, that
result from synergistic actions of the oils various constituents.
To test this postulate, in vivo studies described herein were
undertaken to determine whether the essential oils of turmeric are

2009 American Chemical Society

Article
indeed effective anti-arthritic agents using an animal model of
rheumatoid arthritis (RA), streptococcal cell wall (SCW)-induced
arthritis. Because we previously used this model to characterize
anti-inflammatory properties of essential-oil free, curcuminoidcontaining turmeric extracts (3,4), these experiments also allowed
us to compare the relative in vivo anti-inflammatory efficacy of
each of turmerics two major classes of secondary metabolites.
Moreover, our isolation and in vivo testing of two complex TEO
extracts, a crude hexane extract and a curcuminoid-stripped,
refined fraction of the crude TEO extract, also allowed for
clarification of the independent role of the essential oils (vs
curcuminoids) in preventing intra-articular inflammation.
MATERIALS AND METHODS

Turmeric Essential Oil Preparation. Turmeric essential oil (TEO)

extracts were isolated from dry turmeric rhizome powder as described in
detail in United States Patent 7,205,011. In brief, a crude TEO extract was
prepared from 1 kg of dry Curcuma longa L., Zingiberaceae rhizome
powder (San Francisco Herb, SF, CA) by n-hexane extraction at room
temperature for 24 h. The resultant mixture was filtered, the filtrate was
combined with an n-hexane wash of the marc, and the solvent was stripped
under vacuum for 24 h to obtain a crude turmeric oil extract (yield, 3.7%).
HPLC analysis revealed minor curcuminoid contamination of this essential oil-enriched extract. The crude TEO (36 g) was processed further by
silca gel column chromatography. Elution was by the gravity method. The
column was eluted with n-hexane (100%). Fractions (25-30 mL each)
were collected until the color of the eluant changed from colorless to yellow
to colorless again. The column was then eluted with a mixture of n-hexane
and ethyl acetate (17:1 v/v) for a total of 15 L. The column was finally
leached with 100% ethyl acetate (1 L), and elution was discontinued. The
solvent from the 15 L fraction was stripped off under vacuum using a
rotary evaporator, and the resulting refined TEO was then left under
vacuum for a minimum of 24 h (66-74% yield, by weight).
Chemical Analysis of Turmeric Essential Oil Extract. The major
components of crude and refined TEO extracts were characterized
chemically by GC-MS and HPLC. Identification of the three major
constituents of the essential oils was achieved by comparison of their mass
spectral fragmentation patterns (NIST08 database/WorkStation data
system) and with literature reports (22, 23). GC-MS data were recorded
with Varian Saturn 2100T (Palo Alto, CA). The gas chromatograph was
fitted with a Chrompack capillary column (CP Sil 8 CB; 30 m
0.25 mm).
Operating conditions: column oven temperature programmed at 80 C for
5 min and then to 280 at 10 C/min; injector/transfer line/trap temperatures 250/250/200, respectively; electron voltage, 50-80 eV. UHP helium
was used as the carrier gas at a flow rate of 1.2 mL/min. Each sample (1
mg) was dissolved in CH2Cl2 (0.5 mL) and injected (1 L) directly into the
chromatograph.
The HPLC system for extract analysis was composed of the following:
Agilent 1100 series with a quaternary pump, a degasser, a thermostatted
column compartment, a thermostatted autosampler, a diode array detector and ChemStation for LC 3D, Rev. A.09.03 (1417) software for system
control and data acquisition (Agilent Technologies Inc., Palo Alto, CA).
The samples (20 L) were injected onto a Synergy, 4 m, Hydro-RP 250

4.6 mm column coupled with a SecurityGuard AJO-4287 guard column
(Phenomenex, Torrance, CA). The mobile phases used were as follows: A,
500 L acetic acid/L of Nanopure water; B, 500 L acetic acid/L of
acetonitrile. The organic mobile phase was filtered through a 0.2 m PTFE
membrane filter, and the water based mobile phase was filtered through a
0.2 m cellulose nitrate. The mobile phases were thoroughly degassed
before analysis and throughout the analysis. The eluent was monitored at
425 nm, 370 nm, 280 nm, and 250 nm; the flow rate was 1 mL/min. For
HPLC analysis, triplicates of approximately 1-2 mg of sample were
weighed using an analytical balance, dissolved in a small volume of
dichloromethane, then completed to volume with methanol (5:95) with a
final concentration of approximately 1 ppm. Solutions were transferred
into amber autosampler vials, and 3
20 L was injected onto the HPLC
column.
SCW-Induced Arthritis and Dosing Regimes. All animal experiments were performed in compliance with the University of Arizona

J. Agric. Food Chem., Vol. 58, No. 2, 2010

843

Institutional Animal Use and Care Committee (IACUC). To induce

arthritis, female Lewis rats (110 g, Harlan, Indianapolis, IN) were injected
ip with peptidoglycan-polysaccharide polymers (25 g rhamnose/g body
weight) isolated from the sonicated cell wall of Group A Streptococcus
pyogenes (SCW) (Lee Laboratories, Grayson, GA) or normal saline
(vehicle) (3, 24). Intraperitoneal (ip) or oral (po) administration of TEO
extracts or the appropriate vehicle (0.5 L DMSO/g for ip dosing or
0.5 L 8% saccharine in DMSO/g for oral dosing) began 4 days before or
3 days after SCW injection, as indicated (4). Daily dosing continued until
acute inflammation subsided (10 days post-SCW), at which time frequency
was decreased to 5 days/week. Oral dosing was maintained at this level
until the end of the experimental period, while ip dosing during the final
week decreased again to thrice weekly because of increasing evidence
of toxicity. Treatment groups for arthritis experiments thus included
(1) 2 normal animals treated with vehicle; (2) normal animals treated with
TEO extracts; (3) SCW-injected arthritic animals treated with vehicle; and
(4) SCW-injected arthritic animals treated with TEO extracts.
Joint Inflammation. Joint inflammation was determined in a blinded
fashion by daily assessment of the arthritic index (AI) in each distal limb
using standard criteria (3, 24) (0 = normal; 1 = slight erythema and
edema; 2 = increased edema with loss of landmarks; 3 = marked edema;
4 = marked edema with ankylosis on flexion, for a total possible score of
16/animal).
Real Time RT PCR. RNA was isolated from hind ankle joints as
previously described (3). Equal amounts of RNA were combined from
3 (nonarthritic) or 3 (arthritic) joints/treatment group to make one
sample. Three such pooled RNA samples/treatment group (i.e., total of
9-12 joints analyzed per group) were used for analysis of articular
mRNA expression using real-time RT-PCR TaqMan analysis and
previously described methods (3). Rat specific primers for interleukin 1 (Il-1) (Rn00580432_m1), monocyte-chemotactic protein
1 (MCP-1) (Rn00580555_m1), growth-related oncogene/keratinocyte
chemoattractant (GRO/KC) (Rn00578225_m1), cyclooxygenase 2
(COX-2) Rn00568225-m1), mannan-binding lectin serine peptidase 1
(Rn00434830_m1), properdin (Rn01526084_g1), and an 18S primer (as
an internal control; Hs99999901_s1) were obtained from Applied
Biosystems (Foster City, CA). Data, normalized to the endogenous
reference (18S RNA), were analyzed using the comparative CT method
as previously described (3).
Toxicity. To monitor for possible toxic effects of TEO treatments in
normal or SCW animals, daily weights and mortality were assessed. Liver
and renal function were monitored by assay of alanine aminotransferase
(ALT) and creatinine levels in blood samples obtained at the indicated
times after injection of SCW (or vehicle) using a Hemagen Diagnostics
Endocheck Plus Chemistry Analyzer (Columbia, MD). Circulating white
blood cell counts, hematocrit, and platelet counts in whole blood were also
determined using a Hemavet 880 analyzer (CDC Technologies, Oxford,
Conn.). Necropsies, including histologic assessment of all organs, were
performed in a blinded fashion by a veterinary pathologist. Occult blood in
stools was assessed by Hemocult testing (VWR, West Chester, PA).
Histology. Liver tissue specimens were fixed in 10% formalin and
subsequently embedded in paraffin. SCW-induced hepatic granuloma
formation at day 28 was assessed in H&E stained liver sections using
standard criteria and blinded analysis (3, 24).
Statistical Analysis. Values are presented as mean ( SEM except
where indicated. Accumulative effects of treatment on joint swelling were
determined by assessing the area under the curve (AUC) for each
animal (25). Statistical significance was determined by ANOVA with post
hoc testing, Students t-test, or by Fishers exact test, as indicated, using
InStat software (GraphPad Software, San Diego, CA).
RESULTS AND DISCUSSION

Isolation and Characterization of major components in TEO

Extract. HPLC analysis of the crude hexane extract prepared
from powdered turmeric rhizomes (3.7% yield) revealed slight
contamination of the oils by the 3 major curcuminoids (curcumin,
demethoxycurcumin, and bisdemethoxycurcumin) with retention
times less than 25 min, as well as the presence of 3 major peaks
with retention time more than 25 min. Column fractionation of

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J. Agric. Food Chem., Vol. 58, No. 2, 2010

Figure 1. Effects of ip turmeric essential oil (TEO) extracts on joint

inflammation. Female Lewis rats were injected on day 0 with vehicle or
streptococcal cell wall (SCW, 25 ug/g body weight) to induce arthritis. Joint
swelling in limbs was assessed at indicated times and expressed as mean
arthritic index (AI) (scale 0-3/limb for total possible score of 16/animal)
with statistical significance determined by analysis of variance with post hoc
testing or Mann-Whitney (n = 4-7 animals/group). (A) Intraperitoneal
treatment with crude or refined TEO (normalized to 56 mg refined TEO/g)
or vehicle was begun 4 days prior to SCW injection and continued daily until
10 days after SCW injection, at which time dosing frequency decreased to
3-5 days per week. *, p < 0.05 for refined or crude TEO (vs vehicle). **, p <
0.10 for refined or crude TEO (vs vehicle). , p < 0.05 for crude TEO only
(vs vehicle). z, p < 0.05 for refined TEO only (vs vehicle). (B)
Intraperitoneal treatment with crude TEO (normalized to 56 mg refined
TEO/g) or vehicle was delayed until 3 days after SCW injection and
continued daily until 10 days after SCW injection, at which time dosing
frequency decreased to 3-5 days per week. *, p < 0.05 for crude TEO
(vs vehicle).

the crude TEO extract resulted in a second, refined extract (66%

yield). In the refined TEO, the relative ratios and content of the 3
major terpenoids were retained, while curcuminoids were no
longer detectable (Figures S1 and S2, Supporting Information).
GC-MS data (the retention time order and the MS fragmentation
pattern) of these 3 major peaks were compared with the NIST08
library and with those published of turmeric essential oils (22,23).
This resulted in the identification of the three major HPLC
peaks as ar-turmerone, R-turmerone, and -turmerone (Figure
S1A-G, Supporting Information). These two experimental TEO
extracts were then tested in vivo.
Anti-arthritic Effects of Intraperitoneal TEO Extracts. To
ensure uniform delivery of the two experimental extracts while
establishing and comparing their anti-arthritic bioactivity, TEO
extracts were administered ip to maximize delivery and eliminate
any potential confounding effects of differential gastrointestinal
absorption. Doses of the crude TEO were normalized on the basis

Funk et al.

Figure 2. Dose-dependent inhibition of joint swelling by ip TEO extracts.

Female Lewis rats were injected on day 0 with vehicle or streptococcal cell
wall (SCW, 25 g/g body weight) to induce arthritis. Intraperitoneal
treatment with crude or refined TEO (with indicated extract doses,
beginning as low as 0.28 mg/kg/d, normalized to refined TEO), or with
vehicle, was begun 4 days prior to SCW injection and continued daily until
10 days after SCW injection, at which time dosing frequency decreased to
3-5 days per week. Arthritic index was assessed in TEO-treated and
vehicle-injected animals and is expressed here as percent inhibition relative
to controls (n = 5-30/group) with * p < 0.01 vs untreated. (A) TEO
inhibition of initial, transient joint swelling (day 3). (B) TEO inhibition of joint
swelling during the chronic, joint-destructive phase (day 28).

of the 66% yield of the refined extract and are expressed as mg

equivalents of refined TEO (e.g., 86 mg crude
0.66 = 56 mg
equivalents of refined). Using this approximation, differences in
bioactivity for a given normalized dose of crude vs refined TEO
can be attributed to the additional components present only in the
crude extract. Treatment with either TEO extract (56 mg equivalents of refined/kg/d) beginning prior to SCW injection, profoundly inhibited joint swelling (95-100% inhibition) in animals
with SCW-induced arthritis (Figure 1A). This marked degree of
inhibition by turmerics essential oils is comparable to that
previously demonstrated by our laboratories for an equivalent
dose of turmerics other secondary metabolites, the curcuminoids (3). Indeed, this extract dose was specifically chosen as a
starting point for determining TEOs anti-arthritic efficacy on the
basis of our previous findings with the curcuminoids. When the
start of TEO treatment was delayed until after the peak in acute
inflammation (day 3), the essential oils were also protective
(Figure 1B), although to a lesser degree (64%). These findings

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J. Agric. Food Chem., Vol. 58, No. 2, 2010

845

Table 1. Effects of Crude TEO (56 mg/kg/d) on Articular Gene Expression

non-arthritic
gene type
cytokine
chemokines
complement cascade
arachidonic acid metabolism
c

arthritic

gene

vehicle

TEO

SCW

IL-1beta
GRO/KC
MCP-1
properdin
MBL serine peptidase
COX-2

1.4 ( 0.3
1.0 ( 0.1
1.4 ( 0.6
1.0 ( 0.1
0.9 ( 0.1
1.6 ( 0.3

1.6 ( 0.3
1.6 ( 0.8
0.7 0.3
1.0 ( 0.3
1.2 ( 0.2
1.4 ( 0.6

21.1 6.5b
54.9 11.9d
12.0 7.9
8.1 ( 2.8b
6.5 ( 2.0b
10.3 ( 3.2b

SCW TEO
2.7 ( 0.3c
1.7 0.6e
0.4 0.1
1.0 ( 0.1c
0.9 ( 0.2c
1.9 ( 0.2c

a
Values are expressed as fold change from normal vehicle-treated animals (mean ( SEM for n = 3 samples/group with 3 joints combined per sample). b p < 0.05 vs vehicle.
p < 0.01 vs SCW. d p < 0.001 vs vehicle. e p < 0.001 vs SCW.

Table 2. Screening for TEO Toxicity in Normal and Arthritic Animalsa

nonarthritic animals
vehicle
hepatic granulomas (% incidence)
WBC (K/L)
ALT (U/L)
ALT (% > upper range of normal)
Hct (%)
creatinine (mg/dL)

7.2 ( 0.4
16.0 ( 1.5
0% (0/23)
41.8 ( 0.8
0.18 ( 0.01

crude
10.1 ( 1.2
40.3 13.7
22% (4/18)d
31.3 ( 3.2g
0.21 ( 0.02

arthritic animals
refined
7.4 ( 0.5
24.6 ( 2.0
10% (3/29)
36.1 ( 1.4g
0.22 ( 0.02

SCW vehicle

SCW crude

SCW refined

68% (34/50)
23.7 ( 2.5c
9.5 ( 1.0
0% (0/25)
32.3 ( 1.2c
0.17 ( 0.02

15% (4/26)
13.6 ( 1.5d,e
64.6 ( 41.3
20% (3/15)d
33.7 ( 2.3d
0.15 ( 0.01

41% (7/17)
18.0 ( 1.4c,f
12.9 ( 1.4
0% (0/21)
32.1 ( 1.4c
0.015 ( 0.01

a
Data, obtained from animals surviving one month of treatment with g28 mg/kg/d TEO (or vehicle) (n = 15-29/group), are expressed as mean ( SEM with statistical
significance determined by ANOVA with post-hoc testing or Fishers test, as appropriate. bp < 0.0001 vs (SCW vehicle). cp < 0.001 vs vehicle. dp < 0.05 vs vehicle. ep < 0.01 vs
(SCW vehicle). fp < 0.01 vs (SCW vehicle). gp < 0.01 vs vehicle.

are again consistent with the results we obtained previously in this

model when evaluating curcuminoid-only turmeric extracts (3).
Dose-dependent inhibition of arthritis was similar for the crude vs
refined TEO (ID50 approximately 10 mg equivalents of refined
TEO/kg/d) during the initial acute phase (day 3) of joint swelling
(Figure 2A), as well as the later, chronic phase of articular
inflammation (day 28) when erosion of articular cartilage and
bone occurs (Figure 2B). These data suggest that additional
components present only in the crude TEO, including, but not
limited to, the very low levels of curcuminoids, were not likely to
have additive or synergistic anti-arthritic effects with turmerics
purified essential oils. In totality, then, our data suggest that each
class of secondary metabolites in turmeric, the essential oils and
the curcuminoids, can act independently and with similar efficacy
and potency to block arthritis using this experimental model and
mode of delivery (ip).
In addition to examining the clinical end point of joint swelling,
inhibitory effects of TEO on articular inflammation were confirmed at the molecular level by assessing the activation of genes
critical to articular inflammation. The early induction of chemokines responsible for the intra-articular recruitment of inflammatory cells (the neutrophil chemokine, GRO/KC, and monocyte
chemokine, MCP-1) and of key inflammatory cytokines (IL1beta) was prevented by TEO treatment. Similarly, activation of
an additional arm of the innate immune response, antibodyindependent complement pathways (induced expression of mannose binding lectin and properdin) (26), and the inducible
expression of other downstream mediators of joint inflammation,
such as COX-2, were blocked in the joints of SCW-injected
animals treated with TEO (Table 1).
Extra-Articular Anti-Inflammatory Effects of Intraperitoneal
TEO Extracts. Anti-inflammatory effects of TEO were not
limited to the joints. TEO also blocked SCW-induced leukocytosis and the granulomatous inflammatory response that occurs
at sites of hepatic SCW deposition (4, 24) (Table 2). As with
arthritis inhibition, these extra-articular anti-inflammatory
effects were dose-dependent, occurring only with TEO doses
g28 mg/kg/day (data not shown). However, unlike the equipotent

Figure 3. Mortality in animals treated with ip TEO. Survival in all animals

(SCW or control) treated ip with g28 mg/mg/d refined (n = 62) or crude
TEO (n = 90) vs vehicle (n = 86) was analyzed by log-rank using
Kaplan-Meier survival curves. Mortality was significantly (p < 0.0001)
increased in animals treated ip with either crude or refined TEO extract as
compared with vehicle.

effects of refined and crude TEO on articular inflammation, the

crude TEO was more effective in blocking nonarticular inflammation (Table 2), suggesting that the additional components
present only in the crude extract were also bioactive with
independent and/or synergistic anti-inflammatory effects.
Toxicity Associated with Intraperitoneal TEO Extracts. Jointprotective and/or anti-inflammatory effects of ip TEO were
associated with an extremely high rate of mortality. Significant
numbers of normal and SCW-injected animals treated ip with
g28 mg/kg/d TEO began to die after 2 weeks of treatment
(Figure 3), resulting in a mortality rate of 20% (crude) or 36%
(refined) by the end of the month-long experimental period. In
contrast, no deaths occurred in animals (control or SCW-injected) treated with vehicle or lower TEO doses (e2.8 mg/kg/d)
that were without evidence of anti-inflammatory effects (data not

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Funk et al.

Figure 4. Hepatotoxicity and anemia associated with ip vs oral TEO. (A) Effects of ip treatment (g26 mg/kg/d) with crude TEO (n = 32) or refined TEO (n =
54) vs vehicle alone (n = 48) on serum levels of alanine aminotransferase (ALT) were determined in SCW or control animals surviving 23-32 days of
treatment. Means are not statistically different by ANOVA. (B) Effects of ip treatment with crude TEO (n = 18) or refined TEO (n = 37) vs vehicle alone (n = 29)
on hemacrit (HCT) were determined in control animals surviving 23-32 days of treatment. p < 0.001, crude vs vehicle. p < 0.01, refined vs vehicle. (C) Effects
of oral treatment (>560 mg/kg/d) with crude TEO (n = 34) or refined TEO (n = 5) vs vehicle alone (n = 23) on serum levels of alanine aminotransferase (ALT)
were determined in SCW or control animals surviving 28-32 days of treatment. p < 0.01, refined vs vehicle. (D) Effects of oral treatment (>560 mg/kg/d) with
crude TEO (n = 11) or refined TEO (n = 5) vs vehicle alone (n = 6) on HCT were determined in control animals surviving 28-32 days of treatment. p < 0.01,
crude vs vehicle.

Figure 5. Time course of adverse effects of ip treatment with refined TEO on liver and hematopoetic parameters. In normal female Lewis rats, the effect of daily
ip treatment with 28 mg/kg/d refined TEO vs vehicle was determined over a 23-day period. Animals (n = 3 vehicle and n = 4 TEO) were sacrificed at the
indicated times for the assessment of hematologic parameters (hematocrit (HCT), total white blood cell (WBC), and platelet (PLT) counts) and liver function
(serum levels of alanine aminotransferase) as well as gross necropsy, which was notable for peritonitis, beginning as early as day 8. Results are presented as
mean ( SEM.

shown). Notable findings in those animals that survived one

month of ip treatment with g28 mg/kg/d TEO included elevations
in serum alanine aminotransferase (ALT), consistent with hepatocellular damage, that was more notable in crude TEO survivors
(Table 2 and Figure 4A). In addition, control animals treated with
either TEO were anemic (Table 2 and Figure 4B), while the anemia
that occurs in SCW-injected animals, which is typically reversed
with effective anti-arthritic treatment (3) neither improved nor

worsened with anti-inflammatory doses of TEO (Table 2). In

contrast to these adverse effects, there was no evidence of damage
to the kidneys (Table 2), a primary site of clearance of terpenes and
their metabolites (27), in surviving animals.
To eliminate survivor bias and identify the time of onset of
TEO-induced anemia and hepatotoxicity, toxicity screening
was performed as a function of time in control animals treated
with the TEO extract causing the highest mortality (refined TEO,

Article
56 mg /kg/d ip). Anemia (Figure 5A) and elevations in ALT
(Figure 5B) began at 2 weeks, a time coincident with the onset of
mortality in arthritis treatment experiments (Figure 3), thus
suggesting possible causality. While elevations in circulating
leukocytes (WBC) (Table 2) and platelets (PLT) (data not shown)
were not detected in nonarthritic animals surviving one month of
TEO treatment, both of these indicators of systemic inflammation began to increase after 2 weeks of TEO administration
(Figure 5C,D), consistent with an inflammatory response to ip
administration of the essential oils.
Evidence of occult gastrointestinal bleeding as a cause of
anemia was sought in animals treated with g28 mg/kg/d TEO
vs vehicle. On random screening, occult blood was documented in
the stools of 38% of crude TEO-treated animals (n = 16, p < 0.05
vs vehicle (9% incidence, n = 22)) and 19% of those treated with
refined TEO (n = 32, p > 0.05 (nonsignificant)). Necropsies performed after 2-4 weeks of treatment with TEO g 28 mg/kg/d
(n = 2, crude TEO; n = 4, refined TEO) were notable for signs of
mild to moderate peritonitis, including evidence of a small
intestine perforation in one crude TEO treated animal. In contrast, vehicle-treated animals (n = 2) had no peritonitis, and
necropsies were only notable for granulomatous lesions in
the liver in those animals also injected with SCW. There was
no evidence of renal toxicity on necropsy. In sum, effective
anti-arthritic doses of both TEOs, when administered ip, were
also associated with peritonitis, gastrointestinal bleeding with
anemia, and hepatocellular damage.
Oral TEO Extract Dosing: Toxicity and Anti-Inflammatory
Effects. The above findings beg the question of whether the toxic
effects of both TEOs were specific to their ip route of administration. Additionally, it is possible that the anti-inflammatory
effects of TEO when administered ip were nonspecific and
simply due to TEO-induced peritonitis inducing tolerance to
the SCW (28). To address both questions, oral TEO dosing
studies were undertaken. TEO doses (560 mg/kg/d) 20-fold higher
than the lowest effective ip doses of crude or refined TEO were
arbitrarily chosen for testing since minimal information is available regarding oral absorption and biodisposition of essential
oils or their terpene components (27). No mortality (data not
shown), evidence of hepatocellular damage (Figure 4C), anemia
(Figure 4D), or occult gastrointestinal bleeding (n = 4-5 animals
screened per treatment) occurred after one month of daily oral
administration of crude or refined TEO. No peritonitis was seen
on necropsy (n = 1 animal per treatment). However, proteus bacteria were detected in the abdominal cavity of the crude TEO
treated animals examined, suggesting the possibility of an otherwise undetected intestinal perforation vs accidental specimen
contamination.
Having verified the relative absence of toxicity with oral (vs ip)
administration of TEOs, the oral anti-inflammatory activity of
crude TEO, the extract having the greatest range of articular and
extra-articular anti-inflammatory effects when administered ip,
was assessed in the SCW model. Oral treatment with crude TEO
(520 mg/kg/d), begun prior to SCW administration (Figure 6A) or
after the peak of acute joint inflammation (Figure 6B), decreased
joint swelling at every time point. This anti-inflammatory effect,
when analyzed on a daily basis, was only statistically significant at
the start of pretreatment (Figure 6A, day 1). However, assessment
of joint inflammation by analysis of the area under the curve
(AUC) during the entire course of TEO treatment demonstrated a
relatively modest but statistically significant 21% inhibition of
joint swelling in response to early or delayed oral crude TEO
treatment (p < 0.02 vs vehicle). In contrast to this joint protective
effect of oral crude TEO and in contradistinction to the articular
and extra-articular anti-inflammatory effects of this extract when

J. Agric. Food Chem., Vol. 58, No. 2, 2010

847

Figure 6. Effects of oral crude TEO extract on joint inflammation. Female

Lewis rats were injected on day 0 with vehicle or streptococcal cell wall
(SCW, 25 g/g body weight) to induce arthritis. Joint swelling in limbs was
assessed at indicated times and expressed as mean arthritic index (AI)
(scale 0-3/limb for total possible score of 16/animal) (n = 4-8 animals/
group). (A) Oral treatment with crude TEO (normalized to 560 mg refined
TEO/kg/d) or vehicle was begun 4 days prior to SCW injection and
continued daily until 10 days after SCW injection, at which time dosing
frequency decreased to 5 days per week. *, p < 0.05 for crude TEO (vs
vehicle). (B) Oral treatment with crude TEO (normalized to 560 mg refined
TEO/kg/d) or vehicle was delayed until 3 days after SCW injection and
continued daily until 10 days after SCW injection, at which time dosing
frequency decreased to 5 days per week.

administered ip, SCW-induced leukocytosis or hepatic granulomatous inflammation was unaltered by oral crude TEO (data
not shown).
In sum, oral crude TEO, at a dose that would correspond
to 5000 mg/d in humans after correcting for body surface
area (29), has a modest anti-inflammatory effect in rats that
was limited to the joints and appeared to be specific in that it
was not associated with evidence of significant concurrent
toxicity. However, the clinical relevance of this finding is unclear
given the magnitude of the dose required to achieve a relatively

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J. Agric. Food Chem., Vol. 58, No. 2, 2010

modest effect. One limitation of this study, then, is the absence

of an oral dose response. However, reported side effects in 2 of
9 human subjects treated orally with a 50-fold lower dose of TEO
in a prior human trial, including possible reactivation of tuberculosis (17), calls into question the safety and practicality of
administering TEO doses higher than or indeed equivalent to
those tested here.
Not specifically tested in these studies are possible additive or
synergistic joint-protective effects of TEO when combined with
curcuminoids, the other class of turmeric rhizome secondary
metabolites. Previous reports of enhanced bioavailability of
curcumin when administered orally with TEO terpenes (30)
suggest that the combined administration of these two secondary
metabolites of turmeric may be clinically useful because of matrix
effects independent of any separate biological activity of the
essential oils. However, it must also be stated that the safety of
long-term use of dietary supplements containing higher doses of
TEO and/or curcuminoids than those typically consumed with
culinary intake (e.g., 4 mg/kg/d curcuminoids or TEO in a typical
Indian diet, given the 3% yield of curcuminoids (4) or TEO from
dried rhizome) remains to be investigated. The toxic effects of
turmeric essential oils when administered ip in these studies
should remain a cautionary finding.
ABBREVIATIONS USED

ALT, serum alanine aminotransferase; ANOVA, analysis

of variance; COX, cyclooxygenase; d, day; GRO/KC, growthrelated oncogene/keratinocyte chemoattractant; HCT, hematocrit; HPLC, high pressure liquid chromatography; ip, intraperitoneal; MCP-1, monocyte-chemotactic protein; NF-B, nuclear
factor-B; PGE2, prostaglandin E2; PLT, platelet; RA, rheumatoid arthritis; SCW, streptococcal cell wall; SEM, standard error
of the mean; TEO, turmeric essential oils; TNF, tumor necrosis
factor; WBC, white blood cell.
Supporting Information Available: HPLC chromatograms
and other chemical analyses identifying major components of the
experimental TEO extracts. This material is available free of
charge via the Internet at http://pubs.acs.org.

Funk et al.

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Received for review September 21, 2009. Revised manuscript received
November 17, 2009. Accepted November 23, 2009. This work was
supported by the Office of Dietary Supplements (ODS) and the
National Center for Complementary and Alternative Medicine
(NCCAM of the NIH (5 P50 AT000474). The contents are solely the
responsibility of the authors and do not necessarily represent the official
views of the NCCAM, ODS or NIH.