Anti-Inflammatory Effects of the Essential Oils of Ginger (Zingiber officinaleRoscoe) in

Experimental Rheumatoid Arthritis

Janet L. Funk, a Je nnife r B. Frye, a Ja nice N. Oyar zo,a Jianlin g Che n,a H uapi ng Zha ng,b and Barb ara N. Ti mm er mann b

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The publis her's fi nal e dited versi on of this articl e is av ailable at Ph arm aNut rition

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Abstract

Graphical Abstract

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1. Introduction
Ginger (Zingiber officinale Roscoe, Zingiberaceae), a commonly used botanical in the United
States [1], is primarily known for its anti-emetic properties [2]. However, it has also been
used medicinally since antiquity as an anti-inflammatory [3-5]. In modern usage, particular
attention has focused on the cyclooxygenase (COX)-inhibiting effects of the gingerols,
phenolic compounds that are responsible for ginger's pungent taste, and their potential use in
treating inflammatory disorders such as arthritis [4,5]. Previously, we demonstrated potent
anti-arthritic effects of gingerol-containing extracts of ginger in an experimental model of
rheumatoid arthritis (RA) [6]. However, crude extracts containing both of ginger's secondary
metabolites, the gingerols and the essential oils, were even more potent in inhibiting joint
swelling than gingerols alone. Having previously demonstrated anti-arthritic effects of both 6

the phenolic and essential oil fractions of turmeric (Curcuma longa L, Zingiberaceae), a plant
that is botanically and chemically related to ginger [7-10], we postulated that the essential oils
of ginger could similarly be bioactive with respect to inhibition of joint inflammation and thus
contribute to ginger's potential anti-arthritic effects.
To test this postulate, studies were undertaken to examine the joint protective effects of the
isolated essential oils of ginger (GEO), secondary metabolites that are responsible for ginger's
characteristic aroma [11,12]. For these studies, the streptococcal cell wall (SCW)-induced
arthritis model of RA previously employed by our laboratory to test other ginger (and
turmeric) extracts was employed to facilitate comparisons with chemically-related extracts
[6-10]. In this model, the inflammatory reaction in response to streptococcal cell wall (SCW)
deposition within joints recapitulates the histopathology of RA; female Lewis rats develop an
initial, transient phase of joint swelling that is characterized by an influx of neutrophils and
other inflammatory cells (acute phase, days 0-5), followed by a recrudescence of joint
swelling that is associated with synovial hyperplasia and progressive destruction of
periarticular bone by the invading synovium (chronic phase, days 10-28) [6-10,13].
Additionally, classic granulomas form within the liver at sites of hepatic SCW deposition
[6,7,10,14], an inflammatory response that can be protective in certain settings, such as
pulmonary tuberculosis where invading bacilli are walled off within granulomas, thus helping
to quell the spread of infection [15,16].
In our previous studies, SCW-induced arthritis and granulomatous inflammation were each
more effectively blocked by a crude ginger extract containing GEO and gingerols as
compared to a gingerol-only fraction [6]. The crude extract almost completely prevented both
phases of joint swelling (93% and 97% inhibition of acute and chronic arthritis, respectively),
while the gingerol-only fraction was less effective (78% and 62% inhibition, respectively) [6].
The crude extract also blocked granulomatous inflammation by 76%, while the gingerol-only
fraction was without effect [6]. Therefore, effects of isolated GEO on joint inflammation and
the granulomatous hepatic response were tested here using the SCW model. In addition,
because estrogenic effects have been reported in vitro for certain monoterpenes present in
GEO [17,18], in vivo treatment effects of GEO in the SCW model were compared to those of
17-β estradiol (E). While joint protective effects of estrogen have previously been reported in
2

pre-clinical RA models and have been postulated for RA itself due to the clinical observation
of improved disease activity during pregnancy [19], effects of estrogen in the SCW-model in
female rats have, to our knowledge, not previously been reported. Go to:

2. Material and Methods

2.1. Preparation of GEO

A crude ginger extract was prepared as previously described by extracting ground ginger
rhizome (2500g) with CHCl (dichloromethane) at 25°C for 36 h (6.4% yield) [6,12]. After
2 2

filtration, washing and work up, 40 g of the resultant extract (“crude ginger extract’) were
applied to a silica gel column and sequentially eluted with solvents of increasing polarity to
yield fractions 1 through 11, which were chemically characterized by HPLC (Figure 1C)
and/or GC-MS and screened in vitro for their ability to inhibit PGE production from an
2

LPS-stimulated human macrophage cell line, as previously described [6,12]. For the studies
reported here, fraction 1, a lipophilic, sesquiterpene-containing gingerol-free fraction was
used (23% yield, “GEO”; Figure 1A) [12]. In previous SCW experiments, essential oil-free
fractions (fractions 4-9) containing gingerols and their derivatives, as identified by HPLC and
GC-MS, were combined to constitute a single “gingerol fraction” (Figure 1B) (approximately
50% yield) that was used for in vivo testing [6,12].
Figure 1

HPLC-UV p rofiles (λ = 250 nm ) of G EO (A ), a gingero l-only fraction (B), and the cr ude DCM extract from which the GEO and gingerol fractions were de rived (C). The 3 majo r ginger ols (1, [6 ]-gi ngerol; 2, [8] -gingero l; 3, [10 ]-g ingerol ) and a prima ry gingero l degradation p roduct (4, [6 ]-s hogaol) a re not pres ent in the G EO f raction.

2.2. Animal Procedures

Animal studies were performed in accordance with institutional guidelines using approved
protocols. Using the identical protocol previously described for assessment of other ginger
extracts [6], female Lewis rats (Harlan, Indianapolis, IN) were administered a single
intraperitoneal (i.p.) injection of vehicle (normal saline) or peptidoglycan-polysaccharide
polymers (25 μg rhamnose/g body weight) isolated from the sonicated cell wall of Group
A Streptococcus pyogenes (Lee Laboratories, Grayson, GA). At the indicated times, control
and streptococcal cell wall (SCW)-treated animals received an intraperitoneal injection of
botanical sample or vehicle (0.5-1 μL/g DMSO) [6]. Intraperitoneal treatments with the GEO
extract or vehicle (1 ul/g DMSO) were administered as in our previous studies beginning 4
days prior to SCW administration and continuing daily until the beginning of the chronic
phase (day 14), when treatment frequency was decreased to five days per week [6]. GEO was
dosed at 28 mg/kg/d in order to: 1) facilitate comparison with the in vivo potency of isolated
gingerols, which were dosed at 25 mg/kg/d [6], and to 2) approximate the GEO dose received
by rats treated with the crude ginger extract in prior experiments (containing approximately
32 mg/kg/d GEO [and 25 mg/kg/d gingerols]) [6]. In separate experiments, in order to
compare the anti-inflammatory effects of GEO to those of estrogen, rats were treated
beginning 4 days prior to SCW injection with 17-β estradiol (E, Sigma, St. Louis, MO) using
2

pharmacologic doses with demonstrated anti-arthritic efficacy in other rat arthritis models
[20-22] that also normalize bone parameters and prevent uterine atrophy in ovariectomized
rats [23]25] (200 μg/kg or 600 μg/kg subcutaneously five times a week, as indicated, vs.
vehicle alone [1 μl/g sesame oil]). Joint inflammation was determined in a blinded fashion by
daily assessment of arthritic index (AI) in each distal limb using standard criteria (0 = normal;
1 = slight erythema and edema; 2 = increased edema with loss of landmarks; 3 = marked
edema; 4 = marked edema with ankylosis on flexion) [19,20,23,24]. Bone mineral density
(BMD) of the total femur was determined using a Piximus densitometer (GE Lunar, Madison,
WI) at end of experiment (days 28-30) as previously described [8]. To monitor for possible
toxic effects of treatments in normal or SCW-treated animals, daily weights were recorded,
and serum creatinine and alanine aminotransferase (ALT) levels in blood samples obtained 28
days after injection of SCW (or vehicle) were determined using a Hemagen Diagnostics
Endocheck Plus Chemistry Analyzer to monitor for possible renal- or hepatotoxicity,
respectively [6,8]. Circulating white blood cell counts and hematocrit in whole blood were
assayed using a Hemavet 880 analyzer (CDC Technologies, Oxford, CT), with manual
determination of differential WBC counts [6,8,25].

2.3. Histology
Liver specimens obtained 28 days after SCW injection were fixed in 10% formalin and
embedded in paraffin [6,7,10,14]. Hepatic granuloma formation was assessed in hematoxylin
and eosin (H&E) stained sections of liver in a blinded fashion using standard criteria
[6,7,10,14].

2.4. Statistical Analyses

All values are presented as mean ± the SEM, unless otherwise stated, with statistical
significance determined by ANOVA or non-parametric Kruskal-Wallis analysis with post hoc
testing, non-parametric Mann Whitney analysis, or Fisher's Exact Test, as appropriate, using
Instat 3.0b software (Graphpad, San Diego, CA). Go to:

3. Results

3.1. Isolation and characterization of GEO

Fraction 1, a lipophilic sesquiterpenoid-containing essential oil fraction isolated as described
in the methods from a crude ginger extract (23% yield), was gingerol-free, as determined by
HPLC (“GEO”, Figure 1A). The HPLC profile of the GEO can be compared to that of a
previously tested: 1) GEO-free fraction containing gingerols and their derivatives (“gingerol
fraction” [50% yield], 38% by weight of the 3 most common gingerols, Figure 1B) [6], or 2)
the crude ginger extract itself (“crude extract”, 18% by weight of the 3 most common
gingerols, Figure 1C) [6]. The IC for inhibition of PGE production in vitro by the GEO
50 2

fraction (IC = 0.411 μg extract/ml) was 20-fold lower than those previously reported for the
50

gingerol fraction or gingerol-containing crude ginger extract (IC = 0.06-0.07 μg extract/ml or

50

0.01-0.02 μg gingerols/ml) [6].

3.2. In vivo effect of ginger essential oils (GEO) on joint inflammation

A GEO dosing scheme (28 mg/kg/d beginning 4 days prior to SCW injection) similar to that
previously used for the crude extract (25 mg gingerols/kg with GEO ≈ 32 mg/kg/d) or
gingerol fraction (25 mg gingerols/kg/d) was employed to explore whether the excess joint
protection previously documented with the crude (vs. gingerol only) extract in SCW-induced
arthritis could be attributable to bioactivity of its GEO content [6]. Acute joint swelling in
SCW-injected rats was unaltered by GEO treatment (Figure 2A, day 3). In contrast, GEO
treatment significantly inhibited the chronic phase of arthritis (days 13-28), decreasing joint
swelling by 38% on day 28 (Figure 2A).
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Figure 2

Effects of G EO or E2 on joint in flammation . Female Lewis rats were i njected on day 0 with SCW (or veh icle) to induce ar thrit is with daily G EO, E2, or vehicle t reatments s tarting 4 days prio r to SCW in jection as des cribed in M ethods and M aterials . J oint s welling in limbs o f SCW-injected rats was as s es s ed at times indicated and expres s ed as mean arthritic index (A I, mean ± SEM ) ) (s cale 0-4/li mb fo r total pos s ible s core of 16 ) with s tatis tical s igni ficance as s ess ed by ANOVA with M ann Whitney analys is . *p < 0.0 5, treated vs . vehicle cont rol. A. G EO (28 m g/kg, n = 19) o r vehicle alone (DM SO, 1 μl/g, n = 18) were dos ed ip daily 5 -7 times a week. B. E2 (200 μg/kg , n = 9 ) or vehicle alone (s es ame s eed oil, μl/g, n = 9) we re dos ed s c daily 5 times a week. C. E2 (600 μg/kg, n = 9) o r vehicle alone (s es ame s eed oil, μl/g, n = 9 ) were dos ed s c daily 5 times a week.

3.3. In vivo effect of GEO on hepatic granuloma formation

In previous experiments, the crude ginger extract decreased the incidence of granuloma
formation at site of SCW deposition in the liver by 76% while gingerol-only extracts were
without effect [6], suggesting that blockade of granuloma formation by the crude extract
might be attributable to its the GEO content. However, treatment of SCW-injected animals
with GEO in isolation did not alter the granulomatous response; the incidence of granuloma
formation in the livers of SCW-injected rats was unchanged in GEO vs. vehicle-treated
animals (Figure 3A).
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Figure 3

Effects of G EO or E2 on incidence of hepatic granu loma fo rmation. Female Lewis rats were injected on day 0 with SCW (or vehicle ) to induce arth ritis with daily GEO, E2, o r vehicle treatments s tarti ng 4 days prio r to SCW injectio n as des cribed in M ethods and M aterials . The incidence of granu loma fo rmation on day 28-30 was as s es s ed his tologically as des cribed in SCW-injected animals . Statis tical s ignificance was determined by Fis her's exact tes t. NS = non-s igni ficant. A. G EO (28 mg/kg, n = 8) o r vehicle alone (DM SO, 1 μl/g, n = 8) were dos ed ip dai ly 5-7 t imes a week. B. E2 (200 μg/kg or 600 μg/kg, n = 12) o r vehicle alone (s es ame s eed oil, μl/g, n = 10) were dos ed s c daily 5 times a week. The E2 dos es tes ted did not alter g ranuloma incidence when analyzed s eparately (data no t s hown) or in combinati on, as demons trated here.

3.4. Tolerability of GEO

Mortality, which only occurred in SCW-injected animals, was not altered by GEO (vs.
vehicle) treatment in control or SCW-injected rats (Table1). GEO treatment did not alter body
weight, which tends to decrease in SCW-treated rats, in control or SCW-injected rats (Table
1). Circulating leukocyte counts, which are elevated in SCW-injected rats, were unchanged by
GEO treatment in control or SCW-injected rats (Table 1). Neutrophil, monocyte and
lymphocyte counts were similarly unaltered by GEO treatment in control or SCW-injected
animals (Table 1). Blood hematocrit values, which decrease in SCW-injected animals, were
unchanged by GEO treatment in SCW-injected or control animals (Table 1). Hepatic function,
which remained normal in SCW-injected animals as assessed by ALT, was unaltered by GEO
treatment in control or SCW-injected animals (Table 1). Creatinine levels, which were
unchanged by GEO treatment in control animals, were slightly but statistically increased in
SCW-injected animals treated with GEO, a change that is of questionable physiologic
significance given its small magnitude (Table 1).

Table 1

Tolerability of GEO in Normal and Arthritic Rats

Toxi Con GEO SCW SCW + GEO

city tro

Moni l

tori (ve

ng hic

le)

Mort 0% 0% 4% (1/23) 7% (2/29)

alit (0/9 (0/5

y ) )

(num

ber

died

/tot

al)

Body 159. 158. 146.7 ± 3.5 143.3 ±

Weig 7 ± 6 ± 2.5

ht 4.6 6.8

a
(g)

ALT 17.6 13.9 13.4 ± 1.6 13.1 ± 2.5

(U/L ± ±

) 3.0 3.3

Crea 0.24 0.26 0.28 ± 0.02 0.34 ±

tini ± ± 0.02

ne 0.02 0.05

a
(mg/

dL)

COMP

LETE

BLOO

COUN

WBC 9.1 10.2 18.04 ± 18.2 ±

(K/ ± ± 1.5 2.0

μl) 1.3 1.9

bb
Neut 1.6 2.9 8.0 ± 7.2 ±

roph ± ± 1.2 1.0

ils 0.5 1.1

bb
(K/
T oxi Con GEO SCW SCW + GEO

city tro

Moni l

tori (ve

ng hic

le)

μl)

Lymp 6.5 6.9 9.1 ± 0.7 10.1 ± 1.2

hocy ± ±

tes 1.0 0.9

(K/

μl)

Mono 0.24 0.35 0.90 ± 1.21 ±

cyte ± ± 0.09 0.21

s 0.03 0.07

ac
(K/

μl)

Hema 44.9 39.9 34.7 ± 34.3 ±

tocr ± ± l.2 1.2

it 1.3 4.0

bb
(%)

Values are expres s ed as mean ± SEM with s tatis tical s ignificance of dif ferences between all groups determi ned by ANOVA with pos t -hoc tes ting or Fis her's exact tes t, as appropriate.

ap < 0.05 vs . vehicle

bp<0.01 vs . vehicle

cp<0.001. vs . vehicle.

3.5. In vivo effect of 17ß estradiol on joint inflammation and hepatic granuloma formation
Using the same dosing strategies as for GEO (treatment beginning 4 days prior to SCW
injection), treatment with 200 ug/kg E , a dose that normalizes BMD and prevents uterine 2

atropy in OVX rats [23], had no effect on acute joint swelling (Figure 2B, day 3) but
decreased chronic joint inflammation by 31% (Figure 2B, day 28) in ovary-intact
SCW-injected female rats. A three-fold higher E dose (600 ug/kg) had exactly the same effect 2

(Figure 2C); the initial phase of joint swelling was unchanged (Figure 2C, day 3) while
chronic inflammation was significantly decreased (Figure 2C, day 28). The magnitude of E's 2

inhibitory effect on chronic joint inflammation was the same for both doses, blocking joint
swelling by one third (Figures 2B and 2C), suggesting that this was the maximal achievable
anti-arthritic effect. Hepatic granuloma formation was unaltered by treatment with either dose
of E (Figure 3B). 2

3.6. Comparative effects of GEO and 17-ß estradiol on classic estrogen-responsive

tissues
Because GEO effects on joint and granulomatous inflammation in the SCW model mirrored
those of E, the effects of both treatments on classic estrogen-responsive tissues were 2

compared to explore the possibility that in vivo effects of GEO could be attributed to ER
agonist activity. In non-arthritic control animals, femoral BMD was unchanged after one
month of GEO treatment (Figure 4A, GEO vs. control). In contrast, E treatment of the same 2

duration at low (200 ug/kg/d) or high doses (600 mg/kg/d E ) increased BMD by 4% or 10%, 2

respectively (Figure 4B&C, control vs. E2). Femoral BMD in arthritic SCW-injected animals,
which decreased by 28-37% as compared to non-arthritic control animals in these
experiments, was unaltered by GEO (Figure 4A, SCW vs. SCW + GEO) but was significantly
increased (11%) by treatment with either dose of E (Figure 4B&C, SCW vs. SCW + E).
2 2
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Figure 4

Effects of G EO or E2 on femoral BM D. Female Lewis rats were injected on day 0 wi th vehicle (cont rol) or SCW to i nduce arthri tis with daily G EO, E 2, or vehicle t reatments s tarting 4 days pr ior to SCW injection as des cribed in M ethods and M aterials . Femoral BM D (mean ± SEM , n = 8-36 /group ) was as s ess ed at time of s acrifice on day 28-3 0 us ing a Pixi mus dens itometer as des cribed in M aterials and M ethods . Statis tical s ignificance was determined by ANOVA w ith pos t-hoc tes ting. * p < 0.05 vs . control. ** p < 0.01 vs . SCW. *** p < 0.001 vs . cont rol or SCW, as indicated. NS = non s ign ificant. A. G E O (28 mg/kg ) or vehicle alone (DM SO, 1 μl /g) were dos ed ip dai ly 5-7 t imes a week. B. E2 (200 μg/kg) o r vehicle alone (s es ame s eed oil, μl/g) we re dos ed s c daily 5 times a week. C. E2 (600 μg/kg) or vehicle alone (s es ame s eed oil, μl/g) were dos ed s c daily 5 times a week.

In ovary intact female rats injected with SCW, uterine weights tended to decrease slightly, a
trend of small magnitude, compared to OVX rats [23,26], that achieved statistical significance
in certain experiments (Figure 5A and B, SCW vs. control). Uterine weights, while lower in
GEO treated animals, were statistically unchanged by GEO treatment in SCW-injected rats
(Figure 5A). In ovary–intact SCW-injected animals treated with a 17-β estradiol dose that
prevents uterine atrophy in OVX animals (200 ug/kg/d) [23], an opposing trend towards
increased uterine weight did not achieve statistical significance. To more clearly assess
possible in vivo estrogenic effects of GEO on the uterus, effects of GEO on uterine weights
were determined in ovariectomized (OVX) Lewis rats [26]. Uterine weights in OVX Lewis
rats, which decreased to 11% of sham controls after one month (sham, 3.84 ± 0.37 mg
uterus/kg body weight vs. OVX, 0.43 ± 0.03 mg/kg), were unaltered by one month of GEO
treatment using the same dosing scheme as in the arthritis experiments (OVX + GEO, 0.55 ±
0.06 mg/kg body weight, p > 0.05 vs. OVX).
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Figure 5

Effects of G EO or E2 on u terine weights . Female Lewis rats were injected on day 0 w ith vehicle (cont rol) or SCW to induce arthr itis with daily G EO, E2, or vehicle t reatments s tarting 4 days p rior to SCW injection as des cribed in M ethods and M aterials . Uterine weights , expres s ed as a ratio of body we ight (m g uterus /kg bod y weight, average ± SEM with n = 8-1 3/group ), were as s es s ed at time of s acrifice on day 28-30. Statis tical s ignif icance was determined by ANOVA with pos t- hoc tes ting. ** p < 0 .01 vs control. * ** p < 0.00 1 vs control. N S = non s igni ficant. A. G EO (28 m g/kg) o r vehicle alone (DM SO, 1 μl/g) were d os ed ip daily 5-7 times a week. B. E2 (200 μg/kg) or vehicle alone (s es ame s eed oil, μl/g) were dos ed s c daily 5 times a week.

Consistent with previous reports, lymphocyte counts were decreased in estrogen-treated

animals [27], an effect that achieved statistical significance in SCW-injected animals treated
with either E dose (Figure 6B and C). This effect was lymphocyte-specific as total white 2

counts, which are primarily elevated in SCW-injected animals due to increased neutrophils
(Table 1), were unchanged by E-treatment (data not shown). In contrast, lymphocyte counts
2

in SCW-injected animals treated with GEO tended to be higher, but were statistically
unchanged (Figure 6A).
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Figure 6

Effects of G EO or E2 on ci rculating ly mphocyte counts . Female Lewis rats were injected on day 0 wi th vehicle (cont rol) o r SCW to in duce arthri tis with daily G EO, E 2, or vehicle t reatments s tarting 4 days pr ior to SCW injection as des cribed in M ethods and M aterials . Lymphocy te counts (mean ± S EM , n = 4 -16/g roup) we re as s es sed at time of s acrifice on day 28 -31 as des cribed in M aterials and M ethods . Statis tical s ignificance was determined by ANOVA wi th pos t-hoc tes ting. * p < 0.05 vs . cont rol or SCW, as indica ted. ** p < 0.01 vs . control o r SCW, as indicated. NS = n on s ignificant. A. G EO ( 28 mg/k g) or vehicle alone (DM SO, 1 μl/g ) were dos ed ip daily 5-7 ti mes a week. B. E2 (200 μg /kg) o r vehicle alone (s es ame s eed oil, μl/g) were d os ed s c daily 5 times a week. C. E2 (600 μ g/kg) o r vehicle alone (s es ame s eed oil, μl/g) we re dos ed s c daily 5 times a week.

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4. Discussion
Previous arthritis studies by our laboratory demonstrating a greater joint protective effect of a
crude ginger extract containing gingerols and GEO as compared to gingerols alone (when
normalized to gingerol content) suggested that the crude extract's GEO content could account
for its enhanced anti-inflammatory effect in an experimental model of rheumatoid arthritis
(RA) [6]. Consistent with this hypothesis, isolated administration of GEO in the experiments
described here did significantly reduce joint swelling in experiments using the same
SCW-induced model of RA. However, unlike the protective effects of gingerol-containing
extracts, which prevented both early and late manifestations of joint inflammation [6], GEO
treatment only offered protection during the later chronic, joint-destructive phase of arthritis.
With respect to protection during the later stages of arthritis in this model, the totality of
experimental findings suggests that a possible additive effect of GEO (38% inhibition) when
combined with gingerols (62% inhibition) [6] could explain the increased effectiveness of the
GEO- + gingerol-containing crude extract, which blocked chronic joint inflammation by 97%
when administered at a dose delivering similar amounts of each of the secondary metabolites
[6]. The complete lack of effect of GEO in limiting acute joint inflammation suggests a
possible divergence in the mechanism of GEO's joint protective effects as compared to the
gingerols, whose anti-arthritic efficacy was, in fact, greater during acute (vs. chronic) arthritis
[6].
Previously, to our knowledge, only two interventions have been reported to prevent chronic
(but not acute) joint inflammation in the SCW-model: 1) depletion/inactivation of T cells
[28,29], or 2) treatment with human chorionic gonadotropin (hCG), which protects via an
unknown mechanism [30]. In the SCW model, T cells are required for generating the chronic
(but not acute) recrudescence of joint inflammation in the SCW model and are also key
mediators of the granulomatous response at sites of SCW deposition in the liver; both of these
inflammatory responses are inhibited by T cell depletion/inactivation [14,28,29]. In contrast,
GEO inhibited chronic joint inflammation but had no effect on granulomatous inflammation
in the SCW model; thus, it is unlikely that the joint protective effects of GEO can be
attributed to in vivomodulation of T cell function.
While neither GEO- nor gingerols-only [6] altered granulomatous inflammation in the SCW
model, the crude ginger extract profoundly inhibited this response [6]. There are two possible
explanations for this constellation of findings: 1) either a combination of GEO + gingerols is
required for inhibition of granulomatous inflammation, or 2) additional chemical moieties,
such as the polar polysaccharides that are also uniquely present in the crude extract (e.g.
column chromatography fraction 11) [12], mediate this anti-inflammatory effect. Support for
the later explanation can be found in our previous studies examining the effects of turmeric
extracts in this same model of RA; only turmeric extracts containing polar compounds were
capable of inhibiting granulomatous inflammation [7,10]. Immunomodulatory effects of
polysaccharide moieties have previously been reported in other settings [31-33]. In the
context of RA, where reactivation of tuberculosis has been reported in response to certain
anti-inflammatory treatments [16], inhibition of the protective granulomatous response by
ginger- (or turmeric-) derived polysaccharides could similarly result in untoward clinical
effects, making polysaccharide-containing ginger extracts less desirable.
The joint protective effects of essential oils derived from ginger, which modestly (38%)
inhibited chronic joint inflammation, are very different from those previously reported by our
laboratories for the essential oils of turmeric, which profoundly blocked joint inflammation
throughout the entire course of disease (90-100%) [10]. Sesquiterpenes are major constituents
of the essential oils of turmeric (ar-turmerone, α-turmerone and ß-turmerone) and ginger
10

(zingiberene and ar-curcumene) [34-43], while the monoterpene isomers, neral and geranial,
collectively referred to as citral, are only present in GEO and comprise a variable but
significant amount by weight (16-75%) [35,37,39-43]. Because estrogenic effects have been
reported previously for citral in vitro [17,18], including low affinity estrogen receptor (ER)
binding and estrogenic activity, the in vivo effects of GEO were compared to those of
17ß-estradiol in the SCW model. The anti-inflammatory effects of GEO vs. estradiol were
identical; both provided a similar magnitude of protection against chronic (but not acute) joint
inflammation while leaving hepatic granuloma formation untouched. This similarity in
anti-inflammatory activity suggests that joint protection by GEO could be attributable to
ER-mediated effects of its monoterpene moieties. Running contrary to this postulate, however,
GEO treatment in SCW or OVX rats did not recapitulate estrogen's effects on classic estrogen
target tissues; GEO did not enhance uterine weight in intact or OVX rats, increase bone
density or suppress lymphocyte number. This lack of GEO effect in vivo on classic estrogen
target organs mirrors that reported in vivo for purified citral [18], an outcome that could be
explained by the ≥10,000-fold lower ERα and ERß binding affinity of these monoterpenes as
compared to E itself [18]. At the same time, the possibility that GEO could be acting as a
2

selective estrogen receptor modulator (SERM), having tissue specific effects that allowed for
estrogenic effects in inflamed joints, but not uterus or bone, cannot be discounted by these
data [44].
In conclusion, the totality of our experimental studies exploring the anti-inflammatory effects
of ginger extracts containing one or both of ginger's secondary metabolites, suggest that the
enhanced anti-arthritic efficacy of chemically complex ginger extracts (vs. gingerol-only or
essential oil-only fractions) [6] is likely due to additive effects of both classes of secondary
metabolites present in the crude extract, the gingerols and essential oils, while its ability to
block granulomatous inflammation is likely attributable to the polar compounds that are also
present in the crude extract. Because reactivation of tuberculosis due to inhibition of
granulomatous inflammatory processes can be an unwanted side effect of anti-inflammatory
arthritis treatments [16], these findings suggest that a ginger-derived extract that contains
joint-protective gingerols and essential oils but lacks polysaccharides and other polar
compounds, may have the best efficacy and safety profile for use in arthritis. While we cannot
rule out the possibility that ginger's essential oils may be acting as phytoestrogens in blocking
joint inflammation in this model due to the similarly of their anti-arthritic effects with those of
estrogen, the complete lack of evidence of classic in vivo effects of GEO in other
estrogen-responsive tissues makes this less likely, unless GEO have SERM-like properties. In
this event, a careful evaluation of potential adverse (estrogenic) effects of GEO on mammary
tissue would also be of great importance prior to clinical use given the possible breast cancer
promoting effects of estrogens [44].
One major limitation of the studies presented here is the use of an ip (not oral) dosing strategy
to facilitate a comparison of the bioactivity of different ginger (and turmeric) constituents
independent of differences in their oral bioavailability. While turmeric, one of the top selling
herbs in the United States [45], has been frequently proposed and studied as an anti-arthritic
agent [46,47] and patients will RA are frequent users of alternative treatments [48], much less
is known about the pharmacokinetics and potential anti-arthritic benefits of ginger. Human
studies have demonstrated good tolerability following oral administration of up to 2 g/d of a
hydroalcoholic ginger root extract containing 5% gingerols (rat equivalent dose of 12 mg
gingerols/kg/d) and an unknown quantity of essential oils [49], suggesting that the gingerol
doses evaluated in our previous experimental arthritis studies utilizing crude and essential oil
depleted extracts are clinically relevant [6]. Oral doses of GEO up to 500 mg/kg/d in rats have
been demonstrated to be well tolerated with anti-inflammatory effects (human equivalent dose
of 4 g/d) [50,51]. While oral doses of GEO were not tested here, oral doses of turmeric
essential oils in a similar dosing range (560 mg/kg/d) were well tolerated with anti-arthritic
efficacy in the SCW model, although their protective effect was much more attenuated as
compared to ip dosing [10]. Clinical studies evaluating the anti-arthritic efficacy of ginger
preparations in RA are completely lacking, while a small number of studies in osteoarthritic
patients evaluating poorly characterized extracts containing ginger, alone or in combination
with other herbs, report biological responses [52-55]. The studies presented here suggest that
clinical investigation of the utility and safety of ginger dietary supplements to quell joint
disease in inflammatory arthritis may be warranted, but should clearly include careful
consideration and characterization of the chemical content of the products to be tested.