Author’s Accepted Manuscript

The hidden mechanism beyond ginger (Zingiber

officinale Rosc.) potent in vivo and in vitro anti-
inflammatory activity

Shahira M. Ezzat, Marwa I. Ezzat, Mona M. Okba,

Esther T. Menze, Ashraf B. Abdel-Naim
www.elsevier.com/locate/jep

PII: S0378-8741(17)32876-3
DOI: https://doi.org/10.1016/j.jep.2017.12.019
Reference: JEP11150
To appear in: Journal of Ethnopharmacology
Received date: 31 July 2017
Revised date: 7 December 2017
Accepted date: 14 December 2017
Cite this article as: Shahira M. Ezzat, Marwa I. Ezzat, Mona M. Okba, Esther T.
Menze and Ashraf B. Abdel-Naim, The hidden mechanism beyond ginger
(Zingiber officinale Rosc.) potent in vivo and in vitro anti-inflammatory activity,
Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2017.12.019
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The hidden mechanism beyond ginger (Zingiber officinale Rosc.) potent
in vivo and in vitro anti-inflammatory activity
Shahira M. Ezzata,b*, Marwa I. Ezzata, Mona M. Okbaa, Esther T. Menzec, Ashraf B.
Abdel-Naimd
a
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Ainy
Street, Cairo 11562, Egypt.
b
Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Science
and Arts (MSA), 6th October, 12566 Egypt
c
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams
University, Cairo 11566, Egypt
d
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz
University, Jeddah, Saudi Arabia

*
Corresponding author, Shahira M. Ezzat, Professor, Department of Pharmacognosy,
Faculty of Pharmacy, Cairo University, Kasr-el-Aini street, 11562 Cairo, Egypt. Tel.:
+201000781836. shahira.ezzat@pharma.cu.edu.eg

Abstract
Ethnopharmacological relevance
Ginger (Zingiber officinale Roscoe) is a well known anti-inflammatory drug in the
Egyptian, Indian and Chinese folk medcines, yet its mechanism of action is unclear.
Aim of the study
to explore its mechanism of action and to correlate it to its biophytochemicals.
Materials and methods
Various extracts viz. water, 50%, 70%, 80%, and 90% ethanol were prepared from ginger
rhizomes. Fractionation of the aqueous extract (AE) was accomplished using Diaion HP-
20. In vitro anti-inflammatory activity of the different extracts and isolated compounds
was evaluated using protein denaturation inhibition, membrane stabilization, protease
inhibition, and anti-lipoxygenase assays. In vivo anti-inflammatory activity of AE was
estimated using carrageenan-induced rat paw oedema in rats at doses 25, 50, 100 and 200
mg/kg b.wt.

1
Results
All the tested extracts showed significant (p< 0.1) in vitro anti-inflammatory activities.
The strongest anti-lipoxygenase activity was observed for AE that was more significant
than that of diclofenac (58% and 52%, respectively) at the same concentration (125
μg/ml). Purification of AE led to the isolation of 6-poradol (G1), 6-shogaol (G2); methyl
6- gingerol (G3), 5-gingerol (G4), 6-gingerol (G5), 8-gingerol (G6), 10-gingerol (G7),
and 1-dehydro-6-gingerol (G8). G1, G2 and G8 exhibited potent activity in all the
studied assays, while G4 and G5 exhibited moderate activity. In vivo administration of
AE ameliorated rat paw edema in a dose-dependent manner. AE (at 200 mg/kg) showed
significant reduction in production of PGE2, TNF-α, IL-6, monocyte chemoattractant
protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted
(RANTES), myeloperoxidase (MPO) activity by 60, 57, 60, 41, 32 and 67%,
respectively. AE at 100 and 200 mg/kg was equipotent to indomethacin in reduction of
NOx level and in increasing the total antioxidant capacity (TAC). Histopathological
examination revealed very few inflammatory cells infiltration and edema after
administration of AE (200 mg/kg) prior to carrageenan.
Conclusions

Ginger anti-inflammatory activity is mediated by inhibiting macrophage and neutrophils

activation as well as negatively affecting monocyte and leukocyte migration. This was
evidenced by the dose-dependent decrease in pro-inflammatory cytokines and
chemokines and replenishment the total antioxidant capacity.

Graphical abstract

2
Keywords

Anti-lipoxygenase activity, inflammatory markers, 1-dehydro-6-gingerol, 6-shogaol.

1. Introduction
Ginger (Zingiber officinale Roscoe; family Zingiberaceae) is a monocotyledonous, which
is native to India or Southeast Asia, from where it was introduced to other parts of the
world (Mabberley, 1997; Ravindran, et al., 1994). Both fresh and dried ginger rhizomes
are used worldwide as a spice, ginger extracts are used extensively as beverage, and in
confectionary industries in the production of marmalade, pickles, chutney, liquors,
biscuits, and other bakery products (Wohlmuth et al., 2005). The unique flavor properties
of ginger arise from the combination of pungency of its oleoresin and its aromatic
essential oil (Tyler, 1993).
The main pungent compounds in fresh ginger are a series of homologous phenolic
ketones known as gingerols. The major gingerol is [6]-gingerol, while [8]- and [10]-
gingerol occur in smaller quantities. The gingerols are thermally unstable and are
converted under high temperature to [6]-, [8]-, and [10]- shogaol (He et al., 1998).

3
Shogaols, which are more pungent than gingerols, are the major pungent compounds in
the dried ginger rhizome

In Chinese and Unani-Tibb systems of medicine, ginger is used to treat catarrh,

rheumatism, nervous diseases, gingivitis, toothache, painful menstruation, asthma, stroke,
constipation, diabetes and migraine (Mustafa and Sirivastava, 1990). In Asian medicine,
it is used as a carminative (digestive aid), stimulant, diuretic and anti-emetic (Tyler,
1993). Chronic inflammation has been associated with a number of human diseases
including chronic obstructive pulmonary disease, asthma and rheumatoid arthritis. While
‘‘conventional’’ treatments have met with some success, patients suffering from diseases
with associated chronic inflammation are turning to alternatives for relief of their
symptoms or as prophylactic treatments. These alternatives include dietary supplements
that are purported to have anti-inflammatory actions. However, the efficacy and potency
of these supplements have not been studied in great detail. Plants (or supplements derived
from the plants) that have received attention as being useful for chronic inflammation
include ginger (Wohlmuth et al., 2005). Several studies have indicated that compounds
found in ginger are effective in relief of symptoms from chronic inflammatory diseases.
Administration of ginger has resulted in decreased symptoms of rheumatoid arthritis
(Srivastava and Mustafa, 1992). Gingerols have been reported to cause suppression of
both cyclooxygenase and lipooxygenase metabolites and arachidonic acid (Kiuchi et al.,
1992; Srivas, 1984; Tjendraputra et al., 2001).
Inflammation is associated with a large range of mediators that initiate the
inflammatory response, recruit and activate other cells to the site of inflammation and
subsequently resolve the inflammation (Gallin and Snyderman, 1999). In general, the
chemical mediators can be divided into two large classes: cytotoxins and arachidonic acid
metabolites. Products produced by the metabolism of arachidonic acid include both
cyclooxygenase products (prostaglandins, thromboxanes) and lipooxygenase products
(leukotrienes). The target of our study is to search for the mechanism of the anti-
inflammatory potential of the aqueous extract of Ginger rhizome and to correlate it to its
biophytochemicals through a bioguided fractionation protocol using various in vitro and
in vivo antiinflammatory experiments.

4
2. Material and Methods
2.1. General

Silica gel 60 (70 - 230 mesh ASTM; Fluka, Steinheim, Germany) and Diaion HP-20 AG
for column chromatography (75-150 µm, Mitsubishi Chemical Industries Co. Ltd).
Thin-layer chromatography (TLC) was performed on silica gel GF254 precoated plates
(Fluka) using the following solvent systems: S1: n-Hexane-acetone (8: 2 v/v); S2;
dichloromethane-methanol (9: 1 v/v); S3; dichloromethane-methanol (8.5: 1.5 v/v). The
chromatograms were visualized under UV light (at 254 and 366 nm) before and after
spraying with 10% H2SO4 spray reagent. The mass of the compounds were detected
from m/z 100 to 1000 using a MS QQQ mass spectrometer equipped with an
electrospray ion source in negative ion mode. Bruker NMR was used for 1H NMR (400
13
MHz) and C NMR (125 MHz) measurements. The NMR spectra were recorded in
CD3OD and chemical shifts are given in δ (ppm) relative to TMS as internal standard.

2.2. Plant material

Rhizomes of Z. officinale was obtained from Technology Park Malaysia (TPM)

Corporation Sdn. Bhd. - Herbal Biotech Centre of TPM Biotech Sdn Bhd (TPMB). The
plant was kindly identified by Forest Research institute, Malaysia. A vouched specimen
NO. 9112015 is kept at the herbarium of Pharmacognosy Department, Faculty of
Pharmacy, Cairo University.
2.3. Preparation of the crude extracts
The air-dried powdered rhizomes of Z. officinale were extracted using water, 50%, 70%,
80% and 90% ethanol (50 g powder for each solvent). The liquid-material ratio was 60:1
in a three-stage procedure (each in a ratio of 20:1), each time continued for 30 minutes
using ultrasonic bath at 60ºC. The aqueous extract was lyophilized to yield 8.55 gm solid
residue. The other extracts for each solvent were concentrated under reduced pressure
using rotary evaporator (at 40ºC) to yield solid residues weighing 2.2, 7.55, 5.03 and
4.39 gm of 50%, 70%, 80% and 90% ethanol extracts.

2.4. Preparation and fractionation of the aqueous extract

5
The aqueous extract of air-dried powdered rhizomes of Z. officinale (AE) was prepared as
mentioned before on large scale using (5 kg of the rhizomes). Five hundred grams of the
rhizomes extract were suspended in 600 ml distilled water was loaded to a diaion HP-20
AG (500 g) packed in water. Elution was carried out with water, followed by methanol-
water (50%), methanol (100%) and acetone to give four fractions, which were monitored
by TLC, using different solvent systems (S1-S3), the chromatograms were examined
under UV light at 365 nm and 254 nm before and after spraying with 10% H2SO4
followed by heating at 110°C for 5-10 min. The solvent in each case was evaporated
under reduced pressure (using rotary evaporator) at 40°C to yield solid residues weighing
106, 55, 22 and 14 g of four fractions water fraction (WF), 50 % methanol-water (MWF),
100% methanol (MF) and acetone (AF), respectively.

2.5. Isolation of the major constituents

Upon examination of WF and MWF using TLC, several minor spots were detected. AF
and MF showed better TLC chromatograms with major spots so were used for further
fractionation.

AF (10 g) was subjected to fractionation over a silica gel column (100 g) and eluted with
acetone -n-hexane. Gradient elution was performed using n-hexane, acetone -n-hexane
mixtures. The polarity was increased by 1 % every 100 ml till 20% acetone. Fractions
(100 ml, each) were collected to obtain 40 fractions which were then monitored by TLC
(precoated silica gel plates 60 F254) using solvent system (S1). Sub-fraction (7% acetone
in n-hexane) was chromatographed over a silica gel column. The elution carried out using
n-hexane – acetone (9.9:0.1 v/v) as eluent, to give compound G1 (339.2 mg). Sub-
fraction (9-10% acetone in n-hexane) was chromatographed over a silica gel column. The
elution carried out using n-hexane – acetone (9.8:0.2 v/v) to give compounds G2 (423.5
mg) and G3 (13.5 mg). Sub-fraction (13-15% acetone in n-hexane) was chromatographed
over a silica gel column. The elution carried out using n-hexane – acetone (9.8:0.2 v/v) to
yield one pure compound G4 (227.2 mg).

A weighed amount (15 g) of MF was subjected to fractionation over a silica gel

column (150 g). Gradient elution was performed using dichloromethane, methanol -
dichloromethane mixtures and methanol. The polarity was increased by 5 % every 200 ml

6
till 100% methanol. Fractions (200 ml, each) were collected to obtain 20 fractions which
were then monitored by TLC using solvent system (S2 and S3). Methanol sub-fraction
(30-40% methanol in dichloromethane) was chromatographed over a silica gel column.
The elution carried out using dichloromethane – methanol (9.8:0.2 v/v). Similar fractions
showing pure spots were pooled together to yield three pure compounds G5 (215.2 mg),
G6 (14.4 mg) and G7 (13.7 mg). Sub-fraction (45-60% methanol in dichloromethane)
was chromatographed over a silica gel column using dichloromethane – methanol
(9.8:0.2 v/v) as eluent to yield one pure compound G8 (433.3 mg).

2.6. Evaluation of in vitro anti-inflammatory activity

2.6.1. Protein denaturation inhibition
Protein denaturation inhibition was evaluated according to the method of (Mizushima and
Kobayashi 1968; Sakat et al., 2010) with slight modification. One % bovine serum
albumin (500 μL) was added separately to 100 μL of Z. officinale extracts, fractions, and
isolated compounds dissolved in Tween 80. This mixture was kept for 10 minutes at
room temperature, followed by 20 minutes heating at 51°C. Then the solution was cooled
down and absorbance was measured at 660 nm.
2.6.2. Membrane stabilization
Red blood cell (RBC) suspension preparation
Anticoagulated human blood was obtained from El- Kasr El-Ainy Hospital, Cairo, Egypt.
The blood was centrifuged at 2800 rpm for 10 minutes. Then the solution was washed
several times with saline. RBC layer was then diluted to 10% v/v using phosphate buffer
saline (PBS) (Saket et al., 2010; Sadique et al., 1989).
2.6.3. Heat induced haemolysis
One hundred μL of RBC (10%) was added to equal volume of the tested samples and the
solution was heated at 56°C for 30 min. Then it was centrifuged at 2500 rpm for 10
minutes at room temperature. Supernatant was collected, and absorbance was recorded at
560 nm. Membrane stabilization percentage was calculated according to (Saket et al.,
2010; Shinde et al., 1999).
2.6.4. Protease inhibition assay
Trypsin inhibition was evaluated by Oyedepo and Femurewas (1965) and Sakat et al.
(2010) method. Bovine serum albumin (100 mL) was added to 100 μL tested sample.

7
This was incubated for 5 minutes at room temperature. Trypsin (250 μL) was added to
inhibit the reaction followed by centrifugation. The supernatant was collected, and
absorbance was read at 210 nm.
2.6.5. Anti-lipoxygenase activity
Anti-Lipoxygenase activity was evaluated using according to the method of Shinde et al.,
1999. Lipoxidase from Glycine max (soybean), as enzyme and linoleic acid as substrate
were purchased from Sigma-Aldrich (SigmaMarker, Sigma-Aldrich, St. Louis, MI,
USA). Tested samples were dissolved in 0.25 mL of borate buffer (2M) pH 9.0 and
0.25ml of lipoxidase enzyme (20,000U/mL) was added and incubated at 25ºC for 5 min.
After that, 1mL of lenoleic acid (0.6mM) was then added, mixed well and absorbance
was observed at 234nm.
All the above experiments were done in triplicates and % inhibition was calculated as
follows:
% Inhibition=100−[(Aa-Ab)/Ac)x100]
Where Aa: absorbance of the sample, Ab: absorbance of the product control and Ac:
positive control absorbance. Diclofenac sodium was used as a positive control.
A dose response curve was plotted to determine the IC50 values. IC50 is defined as the
concentration sufficient to obtain 50% of a maximum scavenging capacity.
2.7. In vivo anti-inflammatory activity
2.7.1. Animals
Adult male Sprague- Dawley rats (150–175 g) were obtained from Misr University for
Science and Technology, 6th of October city, Egypt Animal Facility. Animals were
housed at 23 ± 2ºC with free access to standard food pellets and water. The study was
conducted according to the institutional guidelines for experimental animals care and use.
2.7.2. LD50
LD50 was determined using acute toxic class method as per OECD guideline
(Organization for Economic Co-operation and Development, Guideline-423, adopted on
17th December, 2001). Then, a dose response study was conducted to determine the
median effective dose.
2.7.3. Assessment of rat paw edema inhibition activity of AE

8
Rats were randomly divided into seven groups, assigned I–VII. Animals were fasted,
with free access to water, 16 h before the experiment. Groups I and II were given the
vehicle by intragastric tube, while group III-VI were orally treated with the aqueous
extract of Z. officinale rhizomes (AE) (at doses 25, 50, 100 and 200 mg/kg b.wt). Dose
levels were chosen according to previous studies (El-Abhar et al., 2008; Shirpoor et al.,
2017), independent on the concentration of specific active ingredient. Animals in group
VII received indomethacin as standard anti-inflammatory drug. Dosing volume was kept
constant10 ml/kg. Thirty minutes after oral treatment, group I received 0.05 ml saline,
while groups II–VII received subplantar injection of carrageenan (0.05 ml of a 1%
solution in saline) in the right hind paw. The right hind paw volume was measured
immediately after carrageenin injection by water displacement using UGO-BASILE 7140
plethysmometer (Comerio, Italy) (Winter et al., 1962). Edema volume was re-measured
1, 2 and 3 h after carrageenin injection and immediately before decapitation. Edema
inhibitory activity was calculated according to the following formula:
% Inhibition = (Ct – C0) control – (Ct – C0) treated/(Ct – C0) control x 100
Where Ct = paw volume at time t, C0 = paw volume before carrageenan injection
The following markers were estimated in the in paw edema exudates
2.7.4. Determination of PGE2 level
At 3hr after carrageenan challenge, animals were sacrificed and right hind paws were cut.
Saline containing 10 µM indomethacin (0.1 ml) was injected. Paws were incised with a
scalpel and suspended off the bottom of polypropylene tubes to facilitate drainage of the
inflammatory exudates. Then paws were centrifuged at 1800 g for 15 min (Mnich et al.
1995). PGE2 was quantified in the collected exudates using a quantitative binding PGE2
enzyme linked immunosorbent assay (ELISA) kit (R and D Systems Inc., Minneapolis,
USA). A substrate solution is added to the wells to determine the bound enzyme activity.
All samples, controls, and standards were assayed in duplicate. All the reagents,
standards, and samples were prepared as indicated in the kit insert. The color
development is stopped, and the absorbance is recorded at 450 nm. The color intensity is
inversely proportional to the PGE2 concentration (Virella G., 1998). A microplate reader
(ChroMate-4300, FL, USA) with primary filter set to 450 nm and differential filter set to
545 nm was used to determine the optical density (Virella G., 1998).

9
 2.7.5. Assessment of cytokine levels in paw edema exudates
Signosis’ Rat Inflammation ELISA Strip (Signosis Inc., CA, USA) was used to estimate
the levels of 8 different cytokines in rat paw edema exudates. In each strip well, a
primary antibody against a specific inflammation cytokine is coated. The test sample is
allowed to react simultaneously with pairs of two antibodies. Standard, control, or sample
(100μl) were separately added per well and incubated for 1 hour at room temperature
with gentle shaking. Then the wells content was aspirated and washed by adding 200μl of
1X Assay wash buffer. The previous process was repeated three times for a total of three
washes with complete removal of liquid at each wash. An aliquot of 100μl of diluted
biotin-labeled antibody mixture was added to each well and incubated for 1 hour at room
temperature with gentle shaking. Aspiration/wash was repeated as mentioned previously.
Then a volume of 100μl of diluted streptavidin-HRP conjugate was added to each well
and incubated for 45 min at room temperature with gentle shaking. After this step
aspiration/wash was performed again as mentioned above. A volume of 100μl substrate
was added to each well and incubated for 5-30 minutes. Then 50μl of Stop solution was
added to each well. The optical density of each well was determined using a microplate
reader (ChroMate-4300, FL, USA) at 450 nm within 30 minutes. The concentrations of
the inflammation cytokines are directly proportional to the color intensity of the test
sample. The optical density is then determined spectrophotometrically at 450 nm.
2.7.6. Assessment of myeloperoxidase (MPO) activity
Tissue samples from each paw were assessed biochemically for the neutrophil infiltration
marker enzyme MPO, using the method of Bradley et al., 1982 with minor modifications.
Paw tissue was homogenized for 10 min in ice bath (10%, w/v) of 50 mM phosphate
buffer (pH 6) containing 0.5% hexadecyltrimethylammonium bromide (HTBA). The
samples were then centrifuged at 2500 g for 30 min at 5 ºC then the solution was assayed
for MPO spectrophotometrically. In brief, sample (40 µl) was mixed with 50 mM
phosphate buffer pH 6 (960 µl), containing 0.167 mg/mL O-dianisidinedihydrochloride
and 0.0005% H2O2. SHIMADZU® UV-1601 spectrophotometer was used to measure the
absorbance change at 460 nm over a period of 2 min at 25 ºC. The maximum change in
absorbency/minute was used to calculate myeloperoxidase activity based on oxidized O-
dianisidinedihydrochloride molar absorbency index (Bradley et al., 1982). Results were

10
expressed as units of activity/mg protein. Protein content was determined according to
Lowery et al., 1951.
2.7.7. Oxidative stress parameters:
2.7.7.1. Determination of tissue nitrate/nitrite content (NOx)
NOx level was determined in paw edema exudates. Briefly, vanadium trichloride solution
(250 µl) was added to air pouch exudates (250 µl) to reduce any nitrate to nitrite. This is
followed by rapid addition of modified Griess reagent (250 µl) to detect total nitrite. A
chromophore is formed by diazotization of sulfanilamide with acidic nitrite followed by
coupling with bicyclic amine N-(1-naphthyl)-ethylenediamine (Miranda et al., 2001). The
mixture was incubated at 37 °C for 30 minutes and then cooled. Absorbance was
measured using a SHIMADZU® UV-1601 spectrophotometer against blank at 540 nm.
2.7.7.2. Total antioxidant capacity (TAC)
TAC was tested in paw edema exudates by the reaction of the sample antioxidants with a
defined amount of hydrogen peroxide (H2O2). The antioxidants in the sample eliminate a
certain amount of the provided H2O2. The residual H2O2 was determined colorimetrically
by the enzymatic reaction involving the conversion of 3,5, dichloro-2-
hydroxybenzensulphonate to a colored product (Koracevic et al., 2001). TAC was
calculated from the following equation: TAC= (AB-AS)*3.33 mM H2O2, Where: AB =
blank absorbance and AS = sample absorbance.
2.7.8. Histopathology
Representative paw tissue from each group (I-VII) was fixed in 10% formol saline for 24
hours. Washing was by tap water then serial alcohol (methyl, ethyl and absolute ethyl)
dilutions for dehydration. Specimens were cleared in xylene, embedded at 56°C degree in
oven for 24 hours. Sections at 4 microns thickness were performed by sledge microtome
using paraffin bees wax blocks. Tissue sections were collected on glass slides then
stained by hematoxylin and eosin. Examination was done through the light electric
microscope (Banchroft et al., 1996).
2.8. Statistical analysis
Data are presented as mean ± SEM. Comparisons were carried out using one way
analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis.
Statistical significance was acceptable to a level of p< 0.05. All statistical analyses were

11
performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc. La
Jolla, CA, USA). Graphs were plotted using GraphPad Prism software, version 5.00
(GraphPad Software, Inc. La Jolla, CA, USA).

3. Results
3.1. In vitro anti-inflammatory assays
3.1.1. Protein denaturation inhibition
The extracts at (125-500 µg/ml) showed high anti-inflammatory activity by inhibiting the
heat induced albumin denaturation. The highest % of inhibition (66%) was exhibited by
the 50% ethanol extract followed by 70 and 90% ethanol extracts (65 and 63%,
respectively) and moderate activity was observed for the 80% ethanol extract and AE (58
and 56%, respectively) (Table 1).
3.1.2. Membrane stabilization
All the tested extracts exhibited significant (p< 0.01) membrane stabilizing activity at a
dose of 62.5 µg/ml) (Table 1). The 80 and 90% ethanol extracts (at 500 µg/ml) showed
maximum human red blood cells membrane stabilization by 37%, a value which is higher
than that caused by the standard reference drug at the same concentration that showed
34% stabilization. The 70% ethanol extract was also more potent than the standard
(showed 35% stabilization). AE and 50% ethanol extract groups, showed slight lower
activity (caused 33% stabilization). This means that certain Z. officinale extracts were as
potent membrane stabilizers as diclofenac sodium or even more.
3.1.3. Protease inhibition assay
Proteinase activity was also significantly inhibited by all tested Z. officinale extracts. The
70-90% ethanol extracts at 62.5 µg/ml showed significant (p< 0.01) activity. The 80 and
90% ethanol extracts (500 µg/ml) showed the maximum inhibition activity of 56%.
3.1.4. Anti-lipoxygenase activity
Z. officinale extracts have been checked for anti-lipoxygenase activity. All the tested
extracts showed significant (p< 0.01) anti-lipoxygenase inhibition at 125 µg/ml. The
strongest inhibition (70%) was obtained by AE (at 500 µg/ml). At the concentration of
250 µg/ml, AE appeared to be even more potent than the standard used. It inhibited the

12
lipoxygenase activity by 62%, while Diclofenac sodium caused 56% inhibition at the
same concentration.
3.2. Isolation of the major constituents of AE

AE was fractionated over a diaion column and the obtained fractions showed
significant potency in albumin denaturation assay, human red blood cells membrane
stabilization assay, proteinase inhibitory, and lipoxygenase inhibitory assays at p< 0.1.
(Tables 1-3). Further purification of the obtained fractions yielded 8 compounds G1-G8.
G1: MS [M-H] - at m/z: 277, MS2 at m/z: 276.9, 261.9, 140.9; G2: MS [M-H] - at m/z:
275. MS2 at m/z: 275.0, 138.9. G3:, MS [M-H] - at m/z: 307. MS2 at m/z: 298, 218, 164,
150. G4: MS [M-H] - at m/z: 279. MS2 at m/z: 279, 193, 178. G5: MS [M-H] - at m/z:
293. MS2 at m/z: 293.1, 193.1, 99.2, 56.9. G6: MS [M-H] - at m/z: 321. MS2 at m/z:
320.9, 193.1, 127.0. G7: MS [M-H] - at m/z: 349. MS2 at m/z: 349.1, 193.1, 155.1, 179.1.
G8: MS [M-H] - at m/z: 291. MS2 at m/z: 291, 151, 136, 141. The 1HNMR and 13CNMR
data is presented in Tables 4 and 5

These compounds were identified as G1: 6-paradol (Tachie et al., 1975); G2; 6-
shogaol (Connell and Sutherland, 1969); G3: methyl- 6-gingerol (Denniff et al., 1980);
G4: 5-gingerol; G5:6-gingerol (Agarwal et al., 2001); G6: 8-gingerol; G7: 10-gingerol
(Shoji et al., 1982); G8:1-dehydro-6-gingerol (Charles et al., 2000). The structures of the
isolates are shown in Fig. 1. The major isolates G1 G2, G4, and G8 were tested for in
vitro anti-inflammatory activity.

G1 G2, G4, and G8 showed high in vitro anti-inflammatory activity by inhibiting

the heat induced albumin denaturation. The highest percentage of inhibition exhibited by
G2 (500 ug/ml) was found to be 62% and for the standard it was 67% at the same
concentration. G1 and G3 also showed relatively high percentage of inhibition 53% and
44%, respectively. G1 (at a concentration 500 µg/ml) was as potent as diclofenac in
stabilization of human red blood cells membrane. Both of them caused stabilization by
33%. High stabilization potency was reported also by G2 and G4. They caused
membrane stabilization by 30 and 29%, respectively.

13
Proteinase activity was also significantly inhibited (51%) by G2 at 500 µg/ml followed
by G1 and G8 their proteinase inhibition was 40% at the same concentration. Diclofenac
protinase inhibition was recorded to be 64% at the same concentration.
The highest anti-lipoxygenase activity was exhibited by G2 and G8 (at 500 µg/ml). Their
lipoxygenase inhibition activity was 66% followed by G5 which inhibited the enzyme
activity by 50%. The lipoxygenase inhibition of diclofenac was 72%.
3.3. In vivo anti-inflammatory assays
3.3.1. LD50 result
No mortality was recorded after 24 h of administration of Z. officinale AE limit dose of
2000 mg/kg. The test was repeated using three additional animals at the same dose and
also no mortality was observed. According to the Acute Toxic Class Method reported in
OECD guideline# 423. The AE is thus considered to be GHS unclassified with LD50 cut-
off >5000 mg/kg.
3.3.2. Inhibition of carrageenan-induced paw edema
Marked increase in paw volume was observed after carrageenan injection (40.8%
compared to baseline after 3h). Pre-treatment with Z. officinale AE (25, 50, 100 and 200
mg/ kg) resulted in significant amelioration of carrageenan-induced edema by about 30%,
46%, 56% and 59%, respectively (Fig.2 i-ii). Indomethacin caused significant inhibition
of edema volume by about 72%. Z. officinale AE stopped the further changes in paw
volume at 2h while in vehicle treated group the increase in paw volume continued till 3h
(Fig.2iii).
3.3.3. Effect on PGE2 production
Carrageenan challenge resulted in more than 7-folds increase in PGE2 concentration in
inflammatory exudates compared to the control group. Animals receiving Z. officinale
AE at doses of (25, 50, 100 and 200 mg/kg) or indomethacin (10 mg/kg) showed
significant reduction of PGE2 production in the inflammatory exudates by about 23, 44,
47, 60, and 80% respectively (Fig.2 iv).

3.3.4. Effect of Z. officinale AE on certain inflammation markers

Injection of rat paws s.c. with carrageenan resulted in a significant increase in TNF-α, IL-
6, IL-1α, IL-1β, INFr, MCP-1, MIP, RANTES, and NOx levels and MPO activity in the

14
paw edema exudates by about 10, 17, 57, 6, 2.5, 7.7, 30, 14, and 62 folds respectively
compared to animals in the control group (Fig. v-xiv). Animals receiving indomethacin
(10 mg/kg) showed significant reduction of all tested inflammation markers levels in the
paw edema exudates except IL-1α level. Z. officinale AE at different doses (25, 50, 100
and 200 mg/kg) showed significant reduction in their levels except for IL-1α also. Z.
officinale AE all tested doses are superior to indomethacin (10 mg/kg) in reduction of IL-
1β. Doses of (25, 50, 100, 200 mg/kg) of Z. officinale AE showed significant reduction of
the IL-1 β level in the inflammatory exudates by about 38, 38, 39, and 61 respectively
compared to carrageenan-only group, while indomethacin (10 mg/kg) reduction is 37%
(Fig. 2viii).
A dose of 100 and 200 mg/kg Z. officinale AE is equipotent or more potent than
the reference standard anti-inflammatory drug indomethacin (10 mg/kg) in reduction of
TNF-α, IL-6, MCP-1, RANTES, and MPO levels (Fig. 2v,vi,x,xii,xiii). Treatment of
animals with Z. officinale AE (100, 200 mg/kg) or indomethacin (10 mg/kg) showed
significant reduction in TNF-α, IL-6, MCP-1, and RANTES levels, and MPO activity in
the inflammatory exudates by about (31, 57 and 32% ) (65, 60 and 57%) (27, 41 and
28%) (23, 32 and 23%) (66, 67 and 67%) respectively compared to carrageenan-only
group.
Treatment of animals with Z. officinale AE (200 mg/kg) is equipotent to indomethacin in
reduction NOx level in the inflammatory exudates (Fig. 2xiv)
Injection of rat paws s.c. with carrageenan resulted in a significant increase by
about 6-folds in INFr level in the paw edema exudates compared to animals in the control
group. Treatment of animals with AE (200 mg/kg) resulted in significant reduction of the
INFr level in the inflammatory exudates by about 35 % compared to carrageenan-only
group (Fig.2ix).
3.3.5. Effect on total antioxidant capacity (TAC)
The Z. officinale AE is highly effective in increasing the TAC in the inflammatory
exudates. A dose of 100 or 200 mg/kg of the extract significantly increase TAC in the
inflammatory exudates by about 64% compared to carrageenan only treated animals. A
value which is equal to the effect of the standard reference indomethacin (10 mg/kg)
showed (Fig. 2xv).

15
 3.3.6. Effect on paw tissue histopathology
Paw tissues specimens assessment from control animals revealed normal
histopathological structure of the epidermal, covering keratin, underlying dermis, and
musculature layers. Carrageenan injection resulted in focal as well as diffuse
inflammatory cell infiltration. This is in addition to detection of oedema and congestion
in the blood vessels in the dermal and subcutaneous connective tissues. Pretreatment with
indomethacin before carrageenan challenge lead to mild inflammatory cell infiltration
and oedema were observed in the dermis only. Examining paw tissue specimens from rats
pretreated with either 25 or 50 mg/kg dose of Z. officinale AE before carrageenan
revealed focal inflammatory cells aggregation and oedema in the dermis. A dose of 100
mg/kg Z. officinale AE before carrageenan challenge resulted in few focal inflammatory
cells infiltration with mild oedema in the dermis. Administration of 200mg/kg of Z.
officinale AE to rats before carrageenan resulted in very few inflammatory cells
infiltration and oedema in the dermis (Fig. 3). The severity of inflammatory reactions in
paw tissue specimens from different groups was scored according to the histopathological
alterations. No histopathological alterations were noticed in the control group.
Inflammatory cell infiltration with oedema and congestion in the blood vessels at the
dermis of paw tissues were very severe in carrageenan and Z. officinale AE (25 mg/kg)
groups. These alterations were severe in Z. officinale AE (50 mg/kg) group and moderate
in Z. officinale AE (100 mg/kg) and indomethacin groups. Administration of 200 mg/kg
of Z. officinale AE to rats before carrageenan resulted in only mild pathological changes.

4. Discussion
The rhizome of the plant Zingiber officinale Roscoe, commonly known as ginger,
has been commonly used as a food additive and spice as well as a phytomedicine since
ancient times (Ghayur and Gilani, 2005). Ginger has been widely studied for its
pharmacological activities and has been reported to exhibit anti-inflammatory,
antipyretic, antimicrobial, hypoglycemic, antimigraine, antischistosomal, antioxidant,
hepatoprotective, diuretic and hypocholesterolemic (Langner et al., 1998).
Phytochemical studies showed the presence of pungent principles, such as gingerol,

16
shogaol, zingerone, and paradol (Connell and McLachlan, 1972), while the main aroma
defining component is zingiberol (Varma et al., 1962).
A number of recent studies have renewed interest in ginger for the treatment of
chronic inflammatory conditions. The current study aimed at getting more insights about
the mechanisms. Denaturation of tissue proteins is one of the well-documented causes of
inflammation as the production of auto antigens in certain inflammatory diseases may be
due to denaturation of proteins in vivo (Opie, 1962). Stabilization of human red blood
cell from hypotonicity induced lysis can be correlated with the antiinflammatory potential
of a drug as histamine from damaged tissues makes capillaries more permeable, and the
lysosomes of damaged cells release their enzymes breaking down damaged tissue and
also cause destruction of nearby healthy tissue (Scanlon and Sanders, 2010).
antiproteinase activity is used for managing inflammatory disorders through the recovery
of proteinase induced tissue damage (Brown and Mackey, 1962). Lipoxygenase is
responsible for the biosynthesis of inflammatory lipid mediators, such as leukotrienes,
lipoxins, hepoxilins, and other hydroxylated fatty acid derivatives (Kühn et al., 2007).
involved in the anti-inflammatory activity of ginger. On this basis the in vitro anti-
inflammatory activity of different Z. officinale extracts were evaluated using albumin
denaturation assay, human red blood cells membrane stabilization assay, proteinase
inhibitory, and lipoxygenase inhibitory activities at different concentrations (62.5, 125,
250, 500 ug/ml). Diclofenac sodium at the same concentration range was used as a
standard drugAll tested extracts showed significant anti-inflammatory activities at p< 0.1.
The results obtained from our study has shown that the different extracts of Z. officinale
rhizomes exhibited a potential anti-inflammatory activity.
The authors selected the aqueous extract for further purification and the isolation
of the major constituents as the aqueous extract is commonly used in folk medicine
specially that the use of organic solvent extraction have several drawbacks in that they
are harmful to the environment and usually leave toxic residues. Thus, we preferred to
work on the aqueous extract, mainly because it is ecofriendly and can produce extracts
that are free from residues. The major phytoconstituents of the aqueous extract was
isolated to estimate their potency and to identify the chemical constituent responsible for
the potent anti-inflammatory activity of the plant. 8-Paradol and 8-gingerol were reported

17
to possess anti COX-1 activity (Tjendraputra et al., 2003) but here we report the
significant anti-inflammatory potency of the isolated gingerols, paradol and shogaol
using the in vitro albumin denaturation assay, human red blood cells membrane
stabilization assay, proteinase inhibitory, and lipoxygenase inhibitory assays at p< 0.1.
The strongest anti-inflammatory activity was recorded for 6-poradol (G1), 6-shogaol
(G2), and 1-dehydro-6-gingerol (G8). G2 showed significant (p< 0.1) anti-inflammatory
activity in all the studied assays at a concentration of 62.5 ug/ml. G1 and G8 exhibited
significant (p< 0.1) anti-inflammatory activity in all studied assays at (250 and 500
ug/ml). On the other hand, 5-gingerol (G4) and 6-gingerol (G5), exhibited moderate
activity.
Ginger rhizome is famous for its vanillyl ketonic constituents. The eight isolated
compounds G1-8 possess vanillyl ketone moiety. These constituents are known to exhibit
strong anti-inflammatory activity (Surh, Park et al. 1999). In addition to the presence of
α,β-unsaturated ketone moiety in the most active anti-inflammatory compounds; 6-
shogaol (G2) and 1-dehydro-6-gingerol (G8). The influence of this α,β-unsaturated
ketone moiety in the anti-inflammatory potency is well documented (Dugasani, Pichika et
al. 2010).

In order to confirm the obtained results the in vivo anti-inflammatory activity of

the aqueous extract was also evaluated. Initially an LD50 study was conducted to
evaluate safety of ginger extract. It was found that no mortality was observed in rats
receiving ginger extract up to 2000 mg/kg. This finding is in line with previous reports
indicated that ginger extract is safe up to 5000 mg/kg (Plengsuriyakarn et al., 2012).

The anti-inflammatory effect of ginger extract was tested in carrageenan-induced

rat paw edema model. Pretreatment of animals with ginger extract ameliorated
carrageenan-induced edema in rat paws. The effect of ginger was dose-dependent and the
percent of paw edema inhibition exhibited by ginger doses 100, 200 mg/kg was
comparable to that of indomethacin. Our results are in line with previous reports (Zakaria
et al., 2010). The study was further substantiated to investigate the potential mechanisms
involved in ginger anti-inflammatory activity. The level of PGE2 was assessed in paw
edema exudates. It was found that pretreatment with ginger significantly counteracted the

18
carrageenan-induced PGE2 production in a dose-dependent manner. This finding gains
support by previous report indicating the ability of compounds isolated from ginger as 6-
gingerol and other structurally related compounds to inhibit PG synthesis in vitro (Kiuchi
et al., 1982). TNF-α is the proinflammatory cytokine that possesses many biological
effects, including induced expression of other cytokines, chemokines and adhesion
molecules, as well as activation of neutrophils (Ko et al., 2001). Accordingly, the effect
of the ginger extract on TNF-α was investigated. It was shown that ginger caused
significant reduction in TNF-α levels. Moreover, ginger extract ameliorated carrageenan-
induced elevation in the pro-inflammatory cytokine IL-6. Ginger was shown to inhibit
acute inflammatory response induced by acetic acid in ulcerative colitis model through
ameliorating the levels of PGE2 and TNF-α (El-Abhar et al., 2008). The level of IL1- α
was not significantly affected after carrageenan challenge which was consistent with a
previous study (Alebouyeh et al., 2002). Measurement of IL1- β revealed that ginger
produced significant amelioration of carrageenan-induced elevation of IL1- β level. This
finding gains support by previous study showed that ginger inhibited LPS-induced
production of IL1- β from macrophages (Tripathi et al. 2008)[43]. The highest dose of
ginger 200 mg/kg was the only dose producing significant reduction in the level of INFr.
Ginger was previously reported to mitigate INFr levels in LPS-induced macrophages
(Tripathi et al., 2008). The aforementioned findings indicated that ginger extract could
inhibit macrophage activation. The study was further extended to evaluate ginger possible
effects on monocyte and leukocyte migration. This was done through assessing the effect
of ginger extract on monocyte chemoattractant protein-1 (MCP-1) and RANTES
(regulated upon activation, normal T-cell expressed and secreted). The monocyte
chemoattractant protein-1 (MCP-1) is a pro inflammatory cytokine responsible in the
appearance of a monocyte rich inflammatory infiltrate (Rollins, 1996). The elevation in
RANTES production is associated with a wide range of inflammatory conditions. It acts
by promoting leukocyte infiltration to the site of inflammation. At high concentrations
RANTES induces activation of T cells (Appay and Rowland-Jones, 2001). The activation
of T cells by RANTES is followed by increased production of IL-2 and IFN-γ. In the
current study ginger extract alleviated carrageenan-induced increase in RANTES
production. This might, at least partly, explain the evidenced decrease in carrageenan-

19
induced INFr production in ginger-treated rats. As inhibition of RANTES production by
ginger extract not only suppresses inflammation, but also restricts T cell activation, as
observed through reduced INFr production. Taken together, ginger extract could
negatively affects monocyte and leukocyte migration as evident from alleviated MCP-1
and RANTES production. The level of macrophage inflammatory protein (MIP) in paw
edema exudates of carrageenan-treated rats was found to be significantly reduced in
ginger pretreated rats.

Activation of neutrophils is known to generate and release a number of tissue-

damaging factors including reactive oxygen species such as superoxide anion, hydrogen
peroxide, hypochlorous acid, as well as enzymes such as Myeloperoxidase (MPO) and
proteases (Elsbach and Weiss 1988). Neutrophils also migrate out of the
microvasculature into the surrounding tissue, resulting in further disruption (Wallace et
al., 1990). In the present work, the effect of ginger extract on neutrophils activation and
infiltration was further substantiated via assessing MPO activity in paw tissues. It was
found that ginger extract exhibited dose-dependent amelioration of carrageenan-induced
MPO activity in paw tissues.

Furthermore, the effect of ginger extract on oxidative status was assessed through
evaluation of the level of NOx and total antioxidant capacity in paw tissues. Ginger
extract significantly alleviated carrageenan-induced enhancement if reactive nitrogen
species and significantly replenished the total antioxidant capacity in paw tissues.
Histopathological examination of specimens from rat paw tissues revealed that
carrageenen caused focal as well as diffuse inflammatory cell infiltration. This is in
addition to detection of oedema and congestion in the blood vessels in the dermal and
subcutaneous connective tissues. Examination of specimens from rats pretreated with
ginger extract at doses of 100, 200 mg/kg alleviated the inflammatory cell infiltration and
edema. Ginger 100 mg/kg dose resulted in inflammation severity score comparable to
indomethacin. This finding supports the observed reduction in MPO activity in the same
groups which is an indicator of neutrophil accumulation (Goulet et al., 1994).

5. Conclusion

20
Ginger anti-inflammatory activity is mediated by inhibiting macrophage and neutrophils
activation as well as negatively affecting monocyte and leukocyte migration. This was
evidenced by the dose-dependent decrease in pro-inflammatory cytokines and
chemokines and replenishment the total antioxidant capacity.

Acknowledgement
The authors are deeply thankful to Prof. Dr. Adel Bakir, Histology Department, Cairo
University for performing the histopathology part.

Conflicts of interest
The authors report no conflicts of interest.

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O OH

RO

OMe

R n
G3: Methyl- 6-gingerol Me 4
G4: 5-gingerol H 3
G5: 6-gingerol H 4

26
 G6: 8-gingerol H 6
G7: 10-gingerol H 8

O O

6 4

HO HO

OMe OMe

G1: 6-paradol G2: 6-shogaol

O OH

HO

OMe

G8: 1-dehydro- 6- gingerol

Fig. 1. Chemical structure of the compounds isolated from Z. Officinale

27
 (i) (ii) (iii)

(iv) (v) (vi)

(vii) (viii) (ix)

(x) (xi) (xii)

(xiii) (xiv) (xv)

28
 Fig. 2. Effect of Z. officinale AE (25, 50, 100, 200 mg/kg) and indomethacin (10 mg/kg) on (i) the percent
change in paw volume (ii) percent edema inhibition (iii) kinetic changes in paw volumes (iv) PGE2 (v) TNF-α (vi) IL-
6 (vii) IL-1α (viii) IL-1β (ix) INFrin (x) MCP-1(xi) MIP (xii) RANTES (xiii) MPO enzyme activity (xiv) NO x (xv)
TAC levels in paw edema exudates in carrageenan-induced rat paw edema model. Data are mean ± SEM, n=6.*
Significantly different vs control at p<0.05 a Significantly different vs normal at p<0.05. b Significantly different vs
carrageenan at p<0.05

29
 K

(i) (ii) (iii) (iv)

(v) (vi) (vii)

Fig. 3. Histopathology of rat paw tissues in (i) control, (ii) carrageenan, (iii) 10 mg/kg
indomethacin, (iv) 25 mg/kg, (v) 50 mg/kg, (vi) 100 mg/kg, (vii) 200 mg/kg Z. officinale
aqueous extract groups. Upper panel (H&E stain x 16) and lower panel (H&E stain x 40).
d: underlying dermis; dm: diffuse inflammatory cell infiltration; fm: focal aggregation; k:
covering keratin; m: few focal inflammatory cells infiltration; ml: musculature; o:
oedema; p: Normal histopathological structure.

Table 1. Effect of different extracts of Z. officinale on protein denaturation, membrane

stabilization, protease and lipoxygenase activities
Dose Protein denaturation
Model µg/mL Inhibition Membrane stabilization Protease inhibition assay Anti-lipoxygenase activity
1 1 1 1
Abs % in. Abs %M.S. Abs % in. Abs % in.
Dic. 500 0.458±0.01** 67 0.21±0.05** 34 0.16± 0.004** 64 0.42±0.02** 72
250 0.48±0.017** 65 0.243±0.03** 31 0.195±0.01** 57 0.66±0.01** 56
125 0.517±0.01** 63 0.29±0.01** 26 0.245±0.02** 46 0.72±0.014** 52

30
 62.5 0.6820.03** 51 0.313±0.01** 24 0.27±0.03** 40 0.77±0.01** 49
AE 500 0.614± 0.02** 56 0.215±0.01** 33 0.296±0.05** 34 0.45±0.02** 70
250 0.659±0.01** 53 0.24±0.01** 31 0.37±0.03* 18 0.58±0.01** 62
125 0.695±0.002** 50 0.29±0.01** 25 0.45±0.04 0 0.63±0.01** 58
62.5 1.195±0.1 15 0.38±0.06** 17 0.47±0.002 -4 1.07±0.07** 29
50% E 500 0.476±0.014** 66 0.216±0.01** 33 0.263±0.01** 42 0.5±0.01** 67
250 0.483±0.01** 66 0.23±0.01** 32 0.36±0.02** 20 0.62±0.015** 59
125 0.515±0.014** 63 0.27±0.02** 28 0.37±0.03 18 0.87±0.012** 42
62.5 0.634±0.015** 55 0.26±0.03** 29 0.43±0.01 4 0.91±0.02** 40
70% E
500 0.491±0.02** 65 0.205±0.002** 35 0.23±0.02** 49 0.54±0.015** 64
250 0.508±0.01** 64 0.25±0.01** 30 0.27±0.01** 40 0.65±0.015** 57
125 0.551±0.01** 61 0.28±0.003** 27 0.28±0.01** 38 0.74±0.012** 51
62.5 0.931±0.03** 34 0.29±0.01** 26 0.35±0.02** 22 0.91±0.02** 40
80% E
500 0.587±0.02** 58 0.18±0.006** 37 0.2±0.004** 56 0.74±0.01** 51
250 0.607±0.01** 57 0.187±0.01** 36 0.24±0.02** 47 0.94±0.01** 38
125 0.661±0.02** 53 0.203±0.002** 35 0.26±0.04** 42 1.03±0.012** 32
62.5 0.882±0.03** 37 0.21±0.01** 34 0.33±0.03** 27 1.28±0.15* 15
90% E 500 0.513±0.004** 63 0.18±0.01** 37 0.2±0.01** 56 0.53±0.01** 65
250 0.514±0.02** 63 0.192±0.01** 36 0.25±0.01** 44 0.73±0.012** 52
125 0.549±0.03** 61 0.21±0.01** 34 0.3±0.01** 33 0.87±0.012** 42
62.5 0.82±0.01** 41 0.21±0.01** 34 0.3±0.03** 33 0.97±0.01** 36
1: mean ± SEM; abs: absorbance; Dic: diclofenac; AE: aqueous extract; E: ethanol extract; in.: inhibition;
M.S.: membrane stabilization; *: significant at p < 0.5; **:significant at p< 0.01.

Table 2. Effect of Z. officinale AE fractions and isolated compounds on protein

denaturation, membrane stabilization, protease and lipoxygenase activities
Dose Anti-lipoxygenase
Model µg/mL Protein denaturation Inhibition Membrane stabilization Protease inhibition assay activity
1 1 1 1
Abs % in. Abs %M.S. Abs % in. Abs % in.
WF 500 0.562±0.02** 60 0.22±0.01** 33 0.24±0.02** 47 0.56±0.002** 63
250 0.748±0.03** 47 0.26±0.01** 29 0.37±0.01* 18 0.73±0.012** 52
125 0.815±0.014** 42 0.29±0.01** 25 0.4±0.01 11 0.84±0.02** 44
62.5 1.017±0.001** 27 0.32±0.01** 23 0.45±0.01 0 1.17±0.03** 23
MWF 500 1.089±0.06** 22 0.48±0.02** 7 0.27±0.01** 40 1.19±0.01** 21
250 1.243±0.13 11 0.511±0.001 4 0.3±0.01** 33 1.36±0.003** 10
125 1.263±0.14 10 0.53±0.01 2 0.54±0.18 -20 1.55±0.01 -3
62.5 1.277±0.03 9 0.55±0.01 0 0.47±0.01 -4 1.66±0.025 -10
MF 500 0.589±0.04** 58 0.19±0.004** 36 0.27±0.02** 40 0.74±0.02** 51
250 0.779±0.01** 44 0.226±0.01** 32 0.33±0.01** 27 0.86±0.015** 43
125 0.882±0.06** 37 0.29±0.003** 26 0.38±0.013 20 0.89±0.01** 41
62.5 1.089±0.08 22 0.3±0.001** 25 0.36±0.012* 16 0.94±0.03** 38
AF 500 1.091±0.04** 22 0.25±0.002** 30 0.29±0.01** 36 0.97±0.03** 36

31
 250 1.233±0.1 12 0.332±0.01** 22 0.33±0.01** 27 1.05±0.03** 30
125 1.266±0.13 10 0.39±0.004** 16 0.39±0.03 13 1.29±0.01** 15
62.5 1.288±0.1 8 0.42±0.01** 13 0.47±0.02 -4 1.57±0.06 -4
G1 500 0.656±0.02** 53 0.22±0.008** 33 0.27±0.01** 40 0.91±0.02** 40
250 0.771±0.01** 45 0.239±0.01** 31 0.34±0.02** 24 1.16±0.01** 23
125 0.867±0.01** 38 0.258±0.01** 29 0.48±0.04 -7 1.26±0.02** 17
62.5 1.008±0.003** 28 0.27±0.001** 28 0.52±0.004 -16 1.38±0.04** 9
G2 500 0.527±0.01** 62 0.252±0.01** 30 0.22±0.013** 51 0.52±0.02** 66
250 0.725±0.004** 48 0.264±0.02** 29 0.27±0.01** 40 0.65±0.014** 57
125 0.725±0.02** 47 0.303±0.002** 25 0.315±0.02** 30 0.71±0.01** 53
62.5 0.725±0.013** 47 0.35±0.01** 20 0.37±0.01** 18 0.8±0.01** 47
G4 500 1.177±0.022** 16 0.26±0.004** 29 0.29±0.003** 36 0.92±0.02** 39
250 1.216±0.12 13 0.26±0.02** 29 0.27±0.01** 40 1.02±0.01** 32
125 1.237±0.11 12 0.35±0.01** 20 0.402±0.01 11 1.14±0.05** 25
62.5 1.311±0.17 6 0.41±0.002** 14 0.53±0.003 -18 1.28±0.01* 15
G5 500 1.248±0.1 11 0.29±0.007** 25 0.3±0.01** 33 0.75±0.03** 50
250 1.292±0.005 8 0.343±0.02** 21 0.32±0.004** 29 1.05±0.03** 30
125 1.331±0.1 5 0.44±0.02** 11 0.35±0.01* 22 1.4±0.01* 7
62.5 1.348±0.13 4 0.51±0.004 4 0.36±0.01* 20 1.45±0.01 4
G8 500 0.783±0.04** 44 0.291±0.0115** 26 0.27±0.02** 40 0.513±0.02** 66
250 0.996±0.01** 29 0.35±0.02** 20 0.3±0.04** 33 0.71±0.71** 53
125 1.117±00* 20 0.48±0.01** 7 0.35±0.01* 22 0.91±0.01** 40
62.5 1.123±0.06* 20 0.52±0.0001 3 0.34±0.03** 24 0.98±0.02** 35
1: mean ± SEM; abs: absorbance; Dic: diclofenac; in.: inhibition; M.S.: membrane stabilization; WF: water fraction;
MWF: 50% methanol; MF: methanol; AF; acetone diaion fractions of Z. officinale aqueous extract; G1: 6-paradol; G2;
6-shogaol; G4: 5-gingerol; G5:6- gingerol; G8:1-dehydro-6-gingerol; *: sig and p< 0.5; **:sig and p< 0.01.
Table 3. IC50 of Z. officinale different extracts and aqueous extract fractions and isolated
compounds

IC50 (ug/ml)
protein membrane Proteinase Anti-
Model Denaturation stabilization Inhibition lipoxygenase
Diclofenac 60 1217.4 209.85 96.05
a
AE 121 956.4 664.45 168.93
50% E 54a 2064.54 609.45 217.39
70% E 103 1284.26 487.37 174.43
80%E 118 2402.98 363.91 458.79
90%E 148 2263.01 390.74 254.23
WF 330 1284.65 539.21 292.78
MWF 1383 3224.49 537.60 889.63
MF 367 1007.66 a 678.36 466.54
a
AF 1350 1008.57 571.88 615.34
G1 1615 2027.27 546.82 647.49

32
 G2 645 1457.82 450.98 93.03
G4 2187 1086.43 541.22 680.7
G5 2788 1006.63 a 1019.67 479.36
G8 610 919.41 a 742.75 256.71

WF: water fraction; MWF: 50% methanol; MF: methanol; AF: acetone diaion fractions of Z. officinale
aqueous extract;G1: 6-paradol; G2; 6-shogaol; G4: 5-gingerol; G5:6- gingerol; G8:1-dehydro-6-gingerol;
AE: aqueous extract; E: ethanol extract; a: Significance level at p˂ 0.001, compared to control.

Table 4. 1H NMR data of compounds G1- G8 (1H 400MHz; δ in ppm, J in Hz)

Position G1 G2 G3 G4 G5 G6 G7 G8

1 2.84 (2H, 2.84 (2H, m) 2.81 (2H, 2.87 (2H, 2.81 (2H, 2.80 (2H, 2.85 (2H, 7.50 (1H, d,
t, J = 7.6) brd, J=6.8) brd, J=6.8) brd, J=6.8) brd, brd, J = 16)
J=6.8) J=6.8)

2 2.71 (2H, 2.84 (2H, m) 2.73 (2H, 2.79 (2H, 2.73 (2H, 2.78 (2H, 2.79 (2H, 6.63 (1H, d,
t, J = 7.6) brd, J=6.8) brd, J=6.8) brd, J=6.8) brd, brd, J = 16)
J=6.8) J=6.8)

4 2.39 (2H, 6.83 (1H, d, 2.52-2.54 2.52 –2.54 2.52 -2.54 2.48-2.55 2.42-2.65 2.4-2.65
t, J = 7.6) J=16.0) (2H, m) (2H, m) (2H, m) (2H, m) (2H, m) (2H, m)

5 1.56 (2H, 6.09 (1H, dt, 4.03 (1H, m) 4.03 (1H, m) 4.03 (1H, 4.05 (1H, 4.07 (1H, 3.77 (1H, m)
brt, J = J=16, 1.6) m) m) m)
6.4)

6 2.17 (2H, m) 1.49 (2H, 1.49 (2H,

m) m)

7 1.42 (2H, m)

9 0.9 (3H, t,
J=6.8)

10 0.89 (3H, 0.88 (3H, t, 0.88 (3H, t, 0.88 (3H, 0.88 (3H, t,
t, J = 6.8) J=6.8) J=6.8) t, J=6.8) J = 6.5)

12 0.90 (3H,
t, J=6.4)

14 0.87 (3H,

33
 t, J=6.4)

CH2 6-9 8-9 6-9 6-8 6-9 7-11 7-13 6-9

1.27-1.32 1.26-1.31 1.23-1.68 1.27-1.42 1.23-1.68 1.27-1.46 1.31-1.46 1.2–2.4 (8H,
(8H, m) (4H, m) (8H, m) (6H, m) (8H, m) (10H, m) (14H, m) m)
2՝ 6.71 ( 1H, 6.70 (1H, d, 6.67 (1H, d, 6.69 (1H, d, 6.67 (1H, 6.7 (1H, 6.71 (1H, 7.06 (1H, d,
d, J=1.9) J= 1.6) J= 2) J=2) d, J=2) d, J= 2) d, J= 2) J = 2)

5՝ 6.83 (1H, 6.81 (1H, d, 6.81 (1H, d, 6.72 (1H, d, 6.81 (1H, 6.84 6.84 (1H, 6.93 (1H, d,
d, J = 8) J= 7.6) J=8) J=8) d, J=8) (1H,d, d, J= 8) J = 8)
J=7.6)

6՝ 6.67 (1H, 6.66 (1H, 6.64 (1H, 6.66 (1H, 6.64 (1H, 6.68 (1H, 6.67 (1H, 7.11 (1H,
dd, J = 1.9, dd, J=8, 2) dd, J=8, 2) dd, J=8, 2) dd, J=8, 2) dd, J=7.6, dd, J=8, dd, J = 8, 2)
8) 2) 2)

OCH3 3.88 (3H, 3.82 (3H, s) 3.84 (3H, s, 3.84 (3H, s) 3.84 (3H, 3.89 (3H, 3.88 (3H, 3.94 (3H, s)
s) H-3՝ ) s) s) s)

3.92 (3H, s,
H-4՝ )

Table 5. 13C NMR data of compounds G1- G8 (13C 125MHz; δ in ppm)

Position G1 G2 G3 G4 G5 G6 G7 G8

1 31.5 31.1 31.7 31.7 31.7 31.7 31.7 144.8

2 41.7 41.5 45.0 45.0 45.0 45.0 45.0 130.2

3 209.0 198.9 209.0 209.0 209.0 209.0 209.0 199.6

4 44.1 130.3 50.0 50.0 50.0 50.0 50.0 41.7

5 23.6 147.3 67.5 67.5 67.5 67.5 67.5 76.8

6 29.0 32.2 37.2 37.2 37.2 37.2 37.2 32.5

7 28.8 27.5 25. 6 29.0 25. 6 25.6 25. 6 27.1

8 29.3 29.6 29.0 22.3 29.0 29.0 29.3 29.5

9 22.3 22.2 22.3 13.4 22.3 29.0 29.3 22.6

10 13.4 13.4 13.4 - 13.4 29.3 29.0 14.1

34
 11 - - - - - 22.3 29.0 -

12 - - - - - 13.4 29.0 -

13 - - - - - - 22.3 -

14 - - - - - - 13.4 -

1՝ 132.7 132.8 132.7 132.7 132.7 132.7 132.7 114.4

2՝ 111.9 111.5 111.9 111.9 111.9 111.9 111.9 119.6

3՝ 147.3 147.1 147.3 147.3 147.3 147.3 147.3 147.7

4՝ 144.0 144.7 144.7 114.7 144.7 144.7 144.7 145.4

5՝ 114.7 114.8 114.7 144.7 114.7 114.7 114.7 127.8

6՝ 120.5 120.6 120.5 120.5 120.5 120.5 120.5 134.5

OCH3-3՝ 55.3 55.5 55.3 55.3 55.3 55.3 55.3 56.3

OCH3-4՝ - - 55.9 - - - - -

35