Anti-Inflammatory and Anti-Arthritic Activity of Type-A Procyanidine

Available online at www.sciencedirect.com

Food Science and Human Wellness 2 (2013) 59–67

Anti-inﬂammatory and anti-arthritic activity of type-A procyanidine

polyphenols from bark of Cinnamomum zeylanicum in rats

a a,∗ b b

Sachin Vetal , Subhash L. Bodhankar , Vishwaraman Mohan , Prasad A. Thakurdesai

Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune 411 038, India

Indus Biotech Private Limited, 1, Rahul Residency, Off Salunke Vihar Road, Kondhwa, Pune 411 048, India

Received 27 December 2012; received in revised form 26 February 2013; accepted 20 March 2013

Abstract

Type-A procyanidine polyphenols (TAPP) are reported to have immunomodulatory and anti-inﬂammatory potential in vitro. The objective of

present work is to evaluate potential of TAPP extracted from Cinnamon (Cinnamomum zeylanicum) bark in animal models of inﬂammation and

rheumatoid arthritis in rats. Carrageenan-induced rat paw edema (CPE) and adjuvant induced established arthritis (AIA), in rats were used as

the experimental models for inﬂammation and arthritis respectively. Analgesic activity was evaluated in Randall–Selitto assay in AIA rats. TAPP

showed signiﬁcant anti-inﬂammatory effect at dose of 4, 8 and 25 mg/kg, p.o. but not at 2 mg/kg, p.o. dose in CPE model. The dose of 8 mg/kg, p.o.

was selected for the evaluation of anti-arthritic activity in AIA model. TAPP (8 mg/kg, p.o., daily from day-12 to day-21) treatment in established

arthritic rats showed signiﬁcant reversal of changes induced in AIA with respect to body weight drop (cachexia), ankle diameter, arthritic score,

serum C-reactive protein (CRP) levels. Moreover, TAPP was found to be non-ulcerogenic as compared to AIA control rats. However, TAPP did not

show analgesic effect on AIA-induced pain as seen in Randall–Selitto assay. In conclusion, TAPP showed disease-modifying potential in animal

models of inﬂammation and arthritis in rats.

Keywords: Type-A procyanidine polyphenols; Cinnamon bark; Anti-inﬂammatory; Rheumatoid arthritis

1. Introduction and limits in the functions of the joints. The systemic ram-

iﬁcations of the disease, apart from morbidity and mortality,

Rheumatoid arthritis (RA) is a chronic, inﬂammatory, include cardiopathy, nephropathy, vasculopathy and pulmonary

autoimmune disease, affecting freely movable joints, such as and cutaneous disorders [1]. Although the cause of rheumatoid

hand, knee and shoulder joints. The symptoms of active RA arthritis is unknown, autoimmunity plays a pivotal role in both its

include pain, swelling, morning stiffness, warmth, redness, chronicity and progression, and RA is considered as a systemic

autoimmune disease.

Existing treatment therapies for RA usually focused on

anti-inﬂammatory activity. They help to check the process of

Abbreviations: TAPP, type-A procyanidine polyphenols; CPE, carrageenan-

inﬂammation and thus may also help in the repair process. Med-

induced rat paw edema; AIA, adjuvant induced established arthritis; CRP,

ications like non-steroidal anti-inﬂammatory drugs (NSAIDs),

C-reactive protein; FCA, Freund’s complete adjuvant; DTH, delayed-type

hypersensitivity; LPS, lipopolysaccharide; RA, rheumatoid arthritis; NSAID, corticosteroids and analgesics are used to suppress the symptoms

non-steroidal anti-inﬂammatory drug; DMARD, disease-modifying anti- while disease-modifying anti-rheumatic drugs (DMARDs) and

rheumatic drugs.

∗ biological response modiﬁers often required to inhibit or halt

Corresponding author at: Department of Pharmacology, Poona College of

the underlying immune process and prevent long-term damage.

Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Paud Road,

Although NSAIDs, DMARDs and corticosteroids appear to

Pune 411 038, India. Tel.: +91 20 24537237x29; fax: +91 20 25439386.

E-mail address: [email protected] (S.L. Bodhankar). be highly efﬁcient drugs in the treatment of rheumatoid arthritis,

Peer review under responsibility of Beijing Academy of Food Sciences. they may cause side effects that can range in severity from mild

to serious. The major adverse drug reactions (ADRs) associ-

ated with NSAIDs are gastrointestinal (ulceration or bleeding),

and cardiovascular (myocardial infraction) with effects on other

systems. Despite of its wide clinical use, gastrointestinal (GI)

toxicity, upper GI adverse events such as perforation, ulceration

http://dx.doi.org/10.1016/j.fshw.2013.03.003 and bleeding in about 20% of patients taking long-term NSAIDs

60 S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67

is a major clinical limitation. Besides, some NSAIDs were with- with unknown mechanism. Further, TAPP has been shown

drawn from the market because of the risk of heart attacks and anti-inﬂammatory effects in cell culture studies in vitro [16].

stroke in some and some others had “Black box” warning in the Recently, we have demonstrated ameliorative effects of TAPP

package insert addressing risk of suffering from heart attacks from cinnamon bark in animal model of allergic asthma [19].

and/or stroke [2]. On the other hand, other therapies against However, TAPP has not yet been evaluated in vivo using animal

arthritis such as DMARDs and biological agents had a risk of models of rheumatoid arthritis (RA). The objective of present

immunosuppression and serious infection respectively on long- work is to evaluate efﬁcacy and safety of TAPP isolated from C.

term usage. Therefore, search for safer drugs for management zeylanicum in animal model of acute inﬂammation and RA in

of rheumatoid arthritis for chronic use is still going on. rats.

Cinnamon (Cinnamomum zeylanicum Syn C. verum, family:

Lauraceae) bark is one of the oldest herbal medicines mentioned

2. Materials and methods

in many traditional text for inﬂammatory [3], and pain related

to disorders such as enteralgia (acute intestinal pain), bronchi-

2.1. Materials

tis, and rheumatism [4]. It is native to Sri Lanka, Mayanmar

(Burma) and the southern coastal strip of India. In Chinese

Wistar rats of either sex (130–200 g) were obtained from

traditional medicine, cinnamon is indicated as an analgesic

National Toxicology Center, Pune, India. The rats were divided

and antipyretic agent against cold, fever, headache, myalgia

into groups having six rats per group and housed in polypro-

(mascular pain), arthralgia (arthritic pain) and amenorrhea (fail- ◦

pylene cages at a temperature of (24 ± 1) C with 12 h:12 h

ure of menstruation). In Indian traditional literature including

dark–light cycle, with free access to standard pellet feed (Chakan

Ayurveda, many other valuable actions are attributed to cinna-

Oil Mill, India) and ﬁltered water. All experiments were carried

mon bark [5].

out between 08:00 am and 17:00 pm in a quiet laboratory. The

Many scientiﬁc pharmacological investigations are also

research protocol was approved (No. CPCSEA/48/08) by Insti-

reported on anti-inﬂammatory potential of the bark of many

tutional Animal Ethics Committee (IAEC) and as per Indian

species of cinnamon [4,6,7]. The anti-inﬂammatory action of

norms laid down by Committee for the Purpose of Control

the Japanese species Cinnamomum seiboldii [8] and Cinnamomi

and Supervision of Experimental Animals (CPCSEA), New

cortex [9] has been attributed to a series of tannins. The antinoci-

Delhi.

ceptive activity (analgesic) [10] and antipyretic (fever reducing)

The test compound, TAPP was provided by Indus Biotech Pri-

activity C. verum bark [8] were also been reported.

vate Limited, Pune, India, after characterization as per reported

An interesting factor about cinnamon is that it can act both

procedures [19–21]. The TAPP is a standardized extract of C.

as an immunostimulant and a suppressant depending on species

zeylanicum bark with pentameric type-A procyanidine ﬂavonoid

and doses [11]. Another important activity is the immunomod-

as a marker compound (75.9% purity) with presence of trimer

ulatory effects exerted by cinnamon bark. In vitro inhibitory

and tetramer. The solutions of TAPP was freshly prepared daily

activity against complement formation has been documented for

by dissolving in mixture of propylene glycol and distilled water

cinnamon cortex and oil [11]. The extract of cinnamon bark is

in ratio of 30:70 to make solution of 1 mg/mL concentration and

reported to have anti-complementary [12] and immunosuppress-

administered orally.

ive [13] activity. Cinnamon bark’s potential for inﬂammation,

pain and immune system makes it a good candidate as an anti-

arthritic agent [14]. However, the component (s) responsible for 2.2. Anti-inﬂammatory activity against CPE model

these activities is not known. Cinnamon bark polyphenol extract

had been shown anti-inﬂammatory properties in vitro and ther- Anti-inﬂammatory activity was evaluated on the basis of the

apeutic potential for prevention and treatment of inﬂammation inhibition of the CPE [22,23]. Each rat in the experimental group

related diseases [15,16]. (six rats each) was treated orally with 2, 4, 8 and 25 mg/kg

The oligomeric end products of the ﬂavonoid biosynthetic body weight, 1 h before an edematogenic agent, carrageenan

pathway in Cinnamon are procyanidins whose building blocks (Sigma–Aldrich, USA) injection. Carrageenan (0.1 mL of a 1%

−

are (+)-catechin and ( )-epicatechin [17]. Proanthocyani- suspension prepared in 0.9% NaCl) was injected into the sub-

din (procyanidin, oligomeric proanthocyanidins, leukocyanidin, planter aponeurosis of the left hind paw of rats with 26-gauge

leucoanthocyanin and condensed tannins) is a class of ﬂavanols, needle. One group of rats was treated orally with 5 mg/kg body

are now identiﬁed and recognized for their favorable effects in weight of diclofenac sodium as a standard drug. Separate vehicle

human beings. control group was also maintained. Paw volume was measured

The anti-inﬂammatory action has been attributed to polyphe- immediately after carrageenan injection (time 0) and at 1, 3,

nolic components such as tannins [8] and procyanidins [17] from 6 and 24 h using a plethysmometer (Model 7150, Ugo Basile,

a variety of natural products. Based on the linkage between Italy). Mean difference in paw volume (mL) was calculated

the successive monomeric units, procyanidins are classiﬁed with baseline (0 h) values for each rat. Percent (%) inhibition

as Types A, B or C procyanidine polyphenols. Type-A pro- of paw volume at 3 h were calculated with a reported formula

cyanidine polyphenols (TAPP) from cinnamon bark has been [24] from mean difference in mean paw volume (mL) in treated

reported to exhibit activities against microorganisms [18], sug- (Vt) and control (Vc) group according to the equation: % inhibi-

gesting a potential role of TAPP in regulating immune function tion = (Vt/Vc) × 100.

S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67 61

2.3. Effect on adjuvant-induced established polyarthritis (end of the study). Pain latencies were measured by the threshold

(AIA) stimulus for reaction (escape or paw withdrawal) using a weight

of 500 g applied to the pads of hind paws.

Arthritis was induced according to method described ear-

lier [25] and as modiﬁed for therapeutic schedule. AIA was 2.6. Serum turbidity measurements

induced in 6–8 week-old rats by a single intradermal injection

of 0.1 mL Freund’s complete adjuvant (FCA) into the footpad On day-21, rats were sacriﬁced by cervical dislocation

of the right hind paw. FCA contains 0.6 mg heat-inactivated after administration of light ether anesthesia. Blood was with-

Mycobacterium tuberculosis H37Ra (Difco Laboratory, Detroit, drawn by cardiac puncture. The serum turbidity of sacriﬁced

MI, USA) emulsiﬁed in a sterile mixture of parafﬁn oil, saline arthritic rats on day-21 was measured by earlier reported

and Tween-80. Arthritis was allowed to develop for next 12 d. method [32]. To 0.1 mL of nonhaemolyzed serum, 2.9 mL

On day 12, animals were randomly assigned to one of the follow- of 0.067 mol/L sorenson phosphate buffer composition: 2 mL,

ing treatment groups: arthritic untreated controls (AIA controls), 0.067 mol/L monopotassium phosphate + 98 mL, 0.067 mol/L

arthritic rats treated with diclofenac sodium (1 mg/(kg·d)) and disodium phosphate in saline (pH 5.2) was added and gently

arthritic rats treated TAPP (8 mg/(kg·d)). These groups of rats agitated. The mixture was allowed to stabilize at room tempera-

were orally treated once daily with vehicle (distilled water), ture for 15 min before being incubated in a constant temperature

◦

diclofenac sodium or TAPP respectively from the day-12 to water bath at 69 C for 30 min. The samples were cooled in an

day-21 by oral feeding needle. Body weight and pain latency ice bath and absorbance was read as serum turbidity in a spec-

of inﬂamed paw, ankle diameter, arthritis score and serum C- trophotometer (Shimadzu, Japan) at the wavelength of 645 nm.

reactive protein (CRP) levels were measured on day-0 (baseline,

before arthritis induction), day-12 and day-21 of the study. Total 2.7. Microscopic study

body weight was recorded every 3rd day from day 3 to day 21.

The increase in body weight was calculated relative to that at Microscopic evaluation was done by analyzing the histology

day 0 allowing monitoring of the decrease in body weight gain of stomach sections [60]. Freshly excised stomach of one ani-

associated with arthritis [26]. The diameter of the inﬂamed paw mal from each group was washed with saline and preserved

joint (ankle) was measured by Vernier Caliper [27]. The joint in 10% formaldehyde solution for histopathological studies.

was compressed by pressing the gauge till pain was elicited as They were processed for 12 h using isopropyl alcohol, xylene

indicated by squeaking or leg withdrawal. The distance moved and parafﬁn. Then sections of stomach were cut (5 ␮m thick-

by the screw gauge was recorded. ness) with microtome. Sections were deparafﬁnation and stained

Swelling in hind paws were recorded by using a plethys- using hematoxylin and eosin (H&E) stain. The sections were

mometer (UGO Basile Italy.) on day-0, day-12 and day-21 then observed and photomicrographs were taken for histology

after the FCA injection [28]. The rats were scored regularly assessment.

for arthritis severity score based on swelling of paws [29] by

two investigators who were blind to the treatment. Each paw 2.8. Statistical analysis

was graded according to the severity and extent of erythema

and swelling of periarticular soft tissues in the enlargement and Data were obtained for each set of parameters separately

distortion of the joints. The arthritis scores were ranged from 0 for experiments namely CPE and AIA model and expressed as

(no sign) to 4 (severe lesions), yielding a maximum score of 16 means SEM. Data were analyzed by two-way repeated meas-

per animal. Pain stimulus was measured by the method reported ures ANOVA followed by Bonferroni posttests separately for

earlier [30]. each parameter. Data for serum turbidity measurement were ana-

lyzed by one-way ANOVA followed by Dunnett’s test. Level of

2.4. Biochemical estimations signiﬁcance was set at P < 0.05. All statistical tests were carried

out using Prism 5.0 (Graph Pad, San Diego CA, USA) statistical

The measurement of C-reactive protein was carried out software.

according to earlier reported method [31]. Blood samples were

withdrawn retro orbital puncture under light anesthesia on day- 3. Results

0, day-12 and day-21 of the study. The serum was separated

and samples were analyzed for CRP by commercially avail- 3.1. Effect of TAPP in CPE model

able enzyme-linked immunesorbent assay (ELISA) kit for CRP

(R&D system, USA) according to the manufacturers’ recom- Subplanter injection of carrageenan produced signiﬁcant

mendations. amount of edema (increase in paw volume) in all the groups of

animals. Increase of paw volume (edema) was observed within

2.5. Randall–Selitto assay an hour (paw volume increases by 0.69 mL). This edema was

sustained up to 6 h (increase by 1.65 mL) (Table 1). The standard

Pain latencies were measured using Randall–Selitto NSAID, diclofenac sodium (5 mg/kg, p.o.) caused signiﬁcant

Analgesymeter (Ugo basile Model 7200) during AIA model on reduction in edema (anti-inﬂammatory effect) compared with

day 0 (induction of AIA), day-12 (start of treatments) and day-21 vehicle treated rats (P < 0.001) at 1 h of treatment until 6 h.

62 S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67

Table 1

Effect of various doses of TAPP against carrageenan-induced rat paw edema (CPE) in rats.

Treatment (dose in mg/kg, oral) Difference of paw volume (mL) % Inhibition of edema at 3 h

1 h3h 6 h 24 h

Vehicle control 0.69 ± 0.02 1.42 ± 0.07 1.65 ± 0.11 0.89 ± 0.14 –

*** *** *** ***

± ±

Diclofenac sodium (5) 0.14 0.03 0.17 0.05 0.24 0.11 0.17 0.02 88.02

± ± ±

TAPP (2) 0.58 0.09 1.31 0.07 1.55 0.09 0.74 ± 0.12 7.74

* *** ***

TAPP (4) 0.86 ± 0.04 1.03 ± 0.04 1.01 ± 0.13 0.01 ± 0.02 27.47

*** *** ***

TAPP (8) 0.67 ± 0.03 0.92 ± 0.04 1.04 ± 0.13 0.46 ± 0.07 35.21

* *** *** ***

TAPP (25) 0.32 ± 0.08 0.52 ± 0.13 0.76 ± 0.18 0.28 ± 0.06 63.38

Data are represented as Mean change paw volume (mL) ± SEM, n = 6. The difference of paw volume for each rat was calculated with respect to baseline readings

and was analyzed by Two-way repeated measures ANLOVA followed by Bonferroni posttests. Compared to vehicle control at respective time point, signiﬁcance

represented as: * P < 0.05; **P < 0.01; *** P < 0.001.

Oral treatment of the test compound, TAPP showed signiﬁcant 3.2.2. Effect on ankle diameter in AIA rats

(P < 0.001) anti-inﬂammatory effects at 4 mg/kg, 8 and 25 mg/kg Ankle diameter of inﬂamed paw in AIA rats were found to

dose levels. However, at 2 mg/kg, p.o. dose, TAPP did not show increase signiﬁcantly (P < 0.001) in all rats on day-12 compared

anti-inﬂammatory effect. The onset of effect for TAPP was at with day-0 of the study (Table 2). On day-21 of the study, mean

3 h, 3 and 1 h for dose levels of 4, 8 and 25 mg/kg respectively. ankle diameters of rats with AIA rats treated with vehicle did not

At 3 h, TAPP (8 and 25 mg/kg) showed 27.47%, 35.21% and have signiﬁcant change (Table 2) compared with day-12. On the

63.38% inhibition of edema respectively compared to 88.02% other hand, signiﬁcant (P < 0.001) reduction in ankle diameter

inhibition showed by diclofenac sodium (5 mg/kg) treatment. of AIA rats was found on day-21 compared with day-12 with

treatment of diclofenac sodium (from 7.64 mm to 6.82 mm) and

TAPP (from 8.30 mm to 7.30 mm) (Table 2).

3.2. Effect of TAPP on AIA in rats

3.2.1. Effect on body weight reduction (Cachexia) in AIA 3.2.3. Effect on serum C-reactive protein levels in AIA in

rats rats

Single intradermal injection of FCA into the footpad of the The C-reactive protein (CRP) level in serum samples were

rat hind paw brought signiﬁcant (P < 0.001) reduction in body found to increase on day-12 of the study in all rats (Table 2).

weight (cachexia) in all the rats on day-12 of the study. In The increase in serum CRP levels was sustained on day-21 in

AIA control rats treated with vehicle, the weights were further AIA control rats. On day-21, signiﬁcant reduction of CRP levels

reduced (from 202.50 g to 184.67 g) during day-12 to day-21 were found in serum samples of AIA rats treated with diclofenac

(Table 2). In the same period (day-12 to day-21), subacute sodium (from 10.24 mg/mL to 7.47 mg/mL) and TAPP (from

oral administration of diclofenac sodium (1 mg/kg) halted the 10.28 mg/mL to 8.15 mg/mL) (Table 2).

cachaxia of AIA (P < 0.01). The body weights were increased

from 197.17 g to 206.5 g after diclofenac sodium treatment. Sim- 3.2.4. Effect on total arthritis score in AIA rats

ilar cachexia reversal effect was found in rats after subacute The total arthritis score in inﬂamed paw was increased sig-

oral treatment (day-12 to day-21) with TAPP in AIA. TAPP niﬁcantly in 12 d after induction of arthritis (Table 3). Further,

(8 mg/kg) showed signiﬁcant (P < 0.05) increase in body weight increase in total arthritis score was found in AIA control rats

from 196.50 g to 208.33 g. on day-21 of the study (Table 3). However, signiﬁcant reduction

Table 2

Effect of TAPP (8 mg/kg, p.o.) on body weight, ankle diameter and serum C-reactive protein (CRP) levels against FCA-induced established polyarthritis (AIA) in

rats.

Parameter Day of study AIA control Diclofenac sodium (1) TAPP (8)

Body weight (g) 0 215.67 ± 2.04 213.50 ± 3.41 218.17 ± 4.16

### ### ###

12 202.50 ± 1.48 197.17 ± 1.01 196.50 ± 1.77

*** ** *

21 184.67 ± 0.95 206.50 ± 1.57 208.33 ± 2.11

Ankle diameter (mm) 0 5.82 ± 0.23 5.70 ± 0.30 5.81 ± 0.17

### ### ###

12 7.71 ± 0.10 7.64 ± 0.38 8.30 ± 0.15

ns *** ***

21 7.88 ± 0.07 6.82 ± 0.38 7.30 ± 0.12

Serum C-reactive protein (CRP) 0 7.33 ± 0.03 7.01 ± 0.09 7.39 ± 0.53

# ## ##

(mg/mL) 12 9.38 ± 0.18 10.24 ± 0.14 10.28 ± 0.43

ns * **

21 8.77 ± 0.04 7.47 ± 0.24 8.15 ± 0.05

Data are represented as mean ± SEM, n = 6. Data for each parameter were separately analyzed by two-way repeated measures ANOVA followed by Bonferroni

posttests. Compared to day-12 for respective treatment group, signiﬁcance represented as: * P < 0.05; ** P < 0.01; *** P < 0.001. Compared with day-0 reading for

# ## ###

respective treatment group, signiﬁcance represented as: P < 0.05; P < 0.01; P < < 0.001.

ns - not signiﬁcant as compared to day-12 for respective treatment group.

S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67 63

Table 3

Effect of TAPP (8 mg/kg, p.o.) on total arthritis score and pain latency against FCA-induced established polyarthritis (AIA) in rats.

Parameter Day of study AIA control Diclofenac sodium (1) TAPP (8)

Total arthritis score 0 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

# # #

± ±

12 6.67 0.33 6.83 0.17 6.17 0.17

* * *

± ± ±

21 8.50 0.22 2.67 0.33 2.00 0.26

Pain latency (s) in Randall–Sellito 0 16.83 ± 0.70 18.08 ± 0.70 19.50 ± 0.52

# # #

assay 12 3.83 ± 0.21 4.08 ± 0.30 5.42 ± 0.79

ns ns ns

21 2.25 ± 0.50 3.58 ± 0.45 6.92 ± 0.61

Data are represented as mean ± SEM, n = 6. Data of each parameter were analyzed separately by two-way non-parametric ANOVA followed by Bonferroni “t” test.

Compared to day-12 for respective treatment group, signiﬁcance represented as: P < 0.05. Compared with day-0 reading for respective treatment group, signiﬁcance

represented as: P < 0.05.

ns - not signiﬁcant as compared to day-12 for respective treatment group.

in arthritis score of AIA was brought by subacute treatment of increase (from 0.1295 to 0.1621, P < 0.05) in absorbance com-

diclofenac sodium (from 6.83 to 2.67) or TAPP (from 6.17 to pared with AIA control (Fig. 1).

2.00) as observed on day-21 of the study (Table 3).

3.2.7. Histology of gastric mucosa in AIA rats

Gastric mucosal superﬁcial layer of stomach samples of rats

3.2.5. Effect on pain latency of inﬂamed paw (Randall and

removed from normal non-arthritic (Fig. 2A) rats were found

Selitto assay) in AIA rats

to be smooth. The mucosal epithelial layer of stomach sections

The pain latency tested in Randall and Selitto analgesiometer

of AIA control rats treated with vehicle (Fig. 2B) or TAPP at

was found to decrease signiﬁcantly in all rats. The reduction

oral dose of 8 mg/kg (Fig. 2D) were also found to be normal and

was found to be as high as 19.50 to as low as 3.83 (Table 3).

smooth with mild capillary dilation. However, superﬁcial layer

The pain latencies were found to further decrease to 2.25 sec in

of gastric mucosa of AIA rat treated with subacute treatment of

AIA control rats. Subacute administration of diclofenac sodium

diclofenac sodium (1 mg/kg, p.o.) had multiple areas of erosions

or TAPP in AIA rats did not change pain latencies on day-21

and hemorrhage (Fig. 2C).

compared to day-12 (Table 2).

4. Discussion

3.2.6. Effect on change in serum turbidity in AIA rats

Absorbance of various serum samples on day-21 in AIA

The cinnamon bark polyphenol extract is water-soluble and

control rats are presented in Fig. 1. The serum samples of non-

can be standardized to type-A proanthocyanidins [33]. In the

arthritic rats showed absorbance of 0.3493 whereas AIA control

present study, we are reporting anti-inﬂammatory and anti-

rats (vehicle treated) reduced absorbance to 0.1295 (P < 0.001).

arthritic potential of type-A proanthocyanidins from cinnamon

The subacute treatment with diclofenac sodium in AIA con-

bark, TAPP, in animal models.

trol rats from day-12 to day-21 did not cause any change in

The recognition of different mediators for different phases

absorbance compared with serum samples of AIA control rats.

of CPE has important implications for interpreting the anti-

The serum samples of AIA rats treated with TAPP showed mild

inﬂammatory effect of the drugs. The development of such

edema in the rat paw is a biphasic event. The initial phase of

the edema has been attributed to the release of histamine and

serotonin which is then maintained during the plateau phase

because of kinin-like substances [34]. Further, involvement of

histamine and serotonin with other mediators in the pathogenesis

of rheumatoid arthritis was also reported by many researchers

[35–37]. Effectiveness of the test compounds in the 1st hour

after carrageenan injection is indicative of their antihistaminic

and/or anti-serotonin action. The 2nd accelerating phase of car-

rageenan edema is attributed to release of prostaglandins [38].

Our test compound, TAPP was not effective in the 1st hour of

treatment but was found effective in the 2nd phase at 8 mg/kg

and higher dose (observed at 3 h) thus suggesting prostaglandins

inhibition effect of TAPP (Table 1). However, possibility of other

Fig. 1. Effect of TAPP (8 mg/kg) on serum turbidity of FCA-induced arthritic

mechanism such as inhibition of leukocyte emigration by TAPP

rats on day-21. Data are represented as absorbance at 645 nm ± SEM and was

cannot be ruled out.

analyzed by one-way ANOVA followed by Dunnett’s “t” test. Absorbance of

### The ability of anti-inﬂammatory test compounds to offer

serum samples was measured on day-21 of the study. n = 6. P < 0.001 as

compared with non-arthritic rats; *P < 0.05 as compared with arthritic control beneﬁts in clinical management of rheumatic diseases and so

group. modiﬁcation of arthritic syndromes in laboratory animals is an

64 S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67

Fig. 2. Photomicrograph of stomach on day-21 of the study. The ﬁgure shows sections of stomach of (A) normal rats, (B) AIA control rats with treatment from

day-12 to day-21 of vehicle (distilled water), (C) diclofenac sodium (1 mg/kg, p.o.) treated arthritic rats shows erosion (arrow) with hemorrhage at the superﬁcial part

of the mucosa and (D) TAPP (8 mg/kg, p.o.) treated arthritic rats; M. mucosa, MM. muscularis mucosa, and L. lumen. Stained with hematoxylin and eosin (H&E)

at 40×.

important criterion in anti-inﬂammatory screening procedure. to AIA rats reduced ankle diameters of arthritic paw. There-

In 1963, AIA in rats was used as primary animal model for RA fore, the anti-inﬂammatory action of TAPP may be mediated

using intradermal administration of Freund adjuvant in oil emul- by prostaglandin and/or cytokine inhibition. These results are

sion [39]. AIA is a rather aggressive and monophasic form of also in line with reports that anti-inﬂammatory action of cinna-

arthritis. Usually, the AIA is severe and ﬁnally leads to complete mon bark has been attributed to polyphenolic component such

ankylosis and permanent joint deformations similar to progres- as tannins [8] and procyanidines [17]. The inhibition of lipid

sive symptoms of RA patients. AIA has been used extensively as peroxidation, capillary permeability and fragility, and enzymes

an experimental model for studying immune-inﬂammatory pro- such as phospholipase A2, cyclooxygenase, and lipoxygenase

cesses of arthritic diseases in humans, in particular RA, as well as were reported as alleged mechanism for cinnamon tannins and

for screening and testing novel anti-arthritic agents [40]. There- polyphenols [8,17].

fore, we tested TAPP against established polyarthritis model of Analgesia is an important ancillary property of all anti-

AIA rats using therapeutic schedule of treatment [25,41]. We inﬂammatory agents. Most of the anti-inﬂammatory drugs

induced AIA in rats, allowed it to develop for 12 d and evalu- increased pain threshold in various animal models [44]. This

ated effects of TAPP on day-21 of the study. The progression is natural because many endogenous chemical mediators of

of arthritis was conﬁrmed in our study by scoring total arthritis inﬂammation play a part in generating pain impulses (for exam-

lesions. The diseases progressed till day-12 and then became ple, histamine, serotonin and prostaglandins) and some other

quiescent. TAPP halted the progression which was evident by mediators such as bradykinin and cytokines, are involved in

signiﬁcant decrease shown in the total arthritic score (Table 3). prolongation of the sensation of the pain [45].

The inﬂammation associated with AIA is mainly depend- RA is an inﬂammatory condition of the joint that is associated

ent on prostaglandin E2 (PGE2) generated by cyclooxygenases with hyperalgesia and functional impairment [46]. Joint inﬂam-

(COXs) [40,42]. Besides, the role of cytokines like TNF-␣ and mation induced by AIA is associated with hyperalgesia and

IL-1 has also been implicated in this model [43]. TAPP treatment slight compression of the joint which caused leg withdrawal and

S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67 65

squeaking. The hyperalgesia associated with arthritis has been reported to be strongly correlated with decreases in serum tur-

reported to involve prostaglandin synthesis [47]. Increased levels bidity in AIA [59]. Therapeutically useful anti-inﬂammatory

of substance P, calcitonin gene-related peptide and up-regulation drugs were shown to reverse this decrease in serum turbidity

of neurokinin-1 receptor in the dorsal horn of the rat spinal [32]. Taken together, our results strongly pointed to disease-

cord have been reported to play an important role in modulat- modifying properties of TAPP and its potential to slow down

ing both inﬂammation and hyperalgesia in the FCA model [48]. the RA progression.

However, in the present study, TAPP did not prevent reduction One of the most prominent and serious side effects of

pain latency in Randall–Selitto assay. Therefore, nociception- NSAIDs is occurrence of gastrointestinal damage (ulcerogenic-

related mediators (substance-P, calcitonin gene-related peptide ity). Hence, it was necessary to test our compounds for their

and neurokinin-1) are not involved in mechanism of action of ulcerogenicity potential. In the present study, subacute treat-

TAPP. This also pointed toward the possibility of disease mod- ment of TAPP (8 mg/kg, p.o.) did not cause severe ulcerogenicity

iﬁcation as possible mechanism of action of TAPP against RA. as compared to signiﬁcant ulcerogenicity shown by diclofenac

AIA has been generally believed to be the result of a delayed- sodium (1 mg/kg, p.o.) in established AIA in rats.

type hypersensitivity (DTH) response to a disseminated antigen The absence of the analgesic and the ulcerogenicity poten-

probably derived from the injected bacterial cell wall [49]. AIA tial with reduction in serum CRP levels in AIA rats qualiﬁes

in rats is a useful model of inﬂammatory cachexia that mim- TAPP as a disease-modifying anti-rheumatic drug (DMARD).

ics the human pathophysiology in many important ways [50]. DMARDs suppress the rheumatoid process and bring about a

AIA induces inflammation as primary lesion which reached to remission, but do not have nonspecific anti-inflammatory or

peak after 3–5 d with secondary lesions occur after a delay of analgesic action. TAPP offer added advantage of moderate anti-

about 11–12 d. Secondary reactions in AIA are characterized inﬂammatory effects at daily dose of 8 mg/kg, p.o. in arthritic

by immune responses, inﬂammation and decrease of weight rats. The DMARD effects shown by TAPP can also be substanti-

(cachexia). In our study, the test compound, TAPP signiﬁcantly ated by earlier reports on cinnamon bark extracts. For example,

protected rats from cachexia because of arthritis induction. cinnamon bark was shown to inhibit gastric secretions [11] and

In the past, many plant-derived procyanidines reported to eradicate H. pylori (a bacteria known to exacerbate gastric acid-

have cytokine inhibitory effect in vitro [51,52]. Similar effects ity and ulcers) [61,62]. TAPP is also expected to be protective on

were also attributed to Cinnamon bark extract and polyphenols the cardiovascular system as cinnamon polyphenols especially

[16,53]. In vitro pre-challenge with cinnamon extract is reported TAPP by virtue of its antioxidant activity [63,64].

to suppress lipopolysaccharide (LPS)-induced cytokines expres-

sion [54]. Recently, immune regulatory action of TAPP in mouse

5. Conclusion

3T3-L1 adipocytes is attributed to TTP (tristetraprolin) [55].

Besides, TAPP is capable of affecting immune responses by

In conclusion, TAPP, type-A procyanidine polyphenols iso-

regulating anti-inﬂammatory mediators as well as the TTP gene

lated from the bark of C. zeylanicum showed anti-inﬂammory

expression in macrophages [16]. Taken together, cytokine inhi-

and anti-arthritic effects in animal models without ulcerogenic-

bition emerges as a probable mechanism of TAPP responsible

ity potential. Lack of analgesic activity in present study and

for cachexia reducing effects in AIA-induced established pol-

reports of immunomodulatory potential suggested TAPP as

yarthritis in rats. This probability is also supported by our result

potential DMARD. Further studies are required to explain the

that TAPP reduces serum C-reactive protein (CRP) levels of

mechanism of action of TAPP toward autoimmune and inﬂam-

rats on day-21 compared to day-12 levels (Table 2) whereas

matory disease processes.

AIA control rats showed sustained increase in CRP till day-21

(Table 2).

Acknowledgements

CRP is an acute-phase protein and has been identiﬁed as

an important biomarker for various inﬂammatory, degenerative,

The authors would like to acknowledge Dr. K.R. Mahadik,

and neoplastic diseases [56,57]. Elevated levels of CRP have

Principal, Poona College of Pharmacy, Bharati Vidyapeeth

been found in the blood during almost all diseases associated

Deemed University, Pune, India for providing necessary facil-

with active inﬂammation or tissue destruction, particularly in

ities to carry out the study. Research support was provided by

RA patients [57,58]. Sustained increase in serum CRP levels

Indus Biotech Private Limited but played no role in collection,

suggests a lasting production and stimulation of acute-phase

analysis and interpretation of data.

proteins during disease progression.

In our study, serum turbidity (showed by absorbance of

serum samples) of AIA control rats was found to be signiﬁ- References

cantly reduced compared with normal rats (Fig. 1). Treatment

[1] G.S. Firestein, VIP: a very important protein in arthritis, Natural Medicines

from day-12 to day-21 with TAPP but not diclofenac sodium

7 (2001) 537–538.

(an NSAID) prevented AIA-induced fall in turbidity (Fig. 1).

[2] E.J. Pennings, R.J. Verkes, J. de Koning, et al., Tranylcypromine intoxica-

The decrease in serum turbidity in AIA control rats, is known

tion with malignant hyperthermia, delirium, and thrombocytopenia, Journal

to be caused by denatured products and was shown to be cor-

of Clinical Psychopharmacology 17 (1997) 430–432.

related with severity [59] and immune response seen in AIA [3] P.K. Warrier, C. Ramankutty, V.P.K. Nambiar, et al., Indian medicinal

[60]. Further, increased levels of serum lysozyme levels were plants: a compendium of 500 species, Orient Longman, Madras, 1993.

66 S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67

[4] K.R. Kirtikar, B. Basu, E. Blatter, Indian medicinal plants, Bishen Singh [26] M. Koufany, D. Moulin, A. Bianchi, et al., Anti-inﬂammatory effect of

Mahendra Pal Singh, Dehra Dun, 1975. antidiabetic thiazolidinediones prevents bone resorption rather than carti-

[5] R.N. Khory, N.N. Katrak, Materia medica of India and their therapeutics, lage changes in experimental polyarthritis, Arthritis Research and Therapy

Neeraj Publishing House, Delhi, 1903. 10 (2008) R6.

[6] S.P. Ambasto, The useful plants of India, in: Publications and Informa- [27] V.L. Kumar, S. Roy, R. Sehgal, et al., A comparative study on the efﬁcacy

tion Directorate, Council of Scientiﬁc and Industrial Research, New Delhi, of rofecoxib in monoarticular arthritis induced by latex of Calotropis pro-

1986. cera and Freund’s complete adjuvant, Inﬂammopharmacology 14 (2006)

[7] L.V. Asolkar, R.N. Chopra, K.K. Kakkar, et al., Second supplement to 17–21.

glossary of Indian medicinal plants with active principles. Part 1 (A–K) [28] J. Hua, S. Suguro, S. Hirano, et al., Preventive actions of a high dose of

(1965–1981), in: Publications & Information Directorate, CSIR, New glucosamine on adjuvant arthritis in rats, Inﬂammation Research 54 (2005)

Delhi, 1992. 127–132.

[8] C.A. Newall, L.A. Anderson, J.D. Phillipson, Herbal medicines: a guide [29] L. Philippe, P. Gegout-Pottie, C. Guingamp, et al., Relations between func-

for health-care professionals, Pharmaceutical Press, London, 1996. tional, inﬂammatory, and degenerative parameters during adjuvant arthritis

[9] M. Kubo, S. Ma, J. Wu, H. Matsuda, Anti-inﬂammatory activities of 70% in rats, American Journal of Physiology 273 (1997) R1550–R1556.

methanolic extract from Cinnamomi cortex, Biological and Pharmaceutical [30] L.O. Randall, J.J. Selitto, A method for measurement of analgesic activity

Bulletin 19 (1996) 1041–1045. on inﬂamed tissue, Archives Internationales de Pharmacodynamie et de

[10] A.H. Atta, A. Alkofahi, Anti-nociceptive and anti-inﬂammatory effects of Therapie 111 (1957) 409–419.

some Jordanian medicinal plant extracts, Journal of Ethnopharmacology [31] X. Cai, Y.F. Wong, H. Zhou, et al., The comparative study of

60 (1998) 117–124. Sprague-Dawley and Lewis rats in adjuvant-induced arthritis, Naunyn-

[11] P.N. Ravindran, K. Nirmal Babu, M. Shylaja, Cinnamon and cassia: the Schmiedebergs Archives of Pharmacology 373 (2006) 140–147.

genus Cinnamomum, in: Medicinal and aromatic plants – industrial pro- [32] D. Kosersky, J. Brown, M. Malone, Effects of cryogenine on adjuvant-

ﬁles, vol. 36, CRC Press, Boca Raton, 2004, p. 361. induced arthritis and serum turbidity in rats, Journal of Pharmaceutical

[12] H.M. Chang, P.P.H. But, Pharmacology and applications of Chinese materia Sciences 62 (1973) 1965–1968.

medica, vol. 2, World Scientiﬁc, Singapore, 1986. [33] R.A. Anderson, C.L. Broadhurst, M.M. Polansky, et al., Isolation and

[13] W. Tang, G. Eisenbrand, Chinese drugs of plant origin: chemistry, phar- characterization of polyphenol type-A polymers from cinnamon with

macology, and use in traditional and modern medicine, Springer-Verlag, insulin-like biological activity, Journal of Agricultural and Food Chemistry

Berlin, 1992. 52 (2004) 65–70.

[14] K. Joshi, S. Awte, P. Bhatnagar, et al., Cinnamomum zeylanicum extract [34] W.G. Spector, D.A. Willoughby, The pharmacology of inﬂammation, in:

inhibits proinﬂammatory cytokine TNF-alpha: in vitro and in vivo studies, Modern medicine, English Universities, London, 1968.

Research In Pharmaceutical Biotechnology 2 (2010) 13–21. [35] J.N. Sharma, Involvement of the kinin-forming system in the physiopathol-

[15] H. Cao, R.A. Anderson, Cinnamon polyphenol extract regulates triste- ogy of rheumatoid inﬂammation, Agents Actions 38 (Suppl. (Pt 3)) (1992)

traprolin and related gene expression in mouse adipocytes, Journal of 343–361.

Agricultural and Food Chemistry 59 (2011) 2739–2744. [36] D.T. Scott, F.Y. Lam, W.R. Ferrell, Acute joint inﬂammation – mechanisms

[16] H. Cao, J.F. Urban Jr., R.A. Anderson, Cinnamon polyphenol extract affects and mediators, General Pharmacology 25 (1994) 1285–1296.

immune responses by regulating anti- and proinﬂammatory and glucose [37] S. Tanaka, S. Sohen, K. Fukuda, A role for histamine receptors in rheuma-

transporter gene expression in mouse macrophages, Journal of Nutrition toid arthritis, Seminars in Arthritis and Rheumatism 26 (1997) 824–833.

138 (2008) 833–840. [38] J.G. Lombardino, Nonsteroidal anti-inﬂammatory drugs, Wiley, New York,

[17] A.M. Fine, Oligomeric proanthocyanidin complexes: history, structure, and Chichester, 1985.

phytopharmaceutical applications, Alternative Medicine Review 5 (2000) [39] C.M. Pearson, Experimental joint disease observations on adjuvant-

144–151. induced arthritis, Journal of Chronic Diseases 16 (1963) 863–874.

[18] B. Shan, Y.Z. Cai, J.D. Brooks, et al., Antibacterial properties and major [40] M. Billingham, Models of arthritis and the search for anti-arthritic drugs,

bioactive components of cinnamon stick (Cinnamomum burmannii): activ- Pharmacology & Therapeutics 21 (1983) 389–428.

ity against foodborne pathogenic bacteria, Journal of Agricultural and Food [41] B.B. Newbould, Chemotherapy of arthritis induced in rats by mycobacterial

Chemistry 55 (2007) 5484–5490. adjuvant, British Journal of Pharmacology and Chemotherapy 21 (1963)

[19] A.D. Kandhare, S.L. Bodhankar, V. Singh, et al., Anti-asthmatic effects 127–136.

of type-A procyanidine polyphenols from cinnamon bark in ovalbumin- [42] G.D. Anderson, S.D. Hauser, K.L. McGarity, et al., Selective inhibition of

induced airway hyperresponsiveness in laboratory animals, Biomedicine cyclooxygenase (COX)-2 reverses inﬂammation and expression of COX-2

and Aging Pathology 3 (2013) 23–30. and interleukin 6 in rat adjuvant arthritis, Journal of Clinical Investigation

[20] S.A. Lazarus, G.E. Adamson, J.F. Hammerstone, et al., High-performance 97 (1996) 2672–2679.

liquid chromatography/mass spectrometry analysis of proanthocyanidins [43] Y.P. He, Z.Y. Li, X.D. Jiang, et al., Effects of TNF-alpha receptor blocking

in foods and beverages, Journal of Agricultural and Food Chemistry 47 peptide on adjuvant arthritis in rats, Yao Xue Xue Bao 38 (2003) 889–892.

(1999) 3693–3701. [44] R.A. Scherrer, M.W. Whitehouse, Anti-inﬂammatory agents: chemistry and

[21] S. Bhaskaran, V. Mohan, in: US Patent Ofﬁce (Ed.), A method of manag- pharmacology, Academic Press, New York, London, 1974.

ing broncho-constrictive condition, Indus Biotech Private Limited, Pune, [45] J.M. Besson, The complexity of physiopharmacologic aspects of pain,

India/USA, 2012. Drugs 53 (Suppl. 2) (1997) 1–9.

[22] C.A. Winter, E.A. Risley, W.G. Nuss, Carrageenin-induced edema in hind [46] Y. Nagakura, M. Okada, A. Kohara, et al., Allodynia and hyperalgesia in

paw of the rats as an assay for anti-inﬂammatory drugs, Proceedings of the adjuvant-induced arthritic rats: time course of progression and efﬁcacy of

Society for Experimental Biology and Medicine 111 (1962) 544–547. analgesics, Journal of Pharmacology and Experimental Therapeutics 306

[23] D.K. Roy, K.T. Kumar, S. Karmakar, et al., Pharmacological studies on (2003) 490–497.

Indian black tea (leaf variety) in acute and chronic inﬂammatory conditions, [47] J.P. Portanova, Y. Zhang, G.D. Anderson, et al., Selective neutralization

Phytotherapy Research 22 (2008) 814–819. of prostaglandin E2 blocks inﬂammation, hyperalgesia, and interleukin 6

[24] L.V. Nargund, V. Hariprasad, G.R. Reedy, Synthesis and anti-inﬂammatory production in vivo, Journal of Experimental Medicine 184 (1996) 883–891.

activity of ﬂuorinated phenyl styryl ketones and N-phenyl-5-substituted [48] P.I. Mapp, G. Terenghi, D.A. Walsh, et al., Monoarthritis in the rat knee

aryl-3-p-(ﬂuorophenyl) pyrazolins and pyrazoles, Journal of Pharmaceuti- induces bilateral and time-dependent changes in substance P and calcitonin

cal Sciences 81 (1992) 892–894. gene-related peptide immunoreactivity in the spinal cord, Neuroscience 57

[25] J.R. Ward, R.S. Cloud, Comparative effect of antirheumatic drugs on (1993) 1091–1096.

adjuvant-induced polyarthritis in rats, Journal of Pharmacology and Exper- [49] B.H. Waksman, C.M. Pearson, J.T. Sharp, Studies of arthritis and other

imental Therapeutics 152 (1966) 116–121. lesions induced in rats by injection of mycobacterial adjuvant. II. Evidence

S. Vetal et al. / Food Science and Human Wellness 2 (2013) 59–67 67

that the disease is a disseminated immunologic response to exogenous [57] M.A. van Leeuwen, M.H. van Rijswijk, Acute phase proteins in the mon-

antigen, Journal of Immunology 85 (1960) 403–417. itoring of inﬂammatory disorders, Baillieres Clinical Rheumatology 8

[50] R. Roubenoff, L. Freeman, D. Smith, et al., Adjuvant arthritis as a model (1994) 531–552.

of inﬂammatory cachexia, Arthritis and Rheumatism 40 (1997) 534–539. [58] I. Kushner, C-reactive protein in rheumatology, Arthritis and Rheumatism

[51] T. Mao, J. Van de Water, C. Keen, et al., Modulation of TNF-secretion in 34 (1991) 1065–1068.

peripheral blood mononuclear cells by cocoa ﬂavanols and procyanidins, [59] S.J. Piliero, C. Colombo, Action of antiinﬂammatory drugs on the lysozyme

Developmental Immunology 9 (2002) 135–141. activity and “turbidity” of serum from rats with adjuvant arthritis or

[52] X. Terra, G. Montagut, M. Bustos, et al., Grape-seed procyanidins prevent endocrine deﬁciency, Journal of Pharmacology and Experimental Ther-

low-grade inﬂammation by modulating cytokine expression in rats fed a apeutics 165 (1969) 294–299.

high-fat diet, Journal of Nutritional Biochemistry 20 (2009) 210–218. [60] G. Weissmann, The role of lysosomes in inﬂammation and disease, Annual

[53] B. Qin, H. Dawson, M.M. Polansky, et al., Cinnamon extract attenuates Review of Medicine 18 (1967) 97–112.

TNF-alpha-induced intestinal lipoprotein ApoB48 overproduction by reg- [61] M. Tabak, R. Armon, I. Neeman, Cinnamon extracts’ inhibitory effect

ulating inﬂammatory, insulin, and lipoprotein pathways in enterocytes, on Helicobacter pylori, Journal of Ethnopharmacology 67 (1999)

Hormone and Metabolic Research 41 (2009) 516–522. 269–277.

[54] G. Kanuri, S. Weber, V. Volynets, et al., Cinnamon extract protects against [62] Y. Nir, I. Potasman, E. Stermer, et al., Controlled trial of the effect

acute alcohol-induced liver steatosis in mice, Journal of Nutrition 139 of cinnamon extract on Helicobacter pylori, Helicobacter 5 (2000)

(2009) 482–487. 94–97.

[55] H. Cao, M.A. Kelly, F. Kari, et al., Green tea increases anti-inﬂammatory [63] J.S. Lee, S.M. Jeon, E.M. Park, et al., Cinnamate supplementation

tristetraprolin and decreases pro-inﬂammatory tumor necrosis factor enhances hepatic lipid metabolism and antioxidant defense systems in high

mRNA levels in rats, Journal of Inﬂammatory (London) 4 (2007) 1. cholesterol-fed rats, Journal of Medicinal Food 6 (2003) 183–191.

[56] M.B. Pepys, G.M. Hirschﬁeld, C-reactive protein: a critical update, Journal [64] C.C. Lin, S.J. Wu, C.H. Chang, et al., Antioxidant activity of Cinnamomum

of Clinical Investigation 111 (2003) 1805–1812. cassia, Phytotherapy Research 17 (2003) 726–730.