ORIGINAL ARTICLES

Evaluation of the Ant

i-inflammatory Effectsof Ellagic Acid

Stephanie Corbett, MSN, CRNA, Janice Daniel, MSN, CRNA, CPT,

Rachael Drayton, MSN, CRNA, CPT, Melanie Field, MSN, CRNA,

Rebecca Steinhardt, MSN, CRNA, Normalynn Garrett, COL(R), PhD, CRNA

Few studies have investigated the anti-inflammatory properties of ellagic

This study was

Nurse Anesthetists

Stephanie Corbe

at the VA San Die

Daniel, MSN, CRNA

nity Hospital, FT S

CPT, is a staff CRN

Stewart, GA; Mela

the Manhattan Ca

tem, New York, NY

214

acid and no published studies have examined the effects of ellagic acid

in combination with anesthetic adjuvants. In this study, 54 Sprague-

Dawley rats were assigned to one of six groups: (1) vehicle; (2) ketorolac

and vehicle; (3) meloxicam and vehicle; (4) ellagic acid and vehicle; (5)

ellagic acid, ketorolac, and vehicle; and (6) ellagic acid, meloxicam, and

vehicle. Groups 5 and 6 investigated interactions between ellagic acid

and cyclooxygenase inhibitors. Paw inflammation was induced with 3%

carrageenan and was measured with a plethysmometer at 30 minutes

and 4, 8, and 24 hours after intraperitoneal injection. All rats received

one intraperitoneal injection of equivalent volumes according to group as-

signment. Analysis of covariance followed by post hoc analysis determined

that ketorolac was the only compound to significantly reduce paw edema

at 4 hours (P 5 .019); ellagic acid alone (P5 .038) and the combination

of ellagic acid and ketorolac (P5 .038) were the only compounds to signif-

icantly reduce paw edema at 8 hours. At 24 hours, only ellagic acid was

effective (P 5 .01). Our findings suggest that ellagic acid may be effective

against inflammation, may have a prolonged onset and duration of

action, and may interact with known cyclooxygenase inhibitors.

Keywords: ellagic acid, anti-inflammatory, polyphenol, cyclooxygenase

inhibitor, animal studies, research.

� 2010 by American Society of PeriAnesthesia Nurses. All rights reserved.

THROUGHOUT THE LAST several decades, therehas been a significant escalation in the use of die-

tary supplements such as vitamins, herbals, and

medicinal foods in both the general public and

among military personnel. From 1990 to 1997,

funded by the American Association of

Foundation.

tt, MSN, CRNA, is a staff nurse anesthetist

go Medical Center, San Diego, CA; Janice

, CPT, is a staff CRNA atWill Army Commu-

tewart, GA; Rachael Drayton, MSN, CRNA,

A at Will Army Community Hospital, FT

nie Field, MSN, CRNA, is a staff CRNA at

mpus of the VA NY Harbor Healthcare Sys-

; Rebecca Steinhardt, MSN, CRNA, is a staff

Journ

more than 15 million adults in the United States re-ported using herbal supplements in conjunction

with prescribed medications, which accounted

for a growth of 380%.1 In addition, from 1997 to

2002, there was more than a 50% increase in the

CRNA at the Robert J. Dole VAMedical Center, Wichita, KS; and

Normalynn Garrett, COL(R), PhD, CRNA, is the Project

Director/CRNA for the Geneva Foundation, San Antonio, TX.

Address correspondence to Normalynn Garrett, 14702 War

Admiral, San Antonio, TX 78248; e-mail address: nlgarrett@

akimeka.com.

� 2010 by American Society of PeriAnesthesia Nurses. All

rights reserved.

1089-9472/$36.00

doi:10.1016/j.jopan.2010.05.011

al of PeriAnesthesia Nursing, Vol 25, No 4 (August), 2010: pp 214-220

EVALUATION OF THE ANTI-INFLAMMATORY EFFECTS OF ELLAGIC ACID 215

consumption of dietary supplements.2 A survey on

the use of dietary supplements in active duty

military personnel, as reported by Arsenault and

Kennedy,3 found that 64% ofmale soldiers entering

US Army Special Forces and Ranger TrainingSchool, and 71% of air, land, and sea Navy person-

nel report current use of dietary supplements. The

survey suggested that vitamins and minerals were

most commonly consumed, accounting for 54.6%

of usage, followed by performance enhancers

(24.7%) and herbals (20.7%).

The perianesthesia setting presents unique prob-lems related to the consumption of dietary supple-

ments. Temple and colleagues4 indicated an

increased prevalence of herbal use among patients

presenting for surgery. Their research conveyed

that 51% of surgical patients reported herbal use

2 weeks before surgery. Of these, 27% used an

herb that may inhibit blood coagulation and 30%

consumed an herb that could potentially affectblood pressure, heart rate or rhythm, electrolyte

levels, or mental alertness. Furthermore, 70% or

more of these patients may not disclose the use

of these remedies during routine, preoperative in-

terview. In addition, a 2004 survey of parents of

children presenting for ambulatory surgery found

that 16.6% of children had been administered

herbal preparations at home.5

If the majority of patients who use dietary supple-

ments fail to report use in the preoperative inter-

view, there may be increased risk to the patient

because of unknown interactions between anes-

thetic agents or pain management agents and

these supplements. Because use of these products

increases in the general population, so does thepotential for perianesthesia complications because

it is not known how many of these dietary supple-

ments may interact with anesthetic agents. Herbal

supplements have been shown to cause a variety

of clinical alterations that are undesirable in the

perianesthesia setting. For example, the herbal de-

rivative valerian has been shown to prolong emer-

gence time from anesthesia.6 St. John’s wort hasbeen shown to inducehepatic cytochromeP450en-

zymes,whichmay potentially alter the duration and

metabolism of many commonly used anesthetic

drugs.7 Undisclosed consumption or use of under-

studied dietary supplements may make it difficult

to establish and render safe treatment to patients

during the perianesthesia period.

Ellagic acid, a polyphenol compound extracted

from pomegranate juice, has become an increas-

ingly popular dietary supplement over recent years.

Abundant press have promoted pomegranate juice

as having antimutagenic properties in the treatmentof prostate cancer, reducing the incidence of cardio-

vascular disease, and even preventing erectile dys-

function.8-10 Newsweek reported that US sales of

pomegranate juice rose from $12-91 million an-

nually from 2003 to 2006. They further reported

that some pomegranate product companies have

funded more than $10 million in research projects

and equally as much in marketing.11 The term ella-

gic acid generates more than 575,000 queries in

an Internet search engine, with many Web sites of-

feringproducts purported to contain this derivative.

Popularity of ellagic acid has increased somuch that

the American Cancer Society (ACS) maintains a link

to information regarding ellagic acid on its Web site

(http://www.cancer.org). Various commercial Web

sites offer product comparison, report research find-ings, and suggest a wide variety of daily recommen-

ded dosages ranging from 900 to 2,000 mg/day.

Because dietary supplements are not under the

scrutiny and regulation of the Food andDrugAdmin-

istration, many product Web sites provide no infor-

mation regarding the purity or potency of these

supplements, nor do they post any possible adverse

sideeffects. Themechanismofaction,potential druginteractions, and ultimately the impact of the use of

unregulated dietary supplements is not known.

Very little information exists on the mechanism

of action of ellagic acid. Recently researchers

have begun to show possible involvement in the

inflammatory cascade through inhibition of cyclo-

oxygenase (COX) protein expression, as well asanti-inflammatory effects in the animal model.12

Research conducted by Rogerio et al13 suggests

that the mechanism of action of ellagic acid may in-

volve inhibition of the COX enzyme. If ellagic acid

does affect the COX enzyme it may share pharma-

codynamic effects such as platelet inhibition, with

known COX inhibitors such as nonsteroidal anti-

inflammatory drugs.14 Definitive evidence of ella-gic acid’s mechanism of action and potential side

effects has not yet been established.

If ellagic acid is a COX inhibitor, then it may be

a potent anti-inflammatory, antinociceptive agent.

Whereas several studies have examined the anti-

mutagenic and free radicalescavenging effects of

216 CORBETT ET AL

ellagic acid, fewhave investigated the anti-inflamma-

tory effects in vivo. Rogerio et al13 examined the

anti-inflammatory, antinociceptive effects of ellagic

acid in an animal model. Their findings suggest

that ellagic acid significantly decreases paw edemaas measured by calipers after an injection of 1% car-

rageenan and decreases the number of acid-induced

writhing periods inmice. This reduction inwrithing

periodsmay be fromCOX inhibition or another anti-

nociceptive pathway. In 2007, Beltz et al15 exam-

ined the antinociceptive effects of ellagic acid in

Sprague-Dawley rats using themodifiedhotplate. Al-

though the study by Beltz et al did not examine theefficacy of ellagic acid to reduce edema, their find-

ingswere congruentwith those of Rogerio et al, sug-

gesting that ellagic acidmay have anti-inflammatory,

antinociceptive activity.

Few studies have investigated the anti-inflamma-

tory properties of ellagic acid. We found no studies

that examined the effect of ellagic acid in combina-tion with COX inhibitors. The primary objective of

this study was to investigate the anti-inflammatory

effects of ellagic acid compared with known selec-

tive and nonselective COX inhibitors in male

Sprague-Dawley rats as measured by paw volume

Tissue In

Membrane Pho

Arachidon

Cyclooxygenase 2

Inflammation

Figure 1. Damaging stimuli promote inflammation by start

cell membrane phospholipids by the enzyme phospholipase

tion reaction by cyclooxygenase (COX) enzymes to produc

tively expressed and produces prostaglandins and throm

function. The COX-2 enzyme is inducible by stimuli such as

ciated with inflammation. This figure is available in color on

assessment. The secondary objective investigated

whether there is an interaction between ellagic

acid and the anesthesia adjunct ketorolac or the

COX-2 inhibitor meloxicam. Figure 1 illustrates

the theoretical framework used for this study.

Materials and Methods

Fifty-four mature, age-matched, male Sprague-Dawley rats (mean weight 215 6 8.41 g) were

used in this experiment, with six additional rats

used for model development. The animals were ac-

climatized to vivarium, habituation, and handling

for 10 days before the experiments. On days 6, 7,

and 8, the rats were brought to the testing room

for experimental acclimatization, which involved

exposing the paws to the plethysmometer. Therats were rested on day 9. All animals were housed

separately in cages in the same room and had con-

tinuous access to food and water. Furthermore, all

experiments were conducted in accordance with

the guidelines and approval of the Institutional

Animal Care and Use Committee at the 59th Clini-

cal Research Division, Lackland Air Force Base, in

San Antonio, Texas.

jury

spholipids

ic Acid

Cyclooxygenase 1

Homeostasis

ing a cascade inwhich arachidonic acid is cleaved from

A2. Arachidonic acid is then altered through an oxida-

e prostaglandins. COX-1 is an enzyme that is constitu-

boxane necessary for homeostasis such as platelet

tissue injury and produces prostaglandin effects asso-

line at www.jopan.org.

EVALUATION OF THE ANTI-INFLAMMATORY EFFECTS OF ELLAGIC ACID 217

Inflammation was induced in the left hind paw just

proximal to the foot pads in the subcutaneous

space using a 0.1-mL injection of 3% carrageenan.

The right hind paw similarly was injected with

0.1 mL of 0.9% normal saline to serve as a baselinecovariate for the measurement of paw edema. The

rats were briefly anesthetized using 2.5% to 3%

isoflurane anesthesia for paw injections. Thirty

minutes after paw injections, all animals were

administered one intraperitoneal (IP) injection of

compounds and/or vehicle in equivalent volume.

The compounds administered were based ongroup assignment. Rats were randomly assigned

to one of six groups: (1) vehicle alone (n 5 9); (2)

ketorolac plus vehicle (n 5 9); (3) meloxicam

plus vehicle (n 5 9); (4) ellagic acid plus vehicle

(n5 9); (5) ellagic acid plus ketorolac with vehicle

(n 5 9); and (6) ellagic acid plus meloxicam with

vehicle (n 5 9). All compounds were solubilized

in the same vehicle, dimethyl sulfoxide (DMSO),a solution of less than 7.2% alcohol. Dosages were

determined based on a review of previous dose-

response studies thatprovided informationondoses

that are sufficient to produce a response while

preserving potential selectivity. Ketorolac (Bedford

Laboratories, Bedford, OH) was administered at

10 mg/kg.15,16 Meloxicam (Sigma-Aldrich, St Louis,

Figure 2. Each group was composed of 9 rats. The post h

ketorolac was the only effective compound to significantly

*Indicates a statistically significant difference of P , .05.

MO) was administered at 4 mg/kg.17,18 Ellagic acid

(Sigma-Aldrich) was administered at 75 mg/kg.12,19

Thirty minutes after IP injections, right and left

paw volumes were measured by placing eachpaw into the ITTC 520 Plethysmometer (Wood-

land Hills, CA), up to the level of the heel glabrous

surface, a landmark that wasmarked during isoflur-

ane anesthesia on both right and left hind paws

with an indelible marker. The plethysmometer

measures water displacement and converts it to

milliliters, thus providing a quantifiable measure

of paw edema. For this experiment, each ratserved as its own control so that the difference

found in displacement in milliliters between right

and left paws was used to assess paw edema. The

paw volume measurements were taken on a timed

schedule at 30 minutes and 4, 8, and 24 hours after

IP injection to detect any differences in duration of

compound effect. Because this experiment was

one arm of a three-armed protocol, after this exper-iment the rats were returned to the vivarium.

Upon completion of the entire protocol, rats

were euthanized by exsanguination under deep

isoflurane (5%) anesthesia.

Using analysis of covariance,with the saline-injected

paw serving as the covariate, a significant difference

oc analysis demonstrated that 4 hours after IP injection

reduce paw edema from a 3% carrageenan injection.

ANCOVA F 5 689.72, P 5 .00, post hoc P 5 .019.

218 CORBETT ET AL

was found between the groups (P 5 .001). At 30

minutes after IP injection, there was no significant

difference found between the groups. The least sig-

nificant difference (LSD) post hoc test indicated that

at 4 hours after IP injection, ketorolac alonewas theonly compound to significantly reduce paw edema

(P5 .019). This is graphically represented in Fig 2.

At 8 hours after IP injection, the efficacy of ketorolac

was not demonstrated; however, ellagic acid alone

significantly reduced paw edema from a 3% carra-

geenan injection (P5 .038). In addition, at 8 hours

the combination of ellagic acid and ketorolac signif-

icantly reducedpawedema(P5 .038). This is graph-ically represented in Fig 3. Finally, at 24 hours after

IP injection only ellagic acid was effective in signifi-

cantly reducing paw edema (P5 .01). This is graph-

ically represented in Fig 4.

Discussion

Our finding that ellagic acid is effective againstcarrageenan-induced edema is congruent with the

findings of Rogerio et al,13 Ojewole,20 and Feresin

et al.21 In each of those three studies, either the

ellagic acid derivative or extracts from plants con-

taining ellagic acid were found to have a significant

Figure 3. Each group was composed of 9 rats. The post h

ellagic acid (EA) significantly reduced paw edema from a 3%

and ketorolac significantly reduced paw edema. At 8 hour

a statistically significant difference of P , .05. ANCOVA F5P 5 .038.

effect against chemically induced edema in rodents.

As in our study, Rogerio et al13 found that ellagic

acid inhibited carrageenan-induced paw edema.

Ojewole20 found that extracts from the African

Psidium guajava Linn plant significantly inhibitedegg albumineinduced paw edema in rats. Feresin

et al21 found that carrageenan-induced paw edema

was significantly inhibited by extracts from the

Acaena magellanica plant. Both of these plant

extracts contain ellagic acid.

In our study, ellagic acid was not effective in signif-

icantly reducing paw edema from a 3% carrageenaninjection until 8 hours after IP injection, whereas

ketorolac was effective 4 hours after IP injection.

These findings suggest that under our experimental

conditions, ellagic acid may have a longer onset of

actioncomparedwithketorolac. Although thecom-

bination of ellagic acid and ketorolac significantly

reduced paw edema at 8 hours, this effect may be

caused by the ellagic acid alone. Hence, we canmake no conclusions regarding interactions be-

tween the two compounds. Furthermore, in our

study, meloxicam showed no effect, suggesting

that under our experimental conditions the dose

of meloxicam was insufficient.

oc analysis demonstrated that 8 hours after IP injection

carrageenan injection. Further, the combination of EA

s ketorolac alone was no longer effective. *Indicates

496.09, P5 .00, post hoc EA P5 .038, EA + ketorolac

Figure 4. Each group was composed of 9 rats. At 24 hours after IP injection, only ellagic acid to significantly

reduce paw edema from a 3% carrageenan injection. *Indicates a statistically significant difference of P , .05.

ANCOVA F 5 410.56, P 5 .00, post hoc P 5 .01.

EVALUATION OF THE ANTI-INFLAMMATORY EFFECTS OF ELLAGIC ACID 219

Few studies have investigated the pharmacokinetics

of ellagic acid in rodents or humans. To our knowl-

edge, Teel’s22 study is the only research on the phar-

macokinetics of ellagic acid in rodents. In our study,the effects of ellagic acid were not apparent until

hour 8 and persisted through hour 24. These find-

ings appear to contradict those of Teel’s,whosefind-

ings suggest that the redistribution half-life (t1/2 a)

and the elimination half-life (t1/2 b) of ellagic acid

are 1 and 5 hours, respectively, after IP injections

inmice. Our study differed from Teel’s in two signif-

icant ways. First, Teel’s study specifically investi-gated the metabolism of ellagic acid in pain-free,

edema-free mice rather than rats. Body size and

weight differences between rats and mice suggest

that rats would metabolize most compounds more

slowly than mice. Moreover, although these two an-

imal models have similar cytochrome P450 activity

to humans, there are interspecies differences

between mice and rats.23 Secondly, our study mea-sured the effects of ellagic acid at 30 minutes and

4, 8, and 24 hours, whereas Teel directly measured

radioactively labeled ellagic acid and metabolites

in urine, blood, bile, and various organs removed

at 15, 30, and 60 minutes and 2 and 24 hours.

Carrageenan-induced edema and allodynia peaks

approximately 4 to 6 hours after injection and lasts

24 to 96 hours; hence, collecting data at 8 hours

after IP injection may have enabled us to find signif-

icant effects of ellagic acid.24,25 Moreover, studies

have shown that ellagic acid has a number of

active metabolites with half-lives approximating 48hours.26 Our findings may reflect the activity and ef-

ficacy of ellagic acid plus its active metabolites.

Conclusion

To our knowledge, there have been no studies that

have investigated interactions between ellagic acid

and ketorolac. Future research investigating the

anti-inflammatory effects of ellagic acid might in-

clude additional measures to evaluate changes in

known serum biomarkers of inflammation. Further

examination of interactions between ellagic acid

and other known anti-inflammatory agents such ascorticosteroids and fish oil could also be conducted.

Clearly, the rise in use of dietary supplements,

specifically ellagic acid, poses both potential risks

and benefits during the perianesthesia period and

should be carefully evaluated and considered by

the anesthesia provider during patient assessment.

Acknowledgments

We acknowledge the Wilford Hall 59th Clinical Research

Squadron.

220 CORBETT ET AL

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