Antimicrobial effects of Copaifera langsdorffii oleoresin in infected rat wounds

Masson-Meyers et al.

International Journal of Applied Microbiology Science 2013; 2(3):9-20 9

ISSN-2277-6079

ANTIMICROBIAL EFFECTS OF COPAIFERA LANGSDORFFII

OLEORESIN IN INFECTED RAT WOUNDS

Masson-Meyers DS1,2, Enwemeka CS1*,

Bumah VV1, Andrade TAM2,

Cashin SE1, Frade MAC2

1College of Health Sciences, University of Wisconsin-Milwaukee, 2400 East Hartford Avenue. Enderis

Hall, Milwaukee, WI 53211. USA 2School of Medicine of Ribeirao Preto, University of Sao Paulo, Avenida Bandeirantes, 3900. Monte

Alegre. CEP. 14049-900. Ribeirao Preto, Sao Paulo, Brazil

*Corresponding Author:

Chukuka S. Enwemeka, PhD, FACSM

College of Health Sciences, University

of Wisconsin-Milwaukee 2400 East Hartford

Avenue. USA Phone no: 1-414 229-4712.

Email address: [email protected]

Abstract

We determined the effect of Copaifera langsdorffii (copaiba) cream on wounds infected with

Streptococcus pyogenes or Staphylococcus aureus. Wounds were created on the dorsum of

Wistar rats, then inoculated with either pathogen, and observed through 14 days as they were

treated either with saline, control cream or 10% copaiba cream. Wounds were assessed for

healing on days 2, 7 and 14 post-surgery, and then swabbed and biopsied to quantify bacteria.

By day 14, treatment with 10% copaiba decreased S. pyogenes infection to 0.02%; saline

treatment reduced infection to 17.2%, while control cream increased infection 87.4%. Copaiba

similarly decreased S. aureus infection to 0.3% by day 14, compared with 26.9% for saline and

12.7% for control cream. Consistent with these findings, copaiba treated S. pyogenes infected

wounds re-epithelized 29% by day 2, compared with 15.8% for saline (p = 0.025) and 18.4% for

control cream treated wounds; maintaining a higher rate of re-epithelialization (67.5%) over the

control cream group (54.9%) on day 7 (p = 0.002). Similarly, copaiba enhanced healing of S.

aureus infected wounds, 74.4% by day 7, when compared with saline and control cream

wounds. These findings suggest that copaiba retards bacterial infection resulting in better

healing.

Keywords: Antimicrobial agents, Copaifera langsdorffii, wound healing, wound infection

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Introduction

ound healing is a complex biological process characterized by four distinct, but

overlapping phases: hemostasis, inflammation, proliferation and remodeling [1].

Following injury, various intracellular and intercellular pathways must be activated to restore

tissue integrity and homeostasis [2, 3].

Exposure of subcutaneous tissue following loss of skin integrity provides a moist, warm, and

nutritious environment that fosters microbial colonization and proliferation [4]. Staphylococcus,

Streptococcus, Enterococcus, and facultative Gram-negative bacilli rank high among bacterial

groups often recovered from chronic leg ulcers [5]. And these are well known to (a) promote

healing delay and non-healing, (b) elevate pain and discomfort, and (c) produce life-threatening

illness or outright death [4, 6]; thereby, raising the cost of health care as additional resources are

needed to overcome these complications [5-8].

Studies suggest that medicinal plants may reduce infection and optimize wound healing [9];

making them viable alternatives to contemporary treatment paradigms to which microbial

resistance is common. Antimicrobial compounds from plants may inhibit bacterial growth by

mechanisms that differ from those of standard antimicrobial pharmaceutical agents, and this may

be of significant clinical value in treating resistant strains of bacteria, and reducing the side

effects associated with synthetic pharmaceuticals [9, 10].

Copaifera genus (Leguminosae, Caesalpinioideae) has been used for centuries, particularly in the

Amazon region of Brazil, where its natural medicinal value has been long recognized by the

population. Plants of the Copaifera genus, popularly known as copaiba, are large trees and the

exuded material, traditionally obtained by tapping the trunk of the tree, is called copaiba oil,

oleoresin or copaiba balsam [11-14]. The oleoresin is mainly used as a topical anti-inflammatory

and healing agent. In folk medicine it has been used as an antineoplastic, urinary antiseptic, and

for treating bronchitis, skin diseases, ulcers and syphilis, it is claimed to have gastro-protective,

analgesic, antinociceptive, insect repellant and antimicrobial properties as well. In the cosmetic

industry copaiba oleoresin is used as a component in hair lotions, shampoos, soaps, creams and

bathing foams [13, 15-18].

Phytochemical studies carried out on the oleoresin from Copaifera langsdorffii showed that the

biological activities of Copaifera spp. are due to the presence of sesquiterpenes and diterpenes

[17]. The main sesquiterpene is β-caryophyllene and the main diterpenes are kaurenoic acid and

copalic acid, which is considered to be a typical diterpene of the genus Copaifera. Diterpenes

and sesquiterpenes are known mainly for their antimicrobial, anti-inflammatory, antiseptic and

healing properties [11, 13, 19, 20].

In a previous study we assessed the in vitro antimicrobial activity of Copaifera langsdorffii

oleoresin on bacteria of clinical significance in cutaneous wounds. C. langsdorffii oleoresin

showed antimicrobial activity against Staphylococcus aureus (S. aureus) (ATCC 6538),

Streptococcus pyogenes (S. pyogenes) (ATCC 19615) and Enterococcus faecalis (E. faecalis)

(ATCC 10541), with minimum inhibitory concentration (MIC) of 200 µg/mL, 400 µg/mL and

1100 µg/mL, respectively, indicating its antimicrobial activity [21].

In view of the clinical challenges presented by infected chronic wounds, we hypothesized that

copaiba will exhibit similar antimicrobial effect on infected wounds, and hence promote wound

healing. Therefore, the purpose of this study was to determine the effect of 10% copaiba cream

on infected wounds challenged with Streptococcus pyogenes or Staphylococcus aureus, using a

rat model of wound repair.

W



Materials and methods

Plant material

The copaiba oleoresin used in this study was collected from the trunk of the C. langsdorffii tree

in Tarauacá, Acre, Brazil (latitude 9°41'0" South; longitude 72

°5'0" West), between October,

1999 and May, 2000 as described in our previous papers [21, 22].

Experimental precautions pre-surgery

First, the surgical suite was thoroughly cleaned and disinfected, and before full-thickness wounds

were induced or infected, the surgical room was tested for potential environmental bacterial

contaminants. To achieve this, the room door knob, floor, shelves, surgical table, sink, bio-safety

hoods, counter tops and contact surfaces were swabbed. Swabs were then cultured in Mannitol

Salt Agar (MSA) and also in Tryptic Soy Agar (TSA) supplemented with 5% defribinated sheep

blood. Samples cultured in MSA were incubated at 37°C over a 24 hour period. TSA cultured

samples were incubated at the same temperature over the same 24 hour period in the presence of

5% CO2. The test results showed no evidence of bacterial colonization of any plate; thus ruling

out potential contamination of wounds from environmental sources.

Bacterial culture

S. pyogenes (ATCC 19615) and S. aureus (ATCC 6538) were purchased from American Type

Culture Collection (ATCC) (Manassas, VA, USA). The microorganisms were rehydrated and

cultured according to ATCC protocols and frozen at -20°C until they were used as detailed

below.

S. pyogenes was grown in thioglycollate medium without dextrose or indicator (Becton,

Dickinson and Company, Sparks, MD, USA) incubated at 37°C in the presence of 5% CO2

(Thermo Scientific Napco Series 8000 DH, CO2 Incubator, Thermo Scientific, Wilmington, DE,

USA) for 24 hours and S. aureus was grown in Brain Heart Infusion (Becton, Dickinson and

Company, Sparks, MD, USA) medium incubated at 37°C for 24 hours. The culture cells were

then centrifuged at 7,740 rpm (11,500xg) for 2 minutes at 4°C. The supernatant was discarded

and pellet was suspended in 0.9% sterile saline (Aqualite® System, Lake Forest, IL, USA).

Cultures were then diluted in sterile saline and vortex mixed for homogenization.

Colony-forming units (CFU) per mL for the bacterial suspensions were determined by measuring

the optical density (OD) at 625 nm (Shimadzu Spectrophotometer, UV-2501PC; UV-VIS

Recording Spectrophotometer, Kyoto, Japan). Several dilutions were made to reach a final

concentration that had the same OD (0.08 to 0.10, at 625 nm) as a 0.5 McFarland turbidity

standard (Becton, Dickinson and Company, Sparks, MD, USA), which corresponds to

approximately 108 cells/mL [23].

Surgical procedure, induction of infection and assignment of animals to experimental

groups

Following approval by the Institutional Animal Care and Use Committee (IACUC), Animal Care

Program (Protocol 09-10#20) and by the University Safety & Assurances, Biological Safety

Program (#10-Enwemeka-01), University of Wisconsin-Milwaukee (USA), 54 adult male Wistar

rats (Rattus norvegicus) weighing 300g to 340g were obtained from Charles River Laboratories

(Wilmington, MA, USA). Animals were fed with a standard rodent pellet diet and water ad

libitum and housed in standard sterilized individual polypropylene cages in a room maintained at

a temperature of 22°C, 50% humidity, and 12 hours light-dark cycle, throughout the

experimental period. The animals were allowed to acclimate for one week before the

experiments were performed.



Rats were anesthetized with inhaled isoflurane. Then, the dorsal surface of upper cervico-

thoracic region of each rat was shaved with an electrical clipper and disinfected with 70%

isopropyl alcohol. Four excisional 8 mm punch wounds were then made and inoculated with 20

µl of bacterial suspension containing approximately 108

CFU/mL of either S. pyogenes or S.

aureus. Before inoculation, wounds were swabbed at random and the swabs cultured as

previously detailed, to further rule out the presence of extraneous bacteria. Rats were then

assigned to two main groups according to bacterial species, then subdivided according to

treatment: saline (n=9), control cream (n=9) or 10% copaiba cream (n=9) (Figure 1). Thus, the

active treatment was 10% copaiba in a pharmaceutical base cream, while saline and control

cream were the controls. The control cream was a standard pharmaceutical base cream (Table 1).

We used 10% copaiba cream because our previous studies showed that this concentration of

copaiba was suitable for in vivo studies [22].

Fig. 1. Experimental design

Table 1. Percent composition (w/w) of creams used for wounds treatment

aSelf-emulsifying wax (Cetearyl alcohol; Ceteareth 20; Mineral oil; Lanolin alcohol; Petrolatum)

Component Control Cream (%) 10% Copaiba Cream (%)

Crodabase® CR2

a 25.0 25.0

Propyleneglycol 3.0 3.0

Methylparaben 0.18 0.18

Propylparaben 0.02 0.02

Butylated hydroxytoluene 0.02 0.02

Phenonip®b

0.5 0.5

Octyl stearate 2.0 2.0

Copaiba oleoresin --- 10.0

Distilled water to 100 to 100



bPreservative blend (phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben)

To ensure a closed environment and to facilitate bacterial growth, wounds were dressed with a

polyurethane film (Tegaderm®) held in place with a self-adhesive stretch bandage during the first

24 hours. The Tegaderm® film was replaced with sterile 2"x 2" gauze, similarly held in place

with self-adhesive stretch bandage and wrapped with standard athletic tape from the 2nd

day.

Wound dressing and treatments were done daily. Although non-infected control wounds were

also studied as a reference, the results obtained will not be highlighted in this paper because of

our focus on infected wounds.

Wound healing assessment

Wound healing was evaluated on days 0, 2, 7 and 14 post-wounding, using digital photography.

To ensure consistency of wound magnification, the camera was positioned 30 cm

perpendicularly above each wound. A metric scale placed beside each wound and photographed

with each wound served as a reference. The visible margins of wounds were then traced on each

digital image, and the area computed with an image processing and analysis software (ImageJ,

U.S. National Institutes of Health, Bethesda, MD, USA) [9, 24-27]. Wound healing rates

(WHRs) were then calculated as follows:

WHR (%) = Ao - An (100)/ Ao, where: Ao: wound area on day 0; n= day 2, 7 or 14.

Quantitative microbiological analysis

To quantify bacterial colonization, wounds were swabbed 24 hours after bacterial inoculation,

and also on the 2nd

, 7th

and 14th

day post-wounding. Wound exudates were collected using sterile

rayon tipped swabs (Fisherfinest®

bacteriology culture collection and transport system, Fisher

HealthCare, Ontario, Canada). The swabs were taken from the center of each surgical wound by

rotating the swab clockwise three times before culture [28]. For dry wounds, mainly on the 7th

and 14th

day post-wounding, swabs were moistened with sterile saline immediately before

swabbing [29].

Each swab was then placed in a sterile tube containing 1.0 mL sterile saline and vortex mixed for

1 minute. Four serial 10-fold dilutions were performed (1/10; 1/100; 1/1000; 1/10,000) in sterile

saline. One hundred microliters from each dilution was plated on TSA supplemented with 5%

defibrinated sheep blood plates (Remel®, Lenexa, KS, USA). Plates were then incubated for 24

hours at 37°C, in the presence of 5% CO2 (for samples from wounds infected with S. pyogenes)

and under aerobic conditions (for samples from wounds infected with S. aureus). The colony-

forming units per mL (CFU/mL) were determined by multiplying the number of β-hemolytic

colonies counted on a plate (n) by the dilution (D) and by the sample volume plated (10 accounts

for 100 µL). Thus: CFU/mL = n x D x 10. The bacterial count 24 hours after inoculation in

wounds treated with saline, being 100% of the expected bacteria count for each bacterium (S.

pyogenes or S. aureus) at any time point, was used as the baseline for determining the effects of

treatment with 10% copaiba cream and control cream.

Rats were euthanized with an overdose of isoflurane on each of days 2, 7 and 14 post-wounding.

The wound bed was immediately sampled, using a swab and processed as described above.

Biopsies from the same swabbed wounds were obtained using an 8.0 mm sterile biopsy punch,

and then processed for antimicrobial evaluation. Each tissue sample was placed in a sterile

microcentrifuge pre-weighed tube containing 1.0 mL sterile saline, and kept in ice bath and

processed within 2 hours of collection. Tubes were re-weighed and tissue weight (in grams) was

determined. Tissue samples were aseptically minced with a sterile scalpel and vortex mixed for 2

minutes to dislodge adherent microorganisms. Serial dilutions were performed before plating as

previously detailed for wound exudates. Then, colony-forming units per gram of tissue (CFU/g

tissue) were computed as follows: CFU/g tissue = n colonies x dilution x V x 10/ tissue weight



[28, 29]. The bacterial count on day 2 in wounds treated with saline, being 100% of the expected

bacteria count for each bacterium (S. pyogenes or S. aureus) at any time point, was used as the

baseline for determining the effects of treatment with 10% copaiba cream and control cream.

Statistical Analysis

Descriptive results for wound healing rates are presented as mean ± SEM. A three-way ANOVA

was used to compare groups using the software SPSS version 18. Least Significant Difference

(LSD) post hoc tests were used to pin-point groups that differed statistically. Colony counts were

compared using a two-way ANOVA followed by Bonferroni post hoc tests. The level of

statistical difference was set at p<0.05.

Results

Effect of copaiba on wound healing rate following infection with S. pyogenes

Each wound presented evidence of infection, such as purulent exudate and erythema observed

during the initial three days post-wounding. Erythema continued through the 6th

day in saline and

cream control wounds, and up to the 7th

day in 10% copaiba treated wounds (Figure 2a). Obvious

signs of systemic infection were not observed as the rats remained as active as usual. All wounds

healed progressively over time as expected; by day 2, wound healing rates (WHRs) were 15.8%,

18.4% and 29% for saline, control cream and 10% copaiba cream, respectively (p=0.025, 10%

copaiba cream versus saline); while on the 7th

day WHRs were 65%, 54.9% and 67.5%,

respectively (p=0.002, 10% copaiba cream versus control cream). The corresponding WHRs on

the 14th

day were 92.5%, 99.1% and 100% (Figure 2b).

Fig. 2 (a) Representative photos of wounds infected with S. pyogenes on days 0, 2, 7 and 14

respectively, arranged by treatment: Saline (a-d); Control cream (e-h) and 10% Copaiba cream (i-

l). (b) Effect of topical treatments on wound healing rates in the experimental and control groups

infected with S. pyogenes. Results are presented as mean ± SEM (n=12).

Effect of copaiba on wound healing rate following infection with S. aureus

Each wound presented evidence of infection, such as purulent exudate and erythema observed

during the initial four days post-wounding. Wounds treated with control cream or 10% copaiba

cream were erythematous mainly between the 4th

and 7th

day, while wounds treated with saline

presented erythema between the 1st and 5

th day (Figure 3a). By day two, wound healing rates

were 28.5%, 15.4% and 20.5% for wounds treated with saline, control cream and 10% copaiba



cream, respectively, while on the 7th

day WHRs were 65.2%, 65.7% and 74.4%, and on the 14th

day, 97.6%, 98.7% and 99.9% (Figure 3b).

Fig. 3 (a) Representative photos of wounds infected with S. aureus on days 0, 2, 7 and 14

respectively, arranged by treatment: Saline (a-d); Cream control (e-h) and 10% Copaiba cream (i-

l). (b) Effect of topical treatments on wound healing rates in the experimental and control groups

infected with S. aureus. Results are presented as mean ± SEM (n=12).

Antimicrobial effects of copaiba on wounds infected with S. pyogenes

The inoculation procedure truly produced the desired critical and clinically significant level of

wound infection (>105 CFU/mL) 24 hours after wounds were inoculated with each bacterium

[30]. The relative percentage of bacterial count (% CFU/g tissue) in wounds infected with S.

pyogenes is shown in Figure 4a. Bacterial count in the wound tissue decreased progressively in

the saline treated-wounds, reaching 17.2% (4.3x106/2.5x10

7 CFU/g tissue) on day 14; indicating

that, with time, the immune response of the animals suppressed the rate of infection.

Fig. 4 (a) Percentage of Colony-Forming Units (CFU) in tissue biopsies from wounds infected with

S. pyogenes ,according to treatments: Saline, Control cream and 10% Copaiba cream. (b)

Representative culture plates from biopsies of wounds infected with S. pyogenes on days 2, 7 and 14

respectively, according to treatments: Saline (a-c), Control cream (d-f) and 10% Copaiba cream (g-

i). Treatment with the control cream initially decreased bacterial count to 11.8% (2.93x10

6/ 2.5x10

7

CFU/g tissue) on day 2, but increased bacterial count to 87.4% (2.18x107/ 2.5x10

7 CFU/g tissue)

on the 14th

day (Figure 4a). Bacterial count from control cream-treated wounds obtained from



swabs (Table 2) was relatively high, suggesting that bacteria were mostly within the wound

exudates initially. This finding may explain the initial low level of bacteria in the tissue on the

2nd

day (Figure 4a), not necessarily that the control cream was antimicrobial; rather, in the

absence of active medication, the moist minimally aerobic cream environment presented

favorable conditions for facultative anaerobic S. pyogenes growth; more so than oxygenated

tissue which has immune cells capable of fighting bacterial infection. Over time, the bacteria

would naturally invade the tissue - in this case, with the larger population of bacteria that had

incubated in the control cream laden wound exudate - accounting for the progressive increase in

the amount of bacteria in tissue from day 2 to day 14 (Figure 4a, b). To buttress this finding,

swabs of wound exudates obtained on the 7th

and 14th

days showed a continuous decrease in S.

pyogenes from 16% to 0.02% (% CFU/mL) (Table 2). Thus, the control cream had two effects:

(1) initial limitation of bacterial colonization of tissue, and (2) promotion, rather than inhibition,

of tissue infection from the 2nd

to the 14th

day.

Table 2. Percentage of Colony-Forming Units (CFU) in exudates (Swabs) from wounds infected

with Streptococcus pyogenes according to treatments: Saline, Control cream and 10% Copaiba

cream

% CFU/mL (n/total)

Time 2 days 7 days 14 days

Treatment

Saline 42.8

(2.1x105/4.9x10

5)

81.6

(4x105/4.9x10

5)

2.9

(1.43x104/4.9x10

5)

Control cream 82

(4.02x105/4.9x10

5)

16

(7.87x104/4.9x10

5)

0.02

(102/4.9x10

5)

10% Copaiba cream 5.3

(2.6x104/4.9x10

5)

44.3

(2.17x105/4.9x10

5)

0.17

(833/4.9x105)

Saline-treated group, 24 hours after bacteria inoculation, represents 100% (4.9x105) of the

expected bacteria at any time point

With 10% copaiba cream treatment, there was 35.8% bacteria (8.69x106/ 2.5x10

7 CFU/g tissue)

on day 2, rising to 104.9% (2.62x107/ 2.5x10

7 CFU/g tissue) on day 7 (Figure 4a). However,

between the 7th

and 14th

days, the relative percentage CFU/g tissue (S. pyogenes infection)

diminished significantly, reaching a trace amount of 0.02% (6.47x103/ 2.5x10

7 CFU/g tissue) on

the 14th

day (p<0.05), (Figure 4a). Thus, the antibacterial effect of copaiba was strongly

evidenced by the relatively low amounts of bacteria measured at the end of the experiment

(Figure 4b). Overall, bacterial count in saline-treated wounds declined from 100% (2.5x107

CFU/g tissue) to 48% (1.2x107 CFU/g tissue) to 17.2% (4.3x10

6 CFU/g tissue) at each of days 2,

7 and 14, respectively. Control cream group had the opposite result, as bacterial colonies

increased from 11.7% (2.93x106 CFU/g tissue) to 41.6% (1.04x10

7 CFU/g tissue) and then

87.4% (2.18x107 CFU/g tissue) at each of days 2, 7 and 14; while 10% copaiba cream-treated

group had bacterial counts of 35.8% (8.96x106 CFU/g tissue), 104.9% (2.6x10

7 CFU/g tissue)

and a trace amount of 0.02% (6.47x103

CFU/g tissue) at days 2, 7 and 14 respectively (Figure

4a). The representative culture plates from biopsies of wounds infected with S. pyogenes are

shown in Figure 4b. These findings indicate strong antimicrobial effect of copaiba on

experimental wounds infected with S. pyogenes.

Antimicrobial effect of copaiba on wounds infected with S. aureus

In general, the results obtained from wounds infected with S. aureus followed a similar trend as

those obtained from S. pyogenes infected wounds; but with striking differences. The relative



percentage of bacterial count in wounds infected with S. aureus is shown in Figure 5a. Similar to

S. pyogenes infected wounds, bacterial count in wound tissue decreased overtime in the saline-

treated group, reaching 26.9% (3.96x107/ 1.47x10

8 CFU/g tissue) on day 14 (Figure 5a),

indicating that, as expected, the natural immunity of the animals eventually suppressed S. aureus

infection.

Fig. 5 (a) Percentage of Colony-Forming Units (CFU) in tissue biopsies from wounds infected with

S. aureus, according to treatments: Saline, Control cream and 10% Copaiba cream. (b)

Representative culture plates from biopsies of wounds infected with S. aureus on days 2, 7 and 14

respectively, according to treatments: Saline (a-c), Control cream (d-f) and 10% Copaiba cream (g-

i). In contrast to the data obtained for S. pyogenes, treatment with control cream increased bacterial

count to 142.9% (2.1x108/ 1.47x10

8 CFU/g tissue) compared to saline (baseline; 100%) on the

2nd

day; diminishing progressively to 51.3% (7.55x107/ 1.47x10

8 CFU/g tissue) on the 7

th day

and to 12.7% (1.87x107/ 1.47x10

8 CFU/g tissue) on the 14

th day (Figure 5a). The relative

percentage of bacterial count from the swabs (% CFU/mL) (Table 3) showed a similar trend.

Unlike S. pyogenes, S. aureus evidently colonized the tissue from the beginning. Instead of

limiting bacteria colonization of tissue initially, as was the case with S. pyogenes infection, the

moist control cream background would have only further enabled favourable growth of S. aureus

in tissue, given its aerobic nature.

Table 3. Percentage of Colony-Forming Units (CFU) in exudates (Swabs) from wounds infected

with Staphylococcus aureus according to treatments: Saline, Control cream and 10% Copaiba

cream

% CFU/mL (n/total)

Time 2 days 7 days 14 days

Treatment

Saline 16.9

(1.34x105/ 7.9x10

5)

186

(1.47x106/ 7.9x10

5)

7.7

(6.07x104/ 7.9x10

5)

Control cream 252

(2.07x106/ 7.9x10

5)

60

(4.74x105/ 7.9x10

5)

0.25

(2.03x103/ 7.9x10

5)

10% Copaiba cream 72.5

(5.73x105/ 7.9x10

5)

4.8

(3.83x104/ 7.9x10

5)

0.02

(1.87x102/ 7.9x10

5)

Saline-treated group, 24 hours after bacteria inoculation, represents 100% (7.9x105) of the

expected bacteria at any time point



Treatment with 10% copaiba cream decreased tissue infection, reaching 11.9% (1.75x107/

1.47x108 CFU/g tissue) on the 2

nd day and a trace 0.3% (5.6x10

5/ 1.47x10

8 CFU/g tissue) on

each of days 7 and 14 (Figure 5a). The relative amount of bacterial count from the swabs (%

CFU/mL) obtained from the copaiba treated wounds showed that infection also decreased in the

wound exudate overtime (Table 3). Thus, at each of days 2, 7 and 14, bacterial count in the

saline-treated group was 100% (1.47x108 CFU/g tissue), 20.7% (3.05x10

7 CFU/g tissue) and

26.9% (3.96x107 CFU/g tissue) respectively; control cream-treated group had 142.9% (2.1x10

8

CFU/g tissue), 51.3% (7.55x107 CFU/g tissue) and 12.7% (1.87x10

7 CFU/g tissue); while 10%

copaiba cream-treated group had 11.9% (1.75x107 CFU/g tissue), 0.3% (4.3x10

5 CFU/g tissue)

and 0.3% (5.6x105 CFU/g tissue) at each of days 2, 7 and 14; a statistically significant decrease

compared to each of the other groups at each time point (p < 0.05; Figure 5a). The representative

culture plates from biopsies of wounds infected with S. aureus are shown in Figure 5b.

Thus, consistent with S. pyogenes data, treatment with 10% copaiba cream significantly

diminished tissue S. aureus infection to trace amounts on the 14th

day (p<0.05), indicating that

copaiba had strong antibacterial effect on S. aureus infection. Overall, these findings clearly

show that copaiba is strongly antimicrobial in experimental wounds infected with either S.

aureus or S. pyogenes.

Discussion

Copaiba oleoresins are composed of high amounts of sesquiterpenes, contributing to almost 90%

of the total oleoresins composition, followed by a small amount of diterpenes. β-caryophyllene,

the main sesquiterpene, has been reported to have antimicrobial activity in vitro [17, 19]. Our

findings indicate that in experimental full-thickness cutaneous wounds infected with either S.

pyogenes or S. aureus, topical treatment with 10% copaiba cream suppresses the rate of infection

in vivo, when compared to saline or standard pharmaceutical cream controls. This result is

consistent with previous studies which indicate that copaiba has the potential to promote healing

[11, 13] and kill bacteria in vitro [31, 32].

Moreover, our findings are consistent with our previous work which showed that 10% copaiba

cream enhanced healing of non-infected wounds in the rabbit ear model of wound healing [22].

Similarly, it is congruous with a report which showed that topical application of 4% copaiba

oleoresin on non-infected incisional and excisional wounds augments tensile strength and

promotes wound contraction in rat wounds during the early phase of healing [13]. Our results

extend these findings; showing for the first time that copaiba minimizes infection and improves

healing of wounds infected with two common bacteria - S. pyogenes and S. aureus - when

applied topically as a cream.

Studies on medicinal plants have shown that phytochemical constituents like flavonoids,

triterpenoids and tannins promote wound healing [27, 33]. Terpenoids seem to promote wound

healing because of their astringent and antimicrobial properties, which appear to be responsible

for wound contraction and increased rate of epithelialization [34]. The wound healing activity

may be attributed to their individual activities or the synergistic effect of bioactive molecules

[35]. Furthermore, high resolution gas chromatography-mass spectrometry analyses of the

oleoresin used in our study showed a mixture of sesquiterpenes (75%) and diterpenes (25%). The

main compounds among sesquiterpenes were shown to be β-caryophyllene (51%), followed by

α-humulene (8.52%). Among diterpenes, the main compounds were 11-acetoxy-copalic acid

(5.23%), 11-hydroxy-copalic acid (4.8%), copalic acid (4.69%) and agatic acid (3.32%) [20].

Identifying the specific components of copaiba which account for our findings is beyond the

scope of this study; but our results suggest that copaiba initiates early erythema reaction - i.e.,

inflammation - and suppresses bacterial infection, resulting in faster healing of infected

experimental wounds.



Infection, healing resistance and slow healing are common problems in wound care. An

antimicrobial compound that also promotes healing may be a treatment of choice in clinical

practice. Since topical application of 10% copaiba cream minimizes infection and improves

healing of wounds experimentally infected with two common bacteria, S. pyogenes and S.

aureus, that are known to delay healing, this form of treatment holds promise as a clinical tool.

Acknowledgments

We gratefully acknowledge the financial support provided by the Brazilian Federal Agency for

the Support and Evaluation of Graduate Education (CAPES), Brazil, and the financial support

and infrastructure provided by the College of Health Sciences, University of Wisconsin-

Milwaukee, USA.

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Antimicrobial effects of Copaifera langsdorffii oleoresin in infected rat wounds