Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound

Healing in Male Rats

Muhammad Yanis Musdja

1

, Lina Elvita

2

, Nursetyowati Rahayu

3

1, 2, 3

Department of Pharmacy, Faculty of Medicne and Health Sciences, State Islamic University,

Syarif Hidayatullah, Jakarta, Indonesia.

Keywords: Catechins, Burn wound healing, Male rats Uncaria gambir,

Abstract: Background: Indonesia is the largest Uncaria gambir, Roxb (gambir) producing country in the world, but its

benefits as a medicinal plant has not been optimally developed. Traditionally, gambir plants have been

widely used by a lot of people as burn wound healing. Objective: This study aims to determine the effect of

gambir catechins crude for burn wound healing through histopathological observations on white rats male.

Methods: Gambir Catechins crude was isolated by using partition method with ethyl acetate solvent. The

rats were made to suffer burn wound based on Akhoondinasab methods et al. A total of 30 male white rats

were divided into 5 groups and given daily test preparations. A total of 3 groups were given gambir

catechins crude gel with variations of concentrations of 1%, 2% and 4%), for positive control group was

used a commercial drug that contains 10% placenta extract and 0.5% Neomycin sulfate and one other group

was used as a negative control. After administration of test preparation for 21 days, histopathology changes

was observed in the burn wound that occurred. Results: Statistical analysis showed that gel gambir

catechins crude with concentrations 2% had effect of decreasing the burn wound area and an increase in

the percentage of healing of burn wound that did not differ significantly from positive controls (P <0.05).

Histopathologic observations showed that catechins crude of gambir were able to reduce the number of

inflammatory cells and increase neocapillary formation compared with the negative control group.

Conclusion: Gambir catechins crude with concentration 2 % could help the healing process of second

degree of burn wound on the inflammatory phase and proliferation.

1 INTRODUCTION

Burn wound is one of the most common and

devastating forms of trauma and a major health

problem of morbidity and mortality worldwide,

because burn wound is very frequent cause of

infection and difficult to cure and often cause

scarring on the burn wound tissue (Kaddoura et al

2017, Church et al 2006). This greatly reduces the

value of skin beauty and the price of these drugs is

very expensive (Rowan et al, 2015) Current

methods to treat burn wound include antibiotics,

antiphlogistics, and silver salt, which possesses

major drawbacks and unwanted side effects,

(Waserman, 2002, Atiyeh et al, 2007) Therefore it

is necessary to find a healing drugs burn wound that

does not cause scarring on the burn wound tissue

and the price is cheaper. Generally drugs that come

from natural ingredients have smaller side effects.

One of the natural ingredients that have been proven

to be better and safer for burn wound healing than

synthetic drugs that are widely used today (1% silver

sulphadiazine cream) is Aloe Vera Gel (Shahzad and

Ahmed, 2013)

According to the research result of Hananeh et

al (2015) the use of propolis cream was the best

overall wound healing effects in full thickness

skin wounds in rats, where in this study, the

inflammatory reaction was weaker in the

propolis treated group than in the other groups

treated with silver sulphurdiazine (SSD) and

bepanthane (Hananeh et al 2015). Besides that

Centella asiatica also has a very good effect on burn

wound healing (Somboonwong et al, 2012)

On the other hand, the use of honey as a topical

treatment of wounds in diabetic patients in both

human and veterinary medicine, revealed that both

natural honey and amikacin enhanced wound

healing in non -diabetic rat patients. Honey

showed more promising results compared to

Musdja, M., Elvita, L. and Rahayu, N.

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats.

DOI: 10.5220/0009845600002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

Copyright

c

1

amikacin in enhancing the healing of full-

thickness punch biopsy wounds in diabetic rats

(Hananeh et al 2015)

Gambir (Uncaria gambir, Roxb) is a kind of

herbal that has been known in Indonesia and other

countries for a long time. Gambir is added on betel

nut chewing as well as additional substance in

traditional herbal medicine. Traditionally gambir has

been used for people with diarrhea, sore throat, burn

wound healing, anti-cancer, immunomodulator,etc

(Musdja et al 2017, Li et al, 2013)

The main content of the gambir is catechins (up

to 51%), tannin (22-50%), and a number of alkaloids

such as gambirtannin, derivatives dihydro and oxo

of gambirtannin. There are 9 types of catechins that

were found of gambir, namely; (+)-Catechin, (+)-

epicatechin, Gambiriin A1, Gambiriin A2,

Gambiriin B2, Epigallocatechin, Catechin-(4α-8)-

ent-epicatechin, Gambirflavan D1 and

Gambirflavan D2 (Taniguchi et al, 2008).

According to research of Musdja et al (2017),

“The (+)–Catechin was the highest level among 9

types of catechins with concentration about 45.66%

of the total catechins in gambir. Therefore (+)-

catechin can be used as a biomaker to determine the

quality of gambir. Therefore in this study only (+)-

catechin (biomarkers) was measured by a

spectrophotometer UV-VIS at wavelength 279

nm” (Musdja et al, 2017, Isnawati, 2010)

Figure 1. (+)–Catechin, main content of gambir (Musdja

et al, 2017)

Extract of gambir could inhibit the growth of

Streptococcus mutans, Staphylococcus aures and

Bacillus subtilis extract of gambir also could inhibit

the growth of Helicobacter phylori that resistent to

antibiotic (Heitzman et al, 2005, Voravuthikunchai,

and Mitchell 2008). The effect of gambir as an

antimicrobial is thought to prevent infection in burns

wound, so it will accelerate on burn wound healing

(Mohamed et al, 2008)

In addition, traditionally, a lot of people in

Indonesia have long used gambir as a drug for burn

wound healing. Based on this reason, we conducted

a study on the effect of gambir catechins on burn

wound healing on male rats.

2. METHODS

Gambir was obtained from Payakumbuh region,

West Sumatra, the largest producer of gambir in

Indonesia, to ensure that the sample used was

Uncaria gambir Roxb, in this research was done

determination of plant taxonomy in Biological

Research Center of Indonesian Institute of Sciences,

Bogor, Indonesia.

Phytochemical screening of gambir extract was

done based on Harbone methods. (Harborne, 1998,

Farsworth, 1969 ).

Catechins isolation from gambir extract was

done based on National Standard Indonesia: SNI 01-

3391-2000, “A total of 500 g of gambir powder was

extracted with a water solvent at temperature of 90 -

96 0C for 15 minutes while stirring. Then the

infusion was filtered in hot conditions using a funnel

coated with filter paper. The extract obtained was

partitioned with ethyl acetate, the ratio of extract

with ethyl acetate (1 : ½.). Ethyl acetate phase was

taken and the water phase was partitioned repeatedly

with ethyl acetate until a clear solution was obtained.

The ethyl acetate phase was condensed with an

evaporator, then washed with cold water and

filtered. Catechins that obtained was dried in an

oven at temperature 70 °C”. Then, The quality of

gambir extract was determined based on National

Standard Indonesia: SNI 01-3391-2000. In this

research, the yield of catechins obtained from the

gambir extract was determined as (+)-catechins by

comparing with standard (+)-catechins and measured

by a spectrophotometer UV-VIS at wavelength 279

nm. (Ferdinal, 2014, Hargono, 1986).

The yield catechins from gambir calculated

by the formula:

weight of acquired isolate

% Yield = x% purity

weight of extracted powder

Preparation of catechins gel was made base on

modification of (Eva et al, 2014, Rini et al 2013 )

methods. In this experiment, crude catechins of

gambir was mixed into gel formulations as below.

Na CMC 3,75%

Propilen glikol 3,75%

Gliserin 7,5%

Nipagin 0,05%

Aquadest ad 60

The crude catechins of gambir in the gel

formula was prepared with concentrations : 1%, 2%

and 4% b/w . Evaluation of this formulation was

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

2

done by determination: organoleptic, homogeneity,

viscosity and pH of the gel formula based on the

provisions of the Drug and Food Control Agency, RI

(Ditjen POM RI, 2000)

Experimental Animal was used male white

rats (Rattus novergicus) Sprague Dawley strain,

aged 2-3 months with a weight of 150-190 gram.

Animals were acclimatized for 1 week in order to

adaptation to the environment and during the

adaptation process was carried out observations of

general conditions and animals weight were weighed

every day. Animals that qualify for the experiment

were selected (Hoekstra et al, 1993, Amini et al,

2010)

Burn wound was made based on a method

undertaken by Akhoondinasab et al, 2014. Testing

the effects of burn wound healing was done on 30

rats. The feather on the dorsal area about 3 cm from

the ear of the rats was shaved and given local

anaesthesia with ketamine. Burn wound induction

process was performed by using a 4x2 cm iron plate

heated for 5 minutes in boiling water and then

attached to the skin of the back for 10 seconds at the

same pressure (Akhoondinasab et al, 2014)

Animals were divided into 5 groups and each

group consisted of 6 rats, each group was used for

testing, positive control, negative control, gambir

catechins crude with doses : 1%, 2% and 4% b/w.

The burns wound were observed and measured,

and then smeared the drug according to each group,

namely, negative control with gel base, positive

control with commercial drug that contains 10%

placenta extract and 0.5% Neomycin sulfate and

three groups with crude catechins of gambir with

concentration of 1%, 2% and 4%.

Giving test gels were done topically as much as

0.2 g for 1x polishing by applying it on the burn

wound in each group of treatment rats. Giving test

gels were done every day, from day 1 to day 21, 2

times a day, in the morning and evening

Histopathological observations were done in

daily from day 1 to day 21 after burns wound to all

treatment rats. Observation was done by looking

directly at the burn wound healing. To assess burn

wound healing, photographs obtained were

processed with Image software and calculated the

percentage of healing (Akhoondinasab et al, 2014)

Skin tissue samples were taken on the 7th day,

from each of the five groups were taken 1 rat. Skins

retrieval were carried out after rats were euthanized

by using ether solution per inhalation. The area skins

of the back would be taken were cleansed from the

hair that starts to grow, the skins were cut with a

thickness of ± 3 mm to the sub-cutaneous with area

of 1-1.5 cm

2

. The skins obtained were fixed with a

10% Formalin Neutral Buffer solution and left at

room temperature for ± 48 hours (Kulac et al 2013,

Prasetyoet al 2010)

The skin tissue obtained was made

histopathological preparations with Hematoxylin-

Eosin dye by the Histopathology team at the

Pathology Laboratory, Faculty of Medicine,

University of Indonesia.

Histopathologic observations were done on skin

tissue preparations taken on day 7. The observations

were done by descriptive light microscopy. These

observations include the parameters that play a role

in burn wound healing namely the presence of

inflammatory cells and neocapillarization. (Jose et al

2010)

Histopathology Observation Scoring Reading

was done by using Hosseini methods, (S.V. Hosseini

et al 2011)

For measurement of percentage of wound

healing, done by looking directly at the wound then

measured the area of burn with image application

and calculated the percentage of healing burns and

calculated with formula in below :

(extent of initial wound-wound end)

% Burn wound healing = X100%

(initial wound area)

The test results data were analyzed using data

processing software and presented in the mean and

standard deviation of each group. The data were

processed using statistical analysis with normality

test, homogeneity test, One Way ANOVA and

Kruskal-Wallis Test.

3. RESULT AND DISCUSSION

The result of determination of plant taxonomy in

Biological Research Center of Indonesian Institute

of Sciences, Bogor, Indonesia stated that the plant

extract used in this research was an extract from

Uncaria gambir Roxb.

The result of screening of chemical group

content of gambir extract based on Harbone

methods was shown in table 1. This was the same

with the research results of Taniguchi et al (2008),

that gambir extract contains groups of chemical

compounds alkaloids, flavonoids, saponins, tannin

and Quinone (Taniguchi et al, 2008).

Gambir quality that was used in this study, when

compared with the provisions of the Indonesian

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats

3

National Standard: (Indonesian National Standard:

SNI 01-3391-2000) as shown in table 2.

Gambir used in this research was number one

quality, or fulfills the requirements for research.

Table 1. The content of groups of chemical compounds in

the gambir extract that was studied

Chemical group

Results

Alkaloids

+

Flavonoids

+

Saponin

+

Tannin

+

Quinone

+

Steroids &

Triterpenoids

-

Essential oil

-

Qoumarine

-

Catechins are flavonoid compounds that include

natural phenolic compounds that are potential as

antioxidants and have bioactivity as a drug (Lucida,

2007). The working mechanism of the flavonoid is

to circulate blood throughout the body and prevent

the occurrence of blockage in the blood vessels

(Handayani, 2015). In addition, catechins are

potentially antibacterial (Arakawa et al 2004)

According to Anggraini et al (2011) flavonoid

compounds have anti-inflammatory effects that

serve as anti-inflammatories and are able to prevent

stiffness and pain (Anggraini et al 2011).

Catechins have activity as antibacterial,

especially gram-positive bacteria tested on

Staphylococcus aureus bacteria where this

bacterium is one of the gram-positive bacteria that

often cause wound infection (Musdja et al 2017 ).

Table 2. Comparison of gambir quality that was used in

this research with gambir quality requirements of

National Standards of Indonesia (Standar Nasional

Indonesia : SNI 01-3391-2000)

Testing of gambir

extract

Quality

number 1

Quality

number 2

Gambir extract

for experiment

a. Physical condition :

• shape

• color

• smell

b. Water content w/w

c. Ash content w/w

d d. d. Catechins

concentration w/w

of dry weight

e. Insoluble material

Intact

Yellow to

brownish

yellow

Specific

≤ 14%

≤ 5%

≥60%

Intact

Yellow brown

to yellow-

black

Specific

≤ 16%

≤ 5%

≥50%

Intact

Yellow to

brownish

yellow

Specific

4%

1.03 %

88.65%

content of :

• water w/w of dry

weight

• alcohol w/w of dry

weight

≤ 7%

≤ 12%

≤ 10%

≤ 15%

3.9%

8.6%

In addition, the presence of antibacterial activity

can suppress pathogenic bacteria and prevent the

growth of pathogenic bacteria in wounds so that

wound healing can be accelerated (Kaddoura et al

2017)

Catechins act as antibacterials by the mechanism

of binding to the peptide unit on the peptidoglycan

component of the cell wall. The occurrence of such

binding can disrupt the integrity of bacterial cell

walls and cause leaks in Gram-positive bacterial

cells (Pambayun et al, 2007)

Therefore, the presence of antibacterial activity

in catechins prevents the possibility of infection in

the event of injury. Moreover, catechins have

activity as an antioxidant that works by breaking the

lipid peroxidase chain that plays a role in free

radicals that can bind certain substances and harmful

to the body (Musdja et al , 2018) and can damage

three types of compounds that are important to

maintain cell integrity, namely: fat, especially

unsaturated fatty acids, which are important

components of cell membrane phospholipids; DNA,

is a genetic device of cells; protein, plays many

important roles such as enzymes, receptors,

antibodies, and cytoskeleton matrix formers

(Ambiyani and Winny, 2013) So the existence of

antioxidant activity in catechins can avoid more

severe cell damage.

After examination of gambir quality, catechin

isolates were formulated in gel preparation form.

The gel preparation was chosen because it has a

cooling, cooling, moisturizing, easy-to-use water

content, easy penetration to the skin, thus providing

a faster healing effect according to the base used. So

it is in accordance with the principle of the main

handling of minor burns that is cooling the burning

wound with water, where the gel content consists

mostly of water (Ansel, 2005). Observation of gel

preparation shown in Table

Tabel 3. Observation of Gel Preparations

Observation

Dose 1%

Dose 2%

Dose 4%

Color

Light brown

Brown

Form

Semisolid

transparent

Semisolid

transparent

Semisolid,

Not

transparent

Smell

Specific

Homogenity

Homogen

Specific

pH

6,51

6,12

5,76

Viscosity

43600

56400

73000

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

4

As the mechanism of action of positive control

containing 10% placenta extract is growing new

tissue and for healing wounds, while neomycin

sulfate 0.5% to prevent gram negative bacterial

infection in the wound area (Niknejad, 2013)

The formation of a scab indicates the process

of wound healing entering the early stage of

proliferation. To observe the formation of scab, in

the wound was seen a network of granulation

characterized by the appearance of a scab. This scab

serves to close the wound and prevent injury from

further contamination by microbes. The release of

scabs signifies the growth of new cells on the skin,

thus helping to accelerate the release of scabs and to

close the wound edges (Niknejad, 2013)

In the positive control group, the average scab

was formed on day 4 and off on day 11. In the

negative control group, the average scab was formed

on day 3 and off on day 14. In the low concentration

test group ( 1%), the average scab formed on the 2nd

day and off on the 11th day. In the medium

concentration test group (2%), the average scab was

formed on day 2 and off on day 12. In the group

high concentration test (4%), the average scab

formed on day 1 and off on day 14.

Table 4 Observation of Scab

Average on the day to

PC

NC

C1%

C2%

C4%

Scab

formation,

on days to

4

3

2

1

Remove

the scab,

on days to

11

14

11

12

14

Note:

PC = Positive Control, NC = Negative Control,

C1% = Concentration Test 1%

C2% = Concentration Test 2%, C4% = Concentration Test 4%,

Based on the observation of the formation of the

scab, it was obtained that the low concentration test

group (1%) had the potential to accelerate wound

healing time as the formation of the most rapidly

formed scab, ie on the 2nd day and released on the

11th day was almost close to the positive control

group compared to the concentration test group

medium (2%) and high concentration (4%) and

negative control. In high concentration tests (4%)

initially could accelerate drying in the wound area

but this drying triggers the formation of scab or dead

tissue that was very hard and thick and attached

tightly to the surface of the wound. This dead tissue

could inhibit the distribution of the active substance

and the absorption of the drug so that the wound

lasts longer. The duration of the process of

formation of new tissue resulted in the length of the

healing period. Therefore the high concentration test

group (4%) had the longest peeling time. In addition,

it could be observed that the negative control group

undergoes a long wound healing process seen from

the time of formation of the scab and the time of loss

of the scab. This matter showed that gel base alone

did not affect the acceleration of wound healing.

The area of the initial injury to the width of the

wound the day after the wound was made and the

extent of the final wound was the area of the wound

on the day of observation.

The area of the initial wound to the calculation of

the percentage of wound healing was the area of the

wound the day after the rat was injured, after 24

hours wound stability occurs. A wound can be said

to be healed if the wound area had undergone

epithelialization thoroughly and no longer requires

treatment. The result of measurement of the

reduction of burn area on all treatment groups on the

1st day until the 21st day using Akhoondinasab

injection method (Akhoondinasab, 2014) as shown s

in Table 5 and Figure 1.

Giving gambir catechins gel with various

concentration levels (1%, 2% and 4%) had an effect

on time and percentage of burn wound healing. The

highest percentage of reduction of burn wound area

(in Cm

2

) on 8th day was in positive control

(59.60%) followed by medium concentration (2%)

was 44.02%. Based on statistical test of medium

concentration test (2%) was significantly different

with high concentration test group (4%) and did not

significantly different with negative control, low

concentration test (1%) and positive control.

The highest percentage of reduction of burn

wound area on 15th day was obtained on positive

control (95,76%) followed by medium concentration

test group (2%) that was 88,32%. Statistical results

on the 15th day showed that the group of medium

concentration test (2%) was significantly different

with the high concentration test group (4%) and did

not significantly different with the low concentration

test group (1%), positive control and negative

control.

Based on the percentage of reduction of burn

wound area on the 21st day, the positive control

group showed the highest result (100%) followed by

medium-concentration (98.02%) and low

concentration test (97.87 %), negative control

(95,98%) and high concentration test (93,99%).

This suggests that the gambir catechins gel on the

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats

5

low concentration test (1%) and the middle

concentration test (2%) had high activity and were

almost equal to the positive controls in the

percentage of second degree burn wound healing.

However, statistically the percentage reducing of

burn wound area indicates that the data were

normal, but not homogeneously distributed so that

the data processing was followed by a non-

parametric Kruskal-Wallis test. The statistical data

of percentage reducing of burn wound area showed

not significantly different (p> 0.05) in all groups, as

shown in Table 5 and Figure 1.

Histopathological Observation was done by light

microscope descriptively, based on Figure 2, at 20

field of view with 400 x magnification.

Histopathologic observations preparations were

performed by scoring system. The scoring method

was done by using the Hosseini methods (Hosseini,

et al, 2011) as shown in Table 6 and Figure 2.

Table 5. Results of Measurement Reduction of Burn

Wound Area (in Cm

2

)

Figure 1. Percentage of Reduction of burn wound of

gambir catechins dose 1%, dose 2%, dose 4%, positive

control and negative control

The observations histopathology were done by

using light microscope (Olympus SZ61)

descriptively at 20 field of view with magnification

200 x and 400 x, as Shown in Figure 2.

Table 6. Results of Histopathology Assessment with

Scoring System

Group

Score

Neocapillari

zation

Infiltration of

inflame matory

cells

Positive

Control

3

4

Negative

Control

0

Consentration

test 1%

1

2

Consentration

test 2%

2

3

Consentration

test 4%

0

1

Neocapillary formation or neovascularization is

the formation of new blood vessels to the wound

area. Inflammation of inflammatory cells is a

process of inflammation of inflamed cells into the

injury site. (Prasetyo, 2010). From the scoring

results it can be observed that the highest

neocapillarization score was found in the positive

control group, followed by medium concentration

test (2%), low concentration test (1%), high

concentration test (4%) and negative control. The

highest inflammatory cell infiltration score was in

Days to :

1 day

8 days

15 days

21 days

Average

wound

area

Average

decreas

ed

wound

area

Average

percent

age (%)

wound

Average

decreased

wound

area

Average

percentage

(%) wound

Average

decreased

wound area

Average

percentage

(%) wound

Concentra

tion 1%

7,34

3,02 ±

0,86

41,14

5,90 ± 1,01

85,83

7,18 ± 0,77

97,87

Concentra

tion 2%

7,36

3,24 ±

1,39

44,02

6,50 ± 1,36

88,32

7,22 ± 1,55

98,02

Concentra

tion 4%

7,31

1,86 ±

0,72

22,44

5,09 ± 1,49

72,78

6,65 ± 0,97

93,99

Positive

Control

7,08

4,22 ±

1,23

59,60

6,78 ± 0,74

95,76

7,08 ± 0,38

100

Negative

Control

7,07

2,85 ±

1,01

40,24

5,73 ± 0,95

81,84

6,78 ± 1,05

95,54

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

6

the negative control group, followed by high

concentration test (4%), medium concentration test

(2%) and positive control.

From the results of microscopic observation, it

was found the presence of inflammatory cells in the

five groups. From the scores of inflammatory cell

infiltration parameters it was found that 2% gambir

catechins gel had the highest score after positive

control compared to the other test group. High

scores on inflammatory cell infiltration parameters

signify at least inflammatory cells. This was because

the catechins act as anti-inflammatory and

antibacterial while control positive contains

neomycin sulfate as an antibacterial so that the role

of inflammatory cells to phagocytes microbes can be

minimized and fast wound cleansing.

Table 7: Observation of Histopathology Scoring Reading

based on Hosseini method, (2011).

In contrast, the lowest scores were in the negative

group and the high concentration test (4%). Low

scoring of inflammatory cell infiltration parameters

signifies the number of inflammatory cells. This was

caused by the absence of active ingredients in the

preparation that could help to eliminate the foreign

particles so it was possible that microbial and tissue

damage must be phagocytized by the cells in the

wound area so that the inflammation rate was still

high. This suggests that inflammation and

phagocytosis were still occurring and indicate that

the two test groups were still in the inflammatory

phase.

In the neocapilarization parameter, the highest

scores were obtained in the medium concentration

test (2%) after positive control compared to the

other test groups. High scoring on neocapilarization

parameters showed an increase in the number of

neocapillaries that signaled the process of wound

healing in the proliferative phase.

The important process of cellular activity in this

phase is repair and heal wounds and was

characterized by cell proliferation (Hosseini et al,

2011). Moreover, inflammatory cell infiltration

scores in the moderate concentration test group (2%)

had high scores. This shows that in the medium

concentration test group (2%) has passed the

inflammatory phase and entered the proliferation

phase.

Group

Magnification

200x

400x

Positive

Control

Negative

Control

Consentration

Test 1%

Consentration

Test 2%

Consentration

Test 4%

Score

Inflammatory cells

Angiogenesis

0

13-15 inflammatory

cells per field of view

There is no

angiogenesis,

there is

congestion,

hemorrhagic and

edema

1

10-13 inflammatory

cells per field of view

1-2 blood vessels

per field of view,

there are edema,

hemorrhagic,

congestion

2

7-10 inflammatory

cells per field of view

3-4 blood vessels

per field of view,

there are edema,

hemorrhagic,

congestion

3

4-7 inflammatory

cells per field of view

5-6 blood vessels

per field of view,

there are edema,

hemorrhagic,

congestion

4

1-4 inflammatory

cells per field of view

More than 7 blood

vessels per field of

view, there are

edema,

hemorrhagic,

congestion

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats

7

Figure 2: Result of Observation of Histopathology of

white rats skin tissue with Hematoxylin-Eosin staining at

200x and 400x magnifications

Note:

Black arrow : Inflammatory cells

Blue arrow : Neocapillarization

From the analysis of all data, it can be concluded

that the activity of gambir catechins in healing

process of second degree burn did not show

significant result on decreasing of burn area and

percentage of wound healing. However, the activity

of catechin isolates of gambir has affects the

healingof burns in the inflammatory phase and

proliferative phases. This was seen in

histopathologic images. where in inflammatory cell,

the medium concentration test group (2%) could

decrease the number of inflammatory cells

compared to the low concentration test group (1%),

high concentration test (4%) and negative control.

This was because gambir catechins have activity as

anti-inflammatory and antibacterial. (Musdja et al

2017, Musdja et al 2018). The existence of these

compounds would indirectly decrease the

stimulation of inflammatory cell migration to the

wound area so as to reduce the number of

inflammatory cells and the process of clearance of

foreign substances can run faster Hosseini et al,

2011). Thus, the moderate concentration test group

(2%) may affect the healing of burns in the

inflammatory phase.

In the neocapillarization parameter, the moderate

concentration test group (2%) showed a higher score

than the low concentration test group (1%), high

concentration test (4%) and negative control. In the

process of tissue repair, the presence of blood

vessels had an important role to provide nutrient

intake for tissue that was regenerating. In addition,

blood vessels also had a role to deliver inflammatory

cells formed in the marrow to close the wounded

tissue so that the inflammatory cells to emigrate. To

support these functions, blood vessels would form

new vascular shoots that will eventually develop into

a new branching on the wound tissue or called

neokapilerisasi (Hananeh et al 2015). Thus, the

number of new blood vessels in the injury site in the

medium concentration test group (2%) indicates that

the wound healing process had proceeded in the

proliferation phase. (Eva et al, 2014)

Anatomic and histopathology observations

showed differences between groups. This difference

explains that in each gel preparation group had a

different work force. The working strength of the gel

preparation was influenced by the active ingredient

content of each gel and the concentration of isolates

that affect its activity both in the inflammatory

phase, the proliferation phase and the maturation

phase. In general, the moderate concentration test

group (2%) showed better results than the low

concentration test group (1%) and the high

concentration test group (4%). This is in line with

the research conducted by Sumoza et al. (2014)

where in the study of Gambir's Influence on Healing

Burns on white mice showed good results in

moderate concentrations.

In the medium concentration test group (2%)

showed the best results microscopically followed by

low concentration test group (1%) and high

concentration test group (4%). However, in the high

concentration test group (4%) showed unsatisfactory

results. This was probably due to the low

concentration of 1% gambir catechins gel having the

active ingredient content below the optimum dose so

that although it diffuses well but the active

ingredient content was not sufficient for wound

healing, medium concentration (2%) had the active

ingredient content in the range optimal dosage for

wound healing while gambir catechins gel with high

concentration (4%) was above the optimal dose for

wound healing. This causes the high concentration

of 4% gambir catechins gel to have high viscosity

and cause the gel to be too thick resulting in the

release of the active substance and the penetration

slowing into the skin.

4. CONCLUSION

Gambir Catechins (Uncaria gambit, Roxb) on

the concentration group of 1%, 2%, 4%, control

positive and control negative did not significantly

different (p>0,05) in reducing the surface area on

second degree burn wound which was given

topically on anatomical pathology observation.

There were differences in infiltration of

inflammatory cells and neocapillarization on day 7

in the 2% concentration test compared with the 1%

concentration test , 4% concentration test and

negative control on microscopic observation, where

the medium concentration group (2%) was better

than low concentration test group (1%) high

concentration test group (4%) of gambir catechins

and negative control.

Gambir catechins could assist in the healing

process of burns in the inflammatory phase and

proliferation.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

8

ACKNOWLEDGEMENTS

We would like to thank to team of

Histopathology, Faculty of Medicine, University of

Indonesia for their help in preparation and

interpretation of Histopathology in this research

REFERENCES

Akhoondinasab MR, Akhoondinasab M, Saberi M. 2014.

Comparison of Healing Effect of Aloe Vera Extract

and Silver Sulfadiazine in Burn Injuries in

Experimental Rat Model. Original article Vol. 3 No. 1;

29-34

Ambiyani, Winny., 2013, (In bahasa) Pemberian Salep

Ekstrak Daun Mengkudu (Morinda Citrifolia L)

Meningkatkan Proses Regenerasi Jaringan Luka Pada

Tikus Putih Galur Wistar (Rattus Norvegicus) Jantan.

(Administration of Noni Leaf Extract (Morinda

Citrifolia L) Improves Wound Tissue Regeneration

Process in Male Wistar (Rattus Norvegicus) Male

White Rat, Thesis. Denpasar: Universitas Udayana

Anggraini T, Tai A, Yoshino T, Itani T, 2011.

Antioxidative activity and catechin content of four

kinds of Uncaria gambir extracts from West Sumatra

Indonesia. African Journal of Biochemistry Research,

5(1), pp 33-38

Ansel, H.C. 2005, (In bahasa), Pengantar Bentuk sediaan

Farmasi (Introduction of Pharmaceutical Dosage

form). Edisi 4. Penerjemah: Farida Ibrahim. Jakarta:

UI Press.

Arakawa H1, Maeda M, Okubo S, Shimamura T., 2004,

Role of hydrogen peroxide in bactericidal action of

catechin. Biol Pharm Bull. Mar;27(3):277-81.

Atiyeh, B.S.; Costagliola, M.; Hayek, S.N., 2007, Effect of

silver on burn wound infection control and healing:

Review of the literature. Burns, 33, pp 139–148.

Ditjen POM RI, (2000), Departement of Health RI,

General Standard Parameters of Medicinal Plant

Extracts. (In bahasa) Parameter Standar Umum

Ekstrak Tumbuhan Obat.

Eva E, Noo C, Dina R. (2014), The Effect of Quercetin

gels on the number of neutrophils and lymphocytes in

the process on second grade burn wound healing in

wistar male rats (In bahasa: Pengaruh pemberian gel

kuersetin terhadap jumlah neutrofil dan limfosit

dalam proses penyembuhan luka bakar derajat ii a

pada tikus jantan galur wistar), Journal

Pharmascience, Vol 1, No. 2, Oct, 38 - 45

Farsworth, N.R., 1969, Biological and Phytochemical

Screening of Plants, Journal Pharmaceutical Science.

55 (3): 255-276.

Ferdinal N, A., 2014, Simple Purification Method of

Catechin from Gambier, International Journal on

Advanced Science Engineering Information

Technology, Vol.4, No. 6, ISSN: 2088-5334

Hananeh WM, Ismail ZB, Alshehabat MA, Ali J, (2015)

Review of animal models used to study effects of bee

products on wound healing: findings and applications.

Bull Vet Inst Pulawy 59, 425-431.

Harborne JB. Phytochemical methods: A guide to

modern techniques of plant analysis. 3rd ed. London:

Academic Press; 1998. p. 192-204.

Handayani Fitri, Siswanto Eka dan Pangesti Lintang A.T.,

2015, (In bahasa) Uji Aktivitas Ekstrak Etanol

Gambir (Uncaria gambir) Terhadap Penyembuhan

Luka Bakar Pada Kulit Punggung Mencit Putih Jantan

(Mus musculus L.) . Jurnal Ilmiah Manuntung I(2),

133-139

Hargono, D, (1986), Galenic preparations, Directorate

General of Drug and Food Control, Department of

Health RI, (In bahasa) Sediaan Galenik, Direktorat

Jendral Pengawasan Obat dan Makanan (BPOM),

Departemen Kesehatan Republik Indonesia, Jakarta.

Heitzman E.M., Neto C.C., Winiaz E., Vaisberg A.J.,

Hammond G.B., 2005, .Ethnobotany, phytochemistry

and pharmacology of Uncaria (Rubiaceae).

Phytochemistry, 66, 5 – 39.

H. Niknejad, G. Paeini-Vayghan, F.A. Tehrani, M.

Khayat-Khoei, H. Peirovi (2013), Side dependent

effects of the human amnion on angiogenesis

J.Placenta 34 340-345,

https://www.placentajournal.org/article/S0143-

4004(13)00071-4/pdf.

Isnawati, A., 2010, (In bahasa) Analisa Kualitatif dan

Kuantitatif Senyawa Katekin dan Kuersetin pada 3

Mutu Ekstrak Gambir. Laporan Akhir. Program

Insentif Riset Terapan : Teknologi Kesehatan dan

Obat. Pusat Penelitian dan Pengembangan Biomedis

dan Farmasi. Badan Litbang Kesehatan Departemen

Kesehatan. http://km.ristek.go.id/asset/files/479/pdf.

Diakses 1-11.2013

Jose P. Sterling, MD, David M. Heimbach, MD, FACS,

and Nicole S. Gibran, MD, FACS, (2010),

Management of the burn wound, Decker Intellectual

Properties,DOI 10.2310/7800.S07C15.

https://pdfs.semanticscholar.org/c0e1/05f2cf48de1720

a749933a8b0bdc43bede50.pdf.

Kaddoura I, Abu-Sittah G, . Ibrahim A, Karamanoukian R

and Papazian N, (2017), Burn injury: review of

pathophysiology and therapeutic modalities in major

burns, journal Ann Burns Fire Disasters, v.30(2); Jun

30, 95–102.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627

559/.

Kulac M1, Aktas C, Tulubas F, Uygur R, Kanter M,

Erboga M, Ceber M, Topcu B, Ozen OA.(2013) J

Mol Histol. (2013) The effects of topical treatment

with curcumin on burn wound healing in rats. J Mol

Histol. Feb;44(1):83-90. Doi:10.1007/s10735-012-

9452-9.

https://www.ncbi.nlm.nih.gov/pubmed/23054142.

Li, J.; Zhao, G.Z.; Chen, H.H.; Wang, H.B.; Qin, S.; Zhu,

W.Y.; Xu, L.H.; Jiang, C.L.; Li, W.J., 2013,

Antitumour and antimicrobial activities of endophytic

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats

9

streptomycetes from pharmaceutical plants in

rainforest. Lett. Appl. Microbiol. 47, 574–580.

Lucida, H., Bakhtiar, A. dan Putri, W.A. 2007, (In bahasa)

Formulasi sediaan antiseptik mulut dari katekin

gambir (Formulation of oral antiseptic preparations

from gambir catechins). Jurnal Sains Tek. Farmasi.

12(1), 7-14

M. J. Hoekstra, P. Hupkens, R. P. Dutrieux, M. M.

C. Bosch, T. A. Brans and R. W. Kreis, (1993),

Comparative burn wound model in the New

Yorkshire pig for the histopathological evaluation

of local therapeutic regimens: silver sulfadiazine

cream as a standard, Brirish Journal ofP/asric

Surgery, 46, 585-589

https://www.ncbi.nlm.nih.gov/pubmed/8252267.

M. Amini , M. Kherad , D. Mehrabani , N. Azarpira , M.

R. Panjehshahin & N.Tanideh (2010) Effect of

Plantagomajor on Burn Wound Healing in Rat,

Journal of Applied Animal, Research, 37:1, 53-56,

DOI: 10.1080/09712119.2010.9707093. To link to this

article:

https://doi.org/10.1080/09712119.2010.9707093.

M. N. Shahzad, N. Ahmed, (2013), Effectiveness of Aloe

Vera Gel compared with 1% silver sulphadiazine

cream as burn wound dressing in second degree burns,

J Pak Med Assoc, Vol. 63, No.2, February, 225 – 230.

http://jpma.org.pk/full_article_text.php?article_id=400

1.

Moenadjat Y., 2009, (In bahasa) Luka bakar masalah dan

tata laksana. Jakarta: Balai Penerbit FKUI.

Mohamed El-Maghawry HAM, Nem WE, Sherif N-A,

Hagag SA., 2008, An interventional study to decrease

healthcare associated burn wound infections in the

burn unit of Al Ahrar Hospital in Zagazig city, Sharkia

Governorate. International Journal of Current

Microbiology and Applied Sciences (2016) 5: 566-

578. 57.

Musdja MY, Hapsari MA and Agusta A, (2017),

Comparison of Activity and Inhibitory Mechanism

between (+)-Catechin and Water Extract of Gambier

(Uncaria Gambir Roxb.) Against Some Bacteria,

Scientific Journal of PPI-UKM, Vol. 4 (2017) No. 2,

55-60. DOI: 10.27512/sjppi-ukm/se/a29012018.

http://www.kemalapublisher.com/index.php/ppi-

ukm/article/view/300.

Musdja MY, Rahman H A & Hasan D, (2018).

Antioxidant Activity of Catechins Isolate of Uncaria

Gambier Roxb in Male Rats. LIFE: International

Journal of Health and LifeSciences, 4(2).34-46. DOI-

https://dx.doi.org/10.20319/lijhls.2018.42.3446.

http://www.grdspublishing.org/index.php/life/article/vi

ew/1388/1172.

National Standard Indonesia: SNI 01-3391-2000,

http://infolpk.bsn.go.id/index.php?/sni_main/sni/detail

_sni/3793.

Ningsih Sri et al., 2014, Evaluation Of Antilipid

Peroxidation Activity Of Gambir Extract On Liver

Homogenat In Vitro. International Journal of

PharmTech Research. ISSN : 0974-4304 Vol.6, No.3,

982-989

Pakki, Dkk., 2009, (In bahasa), Uji Aktivitas Antibakteri

Enzim Papain Dalam Sediaan Krim Terhadap

Staphylococcus Aureus. Majalah Farmasi Dan

Farmakologi Vol.13, No.1 (Issn: 1410-7031).

Pambayun et al., 2007, Phenolic Content of Gambir Leaf

Extract and Antibacterial Activity, (In bahasa)

Kandungan Fenolik Ekstrak Daun Gambir Dan

Aktivitas Antibakterinya. AGRITECH vol. 27 No. 2

Prasetyo, B.F., Wientarsih, I., Priosoeryanto, B.P., 2010,

Activity of Ambon Banana Tree Stem Extract Gel in

the Process of Healing Wounds in Mice, (In bahasa)

Aktivitas Sediaan Gel Ekstrak Batang Pohon Pisang

Ambon dalam Proses Penyembuhan Luka pada

Mencit. Jurnal Veteriner. Vol: 11, No 2 : 70-73

Rowan, M.P.; Cancio, L.C.; Burmeister, D.M.; Rose, L.F.;

Natesan, S.; Chan, R.K.; Christy, R.J.; Chung, K.K.,

2015, Burn wound healing and treatment: Review and

advancements. Crit. Care 19, 243,

doi:10.1186/s13054-015-0961-2.

Rini A, Oktadefitri Y, Lucida H., 2013, Sunscreen cream

formulation of ethyl p-methoxycinamate combination

with catechins, Proceedings of the National Seminar

on the Latest Development of Pharmaceutical

Sciences and Clinics III (In bahasa : Formulasi krim

tabir surya dari kombinasi etil p – metoksisinamat

dengan katekin), . Prosiding Seminar Nasional

Perkembangan Terkini Sains Farmasi dan Klinik III

ISSN: 2339-2592

Tehrani S, Lotfi P, Tehrani S, Jangholi E, Aryan H,

Aidun A, (2016) Healing Effect of Sesame Ointment

on Second degree Burn Wound in Rats, GMJ, 5(2):56-

62, www.gmj.ir, file:///C:/Users/User/Downloads/658-

3565-5-PB.pdf.

Shahzad MN and Ahmed N (2013), Effectiveness of Aloe

Vera Gel compared with 1% silver sulphadiazine

cream as burn wound dressing in second degree burns,

J Pak Med Assoc, Vol. 63, No.2, February, 225-230

Sjamsuhidayat R, Wim de Jong., 1997, (In bahasa) Buku

Ajar Ilmu Bedah. Edisi 2. EGC, Jakarta.

S.V. Hosseini, H. Niknahad, N.Fakhar, A. Rezaianzadeh

and D. Mehrabani, (2011), The Healing Effect of

Honey, Putty, Vitriol and Olive Oil in Pseudomonas

Aeroginosa Infected Burns in Experimental Rat.

Model. Asian Journal of Animal and Veterinary

Advances 6 (6), 572-579 ISSN 1683-9919 / DOI:

10.3923/ajava, 572.579.

Somboonwong J, Kankaisre M, , Boonyong Tantisira B

and Tantisira MH, (2012), Wound healing activities of

different extracts of Centella asiatica in incision and

burn wound models: an experimental animal study,

BMC Complementary and Alternative Medicine 2012

12:103. doi:10.1186/1472-6882-12-103.

http://www.biomedcentral.com/1472-6882/12/103.

Standar Nasional Indonesia (National Standard of

Indonesia : SNI 01-3391-2000.

http://teknologihutan.fkt.ugm.ac.id/userfiles/download

/SNI_01-3391-2000_-_Gambir.pdf

Sumoza Nelsy, Sucidayana, Efrizal, Rahayu Resti., 2014,

(In bahasa) Pengaruh Gambir (Uncaria gambir R.)

Terhadap Penyembuhan Luka Bakar Pada Mencit

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

10

Putih (Mus musculus L.) Jantan [Effect of Gambir

(Uncaria gambir R.) on Healing Burns in Male White

Mice (Mus musculus L.)], Jurnal Biologi Universitas

Andalas 283-288 (ISSN : 2303-2162)

Taniguchi S., Kuroda K., Yoshikado N., Doi K.I., Tanabe

M., Shibata T., Yoshida T., Hatano T., 2008, New

Dimeric Flavans From Gambir, an Extract of Uncaria

gambir. Japan: The Japan Institute of Heterocyclic

Chemistry, Okayama University, 1-11.

http://ousar.lib.okayamau.ac.jp/files/public/3/34205/20

160528031815252292/fulltext.pdf.

Voravuthikunchai, S.P. and Mitchell, H., 2008, Inhibitory

and killing activities of medicinal plants against

multiple antibiotic-resistant Helicobacter pylori. J

Health Sci 54, 1–8.

W. Hananeh, Ismail ZB, Alshehabat MA, Ali J, (2015),

Review of animal models used to study effects of bee

products on wound healing: findings and applications,

Bull Vet Inst Pulawy 59, 425-431, 2015,

DOI:10.1515/bvip-2015-0062.

https://www.degruyter.com/downloadpdf/j/bvip.2015.

59.issue-3/bvip-2015-0062/bvip-2015-0062.pdf.

Wasserman, D., 2002, Criteria for burn severity:

Epidemiology, prevention, organization of

management. Pathol. Biol. 50, pp 65–73

Effects of Gambir (Uncaria gambir Roxb) Catechins on Burn Wound Healing in Male Rats

11