The development of anti-acne products from Eucalyptus globulus and Psidium Guajava oil

Abstract

The aims of this study were to determine the main chemical constituents of volatile oils from leaves of Eucalyptus globulus Labill. (eucalyptus) and Psidium guajava L. (guava) and to develop stable anti-acne preparations from these medicinal plants. Volatile oils from eucalyptus and guava leaves were extracted by hydrodistillation and then were analyzed of their main chemical components by means of GC/MS. The antimicrobial activity of the oils was determined by agar diffusion and micro-dilution methods. The GC/MS results showed that the main chemical constituents in the volatile oils from eucalyptus and guava leaves were gamma-terpinene and alpha-pinene. These volatile oils showed good antimicrobial activity against Propionibacterium acnes (MIC, MBC = 9.38 mg/ml for eucalyptus oil and MIC = 9.38 mg/ml, MBC = 37.50 mg/ml for guava oil). Further, oil- in-water creams incorporating 2% w/w eucalyptus oil and 4% w/w guava oils were prepared. Both of them had satisfied texture and exhibited good stability after stored at -4 ° C and 45 ° C alternately (freeze-thawing) for 4 cycles. In addition, they had antimicrobial activity against P. acnes determined by agar well diffusion method (inhibition zone = 8.0 and 9.0 mm for eucalyptus cream and guava cream, respectively) compared to commercial 5% benzoyl peroxide anti-acne gel.

Full text

Original Article 109
J Health Res 2008, 22(3): 109-113
THE DEVELOPMENT OF ANTI-ACNE PRODUCTS FROM
EUCALYPTUS GLOBULUS AND PSIDIUM GUAJAVA OIL
Sirivan Athikomkulchai
1,*
Rith Watthanachaiyingcharoen
1
Sujimon Tunvichien
1
Panida Vayumhasuwan
2
Paisarn Karnsomkiet
1
Prapan Sae-Jong
1
Nijsiri Ruangrungsi
3,4
1
Faculty of Pharmacy, Srinakharinwirot University, Nakhonnayok
2
PanRajtheveeGroup, Bangkok
3
Faculty of Pharmaceutical Sciences,
4
College of Public Health Sciences, Chulalongkorn University, Bangkok
ABSTRACT: The aims of this study were to determine the main chemical constituents of volatile
oils from leaves of Eucalyptus globulus Labill. (eucalyptus) and Psidium guajava L. (guava) and to
develop stable anti-acne preparations from these medicinal plants. Volatile oils from eucalyptus and
guava leaves were extracted by hydrodistillation and then were analyzed of their main chemical
components by means of GC/MS. The antimicrobial activity of the oils was determined by agar
diffusion and micro-dilution methods. The GC/MS results showed that the main chemical constituents
in the volatile oils from eucalyptus and guava leaves were gamma-terpinene and alpha-pinene.
These volatile oils showed good antimicrobial activity against Propionibacterium acnes (MIC, MBC =
9.38 mg/ml for eucalyptus oil and MIC = 9.38 mg/ml, MBC = 37.50 mg/ml for guava oil). Further, oil-
in-water creams incorporating 2% w/w eucalyptus oil and 4% w/w guava oils were prepared. Both of
them had satisfied texture and exhibited good stability after stored at -4
°
C and 45
°
C alternately
(freeze-thawing) for 4 cycles. In addition, they had antimicrobial activity against P. acnes determined
by agar well diffusion method (inhibition zone = 8.0 and 9.0 mm for eucalyptus cream and guava
cream, respectively) compared to commercial 5% benzoyl peroxide anti-acne gel.
Keywords: Propionibacterium acnes, Psidium guava, Eucalyptus globulus, anti-acne product, volatile oil
INTRODUCTION:
Acne is a skin disease which
causes painful social and psychological effects on
sufferers. It is a problem for many adults as well as for
many teenagers. The primary factors involved in the
formation of acne lesions are increased sebum
production, sloughing of keratinocytes, bacterial growth
and inflammation. Propionibacterium acnes (P. acnes),
an anaerobic pathogen, plays an important role in the
pathogenesis of acne by inducing certain inflammatory
mediators
1)
. In Thailand, there are many powerful
antimicrobial medicinal plants to research and develop
for effective antimicrobial products.
Tea tree oil has been used for almost 100 years in
Australia
but is now available worldwide both as neat oil
and as an active
component in an array of products.
Employed
largely for its antimicrobial properties, tea tree
oil is incorporated
as the active ingredient in many
topical formulations used to
treat cutaneous infections
2)
.
Eucalyptus globulus Labill. (eucalyptus) and Psidium
guajava L. (guava) are the plants in the same family as
tea tree (Myrtaceae). These are the plants cultivated in
Thailand. The essential oil of eucalyptus and guava
show antibacterial activity against wide range of
bacteria including
Propionibacterium acnes (P. acnes)
3-4)
.
Therefore, the aim of this study is to prepare anti-acne
oil-in-water cream incorporated with volatile oil extracted
from the fresh leaves of eucalyptus and guava.
Furthermore, accelerated stability testing by storing the
preparations at 45
°
C/ -4
°
C for 4 cycles was carried out
and antibacterial assay was determined by agar
diffusion, microwell dilution and agar well diffusion
methods.
MATERIALS AND METHODS:
Plant materials
Fresh leaves of Eucalyptus globulus Labill. (euca-
lyptus) and Psidium guajava L. (guava) were collected
from botanical garden of Faculty of Pharmacy, Sri-
nakharinwirot University, Nakhon-nayok. The voucher
∗
To whom correspondence should be addressed.
E-mail: sirivan@swu.ac.th, Tel. 0 2664 1000 ext. 1700, fax. 0 3739 5096

110 Original Article
J Health Res 2008, 22(3): 109-113
specimens (SWU 130507 and SWU 131607) were
deposited in the herbarium at Faculty of Pharmacy,
Srinakharinwirot University, Nakhon-nayok.
Chemicals
The excipients of the preparations (Table 1) were
purchased from Namsiang trading Co., Ltd. Clinda-
mycin (Dalacin C
®
) from Pfizer international Co., Ltd.
and 5% benzoyl peroxide gel (5% Pan-oxyl
®
) from
Stiefel laboratories (Thailand) Ltd. were purchased from
local distributors.
Test organisms
Propionibacterium acnes DMST 14917 were purchased
from NIH, Thailand.
Culture methods
The test organisms were stored on Tryptic soy agar
(TSA) slopes at 2-4
°
C, except for Propionibacterium
acnes, which was stored on Brain Heart Infusion Agar
(BHIA) with 10% sheep blood. The organism used in
screening activities with agar diffusion assays and
microwell dilution assays were twice pass-aged 16-18
hours cultures grown in Brain Heart Infusion broth
(BHIB). The inoculated broths were adjusted to 0.5
McFarland standard turbidity (0.048 M BaCl
2
0.5 ml and
added with 0.18 M H
2
SO
4
99.5 ml)
The essential oils from the leaves of eucalyptus and
guava were determined for antimicrobial activities by
agar diffusion and microwell dilution assay. The
effectiveness of anti-acne products was determined by
agar well diffusion method.
Agar diffusion assay
5-6)
Antimicrobial tests were carried out by agar diffusion
method using 100
μl of suspension containing 10
8
CFU/ml spread on Brain Heart Infusion Agar (BHIA).
The discs (9.0 mm in diameter) were impregnated with
30
μl of each solution (in high soluble compounds) and
placed on the inoculated agar. The diluted solution
impregnated disc was used as negative control.
Clindamycin was used as positive controls to determine
the sensitivity of tested organism. The inoculated plates
were incubated at 37
°
C for 18-24 hours and in
anaerobic condition for Propionibacterium acnes. Anti-
microbial activity was evaluated by measuring the zone
of inhibition against the test organisms.
Microwell dilution assay
7-8)
The minimal inhibition concentration (MIC) values
were also studied for the microorganisms which were
determined as sensitive to the extracted solutions in
agar diffusion assay. The inocula of microorganisms
were prepared from 16-18 hours broth cultures and
suspensions were adjusted to McFarland standard
turbidity number 0.5. The extracted solutions were
diluted in serial 2-fold dilutions. The initial concentration
was the highest compound of essential oils in tested
solution. The 96-well plates were prepared by
dispensing into each well 90
μl of broth, 5 μl of 2, 3, 5
Triphenyl-Tetrazolium Chloride (TTC) solution and 5
μl
of the inoculum. A 100
μl of highest concentrations
were added into the first wells. Then, 100
μl from their
serial dilutions was transferred into 4 consecutive wells.
The final volume in each well was 200
μl. The diluted
solution impregnated discs were used as negative
controls. Clindamycin was used as positive controls to
determine the sensitivity of tested solutions. The plate
was covered with a sterile plate sealer and mixed on
plate shaker at 200 rpm for 1 minute. After that, the
plates were incubated at appropriate condition for 18-24
hours. The first concentration which not showed the red
color was the MIC value. The 5
μl of solution from clear
wells were spread on nutrient agar plates. The MBC
(Minimum Bactericidal Concentration) was defined as
the concentration of the compounds to kill the
microorganisms. So that, the sample concentration was
not exhibited the growth of microorganisms as the MBC
value.
Extraction and Identification of volatile oil
Plant materials were cut into small pieces. They
were extracted by hydrodistillation with clevenger
apparatus. The volatile oil was collected and stored at
2-4
°
C until being used. The chemical analysis was
done by gas chromatography mass spectrometry
(GC/MS) on a Finnigan Trace GC ultra (Thermo
Electron Corpo-ration, USA) with quadrupole mass

Original Article 111
J Health Res 2008, 22(3): 109-113
spectrometer. The column was ZB-5 fused silica linked
methyl silicon capillary column (30 m. x 0.22 mm. i.d.;
0.25
μM); oven temperature programming was 50-
250
°
C at 7
°
C/min; injector and detector temperature
were 250 and 280
°
C, respectively; sample volume
injected was 1
μl; split ratio was 100:1; and the carrier
gas was He (2 ml/min).
Compounds were identified by comparing the
Kovats gas chromatographic retention indices of the
peaks on the HP-5MS column with literature values,
computer matching using the Masslynx database, and
comparison of the fragmentation patterns of the mass
spectra with those reported in the literature
9-10)
.
Preparation of oil in water anti-acne cream (o/w)
An o/w cream was prepared by addition of the
pre-melted lipophilic parts to the aqueous phase
components. Emulsification was achieved by low shear
homogenization using a lab scale mixer (T25 basic S2,
Ika Labortechnik, Staufen, Germany). The preparations
were contained 2% and 4% w/w of volatile oils from
eucalyptus and guava, respectively. The compositions
of the preparations are given in Table 1.
Accelerated stability testing
The effect of freeze thawing on the physical stability
of products was examined. The products were kept at -
4
°
C (48 hours) and 45
°
C (48 hours) alternately for 4
cycles, and then the phase separation was observed. In
addition, the products were also kept at 45
°
C and -4
°
C
for 30 days.
Agar well diffusion method
11)
The agar well diffusion method was the method to
test the anti-acne products. This method was the same
principle as the agar diffusion assay. It was changed
only the application of the sample. The agar well
diffusion method was used the reservoir of the anti-
acne cream instead of the paper disc. And so on, the
antimicrobial activity was determined by the diameter of
inhibition zone against the tested organisms compared
with the reference standard.
RESULTS ANS DISCUSSION: The extraction of
volatile oil from the fresh leaves of eucalyptus and
guava yields 0.4% and 0.2% w/w, respectively. Both
are clear-yellow oil with characteristic odor.
The results of the screening activity of eucalyptus
and guava volatile oil showed antibacterial activity
against P. acnes by agar diffusion method. The mean
diameter of the inhibition zones from eucalyptus and
guava volatile oil in the concentration 62.5 mg/ml were
in the range of 10.0 to 11.0 mm. The broth dilution
results of eucalyptus and guava volatile oil, MIC and
MBC values against P. acnes were determined. MIC
values of eucalyptus and guava volatile oil were 9.375
mg/ml whereas MBC values of eucalyptus and guava
volatile oil were 9.375 and 37.5 mg/ml, respectively.
The MIC and MBC of positive control, clindamycin,
were 0.5 mg/ml.
The GC/MS results in Table 2 and 3 showed that
the major compounds in the volatile oil from the leaves
of eucalyptus and guava were gamma-terpinene and
alpha-pinene, respectively (Figure 1).
Table 1 The compositions of the 2% eucalyptus and 4% guava
oil creams
Excipients
% w/w
2% Eucalyptus 4% Guava
Stearic acid 4.0 4.0
Spermaceti 4.0 4.0
Glycerylmonostearate 6.0 6.0
White petrolatum 1.0 1.0
Silicone oil 9.0 7.0
Carbopol 940 0.3 0.3
Glycerine 2.0 2.0
PEG 1500 1.0 1.0
Citric acid 0.5 0.5
Sodium citrate 0.05 0.05
Sodium metabisulfate 0.1 0.1
Methyl paraben 0.1 0.1
Propyl paraben 0.01 0.01
Triethanolamine 1.5 1.5
Water 68.5 68.5
Figure 1
Chemical structure of gamma-terpinene and
alpha
-
pinene
Gamma-terpinene
A
lpha-pinene

112 Original Article
J Health Res 2008, 22(3): 109-113
Gamma-terpinene is a monoterpene that plays an
important role for antibacterial activity. It might cross the
cell membranes, thus penetrating into the interior of the
cell and interacting with intracellular sites critical for
antibacterial activity
12)
.
Alpha-pinene is found in the oils of many
species of
many coniferous trees, especially the pine. It shows
antimicrobial activity against wide range of bacteria and
fungal
13)
.
Both eucalyptus and guava oil creams showed
good texture and have proper pH to be used topically.
After stored under freeze thaw condition, phase
separation was not observed.
The antimicrobial activities against P. acnes of
eucalyptus and guava oils containing creams were
determined by agar well diffusion method (Table 4).
The results demonstrated the good activities of 2%
eucalyptus and 4% guava oil creams (the mean
diameter of the inhibition zones were in the range of 8.0
to 9.0 mm.) compared to the commercial 5% benzoyl
peroxide anti-acne gel (the mean diameter of the
inhibition zone was 10.0 mm.). Their efficacy was
decreased after stored under accelerated conditions
(-4
°
C, 45
°
C, freeze thawing).
This study indicated that gamma-terpinene and
alpha-pinene were the monoterpenes that having
antimicrobial activity against P. acnes. They are the
major chemical components of volatile oils from
eucalyptus and guava. These results suggested that
the preparations incorporating the volatile oils of
eucalyptus and guava could be used as anti-acne
products, however further clinical research will be
necessary.
ACKNOWLEDGEMENTS:
The authors wish to
thank the Thailand Research Fund (TRF) for financial
support through Industrial and Research Projects for
Undergraduate Students (IRPUS) 2550.
REFERENCES:
1. Brigitte D. 2004. Topical Antibacterial Therapy for
Acne Vulgaris. Therapy In Practice. Drugs 64: 2389-
2397.
Table 2 Chemical constituents of E. globulus
volatile oil from
hydrodistillation
Compounds %Area Retention Time
Monoterpenes
α -Pinene 8.57 5.67
α -Phellandrene 2.78 7.67
α -Terpinene 0.41 8.09
p- Cymene 28.75 8.36
D-Sylvestrene 2.00 8.50
γ
-Terpinene 44.60 9.56
Terpinolene 0.82 10.68
Oxygenated monoterpenes
1,8-Cineol 4.48 8.59
4-Terpineol 5.42 14.24
α -Terpineol 0.55 14.80
Table 3
Chemical constituents of P. guajava volatile oil from
hydrodistillation
Compounds
%Area Retention Time
Monoterpenes
α
-Pinene
23.89 5.65
Sylvestrene
7.85 8.44
Oxygenated monoterpenes
1,8-Cineol
6.32 8.54
Sesquiterpenes
E-Caryophyllene
14.30 24.18
A
romadendrene
3.01 24.96
α
–
Humulene
1.51 25.54
β
-
Bisabolene
2.19 27.74
Oxygenated sesquiterpenes
E-Nerodiol
3.33 29.85
Caryophyllene oxide
17.25 30.59
Caryophylla
-
4(12), 8(13)-dien
-
5-alpha-ol
2.50 32.58
Table 4 The inhibition zone of the preparations under various
conditions
Preparations
Inhibition zone (mm)
2% Eucalyptus
Freshly prepared
8.0
Stored under freeze thaw condition 6.0
Stored at 45°C (30 days)
6.0
Stored at -4°C (30 days)
7.0
4% Guava
Freshly prepared
9.0
Stored under freeze thaw condition 7.0
Stored at 45°C (30 days)
7.0
Stored at -4°C (30 days)
8.0
Cream base
-
5% benzoyl peroxide gel
10.0

Original Article 113
J Health Res 2008, 22(3): 109-113
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การพัฒนาตํารับผลิตภัณฑ

ตานสิวจากน
้
ํามันหอมระเหยของยูคาลิปตัสและฝรั
่
ง
ศิริวรรณ อธิคมกุลชัย
1,
∗
ฤทธิ์ วัฒนชัยยิ่งเจริญ
1
ศุจิมน ตันวิเชียร
1
พนิดา วยัมหสุวรรณ
2
ไพศาล กานตสมเกียรติ
1
ประพันธ แซจง
1
นิจศิริ เรืองรังษี
3,4
1
คณะเภสัชศาสตร มหาวิทยาลัยศรีนครินทรวิโรฒ
2
บริษัท แพนราชเทวีกรุป จํากัด
3
คณะเภสัชศาสตร จุฬาลงกรณมหาวิทยาลัย
4
วิทยาลัยวิทยาศาสตรสาธารณสุข จุฬาลงกรณมหาวิทยาลัย
บทคัดยอ: วัตถุประสงคของงานวิจัยนี้คือการพัฒนาตํารับผลิตภัณฑรักษาสิวและวิเคราะหองคประกอบทางเคมีของน้ํามัน
หอมระเหยจากใบยูคาลิปตัส (Eucalyptus globulus Labill.) และใบฝรั่ง (Psidium guajava L.) ซึ่งไดจากการกลั่นดวยน้ํา
(hydrodistillation) จากนั้นนําไปวิเคราะหหาองคประกอบทางเคมีดวย GC/MS พบวาองคประกอบหลักของน้ํามันหอมระเหย
จากใบย
ูคาลิปตัสและใบฝรั่ง คือ gamma-terpinene และ alpha-pinene ตามลําดับ สําหรับการพิจารณาฤทธิ์ตานสิวของน้ํามัน
หอมระเหยนั้น พิจารณาจากฤทธิ์ในการยับยั้งเชื้อ Propionibacterium acnes (P. acnes) ดวยวิธี agar diffusion และวิธี
microwell dilution จากผลการทดลองพบวา น้ํามันหอมระเหยจากใบยูคาลิปตัส (MIC, MBC = 9.38 mg/ml) และน้ํามันหอม
ระเหยจากใบฝรั่ง (MIC = 9.38 mg/ml, MBC = 37.50 mg/ml) มีผลในการยับยั้งเชื้อ P. acnes ไดดี หลังจากนั้นนําน้
ํามันหอม
ระเหยที่ไดมาพัฒนาตํารับเปนครีมแบบน้ํามันในน้ํา ครีมที่ไดมีลักษณะที่ดี มีความคงสภาพหลังจากเก็บที่ -4°C หรือ 45°C
สลับกัน 4 รอบ ทําการประเมินประสิทธิภาพของครีมในการตานเชื้อ P. acnes โดยวิธี agar well diffusion ตํารับครีมที่ผสม
น้ํามันหอมระเหยจากใบยูคาล
ิปตัสรอยละ 4 w/w มี inhibition zone เทากับ 8.0 มิลลิเมตรและตํารับครีมที่ผสมน้ํามันหอม
ระเหยจากใบฝรั่งรอยละ 2 w/w มี inhibition zone เทากับ 9.0 มิลลิเมตร ในการยับยั้งเชื้อ P. acnes เทียบกับเจลรักษาสิว
5%
benzoyl peroxide
คําสําคัญ: Propionibacterium acnes ยูคาลิปตัส ฝรั่ง น้ํามันหอมระเหย ผลิตภัณฑรักษาสิว
*ติดตอไดที่ sirivan@swu.ac.th โทรศัพท 0 2664 1000 ตอ 1700 โทรสาร 0 3739 5096