Original Article
29
DEVELOPMENT OF PEACOCK FLOWER EXTRACT AS
ANTI-WRINKLE FORMULATION
Suwicha Soisuwan1,2, Warissara Mapaisansin1, Weerasak Samee1,
Adelheid H. Brantner3 and Narisa Kamkaen1,4,*
1Faculty
of Pharmacy, Srinakharinwirot University, Ongkharak, Nakhorn-nayok, 26120, Thailand
of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
3Institute of Pharmaceutical Sciences, University of Graz, A - 8010 Graz, Austria
4Research and Development Institute, Suan Dusit Rajabhat University, Bangkok 10300, Thailand
2Faculty
ABSTRACT: The main objective of this study was to evaluate the antioxidant property of the
ethanolic extract from the petals of Caesalpinia pulcherrima, and to develop an anti-wrinkle
product from the crude extract including the quality assessment. The DPPH radical scavenging
and ABTS cation radical scavenging assay were used to evaluate the anti-oxidant properties.
The results of DPPH radical scavenging assay showed the strongest activity (IC50= 34.74 µg/ml)
of the crude extract from the red petals of C. pulcherrima, followed by the extract of the orange
petals (IC50=35.63 µg/ml) and the yellow petals (IC50=102.27 µg/ml), respectively. The ABTS
cation radical scavenging assay demonstrated the strongest activity (IC50=227.66 µg/ml) for the
orange petals followed by the red petals (IC50=243.01 µg/ml) and the yellow petals (IC50=338.72
µg/ml). Consequently, orange petals were chosen in order to develop an anti-wrinkle product
in an O/W formula (oil in water cream). Quality evaluation of the anti-wrinkle product was
assessed by using heating–cooling cycle’s method. No change of the physical properties was
observed; the pH was in a proper range (approximately pH7). The quantity of gallic acid as
marker of the crude extract and the product was analyzed by HPLC. The amount of gallic acid
in crude extract and finished product was 1.26 and 1.87 µg/ml, respectively. Finally, 21
volunteers participated in the clinical study for a period of one month in order to test the
product’s safety and efficacy. The results demonstrated that the anti-wrinkle product had the
efficacy of enhancing the elasticity of the skin. No irritation could be observed at the volunteer’s
skin.
Keywords: Peacock flower extract, Antioxidant, Antiwrinkle, Gallic acid, Elasticity
INTRODUCTION: Caesalpinia pulcherrima (L.)
Sw. (Caesalpiniaceae) is commonly known as
“Peacock flower.” It is a shrub growing up to 3 m,
native to tropical America. The leaves are
bipinnate, 20-40 cm long, bearing 3-10 pairs of
pinnae, each with 6-10 pairs of leaflets which are
15-25 mm long and 10-15 mm broad. The flowers
are in racemes up to 20 cm long, each flowers
appearing in a variety of colors including yellow,
pink, off-white, and red with yellow margins. The
fruit is a 6-12 cm long pod. This striking
ornamental plant, widely grown in tropical
gardens, is also the national flower of the
Caribbean island Barbados. In India it is found in
the tropical rain forests. With a beautiful
inflorescence in yellow, red and orange, it is called
“Ratnagundhi”. Medicine men in the Amazon
rainforest use C. pulcherrima, which is known as
“Ayoowiri”, in their traditionl medicine. The juice
from the leaves cures fever, the juice from the
flowers is used against sores, the seeds are
applied against cough, breathing difficulties and
chest pains, the roots induce abortion in the first
trimester of pregnancy. A pharmacological study
of C. pulcherrima demonstrated that the crude
extract exhibited antiviral, antibacterial and antiinflammatory activities1,2). Phytochemical investigations
reported that the root, leaves and stem contained
three diterpenoids which are identified as caesalpin,
cassane and dibenzoate3). Previous studies reported
of tannins and flavonoids from the stem and the
aerial parts4). Moreover, in a recent study gallic
acid, catechin, rutin, and ellagic acid as potential
radical scavengers were found5).
In our laboratory, an anti-wrinkle cream containing
the crude extract of C. pulcherrima flower in an oilin-water (O/W) cream was formulated. In order to
assure the efficacy and safety of this cream, the
antioxidant activity and the efficacy were evaluated
in the present study.
∗
To whom correspondence should be addressed.
E-mail: [email protected]
Tel. +66 2244 5280, Fax. +66 2668 7460
J Health Res 2010, 24(1): 29-34
30
MATERIALS AND METHODS
Plant materials
C. pulcherima petal flowers were collected at
Srinakharinwirot University, Ongkharak campus
in March 2007. The voucher specimen was
deposited in the plant herbarium at Faculty of
Pharmacy.
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), and
2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS) were purchased from Sigma-Aldrich (St.
Louis, USA). Gallic acid and rutin were from
Fluka Chemicals (Buchs, Switzerland). Ethanol,
methanol, acetonitrile and phosphoric acid were
from Merck (Darmstadt, Germany). All other
reagents were analytical grade available.
Extraction of the plant material
Air-dried flowers (100 g) of C. pulcherrima were
powdered and macerated with 95% ethanol (2
liters) with an automatic shaker for 3 days. The
extract was filtered and concentrated by rotary
evaporator. The thick red brown residue (extract
yield: 6.55 %) was kept in a desiccator.
Antioxidant activity of the crude extract
The DPPH radical scavenging and ABTS cation
radical scavenging assay were modified to evaluate
the anti-oxidant properties6).
DPPH radical scavenging assay
The 96-wells of a microtiter plate were divided
into 3 sets (set A-C) as follows: each well of set A
(test sample) contained 100 µl of ethanolic test
sample (concentration 25 – 400 µg/ml) and 100 µl
of the ethanolic DPPH radical; each well of set B
(blank of test sample) contained 100 µl of
ethanolic test sample and 100 µl of ethanol; each
well of set C (control) contained 100 µl ethanol
and 100 µl of the ethanolic DPPH radical. After
filling and mixing the solutions in the well, the
plate was incubated at 25oC for 30 min. The
absorbance was measured at a wavelength of 520
nm by using a microplate reader (Anthos Labtech
Instrument, Zenyth 200). %AA was calculated
from the equation below by comparing with the
standards gallic acid and rutin. IC50 was obtained
from the calibration curve between %AA and the
concentration of sample (equation A).
J Health Res 2010, 24(1): 29-34
Original Article
ABTS cation radical scavenging assay
The 96-wells of a microtiter plate were divided
into 3 sets (set A-C) as follows: each well of set A
(test sample) contained 50 µl of the ethanolic test
sample (concentration 50 – 1000 µg/ml) and 1000
µl of the ABTS+ radical; each well of set B (blank of
test sample) contained 50 µl of the test sample
and 1000 µl ethanol; each well of set C (control)
contained 50 µl ethanol and 1000 µl of the ABTS+
radical.
After filling and mixing the solutions in the
well, the plate was incubated at 25oC for 30 min.
The absorbance was measured at wavelength 734
nm by using the microplate reader (Anthos
Labtech Instrument, Zenyth 200). %AA was
calculated from the below equation by comparing
with standard gallic acid and rutin. IC50 was
obtained from the calibration curve between %AA
and the concentration of sample (equation A).
(Equation A)
When Acontrol = absorbance of C (control)
Asample = the difference of absorbance of
A (test sample) and the absorbance of B
(blank of test sample)
Preparation of the anti-wrinkle formulation
The crude ethanolic extract of C. pulcherrima
flower was mixed with an oil-in-water cream base
for the formulation of the herbal anti-wrinkle
formulation. The ingredients of the formula (%,
w/w) are listed in Table 1. The procedure of
making an herbal cream is as follows. The oil
phase (as shown as “A” in Table 1) was melted in
beaker I heated to 70°C using a water bath. The
water phase (as shown as “B” in Table 1) was
heated to 75°C in a separate beaker II using a
water bath. Once the desired temperature was
reached the water phase was added to the oil
phase, using the homogenizer. C. pulcherrima
flower extract was dissolved in SNP-solubilizant in
a separate beaker III. When temperature of the
mixture decreased to 40 °C, liquid germal plus,
triethanolamine and flower extract in SNPsolubilizant were added and the mixture was
homogenized for 30 min.
Original Article
31
Quality assessment of the anti-wrinkle
formulation
Five concentrations of gallic acid (0.02, 1, 2, 4
and 6 µg/ml) were represented as standard
calibration curves. The gallic acid was extracted
from 3.57 g of the anti-wrinkle product (equal to
25 mg crude extract). Methanol was added until a
final volume of 25 ml. The solution was mixed by
using a Vortex (Vortex genie G-560E) for 3 min
followed by sonication for 10 min. The solution
was refrigerated until the oil phase was combined
and centrifuged with 2500 rpm for 10 min. The
clear supernatant was separated, filtered with
filter paper (pore size 0.45 µm) and analyzed by
High Pressure Liquid Chromatography (HPLC;
Agilent 1100 series). The HPLC conditions for the
analysis of the gallic acid in the anti-wrinkle
product are listed in Table 2.
Evaluation of the efficacy of the anti-wrinkle
formulation
The evaluation of the efficiency of the
formulation was conducted by measuring the skin
elasticity and moisture content by using
and
CutometerTM,
of
both
TriplesenseTM
formulations; the herbal cream and the cream
base at week 0, 1, 2, 3, 4. The test products were
applied at a dose of 0.5 g to the desired area (3 x 3
cm2) listed in Table 3 once daily in the evening.
The safety of the products was observed in term of
the skin irritation (edema and erythema). The
data were collected and analyzed with the paired
t-test by using SPSS for Windows 17.0.
Table 1 Ingredients of cream base and herbal cream
formulation
Phase
A
B
C
E
Ingredients
Action
SNP*-Nanowax
SNP*-ICM
emulsifier
thickening
agent
emollient
Diisopropyl
adipate
Vitamin E
Acetate
Carbopol-Ultrez
10
Allantoin
Deionized-water
Triethanolamine
Liquid Germal
Plus
C. pulcherima
flower extract
SNP*solubilizant
Quantity
(%w/w)
Cream
Herbal
base
cream
7.0
7.0
3.0
3.0
2.0
2.0
antioxidant
0.5
0.5
thickening
agent
moisturizer
vehicle
pH adjuster
preservative
0.7
0.7
0.1
79.8
0.95
0.5
0.1
79.65
0.95
0.5
-
0.7
5.6
4.9
active
ingredient
solubilizer
Table 2 HPLC conditions for the gallic acid in the
anti-wrinkle product
Mobile phase
Stationary
phase
Detector
Flow rate
Injection
volume
Run time
H3PO4 (pH=3):CH3CN (9:1)
C18 column (4.6 I.D. x 250 mm)
UV-DAD (λ = 270 nm)
0.8 ml/min
3.0 µl
20 min
Table 3 The desired area for applying the test
products
Skin area
Upper forearm
Middle forearm
Lower forearm
Application
treated with herbal cream
treated with cream base
untreated skin
RESULTS:
Antioxidant activity assay
DPPH radical scavenging assay
The correlation between percent inhibition and
concentration of C. pulcherrima petal extracts using
the DPPH radical scavenging assay was demonstrated
in Figure 1. Among three colors of flower petal
extracts, the red petals exhibited the strongest
antioxidant activity with IC50 of 34.74 µg/ml while
the orange and yellow petals exhibited weaker
activities with IC50 of 35.63 and 102.27 µg/ml,
respectively.
ABTS cation radical scavenging assay
The correlation between percent Inhibition and
concentration (µg/ml) of C. pulcherima petal extracts
using ABTS cation radical scavenging assay was
demonstrated in Figure 2. Among the three colors
of flower petal extracts, the orange petals
exhibited the strongest antioxidant activity with
IC50 of 227.66 µg/ml while the red and yellow
petals exhibited a weaker activity with IC50 of
243.01 and 338.72 µg/ml respectively.
Evaluation of the particle size by scanning
electron microscope (SEM)
After the formulation of the O/W cream, the
particle size of the anti-wrinkle product has been
evaluated using SEM (JEOL JSM-6400). The results
showed that the formulation was homogenous
and consistent in the particle size of 0.1-0.3 µm as
shown in Figure 3.
*SNP = Specialty Natural ProductTM
J Health Res 2010, 24(1): 29-34
32
Original Article
Stability test of the anti-wrinkle formulation
The quality evaluation of the anti-wrinkle
formulation was accessed under accelerated
controlled humidity conditions (75% RH+25% RH)
by using six cycles of heating–cooling (5oC 48 h
and 45oC 48 h)7,8) as shown in Table 4. The
organoleptical tests showed no differences in
consistence and appearance of the stability test.
The pH was in a proper range (approximately pH
7) as well.
Quality analysis using HPLC Technique
The quantity of gallic acid in the crude extract
and the anti-wrinkle product was analyzed by
HPLC. The linear calibration curve (Y=5045.3X–
0.1662, R2=0.9914) in Figure 4 showed the
correlation between AUC of HPLC chromatogram
and the concentration of gallic acid as chemical
marker. Gallic acid was found in the crude extract
and in the finished product at concentrations of
1.26 and 1.87 µg/ml respectively.
Efficacy of the anti-wrinkle product
Elasticity
The skin elasticity of twenty one volunteers
was evaluated by using a cutometerTM. The antiwrinkle product and the cream base were applied
to the right inner forearm; right upper arm (RUA)
and right central arm (RCA), respectively. One
area of equal size on the right lower arm (RLA)
was evaluated for comparison without treatment.
The skin elasticity was measured in week 0, 1, 2,
3, and 4 as shown in Figure 5. RUA enhanced the
skin elasticity in week 1, 2, 3, 4 compared to the
base line. The data of week 3 were statistically
significant (p=0.03). RCA and RLA enhanced the
skin elasticity in week 2 and 3 compared to the
base line.
Figure 1 Percent inhibition and concentration
(µg/ml) of C. pulcherrima petal extracts (yellow, red
and orange) compared to the standards gallic acid
and rutin using DPPH radical scavenging assay
Figure 2 Percent Inhibition and concentration
(µg/ml) of C. pulcherrima petal extracts (yellow, red
and orange) compared to the standards gallic acid
and rutin using ABTS cation radical scavenging
assay
Figure 3 SEM Image of anti-wrinkle product
Calibration curve of gallic acid
Table 4 Stability test of the anti-wrinkle product
using six cycles of heating–cooling
Before
6 cycles
Appearance
Color
pH
Smooth
cream
Yellow
7.10
J Health Res 2010, 24(1): 29-34
After 6 cycles
5oC
(48 h)
Smooth
cream
Yellow
6.94
45oC
(48 h)
Smooth
cream
Yellow
6.95
y = 5045.3x - 0.1662
R2 = 0.9914
35
30
25
AUC 20
15
10
5
0
0
0.001
0.002
0.003
0.005
0.004
0.006
0.007
Gallic acid concentration (mg/ml)
Figure 4 Calibration curve of the standard gallic acid
Original Article
Moisturizing
Skin moisturizing of twenty one volunteers
was evaluated by using triplesenseTM. The antiwrinkle product and the cream base were applied
to the right inner forearm; right upper arm (RUA)
and right central arm (RCA), respectively. One
area of equal size on the right lower arm (RLA)
was evaluated for comparison without any
treatment. The skin moisturizing was measured
in week 0, 1, 2, 3, and 4 as shown in Figure 6.
The skin moisturizing of RUA, RCA, and RLA
improved in week 2 compared to the base.
Figure 5 Elasticity of test persons (n=21) using
the anti-wrinkle product (RUA), cream base
(RCA) in comparison to untreated skin (RLA) at
the week 0, 1, 2, 3, 4
Figure 6 Moisturizing of probands (n=21) using
an anti-wrinkle product (RUA), the cream base
(RCA) in comparison to untreated skin (RLA) in
week 0, 1, 2, 3, 4
33
DISCUSSION:
Antioxidant activity
The antioxidant activity of the peacock petal
extracts were conducted by using DPPH radical
scavenging assay. The results have shown that the
red petals gave the strongest activity with
IC50=34.74 µg/ml, followed by the orange petals
with IC50=35.63 µg/ml. On the other hand, ABTS
cation radical scavenging assay has shown that
the orange petals have the strongest activity with
IC50=227.66 µg/ml, followed by the red petals with
IC50=243.01 µg/ml. The orange petals were
selected to be the raw material for the anti wrinkle
cream because of the high antioxidant activity.
Stability test of the anti-wrinkle product
The stability test was conducted by six cycles
of a heating-cooling method. It was shown that
the anti-wrinkle product had good optical
appearance with a neutral pH (pH = 7) and a
smooth cream without cracking. Moreover, the
standard microbial assays9) using Petri dishes
showed sufficient microbiological stability with a
microbial count less than 500 CFU/g. No
Escherichia coli, Salmonella sp., Staphylococcus
aureus and Pseudomonas aeruginosa that could
be proved neither in the anti-wrinkle product nor
in the cream base.
Quality analysis using HPLC
The quality analysis was conducted by using
HPLC and gallic acid as chemical marker. Gallic
acid was proved in the orange petal extract and
the anti-wrinkle product at concentrations of 1.26
and 1.87 µg/ml, respectively.
Efficacy of the anti-wrinkle formulation
Twenty one healthy volunteers were recruited
in the clinical trial and followed up every week for
four weeks. The efficacy evaluation was conducted
by measuring the skin elasticity and the moisture
content before and after using the products or the
untreated skin. The skin elasticity of the
volunteers increased in week 1, 2, 3 and 4
compared with that of before using the antiwrinkle product. The elasticity in week 3 was
statistically significant. The skin elasticity of
volunteers which applied the anti-wrinkle cream
J Health Res 2010, 24(1): 29-34
34
increased in week 2, 3 and 4 compared to the
group not using the anti-wrinkle product.
The moisture content of the skin decreased
compared to the time before using the product. It
can be concluded that the products had no effect
on the skin moisture because of the cold dry
weather in the laboratory (room temperature
24oC).
CONCLUSION: This study supports the traditional
Thai use of C. pulcherrima as an ingredient in
cosmetic products with antioxidant and with antiwrinkle activity. The best effect was achieved by
the ethanolic extract which corresponds to the
traditional extraction practice. The amount of
phenolic compounds in term of gallic acid
suggests that the flowers of C. pulcherrima may be
a good source of natural antioxidants which may
be incorporated into a range of cosmetics and
health products.
ACKNOWLEDGEMENT: Thailand Research Fund
(TRF) through Industrial and Research Projects
for Undergraduate Studies (IRPUS) 2550 and
University Mobility Asia Pacific (UMAP) 2007 are
acknowledged for providing the partial financial
assistance to this research project. Specialty
Natural Product Co, Ltd. is acknowledged for
providing the SNP materials for the product
formulation.
J Health Res 2010, 24(1): 29-34
Original Article
REFERENCES:
1. Chiang LC, Chiang W, Liu MC, Lin CC.
2003. In vitro antiviral activities of Caesalpinia
pulcherrima and its related flavonoids. J
Antimicrob Chemother 52:194–8.
2. Rao YK, Fang SH, Tzeng YM. 2005. Antiinflammatory activities of flavonoids isolated from
Caesalpinia pulcherrima. J Ethnopharmacol
14:249-53.
3. Roach JS, McLean S, Reynolds WF, Tinto
WF. 2003. Cassane diterpenoids of Caesalpinia
pulcherrima. J Nat Prod 66(10): 1378-81
4. Srinivas KVNS, Rao YK, Mahender I, et al.
2003. Flavonoids from Caesalpinia pulcherrima.
Phytochemistry; 63: 789–93.
5. Samee W, Saereedenchai V, Sornchaithawatwong C and Worarat S. 2007. Simultaneous
Determination of Gallic acid, Catechin, Rutin,
Ellagic acid and Quercetin in Flower Extract of
Ceasalpinia pulcherrima and Nelumbonucifera
Extracts by HPLC, Thai Pharm and Health Sc J
2: 131-7.
6. Siddhuraju P. and Becker K. 2007. The
antioxidant and free radical scavenging activities
of processed cowpea (Vigna unguiculata (L.) Walp.)
seed extracts, Food Chemistry.101: 10–9.
7. Grimm W. 1987. Stability testing of drug
products. Stuttgart. 160-1.
8. U.S. Food and Drug Administration.
Guidance for Industry Q1A (R2) Stability Testing
of New Drug Substances and Products. Accessed
from http:/www.fda.gov/cber/gdlns/ichstab.htm
9. Department of Medical Sciences, Ministry of
Public Health, Tiwanond Road, Nonthaburi
11000, Thailand. 2000. Thai Pharmacopoeia.:
Volume II Part 2.