developm peacock flower_anti wrinkle formulation_soisuwan et al 2010

8/11/2019 Developm Peacock Flower_Anti Wrinkle Formulation_Soisuwan Et Al 2010

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Original Article 29

J Health Res2010, 24(1): 29-34

DEVELOPMENT OF PEACOCK FLOWER EXTRACT AS

ANTI-WRINKLE FORMULATION

Suwicha Soisuwan1,2, Warissara Mapaisansin1, Weerasak Samee1,

Adelheid H. Brantner3and Narisa Kamkaen1,4,*1Faculty of Pharmacy, Srinakharinwirot University, Ongkharak, Nakhorn-nayok, 26120, Thailand

2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

3Institute of Pharmaceutical Sciences, University of Graz, A - 8010 Graz, Austria4Research and Development Institute, Suan Dusit Rajabhat University, Bangkok 10300, Thailand

ABSTRACT:The main objective of this study was to evaluate the antioxidant property of theethanolic extract from the petals of Caesalpinia pulcherrima, and to develop an anti-wrinkleproduct from the crude extract including the quality assessment. The DPPH radical scavengingand 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 g/ml)of the crude extract from the red petals of C. pulcherrima, followed by the extract of the orangepetals (IC50=35.63 g/ml) and the yellow petals (IC50=102.27 g/ml), respectively. The ABTS

cation radical scavenging assay demonstrated the strongest activity (IC50=227.66 g/ml) for theorange petals followed by the red petals (IC50=243.01 g/ml) and the yellow petals (IC50=338.72g/ml). Consequently, orange petals were chosen in order to develop an anti-wrinkle productin an O/W formula (oil in water cream). Quality evaluation of the anti-wrinkle product wasassessed by using heatingcooling cycles method. No change of the physical properties wasobserved; the pH was in a proper range (approximately pH7). The quantity of gallic acid asmarker of the crude extract and the product was analyzed by HPLC. The amount of gallic acidin crude extract and finished product was 1.26 and 1.87 g/ml, respectively. Finally, 21volunteers participated in the clinical study for a period of one month in order to test theproducts safety and efficacy. The results demonstrated that the anti-wrinkle product had theefficacy of enhancing the elasticity of the skin. No irritation could be observed at the volunteersskin.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 tropicalgardens, 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 anti-

inflammatory activities1,2). Phytochemical investigations

reported that the root, leaves and stem contained

three diterpenoids which are identified as caesalpin,

cassane anddibenzoate3). Previous studies reported

of tannins and flavonoids from the stem andthe

aerial parts4). Moreover, in a recent study gallicacid, catechin, rutin, and ellagic acid as potential

radical scavengers were found5).

In our laboratory, an anti-wrinkle cream containing

the crude extract of C. pulcherrimaflower in an oil-

in-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]. +66 2244 5280, Fax. +66 2668 7460


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30 Original Article

J Health Res2010, 24(1): 29-34

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 otherreagents were analytical grade available.

Extraction of the plant material

Air-dried flowers (100 g) of C. pulcherrimawere

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 cationradical 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 l of ethanolic test

sample (concentration 25 400 g/ml) and 100 l

of the ethanolic DPPH radical; each well of setB

(blank of test sample) contained 100 l of

ethanolic test sample and 100 l of ethanol; each

well of set C (control) contained 100 l ethanol

and 100 l of the ethanolic DPPH radical. After

filling and mixing the solutions in the well, the

plate was incubated at 25oCfor 30 min. The

absorbance was measured at a wavelength of 520

nm by using amicroplate reader (Anthos Labtech

Instrument, Zenyth 200). %AA was calculated

from the equation below by comparing with the

standards gallic acid and rutin. IC50was obtained

from thecalibration curve between%AA and the

concentration of sample (equation A).

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 l of the ethanolic test

sample (concentration 50 1000 g/ml) and 1000l of the ABTS+radical; each well of setB (blank of

test sample) contained 50 l of the test sample

and 1000 l ethanol; each well of set C (control)

contained 50 l ethanol and 1000 l 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 thecalibration curve between%AA

and the concentration of sample (equation A).

When Acontrol = absorbance ofC (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 70C using a water bath. The

water phase (as shown as B in Table 1) was

heated to 75C 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,

triethanolamineand flower extract in SNP-

solubilizant were added and the mixture was

homogenized for 30 min.

(Equation A)


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Original Article 31

J Health Res2010, 24(1): 29-34

Quality assessment of the anti-wrinkle

formulation

Five concentrations of gallic acid (0.02, 1, 2, 4

and6 g/ml) were represented as standard

calibration curves. The gallic acid was extractedfrom 3.57 g of the anti-wrinkle product (equal to

25 mg crude extract). Methanol was added until a

final volume of25 ml. The solution was mixed by

using a Vortex (Vortex genie G-560E) for3min

followed by sonication for10 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 m) 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

TriplesenseTM and CutometerTM, of both

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 (3x 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.

RESULTS:

Antioxidant activity assay

DPPH radical scavenging assay

The correlation between percent inhibition and

concentrationof C. pulcherrimapetal extracts using

the DPPH radical scavenging assaywas demonstrated

in Figure 1. Among three colors of flower petal

extracts, the red petals exhibited the strongest

antioxidant activity with IC50of 34.74 g/ml while

the orange and yellow petals exhibited weakeractivities with IC50 of 35.63 and 102.27 g/ml,

respectively.

ABTS cation radical scavenging assay

The correlation between percent Inhibition and

concentration(g/ml) of C. pulcherimapetal 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 g/ml while the red and yellow

petals exhibited a weaker activity with IC50 of

243.01 and 338.72 g/mlrespectively.

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 mas

shown in Figure 3.

Table 2 HPLC conditions for the gallic acid in theanti-wrinkle product

Mobile phase H3PO4(pH=3):CH3CN (9:1)Stationaryphase

C18 column (4.6 I.D. x 250 mm)

Detector UV-DAD (= 270 nm)Flow rate 0.8 ml/minInjectionvolume

3.0 l

Run time 20 min

Table 3 The desired area for applying the testproducts

Skin area Application

Upper forearm treated with herbal creamMiddle forearm treated with cream baseLower forearm untreated skin

Table 1 Ingredients of cream base and herbal creamformulationPhase Ingredients Action Quantity

(%w/w)

Creambase

Herbalcream

A SNP*-Nanowax emulsifier 7.0 7.0SNP*-ICM thickening

agent3.0 3.0

Diisopropyladipate

emollient 2.0 2.0

Vitamin EAcetate

antioxidant 0.5 0.5

B Carbopol-Ultrez10

thickeningagent

0.7 0.7

Allantoin moisturizer 0.1 0.1Deionized-water vehicle 79.8 79.65

C Triethanolamine pH adjuster 0.95 0.95Liquid GermalPlus

preservative 0.5 0.5

E C. pulcherimaflower extract

activeingredient

- 0.7

SNP*-solubilizant

solubilizer 5.6 4.9

*SNP = Specialty Natural ProductTM


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32 Original Article

J Health Res2010, 24(1): 29-34

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 heatingcooling (5oC 48 hand 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 usingHPLC 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 thecorrelation 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 g/ml respectively.

Efficacy of the anti-wrinkle product

Elasticity

The skin elasticity of twenty one volunteers

was evaluated by using a cutometerTM. The anti-

wrinkle 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(g/ml) of C. pulcherrima petal extracts (yellow, redand orange) compared to the standards gallic acidand rutin using DPPH radical scavenging assay

Figure 2 Percent Inhibition and concentration(g/ml) of C. pulcherrima petal extracts (yellow, redand orange) compared to the standards gallic acidand rutin using ABTS cation radical scavengingassay

Figure3SEM Image of anti-wrinkle product

Calibration curve of gallic acidy = 5045.3x - 0.1662

R2

= 0.9914

0

5

10

15

20

25

30

35

0 0.001 0.002 0.003 0.004

0.005 0.006

0.007

Gallic acid concentration (mg/ml)

AUC

Figure4Calibration curve of the standardgallic acid

Table 4 Stability test of the anti-wrinkle productusing six cycles of heatingcooling

Before6 cycles

After 6 cycles

5oC(48 h)

45oC(48 h)

Appearance Smoothcream

Smoothcream

Smoothcream

Color Yellow Yellow Yellow

pH 7.10 6.94 6.95


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Original Article 33

J Health Res2010, 24(1): 29-34

Moisturizing

Skin moisturizing of twenty one volunteers

was evaluated by using triplesenseTM. The anti-

wrinkle 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.

DISCUSSION:

Antioxidant activity

The antioxidant activity of the peacock petal

extracts were conducted by using DPPH radicalscavenging assay.The resultshave shown that the

red petals gave the strongest activity with

IC50=34.74 g/ml, followed by the orange petals

with IC50=35.63 g/ml. On the other hand, ABTS

cation radical scavenging assay has shown that

the orange petals have the strongest activity with

IC50=227.66 g/ml, followed by the red petals with

IC50=243.01 g/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 couldbe proved neither in the anti-wrinkle product nor

in the cream base.

Quality analysis usingHPLC

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 concentrationsof 1.26

and 1.87 g/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 anti-

wrinkle product. The elasticity in week 3 was

statistically significant. The skin elasticity of

volunteers which applied the anti-wrinkle cream

Figure 5Elasticity of test persons (n=21) usingthe anti-wrinkle product (RUA), cream base(RCA) in comparison to untreated skin (RLA) atthe week 0, 1, 2, 3, 4

Figure6Moisturizing of probands (n=21) usingan anti-wrinkle product (RUA), the cream base(RCA) in comparison to untreated skin (RLA) in

week 0, 1, 2, 3, 4


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34 Original Article

J Health Res2010, 24(1): 29-34

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 effecton 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 anti-

wrinkle 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 acidsuggests 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. SpecialtyNatural Product Co, Ltd. is acknowledged for

providing the SNP materials for the product

formulation.

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1.

Chiang LC, Chiang W, Liu MC, Lin CC.

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pulcherrima and its related flavonoids. J

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Rao YK, Fang SH, Tzeng YM. 2005. Anti-inflammatory activities of flavonoids isolated from

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3. Roach JS, McLean S, Reynolds WF, Tinto

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