International Journal of Cosmetic Science, 2011, 33, 269275
Standardized extract of Syzygium aqueum: a safe cosmetic
ingredient
U. D. Palanisamy*, L. T. Ling, T. Manaharan, V. Sivapalan*, T.Subramaniam, M. H. Helme and T. Masilamani
*Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46100 Bandar Sunway, Faculty
of Medicine, University of Malaya, 59100 Kuala Lumpur and Natural Product and Cosmetics Program, SIRIM Bhd, 1, Persiaran Dato Menteri,
40911 Shah Alam, Selangor, Malaysia
Received 5 July 2010, Accepted 12 October 2010
Keywords: anti-cellulite, antioxidant, high phenolic content, skin whitening, Syzygium aqueum
Synopsis
Syzygium aqueum, a species in the Myrtaceae family, commonly
called the water jambu is native to Malaysia and Indonesia. It is
well documented as a medicinal plant, and various parts of the tree
have been used in traditional medicine, for instance as an antibi-
otic. In this study, we show S. aqueum leaf extracts to have a signif-
icant composition of phenolic compounds, protective activity
against free radicals as well as low pro-oxidant capability. Its
ethanolic extract, in particular, is characterized by its excellent
radical scavenging activity of EC50 of 133 lg mL)1 1,1-diphenyl-2-
picryl-hydrazyl (DPPH), 65 lg mL)1 2,2-azino-bis(3-ethylbenzthia-
zoline-6-sulphonic acid) (ABTS) and 71 lg mL)1 (Galvinoxyl), low
pro-oxidant capabilities and a phenolic content of 585670 mg
GAE g)1 extract. The extract also displayed other activities, deem-
ing it an ideal cosmetic ingredient. A substantial tyrosinase inhibi-
tion activity with an IC50 of about 60 lg mL)1 was observed. In
addition, the extract was also found to have anti-cellulite activity
tested for its ability to cause 98% activation of lipolysis of adipo-
cytes (fat cells) at a concentration of 25 lg mL)1. In addition, the
extract was not cytotoxic to Vero cell lines up to a concentration of
600 lg mL)1. Although various parts of this plant have been used
in traditional medicine, this is the first time it has been shown to
have cosmeceutical properties. Therefore, the use of this extract,
alone or in combination with other active principles, is of interest
to the cosmetic industry.
sume
Re
Le Syzygium Aqueum, est une espe`ce de la famille des Myrtacees,
mieux connue sous le nom de pomme deau, originaire de Malaisie
et dIndonesie. Ses qualites medicinales sont largement et diverses
parties de larbre sont utilisees en medecine traditionelle, dautre
part a ete prouve quelle possedait des proprietes antibiotiques. keeping skin inflammation to a minimum for a healthy functioning
Dans cette etude, nous demontrons que les extraits de feuilles du
S. aqueum posse`dent une quantite significative de composes phenoli-
ques, une activite protectrice contre les radicaux libres, ainsi
quune faible incidence pro-oxydante. Son extrait ethanolique, en
particulier, se caracterise par son excellente activite delimination
des radicaux de EC50 de 133 lg mL)1 (DPPH), 65 lg mL)1 (ABTS)
Correspondence: U. D. Palanisamy, Jeffrey Cheah School of Medicine
and Health Sciences, Monash University Sunway Campus, Jalan Lagoon
Selatan, 46100 Bandar Sunway, Malaysia. Tel.: +603 55145840; fax:
+603 55146323; e-mail: umadevi.palanisamy@med.monash.edu.my
2011 The Authors
ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
doi: 10.1111/j.1468-2494.2010.00637.
et 71 lg mL)1 (Galvinoxyl), faible incidence pro-oxydante et un
contenu phenolique de 585670 mg GAE g)1 extrait. Lextrait pre-
sentait aussi dautres proprietes en faisant un ingredient cosme-
tique ideal. Une activite inhibitrice substantielle de la tyrosinase
avec IC50 de 60 lg mL)1 (blanchissement de la peau) a egalement
ete observee. De plus, il a ete prouve que lextrait avait une propri-
ete anti-cellulite, testee pour sa capacite a` causer la lipolyse
dadipocytes (cellules grasses) avec un EC50 de 16 lg mL)1. Egale-
ment, lextrait naffichait aucune cytotoxicite envers les cellules
Vero avec une concentration inferieure a` 600 lg mL)1. Malgre le
fait que diverses parties de cette plante ont ete utilisees en mede-
cine traditionelle, ceci est la premie`re fois que ses proprietes cosme-
ceutiques sont demontrees. Par consequent, lutilisation de cet
extrait, seul ou combine avec dautres principes actifs, est dun
grand interet pour lindustrie cosmeceutique.
Introduction
Phenolic compounds, the secondary plant metabolites, have been
receiving much attention lately being the naturally occurring
inhibitors of oxidation. There exists numerous literature on the
ability of plant extracts to serve as possible free radical scavengers,
such as green tea at protecting against free radical damage in the
gastrointestinal tract [1], bilberry at upregulating the oxidative
stress defence enzymes [2], grape seed extract with its neuroprotec-
tive effects [3], pomegranate juice for its antioxidative effect in dia-
betes [4] and milk thistle in its protective role against burn-induced
oxidant skin injury [5]. There are plenty of opportunities in the
market for these extracts because they are natural ingredients and
have a positive reputation to be readily accepted [6].
There is also a growing research showing that topically applied
antioxidants can help protect from sun damage [7]. These antioxi-
dants also serve as anti-inflammatory agents that are important in
skin. Some cosmetic companies are utilizing antioxidants for their
rejuvenating effects in make-up. Tocopherols and citric acid are
two well-known natural antioxidants commonly used in industry,
and technologists continue to conduct studies to show the efficacy
of other potential natural antioxidants.
Melanogenesis is a physiological process resulting in the synthe-
sis of melanin pigments, which plays a crucial protective role
against skin photocarcinogenesis. In humans and other mammals,
the biosynthesis of melanin takes place in a lineage of cells known
as melanocytes, which contain the enzyme tyrosinase. Tyrosinase
(phenol oxidase) is known to be a key enzyme for melanin
269
Standardized extract of Syzygium aqueum
biosynthesis, and its inhibition has been the subject of many stud-
ies [8, 9]. The search for natural chemical agents that are able to
modulate the metabolism of pigmentation is of great interest to cos-
metic chemists. Arbutin, a naturally occurring beta-d-glucopyrano-
side of hydroquinone, is used traditionally for depigmentation. The
mode of action of arbutin is through the inhibition of the melanos-
omal tyrosinase enzyme [10].
Cellulite afflictions are a stubborn problem causing emotional
and psychological distress to many women. Many treatments for
cellulite have been devised and are directed at reducing the
agglomerations of fatty tissue. Lipolysis is the breaking down of
fatty deposits and is a very desirable mechanism to stimulate in
any cellulite treatment. A variety of plant extracts appear to serve
as possible anti-cellulite agent and are currently available commer-
cially [11, 12].
Syzygium aqueum is a species in the Myrtaceae family, native to
Malaysia and Indonesia. Common names include wax apple, love
apple, java apple, water apple, mountain apple, jambu air (water
guava in Malay), wax jambu, rose apple, bell fruit, macopa and
tambis (Philippines). It is a tropical tree growing to 12 m height,
with evergreen leaves 1025 long and 510 cm broad. Called
Kavika in Fiji, it is well documented as a medicinal plant (particu-
larly the bark of the Kavika tree). Various parts of the tree are used
in traditional medicine, and some have in fact been shown to pos-
sess antibiotic activity [13].
Materials and methods
Chemicals
The chemicals l-ascorbic acid, adenosine deaminase, caffeine, con-
canavalin A, dexamethasone, l-Dopa, DPPH, Folin-Ciocalteu
reagent, gallic acid, galvinoxyl, genistein, 3-isobutylmethylxanthine
(IBMX), kojic acid, melanin, mushroom tyrosinase, myrecetin,
potassium persulfate, theophylline and l-tyrosine were purchased
from Sigma Chemical Co (St. Louis, MO, U.S.A.). Penicillin, strepto-
mycin, essential amino acids, cell culture media and supplements
were obtained from Flow Lab. Ltd. (Irvine, Scotland). The
dl-a-tocopherol and potassium hexacyanoferrate (III) were obtained
from Fluka Biochemika (Buchs, Switzerland). Vitis vital Grape
seed flour was from Agricultural Research Institute Speyer
(Germany), whereas Emblica from EMD Chemicals Inc. (Gibbs-
town, NJ, U.S.A.). Phosphate-buffered saline tablets (PBS) were
obtained from ICN Biomedicals (Aurora, OH, U.S.A.). All solvents
were obtained from Scharlau Chemicals (Germany). The ABTS
diammonium salt was obtained from Amresco (Solon, OH, U.S.A.).
All cell cultures were purchased from American Tissue Culture
Collection (Manassas, VA, U.S.A.). Randox glycerol kit to determine
the release of glycerol was obtained from Roche, Diagnostics GmbH
(Penzberg, Germany).
Plant collection
Fresh leaves of S. aqueum were obtained from Kuala Lumpur. The
plants were authenticated by a botanist from the Kepong Herbar-
ium, Forest Research Institute of Malaysia.
Preparation of plant extracts
The leaves were washed with copious amounts of water followed by
distilled water and then allowed to air dry at room temperature. It
was then placed in an oven at 40C until completely dry, after which
ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
270
U. D. Palanisamy et al.
it was powderized using a Waring blender or milled using the Fritsch
dry miller. Extraction of the powderized leaf was carried out with
deionized water and the solvents ethanol and propylene glycol:
deionized water (80 : 20) at 1 : 10 (w/v) concentrations. Water
extraction was carried out at 40C, whereas solvent extraction at
root temperature for 24 h in an orbital shaker. The suspension thus
obtained was filtered using a 114 Whatman filter paper, and filtrate
was collected. Aqueous filtrate was concentrated using a freeze drier,
whereas solvent filtrate was concentrated using a rotary evaporator.
Extraction using propylene glycol: water (80 : 20) was used as it is.
Standardization of S. aqueum ethanolic extract
High Performance Liquid Chromatography (HPLC) standardization
was performed on a Shidmazu IT-TOFMS Liquid Chromatography
system equipped with LC-20AD/T liquid pump, SPD-m20A diode
array detector, SIL-20A auto-injector, DGU-20A system controller
and CTO-20AC column oven. Chromatographic separation was
achieved on a Waters Bridge C-18 column (50 2.2 mm,
2.5 lm) (Waters, U.S.A.). The mobile phase consisted of solvent A:
0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile: start-
ing from 10% B for 2 min, 100% B for 5 min and finally 3 min
with 100% B for washing. The column was reconditioned to 10%
B. The detection wavelength was 254 nm with a flow rate of
0.5 mL min)1 and an injection volume of 10 lL. Compounds were
analysed on a Shidmazu Prominence UFLC-LCMS-IT-TOF. The
analysis was performed in both the positive and negative modes.
Nuclear Magnetic Resonance spectroscopy (NMR) spectra of the
compound was obtained using a Jeol ECA 400 (400 MHz) NMR
spectrometer and characterized by 1H, 13C, 2D NMR.
Antioxidant assay
Three different free radical scavenging (FRS) assays were performed:
scavenging activity onto DPPH radicals, galvinoxyl radicals and
ABTS radicals, which were assessed according to Palanisamy et al.
[14].
Determination of total phenolic content (TPC)
Total phenolics were determined using the Folin-Ciocalteu method
described by Miliauskas [15]. This assay is based on a colorimetric
oxidation and reduction reaction. First, 1 mL aliquots of the extracts
were added to 5 mL of Folin-Ciocalteu reagent. After 3 min, 4 mL of
7.5% Na2CO3 solution in water was added to the mixture and the
content was thoroughly mixed. The absorbance at 765 nm was read
after 1 h. Blank consisted of Folin-Ciocalteu reagent (5 mL), etha-
nol/distilled water (1 mL) and 7.5% Na2CO3 solution (4 mL). A lin-
ear doseresponse regression curve was generated using absorbance
reading of gallic acid at the wavelength of 765 nm. The calibration
curve using gallic acid was obtained in the same manner as above
except that the absorbance was read after 30 min. Results were
expressed as milligrams of gallic acid equivalent per gram of dry
weight (mg GAE g)1) of extracts. Total content of phenolic com-
pounds in the plant extracts was calculated using this formula:
C A=B
C = expressed as mg GAE/g dry weight of the extract
A = the equivalent concentration of gallic acid established from
calibration curve (mg)
B = the dry weight of extract (g).
2011 The Authors
International Journal of Cosmetic Science, 33, 269275
Standardized extract of Syzygium aqueum
Pro-oxidant assay
Pro-oxidant and antioxidant effects are attributable to the balance.
In a Fenton reaction, Fe2+ reacts with H2O2, resulting in the pro-
duction of hydroxyl radical, which is considered to be the most
harmful radical to biomolecules. In the Fenton reaction, Fe2+ is
oxidized to Fe3+. Many reductants such as ascorbic acid can reduce
the oxidized form of iron (Fe3+) to reduced form (Fe2+). This reac-
tion could enhance the generation of hydroxyl radicals. The pre-
domination of reducing power (on iron ions) over the free radical
scavenging activity results in the pro-oxidant effect. Reducing
power of iron ion was measured according to the method of Tian
and Hua [16] where 500 lL of the extract and 50 lL of 1% potas-
sium ferricyanate [K3Fe (CN6)] were incubated at 50C for 20 min.
An equal volume of 10% trichloroacetic acid was then added, and
mixture was centrifuged at 3000 g for 10 min. The upper layer of
the solution (1 mL) was mixed with 1 mL of distilled water and
0.2 mL of 0.1% ferric chloride (FeCl3), and its absorbance was
recorded at 700 nm. Ethanol or distilled water was used as nega-
tive control, whereas Vitamin C and Emblica (a commercial anti-
oxidant with very low pro-oxidant activity) was used as positive
controls. Results are expressed in comparison with positive controls
at concentration ranging from 0.1 to 0.5 mg mL)1.
Tyrosinase inhibition assay
Tyrosinase activity was determined as described by Bernard and
Berthon [17] with minor modifications using 96-well plates. Ini-
tially, 60 lL of plant extracts were added to the top well. A serial
dilution of the extracts was performed starting from the second well
using 30 lL 0.02 M phosphate buffer pH 6.9. This was followed by
the addition of 30 lL of 1 mM l-DOPA, 30 lL of 1 mM l-tyrosine
and 470 lL of 50 mM of phosphate buffer (pH 6.8). To start the
reaction, 20 lL of mushroom tyrosine (254 U mL)1) was added to
the solution. The plate was left at 37C for 30 min prior to mea-
suring the amount of dopachrome produced. The absorbance was
read against the blank well (without enzyme) at 490 nm using the
ASYS UVM 340 Microplate Reader (Biochrom, Cambridge, UK).
Kojic acid, a commercially available natural tyrosinase inhibitor, at
0.1 mg mL)1 was used as the positive control. All determinations
were performed in triplicates.
The percentage inhibition of tyrosinase activity was calculated
as follows:
% Inhibition A  B=A  100 S. aqueum was determined against International standards; induc-
where
A = absorbance at 490 nm without test sample
B = absorbance at 490 nm with test sample.
Lipolysis activation assay
The ability of S. aqueum extracts to stimulate lipolysis in adipocytes
was investigated. Lipolysis was assayed in fully differentiated 3T3-L1
fat cells. As adipocytes undergo lipolysis, free fatty acids (FFA) and
glycerol are released into the culture medium. The glycerol released
was used to quantify lipolysis activity. Lipolysis stimulation was com-
pared using the positive controls caffeine and theophylline. 3T3-L1
fibroblasts were grown to confluence in 24-well plates (with initial
seeding of 2 104 cells mL)1) in Dulbeccos modified Eagles med-
ium added with 10% calf serum (CS/DMEM), 1% penicillin, 1% strep-
tomycin and 1% glutamine. The post-confluent cells were stimulated
2011 The Authors
ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 269275
U. D. Palanisamy et al.
to initiate differentiation by the addition of FBS/DMEM medium with
0.5 mM 3-IBMX and 0.1 lM dexamethasone. Subsequently, the
medium was replaced with differentiation progression medium which
is FBS/DMEM medium with 1.7 lM insulin for 48 h followed by
treatment only with FBS/DMEM medium. Differentiation was com-
plete after 1520 days. One day prior to the experiment, the cells
were replaced with fresh medium lacking FBS. To measure lipolysis,
fat cells attached in the 24-well plates were treated with Krebs
Ringer bicarbonate (KRB), 30 mM Hepes, 1% BSA, 2.5 mM glucose
as the incubation buffer at pH 7.4 (KRB medium). Adenosine deami-
nase (10 lg mL)1) was added to the incubation medium to prevent
the accumulation of adenosine which inhibits lipolysis. Positive con-
trols and test extracts (25 lg mL)1) were added to the cells and incu-
bated for 24 h at 37C with constant shaking in a CO2 incubator. At
the end of the incubation, supernatant was removed (after standing
for 5 mins) and heated at 70C for 10 min to inactivate enzymes
released by the cells that may interfere with the glycerol test. Glycerol
release was measured using the Randox Glycerol kit and expressed
as lmoles glycerol/l. Glycerol concentration was calculated from a
glycerol standard curve. Untreated cells (without extracts) were
taken as the control (basal lipolysis level). A viability assay was ini-
tially carried out using 25 lg mL)1 of extract and positive controls.
Cytotoxicity studies
Vero cell lines were cultured in minimum essential medium supple-
mented with 10% foetal bovine serum, non-essential amino acid,
and 100 IU mL)1 Penicillin and 100 lg mL)1 Streptomycin. The
cells are cultured in tissue-culture-grade flasks at 37C in a humid-
ified atmosphere containing 5% CO2. Four different extract prepara-
tions of S. aqueum were assessed. The Vero cells were plated on
96-well plate followed by a 24-h incubation to allow cell attach-
ment. The cells were then subjected to various concentrations of
S. aqueum extracts (00.7 mg mL)1) and solvent controls and left
to incubate for 48 h. Cytotoxic effect was assessed using the colori-
metric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, a yellow tetrazole)] assay following the method of Mos-
mann [18]. Viability was presented as the percentage absorbance
at 570 nm of treated against non-treated cells. ZnSO4 was used as
the positive control, and all assays were performed in triplicate.
Heavy metal content determination
Lead, arsenic and mercury content of the powderized rind of
tively coupled plasma - optical emission spectrometry (ICP-OES) for
the determination of arsenic and lead, and atomic absorption spec-
troscopy (AAS) for the determination of mercury.
Statistical analysis
All experiments were performed in triplicate. Means, standard
errors and standard deviations were calculated from replicates
within the experiments, and analyses were conducted using Micro-
soft Excel 2003.
Results
Isolation and standardization of S. aqueum extract
The extraction yields of S. aqueum under ethanolic and aqueous
conditions were observed to be similar, with aqueous extraction
271
Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

3.5

3 Vitamin C

Absorbance (700 nm)

S.aquem ethanolic
2.5 S.aquem aqueous
2 Emblica

1.5
Figure 1 Standardized Syzygium aqueum ethanolic extract on an
1
analytical HPLC detected at 254 nm. Arrow indicates the retention
0.5
time of myricetin.
0
0 0.2 0.4 0.6
Concentration (mg mL1)
yielding 6.1 0.81%, whereas ethanol extraction saw a
6.3 0.66% yield. To ensure the consistency of the S. aqueum leaf
Figure 2 Pro-oxidant capacity of Syzygium aqueum leaf extracts
extracts, we established the standardized HPLC profile of the ethan-
compared with vitamin C and Emblica.
olic extract (Fig. 1).
The compound at retention time 2.2 min was identified to be
myricetin. The MS and NMR data obtained were consistent with Total phenolic content
that reported in previous studies [19, 20]. The amount of myricetin
The TPC of the extracts were determined following the Folin-Ciocal-
in the ethanolic extracts of S. aqueum was quantified to be
teu method (Fig. 3).
0.1 mg g)1 extract.
The TPC of the aqueous and ethanolic extracts of S. aqueum was
comparable to that of grape seed extracts. Aqueous extracts
Antioxidant assays exhibited TPC of 180200 (S. aqueum) and 120150 (grape seed)
mg g)1 GAE, whereas ethanolic extracts showed 520700
The extracts were evaluated for its DPPH, galvinoxyl and ABTS
(S. aqueum) and 510800 (grape seed) mg g)1 GAE. A correlation
scavenging ability, and its activity is as shown in Table I.
between the FRS activity and TPC of S. aqueum and grape seed
As observed in Table I, the leaves of S. aqueum displayed almost
aqueous and ethanolic extracts was evident in that higher TPC cor-
similar FRS ability to grape seed extract. In both S. aqueum and
responded to higher FRS activity.
grape seed, the ethanolic extracts displayed the lowest IC50 values
in all three scavenging assays. Among the three FRS assays, the
ABTS assay was observed to show the lowest IC50 value indicating Tyrosinase inhibition activity
its sensitivity over the other assays.
The ability of the S. aqueum extracts to behave as whitening agents
by preventing melanin production was assessed by its inhibition of
Pro-oxidant assay the enzyme tyrosinase. Kojic acid, a commercially available natural
compound used commonly as a skin-whitening agent, was used as
The pro-oxidant capability of S. aqueum extract was compared with
the positive control (Fig. 4). It was observed that the IC50 of PG
that of vitamin C and Emblica (Merck KGaA, Darmstadt,
(propylene glycol):H2O (80 : 20) and ethanolic extracts of S. aque-
Germany) (Fig. 2), a commercially available plant extract used in
um (57 and 71 lg mL)1, respectively) were comparable to that of
cosmetics and is known for its very low pro-oxidant capacity [21].
kojic acid (52 lg mL)1). PG:H2O (80 : 20) was chosen as the
As expected, vitamin C showed the highest pro-oxidant activity,
extraction solvent for it is often the solvent used in cosmetic prepa-
induced by transition metals, and the positive control Emblica
ration of natural ingredients.
showed the lowest pro-oxidant capacity. Interestingly, aqueous
S. aqueum extracts exhibited low pro-oxidant capacity, comparable
to that of Emblica over the range of concentration tested. The
ethanolic extract, on the other hand, had lower pro-oxidant capac-
ity than vitamin C but higher than that of its aqueous extract.

900
Aqueous
Total phenolic content (mg g1)

Table I Free radical scavenging activity of Syzygium aqueum leaf 800 Ethanolic
extracts 700
600

S. aqueum extract Grape seed extract 500

400

Aqueous Ethanolic Aqueous Ethanolic 300

200
100
DPPH 0.33 0.07 0.21 0.02 0.46 0.18 0.27 0.1
0
Galvinoxyl 0.15 0.04 0.08 0.01 0.62 0.34 0.09 0.03 S.aquem leaf Grape seed
ABTS 0.19 0.07 0.03 0.003 0.19 0.12 0.04 0.01 Extracts

All values expressed in IC50, mg mL)1. Figure 3 Total phenolic content of aqueous and ethanolic extracts
DPPH, 1,1-diphenyl-2-picryl-hydrazyl. of Syzygium aqueum and grape seed.

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272 International Journal of Cosmetic Science, 33, 269275
Standardized extract of Syzygium aqueum
Figure 4 Doseresponse effect of Syzygium aqueum extracts on the
inhibition of tyrosinase.
Table II Activation of lipolysis (%) in adipocyte cells by Syzygium
aqueum extracts
Extracts (25 lg mL)1) Ethanolic Aqueous PG:H2O
S. aqueum 59 9 98 8 ND
Caffeine 92 11
Theophylline 83 9
Caffeine and theophylline were dissolved in DMSO and ethanol, respec-
tively.
ND, not detected.
Untreated cells (without extracts solvents)1) were taken as basal lipolysis
indicators.
Percentage activation was calculated against these cells.
Lipolysis activation activity
Lipolysis is a catabolic pathway, whereby stored triacylglycerides
(TG) in adipocytes (fat cells) break down to yield FFA and glycerol.
Inhibition of lipolysis in adipocytes was observed using the S. aque-
um extracts, whereas positive controls used were caffeine and the-
ophylline (Table II).
Syzygium aqueum displayed highest lipolysis activation in its
aqueous samples (98%), whereas ethanol samples showed almost
60% lipolysis activation at the same sample concentration of
25 lg mL)1. The PG:H2O extracts did not display any lipolytic
activity. The aforementioned results indicate that S. aqueum
extracts have great potential to be used as an anti-cellulite-active
ingredient. Further work to establish its dose-dependent activity
will require to be established.
Cytotoxicity studies
Three different extract preparations (ethanol, aqueous and PG:H2O)
of S. aqueum were assessed for its toxicity towards Vero cells. The
figure below shows the viability of Vero cells in ethanolic extracts
of S. aqueum, where a 70% viability is observed at the highest con-
centration of 0.6 mg mL)1 (Fig. 5). A 75 and 100% viability were
observed in the PG:H2O and aqueous extract, respectively, at a
concentration of 2 mg mL)1 (results not shown).
2011 The Authors
ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 269275
U. D. Palanisamy et al.
Figure 5 Viability percentages of Vero cells following a 48-h treat-
ment in varying concentrations of Syzygium aqueum ethanolic
extract. Data presented as mean SD. ZnSO4 served as the positive
control.
Heavy metal content
Lead, arsenic and mercury content of powdered S. aqueum was
determined against international standards using the ICP-OES
(inductively coupled plasma-optical emission spectroscopy) for the
determination of arsenic and lead, and AAS for the determination
of mercury. The levels of the aforementioned heavy metals in pow-
dered S. aqueum were 0.21 < 0.01 and <0.02 ppm, respectively,
which is far below the permissible levels for nutraceuticals (10, 5
and 0.5, respectively).
Discussion
Standardization of ethanolic extracts of S. aqueum was established
using HPLC. The extracts were seen to contain the hexahydroxyf-
lavone, myricetin, at 0.1 mg g)1 extract. Reynertson et al. [22]
reported the presence of anti-radical phenolic constituents in a
number of edible Myrtaceae fruits which included myricetin. Inter-
estingly, myricetin was either present in trace amounts or not
reported in any of the seven Syzygium species they had studied. In
the FRS assays, ethanolic extracts were observed to exhibit signifi-
cantly higher activity compared to the more polar aqueous
extracts. Similar findings were reported in our and other laborato-
ries where ethanolic or methanolic plant extracts exhibited much
higher antioxidant activity compared to aqueous or highly polar
extracts [14, 2325]. The DPPH assay is one of the most widely
used FRS assay in plant extracts. However, it was observed that
ABTS assay was comparatively more sensitive than DPPH and gal-
vinoxyl assays, both here and by other researchers [24, 26, 27]. In
this study, although the ABTS assay showed the highest sensitivity
compared to DPPH and galvinoxyl assays, the DPPH assay is
favoured owing to its ease of use and reproducibility.
A pro-oxidant is defined as a substance that can produce oxygen
by-products of metabolism that can cause damage to cells. It is
known that vitamin C and other antioxidants at higher concentra-
tions tend to behave like a pro-oxidant [28]. The interaction of
vitamin C with free catalytically active metal ions could contrib-
ute to oxidative damage through the production of hydroxyl and
alkoxyl radicals; whether these mechanisms occur in vivo, however,
is uncertain. Plant extracts with significant antioxidant activity
have been marketed for its health benefits. However, their use as
dietary supplements should be considered with caution as they
could also exhibit pro-oxidant effects [29]. In our studies with
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Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

S. aqueum, ethanolic extracts although having a high antioxidant
activity were seen to have pro-oxidant capacity at concentrations
higher than 0.4 mg mL)1, whereas its aqueous extracts displayed
very low pro-oxidant capacity over a wide range of concentrations
tested. It is therefore important to take into account the concentra-
tion of plant extracts and its pro-oxidant capacity when formulat-
ing for its cosmetic benefits. The phenolic content of plant extracts
has been shown to correspond closely with its scavenging activity
[3033], and this has been shown to be true with the both the
S. aqueum and grape seed extracts, where the extracts having lower
TPC are seen to have correspondingly lower scavenging activity.
Plant extracts as possible skin-whitening agents have been
widely studied [34] and have been a target of many cosmetic
giants. S. aqueums ability to inhibit the tyrosinase enzyme was
assessed and compared with the commercially available skin-whit-
ening compound, kojic acid. Extracts dissolved in propylene glycol
are commonly used as cosmetic ingredients, and it was interesting
to note that the S. aqueum extract in propylene glycol also exhibited
significant tyrosinase inhibition comparable to that of kojic acid, a
commercially used tyrosinase inhibitor. Myricetin has been shown
to exhibit tyrosinase inhibition activity [35] in an in vitro study;
this compound together with other phenolics may be contributing
to the extracts significant tyrosinase inhibition. Huang [36]
recently showed the ability of myricetin to protect keratinocytes
against UVB damage. This is in fact an added advantage if S. aqueum
extracts were to be used as a skin treatment ingredient for it not
only is an antioxidant, has tyrosinase inhibition activity and may
also possess UVB-blocking ability.
There are basically two pathways that can be targeted to
achieve cellulite reduction: the inhibition of adipogenesis (prevent
fat cell formation) and lipolysis (the active breakdown of fatty tis-
sue under the skin). Both processes support each other in a com-
plimentary way and can provide an extra cosmetic benefit when
applied as a topical treatment. The aqueous extracts of S. aqueum
were observed to be able to promote lipolysis, making it a possible
anti-cellulite ingredient. However, in vivo studies involving the use
of these extracts using non-invasive techniques will require to be
carried out to ensure the efficacy of S. aqueum extracts. It is
important to note here that myricetin, one of the compounds
identified to be present in the S. aqueum extract, was shown to
exhibit lipogenesis activity [37], indicating that other compounds
contained in the extract may be contributing to its lipolysis
activity.
Finally, it was established that the extracts were not cytotoxic to
Vero cell lines. In addition, powderized S. aqueum was shown to
have heavy metal content far below the permissible levels for
nutraceuticals. Our findings in this study support the use of
S. aqueum extract as a possible cosmetic ingredient with its high
TPC, low pro-oxidant capacity, and its ability to scavenge free
radicals, inhibit tyrosinase enzyme and activate lipolysis.
Acknowledgements
This research work was supported in part by research grants from
the Malaysian Ministry of Science, Technology and Innovation.

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