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International Journal of Cosmetic Science, 2011, 33, 269275 doi: 10.1111/j.1468-2494.2010.00637.

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

et 71 lg mL)1 (Galvinoxyl), faible incidence pro-oxydante et un


Synopsis
contenu phenolique de 585670 mg GAE g)1 extrait. Lextrait pre-
Syzygium aqueum, a species in the Myrtaceae family, commonly sentait aussi dautres proprietes en faisant un ingredient cosme-
called the water jambu is native to Malaysia and Indonesia. It is tique ideal. Une activite inhibitrice substantielle de la tyrosinase
well documented as a medicinal plant, and various parts of the tree avec IC50 de 60 lg mL)1 (blanchissement de la peau) a egalement
have been used in traditional medicine, for instance as an antibi- ete observee. De plus, il a ete prouve que lextrait avait une propri-
otic. In this study, we show S. aqueum leaf extracts to have a signif- ete anti-cellulite, testee pour sa capacite a` causer la lipolyse
icant composition of phenolic compounds, protective activity dadipocytes (cellules grasses) avec un EC50 de 16 lg mL)1. Egale-
against free radicals as well as low pro-oxidant capability. Its ment, lextrait naffichait aucune cytotoxicite envers les cellules
ethanolic extract, in particular, is characterized by its excellent Vero avec une concentration inferieure a` 600 lg mL)1. Malgre le
radical scavenging activity of EC50 of 133 lg mL)1 1,1-diphenyl-2- fait que diverses parties de cette plante ont ete utilisees en mede-
picryl-hydrazyl (DPPH), 65 lg mL)1 2,2-azino-bis(3-ethylbenzthia- cine traditionelle, ceci est la premie`re fois que ses proprietes cosme-
zoline-6-sulphonic acid) (ABTS) and 71 lg mL)1 (Galvinoxyl), low ceutiques sont demontrees. Par consequent, lutilisation de cet
pro-oxidant capabilities and a phenolic content of 585670 mg extrait, seul ou combine avec dautres principes actifs, est dun
GAE g)1 extract. The extract also displayed other activities, deem- grand interet pour lindustrie cosmeceutique.
ing it an ideal cosmetic ingredient. A substantial tyrosinase inhibi-
tion activity with an IC50 of about 60 lg mL)1 was observed. In
Introduction
addition, the extract was also found to have anti-cellulite activity
tested for its ability to cause 98% activation of lipolysis of adipo- Phenolic compounds, the secondary plant metabolites, have been
cytes (fat cells) at a concentration of 25 lg mL)1. In addition, the receiving much attention lately being the naturally occurring
extract was not cytotoxic to Vero cell lines up to a concentration of inhibitors of oxidation. There exists numerous literature on the
600 lg mL)1. Although various parts of this plant have been used ability of plant extracts to serve as possible free radical scavengers,
in traditional medicine, this is the first time it has been shown to such as green tea at protecting against free radical damage in the
have cosmeceutical properties. Therefore, the use of this extract, gastrointestinal tract [1], bilberry at upregulating the oxidative
alone or in combination with other active principles, is of interest stress defence enzymes [2], grape seed extract with its neuroprotec-
to the cosmetic industry. 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
sume
Re
market for these extracts because they are natural ingredients and
Le Syzygium Aqueum, est une espe`ce de la famille des Myrtacees, have a positive reputation to be readily accepted [6].
mieux connue sous le nom de pomme deau, originaire de Malaisie There is also a growing research showing that topically applied
et dIndonesie. Ses qualites medicinales sont largement et diverses antioxidants can help protect from sun damage [7]. These antioxi-
parties de larbre sont utilisees en medecine traditionelle, dautre dants also serve as anti-inflammatory agents that are important in
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 skin. Some cosmetic companies are utilizing antioxidants for their
S. aqueum posse`dent une quantite significative de composes phenoli- rejuvenating effects in make-up. Tocopherols and citric acid are
ques, une activite protectrice contre les radicaux libres, ainsi two well-known natural antioxidants commonly used in industry,
quune faible incidence pro-oxydante. Son extrait ethanolique, en and technologists continue to conduct studies to show the efficacy
particulier, se caracterise par son excellente activite delimination of other potential natural antioxidants.
des radicaux de EC50 de 133 lg mL)1 (DPPH), 65 lg mL)1 (ABTS) Melanogenesis is a physiological process resulting in the synthe-
sis of melanin pigments, which plays a crucial protective role
Correspondence: U. D. Palanisamy, Jeffrey Cheah School of Medicine against skin photocarcinogenesis. In humans and other mammals,
and Health Sciences, Monash University Sunway Campus, Jalan Lagoon the biosynthesis of melanin takes place in a lineage of cells known
Selatan, 46100 Bandar Sunway, Malaysia. Tel.: +603 55145840; fax: as melanocytes, which contain the enzyme tyrosinase. Tyrosinase
+603 55146323; e-mail: umadevi.palanisamy@med.monash.edu.my (phenol oxidase) is known to be a key enzyme for melanin

2011 The Authors


ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie 269
Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

biosynthesis, and its inhibition has been the subject of many stud- it was powderized using a Waring blender or milled using the Fritsch
ies [8, 9]. The search for natural chemical agents that are able to dry miller. Extraction of the powderized leaf was carried out with
modulate the metabolism of pigmentation is of great interest to cos- deionized water and the solvents ethanol and propylene glycol:
metic chemists. Arbutin, a naturally occurring beta-d-glucopyrano- deionized water (80 : 20) at 1 : 10 (w/v) concentrations. Water
side of hydroquinone, is used traditionally for depigmentation. The extraction was carried out at 40C, whereas solvent extraction at
mode of action of arbutin is through the inhibition of the melanos- root temperature for 24 h in an orbital shaker. The suspension thus
omal tyrosinase enzyme [10]. obtained was filtered using a 114 Whatman filter paper, and filtrate
Cellulite afflictions are a stubborn problem causing emotional was collected. Aqueous filtrate was concentrated using a freeze drier,
and psychological distress to many women. Many treatments for whereas solvent filtrate was concentrated using a rotary evaporator.
cellulite have been devised and are directed at reducing the Extraction using propylene glycol: water (80 : 20) was used as it is.
agglomerations of fatty tissue. Lipolysis is the breaking down of
fatty deposits and is a very desirable mechanism to stimulate in
Standardization of S. aqueum ethanolic extract
any cellulite treatment. A variety of plant extracts appear to serve
as possible anti-cellulite agent and are currently available commer- High Performance Liquid Chromatography (HPLC) standardization
cially [11, 12]. was performed on a Shidmazu IT-TOFMS Liquid Chromatography
Syzygium aqueum is a species in the Myrtaceae family, native to system equipped with LC-20AD/T liquid pump, SPD-m20A diode
Malaysia and Indonesia. Common names include wax apple, love array detector, SIL-20A auto-injector, DGU-20A system controller
apple, java apple, water apple, mountain apple, jambu air (water and CTO-20AC column oven. Chromatographic separation was
guava in Malay), wax jambu, rose apple, bell fruit, macopa and achieved on a Waters Bridge C-18 column (50 2.2 mm,
tambis (Philippines). It is a tropical tree growing to 12 m height, 2.5 lm) (Waters, U.S.A.). The mobile phase consisted of solvent A:
with evergreen leaves 1025 long and 510 cm broad. Called 0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile: start-
Kavika in Fiji, it is well documented as a medicinal plant (particu- ing from 10% B for 2 min, 100% B for 5 min and finally 3 min
larly the bark of the Kavika tree). Various parts of the tree are used with 100% B for washing. The column was reconditioned to 10%
in traditional medicine, and some have in fact been shown to pos- B. The detection wavelength was 254 nm with a flow rate of
sess antibiotic activity [13]. 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.
Materials and methods
Nuclear Magnetic Resonance spectroscopy (NMR) spectra of the
compound was obtained using a Jeol ECA 400 (400 MHz) NMR
Chemicals
spectrometer and characterized by 1H, 13C, 2D NMR.
The chemicals l-ascorbic acid, adenosine deaminase, caffeine, con-
canavalin A, dexamethasone, l-Dopa, DPPH, Folin-Ciocalteu
Antioxidant assay
reagent, gallic acid, galvinoxyl, genistein, 3-isobutylmethylxanthine
(IBMX), kojic acid, melanin, mushroom tyrosinase, myrecetin, Three different free radical scavenging (FRS) assays were performed:
potassium persulfate, theophylline and l-tyrosine were purchased scavenging activity onto DPPH radicals, galvinoxyl radicals and
from Sigma Chemical Co (St. Louis, MO, U.S.A.). Penicillin, strepto- ABTS radicals, which were assessed according to Palanisamy et al.
mycin, essential amino acids, cell culture media and supplements [14].
were obtained from Flow Lab. Ltd. (Irvine, Scotland). The
dl-a-tocopherol and potassium hexacyanoferrate (III) were obtained
Determination of total phenolic content (TPC)
from Fluka Biochemika (Buchs, Switzerland). Vitis vital Grape
seed flour was from Agricultural Research Institute Speyer Total phenolics were determined using the Folin-Ciocalteu method
(Germany), whereas Emblica from EMD Chemicals Inc. (Gibbs- described by Miliauskas [15]. This assay is based on a colorimetric
town, NJ, U.S.A.). Phosphate-buffered saline tablets (PBS) were oxidation and reduction reaction. First, 1 mL aliquots of the extracts
obtained from ICN Biomedicals (Aurora, OH, U.S.A.). All solvents were added to 5 mL of Folin-Ciocalteu reagent. After 3 min, 4 mL of
were obtained from Scharlau Chemicals (Germany). The ABTS 7.5% Na2CO3 solution in water was added to the mixture and the
diammonium salt was obtained from Amresco (Solon, OH, U.S.A.). content was thoroughly mixed. The absorbance at 765 nm was read
All cell cultures were purchased from American Tissue Culture after 1 h. Blank consisted of Folin-Ciocalteu reagent (5 mL), etha-
Collection (Manassas, VA, U.S.A.). Randox glycerol kit to determine nol/distilled water (1 mL) and 7.5% Na2CO3 solution (4 mL). A lin-
the release of glycerol was obtained from Roche, Diagnostics GmbH ear doseresponse regression curve was generated using absorbance
(Penzberg, Germany). 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
Plant collection
expressed as milligrams of gallic acid equivalent per gram of dry
Fresh leaves of S. aqueum were obtained from Kuala Lumpur. The weight (mg GAE g)1) of extracts. Total content of phenolic com-
plants were authenticated by a botanist from the Kepong Herbar- pounds in the plant extracts was calculated using this formula:
ium, Forest Research Institute of Malaysia.
C A=B
Preparation of plant extracts
C = expressed as mg GAE/g dry weight of the extract
The leaves were washed with copious amounts of water followed by A = the equivalent concentration of gallic acid established from
distilled water and then allowed to air dry at room temperature. It calibration curve (mg)
was then placed in an oven at 40C until completely dry, after which B = the dry weight of extract (g).

2011 The Authors


ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
270 International Journal of Cosmetic Science, 33, 269275
Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

to initiate differentiation by the addition of FBS/DMEM medium with


Pro-oxidant assay
0.5 mM 3-IBMX and 0.1 lM dexamethasone. Subsequently, the
Pro-oxidant and antioxidant effects are attributable to the balance. medium was replaced with differentiation progression medium which
In a Fenton reaction, Fe2+ reacts with H2O2, resulting in the pro- is FBS/DMEM medium with 1.7 lM insulin for 48 h followed by
duction of hydroxyl radical, which is considered to be the most treatment only with FBS/DMEM medium. Differentiation was com-
harmful radical to biomolecules. In the Fenton reaction, Fe2+ is plete after 1520 days. One day prior to the experiment, the cells
oxidized to Fe3+. Many reductants such as ascorbic acid can reduce were replaced with fresh medium lacking FBS. To measure lipolysis,
the oxidized form of iron (Fe3+) to reduced form (Fe2+). This reac- fat cells attached in the 24-well plates were treated with Krebs
tion could enhance the generation of hydroxyl radicals. The pre- Ringer bicarbonate (KRB), 30 mM Hepes, 1% BSA, 2.5 mM glucose
domination of reducing power (on iron ions) over the free radical as the incubation buffer at pH 7.4 (KRB medium). Adenosine deami-
scavenging activity results in the pro-oxidant effect. Reducing nase (10 lg mL)1) was added to the incubation medium to prevent
power of iron ion was measured according to the method of Tian the accumulation of adenosine which inhibits lipolysis. Positive con-
and Hua [16] where 500 lL of the extract and 50 lL of 1% potas- trols and test extracts (25 lg mL)1) were added to the cells and incu-
sium ferricyanate [K3Fe (CN6)] were incubated at 50C for 20 min. bated for 24 h at 37C with constant shaking in a CO2 incubator. At
An equal volume of 10% trichloroacetic acid was then added, and the end of the incubation, supernatant was removed (after standing
mixture was centrifuged at 3000 g for 10 min. The upper layer of for 5 mins) and heated at 70C for 10 min to inactivate enzymes
the solution (1 mL) was mixed with 1 mL of distilled water and released by the cells that may interfere with the glycerol test. Glycerol
0.2 mL of 0.1% ferric chloride (FeCl3), and its absorbance was release was measured using the Randox Glycerol kit and expressed
recorded at 700 nm. Ethanol or distilled water was used as nega- as lmoles glycerol/l. Glycerol concentration was calculated from a
tive control, whereas Vitamin C and Emblica (a commercial anti- glycerol standard curve. Untreated cells (without extracts) were
oxidant with very low pro-oxidant activity) was used as positive taken as the control (basal lipolysis level). A viability assay was ini-
controls. Results are expressed in comparison with positive controls tially carried out using 25 lg mL)1 of extract and positive controls.
at concentration ranging from 0.1 to 0.5 mg mL)1.
Cytotoxicity studies
Tyrosinase inhibition assay
Vero cell lines were cultured in minimum essential medium supple-
Tyrosinase activity was determined as described by Bernard and mented with 10% foetal bovine serum, non-essential amino acid,
Berthon [17] with minor modifications using 96-well plates. Ini- and 100 IU mL)1 Penicillin and 100 lg mL)1 Streptomycin. The
tially, 60 lL of plant extracts were added to the top well. A serial cells are cultured in tissue-culture-grade flasks at 37C in a humid-
dilution of the extracts was performed starting from the second well ified atmosphere containing 5% CO2. Four different extract prepara-
using 30 lL 0.02 M phosphate buffer pH 6.9. This was followed by tions of S. aqueum were assessed. The Vero cells were plated on
the addition of 30 lL of 1 mM l-DOPA, 30 lL of 1 mM l-tyrosine 96-well plate followed by a 24-h incubation to allow cell attach-
and 470 lL of 50 mM of phosphate buffer (pH 6.8). To start the ment. The cells were then subjected to various concentrations of
reaction, 20 lL of mushroom tyrosine (254 U mL)1) was added to S. aqueum extracts (00.7 mg mL)1) and solvent controls and left
the solution. The plate was left at 37C for 30 min prior to mea- to incubate for 48 h. Cytotoxic effect was assessed using the colori-
suring the amount of dopachrome produced. The absorbance was metric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
read against the blank well (without enzyme) at 490 nm using the bromide, a yellow tetrazole)] assay following the method of Mos-
ASYS UVM 340 Microplate Reader (Biochrom, Cambridge, UK). mann [18]. Viability was presented as the percentage absorbance
Kojic acid, a commercially available natural tyrosinase inhibitor, at at 570 nm of treated against non-treated cells. ZnSO4 was used as
0.1 mg mL)1 was used as the positive control. All determinations the positive control, and all assays were performed in triplicate.
were performed in triplicates.
The percentage inhibition of tyrosinase activity was calculated
Heavy metal content determination
as follows:
Lead, arsenic and mercury content of the powderized rind of
% Inhibition A  B=A  100 S. aqueum was determined against International standards; induc-
tively coupled plasma - optical emission spectrometry (ICP-OES) for
where the determination of arsenic and lead, and atomic absorption spec-
A = absorbance at 490 nm without test sample troscopy (AAS) for the determination of mercury.
B = absorbance at 490 nm with test sample.
Statistical analysis
Lipolysis activation assay
All experiments were performed in triplicate. Means, standard
The ability of S. aqueum extracts to stimulate lipolysis in adipocytes errors and standard deviations were calculated from replicates
was investigated. Lipolysis was assayed in fully differentiated 3T3-L1 within the experiments, and analyses were conducted using Micro-
fat cells. As adipocytes undergo lipolysis, free fatty acids (FFA) and soft Excel 2003.
glycerol are released into the culture medium. The glycerol released
was used to quantify lipolysis activity. Lipolysis stimulation was com-
Results
pared using the positive controls caffeine and theophylline. 3T3-L1
fibroblasts were grown to confluence in 24-well plates (with initial
Isolation and standardization of S. aqueum extract
seeding of 2 104 cells mL)1) in Dulbeccos modified Eagles med-
ium added with 10% calf serum (CS/DMEM), 1% penicillin, 1% strep- The extraction yields of S. aqueum under ethanolic and aqueous
tomycin and 1% glutamine. The post-confluent cells were stimulated conditions were observed to be similar, with aqueous extraction

2011 The Authors


ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 269275 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.

2011 The Authors


ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
272 International Journal of Cosmetic Science, 33, 269275
Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

Figure 4 Doseresponse effect of Syzygium aqueum extracts on the Figure 5 Viability percentages of Vero cells following a 48-h treat-
inhibition of tyrosinase. ment in varying concentrations of Syzygium aqueum ethanolic
extract. Data presented as mean SD. ZnSO4 served as the positive
control.

Table II Activation of lipolysis (%) in adipocyte cells by Syzygium


aqueum extracts Heavy metal content
Lead, arsenic and mercury content of powdered S. aqueum was
Extracts (25 lg mL)1) Ethanolic Aqueous PG:H2O 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
S. aqueum 59 9 98 8 ND of mercury. The levels of the aforementioned heavy metals in pow-
Caffeine 92 11 dered S. aqueum were 0.21 < 0.01 and <0.02 ppm, respectively,
Theophylline 83 9 which is far below the permissible levels for nutraceuticals (10, 5
and 0.5, respectively).
Caffeine and theophylline were dissolved in DMSO and ethanol, respec-
tively.
Discussion
ND, not detected.
Untreated cells (without extracts solvents)1) were taken as basal lipolysis Standardization of ethanolic extracts of S. aqueum was established
indicators. using HPLC. The extracts were seen to contain the hexahydroxyf-
Percentage activation was calculated against these cells. 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
Lipolysis activation activity
reported in any of the seven Syzygium species they had studied. In
Lipolysis is a catabolic pathway, whereby stored triacylglycerides the FRS assays, ethanolic extracts were observed to exhibit signifi-
(TG) in adipocytes (fat cells) break down to yield FFA and glycerol. cantly higher activity compared to the more polar aqueous
Inhibition of lipolysis in adipocytes was observed using the S. aque- extracts. Similar findings were reported in our and other laborato-
um extracts, whereas positive controls used were caffeine and the- ries where ethanolic or methanolic plant extracts exhibited much
ophylline (Table II). higher antioxidant activity compared to aqueous or highly polar
Syzygium aqueum displayed highest lipolysis activation in its extracts [14, 2325]. The DPPH assay is one of the most widely
aqueous samples (98%), whereas ethanol samples showed almost used FRS assay in plant extracts. However, it was observed that
60% lipolysis activation at the same sample concentration of ABTS assay was comparatively more sensitive than DPPH and gal-
25 lg mL)1. The PG:H2O extracts did not display any lipolytic vinoxyl assays, both here and by other researchers [24, 26, 27]. In
activity. The aforementioned results indicate that S. aqueum this study, although the ABTS assay showed the highest sensitivity
extracts have great potential to be used as an anti-cellulite-active compared to DPPH and galvinoxyl assays, the DPPH assay is
ingredient. Further work to establish its dose-dependent activity favoured owing to its ease of use and reproducibility.
will require to be established. 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-
Cytotoxicity studies
tions tend to behave like a pro-oxidant [28]. The interaction of
Three different extract preparations (ethanol, aqueous and PG:H2O) vitamin C with free catalytically active metal ions could contrib-
of S. aqueum were assessed for its toxicity towards Vero cells. The ute to oxidative damage through the production of hydroxyl and
figure below shows the viability of Vero cells in ethanolic extracts alkoxyl radicals; whether these mechanisms occur in vivo, however,
of S. aqueum, where a 70% viability is observed at the highest con- is uncertain. Plant extracts with significant antioxidant activity
centration of 0.6 mg mL)1 (Fig. 5). A 75 and 100% viability were have been marketed for its health benefits. However, their use as
observed in the PG:H2O and aqueous extract, respectively, at a dietary supplements should be considered with caution as they
concentration of 2 mg mL)1 (results not shown). could also exhibit pro-oxidant effects [29]. In our studies with

2011 The Authors


ICS 2011 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 269275 273
Standardized extract of Syzygium aqueum U. D. Palanisamy et al.

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

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