Tsai et al.

Botanical Studies 2014, 55:78

http://www.as-botanicalstudies.com/content/55/1/78

RESEARCH Open Access

Antioxidant, cell-protective, and anti-melanogenic

activities of leaf extracts from wild bitter melon
(Momordica charantia Linn. var. abbreviata Ser.)
cultivars
Tsung-Hsien Tsai1, Ching-Jang Huang2, Wen-Huey Wu3, Wen-Cheng Huang3, Jong-Ho Chyuan4 and Po-Jung Tsai3*

Abstract
Background: Several wild bitter melon (WBM; Momordica charantia Linn. var. abbreviata Ser.) cultivars were developed
in Taiwan. However, little information is available regarding biological function of WBM leaf. Therefore, the objectives of
this study were to investigate the nutrient content, antioxidant, cell protection and anti-melanogenic properties of wild
bitter melon leaf.
Results: Methanolic leaf extracts were prepared from a variety and two cultivars of WBM. All extracts exerted potent
nitric oxide and hydroxyl radical scavenging capacities. Furthermore, all extracts effectively reduce the production of
reactive oxygen species and prevent cell death in UVB-irradiated HaCaT keratinocytes. The cell protective effect of leaf
extract was also investigated by the prevention of HaCaT cells from sodium nitroprusside or menadione-induced
toxicity, and significant cyto-protective activities were observed for all of them. Additionally, all extracts significantly
suppressed tyrosinase activity and melanin levels in B16-F10 melanocytes.
Conclusions: WBM leaf extract showed significant antioxidant, cyto-protective and anti-melanogenic activities. These
findings suggested that WBM leaves may be beneficial for preventing the photo-oxidative damage and melanogenesis
of skin.
Keywords: Wild bitter melon leaf; Antioxidant; Cyto-protection; Anti-melanogensis

Background radical molecules like hydrogen peroxide (H2O2), singlet

Oxidative stress has been thought to play an important oxygen (1O2), nitric oxide (NO), etc. ROS are involved in
role in the pathogenesis of diseases such as cancer, cardio- the pathogenesis of several skin disorders including photo-
vascular disease, atherosclerosis, diabetes mellitus, and sensitivity diseases and some types of cutaneous
neurodegenerative disorders (Valko et al. 2007). Ultravio- malignancy (Bickers and Athar 2006). Additionally, ROS
let (UV) irradiation is the most well-known environmental may accelerate aging process and cause uneven pigmenta-
skin aggressor. Skin, the largest organ of human body, is a tion. UVB radiation has been shown to augment nitric
physiological barrier that protects the organism against oxide and peroxynitrite formation in keratinocytes
pathogens and chemical or physical damage. Exposure of (Deliconstmtinos et al. 1996). NO production may con-
UV leads to increased reactive oxygen species (ROS) pro- tribute to the regulation of UV-induced pigmentation.
duction, which alters gene and protein structure and func- NO derived from keratinocytes increases the amount of
tion (Masaki 2010). ROS include free radicals such as the melanogenic factor tyrosinase and then induces me-
superoxide anion (O2? −), hydroxyl radical (? OH), and non- lanogenesis (Masaki 2010). Antioxidant agents may
therefore play a protective role during the development
of ROS-mediated skin disorders.
* Correspondence: [email protected]
3
Department of Human Development and Family Studies, National Taiwan
Wild bitter melon (WBM; Momordica charantia L.var.
Normal University, 162 Hoping E. Rd., Sec. 1, Taipei 10610, Taiwan abbreviata Seringe) is a variety of bitter melon (M.
Full list of author information is available at the end of the article

? 2014 Tsai et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
Tsai et al. Botanical Studies 2014, 55:78 Page 2 of 11
http://www.as-botanicalstudies.com/content/55/1/78

charantia) in Taiwan. WBM fruit is commonly consumed in dimethyl sulfoxide (DMSO) to a concentration of
as vegetable and possesses potent antioxidant and free 400 mg/mL for the subsequent experiments. The yields of
radical scavenging activities (Wu and Ng 2008). The WV, HL-1, and HL-2 extracts were 29.1%, 22.3%, and
young shoots and leaves of WBM are traditionally eaten 23.5%, respectively.
as greens by the Amis, one of the indigenous peoples of
Taiwan. The young tender leaves of bitter melon are also
eaten as a vegetable in the Philippines and Indonesia. Be- Nutrient content determination of fresh WBM leaves
sides being as a vegetable, the pounded leaves of bitter Moisture, crude fat, crude protein, crude fiber and ash deter-
melons are applied to the body for skin diseases and burns minations of WBM fresh leaves were conducted following
in Malaysia and India (Lim 2012). In fact, leaf extracts of bit- the procedure of the AOAC ? Official Methods of Analysis?
ter melon have been demonstrated to have broad-spectrum 14th ed et al. (1984). Vitamin C was determined by a colori-
antimicrobial (Khan and Omoloso 1998) and potent antioxi- metric method of Zhang et al. (2009), with modifications.
dant activities (Kubola and Siriamornpun 2008). Two-gram dried ground WBM leaf was stirred with 30 mL
To date, the genetic improvement of WBM has been distilled water for 30 min at room temperature. The
achieved to improve their agronomic characteristics such homogenization was filtered and then centrifuged at
as disease resistance, environmental tolerance and fruit 3000 ? g for 15 min. The supernatant was collected and
quality. Over the years, several WBM cultivars were de- diluted with distilled water to total of 50 mL. A 10:1 di-
veloped in Taiwan (Lu et al. 2011, 2012). WBM fruit ex- lution was made by taking 1.0 mL of this solution and
tract and its components have been shown to possess adding distilled water to 10 mL. A 2 mL volume of
numerous pharmacological actions including the activa- trichloro-acetic acid (10%) was added to this suspension
tion of peroxisome proliferator-activated receptor, anti- and placed for 5 min in an ice bath. Afterwards, 2 mL of
bacterial, anti-inflammatory, and antioxidant activities Folin? Ciocalteu? s phenol reagent was added and vortexed.
(Lu et al. 2011, 2012; Hsu et al. 2012). But scientific lit- After 10 min at room temperature, the absorbance was
eratures concerning chemical and biological properties measured at 760 nm against distilled water as a blank. The
of WBM leaves remain limited. vitamin C content was estimated through the calibration
In this study, we intended to analyze the nutrient con- curve of ascorbic acid.
tents of fresh leaves from a variety and two cultivars of
WBM, and to investigate the total phenolic contents and Total phenolic content of WBM leaf extract
the antioxidant properties of WBM leaf extracts using Total phenolic content of leaf extracts was evaluated using
cell-free and cell-based assays. In addition, the effects of spectrophotometric analysis with Folin-Ciocalteu reagent
WBM leaf extracts on tyrosinase activity and melanin as described by Tsai et al. (2008). Briefly, Folin? Ciocalteu
levels in B16-F10 melanoma cells were determined. Such phenol reagent was added to the reconstituted samples
a study would contribute to the current knowledge relat- and held for 3 min. Then 2 ml of 10% (w/v) sodium car-
ing to the nutrients and biological functions of WBM bonate solution were added and allowed to stand at room
leaf. temperature for 30 min. The absorbance at 765 nm was
measured. The total phenolic content was calculated by a
Methods standard curve prepared with gallic acid and expressed as
Preparation of extracts milligrams of gallic acid equivalents (GAE) per gram of
A variety of WBM (WV) and two cultivars of WBM, solid of extract.
Hualien-1 (HL-1) and Hualien-2 (HL-2), used in the
present study were cultured in the Hualien District Agricul-
tural Research and Extension Station, Hualien, Taiwan. A Determination of DPPH radical-scavenging activity of
voucher specimen has been deposited in the Department of WBM leaf extracts
Human Development and Family Studies, National Taiwan The 2,2-diphenyl-1-picrylhydrazyl (DPPH? ) radical-scav-
Normal University. After washing with water, leaves of enging capacity of each extract was measured as described
WBM were air-dried. They were ground by a blender and earlier (Tsai et al. 2008). Briefly, 20 μL of each sample or
then extracted with methanol. Briefly, 10 g of fine-ground 100% DMSO (as a negative control) were allowed to react
WBM leaves was extracted with 100 mL of methanol at with 200 μL of freshly prepared 200 μM DPPH ethanolic
room temperature for 4 h. After extraction, the mixture solution in a 96-well microplate. The reaction mixture
was filtered, and the residue was re-extracted with 100 mL was mixed and left to stand for 10 min. The absorbance
of fresh methanol by stirring overnight. The combined at 540 nm was determined against a blank of DMSO.
methanol solutions were centrifuged at 12,000 ? g for The DPPH radical-scavenging activity of WBM extract
10 min and evaporated on a rotary evaporator to get meth- was calculated as follows: (1-[A sample ? A blank of sample/
anolic extracts. The methanolic extracts were reconstituted ADMSO ? A blank of DMSO]) ? 100%.
Tsai et al. Botanical Studies 2014, 55:78 Page 3 of 11
http://www.as-botanicalstudies.com/content/55/1/78

Determination of NO-scavenging activity of WBM with 10% heated-inactivated fetal bovine serum (FBS),
leaf extracts penicillin (100 U/mL), and streptomycin (100 μg/mL).
The NO-scavenging activity of each extract was also These cells were incubated at 37?C in a humidified atmos-
measured. Nitric oxide was generated from sodium ni- phere with 5% CO2. The probe 2? , 7? -dichlorofluorescein
troprusside (SNP) and measured by the Griess reagent diacetate (H2DCF-DA; Sigma, St. Louis, MO, USA) was
(Sumanont et al. 2004). Briefly, 50 μL of serial diluted used to monitor the intracellular ROS generation. HaCaT
sample extract (0.5 ~ 20 mg/mL) was pipetted into a 96- cells (2 ? 10 5 cells/well) were seeded on 6-well plates for
well flat-bottomed plate. Following this, 50 μL of 24 h incubation and then pre-treated with WBM leaf ex-
10 mM sodium nitroprusside dissolved in PBS was tracts at the indicated concentrations for additional 24-h
added into each well and the plate was then incubated incubation. After incubation, cells were washed with PBS
under light at room temperature for 150 min. Finally, an and then irradiated with 80 mJ/cm2 of UVB (Vilber
equal volume of Griess reagent was added into each well Lourmat, France). In parallel, non-irradiated cells were
to measure the nitrite content. The NO-scavenging treated similarly and were kept in the dark in an incubator
activity of WBM leaf extract was calculated as follows: for the time of UVB treatment. After UVB irradiation,
(A DMSO ? A sample)/A DMSO ? 100%. cells were harvested and washed twice with PBS, and then
re-suspended in 10 μM H2DCF-DA at 37?C for 30 min
Determination of superoxide-scavenging activity of WBM incubation. Stained cells were washed with PBS and re-
leaf extracts suspended in PBS. ROS generation of HaCaT cells was
The superoxide scavenging activity of WBM leaf extract was determined by flow cytometry (FACscan, Hercules, CA,
measured using non-enzymatic generation of superoxide an- USA) using 488 nm for excitation and 525 nm for emis-
ions (Robak and Gryglewski 1988). Briefly, the reaction sion. Mean fluorescence intensity (MFI) detected by FLl
mixture contained various concentrations of leaf extracts channel was analyzed using the Windows Multiple Docu-
(0.5-20 mg/mL), 80 μM phenazine methosulphate, 624 μM ment Interface software (WinMDI 2.8). Data were
NADH and 200 μM nitro blue tetrazolium (NBT) in phos- expressed as percentages of vehicle control values.
phate buffer after 15 min of the incubation at room To determine the protective effect of WBM leaf ex-
temperature. The result was measured at 560 nm against tracts against UVB-induced cytotoxicity, HaCaT cells
blank samples without NADH. The percentage of scaven- were seeded in 96-well plates at a density of 2 ? 10 4
ging effect was expressed as % of scavenging activity =1- cells/well for 24 h incubation and then pre-treated with
[A560 sample ? A560 blank of sample/A560 control ? A560 WBM leaf extracts at the indicated concentrations for
blank of control] ? 100%. additional 24-h incubation. After incubation, cells were
washed with PBS and then irradiated with 80 mJ/cm2 of
Determination of hydroxyl radical-scavenging activity UVB. In parallel, non-irradiated cells were treated simi-
of WBM leaf extracts larly and were kept in the dark in an incubator for the
The scavenging activity of leaf extracts on hydroxyl radicals time of UVB treatment. After challenge of HaCaT cells
produced by the Fenton reaction was evaluated with their with UVB, the cells were incubated in fresh DMEM with
quenching effects on the chemical luminescence (CL) signal 10% FBS at 37?C for 30 min or 16 h, and collected for
of the Fe(II)? H2O2? luminol system (Cheng et al. 2003). Re- further analysis. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
action mixtures (200 μL) included luminol (4 μM), Fe2+ diphenyltetrazolium bromide (MTT) assay was per-
(4.6 μM)-EDTA (2.3 μM), H2O2 (24 mM), and tested sam- formed to determine cellular viability.
ples (0.02-1 mg/mL of WBM leaf extract). The reaction was
initiated by adding Fe2+-EDTA, luminol followed by H2O2.
The chemiluminescent reaction was performed in a Cell protection effect of WBM leaf extracts against
KH2PO4-NaOH buffer (pH 7.5) at room temperature. Lumi- SNP-induced toxicity
nescence intensity was monitored over wavelengths at The protective effect of WBM leaf extract against cell
460 nm with Synergy HT multidetection microplate reader death induced by sodium nitroprusside (SNP) was de-
(Biotek Instruments, Winooski, VT, USA). The CL peak termined by the method of Bastianetto et al. (2010).
values were recorded in the absence (I0) or presence (Ii) of SNP is a well-known NO releasing with purported toxic
leaf extracts. The inhibitory rate (IR) was calculated as IR = and apoptotic effects in keratinocytes (Bastianetto et al.
(1 ? Ii / I0) ? 100%. 2010). SNP-induced toxicity was performed in HaCaT
cells (5 ? 10 4/well) plated in 96 wells. After 24 h, the
UVB-irradiated HaCaT keratinocytes medium was removed and replaced with medium con-
The immortalized human keratinocyte cell line HaCaT tain SNP (2 mM) in the presence or absence of leaf ex-
was maintained in Dulbecco? s modified Eagle's medium tracts (50 ~ 200 μg/mL). Cell viability was determined
(DMEM, Gibco, Carlsbad, CA, USA) supplemented 24 h later using the MTT assay.
Tsai et al. Botanical Studies 2014, 55:78 Page 4 of 11
http://www.as-botanicalstudies.com/content/55/1/78

Cell protection effect of WBM leaf extracts against MSH and various concentrations (50 ~ 200 μg/mL) of
menadione-induced toxicity leaf extracts for 48 h. The cells were rendered soluble in
The protective effect of WBM leaf extract against cell death 1 N NaOH containing 10% DMSO at 60?C for 30 min,
induced by menadione (2-methyl-1, 4-naphthoquinone) was and then 200 μL portions of crude cell lysate was trans-
measured by the method of Klausc et al. (2010). Menadione ferred into 96-well plates. Melanin concentration was
is highly cytotoxic, strongly induced ROS formation in hu- calculated by the absorbance measured at 405 nm
man HaCaT keratinocytes. Menadione-induced toxicity was through the calibration curve of melanin.
performed in HaCaT cells (5 ? 10 4/well) plated in 96 wells.
After 24-h incubation, the medium was removed and Statistical analysis
replaced with medium contain menadione (50 μM) in All data are presented as the mean ? standard deviation
the presence or absence of WBM leaf extracts (50 ~ (SD). Statistical analyses were performed using Statistical
200 μg/mL). Cell viability was determined 24 h later Package of Social Science version 17.0 for Windows (SPSS
using the MTT assays. Inc., Chicago, Illinois, USA). Analysis of variance was per-
formed by ANOVA procedures. Significant differences be-
Determination of cellular tyrosinase activity in tween means between groups were determined using
melanoma cells Duncan? s multiple range tests at a level of p < 0.05.
The established murine B16-F10 melanoma cell line offers a
melanogenesis model (Yokozawa and Kim 2007). The B16-
Results
F10 cell line (BCRC 60031) was obtained from the Biore-
Nutrient content of WBM leaves
source Collection and Research Center (Hsinchu, Taiwan)
Moisture, crude protein, crude fat, crude fiber, and ash con-
and was cultured in DMEM (Gibco) supplemented with
tent (%) of WBM leaves were reported on dry-weight basis
10% heat-inactivated FBS, penicillin (100 U/mL), and
and given in Table 1. In fresh WBM leaves, crude fat, crude
streptomycin (100 μg/mL) at 37?C in a humidified atmos-
protein, and crude fiber contents ranged from 0.74% to
phere with 5% CO2. The effect of WBM leaf extracts on cell
1.22%, 4.22% to 6.26%, and 1.59% to 1.92% on dry-weight
viability of B16-F10 melanoma cells was first investigated.
basis, respectively. Ash contents in WBM leaves ranged
B16-F10 melanoma cells (1 ? 10 4 cells/well) were seeded in
from 2.5% to 3.76% on dry-weight basis. Vitamin C con-
96-well culture plates for 24 h prior to use. The cells were
tents in WBM leaves ranged from 1647.3 to 2059.0 μg/g
treated with various concentrations of 50 ~ 250 μg/mL of
dry basis.
WBM leaf extracts for 24 h. The cell viability was evaluated
using the MTT assays.
The tyrosinase activity in B16-F10 cells was examined Total phenolic content of WBM leaf extract
by measuring the rate of oxidation of L-DOPA The total phenolic content of WBM leaf extracts was de-
(Yokozawa and Kim 2007). B16-F10 cells (2.5 ? 10 4 termined as gallic acid equivalents (Table 2). Among
cells/well) were plated in 24-well dishes for 24 h incuba- three extracts tested, WV had the highest phenolic con-
tion before the use. The cells were then incubated in the tent (34 mg GAE/g extract), followed by HL-1 and HL-2
presence or absence of 25 ng/mL α-melanocyte stimulat- (26 mg GAE/g extract).
ing hormone (α-MSH) and various concentrations of
WBM leaf extracts for 48 h. In addition, kojic acid [5-hy- Free radical-scavenging activity of WBM extracts
droxy-2-(hydroxymethyl)-4-pyrone], a popular inhibitor The free radical-scavenging properties of leaf extracts were
of tyrosinase (Kahn 1995) was used as a positive control. examined (Figure 1) and then expressed as the IC50 which
The cells were lysed in 200 μL of 50 mM sodium phos- is the concentration of leaf extract that causes 50% inhib-
phate buffer (pH 6.8) containing 1% Triton X-100 and ition of respective radical generation (Table 2). The DPPH
0.1 mM phenylmethylsulfonyl fluoride and then frozen radical scavenging assay is a simple and widely used screen-
at −80?C for 30 min. After thawing and mixing, cellular ing for bioactive compound discovery. The scavenging ac-
extracts were clarified by centrifugation at 12,000 ? g for tivity of WBM leaf extracts on DPPH radicals is in a
30 min at 4?C. The supernatant (80 μL) and 20 μL of L- concentration-dependent manner (Figure 1A). The IC50
DOPA (2 mg/mL) were placed in a 96-well plate, and values of WV, HL-1, and HL-2 extracts on DPPH scaven-
the absorbance at 492 nm was read for 30 min at 37?C ging activity were 4.79, 28.0, and 20.39 mg/mL, respectively
using a micro-plate reader. (Table 2). Among tested leaf extracts, WV is the strongest
DPPH radicals? scavenger. HL-1 extract is the least potent
Determination of melanin content in melanoma cells scavenger of DPPH.
Melanin content was measured as described previously The NO-scavenging activity of leaf extracts were mea-
(Aoki et al. 2007) with modifications. B16-F10 cells were sured to have IC50 values of 0.79, 0.76, and 0.80 mg/mL
incubated in the presence or absence of 25 ng/mL α- for WV, HL-1, and HL-2 extracts, respectively (Figure 1B,
Tsai et al. Botanical Studies 2014, 55:78 Page 5 of 11
http://www.as-botanicalstudies.com/content/55/1/78

Table 1 Mositure, crude protein, crude fat, crude fiber, and ash content and vitamin C of fresh wild bitter melon leaves
(on dry-weight basis)
Variety and cultivars Moisture (%) Crude protein (%) Crude fat (%) Crude fiber (%) Ash (%) Vitamin C (μg/g dry basis)
WV 83.20 5.28 0.99 1.90 3.76 1647.32
HL-1 81.22 6.26 1.22 1.92 2.85 1920.14
HL-2 83.77 4.22 0.74 1.59 2.50 2059.03

Table 2), where there was no statistically significant dif- Cellular protection effect of WBM leaf extracts against
ference observed among the variety and cultivars. oxidants
WBM leaf extracts also caused a concentration-dependent Prior to the determination of cellular protection effect,
inhibition of superoxide anion (Figure 1C). The respective the cytotoxic effect of WBM leaf extracts on HaCaT ker-
IC50 values of WV, HL-1, and HL-2 extracts on superoxide atinocytes were examined. HaCaT cells were incubated
scavenging activity were 5.77, 9.12, and 7.71 mg/mL with WBM leaf extracts at various concentrations for
(Table 2), indicating that WV extract is more potent scaven- 24-h incubation. The cell viability was determined by
ger of superoxide anion than others. MTT methods. All three WBM leaf extracts (up to
The hydroxyl radical-scavenging capacity of leaf extracts 300 μg/mL) had no cytotoxic effect on HaCaT cells (data
is shown in Figure 1D. The respective IC50 values of WV, not shown). On the other hand, treatment of HaCaT
HL-1, and HL-2 extracts were 26, 42, and 22 μg/mL cells with SNP (2 mM) (Figure 3A) and menadione
(Table 2). WV and HL-2 had more effective scavenging ac- (50 μM) (Figure 3B) strongly impaired cell viability. To
tivity than HL-1. determine the cyto-protection activities, HaCaT kerati-
nocytes were simultaneously exposed to cytotoxic agents
either SNP or menadione as well as WBM leaf extracts
WBM leaf extracts inhibit UV-induced ROS production at various concentrations (Figure 3). All three WBM leaf
and keratinocyte death extracts strongly attenuated SNP-induced toxicity at a
As shown in Figure 2A, UVB exposure of HaCaT kerati- concentration of 50 μg/mL (Figure 3A). The HL-2 ex-
nocytes resulted in an immediate and significant eleva- tract was the most effective one in protecting cells
tion of intracellular ROS. Treatment of WBM leaf against SNP-induced toxicity with an EC50 (effective con-
extracts (50 and 100 μg/mL) prior to UVB irradiation centrations) of 31.31 μg/mL, followed by WV (EC50 =
significantly inhibited intracellular ROS generation. All 49.35 μg/mL), and HL-1 (EC50 = 84.72 μg/mL).
leaf extracts effectively inhibited UVB-induced ROS We then performed a cell viability assay to evaluate
production. After UVB exposure, HaCaT cells were fur- the capacity of WBM leaf extracts to protect HaCaT cells
ther incubated at 37?C for 30 min (Figure 2B) or 16 h against the toxicity induced by the ROS releasing mena-
(Figure 2C). The results showed that UVB irradiation dione (Figure 3B). Exposure of HaCaT cells to mena-
did not significantly affect cell viability for further dione resulted in cell death which was reduced by WV
30 min incubation. All three WBM leaf extracts did not (EC50 = 42.05 μg/mL), HL-1 (EC50 = 42.07 μg/mL), and
significantly modulate cell viability of HaCaT cells dur- HL-2 (EC50 = 43.55 μg/mL). According to EC50 values,
ing 30 min incubation (Figure 2B). WBM leaf extract- the three varieties were comparable in their cyto-
pretreated HaCaT cells were exposed to UVB and protective effect against menadione-induced oxidative
incubated for an additional 16 h. MTT assay showed stress.
that cell viability of HaCaT cells was significantly de-
creased after UVB exposure (Figure 2C). Notably, all Anti-tyrosinase and anti-melanogenic properties of WBM
three WBM leaf extracts at concentrations of 25, 50, leaf extracts
and 100 μg/mL revealed a protective effect on the via- To investigate the anti-melanogenic activity in cellular
bility of irradiated HaCaT cells (Figure 2C). system, the cytotoxicity of WBM leaf extracts on B16-
Table 2 Total phenolics content and radical-scavenging capacity of methanolic extracts from wild bitter melon leaves
Variety and Total phenolics IC50 values (mg/mL)
cultivars (mg GAE/g)
DPPH NO Superoxide Hydroxyl radical
b a a a
WV 33.70 ? 0.48 4.79 ? 0.30 0.79 ? 0.01 5.77 ? 0.10 0.026 ? 0.003
a c c b
HL-1 25.86 ? 0.36 28.00 ? 0.83 0.76 ? 0.02 9.12 ? 0.22 0.042 ? 0.004
a b b a
HL-2 25.94 ? 0.35 20.39 ? 1.12 0.80 ? 0.02 7.71 ? 0.35 0.022 ? 0.004
GAE: Gallic acid equivalent. Data are expressed as the mean ? SD. Values in a column followed by the same superscript letter are not significantly diffe rent as
determined by Duncan's multiple tests.
Tsai et al. Botanical Studies 2014, 55:78 Page 6 of 11
http://www.as-botanicalstudies.com/content/55/1/78

WV WV
100
HL-1 HL-1
A C

superoxide scavenging activity

100 HL-2
HL-2
DPPH scavenging activity

80
80

(% of control)
(% of control)

60
60

40
40

20 20

0 0
0 5 10 15 20 0 5 10 15 20
Concentration (mg/mL) Concentration (mg/mL)

B D
100 100
NO scavenging activity

OH- scavenging activity

(% of control)
(% of control)

80 80

60 60

40 40

20 20

0 0
0 5 10 15 20 0.0 0.2 0.4 0.6 0.8 1.0

Concentration (mg/mL) Concentration(mg/mL)

Figure 1 Scavenging activities of WBM leaf extracts against the DPPH (A), nitric oxide (B), superoxide anion (C), and hydroxyl radical
(D). Data are given as the mean ? SD (n = 3). Radical-scavenging capacity of leaf extract was represented as % of vehicle control.

F10 cells was first evaluated after incubated with extracts 1 is higher than those of WV and HL-2, but lower than that
for 24 hr. Our data showed that these three extracts (up of kojic acid.
to 200 μg/mL) did not show any cytotoxic effect on
B16-F10 cells (data not shown). Thus, WBM leaf extract Discussion
(50? 200 μg/mL) was used to examine their anti- In this study, the cultivars of wild bitter melon leaves are
tyrosinase activity and intracellular melanin formation. proved in possessing antioxidant and anti-melanogenic
As shown in Figure 4, all three WBM leaf extracts exhib- properties. Although there are some differences in the
ited significantly inhibitory effect on tyrosinase activity degree they exerted, however, all of them possess anti-
(Figure 4A). However, at a concentration of 50 μg/mL, oxidant to reduce UVB-induced ROS generation and
the anti-tyrosinase effect of WV was not significant. At a prevent cellular death against oxidants in keratinocytes.
concentration of 200 μg/mL, HL-1 extract exhibited Leafy vegetables are good sources of not only minerals
more significant anti-tyrosinase activity than others. The but also vitamins, antioxidants and pigments. The tender
inhibitory effect of HL-1 extract (ranged from 50 to leaf tips and leaves of bitter melon are a rich source of
200 μg/mL) on tyrosinase activity were similar to kojic minerals, vitamin C, folic acid and vitamin A (Zhang
acid, a well-known tyrosinase inhibitor (Figure 4A). et al. 2009; Lim 2012). This study analyzed nutrient con-
All three WBM leaf extracts significantly suppressed mel- tent of WBM leaf and provided further information on
anin formation in B16-F10 cells (Figure 4B). At a concentra- nutrient contents on WBM leaf for nutritionists and the
tion of 200 μg/mL, WV, HL-1, and HL-2 extracts and kojic general public. During the past few decades, the uses of
acid reduced melanin production of 11.29%, 26.63%, 23.44% natural antioxidants and plant extracts for human health
and 41.2%, respectively. The anti-melanogenis effect of HL- have received increasing attention.
Tsai et al. Botanical Studies 2014, 55:78 Page 7 of 11
http://www.as-botanicalstudies.com/content/55/1/78

Figure 2 Effects of WBM leaf extracts on UV-induced ROS

140 A 50 ? g/mL
production and cytotoxicity in HaCaT keratinocytes. After exposure
100 ? g/mL
d
c
of UVB (80 mJ/cm2), the cells were further incubated at 37?C for 30 min
120
b and then ROS generation was determined by flow cytometry. ROS
ab b b
ab
a
production was represented as % of control without UVB irradiation.
100
(A) After exposure of UVB (80 mJ/cm2), the cells were further incubated at
% of control

80
37?C for 30 min (B) or 16 h (C) and then cell viability was measured using
the MTT assay. Non-irradiated HaCaT keratinocytes were used as the
60 control. Cell viability is expressed as the percentage of control. Data are
presented as the mean ? SD of triplicat e determinations. Values with the
40 same letter are not significantly different as determined by Duncan? s
multiple range tests.
20

0
Control Vehicle WV HL-1 HL-2
UVB (80 mJ/cm2 ) The findings presented here showed that WBM leaf ex-
tracts exhibited antioxidant properties because of their
160 B WV capacity to scavenge various free radicals and to reduce
HL-1
140 HL-2 oxidant-induced cellular death. As Figure 1 and Table 2
shown, WBM leaf extracts exerted hydroxyl radical and
120 a a a a a a a a a a a NO scavenging activities. The most impressive radical-
100
scavenging activity is against hydroxyl radical, which is con-
% of control

sidered to be the most reactive one among ROS. The IC50

80 values for hydroxyl radial ranged from 22 to 42 μg/mL.
60
The wild variety and HL-2 were superior to HL-1.
Leaf extract of Thai bitter melon possesses hydroxyl-
40
radical scavenging activity with an IC50 value of 167 ?
20 0.96 mg/mL (Kubola and Siriamornpun 2008). Lu et al.
(2012) demonstrated fruit extract of the most effective
0
Control Vehicle
25 50 100 25 50 100 25 50 100 WBM cultivar in Taiwan exerted potent hydroxyl-radical
WV (? g/mL) HL-1 (? g/mL) HL-2 (? g/mL)
scavenging activity (IC50, 37 μg/mL). These results further
UVB (80 mJ/cm2)
supported that leaf and fruit of WBM possess strong
120 C hydroxyl-radical scavenging activity.
d WV Numerous phytochemicals such as momordicine,
HL-1
HL-2
kuguacins, and phenolics, have been isolated from bitter
100
melon leaves (Lim 2012; Kubola and Siriamornpun
2008). The new finding triterpenoids isolated from the
80
stems and fruits of bitter melons show their antioxidant
% of control

property (Liu et al. 2010; Lin et al. 2011). Kubola and

60
Siriamornpun (2008) reported that leaf extract of bitter
b b c c c
b
c
c
c c
c melon possess antioxidant activity, based on DPPH
40 b
radical-scavenging activity and ferric acid reducing
a
power. The predominant phenolic compounds in the
20
leaf of bitter melon are gallic acid, followed by caffeic
acid and catechin (Kubola and Siriamornpun 2008). We
0
Control Vehicle
25 50 100 25 50 100 25 50 100 recently investigated phenolic compounds of these three
WV (? g/mL) HL-1 (? g/mL) HL-2 (? g/mL)
WBM leaf extracts using HPLC methods (Kubola and
UVB (80 mJ/cm2) Siriamornpun 2008; Zhang et al. 2009) with some modi-
fications. The phenolic compounds found in WBM
leaves were gallic acid, salicylic acid, cinnamic acid, myr-
icetin, quercetin, and luteolin (Additional file 1). Because
of the diversity and complexity of the natural mixtures
of antioxidants in WBM leaf extracts, it is rather difficult
to characterize all of compounds by HPLC. Further work
is still required to verify the anti-oxidative constituents
of WBM leaves.
Tsai et al. Botanical Studies 2014, 55:78 Page 8 of 11
http://www.as-botanicalstudies.com/content/55/1/78

120
A 50 ? g/mL
100 ? g/mL
h 200 ? g/mL
100 f f
g e
g g
f
e
80
% of control

c
60 c

40

a
20

0
(-) SNP Vehicle WV HL-1 HL-2
SNP (2 mM)

120 B
c
50 ? g/ml
100 100 ? g/ml
200 ? g/ml

80
% of control

b
b b
b b b b
b b
60

40

a
20

0
(-)Menadione Vehicle WV HL-1 HL-2
Menadione (50 ? M)
Figure 3 Effects of WBM leaf extracts against keratinocyte death induced by SNP (A) and menadione (B) treatment. Cells were exposed
to either SNP (2 mM) or menadione (50 μM) in the absence (vehicle) or presence of leaf extracts (50? 200 μg/mL). Cell viability was determined
24 hours later using MTT assay. Data are presented as the mean ? SD of triplicate determinations. Values with the same letter are not significantly
different as determined by Duncan? s multiple range tests.

Skin is constantly exposing to environmental insults, (Evans and Johnson 2010). To elucidate the antioxidant
among all, UV light is thought to be the most harmful activity exerted by WBM leaf extracts, the intracellular
one. UV exposure can cause oxidative stress and inflam- ROS generation in UV-irradiated keratinocyte was carried
mation. Eating plenty of green leafy vegetables has been out using an oxidant-sensitive fluorescent probe DCF-DA.
considered to be beneficial for photoprotection (Mukhtar By measuring the intercellular ROS scavenging activity of
2003). Phytonutrients with antioxidant activity have been WBM leaf extracts, the results has shown that pre-
considered to be beneficial for attenuating UV-caused oxi- treatment of extracts could reduce the intracellular ROS
dative stress and oxidative stress-mediated skin disorders production induced by UV within cells. Moreover, all three
Tsai et al. Botanical Studies 2014, 55:78 Page 9 of 11
http://www.as-botanicalstudies.com/content/55/1/78

A 50 ? g/mL
120 100 ? g/mL
200 ? g/mL
g

Tyrosinase activity(% of positive control)

h f f
h
100 f c c g g
d d
d e d
c e e b
e e
d c
b
80

60 a

40

20

0
(-) α-MSH Vehicle WV HL-1 HL-2 kojic acid

α-MSH

120
B
f
Melanin content (% of positive control)

d d
100 e e d
e e
e
d d

b c c
80 c b
b

a
60

40

20

0
(-) α-MSH Vehicle WV HL-1 HL-2 kojic acid

α-MSH
Figure 4 Effects of WBM leaf extract on cellular tyrosinase activity (A) and melanin level (B) of α-MSH treated B16-F10 melanocytes. Each
value is expressed as the mean ? SD (n = 3). Values with the same letter are not significantly different as determined by Duncan ? s multiple range tests.

WBM leaf extracts showed the protective effect on the via- can be observed herein. Kumar et al. (2010) reported that
bility of UVB-irradiated keratinocytes (Figure 2). Therefore, fruit extract of bitter melon showed significant cytoprotec-
WBM leaf extract could be proposed as a photo-protective tion against oxidants on fibroblasts and keratinocytes. In
agent preventing harmful effects of UVB exposure on hu- this study, WBM leaf extract protect HaCaT cells from
man keratinocytes. damage caused by SNP, which implies that WBM leaf ex-
As WBM leaf extracts showed antioxidant effects against tracts are able to block the harmful events induced by NO
free radicals and intercellular ROS, their cell protective ef- overproduction, a process relevant to premature skin aging
fects on keratinocytes against the cellular damage induced occurring upon long term UV exposure (Weller 2003).
by SNP (a NO donor) and menadione (a ROS donor) were Similarity, all three WBM leaf extracts possessed cyto-
evaluated (Figure 3). The cell protective property of WBM protection against menadione-induced damage on HaCaT
leaf extracts against SNP or menadione-induced cell death keratinocytes, suggesting its ability to reduce superoxide-
Tsai et al. Botanical Studies 2014, 55:78 Page 10 of 11
http://www.as-botanicalstudies.com/content/55/1/78

mediated stress in skin. In considering that ROS plays an the binuclear copper active site and thus gallate derivatives
important role in the pathogenesis of many skin disorders exerted the anti-tyrosinase effect (Tanford 1980). Since
and chronologic skin aging (Bickers and Athar 2006), these gallic acid was found in WBM leaf extracts, it speculated
findings presented in this study suggest that WBM leaf is a that gallic acid may contribute, at least partially, to anti-
good source of natural antioxidants and may prevent the melanogenic activity of WBM leaf extracts. Other unknown
ROS-mediated skin disorders. active constituents present in WBM leaf extract could also
Clinical changes recognizable as photoaging include play critical roles in the biological effects. The identification
wrinkle, inelasticity, telangiectasia and pigmentary change. of these functional or bioactive ingredients in WBM leaf ex-
Although not detrimental in nature, hyperpigmentation tract is an interesting topic for future study.
can be a big cosmetic concern, especially among Asian.
The inhibition of melanogenesis is a critical target for Conclusions
skin-whitening cosmetic and treatment of abnormal pig- In summary, all leaf extracts form WBM variety and
mentation (Yokozawa and Kim 2007). Since tyrosinase is cultivars showed antioxidant, cell protection, and anti-
a rate-limiting enzyme of melanogenesis, inhibition on its melanogenic activities. Among them, HL-1 showed the
action is one major strategy to treat hyperpigmentation. most potent anti-melanogenic activity. These findings
The anti-tyrosinase and anti-melanogenic activity of nat- may be used to develop health foods or cosmetics, and
ural substances have been studied extensively. Fruit ex- to increase the range of applications of traditional agri-
tracts of bitter melon have been shown to exert significant cultural vegetables.
inhibitory effect on mushroom tyrosinase (Kamkaen et al.
2007; Masuda et al. 2007; Baurin et al. 2002). Kamkaen Additional file
et al. (2007) reported that methanolic extract of bitter
melon fruit can exert a significant mushroom tyrosinase Additional file 1: HPLC profiles of WBM leaf extracts (A) and
reference authentic standards (B) detected at 280 nm. Peaks: 1, gallic
inhibition (78.9% compared to positive control of kojic
acid; 2, salicylic acid; 3, caffeic acid; 4, ferulic acid; 5, cinnamic acid; 6,
acid). Masuda et al. (2007) reported that ethanol extract of myricetin; 7, quercetin; 8, luteolin. Chromatographic separations were
bitter melon fruit can show 21.7% of mushroom tyrosin- performed on a C-18 reversed-phase silica Bondclone column (300 ? 3.9 mm i.
d., 10 μm, Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of
ase inhibitory activity at a concentration of 0.5 mg/mL.
solvent A (water/methanol, 98:2), and solvent B (methanol/acetic acid, 98:2)
Similar finding was reported in previous study that used according to a linear gradient elution from 10% B to 80% B during 30 min, at a
propylene glycol/deionized water extract of bitter melon flow-rate of 1 mL/min. The following gradient was used 10-40% B in 15 min;
40-80% B in 15 min.
plants with 32% inhibition of mushroom tyrosinase
(Baurin et al. 2002). In our preliminary study, WBM leaf
Competing interests
extracts exhibited suppressive effect on mushroom tyro- The authors declare that they have no competing interests.
sinase activity at a concentration of 5 mg/mL. It is not yet
known whether WBM leaf extract has anti-melanogenic Authors? contributions
T-TH, H-CJ and T-PJ conceived and designed the experiments, and drafted the
activity in cellular assay. In the experiments reported here,
manuscript. T-TH carried out most of analyses and finished the figures and
HL-1 showed more potent anti-tyrosinase and anti- tables presented in the paper. W-WH carried out the anti-melanogenic assay
melanogenic activities than others. Regarding the inhibi- and performed the statistical analysis. H-WC carried out the HPLC analysis. C-JH
did the culture of wild bitter melon and revised the manuscript. T-PJ wrote and
tory effect on tyrosinase activity, HL-1 extract was similar
revised the manuscript. All authors read and approved the final manuscript.
to kojic acid at each tested concentration (Figure 4). Kojic
acid shows its anti-tyrosinase activity by chelating the cop- Acknowledgements
per ion in the active site of tyrosinase (Kahn 1995). How- We thank the excellent technical assistance of Technology Commons, College
of Life Science, National Taiwan University, Taiwan with flow cytometry. This
ever, the inhibitory mechanism or effective constituents of work was supported by research grants (NSC 98-2321-B-003-001 and NSC
WBM leaf extract is still unclear. Kim et al. (2008), 99-2321-B-003-001) from the National Science Council, Taipei, Taiwan.
Yokozawa and Kim (2007), and Kim (2007) reported that
Author details
the antimelanogenic action of some agents is related to 1
Department of Dermatology, Taipei Municipal Wan Fang Hospital and
their antioxidant activity. Concerning the action mechan- Taipei Medical University, Taipei, Taiwan. 2Institute of Microbiology and
ism of anti-tyrosinase agents, blocking of oxidative path- Biochemistry, and Department of Biochemical Science and Technology,
National Taiwan University, Taipei, Taiwan. 3Department of Human
way (Kim 2007) and binding of enzyme activity sites Development and Family Studies, National Taiwan Normal University, 162
(Curto et al. 1999) may be involved in inhibiting the cata- Hoping E. Rd., Sec. 1, Taipei 10610, Taiwan. 4Hualien District Agricultural
lytic reaction of tyrosinase. Previous studies demonstrated Research and Extension Station, Hualien, Taiwan.

that gallate and its derivatives (Kim 2007; Tanford 1980) Received: 8 July 2014 Accepted: 25 November 2014
and p-alkoxybenzoic acid derivatives (Chen et al. 2005)
can act as tyrosinase inhibitors. The bulky hydrophobic
References
portions of gallate derivatives were considered to interact AOAC ? Official Methods of Analysis? 14th ed., Washington, D.C.: Association of
with tyrosinase? s hydrophobic protein bulk surrounding Official Analytical Chemists; 1984.
Tsai et al. Botanical Studies 2014, 55:78 Page 11 of 11
http://www.as-botanicalstudies.com/content/55/1/78

Aoki Y, Tanigawa T, Abe H, Fujiwara Y (2007) Melanogenesis inhibition by an oolong Sumanont Y, Murakami Y, Tohda M, Vajragupta O, Matsumoto K, Watanabe H
tea extract in B16 mouse melanoma cells and UV-induced skin pigmentation in (2004) Evaluation of the nitric oxide radical scavenging activity of manganese
brownish guinea pigs. Biosci Biotechnol Biochem 71:1879? 1885 complexes of curcumin and its derivative. Biol Pharm Bull 27:170? 173
Bastianetto S, Dumont Y, Duranton A, Vercauteren F, Breton L, Quirion R (2010) Tanford C (1980) The hydrophobic effect: Formation of micelles and biological
Protective action of resveratrol in human skin: possible involvement of membranes. John Wiley & Sons, New York
specific receptor binding sites. PLoS One 5(9):e12935 Tsai TH, Tsai TH, Chien YC, Lee CW, Tsai PJ (2008) In vitro antimicrobial activities
Baurin N, Arnoult E, Scior T, Do QT, Bernard P (2002) Preliminary screening of some against cariogenic streptococci and their antioxidant capacities: A comparative
tropical plants for anti-tyrosinase activity. J Ethnopharmacol 82:155? 158 study of green tea versus different herbs. Food Chem 110:859? 864
Bickers DR, Athar M (2006) Oxidative stress in the pathogenesis of skin disease. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radicals and
J Investig Dermatol 126:2565? 2575 antioxidants in normal physiological functions and human disease. Int J
Chen QX, Song KK, Qiu L, Liu XD, Huang H, Guo HY (2005) Inhibitory effects on Biochem Cell Biol 39:44? 84
mushroom tyrosinase by p-alkoxybenzoic acids. Food Chem 91:269? 274 Weller R (2003) Nitric oxide: a key mediator in cutaneous physiology. Clin Exp
Cheng Z, Yan G, Li Y, Chang W (2003) Determination of antioxidant activity of Dermatol 28:511? 514
phenolic antioxidants in a fenton-type reaction system by chemilumines- Wu SJ, Ng LT (2008) Antioxidant and free radical scavenging activities of wild bitter
cence assay. Anal Bioanal Chem 375:376? 380 melon (Momordica charantia Linn. var. abbreviata Ser.) in Taiwan. LWT Food Sci
Curto EV, Kwong C, Hermersdorfer H, Glatt H, Santis C, Virador V (1999) Inhibitors Technol 41:323? 330
of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of Yokozawa T, Kim YJ (2007) Piceatannol inhibits melanogenesis by its
gentisic acid with other putative inhibitors. Biochem Pharmacol 57:633? 672 antioxidative actions. Biol Pharm Bull 30:2007? 2011
Deliconstmtinos G, Villiotou V, Stawides JC (1996) Alterations of nitric oxide synthase Zhang M, Hettiarachchy NS, Horax R, Chen PY, Kenneth F (2009) Effect of
and xanthine oxidase activities of human keratinocytes by ultraviolet B maturity stages and drying methods on the retention of selected nutrients
radiation. Biochem Pharmacol 51:1727? 1738 and phytochemicals in bitter melon (Momordica charantia) leaf. J Food Sci
Evans JA, Johnson EJ (2010) The role of phytonutrients in skin health. Nutrients 74:C441? C448
2:903? 928
Hsu C, Tsai TH, Li YY, Wu WH, Huang CJ, Tsai PJ (2012) Wild bitter melon doi:10.1186/s40529-014-0078-y
(Momordica charantia Linn. var. abbreviata Ser.) extract and its bioactive Cite this article as: Tsai et al.: Antioxidant, cell-protective, and
components suppress Propionibacterium acnes-induced inflammation. anti-melanogenic activities of leaf extracts from wild bitter melon
Food Chem 135:976? 984 (Momordica charantia Linn. var. abbreviata Ser.) cultivars. Botanical
Studies 2014 55:78.
Kahn V (1995) Effect of kojic acid on the oxidation of L-DOPA, norepinephrine,
and dopamine by mushroom tyrosinase. Pigment Cell Res 8:234? 240
Kamkaen N, Mulsri N, Treesak C (2007) Screening of some tropical vegetables for
anti-tyrosinase activity. Thail Pharm Health Sci J 2:15? 19
Khan MR, Omoloso AD (1998) Momordica charantia and Allium sativum: broad
spectrum antibacterial activity. Korean J Pharmacogn 29:155? 158
Kim YJ (2007) Anti-melanogenic and antioxidant properties of gallic acid. Biol
Pharm Bull 30:1052? 1055
Kim YJ, Kang KS, Yokozawa T (2008) The anti-melanogenic effect of pycnogenol
by its anti-oxidative actions. Food Chem Toxicol 46:2466? 2471
Klausc V, Hartmannb T, Gambinid J, Grafb P, Stahlb W, Hartwigc A, Klotza LO (2010)
1, 4-Naphthoquinones as inducers of oxidative damage and stress signaling in
HaCaT human keratinocytes. Arch Biochem Biophys 496:93? 100
Kubola J, Siriamornpun S (2008) Phenolic contents and antioxidant activities of
bitter gourd (Momordica charantia L.) leaf, stem and fruit fraction extracts
in vitro. Food Chem 110:881? 890
Kumar R, Balaji S, Sripriya R, Nithya N, Uma TS, Sehgal PK (2010) In vitro
evaluation of antioxidants of fruit extract of Momordica charantia L. on
fibroblasts and keratinocytes. J Agric Food Chem 58:1518? 1522
Lim TK (2012) Momordica charantia. In: Lim TK (ed) Edible medicinal and
non-medicinal plants, vol 2. Springer, New York, pp 331? 368
Lin KW, Yang SC, Lin CN (2011) Antioxidant constituents from the stems and
fruits of Momordica charantia. Food Chem 127:609? 614
Liu CH, Yen MH, Tsang SF, Gan KH, Hsu HY, Lin CN (2010) Antioxidant triterpenoids
from the stems of Momordica charantia. Food Chem 118:751? 756
Lu YL, Liu YH, Liang WL, Chyuan JH, Cheng KT, Liang HJ, Hou WC (2011)
Antibacterial and cytotoxic activities of different wild bitter gourd cultivars
(Momordica charantia L. var. abbreviata Seringe). Bot Stud 52:427? 434
Lu YL, Liu TH, Chyuan JH, Cheng KT, Liang WL, Hou WC (2012) Antioxidant
activities of different wild bitter gourd (Momordica charantia L. var. Submit your manuscript to a
abbreviata Seringe) cultivars. Bot Stud 53:207? 214 journal and bene? t from:
Masaki H (2010) Role of antioxidants in the skin: anti-aging effects. J Dermatol Sci
58:85? 90 7 Convenient online submission
Masuda T, Fujita N, Odaka Y, Takeda Y, Yonemori S, Nakamoto K, Kuninaga H 7 Rigorous peer review
(2007) Tyrosinase inhibitory activity of ethanol extracts from medicinal and 7 Immediate publication on acceptance
edible plants cultivated in Okinawa and identification of a water-soluble
inhibitor from the leaves of Nandina domestica. Biosci Biotechnol Biochem 7 Open access: articles freely available online
71:2316? 2320 7 High visibility within the ?eld
Mukhtar H (2003) Eat plenty of green leafy vegetables for photo-protection: 7 Retaining the copyright to your article
emerging evidence. J Invest Dermatol 121:doi:10.1046/j.1523-1747.2003.12377.x.
Robak J, Gryglewski RJ (1988) Flavonoids are scavengers of superoxide anions.
Biochem Pharmacol 37:837? 841 Submit your next manuscript at 7 springeropen.com