Vous êtes sur la page 1sur 25

Authors Accepted Manuscript

Wound healing property of isolated compounds


from Boesenbergia kingii rhizomes
Teeratad Sudsai, Chatchai Wattanapiromsakul,
Supinya Tewtrakul
www.elsevier.com/locate/jep

PII:
DOI:
Reference:

S0378-8741(16)30097-6
http://dx.doi.org/10.1016/j.jep.2016.03.001
JEP10008

To appear in: Journal of Ethnopharmacology


Received date: 13 January 2016
Accepted date: 1 March 2016
Cite this article as: Teeratad Sudsai, Chatchai Wattanapiromsakul and Supinya
Tewtrakul, Wound healing property of isolated compounds from Boesenbergia
kingii
rhizomes,
Journal
of
Ethnopharmacology,
http://dx.doi.org/10.1016/j.jep.2016.03.001
This is a PDF file of an unedited manuscript that has been accepted for
publication. As a service to our customers we are providing this early version of
the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting galley proof before it is published in its final citable form.
Please note that during the production process errors may be discovered which
could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Wound healing property of isolated compounds from Boesenbergia kingii rhizomes

Teeratad Sudsaia,b,c, Chatchai Wattanapiromsakula, Supinya Tewtrakula,b*

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,

Prince of Songkla University, Songkhla 90112, Thailand


b

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of

Science, Prince of Songkla University, Songkhla 90112, Thailand


c

College of Oriental Medicine, Rangsit University, Pathumthani 12000, Thailand

*Corresponding author: Tel.: +66 74 288888; fax: +66 74 428220. E-mail addresses:
supinyat@yahoo.com, supinya.t@psu.ac.th

Abstract
Ethnopharmacological relevance:
Boesenbergia kingii have been traditionally used in the treatment of inflammatory bowel disease,
ulcerative colitis, aphthous ulcer, stomach discomfort, dysentery and abscess. Previously, we
reported the B. kingii extract exert potential wound healing properties. Therefore the search of

responsible constituents for wound healing property from these rhizomes is still relevant.
Aim of study:
This study was aimed to investigate for wound healing property of compounds from this plant in
order to support its traditional uses.
Material and methods:

2
Wound healing activities were tested using in vitro assays including cell proliferation and
migration assays, collagen production and H2O2-induced oxidative stress in mouse broblast
L929 cells. The DPPH assay was also used to determine antioxidant activity.
Results:
Fourteen compounds from the chloroform fraction possessed potent anti-oxidant and wound
healing activities. Compound 11 exhibited the most potent anti-DPPH effect (IC50 = 21.0 M)
and also active against 0.5 mM H2O2-induced oxidative stress by increasing cell survival ability
up to 60.3 % at 10 M. In addition, compounds 3, 8 and 14 at 10 M significantly enhanced
L929 viability with 119.2%, 122.7% and 113.7%, respectively. Compounds 2, 7, 8 and 14
markedly enhanced L929 migration on day 2 up to 60-76% at 10 M, whereas 7 and 14 strongly
stimulated collagen production at 75.0 and 96.7 g/ml compared to the control group (57.5
g/ml), respectively.
Conclusion:
B. kingii is responsible for wound healing property via antioxidative effect, stimulation of
fibroblast proliferation and migration as well as enhancement of collagen production.

Keywords: Boesenbergia kingii; wound healing; sesquiterpenes; diarylheptanoids; flavonoids;


L929 fibroblast cells
1. Introduction
The genus Boesenbergia Kuntze (Zingiberaceae) comprises rhizomatous plants approximately 80
species distributed throughout tropical Asia. Since ancient time, Thai people have been used

these plant species in traditional medicine for the treatment of various ailments.
Kanathum N. (2008), who collected medicinal herbs information, has reported that these
plants contain a wide variety of active principles in book named Medicinal and Lucky
Plant of Thailand (Kanathum, 2008). Boesenbergia longiflora (Wall.) Kuntze is a related crop

3
under this genus which is re-identified by Mood et al. (2013) as Boesenbergia kingii Mood &
L.M. Prince. The rhizomes of B. kingii have been traditionally used in the treatment of ulcerative
colitis, inflammatory bowel disease, aphthous ulcer, stomach discomfort, dysentery and abscess
by decoction with alcohol (Chuakul and Boonpleng, 2003; Kanathum, 2008). Our previous study
demonstrated that the chloroform (CHCl3) fraction of B. kingii significantly enhanced L929
fibroblast migration and collagen production. It also revealed that the isolated compounds form
the CHCl3 fraction inhibited NO production and suppressed mRNA expression of iNOS and
COX-2 genes in dose-dependent manners. Moreover, it has been shown to be potent for antiinflammation in vivo by lowering the rat paw edema induced by carrageenan suggesting their
potential as anti-inflammatory agent. From the phytochemical study, the CHCl3 fraction of B.
kingii was found to contain diarylheptanoids, flavonoids and new sesquiterpenes (Sudsai et al.
2013; Sudsai et al. 2014). These groups of compounds might be responsible for the wound
healing property of this plant. However, the effects of pharmacological agents which modulate
the different phases of the wound healing processes can be assessed by in vitro experiments and
ideally a plant-based remedy should affect at least two different processes before it can be said to
have some scientific support for its traditional use (Houghton et al., 2005).
Wound healing process can be characterized by three overlapping of inflammatory,
proliferative and remodeling phases which repair and organize structure with increased tensile
strength of the damaged tissue partially or completely depending on the severity of wounding
(Wild et al., 2010).

It is well known that when wounding occurs, short-term process of

inflammation caused by the release of the inflammatory mediators and radical oxygen species by
the macrophages mainly impair and delay the process of wound repair (Houghton et al., 2005;
Tam et al., 2011).

Thus, the inhibition of reactive radical production is an important

consideration in recruitment of fibroblast which is attracted into the site to initiate the
proliferative phase of repair or wound healing process. The present study was therefore aimed to
evaluate the wound healing effect of isolated compounds from B. kingii rhizomes by using several

4
in vitro assays.

The assessment of the wound healing parameters including anti-oxidant,

fibroblast proliferation and collagen production were employed for the scientific support.

2. Materials and methods


2.1 Plant material and isolation of active ingredients
The voucher specimen of B. kingii rhizomes (SKP2060200-101) was deposited at the
herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla,
Thailand. The extraction and isolation of B. kingii rhizomes throughout this study are performed
according to the bioassay-guided isolation as previously reported by Sudsai et al. (2014). The
phytochemical study of the CHCl3 fraction was investigated in order to obtain the compounds that
are responsible for the wound healing property. Fourteen compounds (1-14; Fig. 1) were isolated
as 8-hydroxy-dauca-9,11-diene-7-one (longiferone A: 1), dauca-8,11-diene-7-one (longiferone B:
2) and dauca-8,11-diene-7,10-dione (longiferone C: 3), kaempferol-3,7,4-trimethyl ether (4),
kaempferol-7,4-dimethyl
dihydrobisdemethoxycurcumin

ether

(5),
(8),

rhamnazin

(6),

pinostrobin

(7),

1-hydroxy-dihydrobisdemethoxycurcumin

(9),

dihydrobisdemethoxycurcumin-4,4-diacetate (10), curcumin (11), demethoxycurcumin (12),


bisdemethoxycurcumin (13) and -sitosterol-D-glucoside (14).
2.2 2, 2-Diphenyl-1-picrylhydrazyl radical (DPPH) scavenging assay
The methodology described by Jitsanong et al. (2011) was used in this study with slight
modifications in order to assess the DPPH free radical scavenging capacity. The stock solution
(10 mM) of the sample is prepared in DMSO and diluted to concentrations ranging from 1-100
M with absolute ethanol.

The reaction mixture contained 100 l of samples at various

concentrations and 100 l of 610-5 M DPPH in absolute ethanol. The commercial known
antioxidant, butylated hydroxytoluene (BHT) and quercetin were used as positive controls. The
DPPH solution in the absence of sample was used as a control and the absolute ethanol was used
as a blank. The bleaching was measured at 517 nm using a microplate reader after incubation for

5
30 min in the dark condition. The percentage of scavenging activity of the sample against DPPH
radical was calculated according to the following equation and IC50 values were determined
graphically (n = 4):

% Inhibition = [(A control - A sample) / A control] 100

A control = Absorbance of control - Absorbance of control blank


A sample = Absorbance of sample - Absorbance of sample blank

2.3 Cell proliferation and viability assay using L929 fibroblast


L929 fibroblasts in DMEM containing 10% FBS were seeded at 2104 cells/well into 96well plate. After 24 h, cells were exposed to different concentrations (1-100 M) of test samples
and were then incubated for 48 h at 37C in a humidified atmosphere containing 5% CO 2. MTT
solution (10 l, 5 mg/ml) was added directly to the medium in each well, and the plate was then
incubated at 37C for 4 h. All medium was then aspirated and replaced with isopropanol
containing 0.04 N HCl, and the optical density at 570 nm was recorded. The percentage of cell
proliferation was calculated and compared to a negative control.
2.4 Hydrogen peroxide-induced oxidative stress
Hydrogen peroxide (H2O2) induced oxidative stress was determined as previously
described by Jitsanong et al. (2011) with a slight modification. Briefly, broblast L929 cells were
seeded in 96 well plates (2 104 cells/ml) in DMEM medium containing 10% FBS to confluence
and then after 24 h, cells were treated with different concentrations (1-30 M) of test samples.
After pretreatment with different concentrations for 1 h incubation at 37C with 5% CO2, the cells
were co-incubated with 0.5 mM of H2O2 for another 24 h. At the end of the incubation cell
viability was determined by the MTT assay.

2.5 Migration assay


The migration of broblast L929 was examined using a wound healing method. Briefly,
L929 cells (5104 cells/ml) in DMEM containing 10% FBS were seeded into each well of 24
wells plate and incubated at 37C and 5% CO2. After the confluent monolayer of L929 cells was
formed, a horizontally scratched with a sterile pipette tip was generated two scratches (left and
right) in each well. Any cellular debris was removed by washing with PBS and replaced with 1
ml of fresh medium in the absence or presence of test samples. Photographs were taken two
views on the left and right on each well at a 4 magnification using a microphotograph
(Olympus CK2, Japan) on day 0, then plates were incubated at 37C with 5% CO 2 and
photographs were taken at days 1 and 2. To determine the migration of L929 cells, the images
were analyzed using computing software (ImageJ1.42q/Java1.6.0-10). Percentage of the closed
area was measured and compared with the value obtained before treatment (day 0). An increase
of the percentage of closed area indicated the migration of cells (Balekar et al., 2012).

2.6 Determination of soluble collagen production


The soluble collagen productions are determined according to the method described by
Balekar et al. (2012). Fibroblast L929 cells in DMEM containing 10% FBS were seeded at an
initial concentration of 2104 cells/ml in a 96 well plate. After 24 h, the culture medium was
replaced with a fresh medium containing the test samples at various concentrations (0.3-10 M)
and was then incubated for 48 h at 37C with 5% CO2. Cells without a test sample served as
negative controls. After 48 h of incubation, cells generated soluble collagen type I into the
medium, the supernatant (100 l) were collected. The total amount of soluble collagen type I was
assayed using the Sircol Collagen Assay Kit (Bicolor Life Science Assays, Northern Ireland,

7
UK). Briey, 100 l of supernatant was mixed with 1 ml of dye solution at room temperature for
30 min. Then the samples were centrifuged at 15,000 g for 10 min to form a pellet of collagen.
All supernatant was then aspirated and the soluble collagen was dissolved in 1 ml of alkali
reagent. Thereafter, the alkali solutions were transferred to a 96 well plate and the optical density
at 540 nm was recorded. The amount of collagen was calculated based on a standard curve of
soluble collagen (bovine skin collagen type I standard from American disease free animals).

2.7 Statistical analysis of data


For statistical analysis, all data values were calculated using the Microsoft Excel program
and expressed as mean S.E.M of four determinations. The data analysis was performed by oneway analysis of variance (ANOVA), followed by Dunnetts test. The p value < 0.05 was
considered to be significant.

3. Results and discussion


3.1 DPPH radical scavenging activity
DPPH assay is one of the most widely used methods for screening the antioxidant activity
of plant extracts. The assay is based on the measurements of the antioxidant ability to scavenge
the stable radical DPPH. DPPH is a stable nitrogen-centered free radical, which produces violet
color in EtOH solution. DPPH radicals react with suitable reducing agents, during which the
electrons become paired off and the solution loses color depending on the number of electrons
taken up (Mandade et al., 2011). In the experiment, the solution progressively reduced to a
yellow colored product, diphenylpicryl hydrazine, with the addition of the extracts in a
concentration-dependent manner. DPPH radical-scavenging activities of isolated compounds
from B. kingii rhizomes and reference compounds were shown in Table 1.

Among these,

curcumin (11) exhibited the most potent inhibitory effect with an IC50 value of 21.0 M, followed
by bisdemethoxycurcumin (13, IC50 = 29.9 M), rhamnazin (6, IC50 = 31.1 M), kaempferol-7,

8
4-dimethyl ether

(5, IC50 = 39.0 M), demethoxycurcumin (12, IC50 = 53.2 M), and

dihydrobisdemethoxycurcumin (8, IC50 = 95.6 M) respectively. Compounds 5, 6, 8, 11, 12 and


13 exhibited the scavenging activity higher than BHT (IC50 = 104.0 M) but less than quercetin
(IC50 = 9.5 M). The present study showed that two flavonoids (5-6) and four diarylheptanoids
(8, 11-13) significantly decreased the levels of DPPH radical.
Flavonoids are known to possess the ability to scavenge free radicals, including
superoxide and hydroxyl radicals. The structure characteristics of flavonoids that can be effected
radical scavenging activity were as follow; (a) hydroxyl groups, especially on the B-ring, enhance
their antioxidant activity (b) C2-C3 double bond of C-ring appears to increase scavenging
activity, (c) C4-keto double bond in association with C2-C3 double bond increase their activity,
(d) hydroxyl group at C-3 generates an extremely active scavenger, in fact, the combination of
C2-C3 double bond and C4-keto double bond appears to be the best combination, (e) hydroxyl at
C-5 and C-7 groups may also enhance radical scavenging potential (Tapas et. al., 2008). Our
results were in agreement with the structural requirements of flavonids for antioxidant activity.
The antioxidant activity decreased in the order: quercetin > rhamnazin (6) > kaempferol-7,4dimethyl ether (5) > kaempferol-3,7,4-trimethyl ether (4) > pinostrobin (7). In the present study,
the anti-oxidant activities of six natural curcuminoids showed the antioxidant activities that
decreased in the order: curcumin (11) > bisdemethoxycurcumin (13) > demethoxycurcumin (12)
> dihydrobisdemethoxycurcumin (8) > dihydrobisdemethoxycurcumin-4,4-diacetate (10) > 1hydroxy-dihydrobisdemethoxy curcumin (9). These results were in agreement with the structural
requirements of curcuminoid for anti-oxidant activity as follow; (a) the presence of methoxy
groups in the phenyl rings of curcuminoids enhanced anti-oxidant activity, (b) phenolic
substitution is important for anti-oxidant activity and (c) electron delocalization of the double
bonds may not be essential to anti-oxidant activity of curcuminoids (Itokawa et al., 2008).

3.2 Cell viability assay using L929 fibroblasts

9
In vitro cytotoxicity test is based on the idea that toxic chemicals affect basic function of
cells which are common to all cells, and that toxicity can be measured by assessing cellular
damage. Toxicity screening can help for compounds identification whether they can be further
utilized for evaluating biological activity (Balekar et al., 2012). Hence, the cytotoxicity of
isolated compounds from B. kingii rhizomes was evaluated by MTT assay.

The isolated

compounds from the CHCl3 fraction were tested for their enhancement of the growth of L929
fibroblast cells, the results were shown in Table 2. Flavonoids, diarylheptanoids, terpenoids and
sterol found in this fraction have associated the classes of compounds with wound healing
activities by stimulating the growth of fibroblasts. After treatment with compounds 3, 8 and 14,
significant viability-enhancement effects were observed in L929 fibroblasts. Compounds 3 (1-10
M), 8 (1-10 M) and 14 (3-30 M) produced a cell viability of over 100%, while a cell viability
of the other compounds were shown in Table 2. Therefore, the concentration of these compounds
with highest enhancement the growth of L929 fibroblast cells was chosen for subsequent
proliferation and migration of fibroblast cells by in vitro scratch assay. In addition, the range of
concentrations of each isolated compound that produced cells viability more than 80% were
employed in protection of H2O2 induced oxidative stress experiment and collagen production
experiment.
In this study, cytotoxicity effect of isolated compounds from B. kingii rhizomes were
observed at the higher concentrations implies that caution must be taken when using this plant for
treating wounds. However, the acute toxicity study of ethanolic extract and chloroform fraction
were non-toxic at the dose 2000 mg/kg body weight in both male and female Swiss albino mice
(Sudsai et al., 2013).

3.3 H2O2-induced oxidative stress


H2O2, one of the major reactive oxygen species (ROS) transformed from oxygen in the
cellular aerobic metabolism, is well known as a direct oxidant that formed hydroxyl radical to

10
react with all components of the cell. Thus, H2O2-induced oxidative stress is a useful method for
gaining the endogenous antioxidant activity. In this study, treatment with 0.5 mM H 2O2 for 24 h
dramatically decreased cell viability to 20.0%. It is well documented that H2O2-induced L929
cell death is dose-dependent: low concentration of H2O2 (0.4-0.6 mM) caused cell apoptosis,
while higher concentrations (>0.6 mM) caused cell necrosis (Takeyama et. al., 2002; Dash et al.,
2007).

L929 fibroblast cells were pretreated with increasing concentration (1-10 M) of

sesquiterpenes (1-3), flavonoids (4-7), diarylheptanoids (8-13) and sterol (14) 1 h prior to
exposure with 0.5 mM H2O2 for 24 h. The results showed that all pretreated cells with various
isolated compounds were significantly different from untreated cells incubated with 0.5 mM H2O2
(p<0.01, Table 3). Co-culture of isolated compounds from the CHCl3 fraction with 0.5 mM H2O2
showed the protective effect against H2O2-induced cell death at least over a 24 h period by
increasing cell viability.

There are a number of reports showing that diarylheptanoids and

flavonoids suppress H2O2-induced the oxidative stress.

Diarylheptanoid derivatives such as

curcumin has been significantly offered cell protection through inhibition of lipid peroxidation,
increase in endogenous antioxidant defense enzymes, decrease of ROS levels and reduction in
protein carbonyl formation (Kamarehei et al., 2014). In addition, Yokomizo et al. (2014) suggest
that the strong antioxidative flavonoids have both cytoprotective effect owing to the scavenging
of ROS and cytotoxic effect caused by the generation of H2O2. In this study, sesquiterpenes (1-3)
that did not show extracellular antioxidant activity (IC50 > 100 M) by DPPH assay significantly
inhibited H2O2-induced oxidative stress (52.7-57.5% at 10 M) which might involve in the
intracellular free-radical scavenging effect. Many reports have been demonstrated the role of
several antioxidant defense enzymes, including superoxide dismutase, catalase, glutathione
dependent enzymes such as glutathione peroxidase, glutathione reductase, as well as a variety of
non-enzymatic antioxidant by antioxidant compounds such as ascorbic acid, -tocopherol,
glutathione and other dietary antioxidants, which scavenge radicals or neutralize ROS, thus
maintaining

redox balance. Catalase and glutathione peroxidase are involved in the

11
decomposition of H2O2 to water and oxygen and are therefore important in protecting cells
against oxidative stress (Conde de la Rosa et al., 2006; Bak et al., 2012). Thus, sesquiterpenes
presenting in this fraction may also contribute to protect the effect of H2O2-induced oxidative
stress that might affect as enzymatic antioxidants.

Further study for investigation of the

mechanisms of these compounds is still needed.

3.4 Effect of compounds from B. kingii extract on proliferation and migration of L929 cells
Fibroblast proliferation and migration are important steps in wound healing for tissue
regeneration. In the present study, the compounds from B. kingii were determined on the rate of
L929 migration using the scratch assay. Scratch assay is a useful method for gaining an insight
into the potential of an extract or its fractions to repair injured dermis. This assay commonly used
to study cell migration in vitro by creation of an artificial gap on a confluent cell monolayer with
a pipette tip (Balekar et al., 2012). The cellular proliferation and migration of fibroblast cells on
each edge of the gaps move toward to close the scratch area until new cell-cell contacts was
studied on days 0, 1 and 2. The migration effects of selected compounds on L929 fibroblasts
were found in the cells treated with sesquiterpenes (2), flavonoids (7), diarylheptanoids (8) and
sterol (14) as shown in Fig 2. The presence of 2 (10 M), 3 (10 M), 4 (10 M), 7 (30 M), 11
(3 M) and 13 (10 M) significantly enhanced migration of L929 fibroblasts on day 1 by 36.2,
37.9, 40.7, 49.4, 50.9 and 47.3%, respectively. In addition, a more significant increase in percent
migration (p < 0.01) was observed on day 2 by 76.4, 70.7, 72.7, 61.5, 92.4 and 82.7%,
respectively (Table 4). The results demonstrated the significant migration enhancement effect of
the isolated compounds when compared with the control group.

3.5 Effects on soluble collagen production from L929 fibroblast cells


Collagens are the most abundant family of protein in the body that provide strength and
integrity to all tissues and also play a vital role in wound healing (Enoch & Leaper, 2008).

12
Collagen type-I is the main structural component of extracellular matrix, skin and newly healed
wounds. High level of skin collagen was shown to be involved in stimulating improvements on
skin elasticity (Balekar et al., 2012). The study on the effect of isolated compounds on type-I
collagen production by fibroblast was performed using Sircol collagen assay kit. The results
showed that collagen type I production in L929 cells increased significantly after treatment with
some compounds at various concentrations (Table 5). The amount of collagen produced by the
cells was between 50 and 200 g/ml. The presence of various concentrations (0.3-10 M) of
flavonoids (4, 6, and 7) and sterol (14) significantly increased collagen production by fibroblast
cells in a concentration manner. Compounds 4, 6, 7 and 14 at concentration of 10 M exhibited
higher effect (86.7-121.3 g/ml) than the positive control, ascorbic acid (85.0 g/ml). Moreover,
the isolated diarylheptanoids exhibited the promising effects in the collagen production, as high
activities at low concentrations. The present study showed that collagen type-I production in
L929 cells increased significantly after treatment with the isolated flavonoids. Flavonoids have
been shown to increase collagen synthesis, promote the cross-linking of collagen, decrease the
degradation of soluble collagen, accelerate the conversion of soluble collagen to insoluble
collagen, and inhibit the catabolism of soluble collagen (Lodhi & Singhai, 2013). Isolated
compounds of B. kingii showed the positive effects by stimulating the proliferation and migration
of fibroblast cells, diminishing oxygen free radical over production and increasing the collagen
synthesis that are important factors for wound healing enhancement.

4. Conclusion
The present study shows that the plant extract of B. kingii contains flavonoids,
sesquiterpenes and diarylheptanoids which have anti-inflammatory properties. Flavonoids and
diarylheptanoids act as antioxidant whereas sesquiterpenes and flavonoids promote cell
proliferation and migration of fibroblasts. Moreover, the flavonoids and a sterol could enhance
collagen production. This evidence supports the traditional use of B. kingii for treatment of

13
ulcerative colitis, inflammatory bowel disease, aphthous ulcer and abscess which is related to the
wound healing property.

Conflicts of interest

The authors report no conflicts of interest.

Acknowledgments
This research project is supported by Prince of Songkla University, the Center of
Excellence for Innovation in Chemistry (PERCH-CIC) and the Thailand Research Fund (TRF,
RSA5680012). We also thank the Faculty of Pharmaceutical Sciences, Prince of Songkla
University for providing laboratory facilities.
References
Bak, M.M.J., Jun, M., Jeong, W.-S., 2012. Antioxidant and hepatoprotective effects of the red
ginseng essential oil in H2O2-treated HepG2 cells and CCl4-treated, Int. J. Mol. Sci. 13,
2314-2330.
Balekar, N., Katkam, N.G., Nakpheng, T. Jehtae, K., Srichana, T., 2012. Evaluation of wound
healing potential of Wedelia trilobata (L.) leaves. J. Ethnopharmacol. 141 817-824.
Chuakul, W., Boonpleng, A., 2003. Ethnomedical uses of Thai Zingiberaceous plant (1). Thai J.
Phytopharmacy 10, 33-39.
Conde de la Rosa, L., Schoemaker, M.H., Vrenken, T.E., Buist-Homan, M., Havinga, R., Jansen,
P.L., Moshage, H., 2006. Superoxide anions and hydrogen peroxide induce hepatocyte
death by different mechanisms: Involvement of JNK and ERK MAP kinases. J. Hepatol.
44, 918-929.

14
Dash, R., Acharya, C., Bindu, P.C., Kundu, S.C., 2007. Antioxidant potential of silk protein
sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts. BMB Rep.
41, 236-241.
Enoch S, Leaper DJ., 2008. Basic science of wound healing. Surgery. 26, 31-37.
Houghton, P.J., Hylands, P.J., Mensah, A.Y., Hensel, A., Deters, A.M., 2005. In vitro tests and
ethnopharmacological investigations: wound healing as an example. J. Ethnopharmacol.
100, 100-107.
Itokawa, H., Shi, Q., Akiyama, T., Morris-Natschke, S.L., Lee, K.H., 2008. Recent advances in
the investigation of curcuminoids. Chin. Med. 3, 11-23.
Jitsanong, T., Khanobdee, D., Piyachaturawat, P., Wongprasert, K., 2011. Diarylheptanoid 7-(3,4
dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene from Curcuma comosa Roxb.
protects retinal pigment epithelial cells against oxidative stress-induced cell death.
Toxicol. in Vitro. 25, 167-176.
Kamarehei, M., Yazdanparast, R., Aghazadeh, S. 2014. Curcumin protects SK-N-MC cells from
H2O2-induced cell death by modulation of Notch signaling pathway. Cell Biol. 2014, 3,
72-86.
Kanathum, N., 2008. Medicinal and Lucky Plant of Thailand. Baanlaesuan, Bangkok, Thailand, p
258.
Lodhi, S., Singhai, A.K. 2013. Wound healing effect of flavonoid rich fraction and luteolin
isolated from Martynia annua Linn. on streptozotocin induced diabetic rats. Asian Pac. J.
Trop. Med. 2013, 253-259.
Min, H.-Y., Kim, M.S., Jang, D.S., Park, E.-J., Seo, E.-K., Lee, S.K. Min, H.-Y., Kim, M.S.,
Jang, D.S., Park, E.-J., Seo, E.-K., Lee, S.K. 2009. Suppression of lipopolysaccharidestimulated inducible nitric oxide synthase (iNOS) expression by a novel humulene
derivative in macrophage cells. Int. Immunopharmacol. 9, 844-849.

15
Mood, J.D., Prince, L.M, Veldkamp, J.F., Dey, S., 2013.

The history and identity of

Boesenbergia longiflora (Zingiberaceae) and descriptions of five related new taxa. Gard.
Bull. (Singapore) 65, 47-95.
Odukoya, O.A., Sofidiya, M.O., Samuel, A.T., Ajose, I., Onalo, M., Shuaib, B., 2012.
Documentation of wound healing plants in Lagos-Nigeria: inhibition of lipid peroxidation
as in-vivo prognostic biomarkers of activity. Ann. Biol. Res. 3, 1683-1789.
Sudsai, T., Prabpai, S., Kongsaeree, P., Wattanapiromsakul, C., Tewtrakul, S., 2014. Antiinflammatory activity of compounds from Boesenbergia longiflora rhizomes. J

Ethnopharmacol 154, 453-461.


Sudsai, T., Wattanapiromsakul, C., Nakpheng, T., Tewtrakul, S., 2013. Evaluation of the wound
healing property of Boesenbergia longiflora rhizomes. J. Ethnopharmacol. 150, 223231.
Takeyama, N., Miki, S., Hirakawa, A., Tanaka, T., 2002. Role of the mitochondrial permeability
transition and cytochrome C release in hydrogen peroxide-induced apoptosis. Exp. Cell.
Res. 10, 274, 16-24.

Tam, J.C.W., Jau, K.M., Liu, C.L., To, M.H., Kwok, H.F., Lai, K.K., Lau, C.P., Ko,
C.H., Leung, P.C., Fung, K.P., Lau, C.B.S., 2011. The in vivo and in vitro diabetic
wound healing effects of a 2-herb formula and its mechanisms of action. J.

Ethnopharmacol. 134, 831-838.


Tapas, A.R., Sakarkar, D.M., Kakde, R.B. 2008. Flavonoids as nutraceuticals: a review. Trop. J.
Pharm. Res. 7, 1089-1099.
Wild, T., Rahbarnia, A., Kellner, M., Sobotka, L., Eberlein, T., 2010. Basics in nutrition and
wound healing. Nutrition. 26, 862-866.
Yokomizo, A., Moriwaki, M. 2006. Effects of flavonoids on oxidative stress induced by hydrogen
peroxide in human intestinal Caco-2 cells. Biosci. Biotechnol. Biochem. 70, 1317-1324.

16

17

Table 1. DPPH radical scavenging activity of the isolated compounds from B. kingii rhizomes
%Inhibition against DPPH radical at various
IC50
Sample
concentrations (M)
(M)
1
3
10
30
100
-4.2
4.4
8.5
15.0
Longiferone A (1)
>100
0.7
1.0
0.9*
2.4*
-3.1

5.6

12.9

Longiferone B (2)
>100
1.0
0.5
2.6*
-1.5
1.5
4.4
Longiferone C (3)
>100
3.3
1.2
0.6
Kaempferol-3,7,4-trimethyl ether
-4.5
4.5
12.0
>100
(4)
0.6
3.0
6.4*
-1.2
31.1
88.4
Kaempferol-7,4-dimethyl ether (5)
1.5
2.7**
0.6**
39.0
-7.2
0.3
16.7
35.1
83.5
Rhamnazin (6)
1.2
0.8
2.9*
2.6**
1.1**
35.1
-2.9
Pinostrobin (7)
>100
0.5
-0.4
6.2
12.4
52.9
Dihydrobisdemethoxycurcumin (8)
2.0
1.1
2.4*
3.6**
95.6
1-Hydroxy-6.0
6.4
12.0
>100
dihydrobisdemethoxycurcumin (9)
1.8
1.7
2.3*
Dihydrobisdemethoxycurcumin-4.8

6.7

10.2

26.3

>100
4,4-diacetate (10)
1.6
1.4
1.3*
0.9**
-1.6 7.1
23.2
59.2
86.5
Curcumin (11)
1.1
1.2
2.1**
1.9**
1.4**
21.0
2.3 7.6
17.4
31.4
71.6
Demethoxycurcumin (12)
1.4
1.6
2.6**
1.6**
1.4**
53.2
-0.7 2.5
10.6
46.9
85.5
Bisdemethoxycurcumin (13)
0.6
1.0
3.2*
0.7**
1.5**
29.9
-1.1
>100
-Sitosterol-D-glucoside (14)
2.6
-1.1 28.7
40.1
93.8
92.7
Quercetin
1.5
4.2**
4.8**
1.6**
1.0**
9.5
-8.5
0.5
22.5
54.8
BHT
2.7
2.1
0.6**
1.8**
104.0
Value represents mean S.E.M. (N=4). Significantly different from the control (0 M), *p<0.05,
**p<0.01. (-) = not determined

Table 2. Effect of isolated compounds from B. kingii rhizomes on L929 cells viability
%Viability of L929 cells at various
Sample
concentrations (M)

18

0
1
3
10
30
100
100.0
102.7 103.4 107.1 110.0 84.9
Longiferone A (1)
2.8
4.5
4.7
5.3
6.9*
3.3
100.0
101.6
98.3 100.1
91.9 84.8
Longiferone B (2)
2.8
2.2
5.0
4.0
2.3
3.8
100.0
111.5
97.2 119.2 101.9 89.1
Longiferone C (3)
2.8
6.8*
3.2
7.4**
1.9
1.2
Kaempferol-3,7,4-trimethyl ether 100.0
85.8
76.6
93.6
81.4 77.4
(4)
6.9
0.7
2.0**
3.1
1.2*
1.6**
Kaempferol-7,4-dimethyl ether
100.0
79.5
87.5
90.5 100.1 72.1
(5)
6.9
2.8*
1.8
3.6
5.3
3.3**
100.0
93.9
99.9 101.5
92.9 76.6
Rhamnazin (6)
1.8
5.9
4.5
5.4
3.6
4.3**
100.0
86.4
89.1
97.9 105.5 83.5
Pinostrobin (7)
6.9
1.2
1.8
2.8
1.6
0.5
Dihydrobisdemethoxycurcumin
100.0
109.5 117.8 122.7
92.4 84.4
(8)
1.8
1.2*
4.6**
4.9**
4.0
1.9
1-Hydroxy100.0
102.1
94.1
97.2
74.8 68.6
dihydrobisdemethoxycurcumin (9) 1.8
4.3
2.6
2.0
2.4**
0.7**
Dihydrobisdemethoxycurcumin100.0
94.5 103.1
96.3
91.2 83.3
4,4-diacetate (10)
1.8
4.2
1.7
5.2
5.2
5.4
100.0
83.9
91.2
83.0
73.9 39.6
Curcumin (11)
6.9
2.0*
4.0
5.2*
2.2**
5.7**
100.0
81.2
75.2
89.2
85.3 74.6
Demethoxycurcumin (12)
6.9
1.0*
3.0**
4.8
5.4
6.2**
100.0
85.4
83.7
92.9
79.4 51.6
Bisdemethoxycurcumin (13)
6.9
2.2
2.2
6.2
2.5*
4.7**
100.0
97.9 102.7 113.7 112.1 82.7
-Sitosterol-D-glucoside (14)
6.9
1.5
4.3
1.4*
1.5*
5.8*
Value represents mean S.E.M. (N=4). Significantly different from the control (0 M),
*p<0.05, **p<0.01

Table 3. Protective effect of isolated compounds from B. kingii rhizomes on 0.5 mM H2O2induced L929 fibroblast death
%Viability of L929 cells at various concentrations
Sample
(M)

Longiferone A (1)
Longiferone B (2)
Longiferone C (3)
Kaempferol-3,7,4-trimethyl ether (4)
Kaempferol-7,4-dimethyl ether (5)
Rhamnazin (6)
Pinostrobin (7)
Dihydrobisdemethoxycurcumin (8)

Control

H2O2

100.0
2.1
100.0
2.1
100.0
2.1
100.0
2.1
100.0
2.1
100.0
2.1
100.0
2.1
100.0
2.2

26.0
0.9
26.0
0.9
26.0
0.9
26.0
0.9
26.0
0.9
26.0
0.9
26.0
0.9
23.8
0.7

1
47.9
1.9**
38.9
1.1*
34.3
4.5*
47.9
1.3**
49.2
2.5**
42.8
4.3**
38.6
1.3*
39.9
2.4*

3
48.3
0.5**
48.1
0.5**
44.9
2.8**
61.0
1.4**
54.3
1.1**
54.8
0.9**
50.1
1.0**
46.7
2.6**

10
57.5
2.9**
52.7
1.6**
54.8
1.8**
71.6
0.5**
57.2
1.3**
59.4
1.5**
61.6
1.2**
62.3
3.1**

19
1-Hydroxydihydrobisdemethoxycurcumin (9)
Dihydrobisdemethoxycurcumin-4,4diacetate (10)

100.0
23.8
32.1
42.5
47.1
2.2
0.7
3.5*
0.6**
1.2**
100.0
23.8
33.3
36.7
46.6
2.2
0.7
1.2*
2.5*
1.0**
100.0
23.8
37.8
47.8
60.3
Curcumin (11)
2.2
0.7
2.0**
1.2**
2.1**
100.0
23.8
33.7
48.6
53.7
Demethoxycurcumin (12)
2.2
0.7
1.1*
1.7**
1.9**
100.0
23.8
31.7
44.6
56.7
Bisdemethoxycurcumin (13)
2.2
0.7
2.5*
4.2**
1.7**
100.0
23.8
33.3
36.3
46.6
-Sitosterol-D-glucoside (14)
2.2
0.7
1.5*
2.5*
1.0**
Value represents mean S.E.M. (N=4). Significantly different from the control (0 M), *p<0.05,
**p<0.01.

Table 4. Effect of isolated compounds from B. kingii rhizomes on in vitro scratch assay
using fibroblast L929

20
Value represents mean S.E.M. (N=4). Significantly different from the control (0 M), *p<0.01,
(-) = not determined.

Sample

Dose

Length between the scratch


(m)

(M) Day 0

Day 1

Day 2

%Migration rate
of cells
Day 1

Day 2

760.7 509.6 356.0


33.0
52.9
7.7
3.1*
7.5*
0.4
1.0
703.7 386.2 232.8
45.2
66.9
Longiferone A (1)
30
14.0
17.6
10.6
2.5*
1.5*
672.0 428.6 158.7
36.2
76.4
Longiferone B (2)
10
23.1
9.2
18.3
1.4
2.7*
682.5 444.4 105.8
37.9
70.7
Longiferone C (3)
10
27.5
17.3
10.6
2.7
3.4*
Kaempferol-3,7,4-trimethyl ether
688.3 408.5 184.9
40.7
72.7
10
(4)
1.0
6.2*
14.3*
0.9
2.1*
Kaempferol-7,4-dimethyl ether
612.1 423.0
312.0
30.9
49.1
30
(5)
16.1
11.3*
5.2*
1.8
0.8
555.9 399.3
209.9
28.1
61.6
Rhamnazin (6)
10
3.7
13.4*
0.5*
2.4
0.6*
658.3 333.1
233.7
49.4
61.5
Pinostrobin (7)
30
5.5
12.6*
19.7*
1.9*
3.0*
674.5 483.0 264.8
28.4
60.0
Dihydrobisdemethoxycurcumin (8) 10
8.9
9.2*
6.0*
1.4
3.9*
1-Hydroxy620.8 386.4 302.3
37.8
51.7
1
dihydrobisdemethoxycurcumin (9)
3.1
16.4*
4.8*
2.6
0.8
Dihydrobisdemethoxycurcumin688.0 404.3 286.0
41.1
57.1
3
4,4-diacetate (10)
5.2
14.7
11.4*
2.1
1.7
660.8 324.8
52.5
50.9
92.4
Curcumin (11)
3
5.5
10.2
3.2*
1.5*
0.5*
514.8 268.3 156.1
47.9
66.0
Demethoxycurcumin (12)
10
7.8
15.8
21.3*
3.1*
4.1*
615.8 324.8 104.9
47.3
82.7
Bisdemethoxycurcumin (13)
10
6.2
8.7
3.5*
1.4*
0.6*
620.8 441.0 194.9
28.9
69.7
10
-Sitosterol-D-glucoside (14)
9.0
10.2
15.1*
1.6
2.4*
663.3 358.1 247.3
46.0
61.1
Ascorbic acid
10
12.5
7.2
19.0*
1.1*
2.9*
Table 5. Collagen type-I production in L929 cells when treated with isolated compounds
of B.kingii rhizomes
Control

Sample

Longiferone A (1)

0
57.5
3.8
-

Longiferone B (2)

Control

Collagen production (g/ml) at various


concentrations (M)
0.3
1
3
10
<2.5
<2.5

<2.5
5.8
1.1**

3.3
2.2*
20.0
1.5*

29.6
3.2*
75.0
1.0*

21
18.3
7.5
<2.5
<2.5
2.0*
2.0*
56.7
66.7
69.6
86.7
Kaempferol-3,7,4-trimethyl ether (4)
3.5
3.5
1.4*
2.7*
50.0
48.8
47.1
51.7
Kaempferol-7,4-dimethyl ether (5)
4.7
3.0
3.2
2.0
70.4
89.2
105.8
120.8
Rhamnazin (6)
2.2*
1.1*
1.1*
2.5*
60.4
72.5
82.9
96.7
Pinostrobin (7)
2.2
1.1*
2.8*
3.0*
62.1
61.7
44.6
19.6
Dihydrobisdemethoxycurcumin (8)
5.0
3.9
4.0
2.1*
1-Hydroxy84.2
75.8
59.2
18.8
dihydrobisdemethoxycurcumin (9)
1.4*
1.1*
1.1
1.0*
Dihydrobisdemethoxycurcumin-4,488.8
72.9
62.9
33.8
diacetate (10)
0.4*
3.6*
2.2*
2.7*
65.0
68.8
41.7
13.8
Curcumin (11)
5.7
4.2
3.6*
3.2*
69.6
74.6
64.6
24.6
Demethoxycurcumin (12)
2.8*
3.3*
0.8
4.3*
72.9
60.8
36.3
13.8
Bisdemethoxycurcumin (13)
0.8*
4.2
3.6*
3.4*
53.3
76.7
115.8
121.3
-Sitosterol-D-glucoside (14)
1.8
1.2*
1.4*
4.3*
51.7
54.6
60.4
85.0
Ascorbic acid
1.8
2.8
4.6
1.8*
Value represents mean S.E.M. (N=4). Significantly different from the control (0 M), *p<0.01,
(-) = not determined.
Longiferone C (3)

OH

OH
HO

OH

Dihydrobisdemethoxycurcumin (8)

OH

OH

HO

OH

1-Hydroxy-dihydrobisdemethoxycurcumin (9)
H

Longiferone A (1)
Longiferone C (3)

OH

Longiferone B (2)
AcO

OAc

Dihydrobisdemethoxycurcumin-4, 4-diacetate
(10)
OCH3
O

H3CO

OH

H3CO

OCH3

OCH3
OH

HO

OH

Curcumin (11)

22
OCH3
H3CO

OH
OH

OH

H3CO

HO

OH

Demethoxycurcumin (12)

Kaempferol-3, 7, 4-trimethyl ether (4)


Kaempferol-7, 4-dimethyl ether (5)

OH

OCH3
OH
H3CO

CH3

H 3C
CH3

OH
OH

CH3
H

H3CO

CH3
H

CH3

H
H

O
Glc

OH

Rhamnazin (6)

Pinostrobin (7)

-Sitosterol-D-glucoside (14)

Figure 1. Chemical structures of 1-14 isolated from


the rhizomes of B. kingii

HO

OH

Bisdemethoxycurcumin (13)

23
2
Control

14

Ascorbic
acid

Day 0

Day 1

Day 2

Figure 2. Effect of selected compounds from B. kingii rhizomes on fibroblast L929 migration. Images
were captured at day 0 and then treated with 2 (30 M), 7 (10 M), 8 (30 M), 14 (10 M), ascorbic acid
(10 M) and control without treatment. Another set of images were captured at day 1 and 2 after
incubation. Quantitative analysis of the migration rate was quantified by computing software.


Boesenbergia kingii
rhizomes

In vitro wound healing assays

Phytochemical study

OH

Fibroblast

HO

OH

Day 0

Dihydrobisdemethoxycurcumin
OCH3

H3CO

Day 1

OH

OH
OH

Longiferone B

Rhamnazin

Day 2

Control

Longiferone B