Author’s Accepted Manuscript

Wound healing property of isolated compoundsfrom Boesenbergia kingii rhizomes

Teeratad Sudsai, Chatchai Wattanapiromsakul,Supinya Tewtrakul

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

To appear in: Journal of Ethnopharmacology

Received date: 13 January 2016Accepted date: 1 March 2016

Cite this article as: Teeratad Sudsai, Chatchai Wattanapiromsakul and SupinyaTewtrakul, Wound healing property of isolated compounds from Boesenbergiakingii rhizomes, Journal of Ethnopharmacology,http://dx.doi.org/10.1016/j.jep.2016.03.001

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Wound healing property of isolated compounds from Boesenbergia kingii rhizomes

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

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

Prince of Songkla University, Songkhla 90112, Thailand

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

Science, Prince of Songkla University, Songkhla 90112, Thailand

cCollege 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:

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Wound healing activities were tested using in vitro assays including cell proliferation and

migration assays, collagen production and H2O2-induced oxidative stress in mouse fibroblast

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 µM)

and also active against 0.5 mM H2O2-induced oxidative stress by increasing cell survival ability

up to 60.3 % at 10 µM. In addition, compounds 3, 8 and 14 at 10 µM 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 µM, whereas 7 and 14 strongly

stimulated collagen production at 75.0 and 96.7 µg/ml compared to the control group (57.5

µg/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

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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 anti-

inflammation 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

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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 ether (5), rhamnazin (6), pinostrobin (7),

dihydrobisdemethoxycurcumin (8), 1-hydroxy-dihydrobisdemethoxycurcumin (9),

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

bisdemethoxycurcumin (13) and β-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

µM with absolute ethanol. The reaction mixture contained 100 µl of samples at various

concentrations and 100 µl of 6×10-5 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

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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 96-

well plate. After 24 h, cells were exposed to different concentrations (1-100 µM) of test samples

and were then incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2. MTT

solution (10 µl, 5 mg/ml) was added directly to the medium in each well, and the plate was then

incubated at 37°C 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, fibroblast 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 µM) of test samples.

After pretreatment with different concentrations for 1 h incubation at 37°C 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.

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2.5 Migration assay

The migration of fibroblast L929 was examined using a wound healing method. Briefly,

L929 cells (5×104 cells/ml) in DMEM containing 10% FBS were seeded into each well of 24

wells plate and incubated at 37°C 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 37°C with 5% CO2 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 2×104 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 µM)

and was then incubated for 48 h at 37°C 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 µl) were collected. The total amount of soluble collagen type I was

assayed using the Sircol® Collagen Assay Kit (Bicolor Life Science Assays, Northern Ireland,

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UK). Briefly, 100 µl 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 one-

way analysis of variance (ANOVA), followed by Dunnett’s 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 µM, followed

by bisdemethoxycurcumin (13, IC50 = 29.9 µM), rhamnazin (6, IC50 = 31.1 µM), kaempferol-7,

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4´-dimethyl ether (5, IC50 = 39.0 µM), demethoxycurcumin (12, IC50 = 53.2 µM), and

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

13 exhibited the scavenging activity higher than BHT (IC50 = 104.0 µM) but less than quercetin

(IC50 = 9.5 µM). 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-7,4´-

dimethyl 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) > 1-

hydroxy-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

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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

µM), 8 (1-10 µM) and 14 (3-30 µM) 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

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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 H2O2 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 µM) 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 µM) by DPPH assay significantly

inhibited H2O2-induced oxidative stress (52.7-57.5% at 10 µM) 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, α-tocopherol,

glutathione and other dietary antioxidants, which scavenge radicals or neutralize ROS, thus

maintaining redox balance. Catalase and glutathione peroxidase are involved in the

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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 µM), 3 (10 µM), 4 (10 µM), 7 (30 µM), 11

(3 µM) and 13 (10 µM) 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).

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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 µg/ml. The presence of various concentrations (0.3-10 µM) 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 µM exhibited

higher effect (86.7-121.3 µg/ml) than the positive control, ascorbic acid (85.0 µg/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

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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.

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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

Sample %Inhibition against DPPH radical at various

concentrations (µM)

IC50

(µM) 1 3 10 30 100

Longiferone A (1) - -4.2 ±

0.7

4.4 ±

1.0

8.5 ±

0.9*

15.0 ±

2.4* >100

Longiferone B (2) - - -3.1 ±

1.0

5.6 ±

0.5

12.9 ±

2.6* >100

Longiferone C (3) - - -1.5 ±

3.3

1.5 ±

1.2

4.4 ±

0.6 >100

Kaempferol-3,7,4´-trimethyl ether

(4) - - -4.5 ±

0.6

4.5 ±

3.0

12.0 ±

6.4* >100

Kaempferol-7,4´-dimethyl ether (5) - - -1.2 ±

1.5

31.1 ±

2.7**

88.4 ±

0.6**

39.0 Rhamnazin (6) -7.2 ±

1.2

0.3 ±

0.8

16.7 ±

2.9*

35.1 ±

2.6**

83.5 ±

1.1**

35.1 Pinostrobin (7) - - - - -2.9 ±

0.5 >100

Dihydrobisdemethoxycurcumin (8) - -0.4 ±

2.0

6.2 ±

1.1

12.4 ±

2.4*

52.9 ±

3.6**

95.6 1-Hydroxy-

dihydrobisdemethoxycurcumin (9) - - -6.0 ±

1.8

6.4 ±

1.7

12.0 ±

2.3* >100

Dihydrobisdemethoxycurcumin-

4´,4´´-diacetate (10) - -4.8 ±

1.6

6.7 ±

1.4

10.2 ±

1.3*

26.3 ±

0.9** >100

Curcumin (11) -1.6 ±

1.1

7.1 ±

1.2

23.2 ±

2.1**

59.2 ±

1.9**

86.5 ±

1.4**

21.0 Demethoxycurcumin (12) 2.3 ±

1.4

7.6 ±

1.6

17.4 ±

2.6**

31.4 ±

1.6**

71.6 ±

1.4**

53.2 Bisdemethoxycurcumin (13) -0.7 ±

0.6

2.5 ±

1.0

10.6 ±

3.2*

46.9 ±

0.7**

85.5 ±

1.5**

29.9 -Sitosterol-D-glucoside (14) - - - - -1.1 ±

2.6 >100

Quercetin -1.1 ±

1.5

28.7 ±

4.2**

40.1 ±

4.8**

93.8 ±

1.6**

92.7 ±

1.0**

9.5 BHT - -8.5 ±

2.7

0.5 ±

2.1

22.5 ±

0.6**

54.8 ±

1.8**

104.0

Value represents mean ± S.E.M. (N=4). Significantly different from the control (0 µM), *p<0.05,

**p<0.01. (-) = not determined

Table 2. Effect of isolated compounds from B. kingii rhizomes on L929 cells viability

Sample %Viability of L929 cells at various

concentrations (µM)

18

0 1 3 10 30 100

Longiferone A (1) 100.0

± 2.8

102.7 ±

4.5

103.4 ±

4.7

107.1 ±

5.3

110.0 ±

6.9*

84.9 ±

3.3 Longiferone B (2) 100.0

± 2.8

101.6 ±

2.2

98.3 ±

5.0

100.1 ±

4.0

91.9 ±

2.3

84.8 ±

3.8 Longiferone C (3) 100.0

± 2.8

111.5 ±

6.8*

97.2 ±

3.2

119.2 ±

7.4**

101.9 ±

1.9

89.1 ±

1.2 Kaempferol-3,7,4´-trimethyl ether

(4)

100.0

± 6.9

85.8 ±

0.7

76.6 ±

2.0**

93.6 ±

3.1

81.4 ±

1.2*

77.4 ±

1.6** Kaempferol-7,4´-dimethyl ether

(5)

100.0

± 6.9

79.5 ±

2.8*

87.5 ±

1.8

90.5 ±

3.6

100.1 ±

5.3

72.1 ±

3.3** Rhamnazin (6) 100.0

± 1.8

93.9 ±

5.9

99.9 ±

4.5

101.5 ±

5.4

92.9 ±

3.6

76.6 ±

4.3** Pinostrobin (7) 100.0

± 6.9

86.4 ±

1.2

89.1 ±

1.8

97.9 ±

2.8

105.5 ±

1.6

83.5 ±

0.5 Dihydrobisdemethoxycurcumin

(8)

100.0

± 1.8

109.5 ±

1.2*

117.8 ±

4.6**

122.7 ±

4.9**

92.4 ±

4.0

84.4 ±

1.9 1-Hydroxy-

dihydrobisdemethoxycurcumin (9)

100.0

± 1.8

102.1 ±

4.3

94.1 ±

2.6

97.2 ±

2.0

74.8 ±

2.4**

68.6 ±

0.7** Dihydrobisdemethoxycurcumin-

4´,4´´-diacetate (10)

100.0

± 1.8

94.5 ±

4.2

103.1 ±

1.7

96.3 ±

5.2

91.2 ±

5.2

83.3 ±

5.4 Curcumin (11) 100.0

± 6.9

83.9 ±

2.0*

91.2 ±

4.0

83.0 ±

5.2*

73.9 ±

2.2**

39.6 ±

5.7** Demethoxycurcumin (12) 100.0

± 6.9

81.2 ±

1.0*

75.2 ±

3.0**

89.2 ±

4.8

85.3 ±

5.4

74.6 ±

6.2** Bisdemethoxycurcumin (13) 100.0

± 6.9

85.4 ±

2.2

83.7 ±

2.2

92.9 ±

6.2

79.4 ±

2.5*

51.6 ±

4.7** -Sitosterol-D-glucoside (14) 100.0

± 6.9

97.9 ±

1.5

102.7 ±

4.3

113.7 ±

1.4*

112.1 ±

1.5*

82.7 ±

5.8* Value represents mean ± S.E.M. (N=4). Significantly different from the control (0 µM),

*p<0.05, **p<0.01

Table 3. Protective effect of isolated compounds from B. kingii rhizomes on 0.5 mM H2O2-

induced L929 fibroblast death

Sample %Viability of L929 cells at various concentrations

(µM)

Control H2O2 1 3 10

Longiferone A (1) 100.0 ±

2.1

26.0 ±

0.9

47.9 ±

1.9**

48.3 ±

0.5**

57.5 ±

2.9**

Longiferone B (2) 100.0 ±

2.1

26.0 ±

0.9

38.9 ±

1.1*

48.1 ±

0.5**

52.7 ±

1.6**

Longiferone C (3) 100.0 ±

2.1

26.0 ±

0.9

34.3 ±

4.5*

44.9 ±

2.8**

54.8 ±

1.8**

Kaempferol-3,7,4´-trimethyl ether (4) 100.0 ±

2.1

26.0 ±

0.9

47.9 ±

1.3**

61.0 ±

1.4**

71.6 ±

0.5**

Kaempferol-7,4´-dimethyl ether (5) 100.0 ±

2.1

26.0 ±

0.9

49.2 ±

2.5**

54.3 ±

1.1**

57.2 ±

1.3**

Rhamnazin (6) 100.0 ±

2.1

26.0 ±

0.9

42.8 ±

4.3**

54.8 ±

0.9**

59.4 ±

1.5**

Pinostrobin (7) 100.0 ±

2.1

26.0 ±

0.9

38.6 ±

1.3*

50.1 ±

1.0**

61.6 ±

1.2**

Dihydrobisdemethoxycurcumin (8) 100.0 ±

2.2

23.8 ±

0.7

39.9 ±

2.4*

46.7 ±

2.6**

62.3 ±

3.1**

19

1-Hydroxy-

dihydrobisdemethoxycurcumin (9)

100.0 ±

2.2

23.8 ±

0.7

32.1 ±

3.5*

42.5 ±

0.6**

47.1 ±

1.2**

Dihydrobisdemethoxycurcumin-4´,4´´-

diacetate (10)

100.0 ±

2.2

23.8 ±

0.7

33.3 ±

1.2*

36.7 ±

2.5*

46.6 ±

1.0**

Curcumin (11) 100.0 ±

2.2

23.8 ±

0.7

37.8 ±

2.0**

47.8 ±

1.2**

60.3 ±

2.1**

Demethoxycurcumin (12) 100.0 ±

2.2

23.8 ±

0.7

33.7 ±

1.1*

48.6 ±

1.7**

53.7 ±

1.9**

Bisdemethoxycurcumin (13) 100.0 ±

2.2

23.8 ±

0.7

31.7 ±

2.5*

44.6 ±

4.2**

56.7 ±

1.7**

-Sitosterol-D-glucoside (14) 100.0 ±

2.2

23.8 ±

0.7

33.3 ±

1.5*

36.3 ±

2.5*

46.6 ±

1.0**

Value represents mean ± S.E.M. (N=4). Significantly different from the control (0 µM), *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 µM), *p<0.01,

(-) = not determined.

Table 5. Collagen type-I production in L929 cells when treated with isolated compounds

of B.kingii rhizomes

Sample Collagen production (µg/ml) at various

concentrations (µM) 0 0.3 1 3 10

Control 57.5 ±

3.8 - - - -

Longiferone A (1) - <2.5 <2.5 3.3 ±

2.2*

29.6 ±

3.2* Longiferone B (2) - <2.5 5.8 ±

1.1**

20.0 ±

1.5*

75.0 ±

1.0*

Sample Dose Length between the scratch

(µm)

%Migration rate

of cells

(µM) Day 0 Day 1 Day 2 Day 1 Day 2

Control - 760.7 ±

7.7

509.6 ±

3.1*

356.0 ±

7.5*

33.0 ±

0.4

52.9 ±

1.0

Longiferone A (1) 30 703.7 ±

14.0

386.2 ±

17.6

232.8 ±

10.6

45.2 ±

2.5*

66.9 ±

1.5*

Longiferone B (2) 10 672.0 ±

23.1

428.6 ±

9.2

158.7 ±

18.3

36.2 ±

1.4

76.4 ±

2.7*

Longiferone C (3) 10 682.5 ±

27.5

444.4 ±

17.3

105.8 ±

10.6

37.9 ±

2.7

70.7 ±

3.4*

Kaempferol-3,7,4´-trimethyl ether

(4) 10

688.3 ±

1.0

408.5 ±

6.2*

184.9 ±

14.3*

40.7 ±

0.9

72.7 ±

2.1*

Kaempferol-7,4´-dimethyl ether

(5) 30

612.1 ±

16.1

423.0 ±

11.3*

312.0 ±

5.2*

30.9 ±

1.8

49.1 ±

0.8

Rhamnazin (6) 10 555.9 ±

3.7

399.3 ±

13.4*

209.9 ±

0.5*

28.1 ±

2.4

61.6 ±

0.6*

Pinostrobin (7) 30 658.3 ±

5.5

333.1 ±

12.6*

233.7 ±

19.7*

49.4 ±

1.9*

61.5 ±

3.0*

Dihydrobisdemethoxycurcumin (8) 10 674.5 ±

8.9

483.0 ±

9.2*

264.8 ±

6.0*

28.4 ±

1.4

60.0 ±

3.9*

1-Hydroxy-

dihydrobisdemethoxycurcumin (9) 1

620.8 ±

3.1

386.4 ±

16.4*

302.3 ±

4.8*

37.8 ±

2.6

51.7 ±

0.8

Dihydrobisdemethoxycurcumin-

4´,4´´-diacetate (10) 3

688.0 ±

5.2

404.3 ±

14.7

286.0 ±

11.4*

41.1 ±

2.1

57.1 ±

1.7

Curcumin (11) 3 660.8 ±

5.5

324.8 ±

10.2

52.5 ±

3.2*

50.9 ±

1.5*

92.4 ±

0.5*

Demethoxycurcumin (12) 10 514.8 ±

7.8

268.3 ±

15.8

156.1 ±

21.3*

47.9 ±

3.1*

66.0 ±

4.1*

Bisdemethoxycurcumin (13) 10 615.8 ±

6.2

324.8 ±

8.7

104.9 ±

3.5*

47.3 ±

1.4*

82.7 ±

0.6*

-Sitosterol-D-glucoside (14) 10 620.8 ±

9.0

441.0 ±

10.2

194.9 ±

15.1*

28.9 ±

1.6

69.7 ±

2.4*

Ascorbic acid 10 663.3 ±

12.5

358.1 ±

7.2

247.3 ±

19.0*

46.0 ±

1.1*

61.1 ±

2.9*

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

21

Longiferone C (3) - <2.5 <2.5 18.3 ±

2.0*

7.5 ±

2.0* Kaempferol-3,7,4´-trimethyl ether (4) - 56.7 ±

3.5

66.7 ±

3.5

69.6 ±

1.4*

86.7 ±

2.7* Kaempferol-7,4´-dimethyl ether (5) - 50.0 ±

4.7

48.8 ±

3.0

47.1 ±

3.2

51.7 ±

2.0 Rhamnazin (6) - 70.4 ±

2.2*

89.2 ±

1.1*

105.8 ±

1.1*

120.8 ±

2.5* Pinostrobin (7) - 60.4 ±

2.2

72.5 ±

1.1*

82.9 ±

2.8*

96.7 ±

3.0* Dihydrobisdemethoxycurcumin (8) - 62.1 ±

5.0

61.7 ±

3.9

44.6 ±

4.0

19.6 ±

2.1* 1-Hydroxy-

dihydrobisdemethoxycurcumin (9) - 84.2 ±

1.4*

75.8 ±

1.1*

59.2 ±

1.1

18.8 ±

1.0* Dihydrobisdemethoxycurcumin-4´,4´´-

diacetate (10) - 88.8 ±

0.4*

72.9 ±

3.6*

62.9 ±

2.2*

33.8 ±

2.7* Curcumin (11) - 65.0 ±

5.7

68.8 ±

4.2

41.7 ±

3.6*

13.8 ±

3.2* Demethoxycurcumin (12) - 69.6 ±

2.8*

74.6 ±

3.3*

64.6 ±

0.8

24.6 ±

4.3* Bisdemethoxycurcumin (13) - 72.9 ±

0.8*

60.8 ±

4.2

36.3 ±

3.6*

13.8 ±

3.4* -Sitosterol-D-glucoside (14) - 53.3 ±

1.8

76.7 ±

1.2*

115.8 ±

1.4*

121.3 ±

4.3* Ascorbic acid - 51.7 ±

1.8

54.6 ±

2.8

60.4 ±

4.6

85.0 ±

1.8* Value represents mean ± S.E.M. (N=4). Significantly different from the control (0 µM), *p<0.01,

(-) = not determined.

HO

OH

HO

HO

O

Longiferone A (1) Longiferone B (2)

Longiferone C (3)

O

OOH

H3CO

OCH3

OCH3

O OH

OHHO Dihydrobisdemethoxycurcumin (8)

O OH

OHHO

OH

1-Hydroxy-dihydrobisdemethoxycurcumin (9)

O OH

OAcAcO Dihydrobisdemethoxycurcumin-4´, 4´´-diacetate

(10)

O OH

OHHO

OCH3H3CO

Curcumin (11)

22

O

OOH

H3CO

OH

OCH3

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

Kaempferol-7, 4´-dimethyl ether (5)

O

OOH

H3CO

OH

OH

OCH3

O

OOH

H3CO

Rhamnazin (6) Pinostrobin (7)

-Sitosterol-D-glucoside (14)

O OH

OHHO

H3CO

Demethoxycurcumin (12)

O OH

OHHO Bisdemethoxycurcumin (13)

Figure 1. Chemical structures of 1-14 isolated from

the rhizomes of B. kingii

CH3

CH3

CH3

H3C

CH3

CH3

O

Glc

H H

H

H

23

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 µM), 7 (10 µM), 8 (30 µM), 14 (10 µM), ascorbic acid

(10 µM) 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.

Control

2 7 8 14 Ascorbic

acid

Day 0

Day 1

Day 2

 

 

                                                                                        

 

 

 

 

 

O OH

OHHO  

O

OOH

H3CO

OH

OH

OCH3

 

Boesenbergia kingii rhizomes

Phytochemical study In vitro wound healing assays

Fibroblast Control Longiferone B

Day 0

Day 1

Day 2  

Dihydrobisdemethoxycurcumin

Longiferone B Rhamnazin

H O