PII:
DOI:
Reference:
S0378-8741(16)30097-6
http://dx.doi.org/10.1016/j.jep.2016.03.001
JEP10008
1
Wound healing property of isolated compounds from Boesenbergia kingii rhizomes
*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.
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).
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).
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.
fibroblast proliferation and collagen production were employed for the scientific support.
ether
(5),
(8),
rhamnazin
(6),
pinostrobin
(7),
1-hydroxy-dihydrobisdemethoxycurcumin
(9),
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):
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).
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
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).
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).
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.
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
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.
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.
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
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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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
(M) Day 0
Day 1
Day 2
%Migration rate
of cells
Day 1
Day 2
Sample
Longiferone A (1)
0
57.5
3.8
-
Longiferone B (2)
Control
<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)
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)
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
Phytochemical study
OH
Fibroblast
HO
OH
Day 0
Dihydrobisdemethoxycurcumin
OCH3
H3CO
Day 1
OH
OH
OH
Longiferone B
Rhamnazin
Day 2
Control
Longiferone B