202 | IAMPS 35 & CU-MPU International Collaborative Research Conference

Available online at http://www.pharm.chula.ac.th/am2019/

The 35th International Annual Meeting in Pharmaceutical Sciences

& CU-MPU International Collaborative Research Conference

Wound healing effects of compounds from Kaempferia marginata

Sirikorn Thanasakdecha, Supinya Tewtrakul

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla Univesity,Hat-yai, Songkhla, Thailand

* Corresponding Author: E-mail address: sirikorn.noon14@gmail.com

Keywords: Kaempferia marginata, wound healing, Zingiberaceae Introduction

Wound healing is the reparation process that has interaction of cells and biochemical substances. The processes include inflammation, proliferation, and tissue remodeling. Aims of wound healing treatment are shortest time of repair, minimal pain and regenerate the tissue. The use of wound healing plants for treatment of gastric ulcers, burns and skin diseases are popular in Thai people. Several Thai herbal plants have been reported the potential on wound healing activity and trend to develop as an alternative treatment for wound healing.

Genus Kaempferia (Zingiberaceae) is a Thai medicinal plant. Seventeen species of Kaempferia were recorded in Thailand. Especially, K. marginata has been used in northeastern Thailand. It is mostly distributed in India, Myanmar, China, Thailand, Laos and Cambodia.1 K. marginata is locally known in Thai as Proh-paa or Toobmoob (I-San local name). It has two large flat leaves that spread on the soil surface. The root and leaves are used as a flavoring for curries and used in the treatment of allergy, fever and swollen leg. It has been reported on anti-inflammation.2

Chemical constituents of K. marginata include cinnamate derivatives, cyclohexane oxide derivatives, chalcone derivatives, monoterpenes, diterpenes and flavonoids.

Eleven compounds have been isolated using silica gel column chromatography and identified by

spectroscopic data including three pimarane- type diterpenes, 1-acetoxysandaracopimaradien-2-one

(2), 1-acetoxysandaracopi-mara-dien-2-ol (3) and 1-acetoxysandaracopimaradien (7); four known

diterpenes, 2-acetoxysandaracopimaradien-1-ol (1), sandaracopimaradien-1α,2α-diol (4), (2R)-ent-2-hydroxyisopimara-8(14),15-diene (6), isopimara-8(14),15-dien-7-one (10); two known pimarane type diterpenes, marginatol (8) and sandaracopimaradiene (9); one kavalactone, desmethoxyyangonin (5); and one diarylheptanoid, bisdemethoxycurcumin (11).3 Structures of compounds 1-11 isolated from K. marginata rhizomes are shown in Figure 1. Methods Plant material and isolation of compounds:

The rhizomes of K. marginata were bought from the Jatujuk market in Bangkok in May 2010 and identified by Mrs. Pranee Rattanasuwan (SKP 206111301). Three kg dried weight of K. marginata were ground and macerated with ethanol at room temperature, 4 times (6 L). The EtOH extract was kept in airtight container under 4°C until use. Eleven compounds were isolated from the rhizomes of K. marginata.

Wound healing assay: 1) Cell proliferation and viability using Human Dermal Fibroblasts or HDF cells were seeded at

1x104 cells/well into 96 well plate in DMEM containing 10% FBS. After 24 h, cells were exposed to different concentrations (0.3-30 µM) of test samples and were then incubated for 48 h at 37°C in humidified atmosphere containing 5% CO2. MTT solution (10 µL, 5 mg/mL) was added directly to the medium in each well, and the plate is 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 compare to negative control.4

203 | IAMPS 35 & CU-MPU International Collaborative Research Conference

2) The migration of HDF cells was examined using a wound healing method. Briefly, HDF cells (5x104 cells/mL) in DMEM containing 10% FBS were seeded into each well of 24 well plates and incubated at 37°C with 5% CO2. After the confluent monolayer of HDF cells was formed, scratched with a sterile pipette tip 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 sample. Photographs on day 0, then plates were incubated at 37°C with 5% CO2 and photographs were taken at day 1,2 and 3. To determine the migration of HDF cells, the images were analyzed using image J computing software. Percentage of the closed area was measured and compared with the value obtained before treatment (day 0).

Figure 1 Structures of compounds 1-11 isolated from Kaempferia marginata rhizomes. Results and discussion

The enhancement effect on HDF cells proliferation was achieved by MTT method. The increasing cell viability of compounds from K. marginata at concentrations (0.3, 1, 3, 10 and 30 µM) are presented in Table 1. KM1 and KM3-8 exhibited potent proliferative effect (102.10-130.97%) at concentrations 10 and 30 µM. While KM2 acted as proliferative inducer (100.24-113.14%) at low concentrations (0.3 and

1 M). Furthermore, the percent cell movements of KM1 and KM5-8 showed the highest migration of

HDF cells at 3 days (85.01-100%) at concentrations 1 and 3 M as shown in Table 2 and Figure 2. Table 1 Effect of K. marginata compounds on HDF cells viability

sample % Viability of HDF cells at various concentrations (Mean ± S.E.M) (µM)

0 0.3 1 3 10 30 KM 1 100.00 ± 6.63 101.24 ± 0.36 98.69 ± 1.15 96.25 ± 1.26 110.25 ± 0.40 115.55 ± 0.68

KM 2 100.00 ± 6.63 113.14 ± 3.06 100.24 ± 1.98 82.51 ± 1.52 75.99 ± 1.39 72.43 ± 0.31

KM 3 100.00 ± 6.63 106.97 ± 1.40 99.14 ± 0.40 98.13 ± 0.48 108.52 ± 1.67 107.04 ± 1.09

KM 4 100.00 ± 6.63 117.25 ± 0.82 103.77 ± 0.77 96.08 ± 2.66 102.10 ± 0.52 116.09 ± 2.39

KM 5 100.00 ± 1.33 117.77 ± 0.81 113.86 ± 0.42 108.40 ± 0.52 121.16 ± 1.52 118.96 ± 1.02

KM 6 100.00 ± 1.33 106.46 ± 0.28 112.73 ± 0.90 117.05 ± 2.83 119.21 ± 3.01 130.97 ± 1.12

KM 7 100.00 ± 1.33 105.47 ± 1.03 111.33 ± 0.74 110.06 ± 0.54 130.65 ± 3.10 129.71 ± 1.29

KM 8 100.00 ± 1.33 109.59 ± 1.22 119.12 ± 0.22 117.06 ± 0.69 126.77 ± 1.97 128.52 ± 0.78

KM 9 100.00 ± 2.82 94.61 ± 1.40 88.21 ± 0.41 97.94 ± 0.08 83.56 ± 0.78 93.77 ± 0.41

KM 10 100.00 ± 2.82 90.71 ± 0.66 104.55 ± 0.90 103.40 ± 0.23 108.41 ± 1.12 104.54 ± 0.51

KM 11 100.00 ± 2.82 100.97 ± 2.86 102.74 ± 2.24 104.40 ± 1.37 98.91 ± 1.18 114.98 ± 0.57

Aloe gel (µg/ml)

100.00 ± 0.76 98.38 ± 1.63 105.88 ± 4.72 107.00 ± 2.67 104.76 ± 4.21 107.03 ± 4.12

204 | IAMPS 35 & CU-MPU International Collaborative Research Conference

Table 2 Effect of K. marginata compounds on HDF cells migration assay

Sample Dose (µM) %Migration rate of cells

Day 1 Day 2 Day 3

Control 17.44±0.79 45.24±4.38 58.79±2.36

KM1 1 42.14±0.57 73.17±0.35 87.59±0.08

3 23.22±0.43 53.40±2.39 85.01±0.31

KM2 0.3 45.31±2.46 71.17±0.66 100.00±0.00

1 47.43±0.86 65.55±3.23 88.48±1.17

KM3 10 45.49±1.46 77.93±1.90 100.00±0.00

30 51.30±1.85 66.14±1.55 100.00±0.00

KM4 0.3 24.90±1.01 57.06±0.86 79.39±0.70

1 42.83±1.32 49.64±3.42 100.00±0.00

KM5 1 50.39±0.75 78.96±0.45 91.60±0.09

3 49.14±1.66 85.85±0.34 100.00±0.00

KM6 1 18.06±3.96 68.32±2.29 100.00±0.00

3 52.44±3.15 81.51±0.35 100.00±0.00

KM7 1 30.34±1.04 68.75±2.17 100.00±0.00

3 40.62±1.56 70.16±2.87 100.00±0.00

KM8 1 33.52±4.29 72.18±2.73 100.00±0.00

3 41.91±2.70 69.04±3.38 100.00±0.00

KM9 0.3 21.98±0.70 57.71±0.94 81.85±1.12

1 37.91±0.33 60.24±0.58 85.73±0.74

KM10 1 37.06±0.42 51.76±0.76 77.72±1.21

3 46.56±0.28 55.48±0.34 78.72±0.51

KM11 1 30.94±0.71 49.22±2.71 69.16±1.45

3 36.30±0.54 54.12±0.71 80.04±2.12

Aloe vera gel 0.3 26.55±1.90 29.48±0.56 54.94±2.74

(µg/ml) 3 29.48±1.08 48.38±0.45 70.48±0.89

Figure 2 Effect of K. marginata compounds on HDF cells migration assay Conclusion In the present study, proliferation activity of KM1 and KM3-8 at concentrations 10 and 30 µM activated cell proliferation more than other concentrations and the positive control group. For the percentage movement of the cells showed the highest apparent wound closure of KM1 and KM 5 at

day 3 (concentrations 1 and 3 M). These findings may support the pharmacological basis of K. marginata as a Thai herbal medicine for the treatment of wound. Thus, compounds of K. marginata may serve as potential resources for further development of wound healing products.

Control KM1 KM5 Aloe vera gel

1 µM 3 µM 1 µM 3 µM 0.3 µg/ml

Day 0

Day 1

Day 2

Day 3

205 | IAMPS 35 & CU-MPU International Collaborative Research Conference

Acknowledgements The authors are appreciative to the Faculty of Pharmaceutical Sciences, Prince of Songkla

Univesity and PERCH-CIC for furnish the equipment and financial support for the fulfilment of this research work. References 1. Saensouk P, Muangsan N, Saensouk S, Sirinajun P (2016). In vitro propagation of Kaempferia marginata Carey

ex Roscoe, a native plant species to Thailand. The Journal of Animal and Plant Sciences, 26(5): 1405-1410. 2. Kaewkroek K, Wattanapiromsakul C, Matsuda H, Nakamura S, Tewtrakul S (2017). Anti-inflammatory activity

of compounds from Kaempferia marginata rhizomes. Songklanakarin Journal of Science and Technology, 39(1): 91-99.

3. Kaewkroek K, Wattanapiromsakul C, Kongsaeree P, Tewtrakul S (2013). Nitric oxide and tumer necrosis factor-alpha inhibitory substances from the rhizomes of Kaempferia marginata. Natural Product Communications,

8(9): 1205-1208. 4. Sudsai T, Wattanapiromsakul C, Nakpheng T, Tewtrakul S (2013). Evaluation of the wound healing property of

Boesenbergia longiflora rhizomes. Journal of Ethnopharmacology, 150:223-231.