The In Vitro and In Vivo Wound Healing Properties of the Chinese Herbal Medicine "Jinchuang Ointment"

Abstract

"Jinchuang ointment" is a traditional Chinese herbal medicine complex for treatment of incised wounds. For more than ten years, it has been used at China Medical University Hospital (Taichung, Taiwan) for the treatment of diabetic foot infections and decubitus ulcers. Three different cases are presented in this study. "Jinchuang" ointment is a mixture of natural product complexes from nine different components, making it difficult to analyze its exact chemical compositions. To further characterize the herbal ingredients used in this study, the contents of reference standards present in a subset of the ointment ingredients (dragon's blood, catechu, frankincense, and myrrh) were determined by HPLC. Two in vitro cell based assay platforms, wound healing and tube formation, were used to examine the biological activity of this medicine. Our results show that this herbal medicine possesses strong activities including stimulation of angiogenesis, cell proliferation, and cell migration, which provide the scientific basis for its clinically observed curative effects on nonhealing diabetic wounds.

Figure 1

The wound area of the patient Mrs. Wu during “Jinchuang ointment” treatment. Wound dimensions measured on the date specified are as follows: May 25, 2013, 26 × 9 cm; Jun 5, 2013, 26 × 9 cm; Sep 4, 2013, 8 × 4 cm; Aug 27, 2014, 2.5 × 3 cm.

Figure 2

The pressure sore of the patient Mr. Tsai during “Jinchuang ointment” treatment. This wound was located at the sacral region and was completely closed after a 50-day treatment.

Figure 3

The previously nonhealing wound of patient Mr. Wang during “Jinchuang ointment” treatment. Wound dimensions were measured on the following dates: Nov 26, 2014, 6.2 × 5.3 × 0.3 cm; Dec 27, 2014, 4.5 × 4 × 0.1 cm; Jan 21, 2015, 2.5 × 1.5 × 0.1 cm; Feb 21, 2015, wound complete closure.

Figure 4

HPLC separation of reference compounds present in extracts of herbal components. HPLC traces of (a) dracorhodin in “dragon's blood,” (b) catechin and epicatechin in catechu, (c) acetyl-11-keto-β-boswellic acid in frankincense, and (d) (E)-guggulsterone in myrrh.

Figure 5

Chiral GC separations of synthetic borneol. The four major peaks from 22.0 to 26.0 min are (+)-isoborneol, (−)-isoborneol, (−)-borneol, and (+)-borneol, respectively.

Figure 6

Wound healing assay with HaCaT cells displaying the increased cell migration induced by “Jinchuang ointment.” Cells were treated with (a) DMSO alone (control), (b) 100 ng/mL EGF (positive control), (c) 200 μg/mL “Jinchuang ointment,” (d) 20 μg/mL “Jinchuang ointment,” and (e) 2 μg/mL “Jinchuang ointment.” Cell migration was documented by phase contrast microscopy over a 24-hour time course where time 0 is the time of wound scratching.

Figure 7

Wound healing assay with HMEC-1 cells displaying the increased cell migration induced by “Jinchuang ointment.” Cells were treated with (a) DMSO alone (control) or (b) 200 μg/mL “Jinchuang ointment.” Cell migration was recorded and cells were stained by microscopy over a 24-hour time course.

Figure 8

The percentage of wound closure with HMEC-1 cells after six hours of treatment in response to reconstituted variants “Jinchuang ointments.” Lard is replaced by sesame oil (group B), synthetic triacylglycerol (group C), coconut oil (group D), and Vaseline (group E). Group A is the original recipe of Jinchuang ointment, and group F is DMSO only (control). Values are the mean ± SD. ^∗ P < 0.05 and ^∗∗ P < 0.01 compared with control.

Figure 9

Proliferation of HaCaT cells measured by a WST-1 assay. Increased cell proliferation of HaCaT cells was observed in the presence of 200 μg/mL, 20 μg/mL, and 2 μg/mL “Jinchuang ointment.” Negative and positive controls are in the presence of DMSO and 10 ng/mL EGF, respectively. Values are the mean ± SD. ^∗∗ P < 0.01 and ^∗∗∗ P < 0.001 compared with control.

Figure 10

Treatment of HaCaT cells with “Jinchuang ointment” leads to altered expression of cell cycle-related proteins.Western blot analysis of (a) Cdc25b, (b) Cdc25c, and (c) cyclic D3 protein expression in HaCaT cell extracts after six and 12 hours of treatment. Lanes one to four are as follows: control, 100 ng/mL EGF, 200 μg/mL “Jinchuang ointment,” and 20 μg/mL “Jinchuang ointment,” respectively.

Figure 11

In vitro tube formation assay displaying the stimulation of angiogenesis by “Jinchuang ointment” in HUVEC cells. Cells were treated with (a) 200 μg/mL “Jinchuang ointment” and (b) DMSO only (negative control).