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

“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.


Introduction
It is well-known that diabetic foot ulcers are extremely difficult to be treated and are the dominant complication leading to amputations [1]. "Jinchuang ointment" is a traditional Chinese herbal medicine complex for treatment of incised wounds. Its recipe was first described in one ancient Chinese book of medicine, Medicine Comprehended, published in 1732. Clinical applications of this herbal medicine for diabetic foot infections and decubitus ulcers have been a successful course of treatment in the Division of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan, for more than ten years. Despite its track record of curative effects, there is no literature published in the English language describing the clinical efficacy of "Jinchuang ointment" [2]. Moreover, neither a biological mechanism nor the compositions of effective components have yet to be systematically investigated. Like many Chinese herbal medicines, "Jinchuang ointment" is also a mixture of natural product complexes. The combination of compounds results 2 Evidence-Based Complementary and Alternative Medicine in complications when it determines the chemical composition and bioactivity of each component [3].
"Jinchuang ointment" is composed of lard, wax, starch, synthetic borneol, camphor, frankincense, dragon's blood, myrrh, and catechu. To further characterize the chemical content of each component in this complex, the ratio of stereoisomers in chemically synthesized borneol used in this study was analyzed by chiral gas chromatography (GC). Meanwhile, the content of reference standards in the herbal components, like frankincense, myrrh, dragon's blood, and catechin, was determined by high performance liquid chromatography (HPLC). Lard is the major component in this complex, and its weight percentage is as high as 67%. It is of great interest to determine the role of lard in this complex. Lard was therefore substituted for synthetic triacylglycerol, coconut oil, Vaseline5, and sesame oil. The activity of these reconstituted complexes was examined in this study.
"Jinchuang ointment" is directly applied to the wound surface during treatment. The components in this complex are neither digested nor absorbed in the gastrointestinal tract. It is therefore reasonable to evaluate its bioactivity by direct addition of this complex into media containing cultured human skin or endothelial cells. Wound healing is a very complicated process. In this study, an in vitro tube formation assay, a wound healing assay, and a cell proliferation test were carried out to examine the activity of "Jinchuang ointment." Here, we report the outcomes of treating patients with "Jinchuang ointment," the results of cell based activity assays, and characterization of herbal components by HPLC. The composition of "Jinchuang ointment" (100 g) is as follows: lard 67.3 g, dragon's blood 2.1 g, catechu 2.1 g, frankincense 2.1 g, myrrh 2.1 g, camphor 6.3 g, borneol 0.1 g, corn starch 8.4 g, and wax 9.5 g. For wound healing and tube formation assays, the DMSO stock solution of "Jinchuang ointment" is prepared as follows: two grams of "Jinchuang ointment" is dissolved in 10 mL DMSO and homogenized by ultrasonication just before use.

Determination of Reference Standard Content in Dragon's
Blood, Catechu, Frankincense, and Myrrh by HPLC. All experiments were carried out on a Hitachi L-7000 HPLC system, equipped with L-7100 quaternary gradient pump and a L-7450 photo diode array detector. Hitachi HSM software was used for machine controlling, data collecting, and processing. A Mightysil, RP-18, 5 m, 250 × 4.6 mm, analytic column (Kanto Chemical Co., Inc., Tokyo, Japan) was used for analysis.
For samples of dragon's blood, catechu, and frankincense, 0.1 g grounded solids were weighed and dissolved in 10 mL of methanol. After ultrasonicating for 30 minutes at room temperature, methanol extracts were transferred to a new glass vial by using disposable glass Pasteur pipettes. 4 mL of methanol was then added and ultrasonicated for another 30 minutes at room temperature. The final volume of the extract was adjusted to 25 mL by adding methanol. Undissolved particles were removed by centrifugation at 2500 ×g for 10 minutes at room temperature and filtrated through a 0.22 m syringe filter. For myrrh, 95% ethanol was used for extraction rather than methanol. Other preparation steps were identical to those of dragon's blood, catechu, frankincense, and myrrh.
The methanol extract of dragon's blood was separated using a gradient elution of solvent A (10% CH 3 CN containing 0.1% formic acid) and solvent B (90% CH 3 CN containing 0.1% formic acid) with a flow rate of 1 mL/min [4]. The elution program is given in Table 1. The UV detection wavelength was 254 nm.
The catechu methanol extract was separated using a gradient elution of A (H 2 O containing 0.1% formic acid), B (10% CH 3 CN containing 0.1% formic acid), and C (90% CH 3 CN containing 0.1% formic acid) with a flow rate of 1 mL/min [5]. The elution program is given in Table 1. The UV detection wavelength was 270 nm.
To determine the content of (E)-guggulsterones in myrrh, an ethanol extract was separated using an isocratic elution of 0.1% H 3 PO 4 : CH 3 CN (45 : 55, v/v) with a flow rate of 1 mL/min for 25 minutes [7]. The UV detection wavelength was 240 nm.

Determination of the Ratio of Stereoisomers in Synthetic
Borneol. Chiral GC was used to determine the ratio of stereoisomers in synthetic borneol. Analysis was performed Evidence-Based Complementary and Alternative Medicine 3 Chromatographic conditions were as follows: helium used as a gas carrier; a constant flow of 1.0 mL/min; 2 L injection volume (splitless model), and a 280 ∘ C injector temperature.

In Vitro Wound Healing Assay. Confluent HaCaT cells in
12-well plates were starved overnight in DMEM medium. The surface of the plate was scraped with a 200 L pipette tip to generate a cell-free zone. Free cells were then removed by two HBSS washes, and cells were incubated in DMEM medium containing 200 g/mL, 20 g/mL, or 2 g/mL "Jinchuang ointment." After 24 hours of incubation, cells were imaged using microscopy. The area of wound closure was quantitatively determined using Image J software (National Institutes of Health, Bethesda, MD). Stimulation effects on in vitro wound healing assay by "Jinchuang ointment" were also examined using human microvascular endothelial cells (HMEC-1). Ibidi Culture-Inserts (Ibidi Gmbh, Martinsried, Germany) were placed on the chamber of 24-well cell culture plates. About 70 L of HMEC-1 (5 × 10 5 cells/mL) was seeded per well and plates were incubated at 37 ∘ C and 5% CO 2 . After 24hour incubation, Ibidi Culture-Inserts were removed, and 1 mL of MCDB131 media containing 1 L DMSO solution of "Jinchuang ointment" was then added into individual wells. The migration of cells was observed by microscopy over a period of 6-24 hours. The gap size was measured by using software Image J.

Cell Proliferation Assay.
HaCaT cells (5 × 10 4 /well) were seeded in 96-well plates. The medium containing "Jinchuang ointment" at various concentrations was then added after cell adhesion. Cells were incubated in DMEM medium for the indicated time. The proliferation of HaCaT cells was subsequently determined by using Cell Proliferation Reagent WST-1 (Roche, Indianapolis, IN, USA). The statistical method used is Student's -test.
2.6. Western Blotting. Confluent monolayers of HaCaT cells were treated with various concentrations of Jinchuang ointment for the indicated time. Equal quantities of cell lysate proteins were separated by 10% SDS-PAGE and electroblotted onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked for 1 h with 5% low-fat milk powder solubilized in phosphate-buffered saline (PBS) containing 0.05% Tween 20. Levels of Cdc25b, Cdc25c, CDK2, CDC D2, Cyclin B, Cyclin D3, and -tubulin were determined by western blotting using specific antibodies and enhanced chemiluminescence detection methods. The intensity of the resulting bands was measured by densitometric analysis using Image J software and presented as the ratio relative to the internal control.

In Vitro Tube Formation Assay. Human umbilical vein endothelial cells (HUVEC) were bought from Bioresource
Collection and Research Center (Hsinchu, Taiwan). 1 mL of HUVEC (1 × 10 5 cells/well) was placed into the wells of a 24-well flat bottomed plates precoated with 200 L Matrigel (BD Biosciences, Bedford, MA, USA). Cells were then mixed with 1 mL medium containing "Jinchuang ointment" (final concentration of 200 g/mL). Cells were incubated at 37 ∘ C for a 24 h exposure [8]. After incubation, cell tube or network formation was observed using a phase-contrast microscope.

Statistical Analysis.
Results are expressed as the means ± SD from at least three independent experiments. Differences between groups were assessed by one-way analysis of variance (ANOVA). A value less than 0.05 was considered statistically significant.

Clinical Treatment Observations on a Nonhealing Diabetic
Wound by Treating with "Jinchuang Ointment". To the best of our knowledge, there are no English language case reports describing "Jinchuang ointment" treatment for nonhealing diabetic wounds. Three different cases are presented in this study. The first subject, Mrs. Wu, is a 75-year-old female patient with type II insulin-dependent diabetes accompanied by peripheral arterial occlusion disease (PAOD) which led to left lateral leg and ankle necrotizing fasciitis. She was treated with percutaneous transluminal angiography (PTA) to improve lower limb circulation on Feb 6, 2013. As a result of reperfusion injury, the ulcer had enlarged with erythema. After examination, a below-knee amputation was immediately scheduled two weeks later at the Surgery Division, the China Medical University Beigang Hospital. As suggested by a doctor from the Division of Chinese Medicine, she decided to use traditional Chinese medicine to treat her wound. She was referred to the Division of Chinese Medicine for wound management. Normal saline was first used to clean the wound. About 2-3 g "Jinchuang ointment" was applied directly to the wound once daily. Pictures depicting wound healing under treatment with "Jinchuang ointment" are shown in Figure 1.
Mr. Tsai, a 71-year-old male, with past history of type II diabetes was diagnosed on September 8, 2013. He had a 3 × 0.8 cm and a 4 × 1.5 cm grade 3 pressure sore in the sacral region. 1 g of "Jinchuang ointment" was applied topically to the wound area once per day. Pictures documenting wound healing under treatment of "Jinchuang ointment" are shown in Figure 2. It is well-known that all treatments for bedsores are to prevent wounds from worsening. Complete wound closure was observed on October 27, 2013.
Mr. Wang, a 64-year-old male with a past history of hypertension, had a chronic wound measuring 6.2 × 5.3 cm which had not healed for more than six months. He received the topical application of "Jinchuang ointment" once per day beginning November 26, 2014. A great improvement was observed after two months of treatment as shown in Figure 3.

Content of Reference Standards Present in Dragon's Blood,
Catechu, Frankincense, and Myrrh. One of the main problems associated with herbal medicine is the high level of batch to batch variation in the amounts of active components. The content of pure chemical reference standards in herbal products is therefore used as an indicator for the purposes of quality control and standardization. Accordingly, the content of reference standards in dragon's blood, catechu, frankincense, and myrrh used was measured by HPLC in this study. All the calibration curves of reference compounds were linear over the concentration range studied ( Table 2). A linear interpolation method was used to calculate the percentage by mass of each reference standard in the herbal extract that we examined.
Dracorhodin is a red anthocyanin pigment that is a major component in "dragon's blood" resin of the plant Daemonorops draco. It possesses antimicrobial, anticancer, and cytotoxic activity [9,10]. Figure 4(a) shows the separation of dracorhodin in dragon's blood. The mass percentage of dracorhodin in the "dragon's blood" used in this study is 0.15%.
Both catechin and epicatechin are phenol-type antioxidants in catechu, an extract of acacia trees. Catechu is a common component of herbal medicine. In addition to their ability to scavenge free radicals in plasma, the health benefits of catechin and epicatechin also include stimulation of fat oxidation, expansion of the brachial artery, and resistance of LDLs to oxidation [11]. At the cellular and molecular level, catechin can enhance the expression of human PTGS2 (a dioxygenase gene), 1L1B (cyclooxygenase-2 gene), SOD (superoxide dismutase gene), MAPK1 (Mitogen-activated protein kinase 1), and MAPK3 [12][13][14]. Figure 4(b) shows the separation of catechin and epicatechin in catechu. The mass percentage of catechin and epicatechin in the catechu used in this study is 24.2% and 1.7%, respectively.
Frankincense is a resin from plants in the genus Boswellia. In Africa and Asia, it is widely used in incense, perfume, and traditional medicine. Boswellic acids, a series of pentacyclic triterpene molecules, are one of the major components of frankincense. Theiranti-inflammatory properties and ability to induce cancer cell apoptosis have been reported in vitro [15][16][17]. The expression of TOP1 (DNA topoisomerase I) and TOP2A (DNA topoisomerase II) genes can be altered in the presence of 11-keto--boswellic acid derivatives and acetyl-boswellic acid [16,18]. Figure 4(c) shows the separation of acetyl-11-keto--boswellic acid in frankincense. The mass percentage of acetyl-11-keto--boswellic acid in the frankincense used in this study is 1.62%.
Myrrh is also a resin from plants in the genus Commiphora. The usage of myrrh is similar to that of frankincense. In fact, both myrrh and frankincense are frequently used in concert in many traditional Chinese medicine recipes. In western medicine, myrrh is also used in liniment and healing salves for minor skin ailments. The chemical composition of myrrh is rather complicated [19]. Notably, both (Z)-and (E)-isomers of guggulsterone possess high affinity toward a variety of steroid receptors [20]. Both isomers seem equipotent as inhibitors of HUVEC tube formation [21]. The mass percentage of (E)-guggulsterone in the myrrh used in this study is 0.02%. Figure 4(d) shows the separation of (E)guggulsterone in myrrh.  is the lowest among the sources discussed above. Previous results show that borneol stereoisomers can interact with GABA receptors [22,23] and possess antimicrobial activity [24]. Chiral GC was used to analyze the composition of borneol stereoisomers in synthetic borneol used in this study.
Evidence-Based Complementary and Alternative Medicine 7  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 g/mL "Jinchuang ointment," (d) 20 g/mL "Jinchuang ointment," and (e) 2 g/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.

In Vitro Wound Healing Assay. Confluent
HaCaT cells were scratched and treated with "Jinchuang ointment." The wound area was measured after 24 hours of treatment. All experiments were performed in triplicate. The percentage of wound closure was calculated as follows: (initial wound area − 24 h posttreatment wound area)/initial wound area × 100%. The wound closure percentage with 200 g/mL, 20 g/mL, and 2 g/mL "Jinchuang ointment" treatment was 78.2±5.3%, 78.2 ± 4.3%, and 63.8 ± 1.7%, respectively. In contrast, the negative control HaCaT cells without "Jinchuang ointment" treatment only showed 64.4 ± 4.0% wound closure. The positive control treated with 100 ng/mL EGF treatment showed 71.8 ± 1.0% wound closure. It is obvious that application of 200 g/mL or 20 g/mL "Jinchuang ointment" is more potent than 100 ng/mL EGF in promoting in vitro wound closure, showing statistically significant differences when compared to both positive and negative controls ( Figure 6).
The wound-healing assay was also used to assess the stimulatory effect of "Jinchuang ointment" on the migration of HMEC-1 cells. The wound closure percentage with 200 g/mL "Jinchuang ointment" 24 hours after treatment was 85.0 ± 12.3%, whereas the wound closure percentage observed with the DMSO control was only 43.3 ± 8.2% (Figures 7(a) and 7(b)). The weight percentage of lard is as high as 67% in "Jinchuang ointment," and sesame oil is used to prepare another famous traditional Chinese herbal ointment, Shiunko. The contribution of lard to the total cell migratory activity was therefore evaluated by reconstituting "Jinchuang ointment" with various fats. It is apparent that the significant cell migration into the wound region is seen with Jinchuang ointment-treated cells when compared to the control group ( Figure 8). When sesame oil, synthetic triacylglycerol, coconut oil, and Vaseline were used as lard substituents, 85%, 91%, 74%, and 105% migration activity can be observed (Figure 8). Unlike natural fat, Vaseline is mainly made from petroleum jelly, a semisolid mixture of hydrocarbons with carbon numbers greater than 25. The chain length of carbon atoms in the synthetic triacylglycerols used in this study is C 8 and C 10 . These results suggest that the carbon chain length of the fat used in the ointment plays a minor role in stimulating cell migration.

The Effect of "Jinchuang Ointment" on HaCaT Cell
Proliferation. The stimulatory effect of "Jinchuang ointment" on HaCaT cell proliferation was evaluated in the presence of 10% fetal bovine serum (FBS) by WST-1 assay. The increased percentages of cell proliferation by 200 g/mL, 20 g/mL, and 2 g/mL "Jinchuang ointment" at 24 and 48 hours were 117±2.66%, 133±12.7%, and 129.7±14.1% and 126.4±3.5%, 127.3 ± 5.8%, and 124.1 ± 1.3%, respectively (Figure 9). These results indicate that treatment with "Jinchuang ointment" leads to an increase in HaCaT cell growth. 3.6. The Effect of "Jinchuang Ointment" on the Expression of G1/S Transition-Related Regulators. To further investigate the underlying mechanisms involved in "Jinchuang ointment"induced effects on cell proliferation in HaCaT cells, the expression of several key cellular proteins involved in cell cycle progression was investigated by western blot analysis. As shown in Figure 10, a six-hour treatment with "Jinchuang ointment" leads to a dose-dependent increase in Cdc25b, Cdc25c, and Cyclin D3 levels. After treatment for 12 hours, significant changes in the expression pattern of those proteins between experiment and control groups were observed,  Lanes one to four are as follows: control, 100 ng/mL EGF, 200 g/mL "Jinchuang ointment," and 20 g/mL "Jinchuang ointment," respectively.

Tube Formation
Assay. The process of wound healing has been divided into three different stages, inflammatory, proliferative, and remodeling phases [25]. Angiogenesis is responsible for new blood vessel formation and oxygen and nutrient supply and plays an important role in the proliferative phase of wound healing [26]. The tube formation assay was used to evaluate the in vitro angiogenic effect of "Jinchuang ointment" on HUVEC cells. As shown in Figure 11, treatment of HUVEC cell with 200 g/mL "Jinchuang ointment" for 24 hours can efficiently induce endothelial cell capillary tubes and network formation.
3.8. Conclusions. "Jinchuang ointment" is a Chinese herbal medicine complex. It has been clinically used in the treatment of diabetic foot infection and decubitus ulcers in China Medical University Hospital for more than ten years. Because of its complicated composition, its biological activities have never been investigated. To further characterize its herb ingredients, the content of reference standards present in dragon's blood, catechu, frankincense, and myrrh was determined by HPLC. Two cell based assay platforms, in vitro wound healing and tube formation, were used to examine activity. Our results show that this herbal medicine possesses potent activities stimulating cell proliferation, migration, and angiogenesis. This provides a scientific rationale to account for the observed clinical curative effects on wound healing by "Jinchuang ointment." According to current pharmaceutical regulations in Taiwan, only traditional Chinese herbal medicine manufactured from cGMP pharmaceutical factories can be sold in drug stores or by hospital marketing channels. However, for homemade traditional Chinese herbal medicine, they can only be administered to patients by the doctors who made the respective medicine. For this reason, "Jinchuang ointment" cannot be widely used in the Taiwan area since many clinicians are not able or unwilling to prepare this remedy. To facilitate the manufacturing process of "Jinchuang ointment" by cGMP factories, it will be very important to find out the activity indicator markers for components, such as dragon's blood, catechu, frankincense, and myrrh.
preparations, clinical treatment, and observations; Shinn-Jong Jiang helped in western blot analysis, cell proliferation, and wound healing assay of HaCaT cells; Guang-Huey Lin and Ming-Chuan Hsieh helped in wound healing assay of HMEC-1 cells; Jai-Sing Yang helped in tube formation assay; Lih-Ming Yiin helped in GC analysis; Hao-Ping Chen helped in HPLC analysis, experiment design, "Jinchuang ointment" preparation, and paper preparation.