Hemostatic, anti-inflammatory and antibacterial effects of Sanqixiantao dressing in vivo and in vitro

Purpose: To study the hemostatic, anti-inflammatory and antibacterial effects of Sanqixiantao dressing. Methods: Sanqixiantao dressing was prepared by mixting with sanqixiiantao extract (8 %) with membrane-forming matrix (5:4:9:2 volume ratio of polyvinyl alcohol: Na CMC: gelatin: glycerol). Rats with local surface wounds were used to evaluate the effects of Sanqixiantao dressing on hemostatic time, wound healing time and infection rate. Serum levels of tumor necrosis factor (TNF)-α and interleukin (IL) 6 were determined. The anti-inflammatory and analgesic effects of Sanqixiantao extracts were assessed by dimethylbenzene-induced ear edema and acetic acid-induced abdominal writhing tests. In in vitro studies, the effect of the extract on blood clotting time, and its antibacterial activities against six pathogenic bacteria (Escherichia coli, Staphylococous aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Clostridium perfringens and Clostridium tetani) were evaluated. Results: Sanqixiantao dressing significantly decreased hemostatic time (p < 0.01), wound healing time (p < 0.01) and infection rate (10 vs 100 %), when compared to control rats. Sanqixiantao extract significantly shortened blood clotting time in vitro (p < 0.01), and showed antibacterial activities against E. coli (minimum inhibitory concentration, MIC: 0.4 mg/mL, MBC: 1.6 mg/mL), S. aureus (MIC: 0.8 mg/mL, minimum bacterial concentration, MBC: 3.2 mg/mL), P. aeruginosa (MIC: 0.8 mg/mL, MBC: 3.2 mg/mL), S. epidermidis (MIC: 1.6 mg/mL, MBC: 3.2 mg/mL). Besides, Sanqixiantao extracts (100, 200, 400 and 600 mg/kg) dose-dependently decreased dimethyl-benzene-induced ear edema and acetic acid-induced abdominal writhes in mice (p < 0.05, p < 0.01, p < 0.01, p < 0.01). Conclusion: The results demonstrate that Sanqixiantao dressing has significant hemostatic, antiinflammatory and antibacterial effects in vivo and in vitro, and thus provide some support for the therapeutic application of Sanqixiantao dressing for treating skin wounds.


INTRODUCTION
The skin is the largest organ of human body and provides physiological barrier against infections [1,2].Acute skin wounds usually occur in field battles, traffic accidents and in daily life [3].Normally, small wounds heal without much medical intervention, but large wounds require timely medical attention [4].Most of the available drugs currently used for wound healing are synthetic chemicals [5].These drugs are expensive and are associated with some sideeffects [6,7].Thus, there is needed to evolve natural, newer and safer methods for treating acute wounds.
In recent years, herbal therapy have been shown to be beneficial in curing various diseases [8,9].In addition, these alternative herbal remedies are reliable, inexpensive and of low toxicities [9,10].
Sanqixiantao dressing is a preparation in Teaching Hospital of Chengdu University of Traditional Chinese Medicine (Chengdu, PR China) and it is used in the hospital for treating acute wounds.The dressing is derived from six herbs (Panax notoginseng, Veronica peregrine, Rhus chinensis, Bletilla striata, Rheum palmatum, Glycyrrhiza uralensis); polyvinylalcohol, carboxymethylcellulose sodium (CMC-Na), gelatin, and glycerol.Although Sanqixiantao dressing is an effective alternative clinical remedy for treating acute skin wounds, there are so far, no experimental data regarding its pharmacological activities.
In this study, the hemostatic, anti-inflammatory and antibacterial effects of Sanqixiantao dressing were investigated.

EXPERIMENTAL Plant materials
The herbs that make up Sanqixiantao extract (Panax notoginseng, Veronica peregrine, Rhus chinensis, Bletilla striata, Rheum palmatum, Glycyrrhiza uralensis) were obtained from the dispensary of Traditional Chinese Medicine of Teaching Hospital of Chengdu University of Traditional Chinese Medicine (Chengdu, China).

Chemicals
Tumor necrosis factor (TNF) α and interleukin (IL) 6 ELISA kits were purchased from the Biosource International Co. (Camarillo, CA, USA).Yunnanbaiyao powder was purchased from the Yunnanbaiyao Co. (Kunming, China).Polyvinyl alcohol, CMC-Na, gelatin and glycerol were purchased from the Sigma China Co. (Shanghai, China).All other used regents were analytical grade.

Bacterial strains and animals
Bacterial strains used in this research were obtained from ATCC (MD.USA).SD rats, KM mice and rabbits were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China).The animal protocols were approved by the Animal Care and Use Committee of Teaching Hospital of Chengdu University of Traditional Chinese Medicine (approval no.201607-JA-034).The animals were handled according to the standard protocols for the use of laboratory animals [11].

Preparation of Sanqixiantao extracts and Sanqixiantao dressings
The six herbal were ground and decocted 3 times by deionized water.Thereafter, the extracts were pooled and filtrated, and the filtrate was dried by vacuum drying apparatus under 50 °C to yield the Sanqixiantao extract.

Determination of therapeutic effects of Sanqixiantao dressing on localized surface wound in rat
A total 30 SD rats were divided into 3 groups namely: control groups, positive group and Sanqixiantao dressing group (n = 10).For the positive group, Yunnanbaiyao powder, a wellknown traumatic drug in China [12], was used as the positive drugs.
Rats with local wound rats were prepared as follows: The rats were anesthetized by intraperitoneal injection (i.p.) of 1 % pentobarbital sodium at doses of 40 mg/kg.Thereafter, a 1.5 × 1.5 cm bleeding wound was made on the backside of the rat.Subsequently, drugs were administered topically on the wounds, and hemostatic time, wound healing time and infection rate were evaluated in over a period of 10 days [13].During the study, blood sample from each rat was collected 5 days after surgery by orbital blood sampling, and the serum levels of TNF-α and IL-6 were determined by commercial ELISA kits.

Determination of blood coagulation time in vitro
Rabbit blood sample was collected form the ear vein using EDTA vacuum anticoagulant tubes.Then, the anti-coagulated plasma was prepared by centrifugation (3000 rpm, for 20 min).Subsequently, clotting time was determined in vitro using plate method for blood coagulation and test tube method according to the previous reports [13].

Antibacterial assay
The antibacterial activities of sanqixiantao extracts against 6 pathogenic bacteria that cause wound infection were determined.These bacteria

Dimethylbenzene-induced ear edema test
The effect of sanqixiantao extract on dimethylbenzene-induced ear edema test in mice was according to the method outlined previously [15].Sanqixiantao extracts and positive drugs (Indometacin) were administered orally.After 40 min of drug treatment, dimethyl-benzene was topically applied to the right ear of mice, and the mice were sacrificed under anesthesia (1 % sodium pentobarbital, 40 mg/kg, ip).The degree of ear edema was determined.

Acetic acid-induced abdominal writhing test
The analgesic effect of Sanqixiantao extract was evaluated by acetic acid-induced abdominal writhing test according to previously reported method [16].The sanqixiantao extract and positive drug (indometacin) were administered orally.After 40 min of the drug treatment, acetic acid (0.75 %, 10 mL/kg) was intraperitoneal administered to mice.The number of writhes was counted, starting 3 min after acetic acid treatment over a period of 12 min.

Statistical analysis
Data are expressed as mean ± SD.One way analysis of variance (ANOVA) was used to compare the means between two groups.Data analysis was carried out by SPSS 17.0 software.P < 0.05 was considered statistically significant.

Effect of Sanqixiantao dressing on localized wound surface
As shown in Table 1, in control rats, the hemostatic time was 95.83 ± 5.74 s; after treatment with sanqixiantao dressing, the hemostatic time significantly decreased (47.31 ± 4.85 s, p < 0.01) when compared with rats in the control group.Furthermore, would healing time was significantly decreased by treatment with sanqixiantao dressing (3.8 ± 0.80 day vs. 5.8 ± 1.32 day, p < 0.01).In addition, sanqixiantao dressing significantly decreased infection rate of the wounds (10 vs 100 %).
Results from ELISA (Table 2) show that serum levels of TNF-α (932.18 ± 374.49pg/mL vs 217.81 ± 65.75 pg/mL, p < 0.01)and IL-6 (596.92 ± 147.36 pg/mL vs 132.86 ± 28.35 pg/mL, p < 0.01) were significantly decreased after treatment with Sanqixiantao dressing, when compared with control rats.This suggests that the dressing possesses significantly antiinflammatory effect, which was better than that of the positive drug (Yunnan Baiyao powder).

Sanqixiantao extract shortened blood clotting time in vitro
The results of the plate method and tube method (Table 3) revealed that sanqixiantao extracts (0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/mL) had significantly higher blood coagulation effects (p < 0.01) when compared with control.This effect was also concentration-dependent.

Anti-inflammatory and anti-analgesic activities of sanqixiantao extract
The results of anti-inflammatory and antianalgesic properties of the extract which were evaluated by dimethyl-benzene-induced ear edema test and acetic acid-induced abdominal writhing test, are shown in Table 5. Sanqixiantao extract showed significant dose-dependent effects on ear edema at 100, 200, 400 and 600 mg/kg, when compared with mice in control group (p < 0.05, p < 0.01, p < 0.01, and p < 0.01, respectively).In addition, the extract (at 100, 200, 400 and 600 mg/kg) dose-dependently decreased abdominal writhes induced by acetic acid, relative to the control group (p < 0.05, p < 0.01, p < 0.01, and p < 0.01, respectively).These results suggest that sanqixiantao extract possesses potential anti-nociceptive antiinflammatory properties.

DISCUSSION
From time immemorial, herbal derived medicines have been applied in the treatment or prevention of various diseases.Herbal medicines are known to produce reliable pharmacological effects [9,17,18].In the present study, hemostatic, antiinflammatory and antibacterial effects of sanqixiantao dressing were demonstrated in vivo and in vitro for the first time.
Studies have indicated that medical dressings have beneficial for wound healing, especially drug-loaded dressings [19,20].These drugloaded dressings not only inhibit the wound infections, but also promote the healing of wounds [20].In the treatment of acute skin wounds, rapid hemostasis is a very important property of the good wound dressings [21,22].In the present study, sanqixiantao dressing and its extract had significant blood coagulation activities in vivo and in vitro.
Infection and inflammatory reactions are the most common complications of skin wounds, and might result in high fever and even sepsis [22,23].Therefore, controlling inflammatory reactions and infections are essential for wound healing.The present study revealed that sanqixiantao extracts alleviated the inflammation and significantly decreased the serum levels of pro-inflammatory cytokines such as TNF-α and IL-6 [24].Bacterial infections usually result in the exacerbation of wounds and severe inflammatory reactions [25].The results also demonstrated that sanqixiantao extract had significant antibacterial activities against six pathogenic bacteria that cause wound infection (E.coli, S. aureus, P. aeruginosa, S. epidermidis, C. perfringens and C. tetani).In addition, the present investigation indicated that sanqixiantao extract possesses strong analgesic activity.Sanqixiantao dressing shortened wound healing time of local surface wound in rats.These results suggest that sanqixiantao dressing is beneficial for treatment of skin wounds.

CONCLUSION
The findings of this study demonstrate that Sanqixiantao dressing has significant hemostatic, anti-inflammatory and antibacterial effects in vivo and in vitro, and thus lends some support for its therapeutic application in the treatment of skin wounds.

Table 1 :
Effect of sanqixiantao dressing on hemostatic time, wound healing time and infection rate on local wound surface in rat

Table 2 :
Effect of sanqixiantao dressing on serum levels of TNF-α and IL-6 in local wounded rat

Table 3 :
Effect of sanqixiantao extracts on clotting time of plasma in rabbit

Table 4 :
Anti-bacterial effects of sanqixiantao extracts in vitro

Table 5 :
Anti-inflammatory and analgesic effects of sanqixiantao extracts in mice Guo CL, Deng HY, Qu HL.A comparative study on application effect of different silver dressings in treatment of patients with chronic wounds.Chin Nurs Res 2015; 29: 1170-1175.20.Zhou ZX, Wei XL, Yao JR, Zhang J, Bian G, Li JH.McFarland AP, Luo S, Ahmed-Qadri F, Zuck M, Thayer EF, Goo YA, Hybiske K, Tong L. Woodward JJ.Sensing of Bacterial Cyclic Dinucleotides by the Oxidoreductase RECON Promotes NF-κB Activation and Shapes a