Biological and Pathological Studies of Rosmarinic Acid as an Inhibitor of Hemorrhagic Trimeresurus flavoviridis (habu) Venom

In our previous report, rosmarinic acid (RA) was revealed to be an antidote active compound in Argusia argentea (family: Boraginaceae). The plant is locally used in Okinawa in Japan as an antidote for poisoning from snake venom, Trimeresurus flavoviridis (habu). This article presents mechanistic evidence of RA’s neutralization of the hemorrhagic effects of snake venom. Anti-hemorrhagic activity was assayed by using several kinds of snake venom. Inhibition against fibrinogen hydrolytic and collagen hydrolytic activities of T. flavoviridis venom were examined by SDS-PAGE. A histopathological study was done by microscopy after administration of venom in the presence or absence of RA. RA was found to markedly neutralize venom-induced hemorrhage, fibrinogenolysis, cytotoxicity and digestion of type IV collagen activity. Moreover, RA inhibited both hemorrhage and neutrophil infiltrations caused by T. flavoviridis venom in pathology sections. These results demonstrate that RA inhibited most of the hemorrhage effects of venom. These findings indicate that rosmarinic acid can be expected to provide therapeutic benefits in neutralization of snake venom accompanied by heat stability.


Introduction
Envenomation resulting from snakebites is an important public health hazard in many regions, particularly in tropical and subtropical regions [1][2][3]. Snake envenomation causes various pathophysiological changes such as inflammation, increased body temperature, hemorrhage, necrosis, nephrotoxicity, cardiotoxicity, haemostatic changes and ultimately death [4]. Hemorrhage is a common symptom associated with local tissue damage in snake poisoning by Viperidae. In severe poisoning, hemorrhage can be observed in many internal organs [5,6]. Hemorrhage can occur as a result of cytotoxicity of the venom on endothelial cells, and a degradation of the basement membrane of the vein due to the venom acting as a metalloproteinase. In many countries, plant extracts have been traditionally used in the treatment of snakebite envenomations [7][8][9], although only a few cases have been scientifically validated. The present study aims to examine the venom neutralization potential of rosmarinic acid (RA) isolated from a methanolic extract of Argusia argentea [10] and its mode of action.
Hemorrhagic toxin-1 (HT-1) was obtained from B. arietans venom [14]. Human and bovine fibrinogens were supplied by Sigma-Aldrich, Tokyo, Japan. Type IV collagen was purchased from Nitta Gelatin Inc. Cryo-preserved human umbilical vein endothelial cells (HUVEC), its respective cell culture media (HuMedia EB-2), other cell culture supplements, and reagents were obtained from Kurabo (Osaka, Japan). The cell counting kit was purchased from Dojindo (Kumamoto, Japan). Other chemicals were of analytical grade from commercial sources. All experiments involving the use of animals were carried out in compliance with the guidelines for animal experiments of Faculty of Pharmacy, Meijo University.

Rosmarinic Acid
Rosmarinic acid (RA) was isolated and purified from methanolic extract of Argusia argentea as reported previously [10]. A voucher sample has been deposited in the Herbarium of the Faculty of Pharmacy, Meijo University, Japan.

Anti-hemorrhagic Activity Assay
Anti-hemorrhagic activity was assayed by the method of Bjarnason and Tu [15] using ddY mice of 20 g average weight. Two groups of four mice were used for the experiment. All crude venom solutions of T. flavoviridis venom, C. atrox venom, G. blomhoffii venom and B. arietans venom, were prepared at a concentration of 0.14 mg/mL in saline. Concentrations of purified hemorrhagic toxin solutions were as follows: HTb (0.41 mg/mL), bilitoxin-2 (2.75 μg/mL), HT-1 (0.29 mg/mL), and Ac 1 -proteinase (1.04 mg/mL). A test solution was prepared by mixing the venom solution or the toxin solution (50 µL) and RA (0.5 mg/mL in 10% DMSO-saline, 50 µL) followed by 10 min incubation at 37 °C. These test solutions (100 µL) were injected subcutaneously (s.c.) in the abdomen of mice.
Similarly, a group of mice which were injected with a venom solution without RA was used as a control group, and also a group which was only injected with 10% DMSO-saline (50 μL) served as a blank group. Prior to this study, effects of DMSO at several concentrations were investigated, and DMSO at less than 10% was found to cause no significant inactivation of venom. After 24 h, mice were euthanized by inhalation of chloroform, the skin covering the abdomen was removed and hemorrhagic lesions were analyzed as follows. The area of the lesion was estimated by major and minor axes measurements, since the shape of the lesions are always amorphous, like an ellipse.

Fibrinogen Hydrolytic Activity Assay
Fibrinogen hydrolytic activity was assayed by the method of Ouyang and Teng [16]. A solution of 0.1% human fibrinogen in 50 mM Tris-HCl buffer (pH 7.5) (1 mL) and a venom solution (50 µL of 0.21 mg/mL of T. flavoviridis venom or 5.5 µg/mL of bilitoxin-2) were incubated in the presence or absence of RA (0.5 mg/mL) at 37 °C. At various time intervals, aliquots of 100 µL of denaturing solution (10 mM phosphate buffer, pH 7.2, containing 10 M urea, 4% sodium dodecyl sulfate (SDS), and 4% β-mercaptoethanol) were added. This solution was incubated at 37 °C for 6 h and then run on 10% polyacrylamide slab gel electrophoresis. Electrophoresis was carried out for 2 h with a current of 25 mA per slab gel. Bromophenol blue (BPB) solution was used as an indicator.

Collagen Hydrolytic Activity Assay
Collagen hydrolytic activity was assayed as follows. Sodium hydrogen carbonate (60 µL, pH 12) was added to 0.3% type IV collagen (0.9 mL) and adjusted to pH 8. Aliquots of type IV collagen were incubated with T. flavoviridis venom (0.21 μg/mL) in the presence or absence of RA (0.5 mg/mL). At various time intervals, aliquots of 100 µL of denaturing solution (10 mM phosphate buffer, pH 7.2, containing 10 M urea, 4% SDS, and 4% β-mercaptoethanol) were added. This solution was boiled for 3 min and run on SDS-PAGE using a 7.5% polyacrylamide slab gel electrophoresis.
Ten microliters of cell counting kit-8 was added to each well, and the microplate was incubated for 1 h, after which cell densities were measured at 450 nm using Bio-RAD Model 550 MicroplateR eader.

Histopathological Study
Histopathological study for RA was performed by intramuscular (i.m.) injection of T. flavoviridis venom solution into the medial aspect of the thigh muscle of ddY strain white mice. Histopathological study of muscle was conducted in three groups. Group A was injected with the venom (0.21 mg/mL, 100 µL), while group B was injected with RA (0.5 mg/mL, 100 µL). Group C was injected with a mixture of the venom (0.41 mg/mL, 50 µL) and RA (0.25 mg/mL, 50 µL). Test solutions were preincubated at 37 °C for 10 min before injection. The mice were killed by chloroform inhalation 24 h after injection. Tissue samples were immediately fixed in buffered formate fixative for 24 h at room temperature. The tissue was then washed for 4 h in running water, dehydrated in an autotechnicon, and stained with hematoxylin and eosin for observation under light microscope.

Heat Stability
RA (0.5 mg/mL) in 10% DMSO-saline was heated at 37 °C, 50 °C and 100 °C for 10 min, respectively. Fifty microliters of each heat-treated RA solution was mixed with T. flavoviridis venom (0.21 mg/mL, 50 µL) and incubated at 37 °C for 10 min. An aliquot of 0.1% human fibrinogen in 50 mM Tris-HCl buffer, pH 7.5 was added to each test tube. The reactions were stopped by adding 100 µL of denaturing solution (10 mM phosphate buffer, pH 7.2, containing 10 M urea, 4% SDS, and 4% β-mercaptoethanol). These solutions were incubated at 37 °C for 6 h and then run on 10% polyacrylamide slab gel electrophoresis. The venom solution without a sample was also subjected to SDS-PAGE for comparison. Moreover, by using RA (0.5 mg/mL), which was treated at 100 °C, antihemorrhage activity was also examined as mentioned above.

Assay for Edema Activity
Hind-paw edema activity was assayed by the method of Ho et al. [20]. Four ddY strain white mice (20-23 g) were individually injected in the right foot pad with Trimeresurus elegans venom (12.5 µg in 50 µL of 10% DMSO-saline). An equal volume of 10% DMSO saline was injected into the left paws as control. Inhibition assays were performed by preincubated rosmarinic acid (0.5 mg/mL in 10% DMSO saline) with toxin for 10 min at 37 °C. The volume of each paw was measured with a slide caliper. The degree of paw swelling was expressed as % increase of the initial paw volume.
RA effectively inhibited the hemorrhagic activities of crude venoms as well as purified hemorrhagic toxins.

Inhibition of Fibrinogen Hydrolytic Activity
When human fibrinogen was incubated with T. flavoviridis venom, the Aα band of the fibrinogen disappeared on SDS-PAGE, whereas the Bβ chain and γ chain were essentially unaffected (Figure 2a).
The venom with RA did not reveal any apparent degradation of human fibrinogen (Figure 2b). RA also inhibited Aα hydrolysis by bilitoxin-2 (Figure 2c and 2d).

Inhibition of Venom Cytotoxic Action on HUVEC
RA alone had no effect on the viability of HUVEC, but it markedly protected HUVEC from the toxic effects of T. flavoviridis venom (0.14 mg/mL) at all concentrations of RA tested (0.50, 0.25, 0.125, 0.06, and 0.03 mg/mL) (Figure 3). The maximum (84.2%) protective effect of RA was exhibited with RA at 0.5 mg/mL.

Inhibition of Type IV Collagen Hydrolytic Activity
Type IV collagen was incubated with T. flavoviridis venom for different periods of time. The venom completely degraded type IV collagen (104 kDa), especially over 1 h, and degradates with smaller molecular weights (43 and 35 kDa) appeared, as shown in Figure 4a. In the presence of RA

Heat Stability of RA
RA, which was heated at various temperatures, was incubated with human fibrinogen and T. flavoviridis venom. RA inhibited the digestion of Aα chain of human fibrinogen after treatment at each temperature ( Figure 6). RA (0.5 mg/mL), which was heated at 100 °C for 10 min, also showed complete inhibition against T. flavoviridis venom.

Inhibition of Venom-Induced Edema
The edema-forming activity was assayed using four mice. T. elegans venom induced an edema of 30% in the mouse footpad, at a dose of 12.5 µg. When T. elegans venom was preincubated with RA

Discussion
RA effectively inhibited snake venom induced hemorrhage by crude venoms of T. flavoviridis, Crotalus atrox, Gloydius blomhoffii, and B. arietans or purified toxins (HTb, bilitoxin-2, HT-1 and Ac 1 -proteinase) [10]. As shown in Figure 7, envenomation by snakebites often produces persistent hemorrhage due to considerable degradation of fibrinogen and other coagulation factors, thus preventing clot formation [21]. The pathogenesis of venom-induced hemorrhage involves direct damage to endothelial cells in microvessels by hemorrhagic toxins [22,23]. Snake venom metalloproteinases (especially snake venom metalloproteinase from T. flavoviridis venom) degrade the most important components of the basement membrane, such as laminin, type IV collagen and nidogen/entactin [24][25][26]. In this study, an attempt was made to determine the protective effects of RA on digestion of human fibrinogen, digestion of type IV collagen and cytotoxic action on HUVEC  Figure 7). It has been reported that RA isolated from Cordia verbenacea (Boraginaceae) inhibits the edema and myotoxic activity induced by crude venom and isolated PLA 2 s [27]. However, the mechanisms of action of PLA 2 s are quite different from those of metalloproteinases. This is the first report of RA that demonstrates the inhibitory mechanism for snake venom induced hemorrhage and protection from snakebite envenomation. In this study, the inhibitory activities of rosmarinic acid from Argusia argentea against the action of snake venom were investigated. The plant is found on tropical shores of the Pacific and Indian oceans and the extract of the leaves are locally used as an antitoxin for snakebites and fish poisoning [28,29]. In the Okinawa islands, the leaves of Argusia argentea are the first medical treatment against snake venom as well as against jellyfish venom [30]. Hemorrhage is a conspicuous sequela of envenomation by Viperidae. Hemorrhage from damage to the vascular endothelium of vital organs causes death [31].
These observations confirmed that RA possesses potent snake venom neutralizing properties.
Furthermore, the heat stability of RA would satisfy a requirement for a first-aid treatment for snakebite.
Because snakebites often occur outdoors far from medical institutions, it is necessary to distribute a drug to distant locations without refrigeration, and the drug must be storable at room temperature.
Further studies are needed to investigate post-administration of RA, and the effects of different methods of administration. After these issues are resolved, RA could become a potent alternative antidote compound for snake envenomation.