Cassia occidentalis: effect on healing skin wounds induced by Bothrops moojeni in mice

Objective: To evaluate the healing activity of an extract from the leaves of Cassia occidentalis on skin wounds induced by the venom of Bothrops moojeni in mice. Methods: A total of 24 Swiss albino mice were divided into 4 groups: Group 1 animals were treated for 7 days with lanette cream; Group 2 animals were treated for 7 days with 10% leaf extract of Cassia occidentalis in lanette cream; Group 3 animals were treated for 14 days with lanette cream; and Group 4 animals were treated for 14 days with 10% leaf extract of Cassia occidentalis in lanette cream. To induce wounds, the animals were anesthetised intramuscularly after the dorsum cervical region was shaved and inoculated intradermally with 4 μg Bothrops moojeni venom. The gross and histological evaluations were assessed 7 and 14 days after inoculation of the poison. Results: Extract from the leaves of Cassia occidentalis decreased inflammation and epidermal hyperplasia and increased the vascular proliferation and reepithelialisation of wounds induced by Bothrops moojeni venom. Conclusions: These data suggest that the leaves of Cassia occidentalis stimulated the healing of wounds induced by the dermal venom of Bothrops moojeni in mice, and they can be considered an alternative product to treat wounds caused by this snakebites.


Background
The genus Bothrops is found in the Americas and is characterised by its adaptation to different types of environments. It is found in diverse ecosystems, even in flooded regions, and is responsible for the majority of snakebites in Brazil [1,2]. Bothrops moojeni is a species that has been attracting increased medical attention because its venom is composed of a complex mixture of proteins with biological activity, and it is used as a model to evaluate new drugs [3].
Snake bites can result in severe pain, oedema, bruising, redness and haemorrhagic blisters, which can progress to necrosis, which affects the skin, muscles and tendons [4] and is a major cause of loss of organ function.
Studies have been performed with various substances and therapies to minimise the local effects caused by Bothrops poisoning. Of these treatments, medicinal plants, such as Cassia occidentalis, are popularly used for snakebites [5].
C. occidentalis is an herbaceous species that is native to the Americans. It belongs to the family Fabaceae (Leguminosae) and subfamily Caesalpinioideae [6]. It is used in various traditional medicines for curing various diseases, with antibacterial, antifungal, anti-diabetic, anti-inflammatory, anticancer, antimutagenic, and hepatoprotective activities, and it has also been used to treat snakebites [7].
There are no data in the scientific literature on the action of medicinal plants on the side effects arising from snakebites. This study is aimed to evaluate the healing effect of extracts of leaves of Cassia occidentalis on skin wounds induced by the venom of Bothrops moojeni in mice. doi: 10.7243/2050-120X-2-10 weighing 20 to 40 g from the Central Biotery of the UFG were used in the study.
The animals were acclimitisedated in the bioterium of the Centre of Studies and Research Toxic and Pharmacological (NEPET-UFG), Faculty of Pharmacy, UFG, for 15 days. They were kept in individual polyethylene cages lined with wood shavings under controlled environmental conditions (temperature 23 ± 2 ºC, relative humidity between 50 and 70% and a 12 h light/dark photoperiod).Water and food were provided ad libitum. -The ethical principles in animal testing recommended by the Brazilian Society of Science in Laboratory Animals SBCAL -COBEA were follwed.

Collection of plant material and the preparation of extract from the leaves of Cassia occidentalis
The leaves of C. occidentalis were collected in the Goiânia region in December 2010 (15° 01`11.8`` south and 49º 52` 32.2`` west) at an altitude of 756 meters.
The hydroalcoholic extract was obtained through percolation. -A glass percolator and 95% ethanol (p/v) was used as the extraction liquid. The process was monitored by the identification of glycosides anthraquinones with the Borntraeger reaction [8]. The extracts were concentrated with a rotary evaporator at a temperature below 40 °C at a 1:4 ratio, resulting in extracts of the leaves (EF) of C. occidentalis.To obtain the herbal formulation, 10% of the extract from the leaves of C. occidentalis was incorporated into lanette cream (cream base) and packed in a plastic pot.

Preparation of bothrops venom
The crude venom of Bothrops moojeni was prepared according to the method proposed by [9]. The animals were intradermally inoculated with 4 µg of venom diluted in 10 ml of saline applied 0.1 ml in each mouse to cause necrosis with a 10 mm diameter. After 24 hours, the wound was treated with the topical application of 100 µL of ethanolic extract in the treated group and 100 µL of saline in case of untreated group.

Experimental design
The animals were weighed and randomly divided into four groups (n=6): Group 1 -the animals were treated for seven days with lanette cream. Group 2 -the animals were treated for seven days with lanette cream containing 10% of the leaf extract of C. occidentalis in (EF). Group 3 -the animals were treated for fourteen days with lanette cream. Group 4 -the animals were treated for fourteen days with lanette cream containing 10% of the leaf extract of C. occidentalis (EF).

Wound induction
To induce the wound, the animals were anesthetised intramuscularly with a decloridrato combination of ketamine (70 mg kg -1 ) and xylazine (10 mg kg -1 ). After shaving the dorsal neck region, the mice were inoculated intradermally with 4 μg B. moojeni venom. Paracetamol was administered orally as an analgesic, and the dose was calculated by allometry. After 24 hours, topical treatments were initiated and performed daily at the same time.

Wound evaluation
Wound induction and treatment was performed without complications. There was no complication in relation to anaesthesia and the animals demonstrated good general motor activity and normal behaviour for the species after the procedures.
All animals were examined daily for general appearance. Besides, macroscopic evaluation was done through daily observation of the wound, and the data was recorded individually.

Euthanasia of animals
At the end of the experiment, the animals were weighed and euthanised by anaesthesia with a combination of ketamine hydrochloride (100 mg kg -1 ) and xylazine hydrochloride (30 mg kg -1 ).

Histological evaluation
For histological evaluation, a fragment of each wound was removed, fixed in 10% formalin, and processed and stained with haematoxylin and eosin (HE).
The histological examination was conducted by the Division of Pathology, Veterinary School, Federal University of Goiás (Brazil). The following variables were evaluated on day 7: fibrin, haemorrhage, oedema, hyperaemia, inflammatory infiltration of polymorphonuclear cells, the infiltration of fibroblasts and vascular proliferation. On day 14, the following parameters were evaluated: reepithelialisation, epithelial hyperplasia, collagen, vascular proliferation and inflammatory infiltrates. Adapted scores were used [10].

Statistical analysis
The results were submitted to statistical treatment by the use of GraphPad InStat software (Version 3.05 for Windows). From the Kolmogorov-Smirnov test for normality, histological analysis data were performed a semi-quantitative assessment using the Kruskal-Wallis test, and the least significant differences were determined using the Dunn test. The statistics were considered significant when the p value was less than 0.05 (p <0.05).

Animal weights
The results pertaining to the weight of the animals at the beginning and end of the assessment period are shown in  in the final weight.

Macroscopic analysis
In the macroscopic analysis, the wounds treated with leaf extract were dry, thinner and had few signs of inflammation on the 3rd day, and they disappeared on the 6th day after inoculation of the poison (Figure 1). In the groups that were not treated with the plant extract, the wounds appeared red. On the 3rd day, the animals showed a reaction of pain to the touch, and the wounds only disappeared on the 9th day after inoculation of the poison (Figure 1). Area of skin wounds decreased during the healing process. In mice treated with the plant extract, all wounds were completely healed on the 14th day after inoculation of the poison. In mice not treated with the plant extract, only 60% of the animals showed complete healing on the 14th day (Figure 1).
The macroscopic evaluation of wounds in animals treated with leaf extracts revealed that 100% of the mice showed complete reepithelialisation of wounds on 14th day. In Mice not treated with plant extract, partial reepithelialisation was observed. 40% of the animals had open wounds even on the 14th day after inoculation of the poison (Figure 1).

Microscopic analysis
Results of histological analyses are presented in       Figure 3).

Discussion
In this study it was observed that leaf extract of C. occidentalis decreased inflammatory processes and epidermal hyperplasia and increased vascular proliferation and the reepithelialisation of wounds induced by Bothrops moojeni in mice.
Bothrops snake bites are an important public health problem in tropical regions around the world. The local reaction caused by bothropic venom has been the focus of several investigations. The use of substances with the therapeutic potential of minimising these reactions has been studied, with medicinal plant, such as C. occidentalis, which is popularly used for snakebites [11].
From this work, it is clear that the cutaneous wounds of the animals in the mice treated with plant extract were dry and had few signs of inflammation. Scientific studies have shown that Cassia occidentalis has an enormous biological potential and may have anti-inflammatory, antiplatelet, muscle relaxant, and anti-haemolytic activities and inhibit lipid peroxidation. These effects may occur probably as it is rich in derivativeslike anthraquinônes [5].
The reduction in epidermal hyperplasia observed in the groups treated with plant extract may be associated with a positive effect of C. occidentalis on the inflammatory phase. Moreover, the venom of snakes of the genus Bothrops has high proteolytic activity, which is responsible for the primary characteristic systemic effects observed after snakebites [12].
Wound healing occurs in overlapping and interdependent phases [13], including inflammation, repair, and maturation. Many processes occur during the proliferative phase of wound repair, including angiogenesis, fibroplasia and epithelialisation [14], highlighting that angiogenesis is one of the most important histological features in the granulation tissue in the healing process of the skin [15]. In this study, animals treated with plant extract showed complete epithelialisation and angiogenesis on the 7th and 14th day after wound induction by poison.
Angiogenesis is the growth or formation of new blood vessels from pre-existing vessels at the ends of the wound that were previously devoid of blood vessels [16]. One way to enhance the healing process is to stimulate angiogenesis, which can be established by removing debris and supplying essential nutrients and oxygen to the wound site. The results of this study corroborate other studies [10,17,18,19,20]. Similar effects have been observed in the study, which justifies the macroscopic findings of the best resolution of the healing process.
The microscopic analyses revealed a decrease in epidermal hyperplasia on the 14th day in the group treated with the extract of leaves of C. occidentalis. Hyperplasia corresponds to the increased cellularity of a tissue due to a higher functional demand or hyperstimulation [21]. During the inflammatory process, hyperplasia can occur through phenomena that are intrinsic to inflammation, such as hyperaemia and the synthesis of substances that stimulate cell division. Thus, the more intense and long-lasting the inflammatory response, the greater the hyperplastic response of the cells involved in the process [22].
During the healing process, when there is a delay in the elimination of the initial cause of the lesion by the inflammatory response, there is a higher number of hyperplastic cells [23], and leaf extracts of C. occidentalis can act to minimise the inflammatory effects of Bothrops moojeni poison and accelerate the healing process.

Conclusion
These data suggest that extract of leaves of C. occidentalis stimulate the healing of wounds induced by the venom of B. moojeni in mice, and it can be considered an alternative product to treat snakebite wounds.