Effect of Broccoli (Brassica oleracea L. var. italica) Extract on Bleeding Time in Male White Mice (Mus musculus L.)

External bleeding is bleeding originating from an open wound so that it can be seen from a physical examination (Guo & DiPietro, 2010). Bleeding is a process of bleeding from blood vessels that can cause damage to blood vessel walls caused by trauma or disease (Xu et al., 2019). The normal functioning hemostatic system is important for the life of the organism because if the hemostatic is disrupted, even a small wound can cause life-threatening bleeding (Periayah et al., 2017). The normal innate hemostatic mechanism of the body is sufficient to repair damage and stop the secretion of blood from these delicate microcirculation vessels (Chen et al., 2018). The body's mechanism in stopping bleeding involves three main steps, including vascular spasm, formation of platelet plugs, and blood coagulation (formation of blood clots) (Sherwood, 2015). Hemostatic failure causes bleeding and is a dangerous clinical problem (Pierce & Pittet, 2014). An example of a well-known failure of the hemostatic process is hemophilia. Hemophilia is a very common disease which refers to the tendency to experience severe excessive bleeding (Curnow et al., 2016). In the United States, about 1 in 10,000 people suffer from hemophilia with severe severity. From all the case, four out of five cases are caused by factor VIII deficiency (Sacher, 2012). Wound healing is influenced by many factors including the type of medication used. The use of drugs for wound Effect of Broccoli (Brassica oleracea L. var. italica) Extract on Bleeding Time in Male White Mice (Mus musculus L.)


INTRODUCTION
External bleeding is bleeding originating from an open wound so that it can be seen from a physical examination (Guo & DiPietro, 2010). Bleeding is a process of bleeding from blood vessels that can cause damage to blood vessel walls caused by trauma or disease (Xu et al., 2019). The normal functioning hemostatic system is important for the life of the organism because if the hemostatic is disrupted, even a small wound can cause life-threatening bleeding (Periayah et al., 2017).
The normal innate hemostatic mechanism of the body is sufficient to repair damage and stop the secretion of blood from these delicate microcirculation vessels (Chen et al., 2018). The body's mechanism in stopping bleeding involves three main steps, including vascular spasm, formation of platelet plugs, and blood coagulation (formation of blood clots) (Sherwood, 2015). Hemostatic failure causes bleeding and is a dangerous clinical problem (Pierce & Pittet, 2014).
An example of a well-known failure of the hemostatic process is hemophilia. Hemophilia is a very common disease which refers to the tendency to experience severe excessive bleeding (Curnow et al., 2016). In the United States, about 1 in 10,000 people suffer from hemophilia with severe severity. From all the case, four out of five cases are caused by factor VIII deficiency (Sacher, 2012).
Wound healing is influenced by many factors including the type of medication used. The use of drugs for wound Abstract Cessation of bleeding is influenced by many factors including the type of medication used. The use of drugs to stop bleeding can be done in various ways, one of which is the use of traditional medicine. One of the traditional plants that can be used as a stop bleeding is broccoli (Brassica oleracea L. var. italica.) which has a fairly high vitamin K content, where is efficacious as hemostatic. This study aims to determine the effect of broccoli on the bleeding time of mice tail cuts. The first group was negative control (Na-CMC), the second group was positive control (tranexamic acid), while groups three, four, and five were broccoli extract dose of 20, 40, and 60 mg/kg BW, respectively. All treatments were given orally for seven days. Bleeding time was calculated from the initial blood loss until the blood stopped (tail bleeding I method) and the data were analyzed with the ANOVA one way test and the post hoc Least Significant Difference (LSD) statistical test. The results showed that there were differences in the time to stop bleeding in the treatment control group at a dose of 20 mg/kg BW (122.60 ± 29.535 seconds), 40 mg/kg BW (102.40 ± 9.607 seconds) and 60 mg/kg BW (90.40 ± 3.845 seconds). From these results, it can be concluded that the extract of broccoli at a dose of 60 mg/kg BW gives the best results as hemostatic, while the effect is almost similar to the positive control group.
healing can be done with a variety of types and types, one of which is the use of traditional medicine (Guo & DiPietro, 2010;Saghazadeh et al., 2018). The use or treatment is traditionally increasingly preferred because in general there are fewer side effects as well as drugs from chemicals (Yuan et al., 2016).
Broccoli (Brassica oleracea L. var. italica.) is a family of Brassicaceae that contains good phytochemicals such as glucosinolates, phenolic compounds, fiber and antioxidant compounds such as vitamins C and E and minerals (Ca, Mg, Se, and K) (Raiola et al., 2017).
According to United States Department of Agriculture (2012), compared with other vegetables including carrots, cabbage, and spinach, the vitamin K content in broccoli is higher at 101.6 mg or 85% greater than other vegetables.
Based on this background, this study aims to determine the effect of broccoli on the bleeding time of mice tail cuts.

Materials and tools
The material used in this study were broccoli, tranexamic acid (Kalnex®), Na-CMC, natrium chloride 0.9%

Extracting preparation
Broccoli used in this research was 5 kg of fresh broccoli obtained from Samarinda market in Batam City, which has been identified in Herbarium of Universitas Andalas, Padang. Broccoli that has been prepared is then washed using running water. After washing, broccoli is cut into small pieces. Broccoli chunks are then dried in the morning sun for three consecutive days. After drying, broccoli is extracted by maceration method using 70% ethanol for three days while stirring occasionally. The extract obtained was then evaporated using a vacuum rotary evaporator at 40°C to obtain a thick extract.

Alkaloid test
A total of 2 ml of the extract solution was evaporated in a porcelain cup. The residue obtained is then put into a test tube and 5 ml of 2 N HCL is added. The solution is then divided into 2 tubes. Tube 1 is added 2-3 drops of Dragendorff reagent, while tube 2 is added Mayer reagent. Positive results of the alkaloid content are indicated by the formation of red brick, red, or orange colors with the Dragendorff reagent, and white or yellow deposits with the Mayer reagent (Auwal et al., 2014).

Saponin test
As much as 0.5 g of broccoli extract is added to 0.5 ml of hot water. Cool the mixture first to room temperature then shake vigorously for 10 seconds to produce solid foam as high as 1-10 cm. Then 1% HCl is added and waited for 10 minutes. Positive results from the saponin content are shown if the foam does not disappear (Hossain et al., 2013).

Tannin test
Broccoli extract is boiled with 20 ml of water then filtered with filter paper and then added a few drops of 10% FeCl3. A positive result of the tannin content is shown if the solution produces a greenish brown or black-blue color (Batool et al., 2019).

Flavonoid test
As much as 0.5 g of broccoli extract is added to a small amount of Mg powder and then shaken until mixed. The mixture is then added with a few drops of concentrated HCl. The positive results of flavonoids are marked by the formation of orange, red, or yellow (Panche et al., 2016).

Animal care and handling
Test animals used in this study were Swiss Webster male mice selected by purposive sampling with age requirements of around 2-3 months with a body weight of about 20-30 g. The selection of test animals is done by simple random sampling (Samanta et al., 2016).

Provision of test treatment
After going through the acclimation process, test animals are given treatment based on each test group. The negative control group was given 0.5% Na-CMC, the positive control group was given tranexamic acid as much as 102.74 mg/20 g BW. While treatment groups I, II and III, each given a dose of broccoli extract of 20 mg/kg BW, 40 mg/kg BW, and 60 mg/kg BW, respectively.

Bleeding time
Determination of bleeding time is done by calculating the time needed starting from the wound starting to drip blood until the blood stops dripping from the wound.
First the rats' tails were cleaned with 70% alcohol then cut 1 cm from the tail end. The cut tail is inserted into a tube containing warm NaCl (37°C). The duration of bleeding is calculated using a stopwatch from the onset of blood droplets from injured blood vessels until the blood stops flowing out of the blood vessels (Liu et al., 2012). The time interval from the first drop until the blood stops dripping is the bleeding time.

Data analysis
The data obtained were analyzed using SPSS version 21.
To determine the normality of data distribution, tests were performed using the Shapiro-Wilk test.
Homogeneity test was performed using the Levene variant test. Then the Least Significant Difference (LSD) test is performed to see the differences between each treatment group.

Phytochemical screening
Phytochemical  Table I.

Bleeding time
The parameter observed in this test is the bleeding time. inhibiting the breakdown of polymer fibrin by plasmin, so that hemostasis can occur more effectively (Levy et al., 2018 Table II, while the comparison for average bleeding time is presented in Figure 1.   (Periayah et al., 2017).

Statistical analysis
Determination of the normality of data distribution on the percentage of stopping bleeding is done using the

CONCLUSION
Based on the results of this study it can be concluded that the extract of broccoli at a dose of 60 mg/kg BW gives the best results as hemostatic, while the effect is almost similar to the positive control group. Further study needed for further observe the wound healing effect of broccoli extract and from its active metabolites.

ACKNOWLEDGMENT
The author thanks to Pharmacology Laboratory of Sekolah Tinggi Ilmu Kesehatan Mitra Bunda Persada Batam, which has provided facilities and infrastructure so that this research can run smoothly.