Cardioprotective Effect of Chloroform Extract of Arcangelisia flava on Doxorubicin-induced Cardiomyopathy

Long-term use of doxorubicin as cancer chemotherapeutic agent would cause tissue toxicity, including cardiotoxicity. Arcangelisia flava is suggested to have cardio protective effect. This study aimed to determine the effect of chloroform extract of A. flava leaves on cardio histopathology of doxorubicin-treated Wistar white male rats. Wistar male rats were divided into four groups (1) control group; (2) doxorubicin 7.5 mg/kgBW intraperitoneally twice (day 1 and 6); (3) doxorubicin + chloroform extract of A. flava leaves 250 mg/kgBW/day orally for 11 days; (4) Chloroform extract of A. flava leaves 250 mg/kgBW/day orallly for 11 days. At the 12th day, the rats were sacrificed; the heart organ was taken to make histopathological preparations and analyzed using HE staining. Vacuolization and necrosis are the parameters used in evaluating this effect. The phytochemical screening was also done to determine the compounds in chloroform extract of A. flava leaves. Based on the HE staining, chloroform extract of A. flava leaves decreased the cardiotoxicity caused by doxorubicin. The phytochemical screening showed that chloroform extract of A. flava leaves contains flavonoid, tannin, alkaloid, and triterpenoid. The cardioprotective effect of chloroform extract of A. flava leaves was suggested to be contributed by the flavonoid, tannin, and triterpenoid.


INTRODUCTION
Cancer is caused by the abnormal and uncontrolled body tissue cells growth. WHO reports that 1 cancer patient dies every 11 minutes and new cancer case appears every 3 minutes (Brundtland, 2003). Cancer could be prevented by avoiding its triggers and improving diet. Cancer treatments mostly applied are chemotherapy, radiotherapy, surgery, and hormones therapy (Fong & Tse-Ling, 2002). Chemotherapy is a method uses chemical compounds to suppress or stop cell proliferation, and destroy cancer cells (cytotoxic). Doxorubicin is an anthracycline antibiotic that is considered to be effective as an anticancer chemotherapy agent and is widely used. But it provides several side effects including immune system depletion, hair loss, sore throat, hepatotoxicity, and irreversible cardiotoxicity or hepatotoxicity (Frias et al., 2010).
The search of anticancer agent is still progressing. One potentially useful plant for anticancer therapy is Arcangelisia flava (Keawpradub et al., 2004), and endemic plant found in Meru Betiri National Park, Jember. Based on Keawpradub et al. (2004), chloroform extract of A. flava leaves has antioxidant and cytotoxic effect on brine shrimp larvae and MCF-7 breast cancer cells. chloroform extract of A. flava leaves contains berberine, palmatin, and jatrorrhizin, flavonoids, and saponins (Maryani et al., 2013). The saponin in chloroform extract of A. flava leaves is known to provide hepatoprotective activity in rat liver induced by paracetamol with an antioxidant mechanism (Achmadi et al., 2006). Flavonoids are able to suppress the formation of reactive oxygen species (ROS), and chelate metal ions (Scalbert et al., 2005). Chloroform extract of A. flava leaves is potential as a co-chemotherapy agent to reduce the cardiotoxic effects of doxorubicin.
This study aimed to determine the effect of chloroform extract of A. flava leaves on the cardio histopathology on white male Wistar strain rat induced by doxorubicin.

Materials
A. flava leaves were obtained from Meru Betiri National Park, doxorubicin was obtained from the Regional Hospital dr. Soebandi Jember, solvent nhexane, chloroform, formalin 10%, hematoxylin eosin, xylol, and liquid paraffin. The test animals were Wistar strain male white rats, aged 8-10 weeks, weighing 140 ± 30 grams. The test animals were kept in standard conditions and fed with standard commercial pellets and ad libitum as drink.

Preparation of chloroform extract
Chloroform extract of A. flava leaves was made based on research by Keawpradub et al. (2004) with a slight modification. A. flava leaves were sorted and dried, then grinded into powder form. A total of 1 kg dried leaf powder was extracted successively with nhexane and chloroform, each with 3 replications. The chloroform extract was then concentrated using a rotary evaporator and dried in an oven at 50⁰C. A suspension of chloroform extract of A. flava leaves was made in 0.5% CMC Na before use.

Animal experimental
All test animals were acclimatized for 10 days in a cage before given any treatment. Test animals were divided into 4 groups of 5 mice each. Group I, the control group, was given a 0.5% CMC Na solution (po) for 11 days. Group II was given doxorubicin (7.5 mg/kg BW) (ip) twice in 11 days (day 1 and day 6). Group III was given chloroform extract of A. flava leaves (po) at a dose of 250 mg/kg BW/day every day for 11 days and doxorubicin (7.5 mg/kg BW) (ip) twice in 11 days (day 1 and day 6). Group IV was given chloroform extract of A. flava leaves (po) at a dose of 500 mg/kg BW/day every day for 11 days and doxorubicin (7.5 mg/kg BW) (ip) twice in 11 days (day 1 and day 6) (Raksamiharja et al., 2012).

Cardio histopathology
On the 12 th day, the rats were sacrificed, then dissected and their hearts were taken to make histopathological microscope slide set. The heart was cleaned from the fat, washed with distilled water, dried using tissue, and fixed in 10% formalin solution to make histopathological microscope slide set using the paraffin method with the following procedure: fixation stage using formalin 10%; the dehydration stage using gradient alcohol concentrations (70, 80, 95, to 100%); the clearing stage using xylol; the impregnation stage at a temperature of 56-60⁰C; and the embedding stage using paraffin at 56-60⁰C. The samples were then stored in the freezer until it freezes.
The cutting was carried out using a microtome blade. Then, the tissue was stained using hematoxylin eosin (HE) method. The colors obtained are a blue cell nucleus and red cytoplasm. The final result were observed under a light microscope under 400 and 1,000 X magnification, and photographed. The degree of tissue damage was determined based on the Billingham scoring (1978).

Data analysis
The HE histopathological preparations was observed based on Billingham scoring system (Billingham et al., 1978) as described in Table 1.
Statistical analysis was performed using Kruskall-Wallis, followed by post-hoc tests (using Mann-Whitney) for multi-group comparisons. p < 0.05 was considered as statistically significant. 3 Myocyte cell necrosis >35% of all cells.

Phytochemical screening
Identification of compounds in the chloroform extract of A. flava leaves was done using thin layer chromatography. A total sample of 20 mg was dissolved in 10 ml of chloroform and examined using TLC Stationary phase: kiesel gel GF 254 Mobile phase: toluene -ethanol -NH3 25% (4: 3: 1).

Flavonoid Identification
Given the appearance of ammonia and citroborate vapor stains, evaluated on visual observations using UV-365 nm. The indications of the flavonoids presence in the extract are yellowish-green spots appear on visual observations, and red-orange under UV-365 nm (Robinson, 1995).

Tannin Identification
FeCl3 stain was used to detect tannin. Black color appearance on visual observations indicates the presence of tannin (Fong & Tse-Ling, 2002).

Alkaloid Identification
Given the appearance of dragendorf stain to detect the presence of alkaloids (Wagner & Grevel, 1982). Positive results of alkaloids are shown by the presence of orange-brownish brown stains on visual observation (Pascual et al., 2002).

Triterpenoid identification
Given the appearance of anisaldehyde reagent stains, then the plates are heated, and evaluated on visual observations. If a red-violet color appears, it indicates the triterpenoids presence (Wagner & Grevel, 1982).

Cardio histopathology observation
The results of microscopic observations representing each treatment group are shown in Figure 1. The microscope slide sets are cardiac preparations; cross section; with HE staining which shows the difference in the number of necrotic myocardial cells between treatments. The limit of one value variation to another is not clear so only the number of necrotic myocardial cells and vacuolization are comparable. The cardio histopathology preparation in the doxorubicin group (400X mag.) showed a cytoplasmic image that tends to be eosinophilic (reddish) with homogeneous color features ( Figure 2). Yellow arrows pointing at the coagulated or compacted cell nucleus, which is called picnotics.
The more detailed observations of rat heart muscle cells were carried out at 1.000X magnification (Figure 3). It showed that the cardiac muscle cells that were induced by doxorubicin have vacuolized (red arrows). Observations of cardiac muscle cells were analyzed in accordance with the Billingham scoring degree (1978) as seen at Figure 4. The higher the score value, the more severe the damage occurs. The group that received doxorubicin had more severe cell damage (necrosis and vacuolization) than that of the control group. Chloroform extract of A. flava leaves was proven to reduce damage on heart muscle cells. Due to administration of doxorubicin, the decreasing damage of heart muscle cells was in line with chloroform extract of A. flava leaves dose. The greater the chloroform extract of A. flava leaves dose, the greater the heart muscle cells repair. The chloroform extract of A. flava leaves dose of 500 mg/kg BW showed a histopathological profile of rat heart muscle cells with no difference from that of the control group. Cancer therapy with chemotherapy agents such as doxorubicin can cause several side effects, including nausea, vomiting, myelosuppression, hair loss, and cardiotoxicity side effects that can limit the potential use of doxorubicin therapy. Cardiotoxicity is the side effect that depends on the high dose of doxorubicin. Cardiotoxicity can also cause permanent damage to the heart organ that leads to the development of deadly heart failure. One of the cardiotoxic effects is cardiomyopathy; a decrease in myocardial function caused by changes in the histological structure of the myocardium. In the cardiomyopathy case, histologically it will show cardiac atrophy, nuclear pycnotic, myocardial edema and cytoplasmic vacuole (Pontes et al., 2010).
Histopathological examination of the normal rat's heart showed a clear picture of the myocardial cell membrane structure (Fig. 1). The heart muscle cells damage caused by doxorubicin is indicated by the presence of necrosis and vacuolization. The picture of necrosis and vacuolization in the doxorubicin group showed significantly different results compared to the control group. The administration of chloroform extract of A. flava leaves was proven to reduce heart muscle cell damage which is in line with the large dose of chloroform extract of A. flava leaves. The greater the chloroform extract of A. flava leaves dose, the greater the heart muscle cells repair.
The chloroform extract of A. flava leaves group with a dose of 500 mg/kg BW did not show significantly different results from that of the control group, meaning that the dose was able to prevent or repair damaged heart muscle cells due to doxorubicin administration. Therefore, it opens up opportunities for the use of chloroform extract of A. flava leaves as a doxorubicin co-chemotherapy agent, primarily aimed at reducing the side effects of cardiotoxicity.

Phytochemical screening
Phytochemical screening was carried out to determine whether chloroform extract of A. flava leaves contains flavonoids, tannins, alkaloids, and triterpenoids by TLC method (Table 2). Phytochemical screening results showed that chloroform extract of A. flava leaves contains flavonoid, tannin, alkaloid, and triterpenoid compounds.
Flavonoid and tannin compounds have antioxidant activity (Scalbert et al., 2005). Tannins have antioxidant effect, therefore it provides cardioprotective activity. Based on Panchal & Brown (2013), the tannin content in Quercus petraea extract can improve the profile of biochemical parameters in rat heart muscle cells exposed to doxorubicin. Tannin cardioprotective activity is caused by free radical scavenging activity, activation of antioxidant enzymes (Mamta & Sashi, 2012), and lipid peroxidation inhibitors inside tannins (Oliboni et al., 2011).   (Scalbert et al., 2005). The pharmacological effects of flavonoids that provide cardioprotective effects on rat heart muscle cells exposed to doxorubicin are antioxidant and chelating effects (van Acker et al., 2001). Those flavonoids activities are associated with the inhibition of oxidative stress due to excessive production of ROS. It causes a Fenton reaction which produces hydroxyl ions (OH + ), which is highly reactive to all of the molecules causing the formation of lipid peroxides, oxidized protein, and DNA oxidation (Takemura & Fujiwara, 2007). The activity of flavonoids as chelating iron will chelate iron Fe 2+ so it would not form Fe which triggers the formation of the Fenton reaction (Mira et al., 2002). Therefore, chloroform extract of A. flava leaves may be used as a doxorubicin cochemotherapy agent because it reduces the cardiotoxic side effects of doxorubicin by reducing damage to heart muscle cells based on the histopathological profile.
Based on the results of phytochemical screening, it showed that the chloroform extract of A. flava leaves contains triterpenoids. According to research by Shanmugarajan et al., (2008), triterpenoids from Ficus hypida could increase the reduction in glutathione (GSH) in rat cardiotoxicity of rat-induced cyclophosphamide chemotherapy agents. GSH is the first defense against ROS derived from endogenous sulfhydryl (SH). Triterpenoid β-amirin compounds play a role in increasing GSH, they can provide cardioprotective effects in the rats exposed to cyclophosphamide.
The results of this study indicate that chloroform extract of A. flava leaves has the potential as a co-chemotherapy agent to reduce the side effects of doxorubicin cardiotoxicity as seen from the histopathological profile of the heart muscle cells of doxorubicin-induced male Wistar strain rats. Based on the research by Shanmugarajan et al. (2008), groups of flavonoid compounds, tannins, and triterpenoids have cardioprotective effects on chemotherapy agents-induced rats. The the presence of flavonoid, tannin, and triterpenoid on chloroform extract of A. flava leaves suggested to contribute on the cardioprotective effects.
Cardioprotective dose which gives a significant change is chloroform extract of A. flava leaves at the dose of 500 mg/kg BW. It proves that the higher the extract dose, the greater the levels of flavonoids, tannins, and triterpenoids, and will give higher cardioprotective effect. The chloroform extract of A. flava leaves has the potential to be developed as a co-chemotherapy agent for doxorubicin. However, whether or not this extract can increase the effectiveness of chemotherapy, further research is needed to find out the chloroform extract of A. flava leaves's safety and toxicity, and also a research using biochemical parameters of cardiac muscle cell damage (CK-MB and troponin).

CONCLUSION
Chloroform extract of A. flava leaves 250 mg/kg BW and 500 mg/kg BW can reduce the cardiotoxicity of doxorubicin in the heart of male Wistar strain rats. The reduction of cardiotoxicity is dose dependent. The dose of chloroform extract of A. flava leaves 500 mg/kg BW showed histopathological profiles of rat heart muscle cells which are similar with the control group.