INTRODUCTION:

Myocardial infarction (MI) is a cardiovascular disorder that is the leading cause of death worldwide. According to data from the World Health Organization 2017, this disease can cause the death of Cisplatin is a chemotherapy drug used to treat certain types of cancer, such as ovarian cancer, testicular cancer, or bladder cancer.

This drug can be used alone or in combination with other anticancer drugs. approximately 17.9 million sufferers every year and has a 31% rate of all death data in the world¹. Cardiotoxicity is a well-known consequence of cancer chemotherapy. Cisplatin is a potent chemotherapy drug that is associated with several side effects such as myelotoxicity, gastrointestinal toxicity, ototoxicity neurotoxicity, cardiotoxicity, and nephrotoxicity^2-5. Cardiotoxic manifestations of cisplatin chemotherapy have been described: heart failure, angina, acute myocardial infarction, thromboembolic events, autonomic cardiovascular dysfunction, both hypertension and hypotension, myocarditis, pericarditis, and severe congestive cardiomyopathy. Manifestations of cisplatin-induced arrhythmias have also been reported in the literature: supraventricular tachycardia, ventricular arrhythmias, atrial fibrillation, occasional sinus bradycardia, and sometimes complete atrioventricular block as well^6-10. Vascular endothelial damage, induction of coronary vasospasm, significant oxidative and nitrosative stress, impaired platelet function and platelet apoptosis (via the ERK signaling pathway), platelet aggregation, hydro-electrolytic imbalance, abnormalities of ventricular repolarization and QT interval, left systolic or diastolic ventricular dysfunction, depression of cardiomyocyte contraction, mitochondrial abnormalities, and increased endoplasmic reticulum stress are cited as causes. Acute cardiovascular toxicity by cisplatin thought to be caused by direct damage to the vascular endothelium due to the increase in von Willebrand factor (a factor released by endothelial cells due to vascular damage) during chemotherapy has been described. Acute insulin resistance has been described as well. Both direct diffuse endothelial damage and an increased incidence of cardiovascular disease risk factors have been described to explain the delayed cardiovascular toxicity of cisplatin, up to years, leading to coronary artery disease and systolic or diastolic left ventricular dysfunction leading to severe congestive cardiomyopathy^11-14. Free radical reactions have been implicated in the pathology of many human diseases including atherosclerosis, ischemic heart disease, the aging process, inflammation, diabetes, immunodepression, neurodegenerative diseases, and other disease states. Radicals and other reactive oxygen species are formed constantly in the human body and are eliminated by enzymatic and non-enzymatic antioxidant defense systems. Oxidative stress that occurs when antioxidant defenses are inadequate can damage lipids, proteins, carbohydrates, and DNA. (Ocimum bacilicum) which belongs to the Lamiaceae family as a rich source of phytochemical compounds that can show countless pharmacological effects. The contents include carotenoids, terpenoids, alkaloids, saponins, flavonoids, tannins and volatile oils which are useful as antioxidants, inhibitors of bacterial growth, digestive disorders, anticarcinogenic, neuroprotective, cardioprotective, anticoagulant, immunomodulator, analgesic/antipyretic, anti-inflammatory, antidiabetic, hepatoprotective, hypolipidemic, anti-stress activity, even essential oil of basil leaf extract is useful as a fungistatic, insecticide, nematicide¹⁵. This study aims to determine the cardioprotective activity of extract ethanol Ocimum basilicum on rats induced cisplatin.

MATERIALS AND METHOD:

Materials:

Ocimum bacilicum dried plant, 96% ethanol, filter paper, aluminum foil, 3ml syringe, 1ml syringe, distilled water, CMC Na, DPPH, methanol, vitamin C, gallic acid, Folin-Ciocalteu solution, sodium carbonate, quercetin, aluminum chloride, sodium acetate, CK-MB, LDH elisa kit.

Plant

Sampling was carried out purposively without comparing with the same plants from other areas. The samples were obtained from the Simpang Limun traditional market in Medan, North Sumatra.

Extraction process Ocimum bacilicum

Ocimum bacilicum extract was made by maceration using 96% ethanol. Put the simplicia into a dark container. Pour 75 parts of the liquid filter, cover, leave for 5 days protected from light while stirring frequently, shred, squeeze, wash the dregs with enough liquid filter. Transfer to a closed vessel, leave in a cool place, protected from light, for 2 days. Pour clear liquid so as not to waste the sediment. The macerate was evaporated with a rotary evaporator at a temperature of ± 40°C until a thick extract was obtained¹⁶. The extract continuously determine that phytochemical content.

Antioxidant Activity of Ethanol Extract of Ocimum bacilicum:

The principle of testing antioxidant activity using the DPPH method is to look at the ability of the test preparation (Ethanol Extract of Ocimum bacilicum) in reducing the oxidation process of DPPH free radicals (1,1 diphenyl-2-picrylhydrazyl) which are free radicals in methanol solution (so that the DPPH color changes from purple to yellow) with an IC50 value (concentration of the test preparation capable of reducing free radicals by 50%) was used as a parameter to determine the antioxidant activity of the test preparation¹⁷.

Total Phenol of Ethanol Extract of Ocimum bacilicum:

Weighed 10.5mg of Ethanol Extract of Ocimum bacilicum then dissolved with methanol, put into a 10 mL volumetric flask, made up to the mark line. 0.5mL of this solution was pipetted and 2.3mL of distilled water and 0.2mL of Folin-Ciocalteu solution were added, vortexed for ±1 minute then allowed to stand for 4-8 minutes, added 2mL of 20% sodium carbonate solution and allowed to stand for 70minutes and the absorbance was measured at a wavelength of 775nm. Measurements were carried out with 5 repetitions. The phenol concentration was calculated from the substitution in the linear equation and expressed as the equivalent number of milligrams of gallic acid in 1 gram of the extract¹⁸.

Total Flavonoid of Ethanol Extract of Ocimum bacilicum:

Weighed as much as 10.5mg of Ethanol Extract of Ocimum bacilicum, dissolved in ethanol, put into a 10 mL volumetric flask, filled with methanol to the mark line. 0.5mL of this solution was pipetted, added 1.5mL of methanol, 0.1mL of 10% aluminum chloride solution, 0.1mL of 1 M sodium acetate and 2.8mL of distilled water and allowed to stand for 30 minutes then the absorbance was measured at wavelength of 436nm. Measurements were carried out with 5 repetitions. The flavonoid concentration was calculated from the substitution in the linear regression equation and expressed as the equivalent number of milligrams of quercetin in 1gram of extract¹⁹.

Cardioprotective activity of Ethanol Extract Ocimum bacilicium:

Each treatment group contained at least 4 male rats. The researcher chose to use 5male rats per group to maintain the death of the experimental animals with the number of treatment groups as many as 6 groups so that the total number of research samples was 30, which were divided into:

a. group 1 (K1), the normal group, were not given any treatment, only given food and drink normally (ad libitum) in their cage.

b. Group 2 (K2), negative control, only induced cisplatin 50mg/kgbb

c. Group 3 (K3), positive control, + quercetin 150 mg/kg BW/day.

d. Group 4 (K4), cisplatin 50mg/kgbw + treatment with ethanol extract of basil leaves at a dose of 50mg/kg BW/day.

e. group 5 (K5), cisplatin 50mg/kgbw + treatment with ethanol extract of basil leaves at a dose of 100mg/kg BW/day.

f. group 6 (K6), cisplatin 50mg/kgBB + treatment with ethanol extract of basil leaves at a dose of 300mg/kg BW/day.

Determination of the treatment dose following the research conducted by conducting a preliminary study.

The research process begins by providing adaptation (acclimatization) to male white rats for 2 weeks. After adaptation, the test animals were then randomly divided into 6 groups, all rats were given cisplatin injection at the end of the first at a dose of 50mg/kg bw except in the normal group. Then on day 2 to day 15, the extract group was given doses of 50, 150, and 300mg/kg and the positive control group was given quercetin 150 mg/kg. On the 16th day, the rats were dissected, and 3 ml of blood was taken from the heart and then centrifuged for 10 minutes at 10,000 RPM then the supernatant area was taken and further analysis was carried out on the levels of CK-MB, and LDH using the elisa kit.

RESULT AND DISCUSSION:

Phytochemical screening of simplisia powders and extracts:

The particle size of Phytochemical screening of simplisia powders and extracts is shown in Table 1. The results show that simplisia and crude contain the same chemical compounds namely alkaloids, flavonoids, tannins, saponins, glycosides in extracts and powders simplisia.

Table 1: Phytochemical screening of simplisia powders and crude

S. No	Screening	Simplisia	Crude
1	Alkaloida	+	+
2	Flavonoid	+	+
3	Tanin	+	+
4	Saponin	+	+
5	Glikosida	+	+
6	Steroid/ triterpenoid	-	-

Flavonoids are phenyl compounds substituted for benzopyran derivates consisting of the basic framework C15 (C6-C3-C6). Some plants containing flavonoid derivatives, have been used as disease prevention and therapeutic agents in traditional medicine in Asia for thousands of years, including as anticancer, immunomodulatory.¹⁴ The presence of flavonoids, saponins, tannins, glycosides in ethanol extract and ethilasetat indicates that the extract has anti-inflammatory and immunomodulatory effect.

Antioxidant result:

Relationship between concentration and percentage of DPPH free radical trapping by Ocimum bacilicum extract can be seen in the table 2.

Table 2: Relationship between concentration and percentage of DPPH inhibition

Concentration (µg/ml)	Absorbance	% Inhibition
0	0.9848	0
20	0.8514	13.54589764
40	0.7473	24.11657189
60	0.6588	33.10316816
80	0.5665	42.47562957
100	0.466	52.68074736

The tabel 2 shows that the concentration were 0, 20, 40, 60, 80, and 100ug/ml and the percentage of inhibition 0, 13.54589764, 24.11657189, 33.10316816, 42.47562957, and 52.68074736. table 3 below shows the IC50 (inhibitory contration) 50 of ethanol extract Ocimum bacilicum dan vitamin c as standart.

Table 3. IC50 of the extract

Samples	Regression equation	IC₅₀ (ppm)
Vitamin C	Y= 17.54872X + 2.7315	3,238032168
Extract	Y= 15,1547X + 0,9285	93,55045422

Table 2 The shows that the regression equation of vitamin c is Y= 17.54872X+2.7315 and the IC50 is 3,238032168 that lower than extract the regression equation is Y= 15,1547X+0,9285 while the IC50 is 93, 55045422. The antioxidant activity of vitamin C is higher than extract.

Total Phenol of Ocimum bacilicum Extract:

The results of total Phenol Ocimum bacilicum extract can be seen in the table 4 below:

Table 3 shows that total phenol of Ocimum bacilicum high, averagely 9,609654292 (GAE/g extract).

Total Flavonoid of Ocimum bacilicum Extract:

The results of total Flavonoid Ocimum bacilicum extract can be seen in the table 4 below:

Table 3 shows that total flavonoid of Ocimum bacilicum high, averagely 12,73895682 (quercetin/g extract).

LDH Level:

The LDH level can be seen in the table 5.

Table 5. LDH Level

Group	LDH Level (ng/mL)
K1	0,954 ± 0,0434*
K2	11,987 ± 0,103
K3	8,446 ± 0,04*
K4	11,137 ± 0,07
K5	8,046 ± 0,014*
K6	7,345 ± 0,064*

Information: *(p<0,05) significantly different with negative group (K2)

Table 5. show that in group negative (K2) increase the level of LDH 11,987±0,103ng/mL meanwhile in group given extract dose 100mg/kgBB (K5) and dose 300 mg/kgBB (K6) show 8,046±0,014ng/mL and 7,345± 0,064ng/mL.

CK-MB Level:

The CK-MB level can be seen in the table 6.

Table 6: CK-MB level

Group	CK-MB Level (ng/mL)
K1	248,324 ± 15,873
K2	1711,429 ± 17,873
K3	1231,38 ± 20,973
K4	1399,31 ± 24,978
K5	1233,113 ± 29,873
K6	1200,427 ± 15,924

Information: *(p<0,05) significantly different with negative group (K2)

Table 6. show that in group negative (K2) increase the level of CK-MB 1711,429±17,873ng/mL meanwhile in group given extract dose 100mg/kgBB (K5) and dose 300mg/kgBB (K6) show 1233,113±29,873ng/mL and 71200,427±15,924ng/mL.

Ocimum bacilicum one of the plants that widely distributed in Indonesia. Generally called as kemangi which consumed by Indonesian as lalapan. Ocimum bacilicum contain many secondary metabolites which have excellent antioxidant properties²⁰. In this study revealed that ethanol extract of Ocimum bacilicum has strong antioxidant properties which shown has IC50 93,55045422ppm meanwhile vitamin c as standar has antioxidant properties 3,238032168ppm. Antioxidant has uplifting pharmacological effect of extract because have preventive and curative effect whether in cardiovascular, metabolic and etc²¹. in this study show that ethanol extract of Ocimum bacilicum has cardioprotective effect by reducing CK-MB and LDH level. O. basilicum extract has strong antioxidant activity that protects against oxidative stress by increasing levels of antioxidant defense enzymes along with a significant reduction in lactate dehydrogenase activity and lipid peroxidation. The antioxidant activity of basil extract is attributed to polyphenols, flavonoids, and compounds such as rosmarinic acid, which are present in high amounts. Two biologically active compounds – anthocyanins and phenolic acids, as well as estragole, methyl cinnamate, methyl eugenol, -cadinol, linoleic acid, and -bergamoten also contribute to the antioxidant activity of O. basilicum. The antioxidant activity of basil extract is comparable to that of -carotene showed that rosmarinic acid, the main active constituent found in the ethanolic extract of basil, had superior antioxidant activity compared to vitamin E (α-tocopherol). In this study used cisplatin as inducer of cardiotoxic effect. Cisplatin has a great effect as chemotherapy agent which specifically used as anticancer for ovarium cancer. But lately the rising data shown that cisplatin has side effect specially cardiotoxic effect²². So in this study found that extract ethanol of Ocimum bacilicium has cardioprotective activity which shown in the data dose 300mg/kg BB down lifting the CK-MB and LDH levels which as one of the biomarker which continuously use to confirm cardiac injury. In the future the using of cardioprotective effect will be used to prevent the cardiotoxic effect of cisplatin.

CONCLUSION:

Overall, it can be concluded that extract ethanol of Ocimum bacilicum has cardioprotective activity on rats induced cisplatin by reducing the level of CK-MB and LDH.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENT:

This study supported by Kementerian Pendidikan, Kebudayaan, Riset, dan Teknologi by DRTPM Contract number 057/LL1/LT/K/2022, 454/N/LPPM-UMI/2022.

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Received on 25.08.2022 Modified on 08.10.2022

Research J. Pharm. and Tech 2023; 16(12):5770-5774.

DOI: 10.52711/0974-360X.2023.00934

Name of Sampel	Concentration (ppm)	Vol Sample (L)	Wight Sampel (g)	TOTAL Phenol (GAE/g extract)	TOTAL Phenol (Average)
Ocimum bacilicum 1	9,925619835	0,025	0,0258	9,617848677	9,609654292
Ocimum bacilicum 2	9,743801653	0,025	0,0252	9,666469894
Ocimum bacilicum 3	9,73553719	0,025	0,0255	9,544644304

Nama Samp	Concentration (ppm)	Vol Sample (L)	Wight Sampel (g)	Total Flavonoid (quercetin/g extract)	Total Flavonoid (Average)
Ocimum bacilicum 1	12,96285714	0,025	0,0258	12,56090808	12,73895682
Ocimum bacilicum 2	13,00285714	0,025	0,0252	12,89965986
Ocimum bacilicum 3	13,01142857	0,025	0,0255	12,75630252