Faculty of Medicine, Universitas Islam Indonesia Farida Juliantina Rachmawaty, Laksamana Olan Es Orlando ABSTRAK

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1 THE POTENTIAL OF ETHANOL EXTRACT OF RED BETEL VINE LEAVES (Piper crocatum) AS ANTIOXIDANT IN MICE BALB/C EXPOSED TO CIGARETTE SMOKE (IN VIVO TEST) Farida Juliantina Rachmawaty, Laksamana Olan Es Orlando Faculty of Medicine, Universitas Islam Indonesia ABSTRAK Penelitian ini bertujuan untuk menilai kemampuan ekstrak etanol daun sirih merah sebagai antioksidan untuk tikus yang terpapar asap rokok dengan cara in vivo. Sebanyak 120 perempuan mencit balb/ c yang digunakan dengan 60 tikus untuk percobaan 1 (diberi ekstrak sebelum terpapar asap rokok) dan 60 tikus untuk percobaan 2 (diberi ekstrak setelah menghentikan paparan asap rokok). Tikus-tikus tersebut terpapar asap rokok di pagi dan sore hari. Mereka tikus dibagi secara acak menjadi 4 kelompok. Tiga kelompok menerima dosis ekstrak etanol dari daun sirih merah anggur, dengan konsentrasi yang berbeda (2,28 mg, 11,4 mg dan 22,8 mg). Satu kelompok menjadi kelompok kontrol yang menerima cairan steril. Setelah 10 hari terapi, sekitar 5 tikus dari masing-masing kelompok dihentikan dan paru-paru diambil untuk pemeriksaan histopatologi dengan hematoxylin & Eosin (H & E) pewarnaan. Pengobatan diulang pada hari kedua puluh dan tiga puluh dan tubuh tikus ditimbang setiap 10 hari. Data dianalisis dengan menggunakan uji statistik Kruskal-Wallis dan U Mann Whitney. Pada percobaan 1, berat rata-rata tikus menurun sampai akhir penelitian (kecuali untuk perlakuan dengan ekstrak 2 pada hari ke -20. Sementara itu, pada pemeriksaan histopatologi, hasil uji Kruskal-Wallis tidak signifikan secara statistik (p> 0,05). Dalam percobaan 2, berat badan tikus meningkat lebih signifikan daripada tikus dalam kelompok kontrol. Dari pemeriksaan histopatologi, hasil uji Kruskal Wallis menunjukkan perbedaan yang signifikan (p <0,05), yang kemudian diikuti dengan uji statistik U Mann Whitney. Pada hari kesepuluh pengobatan, ekstrak kelompok 1, 2 dan 3 memiliki perbedaan yang signifikan dibandingkan dengan kelompok kontrol. Sementara itu, dalam pengobatan 20-hari, ekstrak kelompok 3 adalah satu-satunya kelompok yang memiliki perbedaan yang signifikan. Dalam pengobatan 30-hari, ekstrak kelompok 2 dan 3 berbeda nyata (p <0,05). Ekstrak etanol daun sirih merah (P. crocatum) dengan cara in vivo dapat antioksidan untuk tikus Babl / c terkena asap rokok, tetapi tidak bisa antioksidan dalam percobaan 1 karena sumber masalah, yang paparan asap rokok, telah dihentikan. Kata kunci: ekstrak etanol, sirih merah (Piper crocatum), antioksidan, asap rokok ABSTRACT This study aims to assess the ability of ethanol extract of red betel vine leaves as antioxidant for mice exposed to cigarette smoke in the in vivo way. A total of 120 female mice balb/c were used with 60 mice for experiment 1 (given extract before exposed to cigarette smoke) and 60 mice for experiment 2 (given extract after stopping cigarette smoke exposure). The mice were exposed to smoke in the morning and afternoon. Those mice were divided randomly into 4 groups. Three groups received a dose of ethanol extracts from the red betel vine leaves, with different concentrations (2,28 mg, 11.4 mg and 22.8 mg). One group became control group that received sterile liquid. After 10 days of therapy, about 5 mice of each group were terminated and the lungs were taken for histopathological examination by hematoxylin & eosin (H&E) staining. The treatment was repeated in the twentieth and thirtieth day and the mice s bodies were weighed every 10 days. The data were analyzed using the Kruskal-Wallis statistical test and U Mann Whitney. In the experiment 1, the average weight of the mice decreased until the end of the study (except for the treatment with the extract 2 in the twentiethday). Volume 7, No.2, Desember

2 Farida Juliantina Rachmawaty, Laksamana Olan Es Orlando Meanwhile, on the histopathological examination, the result of the Kruskal-Wallis test is not statistically significant (p>0.05). In the experiment 2, the body weight of mice increased more significant than the mice in the controlled group. From the histopathological examination, the result of Kruskal Wallis test showed significant differences (p<0.05), which was then followed by the statistical test of U Mann Whitney. At the tenth day of treatment, extract group 1, 2 and 3 had significant difference compared to the control group. Meanwhile, in the 20-day treatment, extract group 3 is the only group that had significant difference. In the 30-day treatment, extracts group 2 and 3 were significantly different (p< 0.05). The ethanol extract of red betel vine leaves (P. crocatum) in the in vivo way can be antioxidant for mice Babl/c exposed to cigarette smoke, but it cannot be antioxidant in experiment 1 because the source of the problem, which is cigarette smoke exposure, has been stopped. Keywords: ethanol extract, red betel vine (Piper crocatum), antioxidant, cigarette smoke INTRODUCTION Smoking is a serious social and health problem. The number of smokers in Indonesia is the third highest after China and India. In Indonesia, about 824 thousand factory workers rely their lives on tobacco companies (Mboy, 2013). On one hand, many people get health problems because of cigarette smoke, but on the other hand a lot of people get income from the cigarette factory. Whether we realize it or not, smoking is harmful to the health of both active smokers and passive smokers. Much research has shown a link between smoking and incidence of lung cancer, larynx cancer, cardiovascular disease and others. However, the dependence on cigarettes is not easily removed (Kusuma, 2012). Basically free radicals can come from pollution, dust and produced continuously as a consequence of normal metabolism (Sarastani, 2002). But smoking is an activity that intentionally incorporate various kinds of harmful substances that can increase the amount of free radicals in the body. The human body is designed to accept natural intake, so that when it receives input like cigarette smoke, the body will attempt to remove a variety of toxic chemicals from the body through metabolic processes, but this metabolic process is actually generate free radicals (Wikipedia, 2013). Antioxidant is a chemical compound that can reduce the bad influence of free radicals. Antioxidants can be obtained either natural or synthetic. However, the use of these synthetic is gradually limited because of the unintended negative effects. One of natural producers of antioxidants is red betel vine (Piper crocatum). Based on chromatographic studies of red betel vine contain alkaloids, flavonoids, polifenolat compounds, tannins and volatile oil (Safithri & Fahma, 2008; Subarnas, 2008). Red betel vine also contains saponins (Rachmawaty, 2014). Suratmo (2008) in his study has reported that red betel vine (P. crocatum) can serve as a very powerful antioxidant with IC50 values of ppm. The study is an in vitro study. In order to be widely applicable and can be proven scientifically, then this research will test red betel vine in vivo in Balb/c mice. Red betel vine tested form of ethanol extract of red betel vine leaves. The selection of 70% ethanol is done because the compound is predicted to be an antioxidant from polar flavonoids. The use of 70% ethanol is similar to polar nature of water but it is more durable. Meanwhile, ethanol is less toxic when compared with methanol. The purpose of this study is to assess the potential of ethanol extract of red betel vine leaves (P. crocatum) as an antioxidant against Balb/c mice which were exposed to cigarette smoke in the in vivo way. Volume 7, No.2, Desember

3 THE POTENTIAL OF ETHANOL EXTRACT OF RED BETEL VINE LEAVES (Piper crocatum) AS ANTIOXIDANT IN MICE BALB/C EXPOSED TO CIGARETTE SMOKE (IN VIVO TEST) Antioxidants can be obtained either natural or synthetic. However, the use of these synthetic is gradually limited because of the unintended negative effects. One of natural producers of antioxidants is red betel vine (Piper crocatum). Based on chromatographic studies of red betel vine contain alkaloids, flavonoids, polifenolat compounds, tannins and volatile oil (Safithri & Fahma, 2008; Subarnas, 2008). Red betel vine also contains saponins (Rachmawaty, 2014). Suratmo (2008) in his study has reported that red betel vine (P. crocatum) can serve as a very powerful antioxidant with IC50 values of ppm. The study is an in vitro study. In order to be widely applicable and can be proven scientifically, then this research will test red betel vine in vivo in Balb/c mice. Red betel vine tested form of ethanol extract of red betel vine leaves. The selection of 70% ethanol is done because the compound is predicted to be an antioxidant from polar flavonoids. The use of 70% ethanol is similar to polar nature of water but it is more durable. Meanwhile, ethanol is less toxic when compared with methanol. The purpose of this study is to assess the potential of ethanol extract of red betel vine leaves (P. crocatum) as an antioxidant against Balb/c mice which were exposed to cigarette smoke in the in vivo way. Figure 1. Red betel vine leaves, the upper green leaves with silver hue, the bottom dark red leaves (Rachmawaty dkk.,2013). MATERIALS AND METHOD The subjects were mice with strain Balb/c. Mice were obtained from laboratory animal provider Faculty of Pharmacy, Gadjah Mada University with certain requirements. The inclusion criteria were female mice, they are in healthy condition when taken, 8-10 weeks old, with grams of bodyweight. The exclusion criteria were dead mice due to disease outside research. Mice that met the inclusion criteria were weighed and then adapted at home for 1-3 days and they were fed with standard drink and food. A total of 60 mice were divided into 4 groups, each 15 mice for the first test and the same amount for the second test. Added 15 mice as a healthy control. Group I: the group given ethanol extract of red betel vine leaves in accordance with concentrations equivalent to 5 pieces of red betel vine leaves in humans (Sudewo, 2008) called extract 1, which is 2.28 mg. Group II: the group given ethanol extract of red betel vine leaves with a concentration of 5 times the concentration of group I referred to as extract 2, i.e mg. Group III: the group given ethanol extract of red betel leaf with a concentration 10 times the concentration of group I called extract 3, which is 22.8 mg. Group IV: a control group,were given a placebo in the form of distilled water. Group V : a healthy control group Testing I A total of 60 mice that met the inclusion criteria were weighed, adapted at home for 1-3 days, and fed with standard food and drink. One day prior to exposure to the smoke, mice were given treatment according to each group. The next day, they were smoked in a special enclosure with 1 cigarette. Mice were kept in cages for 1 hour. Volume 7, No.2, Desember

4 Farida Juliantina Rachmawaty, Laksamana Olan Es Orlando After 1 hour, the mice were excluded from the special enclosure. Fumigation is done in the morning and afternoon, while the treatment of extract and distilled according groups was given once a day as much as 0.5 ml. After 10 days, in each group 5 mice were terminated and their lungs were taken. Lungs were entered in 10% buffered formalin, then the lungs were taken to Anatomic Pathology Laboratory of Gadjah Mada University to be made slide with H&E staining. Treatment was continued in the remaining mice. After 20 days,5 mice in each group were terminated as in the first termination. Similarly, the remaining 5 mice will get the same treatment after 30 days of treatment. Mice were given food and drink according to the standard. Anatomic Pathology Laboratory of Gadjah Mada University to be made slide for hematoxylin and eosin (H&E) staining. Treatment was continued in the remaining mice. After 20 days, the same thing were done for 5 mice in each group. The rest was terminated after treatment for 30 days. RESULTS Mice body weight were weighed from the early research until the end of the study on the first test as described in Table 1. Mice were weighed at baseline, day 10, 20 and 30. Table 1. Summary of Average Mice Body Weight in Testing I Day 10 (gram) Day 20 (gram) Day 30 (gram) Extract 1 Decreased by 1.06 Decreased by 1.88 Decreased by 1.40 Extract 2 Decreased by 0.89 Increased by 0.24 Decreased by 0.74 Extract 3 Decreased by 1.88 Decreased by 0.26 Decreased by 0.51 Aqua Control Decreased by 1.23 Decreased by 0.47 Decreased by 0.65 Healthy Control Increased by 0.85 Increased by 1.70 Increased by 1.72 Testing II A total of 60 mice that met the inclusion criteria were weighed, adapted at home for 1-3 days, and fed with standard food and drink. Mice were divided into 4 groups with 15 mice in each group. In the exposure process, the mice were divided in 2 different enclosures. Every day, the mice were exposed to cigarette smoke in a special enclosure. Each enclosure is smoked with 1 cigarette. After being exposed to smoke, the mice were kept in cages for 1 hour. Fumigation is done 2 times morning and afternoon. The duration of the smoke exposure is according to the results of preliminary test which is 20 days. After exposure to cigarette smoke is done, the next group of mice were treated in accordance respectively. Ten days after treatment, 5 mice per group were terminated, then the lungs were put in pots containing 10% of buffered formalin. The samples were sent to the From the results weighing process obtained, in most of the all treatment, the average body weight of mice is decreasing. The only increase weight in treatment mice, only in the extract 2-treated mice on day 20. Weight loss of mice during exposure with cigarette smoke in accordance with research of Chiolero et al., (2008 ) that the nicotine contained in cigarettes can increase energy expenditure and decrease the appetite so that the body weight tended to decrease for people who smoke compared to those who do not smoke. In this study, the ethanol extract of red betel vine cannot raise the weight of mice (except the extract 2 treatment on day 20). Because until the end of the study, the mice is exposed to cigarette smoke remains. Pipe used for smoking cigarettes, starting the first 10 days have been blackened. This condition increases in the second 10-day or 20 days (Fig. 2) and increasing up to day 30. Volume 7, No.2, Desember

5 THE POTENTIAL OF ETHANOL EXTRACT OF RED BETEL VINE LEAVES (Piper crocatum) AS ANTIOXIDANT IN MICE BALB/C EXPOSED TO CIGARETTE SMOKE (IN VIVO TEST) It shows the bad chemicals found in cigarettes. It is the outside of a human body in such a way, what if it is in the human body. Figure 2. Pipe used for smoking cigarettes day 20 These results are consistent with research of Aubin et al. (2012) that said an increase in weight will happen after someone stop smoking. In contrast to the first test research, in this study, at the time of ethanol extract of red betel vine leaves, cigarette smoke exposure has been discontinued. The results said, the group with ethanol extract of red betel vine leaves showed a faster rise of weight compared with controls given only sterile aqua. Thus, it can be stated that the therapeutic administration of ethanol extract or decoction of red betel vine can accelerate recovery of mice weight. Table 2. Summary of Average Mice Body Weight in Testing II Treatment Mice body weight in gram Day 10 Day 20 Day 30 Day 40 Day 50 Extract 1 Decreased by Decreased by Increased by Increased by Increased by Extract 2 Decreased by Decreased by Increased by Increased by Increased by Extract 3 Decreased by Decreased by Increased by Increased by Increased by Aqua Control Decreased by Decreased by Decreased by Decreased by Increased by Healthy Control Increased by Increased by Increased by Increased by Increased by In the second test, cigarette smoke exposure is performed prior to 20 days, then after 20 days of exposure, exposure is stopped and therapy is started. In the first 10 days of cigarette smoke exposure, the weight of mice had decreased on average, the decline increased in the second 10 day (20 days) except in group 3. Upon exposure to cigarette smoke was discontinued and mice were given red betel vine leaves extract, in the first 10 days of therapy, it has shown increases in the weight of mice. But it was not happened with mice with aqua (Table 2). In the control group of mice (with aqua) up to second 10 days (20 days), the average of the mice weight was still decreasing. The increase in weight happens in the third 10 days (30 days) after cigarette smoke exposure is stopped. Volume 7, No.2, Desember

6 Farida Juliantina Rachmawaty, Laksamana Olan Es Orlando Table 3. The Result of Histopathology Slide for 10 Day, 20 Day and 30 Day of Testing No. Extract 1 (day) Extract 2 (day) Extract 3 (day) Aqua control (day) Healthy control (day) Description: Scores obtained by making observations on lung preparations by assessing the presence or absence of inflammation in the bronchi, alveoli, and vascular/perivaskuler as well as the presence or absence of carbon and bleeding. Value: 0: no damage (normal), 1: minimal damage, 2: minor damage, 3: moderate damage, 4: real damage, 5: extensive damage. On examination of the lungs of mice preparations performed with hematoxylin eosin staining (HE) obtained results overview lungs of mice after cigarette smoke exposure and after receiving the ethanol extract of red betel vine. The presence of cigarette smoke exposure causes inflammation in the lungs of mice either in the bronchi, alveoli and the perivaskuler (Figure 3). The result is the same with the research conducted by Thatcher et al. (2005). In the first test the antioxidant test (Table 3) the results of the examination preparations lungs of mice with HE staining performed statistical tests Kruskal-Wallis and Mann Whitney U. Results obtained no significant difference between the control and the ethanol extract was given aqua. This can be caused by exposure to smoke is not stopped until the end of the study. In the first 10 days of treatment, the data were tested using Kruskal-Wallis statistical test. The results obtained p<0.05. Thus there are at least 2 groups were significantly different. Testing followed by Mann Whitney U test, the result is there is significant differences in treatment with extract 1, extract 2, 3 and healthy controls with the aqua controls (p<0.05). On the 2nd 10 day (20 days), the data were tested using Kruskal-Wallis statistical test, the results is p<0.05. Thus there are at least 2 groups were significantly different. Testing followed by Mann Whitney U, the results is there are significant differences in treatment with the extract 3 and healthy controls against aqua control (p<0.05). Treatment with extracts 1 and 2 show the results that there were not significantly different than the aqua controls (p>0.05). On the 3rd 10 day (30 days), the data were tested using Kruskal-Wallis statistical test, the result is that the p<0.05. Thus there are at least 2 groups were significantly different. Table 4. The Result of Histopathology Slide for 10 Day, 20 Day and 30 Day Testing II No. Extract 1 (day) Extract 2 (day) Extract 3 (day) Aqua control (day) Healthy control (day) Description: Scores obtained by making observations on lung preparations by assessing the presence or absence of inflammation in the bronchi, alveoli, and vascular/perivaskuler as well as the presence or absence of carbon and bleeding. Value: 0: no damage (normal), 1: minimal damage, 2: minor damage, 3: moderate damage, 4: real damage, 5: extensive damage. Volume 7, No.2, Desember

7 THE POTENTIAL OF ETHANOL EXTRACT OF RED BETEL VINE LEAVES (Piper crocatum) AS ANTIOXIDANT IN MICE BALB/C EXPOSED TO CIGARETTE SMOKE (IN VIVO TEST) Testing continued with the Mann Whitney U test, the results is that there are significant differences in treatment with extracts 2, 3 extracts, and healthy controls compared with aqua controls (p < 0.05). While treatment extract 1 shows the results are not significant (p>0.05). Although the results obtained in this study is not consistent between the 10th, 20th and 30th, but in the second test showed that the ethanol extract of red betel can function as an antioxidant in vivo. The difference between test I and II are on exposure to cigarette smoke. In the first test, smoke still be given until the end of the study and the ethanol extract of red betel vine had been given since the beginning of the study. In the second test presented first and then the smoke stopped and then given ethanol extract of red betel vine. In the first test analogous to those still smoking then administered ethanol extract of red betel vine leaves, the result was not significant because the cause is still there. In the second test analogous to those which are used to smoke then quit smoking, then treated with ethanol extract of red betel vine leaves and the results are meaningful. CONCLUSION Ethanol extract of red betel vine leaves (P. crocatum) in the in vivo way has the ability as an antioxidant for balb/c mice exposed to cigarette smoking. However, it can serve as an antioxidant the exposure to the cigarette smoke has been stopped. REFERENCES Aubin HJ., Farley A., Lycett D., Lahmek P. and Aveyard P Weight Gain in Smokers after Quitting Cigarettes. BMJ, 1-21 Kusuma ARP., Pengaruh Merokok terhadap Kesehatan Gigi dan Rongga Mulut, Fakultas Kedokteran Gigi Universitas Islam Sultan Agung. Mboi N. 2013, Jumlah Perokok: Indonesia Nomor 3 di Dunia, com/2013/01/11/jumlah-perokok-indonesia-nomor-3-di-dunia, diakses 10 Maret 2013 Rachmawaty FJ., Hisyam B., Soesatyo MHNE., Wibawa T Potensi Ekstrak Etanol Daun Sirih Merah (Piper crocatum) sebagai Antimikobakterium. JIFI, 11(1): 60-5 Safithri M. dan Fahma F Potency of Piper crocatum decoction as an antihyperglycemia in rat strain Sprague Dawley. Hayati J Biosci., 15(1): 45. Sarastani D., Soekarto ST., Muchtadi TR., Fardiaz D. dan Apriyanto A Aktivitas Antioksidan Ekstrak dan Fraksi Ekstrak Biji Atung, Jurnal Teknologi dan Industri Pangan, 13(2): Subarnas, A., Susilawati, Y., Mulyasari,E., 2007, Aktivitas Antiinflamasi Ekstrak Etanol Daun Sirih Merah (Piper betle var. rubrum) pada Tikus Putih Jantan. Farmaka, 5 (1): 1-5 Sudewo B Basmi Penyakit dengan Sirih merah. PT Agromedia Pustaka, Jakarta Suratmo Aktivitas Antioksidan dan Antikanker Ekstrak Daun Sirih Merah (P. crocatum). Tesis. Fakultas MIPA Universitas Gadjah Mada. Thatcher TH., McHugh NA., Egan, Chapman RW., Hey JA., Turner CK., Redonnet MR., Seweryniak KE, Sime PJ, Phipps RP. 2005, Role of CXCR2 in Cigarette Smoke, Am J Physiol Lung Cell Mol Physiol, 289: L322-8 Wikipedia Radikal Bebas, wikipedia.org/wiki/radikal_bebas, diakses 10 Maret 2013 Volume 7, No.2, Desember