International Journal of Research in Pharmacy and Biosciences Volume 7, Issue 1, 2020, PP 1-9 ISSN 2394-5885 (Print) and ISSN 2394-5893 (Online)

Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Scholaris

Joushan Ara*, A T M Yusuf and Md. Shahidul Islam Department of Pharmacy, University of Science and Technology Chittagong (USTC), Chattogram, Bangladesh *Corresponding Author: Joushan Ara, Lecturer, Department of Pharmacy, University of Science & Technology Chittagong (USTC), Bangladesh.

ABSTRACT Though some found in nature are poisonous but there are some plants which serve us as source of energy and effective drugs. Considering the medical importance and source of origin, the Alstonia scholaris has been subjected for fractionation with different solvents. The different fractions of ethanolic extract of Alstonia scholaris were evaluated for the antibacterial, antifungal activities and cytotoxic activity as well as biological activity. Phytochemical properties of leaves of Alstonia scholaris were also examined. The different solvent fractions showed the presence of tannins, glycosides, steroids and alkaloids. The different fractions of ethanolic extract of Alstonia scholaris were evaluated for the total phenolic content, total flavonoid content, the antibacterial, antifungal activities and cytotoxic activity. The different fractions of ethanolic extract of Alstonia scholaris were also undertaken to investigate for antibacterial activity using well diffusion method on Gram positive (Staphylococcus aureus) as well as Gram negative bacteria (Eacherichia coli). Crude ethanol fraction exhibited highest activity against S. aureus and E. coli, showed the inhibition zone with diameters 14mm and 13 mm respectively. Whereas ethyl acetate, chloroform and Dia-ion resin adsorbed fraction exhibited lesser activity, while petroleum ether fraction showed no inhibition. For screening biological activities, cytotoxicity test was also performed with various extractives of the Alstonia scholaris. From the result it was observed that ethyl acetate fraction had the highest cytotoxicity activity against Artemiasalina (brine shrimp) with LD50 values 944.730, 2819.016, 83.994, 19.012 and 12.213 ppm after 6, 12, 18, 24 and 30 hours’ treatment respectively. Keywords: Phytochemistry, Cytotoxicity test, Alstonia scholaris Chloroform.

INTRODUCTION the food materials so essential for sustenance of the life of animals but also certain other In the beginning of the nineteenth century, the substances, such as alkaloids, vitamins, mankind stepped into the industrial life, partly glycosides, toxalbumins, essential oils, resins, neglecting the dependence on plant kingdom. As bitter principles etc. which are necessary for a result, the ecological balance of the plant and growth, maintenance and protection of life. the animal life is disturbed and the world is Many of these are essential for metabolic being threatened by the industrial pollution and activities (3), many are medicines to human and hazards (1). This is why the modern world is animal life. Many of these are harmful to animal interestingly tending to go back to the pre- life, at least under certain conditions. Plants industrialized days, when the mankind used to containing medicinal properties are commonly depend on the plant kingdom for their food, known as medicinal plants (4). The plants shelter, medicine and other essential commodities. containing these principles are capable of acting This is perhaps, the only way to protect the deleteriously, are popularly known as poisonous ecological balance. plants. A poisonous plant is one which, as a The raw materials of the plant kingdom as whole or as a part thereof, under all or certain mentioned above are directly or indirectly conditions, and in a manner and in amount likely produced by the plants, but are very seldom to be taken or by brought into contact with an used by themselves and serve human beings in organism, 'will exert harmful effects or cause many ways (2). These are called the secondary death either immediately or by reason of metabolites or the natural products. By the cumulative action of the toxic property, due to metabolic activity of plants produces not only the presence of known or unknown chemical

International Journal of Research in Pharmacy and Biosciences V7 ● I1 ● 2020 1 Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris substances in it (5). Although some of these different ethnic communities in have used plants are once poisons, medicines and food or different species of Alstonia in the treatment of fodder. The genus Alstonia belongs to the various human ailments (7). The common family . It includes totally 43 Bengali name of the plant Alstonia scholaris are species of which two species namely, A. Satiani, Chattin, Chatium. Alstonia scholaris is scholaris (L.) R. Br. and A. venenata R. Br. are widely distributed in dried forest of India, represented in South India (6). These two Western Himalayas, Western Ghats and in the species can be identified with their habits, shape Southern region (8). It grows more or less in all and texture of the leaves, fruit size and papilla districts of Bangladesh. It grows Malaysia, of the seeds. When these plants are used in Australia and Solomon Islands as an ornamental. It herbal formulations, their botanical identity is distributed in Pakistan, Thailand, Philippines, needs to be established beyond any ambiguity. Indonesia (9) and tropical regions of Africa and The genus Alstonia finds a prominent place in Asia Habitat found to be in deciduous and different Indian systems of medicine. The evergreen forests, also in plains (10).

Figure1. Alstonia scholaris plant FLOW CHART OF PLANT EXTRACTION

Figure2. Flow chart for the preparation of different fraction

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METHODS AND MATERIALS yellow or red precipitate was the indication for the presence of tannins. In this study, all the reagents and chemicals were used were purchased from THOMAS BAKER Test for Steroids (MUMBAI, INDIA), BDH (ENGLAND), Libermann-Burchard’s Test FLUKA (SWITZERLAND) and E. MERCK (GERMANY). Rectified spirit and absolute A small amount of different fractionates of the alcohol were available from Carew and company, plant materials was dissolved in 1 mL of Darsana, Chuadanga. The Solvents used mainly in chloroform, 2 mL of acetic anhydride and 1 mL of concentrated sulphuric acid were added to the this work are benzene, acetone, tetrahydrofuran solution. Formation of a greenish color which (THF), ethyl acetate, chloroform, n-hexane, terns blue on standing was the indication for the petroleum ether, methanol, absolute alcohol, presence of steroids. toluene etc. The solvents were dried and distilled when necessary. Test for Alkaloids The Alstonia scholaris plant leaves were Color Test collected from the cultivated neighboring areas About 0.5g of the extract was stirred with 5 mL of BCSIR, Rajshahi. The collected leaves were of 1% hydrochloric acid on a steam bath and cleaned with water. was filtered. 1 mL of the filtrate was treated The whole extraction process is shown in the with a few drops of Dragendorff‟s (Bismuth following flow chart. potassium iodide solution) reagent. Formation of orange-red precipitate was the indication for The Alstonia scholaris leaf extracts of the presence of alkaloids. Petroleum ether, Ethyl acetate, Chloroform, Methanol and Ethanol solvents were used for the Biological Studies are Performed in Two Ways phytochemical analysis for the identification of  Anti-bacterial& Anti-fungal screening (in various classes of chemical compounds using vitro) crude methanol extract and different the standard protocol. fractions. SCREENING PROCEDURE  Cytotoxicity assay study Test for Saponins DETERMINATION OF ANTI-BACTERIAL & ANTI-FUNGAL ACTIVITY OF THE TEST Frothing Test AGENT BY MEASURING THE ZONE OF About 0.1g of different fractionates of the plant INHIBITION materials was taken vigorously with water. The discs were placed in such a way that they Production of a persistent frothing (which were not closer than 15 mm to the edge of the remains stable on heating) was the indication for plate and for enough apart to prevent the presence of saponins. overlapping the zones of inhibition. Test for Glycosides After 24 hours incubation, the antibacterial General Test activities of the antibiotic extracts were determined by measuring the zone of inhibition A small amount of different fractionates of the in term of mm by a transparent scale. Inhibitory plant materials was taken in a test tube and was zone obtained by samples were compared to that dissolved in 1 mL of water. A few drops of of the standard disc. aqueous sodium hydroxide solution were then added to the test tube. Development of yellow DETERMINATION OF MINIMUM INHIBITORY color was the indication for the presence of CONCENTRATIONS OF THE ANTIBACTERIAL glycosides. AGENTS Test for Tannins Minimum inhibitory concentration (MIC) may be defined as the lowest concentration of an Lead Acetate Test antimicrobial drug to inhibit the growth of the About 5 mL of aqueous solution of different organism. The data derived from the test can be fractionates of the plant material was taken in a corrected with the knowledge of expected or test tube and a few drops of a 1% solution of measured the antibiotic level in vivo to predict lead acetate were added to the test tube. A the efficacy of the antibiotic.

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There are two methods for determining the MIC to give 800, 400, 200, 100, 50 and 25ppm; For values. They are as follows: the Dia-ion resin adsorbed fraction, a general concentration was selected as successive doses  Serial tube dilution technique or turbid metric by serial dilution to give 800, 400, 200, 100, 50 assay. and 25ppm; For the Chloroform fraction, a  Paper disc plate technique or agar diffusion general concentration was selected as assay. successive doses by serial dilution to give 400, Here “serial tube dilution technique”. Was followed 200, 100, 50 and 25ppm; For the Ethyl acetate using different broth medium to determine the MIC fraction, a general concentration was selected as values of ethyl acetate extract and antibiotics successive doses by serial dilution to give 400, against the following pathogenic bacteria. 200, 100, 50 and 25ppm. Each concentration was tested in triplicate, 18 or 15 test-tubes per EXPERIMENTATION OF CYTOTOXICITY TEST test fraction. The four extracts (Petroleum ether, Chloroform, PREPARATION OF THE CONTROL GROUP Ethyl acetate and Dia-ion resin adsorbed fractions) of Alstonia scholaris leaves were applied against For each concentration, one test tube containing brine shrimp nauplii. For the fractions were the same volume of DMSO diluted up to 10 mL initially dissolved in general concentration of with sea-water and 30 shrimp nauplii was used pure dimethyl sulfoxide (DMSO) to make as negative control group. It was used to verify hydrophilic before adding of water to get a the validity of the test. When the nauplii in the concentration of for each separately which were control showed a rapid mortality, then the test is used as stock solutions. For A. scholaris leaves considered to be invalid as the nauplii might die Petroleum ether fraction, a general concentration due to reasons other than the cytotoxicity of the was selected as successive doses by serial dilution compounds. RESULTS & DISCUSSION Antibacterial and Antifungal Screening Table1. Antibacterial and antifungal activity of test sample of Alstonia scholaris Diameter of inhibition zone (mm) Dose Name of sample Bacteria Fungi (µg/disc) Staphylococcusaureus Escherichia coli Candida albicans Chloroform fraction 400 0 12 10 Petroleum ether fraction 400 0 0 0 Ethyl acetate fraction 400 6 5 6.5 Dia-ion resin adsorbed fraction 400 0 6 8 Crude ethanol extract 400 14 13 11.5 Kanamycin (k 30) standard 400 22 22 22

C C D D

B B Kanamycin Kanamycin E E

A A

Figure3. Antibacterial test of the crude ethanol Figure4. Antibacterial test of the crude ethanol extract, different fractions and Kanamycin (Standard) extract, different fractions and Kanamycin (Standard) against Staphylococcus aureus against E. coli.

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fraction showed no inhibition zone. Activities of the different fractions were comparable to these C D of the standard antibacterial agent Kanamycin (K30).The difference in the observed activities B Kanamycin of the various fractions may be due to varying E degree of solubility of the active constituents in A the five solvents used. It has been documented that different solvents have diverse solubility Figure5. Antifungal test of the crude ethanol extract, capacities for different phytochemical constituents. different fractions and Kanamycin (Standard) Cytotoxicity Assay Study against Candida albicans. Here, A= Chloroform fraction, B= Petroleum ether The mortality of adult beetles was recorded after fraction, C= Ethyl acetate fraction, D= Dia-ion resin 24, 48 and 72 hours of treatment. The mortality adsorbed fraction, E= Crude ethanol fraction. percentage was corrected using Abbott‟s (1925) formula The antibacterial and antifungal activities of different fractions and crude ethanol extracts Pt= (P0-Pc) / (100-Pc) Where, were examined in the present study. The results Pt = corrected mortality% are given in the table 11.Among the extracts P0 = Observed mortality % and Pc = Control tested, Crude ethanol extract showed broader mortality % spectrum of activity, being active to both S. aureus (Gram-positive), E. coli (Gram-negative) The observed data then subjected to prohibit bacteria and C. albicans (fungal)with range of analysis according to Finney and Busvine J.R. diameters of inhibition zone 14 mm, 13 mm and 1971, using software developed in the 11.5mm respectively. While Chloroform fraction Department of Agricultural and Environmental exhibited maximum inhibition zone, 12 mm and Science, University of Newcastle, Upon Tyne, 10 mm against E. coli bacteria and C. albicans United Kingdom, which adapted the traditional fungal respectively. Whereas Ethyl acetate calculations to automatic computation. No showed the inhibition zone, 6 mm, 5 mm and provisional, graph or tables are required. 6.5 mm against S. aureus &E. coli bacteria and Heterogeneity is tested by a chi squared test. If C. albicans fungal respectively. Dia-ion resin the probability is greater than 5% an automatic adsorbed fraction showed the inhibition zone, 6 correction of heterogeneity is introduced. The mm and 8 mm against E. coli bacteria and C. program also calculated confidence limits for albicans fungal respectively. Petroleum ether LD50. Table2. Effect of different fractions of Alstonia scholaris on Artemiasalinanauplii after the treatment of 6-30 hours No. of cysts killed with time (h) Name of fraction Conc.(ppm) 6 12 18 24 30 800 3 7 13 27 27 400 1 2 5 10 13 200 0 0 6 7 11 Petroleum ether 100 0 1 1 1 3 fraction 50 0 2 3 4 6 25 0 0 1 2 3 Control 0 0 0 0 0 800 1 1 4 24 28 400 1 1 8 16 20 200 2 4 6 7 11 Dia-ion resin 100 2 2 4 4 4 adsorbed fraction 50 0 4 6 7 7 25 0 2 2 3 3 Control 0 0 0 0 0 400 5 12 14 17 22 200 2 7 10 17 19 Chloroform fraction 100 0 4 7 9 11 50 2 3 5 7 7 25 1 2 3 5 5

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Control 0 0 0 0 0 400 11 12 22 25 30 200 3 9 16 21 26 Ethyl acetate 100 3 8 14 19 22 Fraction 50 1 9 16 20 24 25 1 6 10 15 17 Control 0 0 0 0 0

Table3. LD50, 95% confidence limits and regression equations of Alstonia scholarisextracts against A. salinanauplii Exposure LD 95% confidence limits Plant organ 50 Regression equation χ2 value (df) (h) (mg cm-1) Lower Upper 6 - - - - - 12 17522.79 223.168 1375864 Y= 2.042 + 0.697X 2.892(2) Petroleum 18 1808.945 549.113 5959.217 Y = 1.681 + 1.019X 5.513(4) ether fraction 24 412.971 193.762 880.177 Y = 0.607 + 1.679X 18.630(4) 30 298.245 160.427 556.243 Y = 1.345 + 1.476X 12.330(4) 6 0.1825 8.655E-09 3850009 Y= 4.613 -0.523X 0.239(2) Dia-ion resin 12 2.841 1.538E-11 524941.4 Y = 4.216-0.308X 3.424(4) adsorbed 18 2100843 2.853E-02 1.547E+14 Y = 3.530+ 0.232 X 4.799(4) fraction 24 348.415 229.722 528.436 Y = 1.679+ 1.306X 9.397(4) 30 215.823 134.649 345.932 Y = 1.043 + 1.695X 10.559 (4) 6 18210.01 47.643 7960137 Y = 2.308 + 0.632X 0.869(2) 12 872.670 259.430 2935.486 Y = 1.928+ 1.044X 0.668(3) Chloroform 18 509.297 196.593 1319.395 Y = 2.319+ 0.990X 0.127(3) fraction 24 220.943 126.270 386.597 Y = 2.484 + 1.073X 1.520(3) 30 141.424 98.398 203.261 Y = 1.947 + 1.419X 1.038(3) 6 944.730 323.986 2754.797 Y = 0.896 + 1.379X 2.498(3) 12 2819.016 30.251 262695 Y = 3.682 + 0.382X 0.773 (3) Ethyl acetate 18 83.994 41.924 168.279 Y = 3.682 + 0.685X 2.412(3) fraction 24 19.012 4.200 86.050 Y = 4.219 + 0.611X 1.467(3) 30 12.213 2.521 59.149 Y = 4.073+ 0.853X 2.101(2)

Figure6. Probit mortality line of the Petroleum ether fraction of A. scholarisagainst A. salinanauplii after 12-30 hours of exposure

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Figure7. Probit mortality line of the Dia-ion resin adsorbed fraction of A. scholarisagainst A. salinanauplii after 6-30 hours of exposure

Figure8. Probit mortality line of the Chloroform fraction of A. scholarisagainst A. salinanauplii after 6-30 hours of exposure

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Figure9. Probit mortality line of the Ethyl acetate fraction of A. scholarisagainst A. salinanauplii after 6-30 hours of exposure CYTOTOXIC EFFECT OF ALSTONIA to168.279, 4.200 to 86.050 and 2.521 to59.149 SCHOLARIS AGAINST A. SALINA NAUPLII ppm. For 6, 12, 18, 24, and 30h of exposures The LD50 value for Chloroform fraction was The brine shrimp lethality test of A. scholaris 18210.01, 872.670, 509.297, 220.943 and leaves against the brine shrimp (A. salina) nauplii 141.424 ppm; while the regression equations were found promising. The chloroform fraction, were Y = 2.308 + 0.632X, Y = 1.928+ 1.044X, Dia-ion resin adsorbed fraction, Ethyl acetate Y = 2.319+ 0.990X, Y = 2.484 + 1.073X and Y fraction and Petroleum ether fraction were = 1.947 + 1.419X with chi-squared values found promising against the 1 day nauplii of A. 0.869, 0.668, 0.127, 1.520, 1.038for 2, 3, 3, 3 salina and the result has been presented in Table and 3 degrees of freedom and the 95% 13. Ethyl acetate fraction was found the highest confidence limits were 47.643 to 7960137, 259.430 active one. For 6, 12, 18, 24, and 30h of to 2935.486, 196.593 to1319.395, 126.270 to exposures the LD50 values for the Ethyl acetate 386.597 and 98.398 to 203.261ppm. This was fraction are 944.730, 2819.016, 83.994, 19.012 just followed by the Dia-ion resin adsorbed and 12.213ppm; while the regression equations fraction For 6, 12, 18,24 and 30h of exposures were Y = 0.896 + 1.379X, Y = 3.682 + 0.382X, the LD50 values were 0.1825, 2.841, 2100843, Y = 3.682 + 0.685X, Y = 4.219 + 0.611X and Y 348.415 and 215.823ppm; while the regression = 4.073+ 0.853X with chi-squared values 2.498, equations were Y= 4.613 -0.523X, Y = 4.216- 0.773 , 2.412, 1.467 and 2.101 0.308X, Y = 3.530+ 0.232 X, Y = 1.679+ For 3, 3, 3, 3 and 2 degrees of freedom 1.306X and Y = 1.043 + 1.695X; with chi- respectively and the 95% confidence limits were squared values 0.239, 3.424, 4.799, 9.397 and 323.986 to 2754.797, 30.251 to 262695, 41.924 10.559 for 2, 4, 4, 4 and 4 degrees of freedom

8 International Journal of Research in Pharmacy and Biosciences V7 ● I1 ● 2020 Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris and the 95% confidence limits were 8.655E- REFERENCES 09 to 3850009, 1.538E-11 to 524941.4, 2.853E- [1] Shan Zhao, Jie Yuan Liu, Si Yu Chen, Ling 02 to 1.547E+14, 229.722 to 528.436 and Ling Shi, Yu Jun Liu and Chao Ma. 134.649 to 345.932ppm. Depending on toxicity Antioxidant Potential of Polyphenols and the last one For 12, 18, 24 and 30h of exposures Tannins from Burs of Castaneamollissima the LD50 values for the Petroleum ether fraction Blume. Molecules. 16, 8590-8600. 2011. were 17522.79, 1808.945, 412.971 and [2] A. H. M.M. Rahman, M. Anisuzzaman, S. A. 298.245ppm; while the regression equations Haider, Ferdous Ahmed, A. K. M. Rafiul Islam were Y= 2.042 + 0.697X, Y = 1.681 + 1.019X, and A. T. M. Naderuzzaman. Research Journal Y = 0.607 + 1.679X and Y = 1.345 + 1.476X; of Agriculture and Biological Science, 4(1): 70- with chi-squared values 2.892, 5.513, 18.630 74.2008. and 12.330 for 2, 4, 4 and 4 degrees of freedom [3] Channa S, Dar A, Ahmed S, Rahman AU. and the 95% confidence limits were 223.168 to Evaluation of Alstonia scholaris leaves for 1375864, 549.113 to 5959.217, 193.762 to broncho- vasidilatory activity. Journal of ethno 880.177 and 160.427 to 556.243ppm. pharmacology; 97: 469-474.2005. CONCLUSION [4] ArulmozhiS, Mazumder PM, L Narayanan S, Prasad AT. In vitro antioxidant and free fadical The present study investigated the plant Alstonia scavenging activity of fractions from Alstonia scholaris for biological activity of different scholaris Linn. International Journal of Pharm extractives. For this purpose, total phenolic content, Tech Research; 2:18-25. 2010. total flavonoid content, antibacterial, [5] Khyade, M. S. and Vaikos, N. P. cytotoxicity and insecticidal activity tests were „phytochemical and antibacterial properties of performed with four different fractions of the leaves of Alstonia scholaris R. Br‟. African plant. For screening different fractions of the journal of Biotechnology; 8(22):6434-6436.2009. plant A. scholaris to have biological activities, [6] Shan Zhao, Jie Yuan Liu, Si Yu Chen, Ling antibacterial and cytotxicity tests have been Ling Shi, Yu Jun Liu and Chao Ma. performed. In these test all the three extractives, Antioxidant Potential of Polyphenols and Ethyl acetate fraction, Chloroform fraction & Tannins from Burs of Castaneamollissima Dia-ion resin adsorbed fraction showed Blume. Molecules. 16, 8590-8600. 2011. remarkable activities. So, in this study, the cytotoxic potential of the plant with four [7] ShaileshRana, C. Bhatt, N. Kanaki and M. fractions have also been determined by using Zaveri. Advanced Research in Pharmaceuticals brine shrimp lethality assay. Ethyl acetate and Biologicals; Vol 2 (3): ISSN 2250-0774: p extract showed highest toxicity against A. salina 290-295. 2012. (brine shrimp). According to the intensity in the [8] Kulkarni MP, Juvekar AR. Effect of Alstonia efficacy of the extracts against the brine shrimp scholaris (Linn) R.Br. on stress and cognition nauplii the result could be arranged in the following in mice. Indian Journal of Experimental order: Ethyl acetate fraction > Chloroform Biology; 47: 47-52. 2008. fraction>Dia-ion resin adsorbed fraction > [9] Khyade, M. S. and Vaikos, N. P. „phytochemical Petroleum ether fraction. Considering the and antibacterial properties of leaves of antimicrobial and cytotoxicity assay of A. Alstonia scholaris R. Br‟. African journal of scholaris, it can be deduced that this plant contains Biotechnology; 8(22):6434-6436. 2009. useful potent bioactive toxic compounds, which [10] Ogawa, S.; Kimura, H.; Niimi, A.; Katsube, T.; can be harnessed and purified into useful Jisaka, M.; Yokota, K. Fractionation and therapeutic drugs. However, further studies are structural characterization of polyphenolic warranted for more extensive antioxidant and antioxidants from seed shells of Japanese horse biological evaluations to elucidate before bringing chestnut (Aesculusturbinate BLUME). J. Agric. them into commercial use. Food Chem. 56, 12046-12051. 2008.

Citation: Joushan Ara, A T M Yusuf and Md. Shahidul Islam, "Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris", International Journal of Research in Pharmacy and Biosciences, 2020, 7(1), pp. 1-9. Copyright: © 2020 Joushan Ara. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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