شناسایی ترکیبات شیمیایی، قدرت آنتی‌اکسیدانی، محتوای تام فنل و فلاونوئید کل و سمیت سلولی اسانس رزماری

مجله پژوهشهای علوم و صنایع غذایی ایران

دوره 18، شماره 4 - شماره پیاپی 76، مهر و آبان 1401، صفحه 467-482   

نوع مقاله: مقاله پژوهشی فارسی

شناسه دیجیتال (DOI): 10.22067/ifstrj.2021.68369.1013

نویسندگان

محسن ابراهیمی همتی کیخا¹؛ حسین جوینده^{* 1}؛ بهروز علیزاده بهبهانی¹؛ محمد نوشاد²

¹گروه علوم و مهندسی صنایع غذایی، دانشکده علوم دامی و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی خوزستان، ملاثانی، ایران.

²گروه علوم و مهندسی صنایع غذایی، دانشکده علوم دامی و صنایع غذایی، دانشگاه علوم کشاورزی و منابع طبیعی خوزستان، ملاثانی، ایران

چکیده

رزماری با نام علمی Rosmarinus officinalis L. گیاهی از تیره نعناعیان می‌باشد که به دلیل داشتن خواص‌های گوناگون از جمله خواص دارویی، پزشکی و طعم‌دهنده در صنایع مختلف مورد استفاده قرار می‌گیرد. در پژوهش حاضر ترکیبات شیمیایی موجود در اسانس رزماری به‌وسیله دستگاه کروماتوگرافی گازی متصل طیف‌سنج جرمی تعیین گردید. ارزیابی محتوای تام فنلی و فلاونوئید اسانس رزماری به‌ترتیب با استفاده از روش‌های ﻓﻮﻟﯿﻦ- ﺳﯿﻮﮐﺎﻟﺘﻮ و رنگ‌سنجی به کمک آلومینیوم کلراید صورت پذیرفت. ارزیابی اثرات آنتی‌اکسیدانی اسانس رزماری به دو روش DPPH و  ABTS محاسبه شد. میزان اثر سمیت سلولی اسانس رزماری بر رده‌های سلولی سرطان روده بزرگ ( (HT29 به روش MTT مورد بررسی قرار گرفت. ترکیبات گروه‌های عاملی زیست فعال موجود در اسانس با استفاده از طیف‌سنجی مادون قرمز تبدیل فوریه بررسی شد. نتایج حاصل از تجزیه ترکیبات شیمیایی اسانس رزماری شامل 29 ترکیب بودند که در مجموع 22/94% از کل اسانس را شامل شدند. عمده‌ترین ترکیب شناسایی شده در اسانس رزماری ترکیب اوکالیپتول با میزان 13/40% بود. محتوای تام فنلی اسانس رزماری 55/72 میلی‌گرم گالیک اسید در گرم اسانس و میزان فلاونوئید آن برابر با 36 میلی‌گرم کوئرستین اکی‌والان در گرم اسانس بود. میزان سنجش مهار رادیکال آزاد در دو روش DPPH و ABTS به‌ترتیب برابر با 74/78% و 97/81% به‌دست آمد. نتایج مربوط به میزان سمیت سلولی اسانس رزماری نشان داد که اثر سیتوتوکسیک وابستگی زیادی به غلظت داشته و با افزایش غلظت اسانس میزان سمیت سلولی نیز افزایش یافت. تجزیه و تحلیل طیف‌سنجی مادون قرمز تبدیل فوریه حضور ترکیبات آلدهیدها، کتون‌ها، اسیدهای کربوکسیلیک، استرها و آلکن‌ها را اثبات نمود. نتایج مربوط به تمامی آزمون‌های اسانس رزماری نشان داد که این اسانس می‌تواند به‌عنوان یک منبع بالقوه در صنایع داروسازی و غذایی مورد استفاده قرار گیرد

کلیدواژه‌ها

اسانس؛ کروماتوگرافی گازی؛ طیف‌سنجی مادون قرمز تبدیل فوریه؛ رادیکال آزاد

مراجع

1. Alipour, M., Jamal Saharkhiz, M., Niakousari, M. & Seidi Damyeh, M., (2019). Phytotoxicity of encapsulated essential oil of rosemary on germination and morphophysiological features of amaranth and radish seedlings. Scientia Horticulturae, 243, 131–139. https://doi.org/10.1016/j.micpath.2017.11.055
2. Alizadeh Behbahani, B. & Imani Fooladi, A. A., (2018). Evaluation of phytochemical analysis and antimicrobial activities Allium essential oil against the growth of some microbial pathogens. Microbial Pathogenesis, 114, 299-303. https://doi.org/10.1016/j.micpath.2017.11.055
3. Alizadeh Behbahani, B., Noshad, M. & Falah, F., (2019). Study of chemical structure, antimicrobial, cytotoxic and mechanism of action of Syzygium aromaticum essential oil on foodborne pathogens. Slovak Journal of Food Sciences, 13(1), 875-883.
4. Alizadeh Behbahani, , Shahidi, F., Tabataba Yazdi, F., Mortazavi, S. A. & Mohebbi, M., (2017). Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage. International Journal of Biological Macromolecules, 94, 515-26. https://doi.org/10.1016/j.ijbiomac.2016.10.055
5. Alizadeh, L., Nayebzadeh, K., & Shahin, R., (2014). Antioxidant effect of rosemary and ferulago extracts and synthetic TBHQ on oil oxidation during deep-frying. Iranian Journal of Nutrition Sciences & Food Technology, 8(4), 135-143. [In Persian].
6. Alvarez, M. V., Ortega‐Ramirez, L. A., Silva‐Espinoza, B. A., Gonzalez‐Aguilar, A. & Ayala‐Zavala, J. F., (2019). Antimicrobial, antioxidant, and sensorial impacts of oregano and rosemary essential oils over broccoli florets. Journal of Food Processing and Preservation, 13889, 2-10.
7. Amaral, P., deCarvalho, N. R., Barcelos, R. P., Dobrachinski, F., de Lima Portella, R., da Silva, M. H., Lugokenski, T. H., Mundstock Dias, GR., da Luz, S. C. A., Boligon, A. A., Athayde, ML., Villetti, M. A., Soares, F. A. A. & Fachinetto, R., (2013). Protective action of ethanolic extract of Rosmarinus officinalis L. in gastric ulcer prevention induced by ethanol in rats, Food and chemical toxicology, 55, 48-55.
8. Asanova, Zh. K., Suleimenov, E. M., Atazhanova, G. A., Dembitskii, A. D., Pak, R. N., Dar, A. & Adekenov, S. M., (2003). Biological activity of 1, 8-cineole from levant wormwood. Pharmaceutical Chemistry Journal, 37 (1), 28-30.
9. Brand-Williams, W., Cuvelier, M. E. & Berset, C., (1995). Use of a free radical method to evaluate antioxidant activity. LWT- Food Science and Technology, 28(1), 25-30. https://doi.org/10.1016/S0023-6438(95)80008-5
10. Bubonja-Sonje, M., Jasminka Giacometti, & Abram, M., (2011). Antioxidant and antilisterial activity of olive oil, cocoa and rosemary extract polyphenols. Food Chemistry, 127, 1821–1827. https://doi.org/10.1016/j.foodchem.2011.02.071
11. Collins, A. R., (2005). Antioxidant intervention as a route to cancer prevention. European Journal of Cancer, 41(13), 1923- 1930. https://doi.org/10.1016/j.ejca.2005.06.004
12. Cui, W., Phillips, G. O., Blackwell, B., & Nikiforuk, J., (2007). Characterisation andproperties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) Supergum (TM)): Part 4. Spectroscopic characterisation of Acacia senegal var. senegal and Acacia (sen) Supergum (TM) arabic. Food Hydrocolloids, 21(3), 347–352.
13. Dehghan , Barzegar, H., Mehrnia, M. A., & Jooyandeh, H. 2018.  Investigation on the effect of methanolic Bene (Pistachia atlantica) hull extract on oxidative stability of soybean oil. Innovative Food Technologies, 5(3), 499-507. [In Persian].
14. Di Domenico, F., Foppoli, C., Coccia, R. & Perluigi, M., (2012). Antioxidants in cervical cancer: chemopreventive and chemotherapeutic effects of polyphenols. Biochimica et Biophysica Acta, 1822(5), 737-747. https://doi.org/10.1016/j.bbadis.2011.10.005
15. Einbond, L. S., Wu, H. A., Kashiwazaki, R., He, K., Roller, M., Su, T., Wang, X. & Goldsberry, S., (2012). Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. Fitoterapia, 83, 1160-1168.
16. Erkan, N., Ayranci, G. & Ayranci, E., (2008). Antioxidant activities of rosemary (Rosmarinus Officinalis) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry, 110, 76–82. https://doi.org/10.1016/j.foodchem.2008.01.058
17. Esmaeli, F., Tajik, H., Mehdizadeh T., & Mayeli, M., (2017). Determination and comparison of antioxidant activity and phenolic content of Pimpinella affinis hydroethanolic extract and essential oil. The Journal of Urmia University of Medical Sciences, 28(5), 311-320. [In Persian].
18. Faixovà, Z. & Faix, S., (2008). Biological effects of rosemary (Rosmarinus officinalis) essential oil. Folia Veterinaria, 52(3-4), 135-139.
19. Fernandez, J., Zhi, N., Aleson, , Perez, J. A. & Kuri, V., (2005). Antioxidant and antibacterial activites of natural extracts: application in the beef meat balls. Meat Science, 69, 371–380. https://doi.org/10.1016/j.meatsci.2004.08.004
20. Gaya, M., Repetto, , Toneatto, J., Anesini, C., Piwien-Pilipuk, G. & Moreno, S., (2013). Antiadipogenic effect of carnosic acid, a natural compound present in Rosmarinus officinalis, is exerted through the C/EBPs and PPARγ pathways at the onset of the differentiation program Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(6), 3796- 3806. https://doi.org/10.1016/j.bbagen.2013.03.021
21. Haghjoo, L., Barzegar, H., & Jooyandeh, H., (2020). The effect of different drying methods on chemical composition and antioxidant properties of methanolic extract of olive leaf. Journal of food Science and Technology (Iran), 16(97), 149-159. [In Persian].
22. Halliwell, B., (2007). Biochemistry of oxidative stress. Biochemical Society Transactions, 35(pt5), 1147-1150.
23. Halliwell, B., (2011). Free radicals and antioxidants - quo vadis. Trends in Pharmacological Sciences, 32(3), 125-30. https://doi.org/10.1016/j.tips.2010.12.002
24. Hameed, I. H., Ibraheam, I. A. & Kadhim, H. J., (2015). Gas chromatography mass spectrum and fouriertransform infrared spectroscopy analysis of methanolic extract of Rosmarinus oficinalis Journal of Pharmacognosy and Phytotherapy, 7(6), 90-106.
25. Hendel, N., Larous, L. & Belbey, L., (2016). Antioxidant activity of rosemary (Rosmarinus officinalis) and its in vitro inhibitory effect on Penicillium digitatum. International Food Research Journal, 23(4), 1725-1732.
26. Jafarzadeh Khaledi, K., Aghazadeh Meshgi, M., Sharifan, A., & Larijani, K., (2010). Investigation of effect of the Rosemary essential oil on growth of Staphylococcus aureus in commercial instant soup. Journal of Comparative Pathobiology, 7(2), 255-264. [In Persian].
27. Jalali-Heravi, M., Sadat Moazenia, R. & Sereshtib, H., (2011). Analysis of Iranian rosemary essential oil: Application of gas chromatography–mass spectrometry combined with chemometrics. Journal of Chromatography, 1218, 2569–2576. https://doi.org/10.1016/j.chroma.2011.02.048
28. Jamshidi, M., Ashtiani, H. R., Rezazade, S. H ., Fathiazad, F., Mazandarani, M. & Khaki, A. (2010). Evaluation and Comparison of phenolic compounds and antioxidant activity of some plant species in mazandaran. Journal of Medicinal Plants, 34(2), 177-183.
29. Jamshidi, R., Afzali, Z. & Afzali, D., (2009). Chemical composition of hydrodistillation essential oil of Rosemary in different origins in Iran and comparison with other countries. American-Eurasian Journal of Agriculture and Environmental Sciences, 5 (1), 78-81.
30. Jianga, Y., Wua, N., Jie Fua, Y., Wanga, W., Luoa, M., & Jian Zhaoa, C. H., (2011). Chemical composition and antimicrobial activity of the essential oil of Rosemary. Environmental Toxicology and Pharmacology, 32: 63–68. https://doi.org/10.1016/j.etap.2011.03.011
31. Johnson, J. J. (2011). Carnosol: A promising anti-cancer and anti-inflammatory agent. Cancer Letters 2011; 305: 1-7. https://doi.org/10.1016/j.canlet.2011.02.005
32. Kacurakova, , Capek, P., Sasinkova, V., Wellner, N. & Ebringerova, A., (2000). FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydrate Polymers, 43(2), 195-203. https://doi.org/10.1016/S0144-8617(00)00151-X
33. Kak, M., Abdulkadhim, H. M. & Noori, S. I., (2016). Chemical composition and anti-bacterial effects of clove (Syzygium aromaticum) flowers. International Journal of Current Microbiology and Applied Sciences, 5(2), 483-489.
34. Kamkar, A., Shariatifar, N., Jamshidi, A. H., & Mohammadian, M., (2010). Study of antioxidant functional of the water, methanol, and ethanol extracts of Endemic Cuminum cyminum and Cardaria draba L. in the In-vitro Systems. Internal Medicine Today, 16(3), 37-45. [In Persian].
35. Kanth, M. K., Mehta, N., Chatli, M. K., Malav, O. P., Kumar, P., Wagh, R. V. & Panwar, H., (2018). In-vitro Assessment of Antimicrobial, Antibiofilm and Antioxidant Potential of Essential Oil from Rosemary (Rosmarinus officinalis). Journal of Animal Research, 8 (6), 989-998.
36. Kar, S., Palit, S., Ball, WB. & Das, PK., 2012. Carnosic acid modulates Akt/IKK/NF-kB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells. Apoptosis, 17, 735-747. https://doi.org/10.1007/s10495-012-0715-4
37. Khlebnikov, A. I., Schepetkin, A., Domina, N. G., Kirpotina, L. N. & Quinn, MT., (2007). Improved quantitative structure–activity relationship models to predict antioxidant activity of flavonoids in chemical, enzymatic, and cellular systems. Bioorganic and Medicinal Chemistry, 15(4), 1749-1770. https://doi.org/10.1016/j.bmc.2006.11.037
38. Kivanc, M. & Akgül, A., (1998). Effect of some essential oil components on the growth of foodborne bacteria and synergism with some food ingredients. Flavor and Fragrance Journal, 3, 95-98. https://doi.org/10.1002/ffj.2730030209
39. Lü, M., Lin, P. H., Yao Q. & Chen, C., (2010). Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. Journal of Cellular and Molecular Medicine, 14(4), 840- 860. https://doi.org/10.1111/j.1582-4934.2009.00897.x
40. Malakootian, M., & Hatami, B., (2013). Survey of chemical composition and antibacterial activity of Rosmarinus Officinalis Essential oils on Escherichia Coli and Its Kinetic. The Journal of Toloo-e-behdasht, 12(1), 1-13. [In Persian].
41. Melka Abdo, B., Asaminew, G., Mieso, B. & Sisay , (2018). Chemotypic Characterization and Antioxidant Activities of Rosemarinus officinalis Essential Oil from Ethiopian Cultivars. Medicinal and Aromatic Plants 7(6), 1-4.
42. Mohammadi, A. R., (2015). Antioxidant effect of methanolic and ethanolic extracts of Salvia officinalis and Rosmarinus officinalis as compare with synthetic antioxidants TBHQ and BHT in oil fish. Dissertation, Islamic Azad University, Pharmaceutical Sciences Branch. [In Persian].
43. Mohammadi, T., & Hoveizi, E., (2017). Comparison of Rosemary (Rosmarinus officinalis) hydroalcoholic extract on the viability of head and neck cancer cells Line HN5 and neuronal progenitor cells of Mouse. Quarterly Journal of Developmental Biology, 9(3), 13-22. [In Persian].
44. Moore, J., Megaly, M., Macneil, A. J., Klentrou, P. & Tsiani, E., (2016). Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells. Biomed Pharmacother, 83,725-32. https://doi.org/10.1016/j.biopha.2016.07.043
45. Moreno, S., Scheyer, T., Romano, C. S. & Vojnov, A. A., (2006). Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition. Free Radical Research, 40, 223-231.
46. Naji Tabasi, S. & Razavi, S. M. A., 2016. New studies on basil (Ocimum bacilicum) seed gum: Part 2– Emulsifying and foaming characterization. Carbohydrate Polymers, 149, 140-150. https://doi.org/10.1016/j.carbpol.2016.04.088
47. Newman, J., Cragg ,G. M. & Snader, K. M., (2003). Natural products as sources of new drugs over the period 1981- 2002. Journal of Natural Products, 66(7), 1022- 1037.
48. NGO, S. N. T., Williams, B. & Head, R. J., (2011). Rosemary and cancer prevention: Preclinical perspectives. Critical Reviews in Food Science and Nutrition, 51, 946-954.
49. Obeng-Ofori, D., Reichmuth, C. H., Bekele, & Hassanali, A., (1997). Biological activity of 1, 8- cineole, a major component of essential oil of Ocimum kenyese (ayobagira) against stored products beetles. Journal of Applied Entomolog, 121 (2), 237-243. https://doi.org/10.1111/j.1439-0418.1997.tb01399.x
50. Petiwala, S. M. & Johnson, J. J., (2015). Diterpenes from rosemary (Rosmarinus officinalis L) Defining their potential for anti-cancer activity. Cancer letters, 367(2), 93-102. https://doi.org/10.1016/j.canlet.2015.07.005
51. Petti, S. & Scully, C., (2009). Polyphenols, oral health and disease: A review. Journal of Dentistry, 37, 413-423. https://doi.org/10.1016/j.jdent.2009.02.003
52. Pourmorad, F., Hosseinmehr, S. J. & Shahabimajd, N. (2006). Antioxidant activity, phenol and flavonoid contents of some slected Iranian medicinal plants. Journal of Biotechnology, 5(11), 1142-1145.
53. Prochazkova, D., Bousova, I. & Wilhelmová N., (2011). Antioxidant and prooxidant properties of flavonoids. Fitoterapia, 82(4), 513- 523. https://doi.org/10.1016/j.fitote.2011.01.018
54. Rababah, T. M., Hettiarachchy, N. S. & Horax. R., (2004). Total phenolics and antioxidant activities of fenugreek, green tea, black tea, grape seed, ginger, rosemary, gotu kola, and ginkgo extracts, vitamin E, and tert- Butylhydroquinone. Journal of Agricultural and Food Chemistry, 52(16), 5183-5186. https://doi.org/10.1021/jf049645z
55. Rahman, K., (2007). Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging, 2(2), 219-236.
56. Rodrigues, S. E., Machado, A. E. H., Berardi, M., Ito, AS., Almeida, L. M., Santana, M. J. Liao, L. M., (2015). Investigation of protonation effects on the electronic and structural properties of halogenated sulfonated porphyrins. Journal of Molecular Structure, 1084, 284–293. https://doi.org/10.1016/j.molstruc.2014.12.053
57. Roomiani, L., Soltani, M., & Akhondzadeh Basti, A., (2016). Effect of Rosmarinus officinalis essential oil and nisin on Log P% of Streptococcus iniae in BHI broth. Iranian Journal of Medical Microbiology, 10(1), 35-43. [In Persian].
58. Sampaio, R. N., Gomes, W. R., Araujo, D. M. S., Machado, E. H., Silva, R. A., Marletta, A., Borissevitch, A. S., Dinelli, Batista, AA., Zílio, SC., Gonçalves, PJ, & Barbosa Neto., NM. (2012). Investigation of Ground- and Excited-State Photophysical Properties of 5,10,15,20-Tetra (4-pyridyl)- 21H, 23H-porphyrin with Ruthenium Outlying Complexes. The Journal of Physical Chemistry, 116, 18-26. https://doi.org/10.1021/jp205963k
59. Santos, F. A., Silva, R. M., Campos, A. R., de Araújo, R. P., Lima Júnior, R. C. P. & Rao, V. S. N., (2004). 1, 8-cineole (eucalyptol), a monoterpene oxide attenuates the colonic damage in rats on acute TNBS-colitis. Food and Chemical Toxicology, 42 (4), 579-584. https://doi.org/10.1016/j.fct.2003.11.001
60. Santos, R. D., Shetty, K., Cecchini, A. L. & Miglioranza, L. H. S., 2012. Phenolic compounds and total antioxidant activity determination in rosemary and oregano extracts and its use in cheese spread. Semina: Ciências Agrárias, 33(2), 655-666.
61. Selmi, S., Rtibi, k., Grami, D., Sebai, H. & Marzouki, L., 2017. Rosemary (Rosmarinus officinalis) essential oil components exhibitanti-hyperglycemic, anti-hyperlipidemic and antioxidant effects inexperimental diabetes. Pathophysiology, 915: 1-7. https://doi.org/10.1016/j.pathophys.2017.08.002
62. Shahidi, F., Liyana-Pathirana, C. M. & Wall, D, S., (2006). Antioxidant activity of white and black sesame seeds and their hull fractions. Food Chemistry, 99, 478–83. https://doi.org/10.1016/j.foodchem.2005.08.009
63. Shan, B., Cai, Y. Z., Sun, M. & Corke, H., (2005). Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. Journal of Agricultural and Food Chemistry, 53(20), 7749-7759. https://doi.org/10.1021/jf051513y
64. Soltani, M., Ghodratnama, M., Ebrahimzadeh-Mosavi, H. A., Nikbakht-Brujeni, , Mohamadian, S. & Ghasemian, M., (2014). Shirazi thyme (Zataria multiflora Boiss) and Rosemary (Rosmarinus officinalis) essential oils repress expression of sagA, a streptolysin S-related gene in Streptococcus iniae. Aquaculture, 430, 248-252. https://doi.org/10.1016/j.aquaculture.2014.04.012
65. Wang, S., Meckling, K. A., Marcone, M. F., Kakuda, Y. & Tsao, R., (2011). Can phytochemical antioxidant rich foods act as anti-cancer agents. Food Research Internationa, 44(9), 2545- 2554. https://doi.org/10.1016/j.foodres.2011.05.021
66. Wojdyło, A., Oszmianskii, J. & Czemerys, R., (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chemistry, 105, 940-949. https://doi.org/10.1016/j.foodchem.2007.04.038
67. Yesil-Celiktas, O., Bedir, E. & Vardar Sukan, F., (2007). In vitro antioxidant activities of Rosmarinus officinalis extracts treated with supercritical carbon dioxide. Food Chemistry, 101, 1457-1464. https://doi.org/10.1016/j.foodchem.2006.03.055
68. Yesil-Celiktas, O., Girgin, G., Orhan, H., Wichers, H. J., Bedir, E. & Vardar-Sukan, F., (2007b). Screening of free radical scavenging capacity and antioxidant activities of Rosmarinus officinalis extracts with focus on location and harvesting times. European Food Research and Technology, 224,443-51. https://doi.org/10.1016/j.foodres.2011.05.021
69. Yesil-Celiktas, O., Sevimli, C., Bedir, E. & Vardar-Sukan, F., (2010b). Inhibitory effects of rosemary cxtracts, carnosic acid and rosmarinic acid on the growth of various human Cancer Cell Lines. Plant Foods for Human Nutrition, 65, 158-163. https://doi.org/10.1007/s11130-010-0166-4
70. Young, IS. & Woodside, J., (2001). Antioxidants in health and disease. Journal of Clinical Pathology, 54, 176- 186.