مقایسه سمیت سلولی پپتیدهای زیست فعال استخراج شده از عضله ماهی مرکب ببری به سه روش خشک کن انجمادی، پاششی و تحت خلاء بر پروماستیگوت‌های انگل لیشمانیا ماژور

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه پاتوبیولوژی دانشکده دامپزشکی دانشگاه شهید چمران اهواز

2 گروه پاتوبیولوژی و کنترل کیفی، پژوهشکده آرتمیا و آبزی پروری دانشگاه ارومیه

10.22034/fr.2021.42967.1777

چکیده

سابقه و هدف: در سال‌های اخیر، درمان بیماری لیشمانیوزیس بدلیل بروز مقاومت دارویی و مشکلات مربوط به اثرات جانبی داروهای متداول با مخاطراتی مواجهه شده است. این مطالعه با هدف بررسی اثر سمیت سلولی پپتیدهای زیست فعال حاصل از هیدرولیز عضله ماهی مرکب ببری بر پروماستیگوت‌های انگل لیشمانیا ماژور انجام شد. مواد و روش: متغیرهای نوع آنزیم، نسبت غلظت آنزیم به سوبسترا، زمان هیدرولیز و روش خشک کردن بر ویژگی‌های درصد تولید، پروتئین پودر پپتید، ظرفیت آنتی اکسیدانی تام، فعالیت مهار رادیکال‌های DPPH، نیتریک اکساید و گروه‌های تیول مورد مطالعه قرار گرفت. پس از تعیین درصد پروتئین عضله سفید ماهیان مرکب، از آنزیم‌های آلکالاز و پاپائین 5 درصد و زمان 180 دقیقه جهت هیدرولیز استفاده گردید. خاصیت سمیت سلولی بر پروماستیگوت انگل لیشمانیا ماژور با استفاده از روش MTT ارزیابی گردید. یافته‌ها: نتایج نشان داد که درجه هیدرولیز پروتئین در عضله با آنزیم آلکالاز 5 درصد، بیشتر از سایر آنزیم‌های مورد مطالعه بود. با کمک آنزیم آلکالاز 5 درصد در زمان 180 دقیقه و با استفاده از روش خشک کردن پاششی بهترین فعالیت آنتی اکسیدانی در روش‌های سنجش ظرفیت آنتی اکسیدانی تام، سنجش قدرت آنتی اکسیدانتی، فعالیت جاروکنندگی نیتریک اکساید، گروه‌های سولفیدریل کل و بیشترین خاصیت سمیت سلولی حاصل گردید. استنتاج: نتایج نشان داد که با افزایش غلظت پودر پپتید بدست آمده، میزان مرگ و میر پروماستیگوت های انگل لیشمانیا ماژور در تمام گروه‌های مورد مطالعه افزایش یافته است. بهترین میزان IC50 در بین گروه‌های مورد مطالعه در غلظت 10 میلی گرم بر میلی لیتر از هیدرولیزات حاصل از آنزیم آلکالاز 5% با روش خشک کن پاششی نسبت به گروه کنترل مشاهده گردید.

کلیدواژه‌ها


عنوان مقاله [English]

Comparison of cytotoxicity of bioactive peptides extracted from tiger squid muscle by three methods of freeze-drying, spray-drying, and vacuum-drying on Leishmania major promastigotes

نویسندگان [English]

  • Farhad Farhangpazhouh 1
  • Mohammad Hossein Razi Jalali 1
  • Alireza Alborzi 1
  • Somayeh Bahrami 1
  • Mehdi Nikoo 2
1 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz
2 Department of Pathobiology and Quality Control, Artemia and Aquaculture Research Institute, Urmia University
چکیده [English]

Background: Leishmaniasis is one of the intracellular parasitic diseases that is known as the most common infectious disease after AIDS, tuberculosis and malaria. Leishmaniasis is among the 6 most prevalent endemic diseases and is in fact a tropical and subtropical disease caused by intracellular parasites worldwide. In recent years, treatment of leishmaniasis has been associated with risks due to drug resistance and problems related to the side effects of conventional drugs. The disease is found on all continents except Oceania and is endemic to enclosed areas in North Africa, southern Europe, the Middle East, southeastern Mexico, and Central and South America. The aim of this study was to investigate the cytotoxic effect of bioactive peptides obtained from hydrolysis of tiger squid muscle on promastigotes of Leishmania major. Materials and Methods: The variables of enzyme type, ratio of enzyme concentration to substrate, time of hydrolysis and drying method were studied on the characteristics of hydrolysis protein. After determining the percentage of white muscle protein of squid caught from Bushehr port, alkalase and papain enzymes were used at 5% levels for 180 minutes for hydrolysis. Production percentage, protein peptide powder, total antioxidant capacity (TAC), radical scavenging activity of DPPH, nitric oxide (NO) and total thiol groups were measured. Also, the cytotoxicity of Leishmania major promastigotes was evaluated using MTT method. Glucantime was used as a positive control and the untreated group was used as a negative control. Results: Muscle hydrolysis with 5% Alkalase enzyme caused the highest degree of hydrolysis (63%) and in general, the concentration of 5% of enzymes had a higher degree of hydrolysis than the concentration of 2.5%. Analysis of variance showed that the effect of enzyme, drying time and drying method on all measured variables had a statistically significant difference at the level of one percent (p <0.01). The effect of concentration on peptide dry powder, peptide protein, TAC and thiol was significant. However, no statistically significant difference was observed between enzyme concentrations in DPPH and Nitric Oxide. The dual interaction of enzyme × concentration on the variables of dry powder of peptide and Thiol at the level of 1% showed a statistically significant difference (p <0.01). However, the interaction of enzyme × concentration on other variables was not significant. The results showed that the dual interaction of enzyme × time on all studied variables showed a statistically significant difference. (p <0.01). The dual interaction effect of enzyme × drying method was significant only on Nitric Oxide and Thiol variables (p <0.01). Also, statistical results using analysis of variance showed that the dual interaction effect of concentration × time on the variables of dry peptide powder, peptide powder protein and Thiol had a statistically significant difference. (p <0.01). The dual interaction effect of concentration × drying method on the variables of peptide dry powder, peptide powder protein, DPPH and Thiol was also significant (p <0.01). Statistical results showed that the dual interaction of drying time × drying method on all studied variables except TAC showed a statistically significant difference (p <0.01). The results of analysis of variance for the triple interactions of enzyme × concentration × time and enzyme × concentration × drying method showed statistically significant difference only on Thiol at the level of one percent (p <0.01). The triple interaction effect of enzyme × time × drying method on protein peptide powder, DPPH, Nitric Oxide and Thiol showed a statistically significant difference at the level of one percent (p <0.01). The results of analysis of variance showed a statistically significant difference (p <0.01) with the triple interaction of concentration × time × drying method on the protein content of peptide powder, DPPH, TAC, Nitric Oxide and Thiol at the level of one percent. Finally, the results of analysis of variance showed that quadruple interaction effect of enzyme concentration × time × drying method on all studied variables had a statistically significant difference at the level of one percent (p <0.01). The results of comparing the mean of the quadruple interaction effect of enzyme × concentration × time× drying method showed that the highest amount of peptide powder, the lowest protein content and the best antioxidant activity are obtained in TAC, DPPH, Nitric Oxide and Thiol using digestion method with the help of 5% Alkalase enzyme for 180 minutes using spray dryer method. The results showed that the degree of protein hydrolysis in muscle with alkalase enzyme was 5% higher than other enzymes studied. With the help of 5% alkalase enzyme in 180 minutes and using spray drying method, the lowest amount of protein in hydrolyzed powder, the best antioxidant activity were obtained in TAC, DPPH, Nitric Oxide, Thiol and the highest cytotoxicity. The highest amount of muscle hydrolysis powder was obtained in the presence of 5% alkalase enzyme in 180 minutes and cooling drying method. Conclusion: MTT results showed that with increasing the concentration of peptide powder obtained from enzymatic hydrolysis of white tiger squid muscle, the mortality rate of Leishmania major promastigotes increased in all study groups. The best level of IC50 among the studied groups was observed at a concentration of 10 mg / ml of hydrolyzate from 5% alkalase enzyme by spray dryer compared to the control group. The results of this study showed that the hydrolysis protein of Sepia pharaonis white muscle produced with 5% Alkalase enzyme has cytotoxic effect on Leishmania major promastigotes and this effect has shown a stronger activity in hydrolysis prepared using spray drying method than other methods.

کلیدواژه‌ها [English]

  • Alkalase
  • Hydrolysis
  • Leishmania major parasite
  • Peptide
  • Promastigote
  • Tiger squid
ابکا خواجوئی ر، کرامت ج و همدمی ن، 1398. بهینه­­سازی شرایط استخراج آلژینات سدیم از جلبک قهوه­ای ایرانی به روش سطح پاسخ. پژوهش­های صنایع غذایی، شماره 29، صفحه 28-13.
اکبربگلو ز، پیغمبردوست س ه، اولادغفاری ع و سرابندی خ، 1397. اثر دمای هوای ورودی، نوع و غلظت ماده حامل بر ویژگی‌های فیزیکی شیمیایی و ضداکسایشی عصاره مرزنجوش ریزپوشانی شده با خشک کن پاششی. پژوهش­های صنایع غذایی، شماره 28، صفحه 30-15.
جعفری ف و جوادی ا، 1399. اثر پوشش‌دهی با کیتوزان و عصاره برگ گردو در ماندگاری پسته. پژوهش­های صنایع غذایی، شماره 30، صفحه 232-221.
زندی ک، فرسنگی م، نبی پور ح و سلیمانی س م، 2004. شناسائی، استخراج و تخلیص پروتئین 60 کیلو دالتونی ماده بنفش رنگ ترشحه از خرگوش دریایی خلیج فارس Aplysia dactylomela با اثرات ضدسرطانی. دانشگاه علوم پزشکی بوشهر، طب جنوب، شماره 6، صفحه 97-103.‎
نیکو م ع، ماهونک ع، قربانی ص، طاهری م، اعلمی ع  و کمالی م، 1392. بررسی اثر شرایط هیدرولیز برفعالیت ضداکسایشی پروتئین هیدرولیز شده از ماهی کاراس (Carassius carassius). نشریه پژوهش و نوآوری در علوم و صنایع غذایی، شماره 4، صفحه 364-351.
Alder-Nissen J, 1986.Enzymic hydrolysis of food proteins. NY. Elsevier applied science publisher. pp. 110-169.
AOAC, 2005. Official Method of Analysis (17th Ed). Washington, DC: Association of Official Analytical Chemists.
Chalamaiah M, Kumar B D, Hemalatha T and Jyothirmay T, 2012. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review. Food Chemistry 135 (4): 3020-3038.
Chen HM, Muramoto K, Yamauchi F and Nokihara K, 1996. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of agricultural and food chemistry 44 (9): 2619-2623.
Esmaeili Kharyeki M, Rezaei M, Khodabandeh S and Motamedzadegan A, 2018. Antioxidant Activity of Protein Hydrolysate in Skipjack tuna Head. Fisheries Science and Technology 7 (1): 57-64.‏
FAO/WHO, 1991. Protein quality evaluation. (Report of Joint FAO/ WHO Expert Consultation. FAO Food and Nutrition Paper 51). FAO/WHO, Rome, Italy.
Hermann B, Kehrer T, Bauer L and Wenz F, 2005. Intraoperative radiotherapy (IORT) for breast cancer using the intrabeam™ system. Tumori Journal 91 (4): 339-345.
Je JY, Qian ZJ, Byun HG and Kim SK, 2007. Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochemistry 42 (5): 840-846
Karami Z and Akbari-adergani B, 2019. Bioactive food derived peptides: a review on correlation between structure of bioactive peptides and their functional properties. Journal of Food Science and Technology 56 (2): 535-547.
Kasankala L M, Xiong Y L and Chen J, 2012. Enzymatic activity and flavor compound production in fermented silver carp fish paste inoculated with douchi starter culture. Journal of Agricultural and Food Chemistry 60 (1): 226-233.
Kurutas EB, 2015. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutrition journal 15(1): 1-22.
Lobo V, Patil A, Phatak A and Chandra N, 2010. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy reviews 4 (8): 118.
Lowry OH, Rosebrough Farr NJ and Randall RJ, 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193: 265-275.
Ngo DH, Qian ZJ, Ryu B M, Part J W and Kim S K, 2010. In vitro antioxidant activity of a peptide isolated from Nile tilapia (Oreochromis niloticus) scale gelatin in free radical-mediated oxidative systems. Journal of Functional Foods 2:107-117.
Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N and Xu X, 2014. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. Journal of Functional Foods 7: 609-620.
Nikoo M, Benjakul S, Yasemi M, Ahmadi Gavlighi H and Xu X, 2019. Hydrolysates from rainbow trout (Oncorhynchus mykiss) processing by-product with different pretreatments: Antioxidant activity and their effect on lipid and protein oxidation of raw fish emulsion. LWT-Food Science and Technology 108: 120-128.
Senphan T and Benjakul S, 2014. Antioxidative activities of hydrolysates from seabass skin prepared using protease from hepatopancreas of Pacific white shrimp. Journal of Functional Foods 6: 147-156.
Shahidi F, Zhong H J and Ambigaipalan P, 2005. Antioxidants: regulatory status. Bailey's industrial oil and fat products 1-21.
Shaughnessy L M, Hoppe AD, Christensen K A and Swanson JA, 2006. Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles. Cellular microbiology 8(5): 781-792.
Zheng L, Yu H, Wei H, Xing Q, Zou Y, Zhou Y and Peng J, 2018. Antioxidative peptides of hydrolysate prepared from fish skin gelatin using ginger protease activate antioxidant response element-mediated gene transcription in IPEC-J2 cells. Journal of Functional Foods 51: 104-112.