مقايسه سميت سلولي پپتيدهاي زيست فعال استخراج شده از عضله ماهي مركب ببري به سه روش خشك كن انجمادي، پاششي و تحت خلاء بر پروماستيگوت‌هاي انگل ليشمانيا ماژور

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

سابقه و هدف: در سال‌هاي اخير، درمان بيماري ليشمانيوزيس بدليل بروز مقاومت دارويي و مشكلات مربوط به اثرات جانبي داروهاي متداول با مخاطراتي مواجهه شده است. اين مطالعه با هدف بررسي اثر سميت سلولي پپتيدهاي زيست فعال حاصل از هيدروليز عضله ماهي مركب ببري بر پروماستيگوت‌هاي انگل ليشمانيا ماژور انجام شد. مواد و روش: متغيرهاي نوع آنزيم، نسبت غلظت آنزيم به سوبسترا، زمان هيدروليز و روش خشك كردن بر ويژگي‌هاي درصد توليد، پروتئين پودر پپتيد، ظرفيت آنتي اكسيداني تام، فعاليت مهار راديكال‌هاي DPPH، نيتريك اكسايد و گروه‌هاي تيول مورد مطالعه قرار گرفت. پس از تعيين درصد پروتئين عضله سفيد ماهيان مركب، از آنزيم‌هاي آلكالاز و پاپائين 5 درصد و زمان 180 دقيقه جهت هيدروليز استفاده گرديد. خاصيت سميت سلولي بر پروماستيگوت انگل ليشمانيا ماژور با استفاده از روش MTT ارزيابي گرديد. يافته‌ها: نتايج نشان داد كه درجه هيدروليز پروتئين در عضله با آنزيم آلكالاز 5 درصد، بيشتر از ساير آنزيم‌هاي مورد مطالعه بود. با كمك آنزيم آلكالاز 5 درصد در زمان 180 دقيقه و با استفاده از روش خشك كردن پاششي بهترين فعاليت آنتي اكسيداني در روش‌هاي سنجش ظرفيت آنتي اكسيداني تام، سنجش قدرت آنتي اكسيدانتي، فعاليت جاروكنندگي نيتريك اكسايد، گروه‌هاي سولفيدريل كل و بيشترين خاصيت سميت سلولي حاصل گرديد. استنتاج: نتايج نشان داد كه با افزايش غلظت پودر پپتيد بدست آمده، ميزان مرگ و مير پروماستيگوت هاي انگل ليشمانيا ماژور در تمام گروه‌هاي مورد مطالعه افزايش يافته است. بهترين ميزان IC50 در بين گروه‌هاي مورد مطالعه در غلظت 10 ميلي گرم بر ميلي ليتر از هيدروليزات حاصل از آنزيم آلكالاز 5% با روش خشك كن پاششي نسبت به گروه كنترل مشاهده گرديد.

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.