Determination of allomelanins in sunflower husk by the gravimetric method

Background. Melanins are a group of pigments with a complex crystalline structure and high molecular weight, which are widely used in modern medical, perfume, food, and polymer production. They have pronounced antiviral, antibacterial, antifungal, antiradiation effects, and antioxidant activity. Melanins of animal (melanin), microbial (mycomelanin), and plant (phytomelanin) origin are distinguished. At present, the search for new sources of plant melanins and simple methods for their determination is relevant, allowing for the further identification of accessions with a higher content of these substances for various uses. An important role of melanin, which accumulates in the sunflower seed coat, is its role in ensuring the resistance of this crop to the sunflower moth (Homoeosoma nebulella Hb.). The present study was aimed at developing a method and demonstrating its capabilities using accessions from the VIR sunflower collection. Materials and methods. The paper presents a modified method for determining allomelanins (phytomelanins) in husk and seed of sunflower accessions from the VIR collection. Optimal modes of allomelanin extraction, filtration, precipitation and washing from impurities have been developed. Results. The allomelanin content in sunflower husk averaged 5.7% with a minimum of 1.5% and a maximum of 8.7%. The fraction of pigment in the studied sunflower seeds ranged from 0.4% to 2.7% and averaged 1.7%. The accessions with a higher pigment content in husk are k-2776, k-3511, k-3892, k-3901, and k-641884, and those with a lower pigment content are k-3568, k-3647, k-3649, k-3760, and k-3762. A high positive correlation was found between the allomelanin content in husk and seed (r=0.83). Conclusion. A simple method for isolating allomelanins has been developed. It yields highly reproducible results and can be used to screen a collection of plant genetic resources to identify accessions with high melanin content. This method allows the identification of the most promising accessions which can be involved in breeding for resistance to the damaging effects of sunflower moth caterpillars.

1. Alekseeva T.N., Oganesiants L.A., Krasnikova E.V., Rudometova N.V. Study of plant melanin as a food coloring agent for soft drinks (Issledovanie rastitel'nogo melanina kak pishchevogo krasitelya dlya bezalkogol'nykh napitkov). Hranenie i pererabotka sel'khozsyr'ja = Storage and processing of agricultural raw materials. 2008;7:40-43. [in Russian]

2. Alekseeva T.N., Oreshchenko A.V., Kulakova A.V., Durnev A.D., Samusenok L.V., Ogarkov B.N. Effect of plant melanin pigment on the clastogenic effects of chemical mutagens in mice (Vliyanie rastitel'nogo melaninovogo pigmenta na klastogennye effekty khimicheskikh mutagenov u myshey). Eksperimental'naya i klinicheskaya farmakologiya = Experimental and clinical pharmacology. 2001;6:56-59. [in Russian]

3. Baraboi V.A. Structure, biosynthesis of melanins, their biological role and application prospects (Struktura, biosintez melaninov, ih biologicheskaya rol' i perspektivy primeneniya). Uspekhi sovremennoy biologii = Advances in Current Biology. 2001;12:1-12. [in Russian]

4. Butterfield D.A. Spin labeling in disease. In: Biological Magnetic Resonance. L.J. Berliner, J. Reuben (eds.). New York: Springer Science and Business Media; 1982. Vol. 4. p.1-78. DOI: 10.1007/978-1-4615-6540-6_1

5. Chu M., Hai W., Zhang Z., Wo F., Wu Q., Zhang Z., Shao Y., Zhang D.Sh.-zi, Jin L., Shi D. Melanin nanoparticles derived from a homology of medicine and food for sentinel lymph node mapping and photothermal in vivo cancer therapy. Biomaterials. 2016;91:182-199. DOI: 10.1016/j.biomaterials.2016.03.018

6. Duran J.M., Retamal N. Coat structure and regulation of dormancy in Sinapis arvensis L. seeds. Plant Physiology. 1989;135:218-222.

7. Fomenko I.A., Ivanova L.A., Churmasova L.A., Degtyarev I.A. Production of water-soluble phytomelanins using various mineral acids. Bulletin of Biotechnology and Physicochemical Biology named after Yu.A. Ovchinnikov. 2021;17(2):64-68. [in Russian]

8. Gashnikova N.M., Balakhnin S.M., Tepliakova T.V., Anan'ko G.G., Kosogova T.A., Suhih A.S. Antiretroviral activity of melanins from natural and cultivated chaga (Inonotus obliquus) (Antiretrovirusnaya aktivnost' melaninov iz prirodnoy i kul'tiviruemoy chagi (Inonotus obliquus)). Uspekhi meditsinskoy mikologii = Advances in Medical Mycology. 2014;12:299-301. [in Russian]

9. Gerdemann C., Eicken C., Krebs B. The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Accounts of Chemical Research. 2002;35:183-191.

10. Gracheva N.V., Zheltobriukhov V.F. Method for obtaining melanin from sunflower husk and study of its antioxidant activity (Sposob polucheniya melanina iz luzgi podsolnechnika i issledovanie ego antioksidantnoy aktivnosti). Vestnik Tehnologicheskogo universiteta = Bulletin of the Technological University. 2016;19(15):154-157. [in Russian]

11. Grossi G.F., Durante M., Gvalanella G. Effects of melanin on high-LET radiation response of human epithelial cells. Radiation and Environmental Biophysics. 1998;37:63-67.

12. Hill H. The function of melanin or six blind people examine an elephant. BioEssays. 1992;14(1):49-56. DOI: 10.1002/bies.950140111

13. Iuasifov E.Yu. Effect of melanin on the free-radical state of gamma-irradiated proteins and lipids (Vliyanie melanina na svobodno-radikal'noe sostoyanie gamma-obluchennykh belkov i lipidov. Radiobiologiya = Radiobiology, 1987;27(1):8-11. [in Russian]

14. Ivanova L.A., Fomenko I.A., Sergeeva D.A., Churmasova L.A., Kabarzhan Zh.K. Development of technologies for producing phytomelanines on waste oil production. Health, Food and Biotechnology. 2019;1(2):136-146. [in Russian]. DOI: 10.36107/hfb.2019.i2.s245

15. Jana B., Mukherjee S. Notes on the distribution of phytomelanin layer in higher plants – a short communication. Journal of Pharmaceutical Biology. 2004;4(3):131-132.

16. Kablov V.F., Novopoltseva O.M., Gracheva N.V., Zheltobryukho V.F., Dao Ph.K. Prospects of application of melanins as antiaging agents in elastomer compositions. Vietnam Journal of Chemistry. 2019.57(2):255-260. DOI: 10.1002/vjch.201960024

17. Kartushina Yu.N., Kirichenko M.A., Sevriukova G.A. Obtaining melanin based on waste from oil extraction production (Poluchenie melanina na osnove otkhodov masloekstraktsionnogo proizvodstva). Vestnik Tehnologicheskogo universiteta = Bulletin of the Technological University. 2016;19(16):124-126. [in Russian]

18. Korytowski W., Sarna T. Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic dopa-melanin. Biological Chemistry. 1990;265(21):12410-12416.

19. Liakh S.P., Bulgak M.L., Isaev A.G. Astromelanin: a therapeutic agent for melanotherapy: the book is dedicated to the 40th anniversary of the study of the Antarctic black yeast Nadsoniella nigra var. hesuelica and their melanopigment AstroMelanin. Moscow; 2007. [in Russian]

20. Łopusiewicz Ł., Drozłowska E., Trocer P., Kostek M., Śliwiński M., Henriques M.H.F., Bartkowiak A., Sobolewski P. Whey protein concentrate/ isolate biofunctional films modified with melanin from watermelon (Citrullus lanatus) seeds. Materials. 2020;13(17):3876. DOI: 10.3390/ma13173876

21. Novikov D.A., Kurchenko V.P., Azarko I.I. Photoprotective properties of melanins from grape (Vitis vinifera) and black tea (Thea sinensis). Radiation biology. Radioecology. 2001;41(6):664-670. [in Russian]

22. Özdemir Ö., Keleş Y. Extraction, purification, antioxidant properties and stability conditions of phytomelanin pigment on the sunflower seeds. International Journal of Secondary Metabolite. 2018;5(2):140-148 DOI: 10.21448/ijsm.377470

23. Park K.I., Ishikawa N., Morita Y. Choi J.D., Hoshino A., Iida S. A bHLH regulatory gene in the common morning glory, Ipomoea purpurea, controls anthocyanin biosynthesis in flowers, proanthocyanidin and phytomelanin pigmentation in seeds, and seed trichome formation. The Plant Journal. 2007;49(4):641-654. DOI: 10.1111/j.1365-313X.2006.02988.x

24. Perestova T.A. Method of early diagnosis of the incrustation of sunflower achenes: (guidelines) (Sposob ranney diagnostiki pantsirnosti semyanok podsolnechnika). Krasnodar: VNIIMK; 1988. [in Russian]

25. Pralea I.E., Moldovan R.C., Petrache A.M., Ilieș M., Hegheș S.C., Ielciu I., Nicoară R., Moldovan M., Ene M., Radu M., Uifălean A., Iuga C.-A. From extraction to advanced analytical methods: the challenges of melanin analysis. International Journal of Molecular Sciences. 2019;20(16):3943. DOI: 10.3390/ijms20163943

26. Prutenskaia E.A., Vasil'ev A.S., Lebedeva E.Yu., Ushchapovsky I.V., Silchenko V.A. Comparative characteristics of the structure of melanins of different origin (Sravnitel'naya kharakteristika struktury melaninov razlichnogo proiskhozhdeniya). International Scientific Journal. Symbol of Science. 2016;11(3):11-13. [in Russian]

27. Rogers C.E., Kreitner G.L. Phytomelanin of sunflower achenes: a mechanism for pericarp resistance to abrasion by larvae of the sunflower moth (Lepidoptera: Pyralidae). Environmental Entomology. 1983;12(2):277-285. DOI: 10.1093/ee/12.2.277

28. Saha L., Tiwari J., Bauddh K., Ma Y. Recent developments in microbe-plant-based bioremediation for tackling heavy metal-polluted soils. Frontiers in Microbiology. 2021;12:731723. DOI: 10.3389/fmicb.2021.731723.

29. Sava V.M., Yang S.-M., Hong M.-Y., Yang P.-C., Huang G.S. Isolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry. 2001;73:177-184. DOI: 10.1016/S0308-8146(00)00258-2

30. Shkolnikova M.N., Kadritskaya E.A. Rationale for the use of buckwheat husk for the production of functional food colors. Scientific Journal NRU ITMO. Series: Processes and Food Production Equipment. 2020;4:22-28. [in Russian]. DOI: 10.17586/2310-1164-2020-10-4-22-28

31. Shoeva O.Y., Mursalimov S.R., Gracheva N.V., Glagoleva A.Y., Börner A., Khlestkina E.K.. Melanin formation in barley grain occurs within plastids of pericarp and husk cells. Scientific Reports. 2020;10(1):179. DOI: 10.1038/s41598-019-56982-y

32. Solano F. Melanins: Skin Pigments and Much More – Types, Structural Models, Biological Functions, and Formation Routes. New Journal of Science. 2014(5):1-28. DOI: 10.1155/2014/498276

33. Varga M., Berkesi O., Darula Z., May N.V., Palágyi A. Structural characterization of allomelanin from black oat. Phytochemistry. 2016;130:313-320. DOI: 10.1016/j.phytochem.2016.07.002

34. Vorontsova Z.A., Ivanov A.A., Nikityuk D.B., Avanesova A.A. Some morphoclinical evidence about radioprotective melanin character (literature report). Bulletin of New Medical Technologies. 2016;4:295-302. [in Russian]. DOI: 10.12737/22219