Nova Biotechnol Chim (2022) 21(1): e1246 DOI: 10.36547/nbc.1246 1 Nova Biotechnologica et Chimica Low abundant bovine colostrum proteins in combination with amaranth oil reveal topical analgesic activity Nail J. Sagdiev1, Jamolitdin F. Ziyavitdinov1, Nodir Sh. Berdiev1, Soyib S. Bozorov1, Tohir A. Khudoyberdiev1, Shukhratjon S. Olimjonov1, Natalya L. Vypova1, Akmal M. Asrorov1, 2,  1Institute of Bioorganic Chemistry of Uzbekistan Academy of Sciences, 100125, 83, M. Ulughbek Str., Tashkent, Uzbekistan 2Ajou University in Tashkent, 100204, 113A, Asalobod Str., Tashkent, Uzbekistan  Corresponding author: akmal84a@gmail.com Article info Article history: Received: 21st October 2021 Accepted: 1st December 2021 Keywords: Amaranth oil Analgesic activity Bovine colostrum Low-abundant proteins Water-alcohol-soluble fraction Abstract Colostrum is an arsenal of proteins and peptides required at the earliest stage of a newborn development. Proteins involved in all biological processes of an infant development have been found in its composition. It is expected as cost-effective source of biologically active proteins and peptides. In this work, we separated water- alcohol-soluble low abundant proteins from bovine colostrum in preparative amounts that were further utilized in combination with amaranth oil for topical cream composition. The ratio of the obtained proteins’ fraction made a thousandth of the colostrum dry mass. The partial sequences of 37 identified proteins were established by mass-spectrometer and using BLAST search in NCBI database. In our previous work, we established the chemical composition of amaranth seed oil with ~6 % squalene by mass. The physical mixtures of these natural resources were fabricated into cream using hyaluronic acid as moisturizing agent and their analgesic activities were established. The optimal ratio of proteins and oil was determined in terms of their effects as analgesic means by experiments carried out on mice. Several proteins could possibly be responsible for the revealed biological efficacy. Among them, G-protein coupled receptor and synaptotagmin were previously linked with analgesic activity. Establishing an optimum ratio of ingredients proved also the contribution of higher quantity of amaranth oil, a rich source of squalene and unsaturated fatty acids. Introduction Colostrum is a rich source of proteins, fats, carbohydrates, and other ingredients. As a product secreted after birth, it has a higher content of proteins than milk. Immunoglobulins, lactoferrin, lysozyme, and different growth factors compile the main content of colostrum (Silva et al. 2019). Albumin, heavy chains of immunoglobulins M and G, antitestosterone antibody, lactotransferrin, and polymeric immunoglobulin receptors were demonstrated as the main content of the colostrum whey (Zhang et al. 2011). By two-dimensional analysis, IGHG1 (immunoglobulin heavy constant gamma 1), C1 with uncharacterized function and VI1a protein were found as low abundant proteins in bovine colostrum whey (Golinelli et al. 2011). Recent research analysis revealed that exosomes of colostrum are enriched with proteins regulating the immune response and growth. The biological roles mailto:akmal84a@gmail.com Nova Biotechnol Chim (2022) 21(1): e1246 2 of the proteins from exosomes were found to belong to immune regulation, growth and infant development (Samuel et al. 2017). A number of colostrum-based products have reached the market as cosmetics, bioadditives, and other means. In combination with other ingredients, colostrum has been patented as a pharmaceutical and cosmetic composition (Gobbi 2007). In another document, different ratios of horse colostrum and milk were earlier patented with other ingredients as pain- relieving agents against sun burns and fire burns (Gobbi 1993). In-depth analysis of bovine colostrum with 2D-LC- MS/MS analysis identified 403 proteins in the nonfractionated colostrum (Nissen et al. 2012). In the fluid phase, 69 additional proteins were found. Among all identified proteins, 38 % of them belonged to cellular processes, biological regulations, response to stimulus, and regulations of biological processes. Studying the proteomic changes in bovine colostrum and milk revealed that the highest number of proteins belonged to immunity, transport and cell processes both in colostrum and milk (Zhang et al. 2015). Number of proteins in colostrum involved in cellular process, response to stimulus, metabolic processes, and regulation of biological processes were found as the highest both in bovine colostrum and milk; but all of them made lower number in the composition of milk (Nissen et al. 2017). A study on the effects of heat treatment on bovine colostrum protein profile demonstrated that low abundant proteins, involved in cellular processes and immune response, are affected by heat (Tacoma et al. 2017). Besides, higher number of low abundant proteins was found to belong to metabolic processes, biological regulation, multicellular organismal processes and response to stimulus. Recent advances in research allowing find low and very low abundant proteins from colostrum and milk. Using so-called combinatorial peptide library technology (CPLL) treatments made it possible to detect the signal of proteins masked by major proteins that constitute >95 % of total bovine colostrum protein mass (Altomarea et al. 2016). Another tandem with LC-MS/MS in this approach used isobaric tag for relative and absolute quantification (iTRAQ) (Yang et al. 2017). Thus, the method allowed to compare differentially expressed proteins in whey of human and bovine colostrum and milk. In this work, we identified the low-abundant proteins in bovine colostrum after enrichment and by eluting with water-alcohol solution. The enrichment process was carried by trapping the proteins in polytetrafluoroethylene sorbent that was further eluted with water-alcohol solution. The idea came from the isolation of the ones of more hydrophobic nature. The proteins of hydrophobic interaction could have higher membrane positioning and thus cross the membrane more easily (Lomize et al. 2017). Sequences of the isolated proteins and peptides were established by mass-spectrometric analysis. Further analgesic activity of the fraction was studied in combination with amaranth oil and hyaluronic acid; optimum mass ratio of the ingredients were established. In our previous work, we demonstrated high hyperlipidemic efficiency of amaranth oil that could be resulted from high concentration of unsaturated fatty acids and squalene (Bozorov et al. 2018). Oil samples obtained from local amaranth varieties (Olimjonov et al. 2020) can contribute to economic competitiveness. Skin surface lipids contain up to 13 % squalene (Passi et al. 2002) that can contribute to higher interaction with squalene that ease the penetration of active compounds. Experimental Separation of proteins from bovine colostrum Bovine colostrum samples obtained from cattle (Bos taurus) on the 1st, 2nd, and 3rd days of the birth were gathered and the mixture was further used for analysis. To precipitate high-molecular-weight proteins, 60 mL of 54 % (w/v) perchloric acid (HClO4) solution was added to 945 mL of colostrum and kept in the refrigerator for 24 h capped. The precipitate was centrifuged and supernatant was collected, the volume was 600 mL. The excess amount of HClO4 in the obtained supernatant was neutralized with potassium alkali until pH reached 7.5 and kept in refrigerator for 10 h. The resulted mixture was centrifuged to remove excess of KClO4. Supernatant volume compiled was 550 mL. It was left in the refrigerator overnight and used for column chromatography. Nova Biotechnol Chim (2022) 21(1): e1246 3 Column chromatography Hydrophobic chromatography with a sorbent polytetrafluoroethylene (polychrome-1) filled in a 400 mL column connected to LKB Bromma pump chromatography system was used to separate the fractions in a 550 mL mixture and desalinate the solution. For that, the obtained solution was first passed through a column. The column was washed out with water; the elution of the trapped proteins from the sorbent was carried out using an aqueous solution of 60 % ethyl alcohol (120 mL solution was used once with a flow rate 60 mL.h-1). The eluents were collected and the excess alcohol was removed using a rotory evaporator (RE 2000A). Further, the solutions were gathered and lyophilized (The Virtis Automatic Freeze-Dryer, 10-010). Mass spectroscopy analysis of low-abundant fraction of proteins For mass-spectral analysis, 2 – 5 µL of digested protein samples were subjected to CHIP (150 µm × 43 mm) Zorbax SB C18 column in Agilent Technologies 1260 Cap Pump with a flow rate of 4 µL per min, elution rate made 0.6 µL per min. B solution was managed as following: 5 %, 3 min; 80 %, 25 – 30 min. The solution was degassed in Agilent Technologies 1260 µ-degasser. Mass detection was performed in CHIP-Q-TOF LC-MS Agilent Technologies 6520В Series mass spectrometer. Mass data was recorded in positive ionization mode (ionization source – CHIP interface, gas flow rate – 4 L.min-1 at 350 oC, skimmer current power – 65 V, MS/MS – 50 – 2400 m/z, mobile phase: A – 0.1 % formic acid, B – acetonitrile + 0.1 % formic acid) and partial sequences were identified with Spectrum Mill database. For functional annotations, obtained fragments were further searched by BLAST analysis in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi): taxid: 9903 (belonging to Bos taurus) identity was chosen as organism. For functional annotations and reliability, NCBI search results of the identified peptides with percentage identity higher than 85 % were checked with higher identities in the proteome of other mammals. Establishment of amaranth oil composition The chemical composition of amaranth oil was established as described in our previous work (Bozorov et al. 2019). The seeds of Helios variety were ground in a porcelain mortar using quartz sand. The oil was extracted from the resulting powder with acetone by stirring for 30 min. The squalene content in the oil sample was 5.7 %. Fabrication of the cream The basic components of the cream included 5 g of T-1 emulsifier and 0.2 g of sorbic acid as antibacterial means in a total mass of 100 g cream. In order to establish optimum ratio of putative biologically active ingredients, we suggested 10 compositions using various ratios of bovine colostrum proteins, amaranth oil (Lacatusu et al. 2018) and hyaluronic acid (Juncan et al. 2021) (Table 1). The composition variants were added to the cream basis respectively, and filled up with water to a final mass of 100 g and further emulsified using a mixer. The obtained cream was of a dairy colour that is stable for more than a year when stored at 4 oC. Analgesic activity The efficiencies of the compositions were determined by the analgesic activity during contact- thermal irritation in the hot plate test following a commonly used method as described by Deuis et al. (2017). The thermal irritation model reveals a central component in the mechanism of the analgesic action of a substance. The analgesic activity was established by the change in the threshold of pain sensitivity in animals during contact-thermal stimulation under the influence of the cream. The initial reaction of the white outbred mice (the time to start licking the hind limbs) was determined when they were placed on a standard plate with a temperature of 55 °C. Ethical norms of evaluating pain behaviors, described by Deuis et al. (2017), were followed when analgesic activity of cream samples was determined. Then the rate of the same reaction was taken into account after certain time of applying the cream compositions to the animals on the hind and fore paws (15, 30 and https://blast.ncbi.nlm.nih.gov/Blast.cgi Nova Biotechnol Chim (2022) 21(1): e1246 2 45 min before the study), and its measurements were expressed as a percentage relative to the initial value. Animal experiments were carried out according to requirements used for scientific purposes (European Directive 2010/63/EU). Table 1. Establishment of optimum ratio of ingredients to fabricate analgesic means. Compositions Amaranth oil [g] Bovine colostrum proteins [mg] Hyaluronic acid [g] 1 5 0 0 2 0 10 0 3 0 0 2 4 6 8 1 5 5 10 2 6 4 7 3 7 3 10 1 8 2 10 2 9 5 8 3 10 1 9 4 Samples that revealed highest analgesic activity are indicated in bold. General and acute toxicity The study of the acute toxicity and general action of the investigated cream was carried out on white outbred mice weighing 20 ± 1.5 g. Five mice were included in each group. The tested substance was orally administered at doses up to 5,000 mg.kg-1; cream samples were dermally used at doses up to 2500 mg.kg-1. The observation was carried out 3 times a day for 2 – 3 days in a laboratory condition and further for 10 days in a vivarium. Results Proteins were isolated from bovine colostrum as above described. Obtained fraction of the water- alcohol-soluble fraction of low-abundant proteins made by dry mass approximately a thousandth of the colostrum dry mass. Proteins, highly abundant in colostrum such as casein, immunoglobulins, and lactoglobulins, are mainly transport proteins and/or ones involved in immune processes (Nissen et al. 2017). Our hypothesis originated from isolating the ones that interact with hydrophobic sorbent polytetrafluoroethylene – polychrome (despite fluorine is electronegative, the polymerized sorbent is of hydrophobic nature). In total, ~1.4 g of protein fraction of low abundant ones were obtained and subjected to sequence analysis. In Table 2, were provided the sequence of the identified proteins, NCBI annotation as well as accession number, query cover, and percentage identity. Query cover only higher than 90 % were selected. Our results by analgesic activity showed the initial reaction of mice in the control group occurred after 9.3 ± 0.7 seconds. Some of the cream samples that did not contain either amaranth oil alone or amaranth oil and bovine colostrum fractions (samples 2 and 3) revealed lowest effect that could be attributed to their presence. They enhanced analgesic activity in 45 min by 175.3 and 207.5 %, respectively. The effect of hyaluronic acid in pain relief can be noticed in samples 6 and 10 that contain 3 and 4 g of hyaluronic acid, respectively. Samples 7, 8, and 10 turned out to be moderately active despite the highest quantity of protein fraction included. When these samples were applied, analgesic activity in 45 min increased by 296.8 % (36.9 ± 3.6 s), 267.7 % (34.2 ± 3.2 s) and 284.9 % (35.8 ± 4.3 s), respectively, compared to the initial latent period. The most active ones were samples 5 and 9, the analgesic activity of which enhanced almost 3.84 % (45.0 ± 3.8 s) and 3.6 times (42.6 ± 4.1 s), respectively (Table 3). The observed time lag in pain relief can be concluded with highest quantity of amaranth oil, bovine colostrum protein fraction, and hyaluronic acid. Based on the obtained results of the studied samples, the most optimal choice in terms of the ratio of components was sample 5 that contained 5 g of amaranth oil, 10 mg of bovine colostrum protein fraction and 2 g of hyaluronic acid (Table 1). In relation to the initial indicators, this composition increased the analgesic activity by 131.2 %, 230.1 % and 383.9 % after 15, 30, 45 min of topical application, respectively (Table 3). Based on the data obtained, we developed cosmetic cream that was named "Zumara". The cream consists of T1 emulsifier, hyaluronic acid, bovine colostrum proteins, amaranth oil, ethyl alcohol, sorbic acid and purified water. The highest biological activity among samples belonged to samples 5 and 9 that could be attributed to higher content of bovine colostrum protein fraction, amaranth oil that contains 4 Nova Biotechnol Chim (2022) 21(1): e1246 3 Table 2. Identified sequences of low-abundant proteins in bovine colostrum and their accession numbers, query covers and percentage identities in NCBI database. Sequences identified by MASS analysis Accession number Query cover Per. Identity Proteins name/NCBI annotation 1. RLVRFYPRADRVMSVCLRVELYGCLWKAGLXSTQTG XP_019841431.1 100 % 100 % Discoidin domain receptor family, member 1 2. VLLGWGSLLIMLKNEGFYSSMCPAENSTNSTQDEQRR XP_027418094.1 91 % 85.29 % Neutral amino acids transporter small subunit 3 3. NISYGGCMAQMFFLSWSVAAEVLLFTAMAYDRYVA XP_027385372.1 100 % 77.14 % Olfactory receptor 13A1-like 4. AIYHCREQPASDWPEAVCLLIGRQDLSKQA XP_005891645.1 100 % 100 % Anaphase-promoting complex subunit 1 isoform X1 5. VLSVELPGLLAQLARSFALLLPVYALGYLGLSFSWVLLALALLAW XP_027394252.1 97 % 88.64 % Extended synaptotagmin-2 6. SIGGTPIRGALQI XP_027405056.1 100 % 100 % Tyrosine phosphatase, receptor type, δ isoform 1 precursor 7. SIPHLLLPLA NP_001192519.1 90 % 87.50 % Integrin alpha-10 8. QLLSQWSAHAMEVYSWKLVHPTD ELR61599.1 100 % 95.95 % Cytoplasmic FMR-1 interacting protein 9. VLVVASFDELQRRASEARGKIIVYNQP ELR52717.1 100 % 77.78 % Plasma glutamate carboxypeptidase 10. QLEKSWSQYLACPEDISGLLHS XP_005892933.1 100 % 95.45 % Huntingtin interacting protein 1 11. SSGPYLTSVMGKAAGEREF ABG67017.1 94 % 77.78 % Transcobalamin II precursor 12. GLLCVCWSPDGKYIVTGGEDDLVTV XP_024837551.1 100 % 96 % WD repeat containing protein 20 13. PKETPFLVSFIIAQIVTTCSGILSYPF AIC38345.1 100 % 92.59 % ADP/ATP translocase 4 14. GEPGKTGPPGLPGAGGSGAISTATYTTVPRVAFYAGLKNPH ELR50847.1 100 % 100 % CRF, C1q-related factor 15. NPVACELKAFV DAA15478.1 90 % 80 % DNA-directed RNA polymerase III 16. SFQTGSWLLDHCQESYCEPTVCQ NP_001074209.1 100 % 86.96 % Keratin associated protein 11-1 17. LYRWQGIFVPFEDLSIE XP_005907599.1 100 % 100 % Very large G protein-coupled receptor 18. SLPPPPLQP XP_027384342.1 100 % 100 % The peroxisome proliferator-activated receptor γ coactivator-1 5 https://www.ncbi.nlm.nih.gov/protein/XP_019841431.1?report=genbank&log$=prottop&blast_rank=1&RID=NBNVKGTZ016 https://www.ncbi.nlm.nih.gov/protein/XP_027418094.1?report=genbank&log$=prottop&blast_rank=1&RID=NES9VPM3014 https://www.ncbi.nlm.nih.gov/protein/XP_027385372.1?report=genbank&log$=prottop&blast_rank=1&RID=NM3GV1BN014 https://www.ncbi.nlm.nih.gov/protein/XP_005891645.1?report=genbank&log$=prottop&blast_rank=1&RID=NET27GZV014 https://www.ncbi.nlm.nih.gov/protein/XP_027394252.1?report=genbank&log$=prottop&blast_rank=1&RID=NET63XYT014 https://www.ncbi.nlm.nih.gov/protein/XP_027405056.1?report=genbank&log$=prottop&blast_rank=2&RID=NETATM43014 https://www.ncbi.nlm.nih.gov/protein/NP_001192519.1?report=genbank&log$=prottop&blast_rank=3&RID=NETSUD3A014 https://www.ncbi.nlm.nih.gov/protein/ELR61599.1?report=genbank&log$=prottop&blast_rank=4&RID=NKYJYWHF016 https://www.ncbi.nlm.nih.gov/protein/ELR52717.1?report=genbank&log$=prottop&blast_rank=1&RID=NEUFTM8F01R https://www.ncbi.nlm.nih.gov/protein/XP_005892933.1?report=genbank&log$=prottop&blast_rank=2&RID=NEUMMZS101R https://www.ncbi.nlm.nih.gov/protein/ABG67017.1?report=genbank&log$=prottop&blast_rank=2&RID=NHSTF1SC016 https://www.ncbi.nlm.nih.gov/protein/XP_024837551.1?report=genbank&log$=prottop&blast_rank=2&RID=NEVARXDD014 https://www.ncbi.nlm.nih.gov/protein/AIC38345.1?report=genbank&log$=prottop&blast_rank=3&RID=NEVFVBBJ016 https://www.ncbi.nlm.nih.gov/protein/ELR50847.1?report=genbank&log$=prottop&blast_rank=1&RID=NEW1HFGB016 https://www.ncbi.nlm.nih.gov/protein/DAA15478.1?report=genbank&log$=prottop&blast_rank=1&RID=NEX9NCNC016 https://www.ncbi.nlm.nih.gov/protein/NP_001074209.1?report=genbank&log$=prottop&blast_rank=3&RID=NEY2YC5Z016 https://www.ncbi.nlm.nih.gov/protein/XP_005907599.1?report=genbank&log$=prottop&blast_rank=3&RID=NEYCVSND016 https://www.ncbi.nlm.nih.gov/protein/XP_027384342.1?report=genbank&log$=prottop&blast_rank=1&RID=NEYTT5V9014 Nova Biotechnol Chim (2022) 21(1): e1246 2 Table 2. Identified sequences of low-abundant proteins in bovine colostrum and their accession numbers, query covers and percentage identities in NCBI database – continued. Sequences identified by MASS analysis Accession number Query cover Per. Identity Proteins name/NCBI annotation 19. MLENYSNLVFLGLAVSKPQLVTFLEQR ELR60893.1 96 % 80.77% Zink finger protein ZNF720 20. IQVTDDDINEIIQIDGTGDNSSAEEG XP_027411025.1 100 % 72 % Transcription factor IIA α/β 21. TSSIHSKEIFHSLT XP_027422939.1 100 % 92.7% DNA polymerase epsilon (ε) catalytic subunit A 22. IQGAYYPRRGSSEIAFHTIPLIQR XP_027411356.1 96 % 86.4 % Retinol saturase 23. EFRRRDQFPLTRGRAIQECRSPVPPPA XP_027369964.1 96 % 95.8 % Striated Muscle Preferentially Expressed Protein Kinase (SPEG) 24. QGVEYAKAVPLGTPIQS NP_991380.2 100 % 94.45 % γ-Protocadherin-C3 isoform 25. TILLGPMSTLVHTDQISTPETP NP_991380.2 100 % 95.45 % Hepatocyte nuclear factor 4 γ 26. MISQLVLEQFLLIGHCKD XP_024835263.1 94 % 64.7 % Zinc finger and SCAN domain containing protein 4D 27. QKLEQQLKVVPRFQPISEHQT XP_024846738.1 100 % 95.24 % A kinase anchor protein 9 28. DSTFANISKDDSDLIYSTYGEDPDLPSDFSIH XP_005218672.1 100 % 100 % Bromodomain containing protein 7 29. HIVIYSSIEEKTTLKDKNALHLFSINGKYLGSQI XP_027420629.1 100 % 85.3 % Neurobeachin-like protein 1 30. PRGPKGHMGDSVIGQKGER NP_001160001.2 100 % 84.21 % Collagen 4 alpha 3 31. VHGEPLGYGV XP_027413406.1 100 % 80 % Serine/Threonine-protein kinase LATS2 32. NLLDLLLNHQVLVPGCLDPFPLLSAYV DAA13671.1 100 % 100 % Glutathione S-transferase P 33. SIQSQSSLTITVSSTP NP_776434.1 93 % 73.33 % Hematopoietic progenitor cell antigen CD34 34. APNVQSRELNYD NP_001179782.1 100 % 83.33 % Parathyroid Hormone B1 35. GHRNTVLLTWKPPD XP_027370093.1 100 % 100 % Obscurin-like 1 36. SDKIHFPS E1BPQ3.1 100 % 87.5 % G-protein coupled receptor family C gr. 6 member A 37. SVNSEVLGATRVKRHKNLLAERWEAHIYA XP_005209316.1 96 % 75 % Mitotic interactor and substrate of PLK1 6 https://www.ncbi.nlm.nih.gov/protein/ELR60893.1?report=genbank&log$=prottop&blast_rank=1&RID=NEYUEEA2014 https://www.ncbi.nlm.nih.gov/protein/XP_027411025.1?report=genbank&log$=prottop&blast_rank=1&RID=NM2U02VZ014 https://www.ncbi.nlm.nih.gov/protein/XP_027422939.1?report=genbank&log$=prottop&blast_rank=2&RID=NEZ4S03N016 https://www.ncbi.nlm.nih.gov/protein/XP_027411356.1?report=genbank&log$=prottop&blast_rank=3&RID=NEZABEDJ016 https://www.ncbi.nlm.nih.gov/protein/XP_027369964.1?report=genbank&log$=prottop&blast_rank=1&RID=NEZHAYER014 https://www.ncbi.nlm.nih.gov/protein/NP_991380.2?report=genbank&log$=prottop&blast_rank=1&RID=NEZS3BH2016 https://www.researchgate.net/publication/306270257_The_g-Protocadherin-C3_isoform_inhibits_canonical_Wnt_signalling_by_binding_to_and_stabilizing_Axin1_at_the_membrane https://www.ncbi.nlm.nih.gov/protein/NP_991380.2?report=genbank&log$=prottop&blast_rank=1&RID=NM3VE0TZ014 https://www.ncbi.nlm.nih.gov/protein/XP_024835263.1?report=genbank&log$=prottop&blast_rank=3&RID=NF012VJ5014 https://www.ncbi.nlm.nih.gov/protein/XP_024846738.1?report=genbank&log$=prottop&blast_rank=3&RID=NF01WSWA016 https://www.ncbi.nlm.nih.gov/protein/XP_005218672.1?report=genbank&log$=prottop&blast_rank=3&RID=NF063A2K016 https://www.ncbi.nlm.nih.gov/protein/XP_027420629.1?report=genbank&log$=prottop&blast_rank=2&RID=NF0BMU5Y016 https://www.ncbi.nlm.nih.gov/protein/NP_001160001.2?report=genbank&log$=prottop&blast_rank=2&RID=NF0F024B016 https://www.ncbi.nlm.nih.gov/protein/XP_027413406.1?report=genbank&log$=prottop&blast_rank=1&RID=NF0JTNU3016 https://www.ncbi.nlm.nih.gov/protein/DAA13671.1?report=genbank&log$=prottop&blast_rank=3&RID=NF0KAC5C016 https://www.ncbi.nlm.nih.gov/protein/NP_776434.1?report=genbank&log$=prottop&blast_rank=2&RID=NF0RHTP3014 https://www.ncbi.nlm.nih.gov/protein/NP_001179782.1?report=genbank&log$=prottop&blast_rank=2&RID=NHRZZRWG014 https://www.ncbi.nlm.nih.gov/protein/XP_027370093.1?report=genbank&log$=prottop&blast_rank=1&RID=NHS6D0FR016 https://www.ncbi.nlm.nih.gov/protein/E1BPQ3.1?report=genbank&log$=prottop&blast_rank=1&RID=NHS5TW7U016 https://www.ncbi.nlm.nih.gov/protein/XP_005209316.1?report=genbank&log$=prottop&blast_rank=1&RID=NHSD5Y33016 Nova Biotechnol Chim (2022) 21(1): e1246 3 Table 3. Effects of the fabricated creams on the latency period and analgesic activity in the thermal nociceptive hot plate test (M  m; n = 6). Fabricated cream compositions Time with the moment of cream application [min] 15 30 45 Latent period (seconds) Analgesic activity (percentage increased by) Latent period (seconds) Analgesic activity (percentage increased by) Latent period (seconds) Analgesic activity (percentage increased by) Control (initial) 9.3 ± 0.7 9.3 ± 0.7 9.3 ± 0.7 1 18.2 ± 1.8 95.7 20.0 ± 2.0 115.1 22.5 ± 3.4 141.9 2 10.5 ± 2 12.9 12.2 ± 0.7 31.2 25.6 ± 2.8 175.3 3 15.3 ± 1.5 64.5 17.5 ± 1.8 88.2 28.6 ± 3.2 207.5 4 20.3 ± 1.5 118.3 25.6 ± 1.5 175.3 40.2 ± 3.5 332.3 5 21.5 ± 1.8 131.2 30.7 ± 2.2 230.1 45.0 ± 3.8 383.9 6 20.3 ± 2.0 118.3 25.7 ± 0.7 176.3 38.7 ± 2.8 316.1 7 19.6 ± 1.8 110.8 24.5 ± 2.0 163.4 36.9 ± 3.6 296.8 8 18.4 ± 1.5 97.8 23.6 ± 1.5 153.8 34.2 ± 3.2 267.7 9 22.5 ± 1.8 141.9 31.2 ± 2.2 235.5 42.6 ± 4.1 358.1 10 19.6 ± 2.0 110.8 24.6 ± 0.7 164.5 35.8 ± 4.3 284.9 Р < 0.01 – reliability in relation to the mean value of initial control. Samples revealing highest activity and their effects, respectively, are shown in bold letters. ± bars indicate standard deviation value of a data set. squalene up to 5.7 % in the composition and hyaluronic acid. Further analysis will prove our hypothesis. Discussion Previous researchers demonstrated different mechanisms of analgesic activities of peptides. Dooley et al. (1994) reported RFWINK peptide that do not resemble to any known opioid peptide. Reported six amino acid-long peptide was demonstrated to induce long-lasting analgesia in mice. The peptide was suggested to cross blood brain barrier. In another work, GYCAEKGIRCDDIHCCTGLKCKCNASGYNCV CRKK peptide, isolated from Alopecosa marikovskyi spider venom, was found to possess analgesic activity by influencing purinergic P2X3 receptors. The maximum bioactivity of the peptide, studied by thermal hypersensitivity test, was 0.5 mg.kg-1when administered intravenously (Palikova et al. 2019). ZRCCNGGCSSRWCRDHSRCC peptide (SIIIA) and similar structures are expected to be potent blocking agents of TTX-R and TTX-S sodium channels (Bulaj et al. 2005; Green et al. 2007). μ-conotoxins were established to block voltage- gated sodium channels (VGSC) and more than twenty sequences belonging to conotoxins were already identified that interact with VGSC subtypes (Green et al. 2014). Another mechanism of analgesic effect of peptides was shown to be connected to a linkage with clathrin heavy chain. Synthetic peptide, obtained by modifying salmon calcitonin sCT16-21 fragment retained the activity of a full molecule (Kotin et al. 2019). Some peptides, identified in this work, were reported to be associated with analgesic mechanisms. Very large G-protein coupled receptor 1 (VLGR1) is one of these peptides included in these analgesic processes. The large ectodomain of VLGRl contains calcium exchanger repeats that possess resemblance to sodium-calcium exchanger proteins. Very large G protein-coupled receptor 1 (VLGR1), having a large ectodomain, contains multiple calcium exchanger repeats resembling regulatory domains of sodium-calcium exchanger proteins. (McMillan et al. 2002). G protein- coupled receptors mediate (GPCR) human sense of vision, smell, taste and pain, and are involved in 7 Nova Biotechnol Chim (2022) 21(1): e1246 2 cell recognition processes. They structurally are classified as GPCR possessing short N-terminal ectodomain (50 – 80 residues) and long ectodomain (80 – 600 residues). The long N-terminus can be involved in ligand recognition; the bound ligand can possibly move to the transmembrane region to activate G protein (Vaidehi et al. 2002). VLGR1 is a core component for the development of ear inner cells. Mutations in VLGR1 genes were defined to cause Usher syndrome with symptoms congenital hearing loss and progressive retinitis pigmentosa (Hu et al. 2014). We suggested synaptotagmin-2 (Syt-2) protein as the next one involved in analgesic processes. Syt-2 was reported to be involved in neuronal processes (Bouhours et al. 2017). Extended synaptotagmin- like proteins, are a family of proteins that are involved in Ca2+-regulated secretion and characterized by an N-terminal transmembrane sequence and two C-terminal C2 domains (C2A and C2B) (Rizo and Rosenmund 2008). E-Syts consist of a short, nonconserved N-terminal transmembrane region and C2 domains. Their C2 domains generally act as Ca2+- and phospholipid- binding domains and/or as protein–protein interaction domain (Min et al. 2007). The E-Syt2 C2A domain binds up to four Ca2+ ions, whereas the C2B domain does not bind Ca2+ (Xu et al. 2014). Other proteins that could be involved in analgesic activity among the ones, identified in this work, are receptor protein tyrosine phosphatase (RPTP) delta isoform, corticotropin releasing factor, and γ- protocadherin C3. Since analgesic effects of these proteins were not conducted separately, our suggestions are speculative only. However, obtained fraction of water-alcohol soluble proteins, possibly of more hydrophobic nature, has been demonstrated as proper natural resources that can be obtained in preparative amounts. Further researches will clarify deeper fundamental basis of the work. Amaranth oil is another factor contributing to the efficiency of the cream. Beneficial effects of squalene in the composition of creams and other means of cosmetic dermatology provide advantages to amaranth oil over other sources (Huang et al. 2009). Up to 13 % concentration among the components of the skin surface lipids (Passi et al. 2002) causes higher interaction of the cream composition with a high ratio of squalene that contributes to the penetration of active compounds. The study of the general and acute toxicity of the substances and the cosmetic cream showed that they belong to practically harmless substances. LD50 level of the product reached more than 25,000 mg.kg-1 with cutaneous and oral administration in mice and rats. The study of the specific toxicology of the substance and the cream showed that they do not have a local irritating effect on the skin of rats and the conjunctiva of the eyes of rabbits and do not have an allergenic effect. The study of the chronic toxicity of the substance and the developed cream showed that the cream revealed prolonged action and did not lead to any changes in the biochemical parameters of the peripheral blood and internal organs of the experimental animals (results are not shown). The study of specific activity showed that the cream smoothes the skin by retaining moisture resulted from hyaluronic acid (Sundaram et al. 2018; Lubart et al. 2019). Hyaluronic acid, another significant component in the composition of the product, is found in many connective tissues (Gupta et al. 2019; Chang et al. 2021). It is mostly used moisturizing agent used in cosmetology. Hyaluronic acid is responsible for the regeneration process of the skin: it helps the synthesis of collagen (Silva et al. 2017) and elastin (Joddar and Ramamurthi 2006), thus contributing to their correct positioning. Thanks to this, the turgor and elasticity of the skin are improved. In addition, the unique ability of hyaluronic acid to retain water provides stability to the product. In the upper layers of the skin, it works as a conductor of nutrients and facilitates their diffusion (Dovedytis et al. 2020). Conclusion In this work, we identified 37 low abundant proteins in bovine colostrum that are soluble in 60 % ethanol. The identified peptides contribute to the extension of proteomic data of bovine colostrum. The used method can serve to identify low- abundant proteins from the colostrum and milk of other mammals. The sum of these peptides was found likely to possess analgesic effects when used as topical cream. We hypothesized the observed 8 Nova Biotechnol Chim (2022) 21(1): e1246 3 analgesic activity might be attributed to at least two proteins: VLGR1 and synaptotagmin-2. Amaranth oil containing high amount of squalene was concluded as efficient additive that likely contributes to biological efficiency. Acknowledgement The work was supported with innovative project I-2016-5- 16/3 “Introduction of technology for complex processing of amaranth plants to produce feed for livestock, and also oil cake and flour for the needs of the pharmaceutical and food industry” by Academy of Sciences of Uzbekistan. Conflict of Interest The authors declare that they have no conflict of interest. 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