Wild-Grown Romanian Helleborus purpurascens Approach to Novel Chitosan Phyto-Nanocarriers—Metabolite Profile and Antioxidant Properties
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening and Classification of the Differential Metabolites
2.2. Phyto-Nanocarriers
2.3. FT-IR Spectroscopy
2.4. X-ray Diffraction Spectroscopy
2.5. Scanning Electron Microscopy (SEM)
2.6. Dynamic Light Scattering
2.7. Thermal Behavior Study
2.8. Screening of the Antioxidant Activity
2.9. DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Free Radical Scavenging Assay
2.10. Total Polyphenolic Contents (TPCs) Assay—Folin–Ciocalteu
2.11. Phosphomolybdate Assay (Total Antioxidant Capacity)
2.12. Iron(III)-Phenanthroline Antioxidant Assay
2.13. Encapsulation Efficiency, Loading Capacity and Encapsulation Yield (EY%)
3. Materials and Methods
3.1. Phyto-Carrier-System Components’ Preparation
Plant Samples’ Preparation for Chemical Screening
3.2. GC-MS Analysis
GC–MS Separation Conditions
3.3. Mass Spectrometry
3.3.1. Phyto-Carrier System Preparation (Hellebore-AgNPs System)
3.3.2. Chitosan Phyto-Nanocarrier Preparation (Encapsulation Procedure)
3.4. Characterization of Nanocarriers
3.4.1. Fourier Transform Infrared (FT-IR) Spectroscopy
3.4.2. XRD Spectroscopy
3.4.3. Scanning Electron Microscopy (SEM)
3.4.4. Dynamic Light Scattering (DLS) Particle Size Distribution Analysis
3.4.5. Thermal Analysis
3.4.6. Antioxidant Activity
3.5. Total Polyphenol Content Using the Folin–Ciocalteu Method
3.6. DPPH Radical Scavenging Assay
- A0 = vehicle control absorbance;
- Al = sample absorbance.
3.7. Phosphomolybdate Assay (Total Antioxidant Capacity)
3.8. Iron(III)-Phenanthroline Antioxidant Assay (OPM)
3.9. Encapsulation Yield, Encapsulation Efficiency and Loading Capacity
3.10. Encapsulation Efficiency (EE%)
3.11. Preparation of the Curves of the Concentrations of the Loaded Materials (Hellebore and Hellebore-AgNPs System)
3.12. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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No. | Retention Time (RT) (min) | Retention Index (RI) Determined | Adams Index (AI) | Area (%) | Compound Name | Ref. |
---|---|---|---|---|---|---|
1 | 11.43 | 1972 | 1983 | 2.18 | palmitic acid | [47,48,49] |
2 | 11.98 | 1598 | 1602 | 5.32 | hexadecane | [47] |
3 | 14.61 | 3160 | 3168 | 6.76 | stigmasterol | [47,50] |
4 | 15.87 | 3195 | 3213 | 9.85 | sitosterol | [47,50] |
5 | 17.22 | 2543 | 2634 | 8.53 | stearic acid | [47,48,49] |
6 | 25.96 | 2078 | 2123 | 21.84 | phytol | [47,50] |
7 | 26.88 | 2848 | 2861 | 35.69 | squalene | [47,50] |
8 | 28.45 | 2688 | 2694 | 6.58 | heptacosane | [47,48] |
No. | m/z Detected | Theoretic m/z | Formula | Tentative of Identification | Category | Ref. |
---|---|---|---|---|---|---|
1 | 76.07 | 75.07 | C2H5NO2 | glycine | amino acids | [3,4,11] |
2 | 89.88 | 89.09 | C3H7NO2 | alanine | amino acids | [4,11,13] |
3 | 96.09 | 96.08 | C5H4O2 | protoanemonin | lactones | [2,6,11] |
4 | 116.15 | 116.16 | C6H12O2 | caproic acid | fatty acids | [47] |
5 | 119.11 | 119.12 | C4H9NO3 | threonine | amino acids | [3,4,11] |
6 | 131.17 | 131.17 | C6H13NO2 | isoleucine | amino acids | [3,4,11] |
7 | 132.15 | 132.16 | C5H12N2O2 | ornithine | amino acids | [3,4,11] |
8 | 147.13 | 146.14 | C5H10N2O3 | alanylglycine | peptides | [4,11] |
9 | 148.12 | 147.13 | C5H9NO4 | glutamic acid | amino acids | [3,4,11] |
10 | 151.14 | 150.13 | C5H10O5 | arabinose | carbohydrates | [51,52] |
11 | 156.16 | 155.15 | C6H9N3O2 | histidine | amino acids | [3,4,11] |
12 | 155.15 | 164.16 | C9H8O3 | p-coumaric acid | phenolic acids | [3,10,53] |
13 | 166.18 | 165.19 | C9H11NO2 | phenylalanine | amino acids | [3,4,11] |
14 | 167.16 | 166.17 | C9H10O3 | 3-phenyllactic acid | organic acid | [52,53] |
15 | 171.11 | 170.12 | C7H6O5 | gallic acid | phenolic acids | [3,10,53] |
16 | 175.19 | 174.20 | C6H14N4O2 | arginine | amino acids | [4,11] |
17 | 177.18 | 176.17 | C6H12N2O4 | glycylthreonine | peptides | [4,11] |
18 | 181.17 | 180.16 | C9H8O4 | caffeic acid | phenolic acids | [3,10,53] |
19 | 193.16 | 192.17 | C10H8O4 | anemonin | butenolides | [2,3,51] |
20 | 195.18 | 194.18 | C10H10O4 | ferulic acid | phenolic acids | [3,10,52] |
21 | 201.33 | 200.32 | C12H24O2 | lauric acid | fatty acids | [47] |
22 | 203.26 | 202.25 | C9H18N2O3 | alanyl-isoleucine | peptides | [4,11] |
23 | 205.21 | 204.22 | C11H12N2O2 | tryptophan | amino acids | [3,4,11] |
24 | 213.22 | 212.21 | C8H12N4O3 | glycyl-histidine | peptides | [4,11] |
25 | 218.26 | 217.27 | C9H19N3O3 | alanyl-lysine | peptides | [4,11] |
26 | 220.23 | 219.24 | C8H17N3O4 | ornithinoalanine | peptides | [4,11] |
27 | 221.29 | 220.29 | C8H16N2O3S | alanylmethionine | peptides | [4,11] |
28 | 225.29 | 224.30 | C6H12N2O3S2 | cysteylcysteine | peptides | [4,11] |
29 | 227.45 | 226.44 | C16H34 | hexadecane | hydrocarbons | [54] |
30 | 229.23 | 228.24 | C14H12O3 | resveratrol | stilbenoids | [3,10] |
31 | 241.29 | 240.30 | C6H12N2O4S2 | cystine | amino acids | [3,4,11] |
32 | 244.31 | 243.30 | C11H21N3O3 | lysylproline | peptides | [4,11] |
33 | 245.19 | 244.20 | C9H12N2O6 | uridine | nucleosides | [55] |
34 | 249.35 | 248.34 | C10H20N2O3S | valyl-methionine | peptides | [4,11] |
35 | 253.39 | 252.40 | C8H16N2O3S2 | cysteylmethionine | peptides | [4,11] |
36 | 255.28 | 254.29 | C11H18N4O3 | histidylvaline | peptides | [4,11] |
37 | 257.24 | 256.25 | C12H16O6 | phenylglucoside | glycosides | [55] |
38 | 257.43 | 256.42 | C16H32O2 | palmitic acid | fatty acids | [47] |
39 | 260.35 | 259.35 | C12H25N3O3 | leucyllysine | peptides | [4,11] |
40 | 262.27 | 261.28 | C13H15N3O3 | tryptophylglycine | peptides | [4,11] |
41 | 269.33 | 268.33 | C12H16N2O3S | phenylalanylcysteine | peptides | [4,11] |
42 | 270.25 | 269.26 | C10H15N5O4 | asparaginyl-Histidine | peptides | [4,11] |
43 | 271.25 | 270.24 | C15H10O5 | apigenin | flavonoids | [55] |
44 | 271.49 | 270.50 | C17H34O2 | margaric acid | fatty acids | [47] |
45 | 276.31 | 275.30 | C14H17N3O3 | tryptophylalanine | peptides | [4,11] |
46 | 277.25 | 276.24 | C11H16O8 | ranunculin | glycosides | [51,56,57] |
47 | 278.35 | 277.35 | C9H19N5O3S | cysteinyl-arginine | peptides | [4,11] |
48 | 279.34 | 278.35 | C15H22N2O3 | phenylalanylleucine | peptides | [4,11] |
49 | 284.28 | 283.28 | C11H17N5O4 | alanyl-glycyl-histidine | peptides | [4,11] |
50 | 284.49 | 284.50 | C18H36O2 | stearic acid | fatty acids | [46] |
51 | 287.23 | 286.24 | C15H10O6 | kaempferol | flavonoids | [3,10,58,59] |
52 | 287.34 | 286.35 | C11H18N4O3S | histidylmethionine | peptides | [4,11] |
53 | 288.37 | 287.36 | C12H25N5O3 | leucylarginine | peptides | [4,11] |
54 | 291.26 | 290.27 | C15H14O6 | epicatechin | flavonoids | [3,10] |
55 | 294.31 | 293.32 | C14H19N3O4 | phenylalanylglutamine | peptides | [4,11] |
56 | 295.29 | 294.30 | C14H18N2O5 | glutamyl-phenylalanine | peptides | [4,11] |
57 | 296.28 | 295.29 | C13H17N3O5 | tyrosylglycylglycine | peptides | [4,11] |
58 | 297.49 | 296.50 | C20H40O | phytol | terpenoids | [57] |
59 | 303.24 | 302.23 | C15H10O7 | quercetin | flavonoids | [3,59] |
60 | 306.39 | 305.40 | C12H23N3O4S | arginylmethionine | peptides | [4,11] |
61 | 308.36 | 307.37 | C11H21N3O5S | glycyl-threonyl-methionine | peptides | [4,11] |
62 | 309.51 | 308.50 | C20H36O2 | eicosadienoic acid | fatty acids | [54] |
63 | 312.33 | 311.34 | C12H21N7O3 | histidinyl-arginine | peptides | [4,11] |
64 | 320.41 | 319.42 | C13H25N3O4S | leucyl-methionyl-glycine | peptides | [4,11] |
65 | 323.36 | 321.37 | C15H23N5O3 | arginylphenylalanine | peptides | [4,11] |
66 | 333.34 | 332.35 | C16H20N4O4 | glycyl-tryptophanyl-alanine | peptides | [4,11] |
67 | 335.41 | 334.40 | C11H22N6O4S | glycyl-arginyl-cysteine | peptides | [4,11] |
68 | 338.37 | 337.37 | C16H23N3O5 | threonyl-phenylalanyl-alanine | peptides | [4,11] |
69 | 341.59 | 340.60 | C22H44O2 | behenic acid | fatty acids | [47] |
70 | 343.39 | 342.40 | C20H24NO4+ | magnoflorine | alkaloids | [51] |
71 | 349.41 | 348.42 | C12H24N6O4S | arginyl-alanyl-cysteine | peptides | [4,11] |
72 | 355.49 | 354.50 | C24H34O2 | bufadienolide | terpenoids | [60] |
73 | 357.34 | 356.33 | C13H20N6O6 | asparaginyl-seryl-histidine | peptides | [4,11] |
74 | 361.29 | 360.30 | C18H16O8 | rosmarinic acid | phenolic acids | [10] |
75 | 365.41 | 364.40 | C18H28N4O4 | lysyl-phenylalanyl-alanine | peptides | [4,11] |
76 | 368.51 | 367.50 | C17H25N3O4S | alanyl-methionyl-phenylalanine | peptides | [4,11] |
77 | 370.41 | 369.40 | C16H23N3O5S | methionyl-tyrosyl-glycine | peptides | [4,11] |
78 | 374.41 | 373.40 | C18H23N5O4 | phenylalanyl-histidyl-alanine | peptides | [4,11] |
79 | 381.69 | 380.70 | C27H56 | heptacosane | hydrocarbons | [47] |
80 | 383.43 | 382.42 | C16H26N6O5 | leucyl-asparaginyl-histidine | peptides | [4,11] |
81 | 384.37 | 383.36 | C14H21N7O6 | histidyl-asparaginyl-asparagine | peptides | [4,11] |
82 | 367.71 | 386.70 | C27H46O | furostan | terpenoids | [2,3] |
83 | 393.51 | 392.50 | C18H24N4O4S | tryptophanyl-methionyl-glycine | peptides | [4,11] |
84 | 398.49 | 397.50 | C18H27N3O5S | threonyl-phenylalanyl-methionine | peptides | [4,11] |
85 | 401.59 | 400.60 | C27H44O2 | spirostan | saponins | [2,3] |
86 | 406.51 | 405.50 | C15H31N7O4S | lysyl-arginyl-cysteine | peptides | [4,11] |
87 | 408.50 | 407.50 | C20H29N3O6 | glutamyl-isoleucyl-phenylalanine | peptides | [4,11] |
88 | 411.69 | 410.70 | C30H50 | squalene | terpenoids | [47] |
89 | 413.70 | 412.70 | C29H48O | stigmasterol | sterols | [47] |
90 | 415.69 | 414.70 | C29H50O | sitosterol | sterols | [47] |
91 | 417.51 | 416.50 | C24H32O6 | hellebrigenin | terpenoids | [2,3,61] |
92 | 434.49 | 433.50 | C15H31N9O4S | arginyl-arginyl-cysteine | peptides | [4,11] |
93 | 450.51 | 449.50 | C15H27N7O7S | arginyl-glycyl-aspartyl-cysteine | peptides | [4,11] |
94 | 453.49 | 452.50 | C21H36N6O5 | alanyl-leucyl-leucyl-histidine | peptides | [4,11] |
95 | 463.61 | 462.60 | C27H42O6 | shidasterone | phytoecdysteroids | [62,63] |
96 | 465.39 | 464.40 | C21H20O12 | hyperoside | flavonoids | [9] |
97 | 469.51 | 468.50 | C24H32N6O4 | phenylalanyl-arginyl-phenylalanine | peptides | [4,11] |
98 | 481.59 | 480.60 | C27H44O7 | ecdysterone | terpenoids | [2,3,9,64] |
99 | 482.61 | 481.60 | C16H31N7O6S2 | arginyl-threonyl-cysteinyl-cysteine | peptides | [4,11] |
100 | 494.51 | 493.50 | C21H31N7O7 | arginyl-glycyl-aspartyl-phenylalanine | peptides | [4,11] |
101 | 521.59 | 520.60 | C24H40N8O5 | H-Lys-Ala-Phe-Arg-OH | peptides | [4,11] |
102 | 563.59 | 562.60 | C30H42O10 | degluco-hellebrin | terpenoids | [60,64] |
103 | 572.69 | 571.70 | C27H37N7O5S | arginyl-phenylalanyl-phenylalanyl-cysteine | peptides | [4,11] |
104 | 611.49 | 610.50 | C27H30O16 | rutin | flavonoids | [3,9] |
105 | 725.81 | 724.80 | C36H52O15 | hellebrin | terpenoids | [2,3] |
Secondary Metabolites | Wavenumber (cm−1) | Ref. |
---|---|---|
bufadienolides | 3560, 3035, 1740–1760, 1718–1736 | [110,111] |
butenolide | 1770, 1605, 830 | [83,112] |
phytoecdysteroids | 3393, 3446, 1652 | [113,114] |
peptides | 3300, 3100, 1695–1610, 1575–1480,1320–1220, 800–640, 765–625, 605–535 | [115,116] |
terpenoids | 2939, 1740, 1651, 810 | [117] |
fatty acids | 3020–3010, 2924–2915, 2855–2847, 2800–2900, 1746, 1710, 1250, 720 | [118,119] |
flavonoids | 4000–3125, 3140–3000, 1670–1620, 1650–1600, 1600–1500, 1450–1490 | [120,121] |
phenolic acids | 1800–1650, 1734, 1720, 1627, 1522, 1440, 1410, 1420–1300, 1367, 1315, 1255, 1170–1100 | [117] |
phytosterols | 3427, 2937, 1466, 1383, 1192, 1063, 740.5 | [122] |
carbohydrates | 3524, 3324, 2943, 2917, 2874, 1129, 1089, 1048, 1016, 991, 782, 671, 602 | [123] |
amino acids | 3400; 3330–3130; 2530–2760; 2130; 1724–1754, 1687, 1675, 1663, 1652, 1644, 1632, 1621, 1611, 1500–1600 | [124] |
alkaloids | 3362, 1645.9, 1515.7, 1283, 1250.5 | [125] |
nucleoside | 3351, 3104, 2925, 2800, 1670, 1470, 1396, 1269, 1209, 1137. 1095, 1053, 980, 906, 830, 766, 572, 451 | [126] |
glycosides | 3401, 1711, 1450, 1073, 991, 914, 863.5; 769, 743, 684, 630, 432 | [127] |
stilbenoids | 1605, 1583, 1380, 960 | [128,129] |
Sample Name | IC50 (mg/mL) | Total Phenolic Content (mg GAE/mL) | mg AAE/mL | mmol Fe/g |
---|---|---|---|---|
hellebore | 3.86 ± 0.16 | 5.16 ± 0.18 | 91.41 ± 0.012 | 34.91 ± 0.024 |
chitosan | 17.21± 0.09 | 1.82 ± −1.24 | 15.16 ± 0.014 | 9.16 ± 0.021 |
HC phyto-nanocarrier | 4.17 ± 0.12 | 4.85 ± 0.14 | 100.48 ± 0.017 | 27.75 ± 0.013 |
hellebore-AgNPs system | 2.35 ± 0.15 | 9.41 ± 0.17 | 156.87 ± 0.019 | 48.08 ± 0.027 |
HAgC phyto-nanocarrier | 2.56 ± 0.14 | 8.93 ± 0.15 | 186.98 ± 0.021 | 41.34 ± 0.018 |
Sample Name | EE (%) | EC (%) | EY (%) |
---|---|---|---|
CN | - | - | 70.34 ± 0.23 |
HC | 82.45 ± 0. 21 | 81.65 ± 0.35 | 87.45 ± 0.35 |
HAgC | 91.05 ± 0.18 | 92.18 ± 0.28 | 95.34 ± 0.48 |
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Segneanu, A.-E.; Vlase, G.; Vlase, T.; Sicoe, C.A.; Ciocalteu, M.V.; Herea, D.D.; Ghirlea, O.-F.; Grozescu, I.; Nanescu, V. Wild-Grown Romanian Helleborus purpurascens Approach to Novel Chitosan Phyto-Nanocarriers—Metabolite Profile and Antioxidant Properties. Plants 2023, 12, 3479. https://doi.org/10.3390/plants12193479
Segneanu A-E, Vlase G, Vlase T, Sicoe CA, Ciocalteu MV, Herea DD, Ghirlea O-F, Grozescu I, Nanescu V. Wild-Grown Romanian Helleborus purpurascens Approach to Novel Chitosan Phyto-Nanocarriers—Metabolite Profile and Antioxidant Properties. Plants. 2023; 12(19):3479. https://doi.org/10.3390/plants12193479
Chicago/Turabian StyleSegneanu, Adina-Elena, Gabriela Vlase, Titus Vlase, Crina Andreea Sicoe, Maria Viorica Ciocalteu, Dumitru Daniel Herea, Ovidiu-Florin Ghirlea, Ioan Grozescu, and Valentin Nanescu. 2023. "Wild-Grown Romanian Helleborus purpurascens Approach to Novel Chitosan Phyto-Nanocarriers—Metabolite Profile and Antioxidant Properties" Plants 12, no. 19: 3479. https://doi.org/10.3390/plants12193479