Polyphenol Characterization and Skin-Preserving Properties of Hydroalcoholic Flower Extract from Himantoglossum robertianum (Orchidaceae)
"> Figure 1
<p>(<b>A</b>) Flower spike of <span class="html-italic">H. robertianum</span>. (<b>B</b>) Plant habitus in olive grove environment.</p> "> Figure 2
<p>Stereomicroscope (<b>A</b>) and light microscope (<b>B</b>–<b>E</b>) pictures of the flower. (<b>A</b>) Total view showing three sepals, two petals, and a labellum. (<b>B</b>) Central portion of the labellum, showing purple, anthocyanin-rich cells interspersed among unpigmented cells. (<b>C</b>) Short papillose cells in an invagination of the medium-high portion of the labellum lateral arm. (<b>D</b>) Elongated pigmented papillae in the sub-stigmatic zone of the central labellum. (<b>E</b>) Stomata and raphides in the sepal.</p> "> Figure 3
<p>SEM micrographs of flower portions. (<b>A</b>) Overview of the central and lateral regions (arm) of the labellum in which two kinds of papillae are observed. Most prominent papillae were found in the central zone of the labellum (black arrow), while in the lateral zone the papillae become increasingly more flattened (white arrow). (<b>B</b>) Elongated papillae in the substigmatic zone of the central labellum. (<b>C</b>) Magnified view of the labellum arm where gradual flattening of papillose cells is visible.</p> "> Figure 4
<p>Representative RP-LC-DAD chromatogram of HFE (Panel <b>A</b>) and reference standard mix 10 μg/mL (Panel <b>B</b>), acquired at 260, 292, and 330 nm. Peak numbers correspond to compounds listed in Tables 2 and 4.</p> "> Figure 5
<p>Biological activities of HFE. All data are expressed as means ± S.D. (<b>A</b>) Protective effect of pre-incubation with 500 μg/mL HFE on cell viability reduction induced by 500 μM H<sub>2</sub>O<sub>2</sub>, evaluated by MTT assay on HaCaT keratinocytes. Data are formazan absorbance at 570 nm standardized as percent of control (<span class="html-italic">n</span> = 8). Differences between means have been evaluated by t test. (<b>B</b>) Increased wound closure induced by HFE in a scratch wound healing assay conducted in vitro on HaCaT keratinocytes. Data are wound closures measured at 24 h since wounding, and expressed as percent of total closure (<span class="html-italic">n</span> = 30–200). * = <span class="html-italic">p</span> < 0.01 according to Bonferroni test. (<b>C</b>) In vitro inhibition of porcine pancreas elastase by 500 μg/mL HFE. Data are percent inhibition obtained from absorbances at 410 nm (<span class="html-italic">n</span> = 3 independent measures). Statistical comparisons as in B. (<b>D</b>) In vitro inhibition of <span class="html-italic">Clostridium histolyticum</span> collagenase by HFE. Data are percent inhibition obtained from enzyme kinetics derived from absorbances at 345 nm, at the time points of 0 and 15 min (<span class="html-italic">n</span> = 3 independent measures). Statistical comparisons as in B.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Flower Morphological Characterization
2.2. Phytochemical Characterization
2.3. Antioxidant and Free-Radical Scavenging Activity
2.4. Effects of HFE on Keratinocytes and Skin Enzymes
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Reagents and Cells
4.3. Light and Scanning Electron Microscopy Analyses
4.4. Hydroalcoholic Flower Extract (HFE) Preparation
4.5. Chemical Characterization
4.5.1. Total Phenols
4.5.2. Flavonoids
4.5.3. Vanillin Index
4.5.4. Proanthocyanindin and Anthocyanin Content
4.5.5. Polyphenol Profile by Reversed Phase Liquid Chromatography with Diode Array Detection (RP-LC-DAD)
4.6. Antioxidant and Free-Radical Scavenging Activities
4.6.1. Scavenging Activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) Free Radical
4.6.2. Trolox Equivalent Antioxidant Capacity (TEAC)
4.6.3. Ferric-Reducing Antioxidant Potential (FRAP)
4.6.4. Oxygen Radical Absorbance Capacity (ORAC)
4.6.5. β-Carotene Bleaching Assay
4.6.6. Iron-Chelating Activity
4.7. Biological Assays
4.7.1. Collagenase and Elastase Inhibition Assays
4.7.2. Cytotoxicity and Cytoprotection Assays
4.7.3. In Vitro Wound Healing Assay
4.8. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
References
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Phytochemical Screening | HFE |
---|---|
Total phenols (mg GAE 1/100 g FW) | 243.7 ± 26.2 |
Flavonoids (mg QuE 2/100 g FW) | 398.1 ± 9.8 |
Anthocyanins (mg ChE 3/100 g FW) | 4.89 ± 0.05 |
Proanthocyanidins (mg CyE 4/100 g FW) | 0.05 ± 0.001 |
Vanillin index (mg CatE 5/100 gFW) | 3.31 ± 0.03 6 |
Polymerization index | 66.2 |
Peak n. 1 | Compound | Rt 2 (min) | λmax (nm) | mg/100 g FW 3 |
---|---|---|---|---|
Phenolic acids | ||||
1 | Protocatecuic acid | 15.057 | 260; 294 | 7.8 ± 0.06 |
2 | Hydroxybenzoic acid | 25.543 | 255 | 0.09 ± 0.001 |
4 | Chlorogenic acid | 31.399 | 294; 326 | 10.85 ± 0.44 |
5 | Caffeic acid | 33.614 | 232; 323 | 11.52 ± 0.37 |
6 | Vanillic acid | 35.252 | 260; 292 | 0.08 ± 0.002 |
8 | Coumaric acid | 43.811 | 233; 310 | 3.33 ± 0.02 |
Flavan-3-ols | ||||
3 | Catechin | 29.451 | 234; 279 | 2.63 ± 0.02 |
7 | Epicatechin | 42.062 | 232; 280 | 9.87 ± 0.38 |
Flavones | ||||
10 | Isovitexin | 55.300 | 270; 337 | 3.82 ± 0.04 |
11 | Naringenin-7-O-glucoside | 55.878 | 284; 340 | 0.98 ± 0.02 |
12 | Vitexin | 57.145 | 268; 338 | 5.47 ± 0.05 |
13 | Rutin | 59.281 | 256; 356 | 5.08 ± 0.03 |
14 | Kaempferol-3-O-rutinoside | 61.975 | 266; 348 | 21.1 ± 0.25 |
15 | Roifolin | 64.619 | 266; 338 | 9.23 ± 0.08 |
16 | Luteolin | 74.693 | 254; 350 | 0.86 ± 0.04 |
17 | Apigenin | 76.417 | 236; 338 | 3.01 ± 0.07 |
Coumarins | ||||
9 | Scopoletin | 47.373 | 296; 344 | 48.85 ± 0.48 |
Antioxidant Assay | HFE |
---|---|
IC50 1 μg/mL (95% C.L. 2) | |
DPPH | 211.1 (181.1–245.3) 3 |
FRAP | 8.85 (7.69–10.18) |
TEAC | 25.04 (20.41–30.71) |
ORAC | 2.52 (2.19–2.9) |
β-carotene bleaching | 31.43 (22.12–44.66) |
Iron-chelating activity | 440.8 (330.1–588.6) |
Peak n. 1 | Compound | Rt (min) | λmax (nm) | Regression Coefficient (R2) |
---|---|---|---|---|
1 | Protocatecuic acid | 15.055 | 260; 294 | 0.9999 |
2 | Hydroxybenzoic acid | 25.545 | 255 | 0.9997 |
3 | Catechin | 29.453 | 234; 279 | 0.9997 |
4 | Chlorogenic acid | 31.401 | 294; 326 | 0.9999 |
5 | Caffeic acid | 33.617 | 232; 323 | 0.9998 |
6 | Vanillic acid | 35.254 | 260; 292 | 0.9996 |
7 | Epicatechin | 42.065 | 232; 280 | 0.9999 |
8 | Coumaric acid | 43.813 | 233; 310 | 0.9999 |
9 | Scopoletin | 47.375 | 296; 344 | 0.9999 |
10 | Isovitexin | 55.302 | 270; 337 | 0.9997 |
11 | Naringenin-7-O-glucoside | 55.881 | 284; 340 | 0.9998 |
12 | Vitexin | 57.145 | 268; 338 | 0.9996 |
13 | Rutin | 59.282 | 256; 356 | 0.9997 |
14 | Kaempferol-3-O-rutinoside | 61.977 | 266; 348 | 0.9998 |
15 | Roifolin | 64.620 | 266; 338 | 0.9999 |
16 | Luteolin | 74.695 | 254; 350 | 0.9999 |
17 | Apigenin | 76.418 | 236; 338 | 0.9998 |
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Bazzicalupo, M.; Burlando, B.; Denaro, M.; Barreca, D.; Trombetta, D.; Smeriglio, A.; Cornara, L. Polyphenol Characterization and Skin-Preserving Properties of Hydroalcoholic Flower Extract from Himantoglossum robertianum (Orchidaceae). Plants 2019, 8, 502. https://doi.org/10.3390/plants8110502
Bazzicalupo M, Burlando B, Denaro M, Barreca D, Trombetta D, Smeriglio A, Cornara L. Polyphenol Characterization and Skin-Preserving Properties of Hydroalcoholic Flower Extract from Himantoglossum robertianum (Orchidaceae). Plants. 2019; 8(11):502. https://doi.org/10.3390/plants8110502
Chicago/Turabian StyleBazzicalupo, Miriam, Bruno Burlando, Marcella Denaro, Davide Barreca, Domenico Trombetta, Antonella Smeriglio, and Laura Cornara. 2019. "Polyphenol Characterization and Skin-Preserving Properties of Hydroalcoholic Flower Extract from Himantoglossum robertianum (Orchidaceae)" Plants 8, no. 11: 502. https://doi.org/10.3390/plants8110502