Extraction, Physicochemical Properties, Anti-Aging, And Antioxidant Activities Of Polysaccharides From Industrial Hemp Residues Part 2

2.8. Anti-Aging Activity Study

2.8.1. Cell Viability

In vitro, cytotoxicity experiments are frequently used to assess the toxicity of tested samples [37]. The effects of IHRPs on the cell viability of HDF and HEK, which were the commonly used epidermal cell lines, are displayed in Figure 5. IHRPs almost had no cytotoxicity for HDF and HEK. Furthermore, IHRPs could promote cell proliferation between 100 and 800 µg/mL. To ensure the cell viability was not affected by the concentration of the sample, we chose IHRP concentrations below 400 µg/mL for the following experiments.

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2.8.2. Scratch Assay of HDF Cells

The scratch assay is an in vitro method widely used to evaluate the contribution of cellular and molecular mechanisms to cell proliferation and migration [38]. The images of the healing condition of HDF scratches are shown in Figure 6. IHRPs of different concentrations were all beneficial to the healing of cell scratches compared with the control. Moreover,  when the concentration of IHRPs was between 50 and 200 µg/mL, IHRPs could significantly promote the healing of cell scratches. The healing rate of HDF scratches after 24 h was 64.51 ± 3.69% (p < 0.01), 58.03 ± 3.90% (p < 0.05), and 66.21 ± 6.60% (p < 0.01) respectively when the IHRPs concentration was 50, 100, and 200 µg/mL, as displayed in Table 6. Besides, the healing effect of IHRPs was very close to that of the positive control TGF-β (62.29 ± 4.69%, p < 0.01). The results of the scratch assay indicated that IHRPs could accelerate the healing of cell scratches and promote cell proliferation.

2.8.3. qRT-PCR Analysis of HDF Gene Expression

Several studies have been conducted to evaluate the anti-aging potential of different plants [39,40]. The relative quantification of anti-aging genes of HDF is displayed in Figure 7. As shown in the figure, TGF-β, Vc, and HA were used as the positive control. The order of AQP-3 relative quantification was HA > TGF-β > IHRPs > Vc. The order of COL1A1 relative quantification was TGF-β > Vc > HA > IHRPs. The order of COL3A1  relative quantification was Vc > TGF-β > HA > IHRPs. The order of ELASTIN relative quantification was TGF-β > Vc > HA > IHRPs. The order of MMP-1 relative quantification was IHRPs > HA > Vc > TGF-β. Therefore, compared with the positive control, IHRPs nearly had no positive effect on the expression of AQP-3, COL1A1, COL3A1, and ELASTIN. However, IHRPs significantly promoted the expression of MMP-1. MMP-1 is primarily generated by keratinocytes and is mainly used for the decomposition and fragmentation of skin collagen fibers [41]. In summary, IHRPs could promote HDF proliferation and the expression of anti-aging-related genes, indicating the anti-aging and skin repair potentials of IHRPs.

3. Materials and Methods 

3.1. Materials and Reagents

IHR was provided by Yunnan Hempmon Pharmaceuticals Co., Ltd. (Kunming, China). Phenol, α-naphthol, sulfuric acid, carbazole, Coomassie blue G-250, phosphoric acid,  ethanol, chloroform, and monosaccharide control were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Galacturonic acid, glucuronic acid, guluronic acid,  and arbutin were bought from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Human dermal fibroblast (HDF), human epidermal keratinocytes (HEK), and complete cell medium were bought from Sciencell Co., Ltd. (Carlsbad, CA, USA). 12-well plates and 96-well plates were offered by Corning Co., Ltd. (Corning, NY, USA). CCK-8 cell viability assay kit was bought from DOJINDO Biology Co., Ltd. (Tokyo, Japan), and other detection kits were offered by Takara Co., Ltd. (Takara, Japan).

3.2. Optimization of IHRPs Extraction 

3.2.1. Comparison of Different Extraction Methods

Percolation extraction: 100 g IHR was added into 2000 mL deionized water. The extraction was performed with a flow rate of 150 ± 50 mL/h after 2 h. Heating extraction: 100 g IHR was added into 2000 mL deionized water. The extraction was performed at 98 ◦C  for 1 h and the process was repeated twice. Ultrasonic-assisted extraction: 100 g IHR was dissolved in 2000 mL deionized water. The extraction was conducted for 0.5 h at 60 ◦C and the process was repeated twice.

The filtrates were concentrated using a vacuum rotary evaporator. Then the solution was dried by a vacuum drier. The polysaccharide content was determined to choose an appropriate method.

3.2.2. Single-Factor Experiments

For the extraction of IHRPs, 100 g IHR was added to a certain amount of deionized water. Extraction temperature (40, 60, 80, and 98 ± 2 ◦C), solid–liquid ratio (1:6, 1:8, 1:10,  and 1:15), extraction time (0.5, 1.0, 1.5, and 2 h), pH (3, 5, 7, 9, and 11), and several successive extractions (1, 2, 3, 4) were studied separately to evaluate the influence of individual factors on IHRPs extraction.

3.2.3. Orthogonal Experimental Design

Extraction conditions were further optimized by orthogonal experimental design. As shown in Table 7, the four selected variables were extraction temperature (60, 80, and 98 ± 2 ◦C), RS/L (1:6, 1:8, and 1:10), number of successive extractions (1, 2, and 3), and pH (4, 7, and 10). The orthogonal experiments were divided into 9 groups. The weight of polysaccharides was considered as the target to estimate the extraction conditions.

3.3. Screen of IHRPs Alcohol Precipitation Conditions

The extraction solution was obtained by the optimum condition according to 3.2 and 400 mL ethanol was added. The experiments were conducted based on the different rates of adding alcohol, stirring method, and cooling rate in Table 8. The precipitate was obtained after centrifugation and drying. Then the weight and polysaccharide content were acquired to determine the suitable alcohol precipitation conditions.

3.4. Determination of Polysaccharide Yield and Chemical Composition of IHRPs 

The protein content was calculated based on the Bradford method and the bovine serum albumin (BSA) was employed as a standard [42]. The total sugar content of IHRPs was acquired according to the phenol-sulfate method and the glucose was used as a standard [43]. The uronic acid content was obtained by a carbazole-sulfuric acid method [44]. Polysaccharide yield was obtained by Equation (1).

where YP refers to the polysaccharide yield, mA refers to the weight of IHRPs, and mB represents the weight of IHR employed for polysaccharide extraction.

3.5. Purifification of IHRPs

First, 100 g crude IHRPs were prepared by the optimum condition determined in Sections 3.2 and 3.3. The crude IHRPs were further purified by different methods.

Activated carbon adsorption: 10 g crude IHRPs were added into 200 mL deionized water. The activated carbon of 1%, 2%, 4%, and 8% IHRPs weight were mixed with the solution, respectively. Then, the solution was agitated at 60 ◦C for 1 h and filtrated. After washing with deionized water, the filtrate was concentrated and dried.

Membrane filtration: 10 g crude IHRPs were added into 500 mL deionized water. Subsequently, the solution was filtrated by membranes with different molecular weights (30,000 Da, 10,000 Da, 1000 Da, and 500 Da) continuously. The filtrate and retentate were both collected and dried. Finally, IHRPs with different molecular weights were obtained.

Savage method: 1 g crude IHRPs were added into 100 mL deionized water. Then the solution was blended with Sevage reagent (n-butanol: chloroform = 1: 5 (v/v)). The mixture was stirred for 15 min and transferred to the separatory funnel for stratification. The above process was repeated 5 times. Subsequently, the organic phase and the aqueous phase were both dried.

Column chromatography: 10 g weakly-basic anion exchange resin was immersed in deionized water for 2 h, then loaded into a glass column (2 × 30 cm). 10 g crude IHRPs were added into 200 mL deionized water. Subsequently, the solution was slowly passed through the resin layer. The column was rinsed with 100 mL of 30% ethanol. The eluent was concentrated and dried.

3.6. Monosaccharide Composition Analysis 

The monosaccharide composition test was performed according to the previous method with slight modifications [45]; 5 mg IHRPs was hydrolyzed with 1 mL trifluoroacetic acid (TFA, 2 M) for 6 h at 105 ◦C. Subsequently, the solution was dried under a nitrogen atmosphere. The dried hydrolysate was added to 5 mL of deionized water after TFA was removed by methanol. Afterward, 0.5 mL of 0.3 M NaOH solution and 1 mL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution were added to the hydrolysate. The obtained solution was kept in a water bath for 2 h at 70 ◦C for derivatization. Then, 0.5 mL HCl solution (0.3 M) was added for neutralization. The solution was mixed with 1 mL chloroform for HPLC analysis. Several monosaccharides were used as references. Chromatographic conditions: Mobile phase A (82%): 0.1 M KH2PO4 solution. Mobile phase B (18%): acetonitrile. Column: C18 (5 µm, 4.6 × 250 mm). Injection volume: 10 µL. Flow rate: 1.0 mL/min. Detection wavelength: 245 nm. Column temperature: 30 ◦C.

3.7. Fourier Transform Infrared Spectroscopy (FT-IR) 

The samples for FT-IR analysis were prepared by mixing 5 mg IHRPs with 125 mg KBr. The FT-IR spectra of samples were obtained between 4000 and 500 cm−1.

3.8. Antioxidant Activity Study 

3.8.1. DPPH Radical Scavenging Activity 

The DPPH free radical scavenging activity of IHRPs was evaluated based on a reported method with a few modifications [46]. In short, 2 mL sample solution (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) was blended with 2 mL DPPH solution (0.1 mM). The mixture was kept for 30 min in the dark, and then the absorbance (Abs) was obtained at 517 nm. The DPPH  scavenging rate was estimated with the following equation (Equation (2)) and the 50%  effective concentration (EC50) was counted.

where RSD represents the free radical scavenging rate, Ai refers to the Abs of the reaction system (DPPH with the sample), Aj is the Abs of the sample background (solvent with the sample), and A0 is the Abs of the negative control (DPPH with solvent).

3.8.2. ABTS Radical Scavenging Activity

The ABTS scavenging activity of IHRPs was evaluated based on a previous method [47]. In short, the potassium persulfate solution (2.45 mM) was blended with the ABTS solution (7 mM) in the dark for 12 h. The obtained ABTS solution was diluted 50 times to the Abs of 0.70 ± 0.02 at 734 nm. Subsequently, 1 mL sample solution (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL)  was added to 4 mL ABTS solution. The mixture was shaken rapidly for 1 min and placed in the dark for 6 min. Then the Abs was measured at 734 nm and Vc was used as a positive control. The ABTS scavenging rate was calculated with Equation (2).

3.9. Anti-Aging Activity Study 

3.9.1. Cell Culture

Human dermal fibroblast (HDF) and human epidermal keratinocytes (HEK) were purchased from ScienCell Co., Ltd. The cells were cultured with DMEM medium containing 10% FBS. The culture condition was 37 ◦C with 5% CO2.

3.9.2. Cell Viability

Cell Counting Kit-8 (CCK-8) assay was carried out to determine the cytotoxicity of IHRPs against HDF and HEK cells. The cells were cultured in 96-well plates with a  concentration of 2 × 104  cells/well for 48 h. Afterward, 100 µL fresh medium containing samples was added and the wells were incubated for 24 h. Then CCK-8 solution was added to wells according to the instruction of test kits and the plate was incubated for 2 h. The Abs at 450 nm was obtained to calculate the cell viability.

3.9.3. Scratch Assay 

HDF was cultured at 37 ◦C with 5% CO2. Then the cells were seeded into a 12-well  plate at a concentration of 1 × 105  cells/well. After incubation for 48 h, the scratch was created on the HDF cellular layer by the syringe needle. The cell fragments were cleaned by PBS and the samples with different concentrations were added. Subsequently, the healing of cell layer scratches was observed after 24 h to evaluate the effect of IHRPs on the proliferation of HDF. The obtained images were quantified by Image J software [48]. Transforming growth factor-β (TGF-β) was used as the positive control. The healing rate was calculated according to Equation (3).

where A1 represents the initial scratch area and A2 refers to the final scratch area. 

3.9.4. Quantitative RT-PCR (qRT-PCR) Analysis

Then the cells were seeded into 12-well plates with a concentration of 1 × 105  cells/well. After incubation for 48 h, the samples were mixed and cultured for another 24 h. Total RNA was extracted and cDNA was synthesized. The qRT-PCR analyses for the aquaporin gene (AQP-3), collagen gene (COL1A1 and COL3A1), elastin gene (ELASTIN),  and matrix metalloproteinase (MMP-1) were performed using a real-time PCR system (Applied Biosystems Life Technologies, Inc., ABI StepOnePlus). Relative quantification was conducted with the comparative CT method (2-∆∆Ct method). Hyaluronic acid (HA), Vc,  and TGF-β were employed as the positive control.

3.10. Statistical Analysis 

Results were displayed as the mean ± SD (n = 3). Statistical significance was performed by ANOVA. Values of p < 0.05 were considered to be statistically significant.

4. Conclusions

In this work, the polysaccharide extraction from IHR was optimized by single-factor experiments and orthogonal experimental design. The optimum heating extraction conditions were extraction temperature was 98 ◦C, solid–liquid ratio of 1:10, extraction time of 1 h,  number of successive extractions of 2, and pH of 4. The extraction ratio and the polysaccharide content were 20.12% and 12.35% respectively at the conditions. Additionally, the suitable alcohol precipitation conditions were pumping with 2 L/h, stirring continuously, and ice-water bath for 4 h. The crude IHRPs were further purified by column chromatography and the polysaccharide/protein contents of purified IHRPs were 34.44% and 1.61%. The monosaccharide composition of IHRPs was: fucose (1.33%), arabinose (19.60%), rhamnose (10.41%), galactose (20.87%), glucose (27.42%), xylose (4.23%), ribose (3.12%), galacturonic acid (6.22%), guluronic acid (0.28%), and glucuronic acid (2.37%). The FT-IR demonstrated the polysaccharide skeleton of IHRPs. Besides, the EC50 values of ABTS and DPPH radicals were 0.34 and 0.47 mg/mL, showing the great antioxidant activity of IHRPs. IHRPs also could promote the cell proliferation of HDF and HEK and the healing of cell scratches. Moreover, IHRPs could significantly promote the expression of MMP-1. Therefore, it is believed that the polysaccharides from industrial hemp residues could be developed as potential antioxidant and anti-aging products for cosmetics or functional foods.

Author Contributions: T.C., Q.Z., and B.Z. designed the experiments. T.C. and H.L. (Hang Li)  performed the experiments and wrote the initial draft. T.C., H.L. (Hang Li), H.L. (Hongning Lv), X.L., and M.L. analyzed the data. M.T., S.H., Q.Z., and B.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Yunnan Hempmon Pharmaceuticals Co., Ltd. 

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable. 

Data Availability Statement: Not applicable. 

Conflicts of Interest: The authors have declared no conflicts of interest. 

Sample Availability: Samples of the compounds IHR and IHRPs are available from the authors. 

References

1. Kumar, P.; Mahato, D.K.; Kamle, M.; Borah, R.; Sharma, B.; Pandhi, S.; Tripathi, V.; Yadav, H.S.; Devi, S.; Patil, U. Pharmacological properties, therapeutic potential, and legal status of Cannabis sativa L.: An overview. Phytother. Res. 2021, 35, 6010–6029. [CrossRef] [PubMed] 

2. Shoyama, Y.; Yagi, M.; Nishioka, I.; Yamauchi, T. Biosynthesis of cannabinoid acids. Phytochemistry 1975, 14, 2189–2192. [CrossRef] 

3. De Meijer, E.D.; Van der Kamp, H.; Van Eeuwijk, F. Characterisation of Cannabis accessions about cannabinoid content about other plant characters. Euphytica 1992, 62, 187–200. [CrossRef] 

4. Luca, S.V.; Roehrer, S.; Kleigrewe, K.; Minceva, M. Approach for simultaneous cannabidiol isolation and pesticide removal from hemp extracts with liquid-liquid chromatography. Ind. Crop. Prod. 2020, 155, 112726. [CrossRef] 

5. Bonini, S.A.; Premoli, M.; Tambaro, S.; Kumar, A.; Maccarinelli, G.; Memo, M.; Mastinu, A. Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history. J. Ethnopharmacol. 2018, 227, 300–315. [CrossRef] [PubMed] 

6. Hartsel, J.A.; Eades, J.; Hickory, B.; Makriyannis, A. Cannabis sativa and hemp. In Nutraceuticals; Gupta, R.C., Ed.; Academic Press: Boston, MA, USA, 2016; Chapter 53; pp. 735–754. 

7. Palmieri, S.; Mascini, M.; Oliva, E.; Viteritti, E.; Eugelio, F.; Fanti, F.; Compagnone, D.; Sergi, M. Cannabinoid Profile in Cannabis  sativa L. Samples by Means of LC-MRM/IDA/EPI Analysis: A New Approach for Cultivar Classification. J. Agric. Food Chem. 2022, 70, 3907–3916. [CrossRef] 

8. Atalay, S.; Jarocka-Karpowicz, I.; Skrzydlewska, E. Antioxidative and Anti-Inflammatory Properties of Cannabidiol. Antioxidants 2020, 9, 21. [CrossRef] 

9. Small, E. Evolution and classification of Cannabis sativa (marijuana, hemp) about human utilization. Bot. Rev. 2015, 81, 189–294. [CrossRef] 

10. Mazzara, E.; Carletti, R.; Petrelli, R.; Mustafa, A.M.; Caprioli, G.; Fiorini, D.; Scortichini, S.; Dall’Acqua, S.; Sut, S.; Nunez, S.;  et al. Green extraction of hemp (Cannabis sativa L.) using microwave method for recovery of three valuable fractions (essential oil, phenolic compounds, and cannabinoids): A central composite design optimization study. J. Sci. Food Agric. 2022, in press. [CrossRef] 

11. Tan, M.H.; Chang, S.L.; Liu, J.N.; Li, H.; Xu, P.W.; Wang, P.D.; Wang, X.D.; Zhao, M.X.; Zhao, B.; Wang, L.W.; et al. Physicochemical Properties, Antioxidant and Antidiabetic Activities of Polysaccharides from Quinoa (Chenopodium quinoa Willd.) Seeds. Molecules 2020, 25, 3840. [CrossRef] 

12. Zhai, F.G.; Liang, Q.C.; Wu, Y.Y.; Liu, J.Q.; Liu, J.W. Red ginseng polysaccharide exhibits anticancer activity through GPX4  downregulation-induced ferroptosis. Pharm. Biol. 2022, 60, 909–914. [CrossRef] 

13. Zhou, X.; Afzal, S.; Wohlmuth, H.; Munch, G.; Leach, D.; Low, M.; Li, C.G. Synergistic Anti-Inflammatory Activity of Ginger and Turmeric Extracts in Inhibiting Lipopolysaccharide and Interferon-gamma-Induced Proinflammatory Mediators. Molecules 2022, 27, 3877. [CrossRef] [PubMed] 

14. Zou, Y.F.; Li, C.Y.; Fu, Y.P.; Jiang, Q.X.; Peng, X.; Li, L.X.; Song, X.; Zhao, X.H.; Li, Y.P.; Chen, X.F.; et al. The comparison of  preliminary structure and intestinal anti-inflammatory and anti-oxidative activities of polysaccharides from different root parts of Angelica sinensis (Oliv.) Diels. J. Ethnopharmacol. 2022, 295, 115446. [CrossRef] [PubMed] 

15. Huang, Z.; Zong, M.H.; Lou, W.Y. Preparation, structural elucidation and immunomodulatory activity of a polysaccharide from Millettia speciosa Champ. Ind. Crop. Prod. 2022, 182, 114889. [CrossRef] 

16. Cao, W.Y.; Wang, C.X.; Mayhesumu, X.; Pan, L.; Dang, Y.; Yili, A.; Abuduwaili, A.; Mansur, S. Isolation, Structural Elucidation, Antioxidant and Hypoglycemic Activity of Polysaccharides of Brassica rapa L. Molecules 2022, 27, 3002. [CrossRef] [PubMed] 

17. Panggabean, J.A.; Adiguna, S.P.; Rahmawati, S.I.; Ahmadi, P.; Zainuddin, E.N.; Bayu, A.; Putra, M.Y. Antiviral Activities of Algal-Based Sulfated Polysaccharides. Molecules 2022, 27, 1178. [CrossRef] 

18. Baeva, E.; Blaha, R.; Lavrova, E.; Sushytskyi, L.; Copikova, J.; Jablonsky, I.; Kloucek, P.; Synytsya, A. Polysaccharides from Basidiocarps of Cultivating Mushroom Pleurotus ostreatus: Isolation and Structural Characterization. Molecules 2019, 24, 2740. [CrossRef]

19. Xu, J.Q.; Zhang, J.L.; Sang, Y.M.; Wei, Y.N.; Chen, X.Y.; Wang, Y.X.; Xue, H.K. Polysaccharides from Medicine and Food Homology Materials: A Review on Their Extraction, Purification, Structure, and Biological Activities. Molecules 2022, 27, 3215. [CrossRef] 

20. Haustveit, G.; Wold, J.K. Some carbohydrates of low molecular weight present in Cannabis sativa L. Carbohydr. Res. 1973, 29, 325–329. [CrossRef] 

21. Hillestad, A.; Wold, J.K.; Paulsen, B.S. Structural studies of water-soluble glycoproteins from Cannabis sativa L. Carbohydr. Res. 1977, 57, 135–144. [CrossRef] 

22. Groce, J.W.; Jones, L.A. Carbohydrate and cyclitol content of Cannabis. J. Agric. Food Chem. 1973, 21, 211–214. [PubMed] 

23. Wen, Z.-S.; Xue, R.; Du, M.; Tang, Z.; Xiang, X.-W.; Zheng, B.; Qu, Y.-L. Hemp seed polysaccharides protect intestinal epithelial cells from hydrogen peroxide-induced oxidative stress. Int. J. Biol. Macromol. 2019, 135, 203–211. [PubMed] 

24. Bao-yao, B.Y.-g.W.; Jie, T.J.-w.L. Extraction isolation and purification of water-soluble polysaccharides from hemp seed. Food Sci. Technol. 2004, 6, 157–161. 

25. Guo, L.; Kong, N.; Zhang, X.; Ma, H. Multimode ultrasonic extraction of polysaccharides from maca (Lepidium meyenii): Optimization, purification, and in vitro immunoregulatory activity. Ultrason. Sonochem. 2022, 88, 106062. [CrossRef] 

26. Chi, Y.Z.; Li, Y.P.; Zhang, G.L.; Gao, Y.Q.; Ye, H.; Gao, J.; Wang, P. Effect of extraction techniques on properties of polysaccharides from Enteromorpha prolifera and their applicability in iron chelation. Carbohydr. Polym. 2018, 181, 616–623. [CrossRef] [PubMed] 

27. Shi, M.J.; Wei, X.; Xu, J.; Chen, B.J.; Zhao, D.Y.; Cui, S.; Zhou, T. Carboxymethylated degraded polysaccharides from Enteromorpha prolifera: Preparation and in vitro antioxidant activity. Food Chem. 2017, 215, 76–83. 

28. Ren, C.J.; Zhang, Y.; Cui, W.Z.; Lu, G.B.; Wang, Y.W.; Gao, H.J.; Huang, L.; Mu, Z.M. A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin. Int. J. Biol. Macromol. 2015, 72, 951–959. 

29. Wu, J.W.; Li, P.; Tao, D.B.; Zhao, H.T.; Sun, R.Y.; Ma, F.M.; Zhang, B.Q. Effect of solution plasma process with hydrogen peroxide on the degradation and antioxidant activity of polysaccharide from Auricularia auricula. Int. J. Biol. Macromol. 2018, 117, 1299–1304. 

30. Fan, S.H.; Li, J.N.; Bai, B.Q. Purification, structural elucidation, and in vivo immunity-enhancing activity of polysaccharides from quinoa (Chenopodium quinoa Willd.) seeds. Biosci. Biotechnol. Biochem. 2019, 83, 2334–2344. 

31. Sun, Y.J.; Hou, S.T.; Song, S.; Zhang, B.; Ai, C.Q.; Chen, X.F.; Liu, N. Impact of acidic, water and alkaline extraction on structural features, antioxidant activities of Laminaria japonica polysaccharides. Int. J. Biol. Macromol. 2018, 112, 985–995. 

32. Patel, M.K.; Tanna, B.; Mishra, A.; Jha, B. Physicochemical characterization, antioxidant and anti-proliferative activities of a  polysaccharide extracted from psyllium (P. ovata) leaves. Int. J. Biol. Macromol. 2018, 118, 976–987. [PubMed] 

33. Palmieri, S.; Fanti, F.; Oliva, E.; Viteritti, E.; Sergi, M.; Pepe, A.; Compagnone, D. Chemical characterization, and evaluation of antioxidant activity from different cultivars of Cannabis sativa L. of Abruzzo’s region. Nat. Prod. Res. 2022, 1–5. [CrossRef] [PubMed] 

34. Li, H.; Zhao, Q.-S.; Chang, S.-L.; Chang, T.-R.; Tan, M.-H.; Zhao, B. Development of cannabidiol full-spectrum oil/2,6-di-Omethyl-β-cyclodextrin inclusion complex with enhanced water solubility, bioactivity, and thermal stability. J. Mol. Liq. 2022, 347, 118318. 

35. Chen, L.; Yang, W.; Gao, C.; Liao, X.; Yang, J.; Yang, B. The complexes of cannabidiol mediated by bridged cyclodextrins dimers with high solubilization, in vitro antioxidant activity, and cytotoxicity. J. Mol. Liq. 2022, 345, 117017. 

36. Russo, C.; Lavorgna, M.; Nugnes, R.; Orlo, E.; Isidori, M. Comparative assessment of antimicrobial, antiradical and cytotoxic activities of cannabidiol and its propyl analog cannabidivarin. Sci. Rep. 2021, 11, 22494. 

37. Khatun, B.; Baishya, P.; Ramteke, A.; Maji, T.K. Study of the complexation of structurally modified curcumin with hydroxypropyl beta-cyclodextrin and its effect on anticancer activity. New J. Chem. 2020, 44, 4887–4897. 

38. Bobadilla, A.V.P.; Arevalo, J.; Sarro, E.; Byrne, H.M.; Maini, P.K.; Carraro, T.; Balocco, S.; Meseguer, A.; Alarcon, T. In vitro cell migration quantification method for scratch assays. J. R. Soc. Interface 2019, 16, 20180709. [CrossRef]

39. Salem, M.A.; Radwan, R.A.; Mostafa, E.S.; Alseekh, S.; Fernie, A.R.; Ezzat, S.M. Using a UPLC/MS-based untargeted metabolomics approach for assessing the antioxidant capacity and anti-aging potential of selected herbs. RSC Adv. 2020, 10, 31511–31524. 

40. Campa, M.; Baron, E. Anti-aging Effects of Select Botanicals: Scientific Evidence and Current Trends. Cosmetics 2018, 5, 54. 

41. Freitas-Rodriguez, S.; Folgueras, A.R.; Lopez-Otin, C. The role of matrix metalloproteinases in aging: Tissue remodeling and beyond. Biochim. Biophys. Acta Mol. Cell Res. 2017, 1864, 2015–2025. 

42. Bradford, M.M. Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of ProteinDye Binding. Anal. Biochem. 1976, 72, 248–254. [PubMed] 

43. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 1956, 28, 350–356. [CrossRef] 

44. Bitter, T.; Muir, H.M. A Modified Uronic Acid Carbazole Reaction. Anal. Biochem. 1962, 4, 330–334. [CrossRef] 

45. Dai, J.; Wu, Y.; Chen, S.W.; Zhu, S.; Yin, H.P.; Wang, M.; Tang, J.A. Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone. Carbohydr. Polym. 2010, 82, 629–635. 

46. Gao, J.; Zhang, T.; Jin, Z.Y.; Xu, X.M.; Wang, J.H.; Zha, X.Q.; Chen, H.Q. Structural characterization, physicochemical properties and antioxidant activity of polysaccharide from Lilium lancifolium Thunb. Food Chem. 2015, 169, 430–438.

47. Xiao, H.; Fu, X.; Cao, C.; Li, C.; Chen, C.; Huang, Q. Sulfated modification, characterization, antioxidant and hypoglycemic activities of polysaccharides from Sargassum pallidum. Int. J. Biol. Macromol. 2018, 121, 407–414. 

48. Allaw, M.; Manconi, M.; Aroffu, M.; Marongiu, F.; Porceddu, M.; Bacchetta, G.; Usach, I.; Rached, R.A.; Rajha, H.N.; Maroun, R.G.;  et al. Extraction, Characterization, and Incorporation of Hypericum Scruglii Extract in Ad Hoc Formulated Phospholipid Vesicles Designed for the Treatment of Skin Diseases Connected with Oxidative Stress. Pharmaceutics 2020, 12, 1010.

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