Part 2: Why Herbal Extracts Can Be Potential Antioxidant, Anti-Aging, Anti-Inflammatory, And Whitening Cosmeceutical Ingredient
Mar 22, 2022
Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791
CLICK HERE TO PART 1
Conclusions
Herbal extracts from different plant materials varied in external appearance, including color and flavor. Among 16 herbal extracts, SR contained the most significant level of both phenols and flavonoids, so SR possessed the most significant antioxidant activities in both DPPH and FRAP assays (p < 0.05). In addition, RD and PE also possessed antioxidant activities comparable to those of SR (p >0.05). Moreover, SR, RD, and PE showed a promising whitening effect with the most significant anti-tyrosinase activities compared with the others (p < 0.05). However, the herbal extract with the most significant anti-aging activities was EP, which inhibited collagenase, elastase, and hyaluronidase activity by 78.5 ± 0.0%, 69.0 ± 1.4%, and 64.2 ± 0.3%, respectively. Furthermore, MA and MS possessed the most significant anti-inflammatory activity, since it inhibited IL-6 and TNF-α secretion (p < 0.05). Therefore, the various herbal extracts mentioned above have promising beneficial effects on the skin and could potentially be used in cosmetic/cosmeceutical products. Herbal extracts in the form of aqueous solutions can be directly applied on the skin as several forms of cosmetic products, such as toner, facial mist, and facial serum. They could also be developed into several forms of cosmetic products, including cream, lotion, and gel. However, nano-delivery systems are proposed for the delivery of these herbal extracts into a deeper skin layer in order to fulfill their cosmeceutical properties. Furthermore, lyophilization or solvent removal is suggested as a further process for producing a dried extract that not only yields more detail about the amount of extracted material but also can be kept for a longer period of time.
For more information, please click here
Experimental Section
Plant materials
G. extension and M. alba leaves were collected from a local farm in Mae Rim district, Chiang Mai, Thailand, in October 2018. The fresh leaves were washed using tap water and allowed to dry at ambient temperature. The leaves were cut into small pieces and steamed for about 5-10 min. Subsequently, the streamed leaves were roasted at low temperature for 15 min and finally dried in an oven (UF110, Memert, Germany) set at a temperature of 45 oC for 3 days. Furthermore, dried M. alba leaves were prepared without steaming or roasting processes. Dried E. purpurea flowers were purchased from a local farm in Chiang Mai, Thailand. Dried A. elatior and G. pentaphyllum leaves were purchased from the Royal Project Foundation shop in Chiang Mai, Thailand. Dried C. tinctorius, C. morifolium, C. ternatea, H. sabdarifa, and J. sambac flowers, dried P. amaryllifolius leaves, dried R. Damascena flowers, dried S. rebaudiana leaves, dried C. verum bark powder, and dried P. emblica fruit powder were purchased from a local market in Chiang Mai, Thailand. All dried plant materials were ground into a fine powder with a Moulinex blender (Moulinex, Paris, France) and kept in sealed containers as shown in Figure 10 until further use.
Figure 10. Dried herbs (a) and dried powders (b) of various plant materials
Microscopic analysis of dried plant materials
The plant samples were identified and authenticated by Ms. Wannaree Charoensup, a botanist at the Herbarium, Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University. Dried powder of each plant material was analyzed using a Nikon ECLIPSE E200 Microscope (Nikon Solutions Co., Ltd., Konan, Japan) connected with a Canon EOS750D camera (Canon Inc., Tochigi, Japan).[54] Mounting samples in dilute glycerol was used to prepare slides. The microscopic characteristics and cell components of each sample were examined and photographed using a microscope with 400× lens magnifications.
cistanche plant whitening effect on the skin to anti-oxidation
Chemical materials
L-ascorbic acid, kojic acid, gallic acid, quercetin, oleanolic acid, hyaluronic acid, Folin–Ciocalteu reagent, lipopolysaccharide (LPS), bovine serum albumin (BSA), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 2,2’-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-dimethyl thiazolyl-2) -2,5-diphenyl tetrazolium bromide (MTT), collagenase from Clostridium histolyticum (EC 3.4.24.3), elastase from porcine pancreatic (EC 3.4.4.7), hyaluronidase from bovine testis (EC 3.2.1.3.5), N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), dihydrochloride mushroom tyrosinase (EC 1.14.18.1), L-3,4 dihydroxyphenylalanine (L-DOPA), and L-tyrosine were purchased from SigmaAldrich (St. Louis, MO, USA). Tricine and Tris base was purchased from Fisher Chem Alert (Fair Lawn, NJ, USA). Dulbecco modified eagle medium (DMEM), L-glutamine, RPMI-1640, penicillin/streptomycin, and Trypan blue were purchased from Invitrogen™ (Grand Island, NY, USA). Hydrochloric acid and acetic acid of AR grade were purchased from Merck (Darmstadt, Germany). Aluminum chloride (AlCl3), calcium chloride (CaCl2), ferrous chloride (FeCl2), ferric chloride (FeCl3), ferrous sulfate (FeSO4), potassium acetate (CH3CO2K), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), potassium persulfate (K2S2O8), sodium acetate (C2H3NaO2), sodium carbonate (Na2CO3), sodium chloride (NaCl), sodium hydroxide (NaOH), monosodium phosphate (NaH2PO4), and disodium phosphate (Na2HPO4) were purchased from Fisher Chemicals (Loughborough, UK). Ethanol and dimethyl sulfoxide (DMSO) of AR grade were purchased from Labscan (Dublin, Ireland).
Herbal extraction
Herbal plant materials were extracted by infusion in boiled water. Briefly, 1 g of each dried herbal plant powder was packed into a tea bag and immersed into 50 mL of boiled DI water. The teabag was then removed after 1, 3, 5, 10, and 15 min of infusion. The infused extract was left cooling down to room temperature and used in further investigations.
cistanche herba extract
Total phenolic content determination by the Folin–Ciocalteu method
The total phenolic content of each herbal extract was evaluated by the Folin–Ciocalteu method based on a method of Chaiyana et al., [55] which had been modified from the method of Li et al. [56]Gallic acid was used to establish a standard curve. The levels of phenolic compound are presented in terms of milligrams of gallic acid equivalent (GAE) per milliliter of herbal extracts. All experiments were done in triplicate.
Total flavonoid content determination by the aluminum chloride method
The total flavonoid content of each herbal extract was evaluated by the aluminum chloride method based on a method of Do et al.[57] Quercetin was used to establish a standard curve. Flavonoid levels are presented in terms of milligrams of quercetin equivalent (QE) per milliliter of herbal extracts. All experiments were done in triplicate.
2,2’-diphenyl-1-picrylhydrazyl reagent (DPPH) assay
The scavenging activity against DPPH radicals (DPPH) of each herbal extract was evaluated using the DPPH assay based on a method of Chaiyana et al., [55] which had been modified from the method of Blois. [58] The scavenging activity was calculated using the equation
% Scavenging activity = {((A-B)-(C-D))/(A-B)} x 100 (1)
where A is the UV absorbance of the DPPH solution, B is the UV absorbance of the solvents, C is the UV absorbance of the mixture of herbal extracts and DPPH solution, and D is the UV absorbance of the herbal extract solution. The positive control was L-ascorbic acid. All experiments were done in triplicate.
Ferric reducing antioxidant power (FRAP) assay
The ferric reducing antioxidant power of each herbal extract was evaluated using the FRAP assay based on a method of Chaiyana et al.,[55] which had been modified from the method of Saeio et al.[59] FeSO4 was used to establish a standard curve. The ferric reducing power is presented in terms of equivalent capacity (EC1), which was the amount of FeSO4 equivalents per milliliter of the sample. The positive control was L-ascorbic acid. All experiments were done in triplicate.
Anti-tyrosinase activity determination
The inhibitory activity against the tyrosinase enzyme of each herbal extract was evaluated using a spectrophotometric assay based on a method of Laosirisathian et al.,[60] which had been modified from the method of Pomerantz.[61] The anti-tyrosinase activities were calculated using the equation
% Anti-tyrosinase activity = {((A-B)-(C-D))/(A-B)} x 100 (2)
where A is the UV absorbance of a tyrosinase enzyme combined with the substrate, B is the UV absorbance of the solvent, C is the UV absorbance of the herbal extracts combined with a tyrosinase enzyme and the substrate, D is the UV absorbance of the herbal extract solution. The positive control was kojic acid. All experiments were done in triplicate.
Cistanche is a tyrosinase inhibitor
Collagenase inhibitory activity determination by spectrophotometric method
The collagenase inhibitory activity of each herbal extract was evaluated using a spectrophotometric assay based on a method of Chaiyana et al.[44] with slight modifications. Firstly, 0.16 units/mL of collagenase solution was a combination of collagenase from Clostridium histolyticum, 80 mM NaCl, 2 mM CaCl2, and 50 mM Tricine buffer, pH 7.5. Subsequently, 200 µL of the resulting collagenase solution was applied to 20 µL of each herbal extract in a flat-bottom well plate (Costar, Corning Ltd., Sunderland, UK) and left for 15 min. Subsequently, 80 µL of 1 mg/mL of FALGPA in the Tricine buffer, pH 7.5, was applied as a substrate to the enzymatic reaction and further left for 20 min. The UV absorbance of the resulting mixture was measured at 340 nm using a multimode detector (Beckman CoulterDTX880, Fullerton, CA, USA). The collagenase inhibitory activities were calculated using the equation
% Anticollagenase activity = {((A-B)-(C-D))/(A-B)} x 100 (3)
where A is the UV absorbance of collagenase solution combined with the FALGPA solution, B is the UV absorbance of the solvents, C is the UV absorbance of the herbal extracts combined with the collagenase and FALGPA solution, and D is the UV absorbance of the herbal extract solution. The positive control was EGCG. All experiments were done in triplicate.
Elastase inhibitory activity determination by the spectrophotometric method
The elastase inhibitory activity of each herbal extract was evaluated using a spectrophotometric assay based on a method of Chaiyana et al.[44] The elastase inhibitory activities were calculated using the equation
% Antielastase activity = {((A-B)-(C-D))/(A-B)} x 100, (4)
where A is the UV absorbance of elastase solution and AAAPVN solution, B is the UV absorbance of the solvents, C is the UV absorbance of the herbal extracts combined with elastase and AAAPVN solution, and D is the UV absorbance of the herbal extract solution. EGCG was used as a positive control. The positive control was EGCG. All experiments were done in triplicate.
Hyaluronidase inhibitory activity determination by spectrophotometric method
The hyaluronidase inhibitory activity of each herbal extract was evaluated using a spectrophotometric assay based on a method of Chaiyana et al.[44] The hyaluronidase inhibitory activities were calculated using the equation
% Hyaluronidase activity = {((A-B)-(C-D))/(A-B)} x 100 (5)
where A is the UV absorbance of hyaluronidase solution combined with the hyaluronic acid solution, B is the UV absorbance of the solvents, C is the UV absorbance of the herbal extracts combined with the hyaluronidase solution and hyaluronic acid solution, and D is the UV absorbance of the herbal extract solution. The positive control was oleanolic acid. All experiments were done in triplicate.
Anti-inflammatory activity determination by the enzyme-linked immunosorbent assay (ELISA)
The anti-inflammatory activity of each herbal extract was evaluated by means of inhibitory activities against IL-6 and TNF-α secretion based on a method of Chaiyana et al.,[44] which had been modified from the method of Mueller et al.[62] LPS was used to stimulate the inflammatory process in mouse monocyte macrophage RAW 264.7 cells (American Type Culture Collection, ATCCTIB-71). The cell incubated with LPS served as vehicle control, with 100% of the cytokines secreted, while non-treated RAW 264.7 cells served as a negative control. The MTT assay was used to evaluate RAW 264.7 cell viability in conjunction with the ELISA.[62] Inhibition on IL-6 and TNF-α secretion was calculated using the equation
% Cytokine inhibition = {((A-B)-(C-D))/(A-B)} x 100 (6)
where A is the optical density of the herbal extracts from the MTT assay, B is the optical density of the negative control from the MTT assay, C is the optical density of the herbal extracts from ELISA, and D is the optical density of the vehicle control from ELISA. The positive control was dexamethasone. All experiments were done in triplicate.
Statistical analysis
All results are presented in the form of mean ± standard deviation (S.D.). The one-way analysis of variance (ANOVA) was used to determine statistical significance, followed by Turkey's post-hoc tests, using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). P < 0.05 was regarded as statistically significant.
References
[1] M. Kong, X. G. Chen, D. K. Kweon, H. J. Park, ‘Investigations on skin permeation of hyaluronic acid-based nanoemulsion as transdermal carrier’, Carbohydr. Polym. 2011, 86, 837-843.
[2] H. Takigawa, H. Nakagawa, M. Kuzukawa, H. ori, G. Imokawa, ‘Deficient production of hexadecenoic acid in the skin is associated in part with the vulnerability of atopic dermatitis patients to colonization by Staphylococcus aureus’, Dermatology. 2005, 211, 240-248.
[3] S. H. Lee, S. K. Jeong, S.K. Ahn, ‘An update of the defensive barrier function of the skin’, Yonsei Med. J. 2006, 47, 293-306.
[4] F. Bonté, D. Girard, J. C. Archambault, A. Desmouliere, ‘Skin Changes During Ageing’, In Biochemistry and Cell Biology of Ageing: Part II Clinical Science, Springer: Singapore, 2019, 249-280.
[5] A. K. Langton, H. K. Graham, C. E. Griffiths, R. E. B. Watson, ‘Ageing significantly impacts the biomechanical function and structural composition of the skin’, Exp. Dermatol. 2019, 28, 981-984.
[6] T. Schikowski, A. Hüls, ‘Air Pollution and Skin Aging’, Curr. Environ. Health Rep. 2020, 1-7.
[7] N. Amberg, C. Fogarassy, ‘Green Consumer Behavior in the Cosmetics Market’, Resources. 2019, 8, 137.
[8] J. Azmir, I. S. M. Zaidul, M. M. Rahman, ‘Techniques for extraction of bioactive compounds from plant materials: A review, J. Food Eng. 2013, 117, 426-436.
[9] M. Gašperlin, M. Gosenca, ‘Main approaches for delivering antioxidant vitamins through the skin to prevent skin aging’, Expert. Opin. Drug Deliv. 2011, 8, 905-919.
[10] S. M. Salavkar, R. A. Tamanekar, R. B. Athawale, ‘Antioxidants in skin aging–Future of dermatology‘, Int. J. Green Pharm. 2011, 5(3). 161-168.
[11] J. Calleja-Agius, Y. Muscat-Baron, M. P. Brincat, ‘Skin aging ‘, Menopause Int. 2007,13, 60-64.
[12] G. J. Fisher, T.Quan, T. Purohit, Y. Shao, M. K. Cho, T. He, J. Varani, S. Kang, J. J. Voorhees, ‘Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin‘, Am. J. Pathol. 2009, 174, 101-114.
[13] A. C. Weihermann, M. Lorencini, C. A. Brohem, C. M. De Carvalho, ‘Elastin structure and its involvement in skin photoaging, Int. J. Cosmet. Sci. 2017, 39, 241- 247.
[14] S. Abhijit, D. Manjushree, ‘Anti-hyaluronidase, the anti-elastase activity of Garcinia Indica, Int. J. Botany. 2010, 6(3), 299-303.
[15] T. Pillaiyar, M. Manickam, V. Namasivayam, Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 2017, 32, 403-425.
[16] M. Ashawat, M. Banchhor, S. Saraf, S. Saraf, ‘Herbal Cosmetics: Trends in Skin Care Formulation‘ Pharmacogn. Rev. 2009, 3, 82-89.
[17] F. N. Muanda, R. Soulimani, B. Diop, A. Dicko, ‘Study on chemical composition and biological activities of essential oil and extracts from Stevia rebaudiana Bertoni leave’, LWT-Food Sci. Technol. 2011, 44, 1865-1872.
[18] N. G. Baydar, H. Baydar, ‘Phenolic compounds, antiradical activity and antioxidant capacity of oil-bearing rose (Rosa damascena Mill.) extracts’, Ind. Crops Prod. 2013, 41, 375-380.
[19] S. M. Ghoreishi, A. Hedayati, S. O. Mousavi, ‘Quercetin extraction from Rosa damascena Mill via supercritical CO2: Neural network and adaptive neuro-fuzzy interface system modeling and response surface optimization, J. Supercrit. Fluids. 2016, 112, 57-66.
[20] T. E. A. Ardjani, J. R. Alvarez-Idaboy, ‘Radical scavenging activity of ascorbic acid analogs: Kinetics and mechanisms’, Theor. Chem. Acc. 2018. 137, 69.
[21] L. Panzella, ‘Natural Phenolic Compounds for Health, Food and Cosmetic Applications’ Antioxidants. 2020, 9, 427.
[22] D. S. Menamo, D. W. Ayele, M. T. Ali, ‘Green synthesis, characterization and antibacterial activity of copper nanoparticles using L-ascorbic acid as a reducing agent’, Ethiop. j. sci. technol. 2017, 10, 209-220.
[23] N. G. Quilantang, S. H. Ryu, S. H. Park, J. S. Byun, J. S. Chun, J. S. Lee, J. p. Rodriguez, Y-S. Yun, S.D. Jacinto, S. Lee, ‘Inhibitory activity of methanol extracts from different colored flowers on aldose reductase and HPLC-UV analysis of quercetin’, Hortic. Environ. Biotechnol. 2018, 59, 899-907.
[24] S. M. Sabir, R. H. Shah, A. H. Shah, ‘Total Phenolic and Ascorbic Acid Contents and Antioxidant Activities of Twelve Different Ecotypes of Phyllanthus emblica from Pakistan’, Chiang Mai J. Sci. 2017, 44, 904-911.
[25] J. F. Morton, ‘The emblic (Phyllanthus emblica L.)’, Econ. Bot. 1960, 14, 119-128.
[26] N. N. Barthakur, N. P. Arnold, ‘Chemical analysis of the emblic (Phyllanthus emblica L.) and its potential as a food source’, Sci. Hortic. 1991, 47, 99-105.
[27] D. Huang, B. Ou, R. L. Prior, ‘The chemistry behind antioxidant capacity assays’, J. Agric. Food Chem. 2005, 53, 1841-1856.
[28] V. Bondet, W. Brand-Williams, C. L. W. T. Berset, ‘Kinetics and Mechanisms of Antioxidant Activity using the DPPH Free Radical Method’, Food Sci. Technol.- Zurich. 1997, 30, 609-615.
[29] S. Nam, H. W. Jang, T. Shibamoto, ‘Antioxidant activities of extracts from teas prepared from medicinal plants, Morus alba L., Camellia sinensis L., and Cudrania tricuspidata, and their volatile components’, J. Agric. Food Chem. 2012, 60, 9097-9105. [30] J. Harrathi, H. Attia, M. Neffati, ‘Salt effects on shoot growth and essential oil yield and composition in safflower (Carthamus tinctorius L.)’, J. Essent. Oil Res. 2013, 25, 482-487.
[31] A. Mar, A. A. Mar, P. P. Thin, ‘Study on the Phytochemical Constituents in Essential oil of Pandanus amaryllifolious Roxb. Leaves and their Anti-bacterial Efficacy’, Yadanabon Univ. Res. J. 2019, 10, 1-9.
[32] M. Rinnerthaler, J. Bischof, M. K. Streubel, A. Trost, K. Richter, ‘Oxidative stress in aging human skin’, Biomolecules. 2015, 5, 545-589.
[33] N. R. Perron, J. L. Brumaghim, ‘A review of the antioxidant mechanisms of polyphenol compounds related to iron binding’, Cell Biochem. Biophys. 2009, 53, 75-100.
[34] A. Karadag, B. Ozcelik, S. Saner, ‘Review of methods to determine antioxidant capacities’, Food Anal. Methods. 2009, 2, 41-60.
[35] R. J. Wang, M. L. Hu, ‘Antioxidant capacities of fruit extracts of five mulberry genotypes with different assays and principle components analysis, Int. J. Food Prop. 2011, 14, 1-8.
[36] S. Mirunalini, M. Krishnaveni, ‘Therapeutic potential of Phyllanthus emblica (amla): the ayurvedic wonder’, J. Basic Clin. Physiol. Pharmacol. 2010, 21, 93-105.
[37] R. Jakmatakul, R. Suttisri, P. Tengamnuay, ‘Evaluation of antityrosinase and antioxidant activities of Raphanus sativus root: comparison between freeze-dried juice and methanolic extract’, Thai J. Pharm. Sci. 2009, 33. 22-30.
[38] S. J. Lee, W. J. Lee, S. E. Chang, G. Y. Lee, ‘Antimelanogenic effect of ginsenoside Rg3 through extracellular signal-regulated kinase-mediated inhibition of microphthalmia-associated transcription factor’ J. Ginseng Res. 2015, 39, 238-242
