Abstract
Elastase and tyrosinase are important targets both for cosmetics and for dermatological disorders. In this work, ninety herbal products were tested as inhibitors of these two enzymes. Eleven extracts resulted strongly active. Four out of them (Camellia sinensis, Ginkgo biloba, Rhodiola rosea, Vitis vinifera) inhibited both enzymes, five (Glycyrrhiza glabra, Ribes nigrum, Rheum officinale, Salvia officinalis, Tilia platyphyllos) were active against tyrosinase only, and two (Ceterach officinarum and Cinnamomum zeylanicum) proved selectively active against elastase. The IC50 ranged from 3.1 to 104.9 μg/mL and 19.3 to 164.3 μg/mL, against elastase and tyrosinase, respectively. The most active extracts resulted enriched in flavonoids (from 1.47 to 56.47 mg RE/g of extract) and phenolics (from 37.43 to 123.56 mg GAE/g of extract), indicating also an antioxidant potential. Finally, a positive correlation between enzymatic bioactivities and phenolic content was also established.
1. Introduction
The demand for new skincare ingredients, especially based on natural products, strongly increased during the last few decadesCitation1,Citation2. Plant bioactive metabolites are most of the time free from harmful side effects, hence, great importance is given to the research of naturally occurring anti-ageing agents. Several plants proved to be effective to slow down skin ageingCitation3, acting as antioxidants, protecting skin against solar radiationsCitation4, and/or modulating the activity of enzymes involved in the ageing processesCitation5, among which elastase (Ela) and tyrosinase (Tyr) are of remarkable importance.
Elastase activity increases significantly with age and after chronic UV-B irradiation Citation6, resulting in sagging due to loss of skin elasticity, thus inhibition of this enzyme is valuable strategy to slow down the intrinsic and extrinsic ageing processesCitation7. Tyrosinase is responsible for skin hyper-pigmentation, as in case of melasma, freckles, ephelide and senile lentinginesCitation8 being an important target for skin-whitening agents.
This work aimed at identifying herbal products endowed with Tyr and/or Ela inhibitory activity (IA), thus potential anti-ageing agents. The bioactivity screening was carried out on extracts obtained from ninety commercial plants, widely used as ingredients for herbal preparations, including botanicals, herbal teas, and food supplementsCitation9,Citation10. Considering the importance of polyphenols as antioxidant, the total phenolic and flavonoid content of the extracts was also determined and their content was also statistically correlated to the percentages of enzymatic inhibition.
2. Methods and materials
2.1. Plant material and extracts preparation
Plant samples were kindly supplied by Biokyma S.r.l., Anghiari (AR) Italy, and identified by Dr. Franco Maria Bini and vouchers of crude drugs were deposited in Department of Pharmacy and Biotechnology, University of Bologna (via Irnerio 42, Bologna, Italy) and reported in .
Table 1. Plants tested in this study, including their botanical name, family, organ/s used, voucher number, percentage of tyrosinase (Tyr IA %). and elastase inhibitory (Ela IA %) activity at 50 μg/mL, total phenolics (TPC) and flavonoids (TFC) content expressed in µg of gallic acid (GA) equivalent/mg of extract and µg of rutin (R) equivalent/mg of extract respectively.
Thirty mg of dried and powdered plant material were extracted by sonication for 30 min using 1.5 mL of EtOH/H2O (1:1). Crude extracts were obtained as reported by Chiocchio et al.Citation11.
2.2. Enzyme inhibitory assays, total phenolic and flavonoid content
The assays were performed according to the methods described by Chiocchio et al.Citation11, with slight modification for elastase inhibitory assay, where N-succinyl-Ala-Ala-Pro-Phe was used as substrate and p-phenylmethylsulfonyl fluoride (PMSF) from 1 to 250 μg/mL was used as positive control. For PMSF the assay was performed in 5% DMSO, thus, a proper negative control in the same conditions was used for the IC50 calculation.
The kinetic parameters for the enzymatic reactions in the assay conditions were KM = 0.2 mM for both enzymes and Vmax = 6 μmol/min for elastase and Vmax = 10 μmol/min for tyrosinase.
The assays for phenolic and flavonoid contents were performed in Spectrophotometer (Jasco V-530) as described by Chiocchio et al.Citation11.
2.3. Statistical analysis
Values were expressed as the mean ± SD of three independent experiments (each one performed in duplicate). Statistical analyses were performed using R Studio software (version 1.1.463) based on the R software version 3.5.2Citation12.
Samples were compared by one-way analysis of variance (ANOVA) performed with “aov” function using “stats” package, followed by Tukey’s Honestly Difference (HSD) post-hoc test using TukeyHSD function presents in “stats” packageCitation13, considering significant difference at p values < 0.05. In order to determine the correlation between total phenolic and flavonoid content and enzymatic activities, Pearson correlation coefficient (r) was evaluated with “cor.test” function using “stats” packageCitation14.
3. Results and discussion
A first bioactivity screening was carried out on extracts at the fixed concentration of 50 μg/mL. The obtained results () allowed the selection of eleven extracts, whose IA was higher than 30%, namely: Camellia sinensis Kuntze (leaves) (CCS), Ceterach officinarum D.C. (aerials parts) (COF), Cinnamomum zeylanicum Nees (barks) (CZE), Ginkgo biloba L. (leaves) (GBI), Glycyrrhiza glabra L.(roots) (GGL), Rheum officinale Baill. (rhizomes) (ROF), Rhodiola rosea L. (roots) (RRO), Ribes nigrum L. (leaves) (RNI), Salvia officinalis L. (leaves) (SOF), Tilia platyphyllos Scop. (aerial parts) (TPL) and Vitis vinifera L. (leaves) (VVI). Among them, four resulted active against both enzymes (CSI, GBI, RRO, VVI), five showed Tyr IA only (GGL, RNI, ROF, SOF, TPL), and two only Ela IA (COF, CZE).
The IC50 values of Ela IA of the six selected samples ranged from 3.1 ± 1.9 to 104.9 ± 2.1 μg/mL (), and among them, RRO and COF resulted the most potent elastase inhibitors (EI). These results are particularly promising, considering that the positive control (PMSF) used for Ela inhibitory assay showed IC50 of 42 μg/mL (241 μM). The extract of COF resulted the most potent Ela inhibitor of this screening, with an IC50 value of 26 ± 0.3 µg/mL. Regarding VVI, this study reported for the first time its activity against Ela, while its Tyr IA was already knownCitation15. Finally, this study provides new bioactivity data also for CZE, which is generally used as a food additive for its taste and scent. In particular, CZE expressed a selective Ela IA, showing an IC50 value of 104.9 ± 2.1 µg/mL, providing evidence in favour of the use of this herbal product also for skin care.
Figure 1. IC50 values of tyrosinase inhibition (A) and IC50 values of elastase inhibition (B) obtained for the most active extracts. Different letters within the same assay indicate significant differences in ANOVA test (p < 0.05). Results are expressed ad means ± SD of three independent experiments. COF: Ceterach officinarum DC.; CSI: Camellia sinensis Kuntze; CZE: Cinnamomum zeylanicum Nees; GBI: Ginkgo biloba L.; GGL: Glycyrrhiza glabra L.; RNI: Ribes nigrum L.; ROF: Rheum officinale Baill.; RRO: Rhodiola rosea L.; SOF: Salvia officinalis L.; TPL: Tilia platyphyllos Scop.; VVI: Vitis vinifera L.
Concerning Tyr IA, the IC50 values calculated for the nine active extracts ranged from 19.3 ± 1.2 to 164.3 ± 25.5 μg/mL (), and the highest IA was shown by CSI, GGL, RNI, RRO, VVI.
Flowers and bracts of TPL are widely used in southern Europe folk medicineCitation16, our results highlighted its strong Tyr IA, indicating a new potential use of this renowned herbal product as cosmetic ingredient. According to Chen et al.Citation17, RRO acetone extract exerts Tyr IA with an IC50 of 181.8 ± 11.0 µg/mL, resulting remarkably less active then the hydroalcoholic extract tested in this study (IC50 of 19.26 ± 1.16 µg/mL). This difference may be due both to the different assay conditions, particularly incubation time and types of substrate, and to the diverse extraction method employed, since solvent properties strongly affect compounds extraction and consequently the inhibitory effect of a plant extractCitation18,Citation19. The same reasons could justify also the significant difference in Tyr IA found for CSI extract by Chen et al.Citation17 (IC50 = 232.5 ± 3.3 μg/mL) and the one obtained in this study (IC50 = 66.04 ± 1.75 μg/mL).
Among the active principles contained in RRO tyrosol and salidroside are known to posses Tyr IA activityCitation20. Regarding GGL, it is traditionally and commercially used for skin whitening formulations. The pyranoisoflavan glabridin showed promising anti-tyrosinase activity on melanoma cells and anti-melanogenesis activity on B16 murine melanoma cells. Additionally, reduced pigmentation and inflammation induced by UVB on guinea-pig skins at 0.5% w/v concentrationCitation21. Licuraside, isoliquiritin and licochalcone A also showed competitive inhibition on monophenolase activity of mushroom tyrosinaseCitation22, conversely, glabrene and isoliquiritigenin can inhibit both the reactions catalysed by tyrosinaseCitation23.
C. sinensis is widely used in skin care preparations for its peculiar catechines having significant antioxidant, anti-inflammatory and UV-protection activities. Moreover, tea polyphenols, i.e. (–)-epicatechin 3-O-gallate, (–)-gallocatechin 3-O-gallate, and (–)-epigallocatechin 3-O-gallate (EGCG) are known to possess tyrosinase inhibitory potentialCitation24.
Due to its popularity as cosmetic ingredient, CSI can be considered a further positive control in this screening. On this basis COF and RRO resulted very promising Ela inhibitors, being significantly stronger than CSI. While in the case of TI, GGL, RRO, VVI, and RNI showed an activity comparable to CSI.
Considering the antioxidant properties of polyphenols and flavonoids and their reported activity against several enzymesCitation25,Citation26, the total content of these classes of metabolites was evaluated in all the selected samples (). Pearson correlation test was performed to correlate the percentage of IA (showed by extracts at 50 µg/mL) to the phenolic and flavonoids content respectively.
A moderate positive correlation was found between the IA and the total phenolic content, the highest with TI (r = 0.4965449 and p values = 6.442e-07). A similar correlation trend was found in our previous workCitation11, supporting the importance of phenolics in this biological activity. Conversely, for total flavonoid content no correlation was observed with both inhibitory activities. However, the eleven most active extracts resulted interestingly enriched in flavonoids (ranging from 1.47 to 56.47 mg RE/g of extract) and phenolics (ranging from 37.43 to 123.56 mg GAE/g of extract), which indicate also a potential activity as antioxidant agents.
5. Conclusions
Eleven extracts out of ninety resulted promisingly active against enzymes of cosmetic interest, showing IC50 values comparable or even lower than positive controls (PMSF and kojic acid). In particular, four out of them inhibited both enzymes, five tyrosinase only and two acted prominently only against elastase. For C. officinarum (aerial parts), C. zeylanicum (barks), R.nigrum (leaves), T. platyphyllos (flowers and bracts) and V. vinifera (leaves) the activity against one or both enzymes was reported for the first time, providing a new perspective for the use of these plants. Among them, C. officinarum (aerial parts) resulted one of the most potent EI, while R. nigum (leaves) and V. vinifera (leaves) showed the highest inhibitory activity against tyrosinase. The plants active against both enzymes (C. sinensis, G. biloba, R. rosea, V. vinifera) are potentially useful to develop cosmetics endowed with both skin-whitening and anti-wrinkles effect. The five plants active against tyrosinase only (G. glabra, R. nigrum, R. officinalis, S. officinalis, T. platyphyllos) are suitable for skin whitening agents, and the two active only against elastase (C. officinarum and C. zeylanicum) are interesting for selective anti-wrinkles cosmetics. Moreover, these plants resulted also enriched in polyphenols and flavonoids, conferring them additional antioxidant properties relevant for cosmetic ingredients and the total phenolic content showed a linear correlation with the enzymatic inhibitory activities.
Further biological and phytochemical studies are ongoing on the selected plants in order to identify the metabolites responsible for the observed biological activities.
Acknowledgments
Thanks are due to Biokyma s.r.l. (Località Mocaia 44/b 52031 Anghiari (AR), Italy) for kindly providing the herbal products analysed in this work.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- Dorni AC, Amalraj A, Gopi S, et al. Novel cosmeceuticals from plants. An industry guided review. J Appl Res Med Aromat Plants 2017;7:1–26.
- Feetham HJ, Jeong HS, McKesey J, et al. Skin care and cosmeceuticals: attitudes and trends among trainees and educators. J Cosmet Dermatol 2018;17:220–6.
- Mukherjee PK, Maity N, Nema NK, Sarkar BK. Bioactive compounds from natural resources against skin aging. Phytomedicine 2011;19:64–73.
- Roy A, Sahu RK, Matlam M, et al. In vitro techniques to assess the proficiency of skin care cosmetic formulations. Pharmacogn Rev 2013;7:97–106.
- Cefali LC, Ataide JA, Moriel P, et al. Plant-based active photoprotectants for sunscreens. Int J Cosmet Sci 2016;38:346–53.
- Labat-Robert J, Fourtanier A, Boyer-Lafargue B, Robert L. Age dependent increase of elastase type protease activity in mouse skin. Effect of UV-irradiation. J. Photochem. Photobiol. B, Biol 2000;57:113–8.
- Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci 2008;30:87–95.
- Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84:1155–228.
- Lange D, Europe’s medicinal and aromatic plants: their use, trade and conservation. Cambridge (UK): TRAFFIC International; 1998.
- Weiss RF, Fintelmann V, Herbal medicine. Stuttgart (GER): Thieme; 2000.
- Chiocchio I, Mandrone M, Sanna C, et al. Screening of a hundred plant extracts as tyrosinase and elastase inhibitors, two enzymatic targets of cosmetic interest. Ind Crops Prod 2018;122:498–505.
- R Core Team. 2018. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. Available from: https://www.R-project.org
- Yandell BS, Practical Data Analysis for Designed Experiments. London (UK): Chapman & Hall/CRC; 1997.
- Best DJ, Roberts DE. Algorithm AS 89: the upper tail probabilities of Spearman’s rho. J R Stat Soc C-Appl 1975;24:377–9.
- Lin YS, Chen HJ, Huang JP, et al. Kinetics of tyrosinase inhibitory activity using Vitis vinifera leaf extracts. Biomed Res Int 2017;2017:5232680.
- Wichtl M. Tiliae flos. In: Wichtl, M., ed., Herbal drugs and phytopharmaceuticals: a handbook for practice on a scientific basis. 3rd expanded and completely rev. ed. Stuttgart (GER), Medpharm; Boca Raton, FL: CRC Press; 2004.
- Chen CH, Chan HC, Chu YT, et al. Antioxidant activity of some plant extracts towards xanthine oxidase, lipoxygenase and tyrosinase. Molecules 2009;14:2947–58.
- Alasbahi R, Melzig M. The in vitro inhibition of human neutrophil elastase activity by some Yemeni medicinal plants. Sci Pharm 2008;76:471–84.
- Alam N, Yoon KN, Lee KR, et al. Antioxidant activities and tyrosinase inhibitory effects of different extracts from Pleurotus ostreatus fruiting bodies. Microbiology 2010;38:295–301.
- Chiang HM, Chien YC, Wu CH, et al. Hydroalcoholic extract of Rhodiola rosea L. (Crassulaceae) and its hydrolysate inhibit melanogenesis in B16F0 cells by regulating the CREB/MITF/tyrosinase pathway. Food Chem Toxicol 2014;65:129–39.
- Yokota T, Nishio H, Kubota Y, Mizoguchi M. The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation. Pigment Cell Res 1998;11:355–61.
- Nerya O, Vaya J, Musa R, et al. Glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots. J Agric Food Chem 2003;51:1201–7.
- Fu B, Li H, Wang X, et al. Isolation and identification of flavonoids in licorice and a study of their inhibitory effects on tyrosinase. J Agric Food Chem 2005;53:7408–14.
- Mukherjee PK, Biswas R, Sharma A, et al. Validation of medicinal herbs for anti-tyrosinase potential. J Herb Med 2018;14:1–16.
- Kacem R. Phenolic compounds from medicinal plants as Natural anti-elastase products for the therapy of pulmonary emphysema. J Med Plants Res 2013;7:3499–507.
- Pillaiyar T, Manickam M, Namasivayam V. Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J Enzyme Inhib Med Chem 2017;32:403–25.