Antioxidant And Anti-Skin Aging Potential Of Selected Thai Plants: In Vitro Evaluation And In Silico Target Prediction Part 1

Abstract: The skin is the largest organ that performs a variety of the body’s essential functions. Impairment of skin structure and functions during the aging process might severely impact our health and well-being. Extensive evidence suggests that reactive oxygen species play a fundamental role in skin aging through the activation of the related degradative enzymes. Here, the 16 Thai medicinal plant species were screened for their potential anti-skin aging properties. All extracts were investigated for total phenolic and flavonoid contents, antioxidant, anti-elastase, and antityrosinase activities, as well as the binding ability of compounds with target enzymes by molecular docking. Among all the plants screened, the leaves of A. occidentale and G. zeylanicum exhibited strong antioxidants and inhibition against elastase and tyrosinase. Other potential plants include S. alata leaf and A. catechu fruit, with relatively high anti-elastase and anti-tyrosinase activities, respectively. These results are also consistent with docking studies of compounds derived from these plants. The inhibitory actions were found to be more highly positively correlated with phenolics than flavonoids. Taken together, our findings reveal some Thai plants, along with candidate compounds as natural sources of antioxidants and potent inhibitors of elastase and tyrosinase, could be developed as promising and effective agents for skin aging therapy.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a robust scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】

Keywords: phytomedicine; anti-aging; antioxidant; anti-elastase; anti-tyrosinase; proanthocyanidin; Areca catechu; Anacardium occidentale; Glochidion zeylanicum; Senna alata

1. Introduction 

Skin is the largest and most complex organ in the human body. The skin serves as a barrier between the body and the outside environment, and it serves a variety of functions [1]. It has a significant cosmetic role in addition to protecting the body from water loss and microbial infection [2]. In addition, it works to support other body parts, such as the immune, nervous, and endocrine systems [1]. The look of youth and beauty may have a  positive impact on people’s social behavior and human life [1,2]. Hence, the impairment of skin structure and functions that occur as we age might have a severe impact on our health and well-being [2,3]. Thinness, dryness, lack of elasticity, rough texture, wrinkles, and dark pigments are all common characteristics of older skin [4]. Many researchers are currently working on generating potential anti-aging drugs or chemicals, particularly those derived from natural sources for skin aging treatment.

In general, human skin ages in two ways: internally (as a result of chronological aging)  and extrinsically (as a result of environmental variables influenced by environmental factors) [5]. Extensive evidence suggests that oxidative stress, through the formation of reactive oxygen species (ROS), plays a fundamental role in both intrinsic and extrinsic skin aging [6]. ROS causes oxidative damage to skin cells by damaging essential macromolecules such as nucleic acids, enzymatic proteins, and membrane lipids, resulting in cellular malfunction and cell death [7]. Oxidative stress also contributes to the degradation of the extracellular matrix (ECM) by suppressing ECM component synthesis (e.g., elastin)  and activating ECM degrading enzymes (e.g., elastase), which results in loss of skin elasticity [2,8,9]. Moreover, ROS can cause irregular or dark colors in the skin by inducing the production of an α-melanocyte-stimulating hormone (α-MSH) in keratinocytes, thereby triggering the activation of the tyrosinase enzyme and promoting melanin synthesis in melanocytes [10,11]. Therefore, scavenging ROS and inhibiting elastase and tyrosinase activities could be useful in the treatment or even prevention of skin aging.

Natural products are currently receiving much interest as potential alternative medicines for treating several diseases as well as aging and age-associated declines [8,12,13]. Tropical plants could be of interest to explore their potential in skin aging treatments. As reported previously, several Andean and Himalayan plants have been regarded as sources of compounds with potential use as anti-aging ingredients [14,15]. Thailand is a known place for cultivating a wide variety of tropical plants, many of which have not been studied extensively. The goal of this research was to find potential natural sources for developing novel treatments against skin aging. The extracts of 16 Thai medicinal plant species were studied in vitro for their properties related to anti-skin aging, including total phenolic and flavonoid contents, free radical scavenging, anti-elastase, and anti-tyrosinase activities. We also further performed correlation analysis and an in silico molecular docking approach to reveal the promising phytochemical compounds in the three most effective plants with strong inhibition against elastase or tyrosinase enzyme.

2. Results 

2.1. Extraction Yields 

Table 1 shows the scientific name, parts used, source, extraction method/solvent, and percent yield of Thai plants used in this study. The percent yields of the extracts ranged from 2.0% to 36.3% (Table 1). C. calendars had the highest extraction yield (36.3%), followed by M. caldera (18.2%) and A. occidentale (16.0%), whereas H. undatus had the lowest yield (2.0%).

2.2. Total Phenolic Content of Thai Plants

The plant extracts showed a variety of total phenolic content ranging from 14.06 ± 1.55  to 320.14 ± 7.95 mg of gallic acid equivalent (GAE) per g dry weight extract (Table 2). G. zeylanicum (320.14 ± 7.95 mg GAE per g dry weight extract) had the highest phenolic content in all extracts, followed by A. catechu (295.79 ± 11.97 mg GAE per g dry weight extract) and M. caldera (210.99 ± 10.40 mg of GAE per g dry weight extract), respectively. The lowest level was found in C. calendars at 14.06 ± 1.55 mg GAE per g dry weight extract.

2.3. Total Flavonoid Content of Thai Plants

The total flavonoid content of plant extracts varied among the plant species, ranging from 2.61 ± 0.42 to 84.48 ± 18.32 mg of quercetin equivalent (QE) per g dry weight extract (Table 2). Of all extracts, the highest flavonoid content was found in M. caldera (84.48 ± 18.32 mg of QE per g dry weight extract), followed by G. zeylanicum (52.54 ± 7.25 mg of QE per g dry weight extract) and P. dulce (28.01 ± 4.39 mg of QE per g dry weight extract), respectively. On the other hand, the lowest level was found in E. Americana at 2.61 ± 0.42 mg of QE per g dry weight extract.

2.4. DPPH Radical Scavenging Activity of Thai Plants

The DPPH assay is based on the hydrogen-donating capacity of the compound to scavenge the stable DPPH radicals [16]. The antioxidant capacities of plant extracts were expressed as the percent DPPH radical scavenging activity, the mg of vitamin C equivalent antioxidant capacity (VCEAC) per g dry weight extract, and the half-maximal inhibitory concentration (IC50) (Table 3). At 0.1 mg/mL of extracts, the percentages of DPPH scavenging activity ranged from 6.40 to 93.18%. Four plant extracts: G. zeylanicum (93.18%), M. caldera (90.61%), A. occidentale (89.01%), and A. catechu (88.12%), exhibited DPPH  scavenging activity greater than 80%. In contrast, C. ternatea showed the lowest scavenging activity at 6.40%. This rank order was the same when compared based on the relative VCEAC values. However, according to the IC50 values, the antioxidant capacity of the top four plant extracts was changed in the following order: G. zeylanicum > A. catechu > A. occidentale > M. caldera. The obtained results of the DPPH assay show that G. zeylanicum exhibited the strongest antioxidant potential with the highest percentage of scavenging activity and the lowest IC50 value.

2.5. ABTS Radical Scavenging Activity of Thai Plants

The ABTS assay is based on the compound’s ability to transfer hydrogen atoms for neutralizing a stable ABTS radical cation. The antioxidant capacities of plant extracts were expressed as the percent ABTS radical scavenging activity, the mg of VCEAC per g dry weight extract, and the IC50 (Table 4). At 0.1 mg/mL of extracts, the percentages of ABTS  scavenging activity ranged from 13.93% to 99.37%. Eight plant extracts: G. zeylanicum (99.37%), A. catechu (99.31%), A. occidentale (99.23%), M. caldera (98.88%), E. Americana (95.62%), Z. officinale (94.07%), P. dulce (83.99%), and H. undatus (82.16%) exhibited the ABTS  scavenging activity greater than 80%. In contrast, C. calendars showed the lowest scavenging activity at 13.93%. The antioxidant capacity of the top four plant extracts, according to the VCEAC, and IC50 values were found in a similar rank order as follows: G. zeylanicum > A. catechu > A. occidentale > M. caldera. Remarkably, the results of the ABTS assay also showed that the extract with the strongest antioxidant activity was G. zeylanicum leaf, as represented by the highest percentage of scavenging activity and the lowest IC50 value.

2.6. Anti-Elastase Activity of Thai Plants

The elastase inhibitory activity of plant extracts was evaluated using the elastase inhibition assay with N-succinyl-trialanyl-paranitroanilide (SANA) as the substrate. Epigallocatechin gallate (EGCG) (0.1 mg/mL), which was used as a positive control, showed an inhibition level of 45.27%. The elastase inhibitory activities of the extracts are presented in Table 5 (see also Table S1). At 0.5 mg/mL of extracts, the percentages of elastase inhibition ranged from 1.33% to 88.31%. A. catechu had the highest elastase inhibitory effect at 88.31%,  followed by G. zeylanicum (87.43%), A. occidentale (84.78%), and S. alata (73.95%), while S. asper bark had the lowest elastase inhibitory effect at 1.33%. Due to high background absorbance, some plant extracts with no detectable activity were reassayed at 0.1 mg/mL. It was found that M. caldera, P. nigrum, Z. cassumunar, and S. asper leaf, except Z. officinale,  showed low to moderate inhibitory activities ranging from 3.46% to 35.74%. Nevertheless,  the effects of C. calendars, C. asiatica, C. ternatea, E. americana, and H. undatus were not observed even at higher concentrations.

2.7. Anti-Tyrosinase Activity of Thai Plants

Tyrosinase inhibitory activity of plant extracts was evaluated using the dopachrome method with 3,4-dihydroxy-L-phenylalanine (L-DOPA) as the substrate. Kojic acid (KA) (0.02 mg/mL), a positive control, showed an inhibition level of 68.35%. The tyrosinase inhibitory activity of the extracts is presented in Table 6 (see also Table S2). At 1 mg/mL of extracts, the percentages of tyrosinase inhibition ranged from 4.80% to 91.51%. G. zeylanicum had the highest tyrosinase inhibitory effect at 91.51%, followed by A. occidentale (81.01%), M. caldera (76.12%), and A. catechu (75.38%), whereas P. nigrum had the lowest tyrosinase inhibitory effect at 4.80%. Two plant extracts with no detectable activity due to high background absorbance were reassayed at 0.1 mg/mL, and the activities were found at 3.98% (Z. cassumunar) and 21.28% (Z. officinale). However, P. dulce did not exhibit any effect,  even at increased concentration.

2.8. Correlation Analysis

Considering several previous reports of potential elastase and tyrosinase inhibitory action of plant extracts [8,16,17], it was likely that the extracts possess a high antioxidant potential and tend to have strong elastase and tyrosinase inhibitory action. Thus to verify those relationships in this study, we further performed Pearson’s correlation analysis to investigate the relationship between the inhibitory activities of both enzymes and the level of antioxidant contents as well as antioxidant capacities among the extracts used. The strength of the correlation is distributed by correlation coefficient (r-value) as follows: r = 0.910  to 1.000 indicate a very strong correlation, r = 0.710 to 0.900 indicate a high correlation,  r = 0.410 to 0.700 indicate a moderate correlation, r = 0.210 to 0.400 indicate a small correlation, and r = 0.000 to 0.200 indicate a slight correlation [18]. The elastase inhibition had a  high positive correlation to total phenolic content, DPPH, and ABTS radical scavenging activities, as shown in Figures 1a,c, and d, respectively. Similarly, the tyrosinase inhibition showed a very strong positive correlation to total phenolic content and ABTS radical scavenging activity (Figure 2a,d), while it showed a high positive correlation to DPPH radical scavenging activity (Figure 2c). However, the total flavonoid content was moderately positively correlated to both elastase and tyrosinase inhibition (Figures 1b and 2b). These results demonstrated that phenolic compounds and antioxidant activity might have a significant contribution to the inhibition of elastase and tyrosinase enzymes.

2.9. Molecular Docking

We next evaluated the abilities of phytochemical compounds in the three most effective plants with strong inhibition against elastase or tyrosinase enzymes. Molecular docking is generally used to predict the binding affinity of compounds to protein receptors or enzymes compared to known inhibitors. According to our results of in vitro screening assays, we found that A. occidentale was the most potent elastase inhibitor, followed by G. zeylanicum and S. alata, in rank order of IC50 values. Thus, we selected phytochemical compounds derived from these three plants to evaluate their ability to inhibit elastase. The results of interactions between elastase (3HGP) and compounds are presented in Table S4 as binding energy, inhibition constant, the number of hydrogen bonds, amino acid interaction, and bond length. The binding energy between 3HGP and compounds shows scores ranging from −2.26 to −11.95 kcal/mol (Table S4). EGCG, a positive control, showed the binding energy at −9.69 kcal/mol. According to the docking results, five compounds showed lower binding energy than the positive control. Tetramer of proanthocyanidin exhibited the lowest binding energy (−11.95 kcal/mol), which indicated that it has the best affinity compared to other compounds in elastase inhibition, followed by amentoflavone, rutin,  agathisflavone, and kaempferol 3-O-gentiobioside, with the binding energies at −11.81, −10.12, −9.92, and −9.73, respectively.

In addition, the rank order of IC50 values for plant extracts with tyrosinase inhibition was closely similar to elastase inhibition. We found that G. zeylanicum possessed the highest inhibitory effect, followed by A. catechu, and A. occidentale. Hence, the compounds derived from these three plants were selected to investigate their ability to inhibit tyrosinase. The results of interactions between tyrosinase (2Y9X) and compounds are presented in Table S5 as binding energy, inhibition constant, the number of hydrogen bonds, amino acid interaction, and bond length. The binding energy between 2Y9X and compounds showed scores ranging from −4.56 to −10.42 kcal/mol (Table S5). KA, a well-known inhibitor of tyrosinase, showed the binding energy at −4.59 kcal/mol. Based on the docking results,  o-coumaric acid (−10.42 kcal/mol) and tetramer of proanthocyanidin (−10.42 kcal/mol) exhibited the lowest binding energy against elastase when compared to other compounds,  followed by caffeic acid, ferulic acid, and areca tannin A1 with the binding energies at −10.10, −10.00, and −9.94, respectively. Figures 3 and 4 represent the 2D diagrams of ligand–protein interactions for elastase and tyrosinase, for the positive control, and five compounds with the lowest binding energy.

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】