Articles Service
Original
Inhibitory Effects of Vernonia amygdalina Leaf Extracts on Free Radical Scavenging, Tyrosinase, and Amylase Activities
1Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathum Thani 12120, Thailand
2Programme of Agricultural Business and 3Programme of Veterinary Technology and Veterinary Nursing, Faculty of Agricultural Technology, Rajabhat Maha Sarakham University, Maha Sarakham 44000, Thailand
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Prev Nutr Food Sci 2023; 28(3): 302-311
Published September 30, 2023 https://doi.org/10.3746/pnf.2023.28.3.302
Copyright © The Korean Society of Food Science and Nutrition.
Abstract
Keywords
INTRODUCTION
Wrinkles, melasma, freckles, and spots are common skin issues, particularly in aging individuals. These problems are influenced by two main factors: intrinsic factors, such as hormones and oxidative damage, and extrinsic factors, such as pollution, alcohol consumption, smoking, and sunlight exposure (Brincat, 2000; Makrantonaki et al., 2006). These factors contribute to the generation of free radicals in skin cells. Sunlight, a natural energy source of ultraviolet (UV) radiation, plays a crucial role in this process. UV radiation consists of UVA, UVB, and UVC rays, which induce the production of reactive oxygen species (ROS) and stimulate melanin synthesis. ROS can cause oxidative stress and damage various biomolecules, especially the plasma membrane of cells, leading to the loss of skin strength and flexibility and the formation of wrinkles.
Additionally, ROS can enhance the activity of enzymes such as collagenase, elastase, and hyaluronidase, which contribute to skin aging and wrinkle formation. Melasma, a skin condition characterized by pigmentation on the epidermis, is influenced by tyrosinase, an enzyme that controls melanocyte function (Fisher et al., 2002; Maity et al., 2011).
The cosmetic industry uses various whitening agents, including hydroquinone, butylated hydroxytoluene, kojic acid, ascorbic acid, and alpha-arbutin. However, hydroquinone, a skin-lightening compound, has been associated with mutagenic effects, adverse reactions, and skin irritation when used in high doses (Sarkar et al., 2012); hence, strict regulations govern its usage. Kojic acid inhibits tyrosinase but poses challenges owing to its cytotoxicity and instability. Ascorbic acid shows potential as an antityrosinase compound but is prone to decomposition under high temperatures and UV radiation (Bae et al., 2013). The use of pure compounds as cosmetic ingredients increases product value but comes with higher costs. To address these issues, the cosmetic industry incorporates extracts from plants, animals, and marine organisms into their products.
In addition to avoiding external factors that contribute to skin issues, two primary goals are targeted: preventing free radical generation through antioxidants and inhibiting enzymes that cause cellular degradation, including tyrosinase, elastase, hyaluronidase, and collagenase. Natural extracts containing potent antiwrinkle properties are key components of antiaging products. For example, red grape (
The present study focuses on studying the biological activities of
MATERIALS AND METHODS
Extraction of V. amygdalina leaves
The extraction method was adapted from the method of Ekaluo et al. (2015).
-
Figure 1. Schematic diagram depicting the process of
Vernonia amygdalina leaf extraction.
Two groups of extraction solvents were prepared. In group A, 70% (v/v) methanol (BTL70ME), 70% (v/v) ethanol (BTL70ET), and 70% (v/v) acetone (BTL70AC) were used to extract
The mixture in each bottle were stirred at 22
-
Table 1 . The IC50 values of the extracted samples for antityrosinase and antioxidant activities
Sample groups Extracted solvent ID name IC50 value (μg/mL) Antityrosinase activity Antioxidant activity Group A 70% (v/v) methanol BTL70ME 25 16 70% (v/v) ethanol BTL70ET 125 37 70% (v/v) acetone BTL70AC 20 20 Group B Hexane BTL_Hexane >1,000 >160 Ethyl acetate BTL_Ethyl acetate 1,000 9 Isopropanol BTL_Isopropanol 720 4 Ethanol BTL_Ethanol 360 4 Control Ascorbic acid 11 2 IC50, half-maximal inhibitory concentration.
Determination of tyrosinase inhibition property using a modified dopachrome method
The tyrosinase inhibitory effect of the extracts was assessed following the procedure outlined by Patathananone et al. (2019). Each extract powder was dissolved in 5% (v/v) dimethyl sulfoxide (DMSO). Subsequently, 10 μL of various concentrations of each extract were mixed with 100 μL of a 250 unit/mL tyrosinase solution [250 unit/mL tyrosinase in 20 mM phosphate buffer (pH 6.8)] in a 96-well plate. Next, 70 μL of deionized (DI) water was gently added to each well. The mixtures were incubated at 37°C for 10 min, after which 20 μL of 20 mM L-Dopa (HiMedia Laboratories Pvt. Ltd.) was added. The plate was shaken and further incubated at 37°C for 20 min. UV-visible (UV-Vis) absorption measurements were performed at 495 nm to determine the percentage of tyrosinase inhibition.
Antiamylase activity assay
α-Amylase is an enzyme that breaks down starch by hydrolyzing α-(1-4)-glycosidic bonds. The resulting products include short-chain oligosaccharides, trisaccharides, maltose, and glucose. The level of product formation is influenced by several factors, such as enzyme concentration, substrate concentration, pH, and temperature. Inhibition of α-amylase activity is important for controlling sugar production during starch digestion, especially for individuals with hyperglycemia or diabetes. The assessment of reducing sugar products resulting from amylase activity was conducted using a modified version of the method described by Prasathkumar et al. (2021). Reducing sugars react with 3,5-dinitrosalicylic acid (DNS) at 100°C, producing 3-amino-5-nitrosalicylic acid, which exhibits a red-brown color. The absorbance value at a wavelength of 540 nm was measured to quantify the amount of reduced sugar, using the glucose standard curve for the DNS method.
In the experiment, 40 μL of 0.1% (w/v) α-amylase (HiMedia Laboratories Pvt. Ltd.) in 50 mM phosphate buffer at pH 6.8 was mixed with 20 μL of 20 mg/mL of each sample and incubated at 37°C for 10 min. A cuvette containing 1.8 mL of 1.0% w/v starch solution was prepared, and the optical density at a wavelength of 600 nm was measured using a UV-Vis spectrophotometer (UV-Vis-1200, MAPADA). The incubated α-amylase was then added to the starch solution, rapidly mixed, and the optical density at 600 nm (OD600 nm) was recorded every 30 s for 5 min. The reactions were halted through incubation at 100°C for 10 min, followed by a 10 min cooling period in water. Subsequently, 2 mL of 0.01 M DNS solution was added to each reaction, mixed, and boiled at 100°C for 10 min. The absorbance values of each reaction were measured at 540 nm. The quantity of reducing sugar products under each condition was determined using the standard curve equation: y=0.4023x+0.0379 (R2=0.9989).
Determination of antioxidant properties using the DPPH assay
The antioxidant activity of the extracts was assessed and calculated using a modified version of the method reported by Patathananone et al. (2019). The reaction took place in a 96-well plate, where 10 μL of the extracted samples at different concentrations were added to 190 μL of 0.101 M DPPH solution (0.101 mM DPPH in methanol). The mixture was gently mixed, and incubated at 37°C for 30, 45, and 60 min. Ascorbic acid and 5% (v/v) DMSO served as the positive and negative controls, respectively. The absorbance values were measured at 515 nm (Piao et al., 2002; Theansungnoen et al., 2014; Ekaluo et al., 2015) using a microplate reader (EZ-Read 2000, Biochrom).
Determination of total phenolic contents using Folin-Ciocâlteu reagent
The total phenolic content in the extracts was analyzed using a modified experiment based on the method of Dewanto et al. (2002). Gallic acid, dissolved in methanol, was used as the standard agent. Standard gallic acid solutions were prepared at initial concentrations of 0.2, 0.4, 0.6, 0.8, and 1 μg/mL. For the reaction, 3.5 mL of 2.0% (w/v) sodium carbonate solution was mixed with 100 μL of different concentrations of the extracts (BTL70ME, BTL70ET, and BTL70AC), as well as the gallic acid solutions, in test tubes. Subsequently, 400 μL of 10-fold diluted Folin-Ciocâlteu solution was added to each tube. The mixtures were incubated in the dark at room temperature for 30 min. UV-Vis measurements were taken at 750 nm, using a modified version of the method described by Dewanto et al. (2002), to create a standard curve. Sample measurements were taken under the same conditions to determine total phenolic content.
Determination of total flavonoid content using aluminum chloride reagent
To assess the total flavonoid content in the group A extracts, standard quercetin solutions were prepared at different concentrations (20, 40, 60, 80, and 100 μg/mL). A typical mixture was created in a 10 mL volumetric flask by sequentially adding 1.0 mL of the sample solution, 4 mL of distilled water, and 0.3 mL of 5% (w/v) of sodium nitrite solution. The mixture was left at room temperature for 5 min, after which 0.3 mL of 10% (w/v) aluminum chloride solution was added. Following a 6 min incubation period, 0.2 mL of 1 M sodium hydroxide (NaOH) was added, and the total volume of the mixture was adjusted to 10 mL using distilled water. UV-Vis measurements were taken at 510 nm, using a modified version of the method described by Zhishen et al. (1999).
Preliminary analysis of bioactive compounds
The methods for phytochemical screening in the extracts were mini modified according to the report of Usunobun and Ngozi (2016).
Circular dichroism (CD) spectroscopy analysis
The conformational changes of tyrosinase were determined using CD spectroscopy (Jasco J-815 CD Spectrometer, Analytical Lab Science Co., Ltd.). Four solutions were prepared to analyze the CD spectra: (1) 5 μg/mL tyrosinase (E) in 50 mM phosphate buffer (pH 6.8), (2) 125 unit/mL tyrosinase+5 mM L-Dopa (E+S), (3) 5 μg/mL tyrosinase+0.7 mg/mL BTL70ME, and (4) 5 μg/mL tyrosinase+0.7 mg/mL BTL70ME+5 mM L-Dopa (E+I+S). The spectra were analyzed in the 176∼260 nm range for all test conditions. A phosphate buffer was used as the buffer blank.
Statistical analysis
One-way ANOVA was used for the statistical analysis (IBM SPSS Statistics version 29, IBM Corp.), as previously explained by Patathananone et al. (2019). Each biological characteristic’s proportion (means±SD) was used to represent the data. Statistics were computed within the same extracted group and compared to equivalent concentrations for the results depicted in Fig. 2 and 4. At a
-
Figure 2. Antityrosinase activity of the extracts in both groups. (A) Group A and (B) group B. The data are presented as means±SD (n=3). The statistical information was analyzed using one-way ANOVA. When the
P <0.05, the letters (a-d) showed statistically significant differences within the same concentration.
-
Figure 3. Results of antiamylase activity tests. (A) Decrease in optical density at 600 nm of starch solution after incubation with buffer, α-amylase, and the group A and B extracts for 5 min. (B) Quantification of reducing sugar products corresponding to α-amylase activity under each condition. Using one-way ANOVA, the absorbance values of the control and all test groups were compared. *Statistical significance was represented as a
P <0.05. BTL_He, BTL_Hexane; BTL_EA, BTL_Ethyl acetate; BTL_Iso, BTL_Isopropanol; BTL_ET, BTL_Ethanol.
-
Figure 4. The antioxidant potential of both extracted groups: (A) group A and (B) group B. The data are presented as means±SD. The letters (a-d) indicated significance different statistically within the same concentration at
P <0.05.
RESULTS
Tyrosinase-inhibiting properties of extracts
The tyrosinase-inhibiting properties of
Effect of V. amygdalina leaf extracts on α-amylase
The results of the antiamylase activity experiment are presented in Fig. 3. The rate of change in OD600 nm for 50 mM phosphate buffer (pH 6.8), BTL_Hexane, BTL_Ethyl acetate, BTL_Isopropanol, BTL_Ethanol, BTL70AC, BTL70ME, and BTL70ET was 5.2, 2.7, 4.3, 4.5, 2.7, 3.8, 4.7, and 3.8×10−3 unit/s, respectively, and the amount of reducing sugar products under these conditions were 1.41±0.003, 0.94±0.056, 0.46±0.090, 0.57±0.040, 0.85±0.090, 0.43±0.020, 0.58±0.060, and 0.79±0.090 mM, respectively. The number of products observed under treated conditions was lower compared with the control, indicating a decrease in amylase activity. These findings suggest that the bioactive compounds present in
Antioxidant properties of extracts
Antioxidants can decrease oxidative stress and oxidative damage. Primary antioxidants show the mechanism of action as electron donors. This reaction is the beginning step to inhibiting the chain reaction of dangerous free radicals. The antioxidant screening ability of both extracted groups was performed by DPPH assay. The phytochemicals display antioxidant properties by donating the electrons to DPPH (•) radicals, and then the purple color is decreased or changed to a yellow color solution. The colorimetric reaction is determined using the spectrophotometric technique. The results are presented in Fig. 4. The extracts of both groups represented antioxidant activity as dose-dependent. The IC50 values of BTL70ME, BTL70ET, and BTL70AC were 16, 37, and 20 μg/mL, respectively. These results differ from those of the group B extracts. The IC50 value of BTL_Hexane was >160 μg/mL, whereas the IC50 values of BTL_Ethyl acetate, BTL_Isopropanol, and BTL_Ethanol were 9, 4, and 4 μg/mL, respectively. The IC50 values of all extracts are summarized in Table 1. Overall, the data suggest that the group A extraction process should be used to obtain bioactive agents with high antioxidant efficacy.
Total phenolic content of group A extracts
The group A extracts exhibited antityrosinase and antioxidant properties. Previous studies have highlighted the crucial role of phenolic content in plant extracts for expressing durable biological properties. Therefore, the total phenolic content of the group A extracts was determined using Folin-Ciocâlteu reagent with gallic acid as the standard. To calculate the total phenolic content in each sample, the following equation was used: y=0.0157x−0.0068 (R2=0.9984). The summarized total phenolic content results are presented in Table 2. BTL70ME exhibited higher total phenolic content compared with BTL70AC and BTL70ET, although the differences were not statistically significant.
-
Table 2 . Total phenolic and flavonoid contents of the group A extracts
ID name Total phenolic contents (mg gallic/g extract) Total flavonoid contents (mg quercetin/g extract) BTL70ME 72.29±14.14 53.04±5.22 BTL70ET 65.98±11.91 44.35±13.17 BTL70AC 69.37±7.72 61.74±13.17
Total flavonoid contents of group A extracts
Flavonoids are a group of phytochemicals known to possess biological activities and efficacy. In this study, the total flavonoid content of the group A extracts was analyzed using quercetin as the standard. The standard equation y=0.0023x+0.0002 (R2=0.9943) was used to determine the total flavonoid content in each sample, and the results are presented in Table 2. BTL70AC exhibited higher total flavonoid content compared with BTL70ME and BTL70ET. Therefore, flavonoids may play an essential role in inhibiting tyrosinase and reducing the number of free radicals in BTL70AC extract.
Preliminary analysis of bioactive compounds
A variety of experiments were conducted to determine the types of phytochemicals present in group A and group B extracts, and the results are presented in Table 3. The group A extracts contained a similar range of phytochemicals, although coumarins, and cardiac glycosides were not detected in BTL70AC. These findings suggest that these specific phytochemical types contribute to potential antityrosinase and antioxidant activities. It is possible that certain phytochemicals in group A exhibit a synergistic effect that enhances their biological properties. In contrast, the extraction process used for group B separates different types of phytochemicals, which may reduce their potential biological activities. These results support the hypothesis that bioactive compounds in the group A extracts function together synergistically.
-
Table 3 . Preliminary phytochemical compounds analysis of
Vernonia amygdalina leaf extracts in groups A and BBioactive compounds Preliminary analysis BTL70AC BTL70ET BTL70ME BTL_He BTL_EA BTL_Iso BTL_ET Phenols/phenolics + + + – – + + Flavonoids + + + – – + + Coumarins – + + – – – – Saponins + + + + – + + Tannins – – – – – + – Terpenoids + + + – + + + Steroids + + + + + + + Cardiac glycosides – + + – – + – “+” indicates a positive test result.
BTL_He, BTL_Hexane; BTL_EA, BTL_Ethyl acetate; BTL_Iso, BTL_Isopropanol; BTL_ET, BTL_Ethanol.
CD spectroscopy of tyrosinase
The secondary structure of the tyrosinase enzyme [125 unit/mL tyrosinase in 50 mM phosphate buffer (pH 6.8)] was analyzed using CD spectroscopy, and the results are summarized in Table 4. The percentages of helix, beta-sheet, turn, and other structures in the tyrosinase enzyme were determined to be 11.79, 30.75, 14.97, and 42.45%, respectively. After incubating the enzyme with the substrate (L-Dopa), the percentage of helix structure increased, which is consistent with the induced fit hypothesis. However, the percentages of helix and β-sheet structures in tyrosinase treated with BTL70ME differed from the current conditions (tyrosinase+L-Dopa). The presence of phytochemicals may interfere with the binding ability between tyrosinase and the substrate, leading to a decrease in catalytic properties.
-
Table 4 . Percentage of tyrosinase secondary structures measured using circular dichroism spectroscopy
Conditions Percentage of secondary structures (%) Helix Beta sheet Turn Others 125.0 unit/mL tyrosinase enzyme in 50 mM phosphate buffer (pH 6.8) 11.79 30.75 14.97 42.45 125.0 unit/mL tyrosinase enzyme+5.0 mM L-Dopa 12.02 30.47 14.97 42.49 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME 11.40 31.25 14.93 42.33 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME+5 mM L-Dopa 11.25 31.38 14.93 42.39
DISCUSSION
Cell degeneration results from intrinsic and extrinsic factors that induce oxidation reactions, generating destructive free radicals within cells. Enzymes can also contribute to cell degeneration by damaging protein structures. Skin pigmentation enzymes, such as those associated with melasma, freckles, spots, deep and shallow wrinkles, and specific diseases, are particularly relevant. To address these issues, two primary goals have emerged: (1) inhibiting related enzymes using inhibitors from different sources, especially natural sources such as plants (Brincat, 2000; Fisher, 2002; Makrantonaki et al., 2006); (2) countering the destructive effects of free radicals through the use of antioxidants. Numerous studies have focused on bioactive compounds, with particular emphasis on plant-derived sources.
In the present study,
Tyrosinase, a key enzyme involved in melanin synthesis, is influenced by both intrinsic and extrinsic factors. Its activity is closely linked to hyperpigmentation (Kim et al., 2002), melasma, and disorders such as Parkinson’s disease. Therefore, tyrosinase inhibitors have been investigated to develop solutions for these issues. In the present study,
Tyrosinase enzyme inhibitors can be classified into two types based on their characteristics. The first type consists of specific tyrosinase inactivators known as suicide substrates. These inhibitors form covalent bonds with the tyrosinase enzyme, leading to denaturation of the enzyme and dysfunction. The second type includes specific tyrosinase inhibitors that bind to the enzyme structure, reducing its activity (Bae et al., 2013). Tyrosinase is a copper (Cu)-containing enzyme. For example, mushroom tyrosinase (EC. 1.14.18.1) is a conjugated enzyme with an active site containing two Cu atoms. Tyrosinase-Cu2+ exhibits catalytic properties (a holoenzyme), whereas tyrosinase lacking Cu2+ exhibits a nonenzymatic function (an apoenzyme). Thus, the presence, and location of Cu ions affect the enzyme’s activity. Three isoforms of Cu-tyrosinase complexes exist: oxy-tyrosinase, met-tyrosinase, and deoxytyrosinase. Oxytyrosinase, with two Cu2+ ions at the active site, can catalyze both monophenol, and orthodiphenol substrates. Met-tyrosinase, which also binds Cu2+, can only activate orthodiphenol substrates. Deoxytyrosinase lacks the Cu2+ ion and cannot catalyze any substrate (Silavi et al., 2012; Ramsden and Riley, 2014). Therefore, conformational changes to enzyme structures, especially from the oxy-tyrosinase isoform to the met-tyrosinase or deoxytyrosinase isoforms, may decrease enzyme activity. Chelation is one mechanism by which bioactive compounds inhibit tyrosinase (Silavi et al., 2012; Bae et al., 2013). Polyphenolic compounds containing orthodihydroxyl phenol in their molecules can bind to transition metals, displacing, or removing them from the active site. This structural change in tyrosinase leads to its inactivation, hindering the catalytic reaction. Therefore, the ability of BTL70ME and BTL70AC crude extracts to inhibit tyrosinase may be attributed to the presence of polyphenolic/phenolic compounds.
Polyphenolic compounds have also been reported to induce conformational changes in proteins. The CD spectra of tyrosinase incubated with
Controlling blood glucose levels remains a significant concern for diabetic patients. α-Amylase plays a crucial role in hydrolyzing the glycosidic linkage in starch, directly impacting glucose levels. Hence, suppressing α-amylase activity is a key objective in managing blood glucose levels.
Free radicals play a significant role in biomolecule degradation, contributing to the incidence of noncommunicable diseases (Abegunde et al., 2007; Dudonné et al., 2009; Terzic and Waldman, 2011). Consequently, antioxidants are essential agents for protecting biomolecules from high levels of free radicals. In the present study,
Based on the abovementioned results,
ACKNOWLEDGEMENTS
The authors of this research are thankful for all the support provided by the Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi. We also spread our gratitude towards the Research Instrument Centre at Khon Kaen University, which supported the CD spectroscopy for studying this research.
FUNDING
None.
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Concept and design: SP. Analysis and interpretation: SP, WK. Data collection: SP. Writing the article: all authors. Critical revision of the article: SP. Final approval of the article: all authors. Statistical analysis: WK. Overall responsibility: SP.
References
- Abegunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases in low-income and middle-income countries. Lancet. 2007. 370:1929-1938.
- Alara OR, Abdurahman NH, Mudalip SKA, Olalere OA. Phytochemical and pharmacological properties of
Vernonia amygdalina : a review. J Chem Eng Ind Biotechnol. 2017. 2:80-96. - Asante DB, Effah-Yeboah E, Barnes P, Abban HA, Ameyaw EO, Boampong JN, et al. Antidiabetic effect of young and old ethanolic leaf extracts of
Vernonia amygdalina : a comparative study. J Diabetes Res. 2016. 2016:8252741. https://doi.org/10.1155/2016/8252741. - Bae SJ, Ha YM, Kim JA, Park JY, Ha TK, Park D, et al. A novel synthesized tyrosinase inhibitor: (E)-2-((2,4-dihydroxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate as an azo-resveratrol analog. Biosci Biotechnol Biochem. 2013. 77:65-72.
- Baxter RA. Anti-aging properties of resveratrol: review and report of a potent new antioxidant skin care formulation. J Cosmet Dermatol. 2008. 7:2-7.
- Bestari R, Ichwan MF, Mustofa M, Satria D. Anticancer activity of
Vernonia amygdalina Del. extract on WiDr colon cancer cell line. The 2nd Public Health International Conference; 2017 Dec 18-19. pp. 1-5. - Brincat MP. Hormone replacement therapy and the skin. Maturitas. 2000. 35:107-117.
- Chainani-Wu N. Safety and anti-inflammatory activity of curcumin: a component of tumeric (
Curcuma longa ). J Altern Complement Med. 2003. 9:161-168. - Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci. 2009. 10:2440-2475.
- Cronin H, Draelos ZD. Top 10 botanical ingredients in 2010 anti-aging creams. J Cosmet Dermatol. 2010. 9:218-225.
- Dewanto V, Wu X, Adom KK, Liu RH. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem. 2002. 50:3010-3014.
- Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon JM. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem. 2009. 57:1768-1774.
- Ebanks JP, Wickett RR, Boissy RE. Mechanisms regulating skin pigmentation: the rise and fall of complexion coloration. Int J Mol Sci. 2009. 10:4066-4087.
- Ekaluo UB, Ikpeme EV, Ekerette EE, Chukwu CI.
In vitro antioxidant and free radical activity of some Nigerian medicinal plants: bitter leaf (Vernonia amygdalina L.) and guava (Psidium guajava Del.). Res J Med Plants. 2015. 9:215-226. - Erasto P, Grierson DS, Afolayan AJ. Evaluation of antioxidant activity and the fatty acid profile of the leaves of
Vernonia amygdalina growing in South Africa. Food Chem. 2007. 104:636-642. - Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002. 138:1462-1470.
- Garg C. Molecular mechanisms of skin photoaging and plant inhibitors. Int J Green Pharm. 2017. 11:S217-S232.
- Iwo MI, Sjahlim SL, Rahmawati SF. Effect of
Vernonia amygdalina Del. leaf ethanolic extract on intoxicated male Wistar rats liver. Sci Pharm. 2017. 85:16. https://doi.org/10.3390/scipharm85020016. - Kim YJ, Uyama H. Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. Cell Mol Life Sci. 2005. 62:1707-1723.
- Kim YM, Yun J, Lee CK, Lee H, Min KR, Kim Y. Oxyresveratrol and hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of action. J Biol Chem. 2002. 277:16340-16344.
- Maity N, Nema NK, Abedy MK, Sarkar BK, Mukherjee PK. Exploring
Tagetes erecta Linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity. J Ethnopharmacol. 2011. 137:1300-1305. - Makrantonaki E, Adjaye J, Herwig R, Brink TC, Groth D, Hultschig C, et al. Age-specific hormonal decline is accompanied by transcriptional changes in human sebocytes
in vitro . Aging Cell. 2006. 5:331-344. - Oguwike FN, Offor CC, Onubeze DP, Nwadioha AN. Evaluation of activities of bitterleaf (
Vernonia amygdalina ) extract on haemostatic and biochemical profile of induced male diabetic albino rats. IOSR-JDMS. 2013. 11:60-64. - Panigrahy SK, Bhatt R, Kumar A. Targeting type II diabetes with plant terpenes: the new and promising antidiabetic therapeutics. Biologia. 2021. 76:241-254.
- Patathananone S, Daduang J, Koraneekij A, Li CY. Tyrosinase inhibitory effect, antioxidant and anticancer activities of bioactive compounds in ripe hog plum (
Spondias pinnata ) fruit extracts. Orient J Chem. 2019. 35:916-926. - Piao LZ, Park HR, Park YK, Lee SK, Park JH, Park MK. Mushroom tyrosinase inhibition activity of some chromones. Chem Pharm Bull. 2002. 50:309-311.
- Prasathkumar M, Raja K, Vasanth K, Khusro A, Sadhasivam S, Sahibzada MUK, et al. Phytochemical screening and
in vitro antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing attributes ofSenna auriculata (L.) Roxb. leaves. Arab J Chem. 2021. 14:103345. https://doi.org/10.1016/j.arabjc.2021.103345. - Ramsden CA, Riley PA. Tyrosinase: the four oxidation states of the active site and their relevance to enzymatic activation, oxidation and inactivation. Bioorg Med Chem. 2014. 22:2388-2395.
- Sarkar R, Chugh S, Garg VK. Newer and upcoming therapies for melasma. Indian J Dermatol Venereol Leprol. 2012. 78:417-428.
- Silavi R, Divsalar A, Saboury AA. A short review on the structure-function relationship of artificial catecholase/tyrosinase and nuclease activities of Cu-complexes. J Biomol Struct Dyn. 2012. 30:752-772.
- Sumiyoshi M, Kimura Y. Effects of a turmeric extract (
Curcuma longa ) on chronic ultraviolet B irradiation-induced skin damage in melanin-possessing hairless mice. Phytomedicine. 2009. 16:1137-1143. - Terzic A, Waldman S. Chronic diseases: the emerging pandemic. Clin Transl Sci. 2011. 4:225-226.
- Theansungnoen T, Yaraksa N, Daduang S, Dhiravisit A, Thammasirirak S. Purification and characterization of antioxidant peptides from leukocyte extract of
Crocodylus siamensis . Protein J. 2014. 33:24-31. - Usunobun U, Ngozi OP. Phytochemical analysis and proximate composition of
Vernonia amygdalina . Int J Sci World. 2016. 4:11-14. - Yao LH, Jiang YM, Shi J, Tomás-Barberán FA, Datta N, Singanusong R, et al. Flavonoids in food and their health benefits. Plant Foods Hum Nutr. 2004. 59:113-122.
- Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999. 64:555-559.
Article
Original
Prev Nutr Food Sci 2023; 28(3): 302-311
Published online September 30, 2023 https://doi.org/10.3746/pnf.2023.28.3.302
Copyright © The Korean Society of Food Science and Nutrition.
Inhibitory Effects of Vernonia amygdalina Leaf Extracts on Free Radical Scavenging, Tyrosinase, and Amylase Activities
Supawadee Patathananone1 , Mahinthorn Pothiwan2
, Boontida Uapipatanakul1
, Wuttisak Kunu3
1Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathum Thani 12120, Thailand
2Programme of Agricultural Business and 3Programme of Veterinary Technology and Veterinary Nursing, Faculty of Agricultural Technology, Rajabhat Maha Sarakham University, Maha Sarakham 44000, Thailand
Correspondence to:Wuttisak Kunu, E-mail: wut.kunu@gmail.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Cytotoxicity and instability are the limitations when using bioactive compounds in cosmetic and pharmacology products. This study assesses Vernonia amygdalina leaf extracts for their antityrosinase, antiamylase, and antioxidant activities. Group A extracts were obtained using an aqueous solvent system [70% (v/v) of methanol (BTL70ME), ethanol (BTL70ET), and acetone (BTL70AC)]. Group B extracts were obtained using organic solvents of varying polarities. The results displayed that all extracts exhibited antityrosinase, antiamylase, and antioxidant activities in vitro. The most potent antityrosinase activity was observed in BTL70AC, with a half-maximal inhibitory concentration (IC50) value of 20 μg/mL. BTL_Ethyl acetate and BTL70AC showed potential antiamylase activity. BTL_Isopropanol and BTL_Ethanol exhibited potential antioxidant activity, with IC50 values of 4.0 μg/mL. The total phenolic content of BTL70ME, BTL70ET, and BTL70AC was 72.29±14.14, 65.98±11.91, and 69.37±7.72 mg gallic acid/g extract, respectively. The total flavonoid content was 53.04±5.22, 44.35±13.17, and 61.74±13.17 mg quercetin/g extract, respectively. Group A extracts contained polyphenols, flavonoids, saponins, terpenoids, steroids, and cardiac glycosides. These biological properties can potentially be attributed to the types and quantities of phytochemicals present. Bioactive compounds in the extracts may exert synergistic effects in vitro by interfering with the conformational changes of tyrosinase during substrate binding. Both groups of extracts have the potential to suppress biomolecule degradation, promote antiaging and antimelasma effects, and their phytochemicals can help lower blood glucose levels in diabetes.
Keywords: alpha-amylase, antioxidant, antityrosinase, inhibitors, phytochemicals
INTRODUCTION
Wrinkles, melasma, freckles, and spots are common skin issues, particularly in aging individuals. These problems are influenced by two main factors: intrinsic factors, such as hormones and oxidative damage, and extrinsic factors, such as pollution, alcohol consumption, smoking, and sunlight exposure (Brincat, 2000; Makrantonaki et al., 2006). These factors contribute to the generation of free radicals in skin cells. Sunlight, a natural energy source of ultraviolet (UV) radiation, plays a crucial role in this process. UV radiation consists of UVA, UVB, and UVC rays, which induce the production of reactive oxygen species (ROS) and stimulate melanin synthesis. ROS can cause oxidative stress and damage various biomolecules, especially the plasma membrane of cells, leading to the loss of skin strength and flexibility and the formation of wrinkles.
Additionally, ROS can enhance the activity of enzymes such as collagenase, elastase, and hyaluronidase, which contribute to skin aging and wrinkle formation. Melasma, a skin condition characterized by pigmentation on the epidermis, is influenced by tyrosinase, an enzyme that controls melanocyte function (Fisher et al., 2002; Maity et al., 2011).
The cosmetic industry uses various whitening agents, including hydroquinone, butylated hydroxytoluene, kojic acid, ascorbic acid, and alpha-arbutin. However, hydroquinone, a skin-lightening compound, has been associated with mutagenic effects, adverse reactions, and skin irritation when used in high doses (Sarkar et al., 2012); hence, strict regulations govern its usage. Kojic acid inhibits tyrosinase but poses challenges owing to its cytotoxicity and instability. Ascorbic acid shows potential as an antityrosinase compound but is prone to decomposition under high temperatures and UV radiation (Bae et al., 2013). The use of pure compounds as cosmetic ingredients increases product value but comes with higher costs. To address these issues, the cosmetic industry incorporates extracts from plants, animals, and marine organisms into their products.
In addition to avoiding external factors that contribute to skin issues, two primary goals are targeted: preventing free radical generation through antioxidants and inhibiting enzymes that cause cellular degradation, including tyrosinase, elastase, hyaluronidase, and collagenase. Natural extracts containing potent antiwrinkle properties are key components of antiaging products. For example, red grape (
The present study focuses on studying the biological activities of
MATERIALS AND METHODS
Extraction of V. amygdalina leaves
The extraction method was adapted from the method of Ekaluo et al. (2015).
-
Figure 1. Schematic diagram depicting the process of
Vernonia amygdalina leaf extraction.
Two groups of extraction solvents were prepared. In group A, 70% (v/v) methanol (BTL70ME), 70% (v/v) ethanol (BTL70ET), and 70% (v/v) acetone (BTL70AC) were used to extract
The mixture in each bottle were stirred at 22
-
Table 1 . The IC50 values of the extracted samples for antityrosinase and antioxidant activities.
Sample groups Extracted solvent ID name IC50 value (μg/mL) Antityrosinase activity Antioxidant activity Group A 70% (v/v) methanol BTL70ME 25 16 70% (v/v) ethanol BTL70ET 125 37 70% (v/v) acetone BTL70AC 20 20 Group B Hexane BTL_Hexane >1,000 >160 Ethyl acetate BTL_Ethyl acetate 1,000 9 Isopropanol BTL_Isopropanol 720 4 Ethanol BTL_Ethanol 360 4 Control Ascorbic acid 11 2 IC50, half-maximal inhibitory concentration..
Determination of tyrosinase inhibition property using a modified dopachrome method
The tyrosinase inhibitory effect of the extracts was assessed following the procedure outlined by Patathananone et al. (2019). Each extract powder was dissolved in 5% (v/v) dimethyl sulfoxide (DMSO). Subsequently, 10 μL of various concentrations of each extract were mixed with 100 μL of a 250 unit/mL tyrosinase solution [250 unit/mL tyrosinase in 20 mM phosphate buffer (pH 6.8)] in a 96-well plate. Next, 70 μL of deionized (DI) water was gently added to each well. The mixtures were incubated at 37°C for 10 min, after which 20 μL of 20 mM L-Dopa (HiMedia Laboratories Pvt. Ltd.) was added. The plate was shaken and further incubated at 37°C for 20 min. UV-visible (UV-Vis) absorption measurements were performed at 495 nm to determine the percentage of tyrosinase inhibition.
Antiamylase activity assay
α-Amylase is an enzyme that breaks down starch by hydrolyzing α-(1-4)-glycosidic bonds. The resulting products include short-chain oligosaccharides, trisaccharides, maltose, and glucose. The level of product formation is influenced by several factors, such as enzyme concentration, substrate concentration, pH, and temperature. Inhibition of α-amylase activity is important for controlling sugar production during starch digestion, especially for individuals with hyperglycemia or diabetes. The assessment of reducing sugar products resulting from amylase activity was conducted using a modified version of the method described by Prasathkumar et al. (2021). Reducing sugars react with 3,5-dinitrosalicylic acid (DNS) at 100°C, producing 3-amino-5-nitrosalicylic acid, which exhibits a red-brown color. The absorbance value at a wavelength of 540 nm was measured to quantify the amount of reduced sugar, using the glucose standard curve for the DNS method.
In the experiment, 40 μL of 0.1% (w/v) α-amylase (HiMedia Laboratories Pvt. Ltd.) in 50 mM phosphate buffer at pH 6.8 was mixed with 20 μL of 20 mg/mL of each sample and incubated at 37°C for 10 min. A cuvette containing 1.8 mL of 1.0% w/v starch solution was prepared, and the optical density at a wavelength of 600 nm was measured using a UV-Vis spectrophotometer (UV-Vis-1200, MAPADA). The incubated α-amylase was then added to the starch solution, rapidly mixed, and the optical density at 600 nm (OD600 nm) was recorded every 30 s for 5 min. The reactions were halted through incubation at 100°C for 10 min, followed by a 10 min cooling period in water. Subsequently, 2 mL of 0.01 M DNS solution was added to each reaction, mixed, and boiled at 100°C for 10 min. The absorbance values of each reaction were measured at 540 nm. The quantity of reducing sugar products under each condition was determined using the standard curve equation: y=0.4023x+0.0379 (R2=0.9989).
Determination of antioxidant properties using the DPPH assay
The antioxidant activity of the extracts was assessed and calculated using a modified version of the method reported by Patathananone et al. (2019). The reaction took place in a 96-well plate, where 10 μL of the extracted samples at different concentrations were added to 190 μL of 0.101 M DPPH solution (0.101 mM DPPH in methanol). The mixture was gently mixed, and incubated at 37°C for 30, 45, and 60 min. Ascorbic acid and 5% (v/v) DMSO served as the positive and negative controls, respectively. The absorbance values were measured at 515 nm (Piao et al., 2002; Theansungnoen et al., 2014; Ekaluo et al., 2015) using a microplate reader (EZ-Read 2000, Biochrom).
Determination of total phenolic contents using Folin-Ciocâlteu reagent
The total phenolic content in the extracts was analyzed using a modified experiment based on the method of Dewanto et al. (2002). Gallic acid, dissolved in methanol, was used as the standard agent. Standard gallic acid solutions were prepared at initial concentrations of 0.2, 0.4, 0.6, 0.8, and 1 μg/mL. For the reaction, 3.5 mL of 2.0% (w/v) sodium carbonate solution was mixed with 100 μL of different concentrations of the extracts (BTL70ME, BTL70ET, and BTL70AC), as well as the gallic acid solutions, in test tubes. Subsequently, 400 μL of 10-fold diluted Folin-Ciocâlteu solution was added to each tube. The mixtures were incubated in the dark at room temperature for 30 min. UV-Vis measurements were taken at 750 nm, using a modified version of the method described by Dewanto et al. (2002), to create a standard curve. Sample measurements were taken under the same conditions to determine total phenolic content.
Determination of total flavonoid content using aluminum chloride reagent
To assess the total flavonoid content in the group A extracts, standard quercetin solutions were prepared at different concentrations (20, 40, 60, 80, and 100 μg/mL). A typical mixture was created in a 10 mL volumetric flask by sequentially adding 1.0 mL of the sample solution, 4 mL of distilled water, and 0.3 mL of 5% (w/v) of sodium nitrite solution. The mixture was left at room temperature for 5 min, after which 0.3 mL of 10% (w/v) aluminum chloride solution was added. Following a 6 min incubation period, 0.2 mL of 1 M sodium hydroxide (NaOH) was added, and the total volume of the mixture was adjusted to 10 mL using distilled water. UV-Vis measurements were taken at 510 nm, using a modified version of the method described by Zhishen et al. (1999).
Preliminary analysis of bioactive compounds
The methods for phytochemical screening in the extracts were mini modified according to the report of Usunobun and Ngozi (2016).
Circular dichroism (CD) spectroscopy analysis
The conformational changes of tyrosinase were determined using CD spectroscopy (Jasco J-815 CD Spectrometer, Analytical Lab Science Co., Ltd.). Four solutions were prepared to analyze the CD spectra: (1) 5 μg/mL tyrosinase (E) in 50 mM phosphate buffer (pH 6.8), (2) 125 unit/mL tyrosinase+5 mM L-Dopa (E+S), (3) 5 μg/mL tyrosinase+0.7 mg/mL BTL70ME, and (4) 5 μg/mL tyrosinase+0.7 mg/mL BTL70ME+5 mM L-Dopa (E+I+S). The spectra were analyzed in the 176∼260 nm range for all test conditions. A phosphate buffer was used as the buffer blank.
Statistical analysis
One-way ANOVA was used for the statistical analysis (IBM SPSS Statistics version 29, IBM Corp.), as previously explained by Patathananone et al. (2019). Each biological characteristic’s proportion (means±SD) was used to represent the data. Statistics were computed within the same extracted group and compared to equivalent concentrations for the results depicted in Fig. 2 and 4. At a
-
Figure 2. Antityrosinase activity of the extracts in both groups. (A) Group A and (B) group B. The data are presented as means±SD (n=3). The statistical information was analyzed using one-way ANOVA. When the
P <0.05, the letters (a-d) showed statistically significant differences within the same concentration.
-
Figure 3. Results of antiamylase activity tests. (A) Decrease in optical density at 600 nm of starch solution after incubation with buffer, α-amylase, and the group A and B extracts for 5 min. (B) Quantification of reducing sugar products corresponding to α-amylase activity under each condition. Using one-way ANOVA, the absorbance values of the control and all test groups were compared. *Statistical significance was represented as a
P <0.05. BTL_He, BTL_Hexane; BTL_EA, BTL_Ethyl acetate; BTL_Iso, BTL_Isopropanol; BTL_ET, BTL_Ethanol.
-
Figure 4. The antioxidant potential of both extracted groups: (A) group A and (B) group B. The data are presented as means±SD. The letters (a-d) indicated significance different statistically within the same concentration at
P <0.05.
RESULTS
Tyrosinase-inhibiting properties of extracts
The tyrosinase-inhibiting properties of
Effect of V. amygdalina leaf extracts on α-amylase
The results of the antiamylase activity experiment are presented in Fig. 3. The rate of change in OD600 nm for 50 mM phosphate buffer (pH 6.8), BTL_Hexane, BTL_Ethyl acetate, BTL_Isopropanol, BTL_Ethanol, BTL70AC, BTL70ME, and BTL70ET was 5.2, 2.7, 4.3, 4.5, 2.7, 3.8, 4.7, and 3.8×10−3 unit/s, respectively, and the amount of reducing sugar products under these conditions were 1.41±0.003, 0.94±0.056, 0.46±0.090, 0.57±0.040, 0.85±0.090, 0.43±0.020, 0.58±0.060, and 0.79±0.090 mM, respectively. The number of products observed under treated conditions was lower compared with the control, indicating a decrease in amylase activity. These findings suggest that the bioactive compounds present in
Antioxidant properties of extracts
Antioxidants can decrease oxidative stress and oxidative damage. Primary antioxidants show the mechanism of action as electron donors. This reaction is the beginning step to inhibiting the chain reaction of dangerous free radicals. The antioxidant screening ability of both extracted groups was performed by DPPH assay. The phytochemicals display antioxidant properties by donating the electrons to DPPH (•) radicals, and then the purple color is decreased or changed to a yellow color solution. The colorimetric reaction is determined using the spectrophotometric technique. The results are presented in Fig. 4. The extracts of both groups represented antioxidant activity as dose-dependent. The IC50 values of BTL70ME, BTL70ET, and BTL70AC were 16, 37, and 20 μg/mL, respectively. These results differ from those of the group B extracts. The IC50 value of BTL_Hexane was >160 μg/mL, whereas the IC50 values of BTL_Ethyl acetate, BTL_Isopropanol, and BTL_Ethanol were 9, 4, and 4 μg/mL, respectively. The IC50 values of all extracts are summarized in Table 1. Overall, the data suggest that the group A extraction process should be used to obtain bioactive agents with high antioxidant efficacy.
Total phenolic content of group A extracts
The group A extracts exhibited antityrosinase and antioxidant properties. Previous studies have highlighted the crucial role of phenolic content in plant extracts for expressing durable biological properties. Therefore, the total phenolic content of the group A extracts was determined using Folin-Ciocâlteu reagent with gallic acid as the standard. To calculate the total phenolic content in each sample, the following equation was used: y=0.0157x−0.0068 (R2=0.9984). The summarized total phenolic content results are presented in Table 2. BTL70ME exhibited higher total phenolic content compared with BTL70AC and BTL70ET, although the differences were not statistically significant.
-
Table 2 . Total phenolic and flavonoid contents of the group A extracts.
ID name Total phenolic contents (mg gallic/g extract) Total flavonoid contents (mg quercetin/g extract) BTL70ME 72.29±14.14 53.04±5.22 BTL70ET 65.98±11.91 44.35±13.17 BTL70AC 69.37±7.72 61.74±13.17
Total flavonoid contents of group A extracts
Flavonoids are a group of phytochemicals known to possess biological activities and efficacy. In this study, the total flavonoid content of the group A extracts was analyzed using quercetin as the standard. The standard equation y=0.0023x+0.0002 (R2=0.9943) was used to determine the total flavonoid content in each sample, and the results are presented in Table 2. BTL70AC exhibited higher total flavonoid content compared with BTL70ME and BTL70ET. Therefore, flavonoids may play an essential role in inhibiting tyrosinase and reducing the number of free radicals in BTL70AC extract.
Preliminary analysis of bioactive compounds
A variety of experiments were conducted to determine the types of phytochemicals present in group A and group B extracts, and the results are presented in Table 3. The group A extracts contained a similar range of phytochemicals, although coumarins, and cardiac glycosides were not detected in BTL70AC. These findings suggest that these specific phytochemical types contribute to potential antityrosinase and antioxidant activities. It is possible that certain phytochemicals in group A exhibit a synergistic effect that enhances their biological properties. In contrast, the extraction process used for group B separates different types of phytochemicals, which may reduce their potential biological activities. These results support the hypothesis that bioactive compounds in the group A extracts function together synergistically.
-
Table 3 . Preliminary phytochemical compounds analysis of
Vernonia amygdalina leaf extracts in groups A and B.Bioactive compounds Preliminary analysis BTL70AC BTL70ET BTL70ME BTL_He BTL_EA BTL_Iso BTL_ET Phenols/phenolics + + + – – + + Flavonoids + + + – – + + Coumarins – + + – – – – Saponins + + + + – + + Tannins – – – – – + – Terpenoids + + + – + + + Steroids + + + + + + + Cardiac glycosides – + + – – + – “+” indicates a positive test result..
BTL_He, BTL_Hexane; BTL_EA, BTL_Ethyl acetate; BTL_Iso, BTL_Isopropanol; BTL_ET, BTL_Ethanol..
CD spectroscopy of tyrosinase
The secondary structure of the tyrosinase enzyme [125 unit/mL tyrosinase in 50 mM phosphate buffer (pH 6.8)] was analyzed using CD spectroscopy, and the results are summarized in Table 4. The percentages of helix, beta-sheet, turn, and other structures in the tyrosinase enzyme were determined to be 11.79, 30.75, 14.97, and 42.45%, respectively. After incubating the enzyme with the substrate (L-Dopa), the percentage of helix structure increased, which is consistent with the induced fit hypothesis. However, the percentages of helix and β-sheet structures in tyrosinase treated with BTL70ME differed from the current conditions (tyrosinase+L-Dopa). The presence of phytochemicals may interfere with the binding ability between tyrosinase and the substrate, leading to a decrease in catalytic properties.
-
Table 4 . Percentage of tyrosinase secondary structures measured using circular dichroism spectroscopy.
Conditions Percentage of secondary structures (%) Helix Beta sheet Turn Others 125.0 unit/mL tyrosinase enzyme in 50 mM phosphate buffer (pH 6.8) 11.79 30.75 14.97 42.45 125.0 unit/mL tyrosinase enzyme+5.0 mM L-Dopa 12.02 30.47 14.97 42.49 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME 11.40 31.25 14.93 42.33 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME+5 mM L-Dopa 11.25 31.38 14.93 42.39
DISCUSSION
Cell degeneration results from intrinsic and extrinsic factors that induce oxidation reactions, generating destructive free radicals within cells. Enzymes can also contribute to cell degeneration by damaging protein structures. Skin pigmentation enzymes, such as those associated with melasma, freckles, spots, deep and shallow wrinkles, and specific diseases, are particularly relevant. To address these issues, two primary goals have emerged: (1) inhibiting related enzymes using inhibitors from different sources, especially natural sources such as plants (Brincat, 2000; Fisher, 2002; Makrantonaki et al., 2006); (2) countering the destructive effects of free radicals through the use of antioxidants. Numerous studies have focused on bioactive compounds, with particular emphasis on plant-derived sources.
In the present study,
Tyrosinase, a key enzyme involved in melanin synthesis, is influenced by both intrinsic and extrinsic factors. Its activity is closely linked to hyperpigmentation (Kim et al., 2002), melasma, and disorders such as Parkinson’s disease. Therefore, tyrosinase inhibitors have been investigated to develop solutions for these issues. In the present study,
Tyrosinase enzyme inhibitors can be classified into two types based on their characteristics. The first type consists of specific tyrosinase inactivators known as suicide substrates. These inhibitors form covalent bonds with the tyrosinase enzyme, leading to denaturation of the enzyme and dysfunction. The second type includes specific tyrosinase inhibitors that bind to the enzyme structure, reducing its activity (Bae et al., 2013). Tyrosinase is a copper (Cu)-containing enzyme. For example, mushroom tyrosinase (EC. 1.14.18.1) is a conjugated enzyme with an active site containing two Cu atoms. Tyrosinase-Cu2+ exhibits catalytic properties (a holoenzyme), whereas tyrosinase lacking Cu2+ exhibits a nonenzymatic function (an apoenzyme). Thus, the presence, and location of Cu ions affect the enzyme’s activity. Three isoforms of Cu-tyrosinase complexes exist: oxy-tyrosinase, met-tyrosinase, and deoxytyrosinase. Oxytyrosinase, with two Cu2+ ions at the active site, can catalyze both monophenol, and orthodiphenol substrates. Met-tyrosinase, which also binds Cu2+, can only activate orthodiphenol substrates. Deoxytyrosinase lacks the Cu2+ ion and cannot catalyze any substrate (Silavi et al., 2012; Ramsden and Riley, 2014). Therefore, conformational changes to enzyme structures, especially from the oxy-tyrosinase isoform to the met-tyrosinase or deoxytyrosinase isoforms, may decrease enzyme activity. Chelation is one mechanism by which bioactive compounds inhibit tyrosinase (Silavi et al., 2012; Bae et al., 2013). Polyphenolic compounds containing orthodihydroxyl phenol in their molecules can bind to transition metals, displacing, or removing them from the active site. This structural change in tyrosinase leads to its inactivation, hindering the catalytic reaction. Therefore, the ability of BTL70ME and BTL70AC crude extracts to inhibit tyrosinase may be attributed to the presence of polyphenolic/phenolic compounds.
Polyphenolic compounds have also been reported to induce conformational changes in proteins. The CD spectra of tyrosinase incubated with
Controlling blood glucose levels remains a significant concern for diabetic patients. α-Amylase plays a crucial role in hydrolyzing the glycosidic linkage in starch, directly impacting glucose levels. Hence, suppressing α-amylase activity is a key objective in managing blood glucose levels.
Free radicals play a significant role in biomolecule degradation, contributing to the incidence of noncommunicable diseases (Abegunde et al., 2007; Dudonné et al., 2009; Terzic and Waldman, 2011). Consequently, antioxidants are essential agents for protecting biomolecules from high levels of free radicals. In the present study,
Based on the abovementioned results,
ACKNOWLEDGEMENTS
The authors of this research are thankful for all the support provided by the Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi. We also spread our gratitude towards the Research Instrument Centre at Khon Kaen University, which supported the CD spectroscopy for studying this research.
FUNDING
None.
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Concept and design: SP. Analysis and interpretation: SP, WK. Data collection: SP. Writing the article: all authors. Critical revision of the article: SP. Final approval of the article: all authors. Statistical analysis: WK. Overall responsibility: SP.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

-
Table 1 . The IC50 values of the extracted samples for antityrosinase and antioxidant activities
Sample groups Extracted solvent ID name IC50 value (μg/mL) Antityrosinase activity Antioxidant activity Group A 70% (v/v) methanol BTL70ME 25 16 70% (v/v) ethanol BTL70ET 125 37 70% (v/v) acetone BTL70AC 20 20 Group B Hexane BTL_Hexane >1,000 >160 Ethyl acetate BTL_Ethyl acetate 1,000 9 Isopropanol BTL_Isopropanol 720 4 Ethanol BTL_Ethanol 360 4 Control Ascorbic acid 11 2 IC50, half-maximal inhibitory concentration.
-
Table 2 . Total phenolic and flavonoid contents of the group A extracts
ID name Total phenolic contents (mg gallic/g extract) Total flavonoid contents (mg quercetin/g extract) BTL70ME 72.29±14.14 53.04±5.22 BTL70ET 65.98±11.91 44.35±13.17 BTL70AC 69.37±7.72 61.74±13.17
-
Table 3 . Preliminary phytochemical compounds analysis of
Vernonia amygdalina leaf extracts in groups A and BBioactive compounds Preliminary analysis BTL70AC BTL70ET BTL70ME BTL_He BTL_EA BTL_Iso BTL_ET Phenols/phenolics + + + – – + + Flavonoids + + + – – + + Coumarins – + + – – – – Saponins + + + + – + + Tannins – – – – – + – Terpenoids + + + – + + + Steroids + + + + + + + Cardiac glycosides – + + – – + – “+” indicates a positive test result.
BTL_He, BTL_Hexane; BTL_EA, BTL_Ethyl acetate; BTL_Iso, BTL_Isopropanol; BTL_ET, BTL_Ethanol.
-
Table 4 . Percentage of tyrosinase secondary structures measured using circular dichroism spectroscopy
Conditions Percentage of secondary structures (%) Helix Beta sheet Turn Others 125.0 unit/mL tyrosinase enzyme in 50 mM phosphate buffer (pH 6.8) 11.79 30.75 14.97 42.45 125.0 unit/mL tyrosinase enzyme+5.0 mM L-Dopa 12.02 30.47 14.97 42.49 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME 11.40 31.25 14.93 42.33 125.0 unit/mL tyrosinase enzyme+0.7 mg/mL BTL70ME+5 mM L-Dopa 11.25 31.38 14.93 42.39
References
- Abegunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases in low-income and middle-income countries. Lancet. 2007. 370:1929-1938.
- Alara OR, Abdurahman NH, Mudalip SKA, Olalere OA. Phytochemical and pharmacological properties of
Vernonia amygdalina : a review. J Chem Eng Ind Biotechnol. 2017. 2:80-96. - Asante DB, Effah-Yeboah E, Barnes P, Abban HA, Ameyaw EO, Boampong JN, et al. Antidiabetic effect of young and old ethanolic leaf extracts of
Vernonia amygdalina : a comparative study. J Diabetes Res. 2016. 2016:8252741. https://doi.org/10.1155/2016/8252741. - Bae SJ, Ha YM, Kim JA, Park JY, Ha TK, Park D, et al. A novel synthesized tyrosinase inhibitor: (E)-2-((2,4-dihydroxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate as an azo-resveratrol analog. Biosci Biotechnol Biochem. 2013. 77:65-72.
- Baxter RA. Anti-aging properties of resveratrol: review and report of a potent new antioxidant skin care formulation. J Cosmet Dermatol. 2008. 7:2-7.
- Bestari R, Ichwan MF, Mustofa M, Satria D. Anticancer activity of
Vernonia amygdalina Del. extract on WiDr colon cancer cell line. The 2nd Public Health International Conference; 2017 Dec 18-19. pp. 1-5. - Brincat MP. Hormone replacement therapy and the skin. Maturitas. 2000. 35:107-117.
- Chainani-Wu N. Safety and anti-inflammatory activity of curcumin: a component of tumeric (
Curcuma longa ). J Altern Complement Med. 2003. 9:161-168. - Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci. 2009. 10:2440-2475.
- Cronin H, Draelos ZD. Top 10 botanical ingredients in 2010 anti-aging creams. J Cosmet Dermatol. 2010. 9:218-225.
- Dewanto V, Wu X, Adom KK, Liu RH. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem. 2002. 50:3010-3014.
- Dudonné S, Vitrac X, Coutière P, Woillez M, Mérillon JM. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem. 2009. 57:1768-1774.
- Ebanks JP, Wickett RR, Boissy RE. Mechanisms regulating skin pigmentation: the rise and fall of complexion coloration. Int J Mol Sci. 2009. 10:4066-4087.
- Ekaluo UB, Ikpeme EV, Ekerette EE, Chukwu CI.
In vitro antioxidant and free radical activity of some Nigerian medicinal plants: bitter leaf (Vernonia amygdalina L.) and guava (Psidium guajava Del.). Res J Med Plants. 2015. 9:215-226. - Erasto P, Grierson DS, Afolayan AJ. Evaluation of antioxidant activity and the fatty acid profile of the leaves of
Vernonia amygdalina growing in South Africa. Food Chem. 2007. 104:636-642. - Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002. 138:1462-1470.
- Garg C. Molecular mechanisms of skin photoaging and plant inhibitors. Int J Green Pharm. 2017. 11:S217-S232.
- Iwo MI, Sjahlim SL, Rahmawati SF. Effect of
Vernonia amygdalina Del. leaf ethanolic extract on intoxicated male Wistar rats liver. Sci Pharm. 2017. 85:16. https://doi.org/10.3390/scipharm85020016. - Kim YJ, Uyama H. Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. Cell Mol Life Sci. 2005. 62:1707-1723.
- Kim YM, Yun J, Lee CK, Lee H, Min KR, Kim Y. Oxyresveratrol and hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of action. J Biol Chem. 2002. 277:16340-16344.
- Maity N, Nema NK, Abedy MK, Sarkar BK, Mukherjee PK. Exploring
Tagetes erecta Linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity. J Ethnopharmacol. 2011. 137:1300-1305. - Makrantonaki E, Adjaye J, Herwig R, Brink TC, Groth D, Hultschig C, et al. Age-specific hormonal decline is accompanied by transcriptional changes in human sebocytes
in vitro . Aging Cell. 2006. 5:331-344. - Oguwike FN, Offor CC, Onubeze DP, Nwadioha AN. Evaluation of activities of bitterleaf (
Vernonia amygdalina ) extract on haemostatic and biochemical profile of induced male diabetic albino rats. IOSR-JDMS. 2013. 11:60-64. - Panigrahy SK, Bhatt R, Kumar A. Targeting type II diabetes with plant terpenes: the new and promising antidiabetic therapeutics. Biologia. 2021. 76:241-254.
- Patathananone S, Daduang J, Koraneekij A, Li CY. Tyrosinase inhibitory effect, antioxidant and anticancer activities of bioactive compounds in ripe hog plum (
Spondias pinnata ) fruit extracts. Orient J Chem. 2019. 35:916-926. - Piao LZ, Park HR, Park YK, Lee SK, Park JH, Park MK. Mushroom tyrosinase inhibition activity of some chromones. Chem Pharm Bull. 2002. 50:309-311.
- Prasathkumar M, Raja K, Vasanth K, Khusro A, Sadhasivam S, Sahibzada MUK, et al. Phytochemical screening and
in vitro antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing attributes ofSenna auriculata (L.) Roxb. leaves. Arab J Chem. 2021. 14:103345. https://doi.org/10.1016/j.arabjc.2021.103345. - Ramsden CA, Riley PA. Tyrosinase: the four oxidation states of the active site and their relevance to enzymatic activation, oxidation and inactivation. Bioorg Med Chem. 2014. 22:2388-2395.
- Sarkar R, Chugh S, Garg VK. Newer and upcoming therapies for melasma. Indian J Dermatol Venereol Leprol. 2012. 78:417-428.
- Silavi R, Divsalar A, Saboury AA. A short review on the structure-function relationship of artificial catecholase/tyrosinase and nuclease activities of Cu-complexes. J Biomol Struct Dyn. 2012. 30:752-772.
- Sumiyoshi M, Kimura Y. Effects of a turmeric extract (
Curcuma longa ) on chronic ultraviolet B irradiation-induced skin damage in melanin-possessing hairless mice. Phytomedicine. 2009. 16:1137-1143. - Terzic A, Waldman S. Chronic diseases: the emerging pandemic. Clin Transl Sci. 2011. 4:225-226.
- Theansungnoen T, Yaraksa N, Daduang S, Dhiravisit A, Thammasirirak S. Purification and characterization of antioxidant peptides from leukocyte extract of
Crocodylus siamensis . Protein J. 2014. 33:24-31. - Usunobun U, Ngozi OP. Phytochemical analysis and proximate composition of
Vernonia amygdalina . Int J Sci World. 2016. 4:11-14. - Yao LH, Jiang YM, Shi J, Tomás-Barberán FA, Datta N, Singanusong R, et al. Flavonoids in food and their health benefits. Plant Foods Hum Nutr. 2004. 59:113-122.
- Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999. 64:555-559.