A diterpenoid sugiol from Metasequoia glyptostroboides with alpha-glucosidase and tyrosinase inhibitory potential
Abstract
Nowadays use of plant derived natural compounds have become a topic of increasing interest in food and medicine industries due to their multitude of biological and therapeutic properties. In this study, a diterpenoid compound sugiol, isolated from Metasequoia glyptostroboides was evaluated for alpha-glucosidase and tyrosinase inhibitory efficacy in terms of its potent anti-diabetic and anti-melanogenesis potential, respectively. As a result, sugiol at the concentration range of (100-10,000 µg/mL) and (20-500 µg/mL) showed potent efficacy on inhibiting alpha-glucosidase and tyrosinase enzymes in vitro ranging from 12.34-63.5 and 28.22-67.4%, respectively. These findings confirm the therapeutic potential of diterpenoid compound sugiol from M. glyptostroboides as a novel candidate for using in food and medicine industry which may have practical potential to cure skin and diabetes mellitus type-2 related disorders.
Introduction
The early stage of diabetes mellitus type 2 is associated with hyperglycemia due to impaired after-meal acute insulin secretion. Hyperglycemia is believed to increase the production of free radicals and reactive oxygen species, leading to oxidative tissue damage and diabetic complications (Maritim et al., 2003). Glucosidases are a group of digestive enzymes which break down the dietary carbohydrates into simple monosaccharides. Glucosidase inhibitors reduce the rate of carbohydrate digestion and delay the carbohydrate absorption from the digestive tract. Therefore, they have a potential to prevent the development of type 2 diabetes mellitus by lowering the after-meal glucose levels (Liu et al., 2011).
Tyrosinase, a copper-containing polyphenol oxidase, plays a highly critical role in forming melanin pigments (Ozer et al., 2007). Previous reports have shown that tyrosinase might also be involved in neuromelanin production and be associated with Parkinson's disease (Chen et al., 2013). Therefore, inhibiting tyrosinase activity is applicable to skin-lightening and in preventing neurodegeneration (Kwon et al., 2011).
Phytochemicals confer various health benefits, among them, alpha-glucosidase and tyrosinase inhibitory activities have particularly received intensive attention due to the increasing number of patients suffering from diabetes type 2 and skin disorders. Though synthetic alpha-glucosidase and tyrosinase inhibitors have been used effectively, many doubts have been raised on their safety such as increased toxicity and adverse side effects. Hence, attention has been focused on the effective use of plant based compounds which are less toxic and natural in origin.
Although the biological efficacy of M. glyptostroboides derived compounds has been reported previously (Bajpai et al., 2010; Bajpai et al., 2011; Bajpai et al., 2014), there is no report available on alpha-glucosidase and tyrosinase inhibitory effects of sugiol, a diterpenoid isolated from M. glyptostroboides. Hence, the aim of this research is to confirm the therapeutic efficacy of sugiol as a potent alpha-glucosidase and tyrosinase inhibitor.
Materials and Methods
Chemicals and instrument
Kojic acid, acarbose, sodium azide (NaN3), bovine serum albumin, p-nitrophenyl-alpha-D-glucopyranoside, yeast alpha-glucosidase, mushroom tyrosinase, and 3,4-dihydroxy-L-phenylalanine (DOPA) were purchased from (Sigma-Aldrich, USA). All other reagents used were of high analytical grade. Spectrophotometric measurements were done by using a 96-well microplate ELISA reader (Infinite M200, Switzerland).
Plant material
The cones of M. glyptostroboides were collected from Pohang city, Korea, and identified by the morphological features and the database present in the library at the Department of Biotechnology, Daegu University, Korea. A voucher specimen (DUB-0038) was deposited in the herbarium of College of Engineering, Department of Biotechnology, Daegu University, Korea.
Extraction and isolation of sugiol
Dried cones of M. glyptostroboides (2 kg) were milled into powder and then extracted with ethyl acetate at room temperature for 12 days. The extract was evaporated under reduced pressure using a rotary evaporator (EYELA N1000, Japan). The dried ethyl acetate extract (7 g) was subjected to column chromatography over silica gel (mesh 230-400 mesh, Merck, Germany) and was eluted with hexane-ethyl acetate-methanol solvent system to give 20 fractions. Of the fractions obtained, fraction-14 was further purified by preparative thin layer chromatography (TLC) over silica gel GF254 using hexane-ethyl acetate (2:1) as a mobile phase to give one compound (122 mg) which on the basis of spectral data analysis was characterized as sugiol shown in Figure 1 (Bajpai et al., 2011).
Figure 1: Chemical structure of a diterpenoid sugiol isolated from Metasequoia glyptostroboides
Assay of alpha-glucosidase inhibition
alpha-Glucosidase inhibitory activity of sugiol isolated from M. glyptostroboides was evaluated according to the chromogenic method (Liu et al., 2014). Briefly 10 µL of test samples at various concentrations (100, 500, 1,000, 5,000 and 10,000 µg/mL) and 50 uL of yeast alpha-glucosidase, dissolved in 100 mM phosphate buffer (pH 7.0) (containing 2 g/L bovine serum albumin and 0.2 g/L NaN3) were mixed in 96 well microplate and absorbance at 405 nm was measured for titer at zero time with a microplate reader (Tecan, Infinite M200, Switzerland). After 5 min incubation, 50 uL of p-nitrophenyl-alpha-D-glucopyranoside (5 mM) in the same buffer (pH 7.0) was used as a substrate solution and incubated for an additional 5 min at room temperature. Eventually the reaction was terminated by adding 80 uL of 0.2 M sodium carbonate solution. Absorbance of the reaction mixture was measured with a microplate reader at 405 nm. The increase in absorbance from zero time was measured. Inhibitory activity was expressed as 100 minus relative absorbance difference (%) of test compounds to absorbance change of the control, while the reaction system without sugiol was served as a control test. The system without alpha-glucosidase was used as blank, and acarbose at various concentrations (100, 500, 1,000, 5,000 and 10,000 µg/mL) was used as a positive control. Each experiment was conducted in triplicate, and the enzyme inhibitory rate was calculated as follows:
Inhibition (%) = (Control absorption - Sample absorption)/Control absorption x 100
Assay of tyrosinase inhibition
The tyrosinase activity of sugiol was measured by a previously reported method (Fawole et al., 2012). Briefly, 100 uL of different concentrations (20, 40, 100, 200 and 500 µg/mL) of sugiol were mixed with 600 µL of 0.175 M sodium phosphate buffer (pH 6.8). Further, 200 µL of 10 mM L-DOPA solution (L-3,4-dihydroxy-phenylalanine) was added to each well. After that, 200 µL of tyrosinase (110 units/mL in 0.175 M sodium phosphate buffer) was added to the reaction mixture and further incubated at 37°C for 2 min. Then after incubation, the amount of dopachrome produced in the reaction mixture was measured at 475 nm in a 96-well microtiter plate with a microplate reader. Kojic acid (20, 40, 100, 200 and 500 µg/mL) was used as a positive control. The experiment was conducted in triplicate at room temperature, and the enzyme inhibitory rate was calculated as follows:
Inhibition (%) = (Control absorption - Sample absorption)/Control absorption x 100
Statistical analysis
All the data were expressed as mean ± standard deviation of three replicates. Tests of significant differences were determined by one-way ANOVA followed by Duncan's test using SAS software (SAS 9.2, SAS), and the values were considered to be significant (p<0.05).
Result and Discussion
Identification of sugiol
The ethyl acetate cone extract of M. glyptostroboides after column chromatography over silica gel yielded a pure compound, which was obtained as yellow glass crystal with a specific melting point (mp 282-84°C). The 1H NMR data (pyridine-d5, 250 MHz) showing two singlet protons at d 8.39 and 7.16 (each 1H, s), an aliphatic methine signal at d 3.59 (1H, m, signals partially overlapped), and five terminal methyl groups at d 1.35 (3H, d, J = 6.8 Hz), 1.33 (3H, d, J = 6.8 Hz), 1.11 (3H, s, Me-20), 0.83 (3H, s, Me-19), 0.79 (3H, s,Me-18), as well as the 13C NMR data displaying twenty carbon signals including a carbonyl group at d 197.6 strongly suggested that this compound should be an abietane diterpenoid. The structure of this compound was determined to be sugiol (Figure 1) by 1D and 2D NMR analysis, and confirmed by comparing the physical and spectroscopic data with those in the literature (Chang et al., 1990; Bajpai et al., 2011).
Inhibition of alpha-glucosidase
Phytochemicals have become important sources of human therapeutics and potential exists for their use in developing novel diabetes therapies or bio-therapeutic agents (Tiwari et al., 2010). There is a huge ongoing interest in the application of plant-based compounds and/or products for alleviating chronic diseases due to their potent therapeutic and medicinal efficacy. Still the use of phytochemicals within the context of diabetes remains largely unexplored; which has urged the scientists to provide scientific evidences on the development of more effective agents conferring inhibitory effects on intestinal glucosidases. As reported previously, to control hyperglycemia, inhibition of intestinal alpha-glucosidase is an established strategy (Tiwari et al., 2010; Ma et al., 2014). Although there have been reports on the clinical availability of alpha-glucosidase inhibitors, there is still need to develop alternative therapies to inhibit this key enzyme in order to minimize side effects and drug cost efficacy. In this regard, several natural substances from plant origins have been analyzed for their enzymatic inhibitory activities (Tiwari et al., 2010; Ma et al., 2014; Liu et al., 2014).
The α-glucosidase inhibitory activity of sugiol was found to be in a concentration dependent manner. The inhibitory effect of sugiol on alpha-glucosidase has been demonstrated in Figure 2. The sugiol at various concentrations (100, 500, 1,000, 5,000 and 10,000 µg/mL) showed the inhibition of alpha-glucosidase by 12.3, 24.5, 32.2, 51.3 and 63.5%, respectively. In case of standard acarbose at 100, 500, 1,000, 5,000 and 10,000 µg/mL, it displayed alpha-glucosidase inhibitory activity by 19.2, 29.9, 36.7, 57.1 and 65.5%, respectively. In this assay, the test compound sugiol showed inhibitory effect in a dose-dependent manner as did by the standard compound acarbose. Similar findings on alpha-glucosidase inhibitory activity of flavonoid and terpenoid compounds isolated from Agrimonia pilosa were observed by Liu et al. (2014). In addition, sarcoviolins isolated from edible mushroom Sarcodon leucopus were also found to inhibit alpha-glucosidase in vitro (Ma et al., 2014). Currently alpha-glucosidase inhibitors are used for the treatment of type 2 diabetes mellitus, suggesting that sugiol in formulation might be used as an oral hypoglycemic agent.
Figure 2: alpha-Glucosidase inhibitory effect of standard compound acarbose and sugiol isolated from Metasequoia glyptostroboides.
Data are expressed as mean ± SD (n = 3). Values with different superscripts are significantly different (p<0.05)
Inhibition of tyrosinase
Melanin is a polymerized natural coloring determinant in animal, plant, and microorganisms. A number of melanins are produced by multi-stepped enzymatic and non-enzymatic oxidation and polymerization processes. In mammals, the melanin can be divided into eumelanin and pheomelanin due to its solubility in an alkali solution and the color reaction (An et al., 2005). The mechanism of tyrosinase inhibition activity may be an important factor in the skin whitening of a cosmetic composition (An et al., 2005). Melanin biosynthesis steps in the body include L-DOPA by tyrosine as a substrate, followed by its conversion to L-dopaquinone by the successive enzymatic oxidations. Finally, a polymerization reaction takes place (An et al., 2005).
The inhibitiory effect of diterpenoid compound sugiol on the tyrosinase using a mushroom tyrosinase is demonstrated in Figure 3. In this assay, the sugiol at various concentrations (20, 40, 100, 200 and 500 µg/mL) showed 28.2, 39.9, 54.3, 59.5 and 67.4% inhibition of tyrosinase, respectively. Meanwhile, the mushroom tyrosinase inhibitory activity of standard compound kojic acid (20, 40, 100, 200 and 500 µg/mL) were found to be 32.4, 43.4, 60.8, 69.1 and 76.5%, respectively. Batubara et al. (2010) observed that the phytoconstituent taxifolin and some other flavanonol rhamnosides from K. malaccensis also suppressed tyrosinase activity in the range of 5.86-25.95%. Piao et al. (2002) demonstrated tyrosinase inhibitory effect of some chromones derived from a tropical plant aloe in a dose-dependent manner. In addition, the terpenoids isolated from the leaves of Chloranthus tianmushanensis were also found to exhibit tyrosinase inhibitor effect dose-dependently (Wu et al., 2008). Similar findings on dose-dependent tyrosinase activity of sugiol were observed in our study. Since it has been shown that tyrosinase inhibitors can repress the conversion of tyrosine to DOPA, dopaquinone and subsequently melanin, various tyrosinase inhibitors have been isolated and studied as potential candidates to decrease melanin content. Our results suggest that sugiol derived from M. glyptostroboides might be used as a potential treatment of melanin-related disorders to serve as skin- whitening agent (Chai et al., 2012).
Figure 3: Tyrosinase inhibitory effect of standard compound kojic acid and sugiol isolated from Metasequoia glyptostroboides.
Data are expressed as mean ± SD (n = 3). Values with different superscripts are significantly different (p<0.05)
In this research work, a diterpenoid compound sugiol isolated from M. glyptostroboides demonstrated a considerable amount of alpha-glucosidase and tyrosinase inhibitory effects in vitro which may have potential to reduce blood glucose responses and to maintain skill health by inhibiting melanogenesis. The potent alpha-glucosidase and tyrosinase inhibitory efficacy of sugiol in terms of anti-diabetic and skin-whiting efficacy makes it to be a molecule of choice for using in healthcare and drug therapies in the treatment of infectious diseases. However, further studies are needed to demonstrate a precise mode of action of sugiol to confirm its in vivo anti-diabetic and anti-melanin potential.
References
An BJ, Kwak JH, Park JM, Lee JY, Park TS, Lee JT, Son JH, Jo C, Byun MW. Inhibition of enzyme activities and the anti-wrinkle effect of polyphenol isolated from the Persimmon leaf (Diospyros kaki folium) on human skin. Dermatol Surg. 2005; 31: 848-54.
Bajpai VK, Na MK, Kang SC. The role of bioactive substances in controlling foodborne pathogens derived from Metasequoia glyptostroboides Miki ex Hu. Food Chem Toxicol. 2010; 48: 1945-49.
Bajpai VK, Kang SC. Isolation and characterization of biologically active secondary metabolites from Metasequoia glyptostroboides Miki Ex Hu. J Food Safety. 2011; 31: 276-83.
Bajpai VK, Kang SC. Antimycotic potential of a diterpenoid taxoquinone against Candida species isolated from Metasequoia glyptostroboides. Bangladesh J Pharmacol. 2014; 9: 154-60.
Batubara I, Kuspradini H, Mitsunaga T. Anti-acne and tyrosinase inhibition properties of taxifolin and some flavanonol rhamnosides from Kempas (Koompassia malaccensis). Wood Res J. 2010; 1: 45-49.
Chang HS, Cheng KP, Choang TF, Chow HF, Chui KY, Hon PM, Tan FWL, Yang Y, Zhong ZP. Structure elucidation and total synthesis of new tanshinones isolated from Salvia miltiorrhiza Bunge (Danshen). J Org Chem. 1990; 55: 3537-43.
Chai C, Ju HK, Kim SC, Park JH, Lim J, Kwon SW, Lee J. Determination of bioactive compounds in fermente d soybean products using GC/MS and further investigation of correlation of their bioactivities. J Chrom B. 2012; 880: 42-49.
Chen YS, Liou HC, Chan CF. Tyrosinase inhibitory effect and antioxidative activities of fermented and ethanol extracts of Rhodiola rosea and Lonicera japonica. Sci World J. 2013; 1: 1-5.
Fawole OA, Makunga NP, Opara UL. Antibacterial, antioxidant and tyrosinase-inhibition activities of pomegranate fruit peel methanolic extract. BMC Comp Alt Med. 2012; 12: 202-11.
Kwon SH, Hong SI, Kim JA, Jung YH, Kim SY, Kim HC, Lee SY, Jang CG. The neuroprotective effects of Lonicera japonica THUNB. against hydrogen peroxide-induced apoptosis via phosphorylation of MAPKs and PI3K/Akt in SH-SYSY cells. Food Chem Toxicol. 2011; 49: 1011-19.
Liu L, Deseo MA, Morris C, Winter KM, Leach DN. Investigation of α-glucosidase inhibitory activity of wheat bran and germ. Food Chem. 2011; 126: 553-61.
Liu X, Zhu L, Tan J, Zhou X, Xiao L, Yang X, Wang B. Glucosidase inhibitory activity and antioxidant activity of flavornoid compound and triterpenoids compound from Agrimonia Pilosa Ledeb. BMC Comp Alt Med. 2014; 14: 12-16.
Ma K, Han J, Bao L, Wei T, Liu H. Two sarcoviolins with antioxidative and α-glucosidase inhibitory activity from the edible mushroom Sarcodon leucopus collected in Tibet. Nat Prod. 2014; 77: 942-47.
Maritim AC, Sanders RA, Watkins JB. Diabetes, oxidative stress, and antioxidants: A review. J Biochem Mol Toxic. 2003; 17: 24-38.
Ozer O, Mutlu B, Kivcak B. Antityrosinase activity of some plant extracts and formulations containing ellagic acid. Pharma Biol. 2007; 45: 519-24.
Piao LZ, Park HR, Park YK, Lee SK, Park JH, Park MK. Mushroom tyrosinase inhibition activity of some chromones. Chem Pharm Bull. 2002; 50: 309-11.
Tiwari AK, Viswanadh V, Gowri PM, Ali AZ, Radhakrishnan SVS, Agawane SB, Madhusudana K, Rao JM. Oleanolic acid: An α-glucosidase inhibitory and antihyperglycemic active compound from the fruits of Sonneratia caseolaris. J Med Arom Plants. 2010; 1: 19-23.
Wu B, Chen J, Qu H, Cheng Y. Complex sesquiterpenoids with tyrosinase inhibitory activity from the leaves of Chloranthus tianmushanensis. J Nat Prod. 2008; 71: 877-80.
