Diabetes and its complications had affected approximately 415 million people in 2015, and the number of diabetic patients has been projected to grow to 642 million by 2040 (Ogurtsova et al., 2017). An individual with diabetes mellitus has an increase in blood sugar due to faulty insulin from the pancreas or a malfunction in the cells’ response to the insulin (Devaki et al., 2016). As a result of long-term hyperglycemia in diabetes mellitus, advanced glycation end products (AGEs) are formed, which can lead to diabetic complications (Singh et al., 2014). To reduce postprandial hyperglycemia, enzyme inhibitors that act on carbohydrate metabolism, such as α-amylase and α-glucosidase, are effective (Wang et al., 2013; You et al., 2012). Recently, there has been much interest in using antidiabetic and antiglycation products for relieving diabetes and its complications. Synthetic drugs have been reported to cause several side effects. Therefore, many studies have been focused on finding alternative therapies, especially plant-derived natural products that are used in managing diabetes and its complications due to low toxicity and low side effects.
Natural phytochemicals found in plants have a multitude of health-promoting properties, including antidiabetic, antioxidant, anti-inflammatory, antibacterial, antihypertensive, and anti-inflammatory activities. A large genus in Orchidaceae, Dendrobium, has about 1,400 species distributed throughout the Himalayas, Asia, Australia, Tasmania, and the Pacific Islands (Xu et al., 2006). More than 150 species of this plant genus in Thailand have been identified (Seidenfaden, 1985). Recent pharmacological investigations have shown that the plants of the genus Dendrobium produce structurally different components resulting in a wide range of biological properties, including anticancer, antioxidant, antimalarial, and antidiabetic activities (Ng et al., 2012). Inhibitory potential was shown for the whole plant extracts of D. formosum Roxb. ex Lindl. against α-glucosidase and pancreatic lipase enzymes (Inthongkaew et al., 2017). Two new dihydrophenanthrenes, dendroinfundin A and dendroinfundin B, were isolated from the whole plant of D. infundibulum Lindl. and showed strong α-glucosidase inhibitory activity (Na Ranong et al., 2019). In addition, a methanol extract from the root of D. christyanum Rchb.f. exhibited α-glucosidase inhibitory activity and glucose uptake stimulatory effect (San et al., 2020); also, the whole plant of D. scabrilingue Lindl. displayed the most potent α-glucosidase inhibitory activity (Sarakulwattana et al., 2018). A Dendrobium species is considered an important phytochemical used in antidiabetic products; several species have not yet been reported in antidiabetic and antiglycation products. The main aim of this study was focused on investigating the in vitro antidiabetic and AGEs inhibitory activity of the methanol extracts of 20 Dendrobium species.
MATERIALS AND METHODS
Plant materials and plant extracts
The whole plant of Dendrobium orchids was purchased from Chatuchak Market, Bangkok, Thailand, and identified by one of the authors (B. Sritularak). The dried and powdered whole plant of samples was extracted with methanol (MeOH commercial grade) at room temperature by the maceration method. The sample was extracted three times in MeOH using a ratio of 1 kg/10 l. The methanol extracts of 20 species of Dendrobium orchids were obtained after removal of the solvent by using an evaporator and desiccator.
The methanolic extracts of 20 species of Dendrobium orchids were subjected to qualitative chemical tests to detect the presence of various classes of phytoconstituents, including alkaloids, flavonoids, anthraquinones, coumarins, saponins, tannins, terpenoids, steroids, and glycosides. The qualitative analysis of secondary metabolites was carried out by following the methods of Yadav and Agarwala (2011) and Singh et al. (2015).
Quantification of bioactive compounds
The bioactive compounds phenols and flavonoids were quantified according to the standard procedure in a 96-well plate with a microplate reader (BioTek, USA Model Epoch 2 Gen5). A total of 20 species of Dendrobium orchids extracts were subjected to quantify the total phenolic and total flavonoid contents. The total phenolic contents in the Dendrobium orchids extracts were measured using the Folin-Ciocalteu method explained by a modified Miliauskas et al. (2004) method and were reported as mg gallic acid equivalents (GAE) per gram of dry extract. The total flavonoid content of Dendrobium orchids extracts was considered by the aluminum chloride colorimetric assay by a modified Chatatikun and Chiabchalard (2013) method and was reported as mg quercetin equivalents (QE) per gram of dry extract.
In vitro antidiabetic assays
α-amylase inhibitory assay by CNPG3
The in vitro α-amylase inhibition activity of Dendrobium orchids extracts from 20 species was determined based on the 2-chloro-4-nitrophenol (CNP) colorimetric assay using acarbose as the positive control by a modified Kumar et al. (2011) method. The plant extracts were dissolved in a sodium phosphate buffer (pH 6.9) to give concentrations in the range of 6.25–100 μg/ml. The enzyme α-amylase solution was prepared by dissolving of 0.5 U/ml of α-amylase in 40 mmol/l phosphate buffer, pH 6.9. The assays were conducted by mixing 80 μl of Dendrobium orchids extracts, 20 μl of the α-amylase solution, and 50 μl of CNPG3. The mixture was incubated at 37°C for 10 minutes. Finally, 50 μl of a 1 mM Na2CO3 solution was added to terminate the reaction. The absorbance was measured at 405 nm with a microplate reader (BioTek, USA Model Epoch 2 Gen5). A control reaction without the plant extract/acarbose was also conducted. From the graph, the IC50 values for α-amylase inhibition were determined based on the percent inhibition plotted against extract concentration.
α-glucosidase inhibition assay
The activity of α-glucosidase was measured as described previously, with slight modifications (Inthongkaew et al., 2017). By hydrolyzing, specifically by α-glucosidase, p-nitrophenyl-α-D-glucopyranoside (pNPG) can be converted into p-nitrophenol (a yellow-colored product). In brief, 50 μl of 20 species of Dendrobium orchids extracts (25–250 μg/ml) was mixed with 80 μl of a 0.1 M phosphate buffer (pH 6.8), and 20 μl of 0.2 U/ml α-glucosidase in a 0.1 M phosphate buffer (pH 6.8) was added to a 96-well plate, and the mixture was preincubated at 37°C for 10 minutes. Then, 50 μl of 2 mM pNPG was added, and the reaction mixture was further incubated for 20 minutes. Finally, 50 μl of a 0.1 M Na2CO3 solution was added to terminate the reaction. The absorbance was measured at 405 nm with a microplate reader (BioTek, USA Model Epoch 2 Gen5). Acarbose was used as the positive control. IC50 values were calculated by plotting percent inhibition of α-glucosidase against the extract.
Inhibition of AGEs formation
The inhibitory effects of the extracts of 20 Dendrobium orchids species on AGEs formation were analyzed by a modified Jung et al. (2015) method. The AGEs reaction solution that consisted of bovine serum albumin (BSA) (10 mg/ml), D-glucose (0.2 M), D-fructose (0.2 M), and 0.02% w/v of sodium azide in a phosphate buffer (0.05 M, pH 7.4) was prepared. The reaction mixture (900 µl) of the AGEs reaction solution was incubated at 37°C for 7 days (in the dark) with or without 100 µl of Dendrobium orchids extracts (31.25–250 µg/ml) dissolved in the phosphate buffer. Aminoguanidine was used as the positive control (15.625–250 µg/ml). The fluorescence intensity of the reaction products was evaluated using a fluorescence spectrometer (PerkinElmer model LS-55) with respective excitation and emission wavelength at 355 and 450 nm. Based on the graph, the IC50 of the extracts were measured based on the percent inhibition of AGEs formation.
At least three experiments were performed, and the results were expressed as mean ± SD. Statistically significant differences were determined by Student’s t-test, and p-values < 0.05 were considered significantly different.
Various species of Dendrobium orchids in this research are shown in Figure 1. The dried and powdered whole plant of 20 species of Dendrobium orchids was macerated with MeOH to obtain the MeOH extracts. The preliminary phytochemicals were identified in the MeOH extracts using various chemical tests. The presence of phytochemicals is shown in Table 1. The methanolic extract of 20 species of Dendrobium orchids shows the presence of major phytoconstituents like alkaloids, flavonoids, coumarins, tannins, terpenoids, and glycosides.
Total phenolic and flavonoid contents
The total phenolic and total flavonoid contents in the whole plant of 20 Dendrobium orchids extracts are presented in Table 2; it could be noticed that D. formosum Roxb. ex Lindl. showed the highest phenolic compounds at a value of 132.70 ± 3.41 mg GAE/g. The total phenolic contents of D. tortile Lindl., D. williamsonii Day & Rchb. f., D. scabrilingue Lindl., and D. bellatulum Rolfe. were evaluated in the range of 111.48–119.92 mg GAE/g.
The highest total flavonoid content was recorded in the methanol extract of D. brymerianum Rchb.f. at a value of 237.57 ± 1.19 mg QE/g, followed by D. tortile Lindl. and D. scabrilingue Lindl. at the values of 228.82 ± 7.64 mg QE/g and 214.93 ± 1.43 mg QE/g, respectively. Moreover, the total flavonoid content in the high value was found in D. formosum Roxb. ex Lindl., D. williamsonii Day & Rchb. f., and D. ochreatum Lindl. in the range from 179.51 to 197.73 mg QE/g.
In vitro α-amylase inhibitory activity
In the preliminary evaluation, the α-amylase inhibitory activity of Dendrobium orchids extracts at the concentration of 100 µg/ml exhibited higher than 70% inhibition (Fig. 2). The IC50 values were calculated (Table 3). The MeOH extract of D. parishii Rchb.f. exhibited the lowest IC50 of 46.57 ± 0.33 μg/ml, and the IC50 values of the D. albosanguineum Lindl., D. ochreatum Lindl., and D. brymerianum Rchb.f. extracts were 51.87 ± 0.61, 54.45 ± 0.47, and 67.12 ± 0.41 μg/ml, respectively. The standard positive control, acarbose, showed an IC50 of 17.43 ± 0.67 μg/ml.
In vitro α-glucosidase inhibitory activity
Six methanolic extracts of Dendrobium orchids exhibited higher than 50% of α-glucosidase inhibitory activities at the concentration of 250 µg/ml (Fig. 3). The IC50 values were calculated (Table 3). Surprisingly, the α-glucosidase inhibitory potential of D. ellipsophyllum Tang & Wang. (IC50 128.69 ± 1.16 µg/ml) and D. brymerianum Rchb.f. (IC50 138.27 ± 1.60 µg/ml) was significantly higher than acarbose (IC50 166.83 ± 1.96 µg/ml) of the analyzed standard positive control. In addition, the MeOH extract of D. signatum Rchb. f. and D. scabrilingue Lindl. was characterized by the high inhibitory potential with IC50 of 166.86 ± 1.26 and 169.96 ± 0.83 μg/ml, respectively.
The formation of AGEs inhibition
The methanol extracts of all Dendrobium orchids, excluding the five crude extracts of D. tortile Lindl., D. cretaceum Lindl., D. palpebrae Lindl., D. formosum Roxb. ex Lindl., and D. williamsonii Day & Rchb. f., inhibited the formation of AGEs in a dose-dependent manner (data not shown). The IC50 values of AGEs formation inhibitory activities are presented in Table 4 and Figure 4. The inhibition for the formation of AGEs of D. brymerianum Rchb.f. (IC50 34.44 ± 1.29 µg/ml) and D. scabrilingue Lindl. (IC50 46.95 ± 0.91 µg/ml), involved in the inhibition of the sugar-adding reaction of BSA molecules or anti-AGEs, is more effective than aminoguanidine (IC50 49.81 ± 4.50 µg/ml). Moreover, the MeOH extracts of D. infundibulum Lindl., D. albosanguineum Lindl., and D. ellipsophyllum Tang & Wang. were considered by the high AGEs inhibitory potential with IC50 of 51.50 ± 2.25, 55.00 ± 2.06, and 59.05 ± 1.62 μg/ml, respectively. In this report, a strong AGEs inhibitory effect of the nine methanol extracts of Dendrobium orchids was indicated, as well (IC50 lower than 100 µg/ml) (Table 4, Fig. 4).
|Figure 1. Dendrobium orchid species used in this study.|
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|Table 1. Phytochemical constituents of the methanolic extract of 20 species of Dendrobium orchids.|
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|Table 2. Quantitative estimation of phytochemicals.|
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|Figure 2. Dendrobium orchids extracts were detected for α-amylase inhibitory activity. D. parishii Rchb.f. (♦), D. albosanguineum Lindl. (?), D. ochreatum Lindl. (?), and D. brymerianum Rchb.f. (?), whereas the other Dendrobium species were inactive at concentration up to 100 μg/ml.|
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|Table 3. IC50 values of the methanolic extracts of 20 species of Dendrobium orchids for α-amylase and α-glucosidase inhibitory activities.|
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|Figure 3. Dendrobium orchids extracts were detected for α-glucosidase inhibitory activity. D. ellipsophyllum Tang & Wang. (♦), D. brymerianum Rchb.f. (?), D. signatum Rchb.f. (?), D. scabrilingue Lindl. (?), D. williamsonii Day & Rchb.f. (?), and D. cretaceum Lindl. (?), whereas the other Dendrobium species were inactive at concentration up to 250 μg/ml.|
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|Table 4. Inhibition of AGEs formation by the methanolic extracts of 20 species of Dendrobium orchids.|
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In traditional Chinese medicine, the stem of several species of Dendrobium known as “Shi-Hu” has been used as a tonic and antipyretic to promote body fluid production and reduce fever (Lam et al., 2015; Xu et al., 2013;). Previous studies have shown that Dendrobium plants produce many classes of metabolites, including polysaccharides, alkaloids, coumarins, bibenzyls, phenols, phenanthrenes, and terpenoids constituents (Cakova et al., 2017; Xu et al., 2013). Several chemical structures of these compounds showed pharmacological activities, including antioxidant, anticancer, antimicrobial, antifungal, antiherpetic, antidiabetic, anti-inflammatory, and antimalarial activities (Hei et al., 2021). Recently, much research has reported Dendrobium possess outstanding antidiabetic activity, for example, D. nobile Lindl., D. loddigesii, D. crepidatum, D. huoshanense, D. officinale Kimura & Migo, and D. chrysotoxum Lindl. (Hei et al., 2021; Lu et al., 2014; Qian et al., 2014; Xu et al., 2019). In the present investigation, many species of Dendrobium orchids have never been reported for antidiabetic and inhibitory activity against AGEs formation. In this research, a preliminary of phytochemical constituents was indicated, as well. The methanolic extracts of 20 species of Dendrobium orchids were determined to show the presence of major phytoconstituents, like alkaloids, flavonoids, coumarins, tannins, terpenoids, and glycosides (Table 1). Regarding the total phenolic and flavonoid contents (Table 2), it could be noticed that D. formosum Roxb. ex Lindl. contained the highest phenolic compounds at a value of 132.70 ± 3.41 mg GAE/g and the high value of total flavonoid content was recorded in D. brymerianum Rchb.f., D. tortile Lindl., and D. scabrilingue Lindl. at the values of 237.57 ± 1.19, 228.82 ± 7.64, and 214.93 ± 1.43 mg QE/g, respectively. The results support the previous studies; an extract rich in polyphenols from Dendrobium was used in mice to treat diabetes (Li et al., 2018). 12 phenolic compounds were isolated from D. formosum Roxb. ex Lindl. and showed antidiabetic and antiobesity properties (Inthongkaew et al., 2017). Therefore, phytochemical screening serves as the first initial step to predicting the kinds of potential active compounds from Dendrobium plants. Several studies showed that Dendrobium has antidiabetic activity and acts as a good inhibitor of crucial enzymes like α-amylase and α-glucosidase associated with type 2 diabetes (Cakova et al., 2017; Hei et al., 2021; Lam et al., 2015). In this report, the methanolic extracts of Dendrobium orchids also reveal better in vitro enzyme inhibitory activity (α-amylase and α-glucosidase), which are involved in the regulation and absorption of carbohydrates (Table 3). In particular, D. ellipsophyllum Tang & Wang. and D. brymerianum Rchb.f. exhibited potent α-glucosidase inhibitory activities at the IC50 values of 128.69 ± 1.16 and 138.27 ± 1.60 µg/ml, respectively, compared to acarbose (IC50 166.83 ± 1.96 µg/ml). In the cited reference, D. tortile Lindl., D. christyanum Rchb.f., D. infundibulum Lindl., and D. formosum Roxb. ex Lindl. exhibited strong α-glucosidase inhibitory activities, which is inconsistent with this study’s results. The solvent EtOAc partition of separated extract may result in strong α-glucosidase inhibitory effects (Inthongkaew et al., 2017; Limpanit et al., 2016; Na Ranong et al., 2019; San et al., 2020). Besides, persistent hyperglycemia induces increased formation of AGEs, which play an important role in the pathogenesis of diabetic complications. At this point, we investigated the AGEs formation inhibitory activities of 20 species of Dendrobium orchids; several species exhibited potent inhibitory activity against AGEs formation. It is interesting to note that D. brymerianum Rchb.f. (IC50 34.44 ± 1.29 µg/ml) and D. scabrilingue Lindl. (IC50 46.95 ± 0.91 µg/ml) showed higher AGEs inhibitory activities than aminoguanidine, as a reference compound (IC50 49.81 ± 4.50 µg/ml). Furthermore, D. infundibulum Lindl., D. albosanguineum Lindl., and D. ellipsophyllum Tang & Wang. also showed strong AGEs inhibitory potential with IC50 values close to that of the standard substance (Table 4). The results from our investigation have established a basis for the future development of inhibition of diabetes and its complications.
|Figure 4. Inhibition of AGEs formation by the methanolic extracts of Dendrobium orchids. A bar graphical plot of the data expressed as the half maximal inhibitory concentration (IC50). p- values less than 0.05 (p < 0.05) indicate significant differences among data with a different letter.|
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According to the result, the methanol extracts of several Dendrobium species are effective against diabetes and AGEs formation. In particular, D. brymerianum Rchb.f. has potent inhibitory effects on α-amylase and α-glucosidase and anti-AGEs formation. A further benefit of this study is that it opened up the possibility of new drugs for diabetics with antidiabetic and AGEs inhibitory properties.
The authors are grateful to the Department of Biology, Faculty of Science, Ramkhamhaeng University, and Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for providing research facilities.
Boonchoo Sritularak collected and identified plant materials. Boonchoo Sritularak performed the extraction of plant samples. Thaniwan Cheun-Arom initiated and designed the experiments, performed the experiments, and analyzed the data. Thaniwan Cheun-Arom revised the manuscript. The final version of the manuscript was approved by all authors.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
This research was funded by the Research and Development Institute, Ramkhamhaeng University.
This study does not involve experiments on animals or human subjects.
All data generated and analyzed are included in this research article.
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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