Assessment of the anti-diabetic potential of the Cratoxylum formosum subsp. formosum extracts via carbohydrate hydrolyzing enzymes inhibitory activities

Introduction Diabetes mellitus is defined as a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. According to the World Health Organization (WHO) report, the global prevalence of diabetes in adults of age over 18 years has increased from 4.7% in 1980 to 8.5% in 2014. Moreover, the estimated 1.6 million deaths were directly caused by diabetes as reported in 2016 (1,2). The morbidity and mortality are not directly caused by diabetes but relatively resulted from the long-term complications in diabetes patients. Prolonged and uncontrolled hyperglycemia is a major risk, which is associated with diabetic angiopathy, neuropathy, nephropathy, and retinopathy (3). These chronic conditions definitively lead to serious clinical complications, e.g. cardiovascular and cerebrovascular disorders, chronic renal diseases, hypertension, and cataract. Thus, the glycemic control strategy is necessary for the prevention of morbidity and mortality of diabetes (4). Diabetes, especially type 2 diabetes, can be effectively managed by diet control, physical activity changes and anti-diabetic drugs (5). Several types of medications are utilized for therapeutic purposes such as biguanides, http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.36


Introduction
Diabetes mellitus is defined as a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. According to the World Health Organization (WHO) report, the global prevalence of diabetes in adults of age over 18 years has increased from 4.7% in 1980 to 8.5% in 2014. Moreover, the estimated 1.6 million deaths were directly caused by diabetes as reported in 2016 (1,2). The morbidity and mortality are not directly caused by diabetes but relatively resulted from the long-term complications in diabetes patients.
Prolonged and uncontrolled hyperglycemia is a major risk, which is associated with diabetic angiopathy, neuropathy, nephropathy, and retinopathy (3). These chronic conditions definitively lead to serious clinical complications, e.g. cardiovascular and cerebrovascular disorders, chronic renal diseases, hypertension, and cataract. Thus, the glycemic control strategy is necessary for the prevention of morbidity and mortality of diabetes (4). Diabetes, especially type 2 diabetes, can be effectively managed by diet control, physical activity changes and anti-diabetic drugs (5). Several types of medications are utilized for therapeutic purposes such as biguanides, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors and dipeptidyl peptidase-4 (DPP-4) inhibitors. However, these modern anti-diabetic drugs still produce serious adverse effects, for example, hypoglycemia, lactic acidosis, nausea, vomiting, pancreatitis, and metabolic acidosis (6). Therefore, the discovery of anti-diabetic drugs from natural origin with minimal or without any adverse effects has been extensively investigated. Different extracts from medicinal plants have been traditionally used to control diabetes globally, which are well-considered as relatively inexpensive, less toxic and minimal adverse effects (7). Therefore, the discovery of plasma glucose-lowering agents from edible plants is the priority of our research target. The Thai edible plant Cratoxylum formosum (Jacq.) Benth. & Hook. f. ex Dyer subsp. formosum belongs to the family Hypericaceae, which distributes mainly in North-eastern and Southern Thailand (8). The flowers, fresh shoots, and young leaves are served as vegetable in North-eastern and Southern Thai cuisines. Certain parts of C. formosum subsp. formosum have been used as Thai traditional medicines, for example, roots as a diuretic, leaves as for stomach pain relief and stems as a topical treatment for skin conditions. The major phytochemicals of the closest subspecies, C. formosum subsp. pruniflorum, are the prenylated xanthones, anthraquinones and phenolic compounds that have exhibited the topoisomerase I inhibitory, anti-cancer, anti-bacterial and cytotoxic activities (9)(10)(11)(12)(13)(14). To date, there is no report on the inhibitory activity from the flowers of C. formosum subsp. formosum on the carbohydrate-hydrolyzing enzymes. Thus, the objective of this study was to assess the anti-diabetic potential of the extracts from the flower, leaf, stem and root parts of C. formosum subsp. formosum on the inhibition of porcine pancreatic α-amylase (pAA), Saccharomyces α-glucosidase (SAG), and rat intestinal maltase (rIM) and sucrase (rIS).

Plant Materials
Plant samples of C. formosum subsp. formosum were collected from Roi-Et province, Thailand between October 2016 and March 2017. A plant specimen was identified and deposited at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham, Thailand (Voucher no. S. Sedlak 19-1). The leaf, flower, stem, and root parts of the plant were cleaned then dried in a hot-air oven at 50°C for 48 hours before the extraction process.
Extraction procedure Ground plant samples (400 g) were extracted by Soxhlet extractor using methanol as an extracting solvent for 6 hours. Methanolic extracts from each part were concentrated under the vacuum condition by rotary evaporator. The methanol extracts were suspended in deionized water and successfully partitioned with hexane (HEX), dichloromethane (DCM), and ethyl acetate (EtOAc), respectively. The organic solvents were removed to yield HEX, DCM, and EtOAc extracts. The extracts were stored at -20°C until use. The crude extracts were dissolved in 50% DMSO/H 2 O before the enzyme inhibitory assays.
Porcine pAA inhibitory assay The inhibitory assay of the crude extract was evaluated by the amyloclastic method. The assay was modified from the previous report to a 96-well microtiter plate format (15,16). The stock solution of porcine pancreatic αamylase was prepared by dissolving 50 mg of the enzyme in 10 mL of buffer (10mM phosphate saline buffer pH 6.9, 10mM CaCl 2 , and 0.04% NaN 3 ) to yield a 65 U/ mL of the enzyme. The working enzyme solution was diluted from a stock solution to a concentration of 0.1 U/ mL. The reaction mixture consisted of 40 µL of working enzyme solution, 50 µL of 1.25 mg/mL of amylose and 10 µL of crude extract was dissolved in 50% DMSO/H2O and incubated for 10 minutes at 37°C. The reaction was terminated by the addition of the 100 µL of iodine reagent (0.2mM KOI 3 , 4.2mM KI and 1.6mM HCl) and further incubated for 3 minutes. The starch-iodine complex was determined by measurement of absorbance at 655 nm. The inhibition percentage (IP) values of crude extracts were calculated according to the equation; All extracts were screened at the final concentration of 100 µg/mL in triplicate. The IP values from screening assay were expressed as mean ± SD. The IP was plotted against the crude extract concentration to obtain the amount of extract necessary to inhibit porcine pAA by 50% (IC 50 ). The data were obtained from 5 independent experiments and expressed as mean ± SEM, each conducted in triplicate.

SAG inhibitory assay
The α-glucosidase inhibitory assay was performed in a 96-well microtiter plate format (17). The stock enzyme solution of α-glucosidase from S. cerevisiae was prepared by dissolving enzyme in a buffer solution (0.5M phosphate buffer pH 6.5, 0.1% bovine serum albumin and 0.01% NaN 3 ). The working enzyme solution was diluted from a stock solution to a concentration of 3 U/ mL. The reaction mixture consisted of 20 µL of working enzyme solution, 70 µL of buffer solution and 10 µL of crude extract solution was pre-incubated for 5 minutes at 37°C. The reaction mixture contained 10 µL 50% DMSO/ H 2 O instead of a crude extract solution that was set as the blank. The reaction was started by adding 50 µL of 20mM NPG and further incubated for 20 minutes. The reaction was terminated by adding 50 µL of 1 M Na 2 CO 3 . The absorbance was measured at 405 nm against a buffer blank. All extracts were screened at the final concentration of 100 µg/mL in triplicate. The IP values from screening assay were expressed as mean ± SD. The IP and IC 50 values of crude extracts were calculated and plotted in the same manner of the porcine pAA inhibitory assay. The data were obtained from 5 independent experiments and expressed as mean ± SEM, each conducted in triplicate.
rIM and rIS inhibitory assay The rIM and rIS inhibitory assays were performed in a 96-well microtiter plate format (18). The 0.1 g of rat intestinal acetone powder was dissolved in 10 mL of 0.1 phosphate saline buffer pH 6.8 at 4°C overnight. The enzyme suspension was centrifuged (12 000 g at 4°C for 15 minutes) and the supernatant was kept for further enzymatic activity determination. The 30 μL of enzyme solution was incubated with 10 μL of 100mM maltose or sucrose as substrates at 37°C for 10 minutes. The reaction was terminated by heating in a hot bath at 100°C for 3 minutes and cooled down in tab water. The amount of glucose released from maltose and sucrose was assayed by the Glucose LiquiColor reagent kit. The maltase unit was defined as an amount of enzyme to convert 1μM of maltose to 2μM of glucose in 1 minute under the assay condition. The sucrase unit was defined as an amount of enzyme to convert 1μM of sucrose to 1μM of glucose in 1 minute under the assay condition. The enzyme solution was diluted to the concentration of 100 U/mL maltase and sucrose. The reaction mixture consisted of 10 µL of enzyme solution, 10 µL of crude extract dissolved in 50% DMSO/H2O and 30 µL of a substrate (10 mM maltose or 30 mM sucrose) and incubated for 10 minutes at 37°C. The reaction mixture containing 10 µL 50% DMSO/H 2 O instead of crude extract solution was set as blank. The reaction was terminated by incubation in a hot water bath for 5 minutes and cooled down in tap water before the determination of liberated glucose. The IP values of crude extracts were calculated according to the equation; All extracts were screened at the final concentration of 100 µg/mL in triplicate. The IP values from screening assay were expressed as mean ± SD. The IP was plotted against the crude extract concentration to obtain the amount of extract necessary to rIM and rIS by 50% (IC 50 ). The data were obtained from 5 independent experiments and expressed as mean ± SEM, each conducted in triplicate.

Statistical analysis
One-way ANOVA was used to test for overall differences. Significant ANOVA was followed by Duncan's multiple comparisons for consideration of pair-wised differences between treatment groups. A P value of less than 0.05 was considered statistically significant.

pAA inhibitory activities
According to the screening assay, the DCM and EtOAc extracts from the flowers, DCM extract from the roots, and CDM extract from the stems exhibited strong pAA inhibitory activities ( Figure 1A). These extracts were obtained for further determination for the inhibitory activity. The saccharolytic inhibition of the DCM (IC 50 5.4 ± 1.5 µg/mL) and EtOAc (IC 50 10.5 ± 0.6 µg/mL) extracts from the flowers were not significantly different with acarbose (IC 50 7.2 ± 0.4 µg/mL) ( Table 1). All extracts from the leaf part were inactive. The DCM extracts from the roots (IC 50 37.3 ± 1.1 µg/mL) and stems (IC 50 76.7 ± 12.1 µg/mL) were less potent than acarbose. By contrast, the EtOAc extracts from both parts were inactive.

SAG inhibitory activities
The result of SAG inhibitory activities demonstrated that DCM and EtOAc extracts from the flowers, EtOAc extract from the leaves, DCM and EtOAc from the roots, and EtOAc from the stems had significant inhibitory activities ( Figure 1B). The EtOAc extract (IC 50 20.4 ± 0.4 µg/mL) was more active than the DCM extract (IC 50 56.7 ± 8.9 µg/ mL) and acarbose (IC 50 431.4 ± 16.7 µg/mL), the referent compound (P < 0.05) ( Table 1). The EtOAc extract (IC 50 45.0 ± 3.5 µg/mL) from the leaves was also more potent than acarbose (P < 0.05), whereas the DCM extract was not active at the screening concentration (100 µg/mL). The EtOAc extract (IC 50 16.7 ± 3.5 µg/mL) from the roots was more potent than the DCM (IC 50 35.0 ± 6.7 µg/mL) and acarbose (P < 0.05). The inhibitory activity of the EtOAc extract (IC 50 31.1 ± 7.3 µg/mL) form the stems was more potent than acarbose (P < 0.05), while the DCM extract was inactive. rIM and rIS inhibitory activities As the results from intestinal maltase inhibitory assay, DCM and EtOAc extracts from the flowers, DCM and EtOAc extracts from the leaves, DCM and EtOAc extracts from the roots, and DCM and EtOAc extracts from the stem were subjected to evaluation for the rIM inhibitory Gluc se G P lucose (Figure 2A). The DCM (IC 50 8.5 ± 0.2 µg/mL) and EtOAc (IC 50 12.4 ± 0.3 µg/mL) extracts from the flowers were more potent than acarbose (IC 50 38.5 ± 7.2 µg/mL) (P < 0.05) ( Table 1). The DCM extract from the leaves (IC 50 76.8 ± 2.3 µg/mL) was less potent than the EtOAc extract (IC 50 45 ± 2.7µg/mL) and acarbose (P < 0.05). The DCM and EtOAc extracts from the roots were comparable to acarbose in the maltase inhibitory capability. The inhibitory activity of DCM extract (IC 50 67.40 ± 2.2 µg/ mL) from the stems was less active than the EtOAc one (IC 50 40.9 ± 1.5 µg/mL) and acarbose (P < 0.05). The DCM and EtOAc extracts from the flowers, EtOAc extract from the leaves, EtOAc extract from the roots, and EtOAc extract from the stems were obtained for the rIS inhibitory activity assay ( Figure 2B). The effect of CDM extract (IC 50 16.9 ± 1.5 µg/mL) from the flowers was the  same as the acarbose (IC 50 15.5 ± 1.2 µg/mL). By contrast, the EtOAc extract from the flowers (IC 50 39.9 ± 3.1 µg/ mL) was less potent than acarbose (P < 0.05) ( Table 1). The HEX and DCM extracts from the leaves were inactive under the assay condition. The EtOAc extract (IC 50 61. 8 ± 1.5 µg/mL) from the leaves was less active than acarbose (P < 0.05). The HEX and DCM extracts from the roots and stems were inactive. The EtOAc extracts from the leaves (IC 50 60.0 ± 2.5 µg/mL) and stems (IC 50 75.7 ± 4.1 µg/mL) were less active than acarbose (P < 0.05).

Discussion
Certain α-glucosidase inhibitors (acarbose, miglitol, voglibose) are one group of the diabetic drugs for the patient's glycemic control, which competitively inhibit carbohydrate-hydrolyzing enzymes to delay digestion of carbohydrates and absorption of the monosaccharides (19,20). Human pAA is the first intestinal enzyme that catalyses the hydrolysis of 1,4-glucan linkage in the ingested starch to yield maltose, maltotriose, several of α-(1,6) and α-(1,4) oligoglycans, and a small amount of glucose. The 3D structural comparison by X-ray diffraction demonstrated that human pAA had a high degree of structural identity to the porcine enzyme (21). Moreover, the enzyme kinetic characteristics and enzymeinhibitor interaction (acarbose) between human and porcine enzymes were not significantly different (22). Thus, the porcine pAA was reasonable to use this enzyme as the experimental target in this study. These results led to the conclusion that the flowers of C. formosum subsp. formosum possessed the potent pAA inhibitory activity as compared to acarbose. Saccharomyces cerevisiae α-glucosidase is a 1,4-α-glycosidic bond-specific hydrolase widely used as a target for the searching of anti-diabetic agents from natural origins (23). The results revealed that the EtOAc from the roots and flowers were the most active extracts among different extracts from this plant. HEX extracts from all parts were completely inactive under the assay concentration (100 µg/mL). Maltose is the disaccharide that is mainly released from the hydrolysis of starch catalysed by intestinal α-amylase. Maltase is the membrane-bound enzyme located in the intestinal brush border, and is the last enzyme to cleave maltose to release two molecules of glucose before intestinal absorption. Sucrase is another membrane-bound enzyme which hydrolyses ingested sucrose to glucose and fructose (24). The rIM and rIS were acceptable models for the humanliked maltase and sucrase inhibitory assays (25). The results demonstrated that the CDM extracts from the flowers were the most active extracts of this plant in the rIM and rIS inhibitory assays.
The investigation of porcine pAA, SAG, rIM and rIS inhibitory activities demonstrated that the flower part of C. formosum subsp. formosum displayed the strongest anti-diabetic potential. The DCM and EtOAc extracts exhibited the inhibitory activities while the HEX was completely inactive. This result indicated that the nonpolar constituents dissolved in hexane from the flowers were not the active ingredients. There were no phytochemical and biological activities reported from the flowers of this plant. The polar extract, EtOAc extract, from the leaf part of C. formosum subsp. formosum showed potent SAG, rIM and rIS inhibitory activities. Recently, the phenolic compounds from the ethanolic extract of C. formosum subsp. formosum leaves exhibited the in vitro protective effects against H 2 O 2 -induced cell death, and antioxidant activities (26). The DCM root extract exhibited moderate pAA, rIM, and rIS inhibitory activities. Besides, the EtOAc extract from the roots showed strong α-glucosidase inhibitory activity. The phytochemical constituents have not been reported from the roots of this subspecies. Several prenylated xanthones, anthraquinones, flavonols, and phenolic compounds have been isolated and identified from the roots of C. formosum (27,28). Moreover, certain caged and prenylated xanthones were isolated from the roots of C. cochinchinense (29,30). The polar part of the stems, EtOAc extract, showed strong SAG, and potent rIM and rIS inhibitory activities. The chemical constituents from the stems of C. formosum ssp. pruniflorum and C. cochinchinense were prenylated xanthone, benzophenone glycosides and bisanthraquinone (31)(32)(33).

Conclusion
The inhibitory effects of the different parts of the C. formosum subsp. formosum demonstrated the antidiabetes potential. The DCM and EtOAc extracts from the flowers and roots showed the significant saccharolytic inhibitory activities via the pAA, SAG, rIM, and rIS inhibitory activities. The DCM extracts from the leaves and stems also showed potent rIM inhibitory activity, whereas the EtOAc extracts from these parts exhibited the significant SAG, rIM, and rIS inhibitory activities. The obtained results indicated that these active extracts possibly inhibited the carbohydrate-hydrolyzing enzyme through the interferences of catalytic activity. However, the exact mechanisms of enzymatic inhibition were not revealed by the defined assay protocol. Thus, extensive enzyme kinetic studies are needed to clarify the inhibitory mechanisms. Although the in vitro study indicates the possibility of this plant for the prevention and treatment of diabetes, the in vivo study is extensively needed to assess the therapeutic effectiveness and toxicological effects for therapeutic purposes.
Authors' contribution SS collected and identified the plant specimens. KA and BT tested the enzyme inhibition and analysed the data. SS conducted the project and wrote the paper. All authors read and confirmed the publication of the paper.

Conflict of interests
The authors declare no conflict of interest.

Ethical considerations
Ethical issues (including plagiarism, misconduct, data fabrication, falsification, double publication or submission, redundancy) have been completely observed by the authors. The authors affirm that no animal and human subjects were involved in this research.