Pharmacogn Res. 2022; 14(1):89-99

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Original Article

Antidiabetic Activities of Medicinal Plants in Traditional Recipes and Candidate Antidiabetic Compounds from Hydnophytum formicarum Jack. Tubers

Mingkwan Rachpirom1,2, Louis R Barrows3, Suriyan Thengyai4, Chitchamai Ovatlarnporn5, Chonlatid Sontimuang6, Pitchanan Thiantongin7, Panupong Puttarak1,2,*

Mingkwan Rachpirom1,2, Louis R Barrows3, Suriyan Thengyai4, Chitchamai Ovatlarnporn5, Chonlatid Sontimuang6, Pitchanan Thiantongin7, Panupong Puttarak1,2,*

1Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, THAILAND.

2Phytomedicine and Pharmaceutical Biotechnology Excellence Center, Prince of Songkla University, Hat-Yai, Songkhla, THAILAND.

3Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, UT, USA.

4Drug and Cosmetics Excellence Center, Walailak University, Thasala, Nakhonsithammarat, THAILAND.

5Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, THAILAND.

6Department of Thai Massage and Midwifery, Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat-Yai, Songkhla, THAILAND.

7Faculty of Thai Traditional and Alternative Medicine, Ubon-Ratchathani Rajabhat University, Meung, Ubon-Ratchathani, THAILAND.

Correspondence

Assoc. Prof. Dr. Panupong Puttarak

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla-90112, THAILAND.

Email id: [email protected]

History

Submission Date: 31-10-2021;

Review completed: 12-11-2021;

Accepted Date: 21-12-2021

DOI : 10.5530/pres.14.1.13

Article Available online

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Copyright

© 2022 Phcog.Net. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

Introduction: Wang-Nam-Yen hospital recipe used as a traditional antidiabetic for a long time, but its beneficial properties have not been described. Materials and Methods: Antidiabetic mechanisms including anti-α-glucosidase, anti-α-amylase, anti-dipeptidyl-peptidase-4, antioxidant, anti-inflammatory activities, and wound healing effects of 26 medicinal plants that make up the Wang-Nam-Yen preparation were investigated. Results: Interestingly, most plants in this study inhibited α-glucosidase and α-amylase at excellent levels with higher potency than standard acarbose (18.2% ± 0.5). Hydnophytum formicarum, Urceola minutiflora, and Lagerstroemia speciosa, inhibited DPP-4 with more than 70% inhibition at 50 μg/mL (82.8%± 0.8, 71.9% ± 0.3, and 71.1% ± 0.1, respectively compared with standard Diprotin A at 50 μg/mL, 90.1% ± 0.4). Terminalia arjuna showed the highest inhibition in all anti-oxidation assays. Andrographis paniculata inhibited NO production at 90.1% ± 2.4, which was more effective than indomethacin (34.3% ± 2.4). Most of the herbs contained high amounts of terpenoids and flavonoids, which might play an important role in antidiabetic activity. The results demonstrated that H. formicarum extract exhibited the highest anti-DPP-4 activity (IC50 = 33.87 ± 0.02 µg/mL). When H. formicarum ethanolic extract was isolated, Palmitic acid (1) exhibited DPP-4 inhibitory activity at IC50 value of 73.82 ± 2.64 µg/mL, and a mixture of stigmasterol (2) and ß-sitosterol (3) at 78.58 ± 0.92 µg/mL. Conclusion: Many herbs in the Wang-Nam-Yen preparation possessed properties predictive for antidiabetic treatment. The results also suggest the possibility of further use of H. formicarum and its isolated compounds as a standard diabetic drug in the future.

Key words: Anti-DPP-4 activity, Anti-α-glucosidase activity, Anti-α-amylase activity, antioxidant activity, Anti-inflammation activity, Antidiabetic recipe.

Cite this article: Rachpirom M, Barrows LR, Thengyai S, Ovatlarnporn C, Sontimuang C, Thiantongin P, et al. Antidiabetic Activities of Medicinal Plants in Wang Nam Yen Hospital Recipes and Candidate Antidiabetic Compounds from Hydnophytum formicarum Jack. Tubers. Pharmacog Res. 2022;14(1):89-99.

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INTRODUCTION

Diabetes mellitus (DM) is a public health syndrome in which up to 422 million adults worldwide have been diagnosed, and the number of DM patients is rising continuously. DM isa result of a disturbance in glucose metabolism and presents as an elevation of fasting blood sugar. The disease causes substantial morbidity, mortality, and long-term complications and leading to a significant risk factor for various chronic diseases such as cardiovascular disease.[1-4] In the early stages, DM does not distinctly affect the daily life of patients; however, long term elevations of blood glucose often leads to micro-and macro-vascular complications, including retinopathy, nephropathy, neuropathy, peripheral vascular disease, and cerebrovascular disease, all of which are associated with quality of life reduction.[1,5] Usually, the treatment of DM aims to maintain lower blood sugar levels and prevent those complications. Metformin is the first-line drug for mild to moderate type-2 DM (T2DM) as monotherapy or in combination with sulfonylurea and/or /-glucosidase inhibitor, and when unsatisfactory, a third agent, such as dipeptidyl-peptidase-4 (DPP-4), a thiazolidinedione, or glucagon-like peptide-1 (GLP-1) agonist is required.[2,6,7] α-glucosidase and a-amylase are digestive enzymes that play roles in breaking down the polysaccharide to monosaccharide. The inhibition of i-glucosidase and --amylase has proven to be effective in lowering glucose uptake by reducing an absorbable monosaccharide.[2,8,9] Synthetic drugs or dietary supplements with their actions as -glucosidase and -amylase inhibitors have become alternatives in the treatment of DM.[6] Several -glucosidase and --amylase inhibitors are commercially available such as acarbose, voglibose, and miglitol.[9] Other than non-serious gastrointestinal side-effects, these agents provide a helpful action in controlling DM. The incretin hormones, comprised of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP, also known as a gastric inhibitory polypeptide), are secreted from L-cell and K-cell, respectively, in response to food ingestion. They regulate insulin release, improve insulin resistance, and lower gastric emptying time. Unfortunately, GLP-1 and GIP persist in the bloodstream for a very short time after secretion due to the degradation by DPP-4.[2,8,10] Therefore, inhibition of DPP-4 can prevent the hydrolysis of intact GLP-1 levels and has been proven to be an effective mechanism in lowering blood sugar. Presently, DPP-4 is an attractive target for discovering additional oral antidiabetic drugs.

Moreover, it was reported that the antioxidant defense mechanisms in DM patients and in patients with DM-associated metabolic syndrome are attenuated and result in higher levels of oxidative stress in these individuals.[11,12] Increased oxidative stress is reported to promote the development and progression of DM and its complications.[11-13] The formation of advanced glycated end products from elevated glucose can promote free radicals and reactive oxygen species and worsen the disease.[11,12] The advanced glycated end products can activate the NF-kB transcription factor and increase nitric oxide level, contributing to the inflammatory condition.[14,15] Also, DM patients have been in trouble with wound complications from the impairment of microcirculation, immune function, collagen accumulation, the proliferation of keratinocytes, and fibroblast migration.[16]

Many Thai-traditional antidiabetic recipes are extensively accepted as alternatives for holistic care in DM patients to lower blood glucose levels, slowing the progress of DM complications, and even wound healing.[16-19] Although the acceptance by traditional doctors, patients, and routine usage over a decade reflects clinical effectiveness, scientific evidence that describes the biological activities associated with DM benefit of component plants is limited. This study aims to evaluate several antidiabetic activities of individual plant components of Wang Nam Yen recipes, including anti-α-glucosidase, anti-α-amylase, anti-DPP-4, antioxidant, anti-inflammatory, and skin proliferative activity. Twenty-six plants from the Thai-traditional recipe of Wang-Nam-Yen hospital, Thailand, were selected for the study. This preparation has been routinely used in the hospital. In addition, one plant that demonstrated exceptional anti-DPP-4 activity was extracted and fractionated, and pure compounds showing anti-DPP-4 activity were isolated using chromatographic methods. These bioactive compounds may be used as biomarkers for product quality assurance of Wang Nam Yen preparations in the future.

MATERIALS AND METHODS

Chemicals and reagents

DPP-4 enzyme from porcine kidney, trichloroacetic acid, Folin-Ciocalteu reagent, and 1,10-phenanthroline chloride monohydrate were obtained from Merck (Darmstadt, Germany). Indomethacin standard, gallic acid standard, quercetin standard, ascorbic acid standard, 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), gly-pro-p-nitroanilide (GP-pNA) as p-toluene sulfonate salt, lipopolysaccharide (LPS) from Escherichia coli 0111:B4, RPMI-1640 medium, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma-Aldrich (MO, USA). Penicillin, streptomycin, fetal calf serum, trypsin–EDTA, and Dulbecco’s Modified Eagle Medium (DMEM) from Thermo Fisher Scientific (CA, USA) were used for cell culture study. The solvents were purchased from RCI Labscan (Thailand). The mouse fibroblast L929 cells were obtained from the Chinese Academy of Preventive Medical Sciences, Beijing, China. Murine macrophage-like RAW264.7 cell-line (ATCC® TIB-71TM) was kindly provided by the Medical Science Research and Innovation Institute, Prince of Songkla University.

Plant materials and crude extracts preparation

The twenty-six medicinal plants used in this study are listed in Table 1. All plants were bought from local Thai-traditional drug stores in Songkhla, Thailand. Plant materials were authenticated by a botanist from the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University. The plant specimens were also deposited, as shown in Supplement 1. After drying at 50 - 55°C, each dried plant was powdered with an electric grinder and stored the plant powder at -20°C until use. Each plant powder (30 g) was macerated in ethanol (~150 mL) at room temperature for two days. The macerated extract was dried under reduced pressure to yield the ethanolic crude extract. Each sample powder was refluxed in water over 90 min for aqueous extract preparation. After cooling, the extract was filtered through a filtering paper (Whatman® No. 1), and the filtrate was then freeze-dried to obtain the aqueous extract. All extracts were kept at - 20°C and protected from light until use.

Table 1: The list of 26 Thai medicinal plants used in this study.

Abbreviation Scientific name Part of uses
AHW Abutilon hirtum (Lam.) Whole plant
AEW Acanthus ebracteatus Vahl. Whole plant
AMH Albizia myriophylla Benth. Heartwood
APL Andrographis paniculata (Burm. f.) Wall. Ex Nees. Leaves
CMH Capparis micracantha DC. Heartwood
CMT Caryota mitis Lour. Tubers
CRR Cyperus rotundus L. Roots
HPR Harrisonia perforata (Blanco) Merr. Roots
HAS Homalomena aromatic Schott. Stem
HFT Hydnophytum formicarum Jack. Tubers
ICR Imperata cylindrica (L.) P. Beauv. Roots
LSL Lagerstroemia speciosa (L.) Pers. Leaves
OAW Orthosiphon aristatus (Blume) Miq. Whole plant
POS Pandanus odoratissimus L. f. Stem
RNW Rhinacanthus nasutus (L) Kurz. Whole plant
SCH Salacia chinensis L. Heartwood
SCR Smilax corbularia Kunth Rhizome
SGR Smilax glabra Wall. Ex Roxb. Rhizome
SIF Solanum indicum L. Fruits
TAF Terminalia arjuna (Roxb.) Wight & Arn. Fruits
TBF Terminalia bellirica (Gaertn.) Roxb. Fruits
TCF Terminalia chebula Retz. var. chebula. Fruits
TCV Tinospora cripa (L.) Miers ex Hook.f & Thomson. Vines
TTW Tribulus terrestris L. Whole plant
UMV Urceola minutiflora (Pierre) D.J. Middleton. Vines
URV Urceola rosea (Hook. &Arn.) D.J. Middleton. Vines

Phytochemicals analysis Determination of total phenolic content

The Folin-Ciocalteu method was used for determining total phenolic content.[20] A sample solution in MeOH (200 µL) and 0.25 N FolinCiocalteu reagent (1 mL) were mixed in a 2 mL Eppendorf tube using a vortex mixer. The mixture was allowed to react for 3 min and added 1 N Na2 CO3 transferred into 96-well microplate and incubated at room temperature solution (0.8 mL). An aliquot of each mix (200 µL) was for 20 min. The absorbance of the incubated solution was determined at 765 nm using a microplate reader (Bio-Tec instruments, Inc., U.S.A). Total phenolic contents were reported as gallic acid equivalents (mg/g dry mass). Each sample was conducted in triplicate.

Determination of total flavonoid content

The aluminum-chloride colorimetric method[21] was used to determine the total flavonoid content of all samples. Test sample solutions in MeOH (200 µL) were mixed with MeOH (600 µL), 10% w/v aluminum chloride (40 µL), 1 M potassium acetate (40 µL), and distilled water (1,120 µL). The mixture was left standing at room temperature for 30 min, and the absorbance measurement was then carried out at 415 nm using a microplate reader (Bio-Tec instruments, Inc., U.S.A). Total flavonoid contents were summarized as quercetin equivalents (mg/g dry mass). Each sample was conducted in triplicate.

Determination of total anthocyanin content

The total anthocyanin content of all samples was analyzed by the pH differential method.[22] 500 µL of the test sample solution in MeOH was mixed with 3,500 µL of 25 mM KCl buffer solution pH 1. The mixture was left at room temperature for 15 min. Then the absorbance of the mixture was then measured at 510 and 700 nm. Following the same procedure, a test sample (500 µL) was added to 25 mM sodium acetate buffer solution pH 4.5 (3,500 µL), and the absorbance of the mixture was again measured at 510 and 700 nm after 15 min. Total anthocyanin values were calculated using the following equations (mg/L).

Total anthocyanin content (mg/L) = (A×MW×DF×1000) / (ɛ×C) Where A is absorbance of the sample calculated as:

A = (A515 ×A700) pH 1.0 - (A515 ×A700) pH 4.5,

MW = the molecular weight for cyanidin-3-glucoside = 449.2, DF = the dilution factor of the samples,

ε = the molar absorptivity of cyanidin-3-glucoside = 26,900, and C = the concentration of sample buffer in mg/mL

The total anthocyanin content was reported as mg of cyanidin-3-glucoside equivalents (c-3-gE) for 100 g of sample. Each sample was conducted in triplicate.

Determination of total alkaloid contents

Total alkaloid content was determined by using bromocresol green (BCG) reagent to form a yellow-color product.[23] The sample (1,000 µL, 2.5 mg/mL in 2 N HCl) was transferred to a 50 mL separatory funnel and washed with 10 mL of chloroform three times, and the pH was adjusted to neutral by 0.1 N NaOH. Then 5 mL of BCG solution (BCG was prepared by warming BCG (69.8 mg) in 3 mL of 2 N NaOH and diluted to 1,000 mL with distilled water) and 5 mL of phosphate buffer solution (pH 4.7) were mixed. Then the solution was extracted with 3 mL chloroform three times by vigorous shaking. Then, the volume was adjusted to 10 mL with chloroform by the volumetric flask. The absorbance of the solution was then measured at 470 nm with a microplate reader (Bio-Tec instruments, Inc., U.S.A). Total alkaloid values were reported as atropine equivalents (mg/g dry mass). Each sample was conducted in triplicate.

Determination of total terpenoid content

Total terpenoid content was determined by using linalool as a positive control.[24] The sample or linalool solution (200 µL) and 1.5 mL of chloroform were combined in a 2 mL Eppendorf tube and mixed well using a vortex mixer. The mixture was allowed to stand for 3 min and 100 µL of conc. H2SO4 was then added to each tube. The mixture was placed in the dark at room temperature for 120 min (90 min for linalool). MeOH (1.5 mL) was then added and mixed using a vortex mixer. The absorbance of the final solution was measured at 538 nm (Bio-Tec instruments, Inc., USA). The total terpenoid content was reported as linalool equivalents (mg/g dry mass). Each sample was conducted in triplicate.

Anti-α-amylase activity

The α-amylase inhibitory activity method was determined using the colorimetric method.[25,26] Briefly, 2 mg of starch azure was dissolved by boiling in 0.2 mL of 0.05 M Tris-HCl buffer (pH 6.9) containing 0.01 M CaCl2 at 100°C for 10 min and then cooled down to 37°C. The sample (2 mg) was dissolved in a mixture of 1 mL of DMSO and 0.1 mL of porcine pancreas α-amylase (1.6 unit/mL) in 20 mM phosphate buffer (pH 6.9) containing 6.7 mM NaCl solution. The reaction was initiated by adding the sample solutions into a starch azure solution. After incubation at 37°C for 10 min, the chemical reaction was quenched by adding 0.5 mL of 50% acetic acid, and the mixture was centrifuged at 3000 rpm, 4°C for 5 min. The absorbance of the supernatant at wavelength 595 nm was monitored on the microplate reader (DTX 880, Multimode Detector, Beckman Coulter, Inc., Austria). Acarbose was used as a positive standard. Each sample and positive control at the same concentration (25 mg/mL) were screened to establish the % inhibition. All test samples were conducted in triplicate. The % inhibition was calculated according to the following equation;

% Inhibition =[ (Acontrol Asmple )/Acontrol  ]×100

WhereAcontrol = the absorbance of blank

Asample = the absorbance of the sample

Anti α-glucosidase activity

The method of Kumar et al.[27] was used to evaluate α-glucosidase inhibitory activity. The sample solution in DMSO (50 µL) was mixed with 50 µL of α-glucosidase enzyme solution (0.57 unit/mL in 50 mM phosphate buffer, pH 6.9). After incubation at 37°C for 10 min, 50 µL of 5 mM p-nitrophenyl-α-D-glucopyranoside in phosphate buffer, pH 6.9, was added, and the incubation continued at 37°C for 20 min. The reaction was quenched by adding 50 µL of 1 M Na2 CO3 absorbance of the final solution was measured at a wavelength of 405 nm solution. The (DTX 880, Multimode Detector, Beckman Coulter, Inc., Austria). Acarbose was used as a positive standard. Each sample and positive control at the same concentration (25 mg/mL) were screened to establish the % inhibition. All test samples were conducted in triplicate. The % inhibition was calculated according to the following equation;

% Inhibition =[ (Acontrol Asample )/Acontrol  ]×100

WhereAcontrol = the absorbance of blank (without sample)

Asample = the absorbance of the sample

Anti DPP-4 activity

In this study, the method of Rachipirom et al.[18] was used to determine anti-DPP-4 activity. Briefly, each sample was dissolved in 50 mM TrisHCl buffer (pH 7.5) to a final concentration of 50 µg/mL. The sample solution (40 µL) was mixed with 20 µL of DPP-4 enzyme (0.05 U/mL) in a 96-well plate. The 0.2 mM GP-pNA in Tris-HCl (100 µL) was added after incubation for 10 mins at 37°C. The mixture was placed at 37°C in the incubator for 30 min. Then, the reaction was terminated with 30 µL of 25% glacial acetic acid. The absorbance at wavelength 405 nm of the resulting yellow solution was measured on the microplate reader (DTX 880, Multimode Detector, Beckman Coulter, Inc., Austria). Diprotin A was used as a positive standard. The testing was conducted in triplicate for each sample. The % inhibition was calculated according to the following equation;

% Inhibition =[ (Acontrol Asample )/Acontrol  ]×100

WhereAcontrol = the absorbance of blank

Asample = the absorbance of the sample

Antioxidant activity

DPPH radical scavenging assay

DPPH radical scavenging activity was determined by the method of Brand-Williams et al.[28] Briefly, DPPH solution at conc. 24% w/v (170 µL) was mixed with the test sample in MeOH (30 µL). The mixture was allowed to stand at room temperature for 30 min, avoiding light. The absorbance measurement at wavelength 515 nm was executed using MeOH as a blank and ascorbic acid as a positive control. Each sample was conducted in triplicate. The % inhibition was calculated according to the following equation;

% Inhibition =[ (Acontrol Asample )/Acontrol  ]×100

WhereAcontrol = the absorbance of blank (without sample)

Asample = the absorbance of the sample

Ferric reducing antioxidant power (FRAP)

The ferric reducing antioxidant power was determined the potassium ferricyanide method.[29]The 200 µL of 200 µg/mL extract was mixed with 500 µL phosphate buffer (0.2 M, pH 6.6) and 500 µL 1% w/v potassium ferricyanide. After incubation at 50iC for 30 min, 500 µL of 10% w/v trichloroacetic acid was added, and the mixture was then centrifuged at 3000 rpm for 30 min. The supernatant solution (600 µL) was mixed with 600 µL of distilled water and 120 µL 0.1% w/v ferric chloride. The absorbance of the final mixture was measured at a wavelength of 700 nm. The antioxidant activity was reported as quercetin equivalents (mg/g dry mass).

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity (HRSA) was modified from the method of Tian et al.[30] Briefly, the 300 µL of 1.87 mM 1,10-phenanthroline solution, 600 µL of 0.2 M phosphate buffer saline pH 7.4, and 300 µL of 30 µg/mL sample solution were mixed homogenously. The 300 µL of ferrous (II) sulfate solution (1.87mM) was then pipetted into the mixture. The reaction was initiated by adding 300 µL of 0.03% v/v H2 O2 the reaction mixture was measured at wavelength 536 nm. Each sample. After incubation at 37°C for 60 min, the absorbance of was conducted in triplicate. The percentage of HRSA was calculated by the following equation;

%HRSA=[ (ASAn)/(AbAn) ]×100

WhereAb = the absorbances of blank,

An = the absorbances of negative controls, and As are the absorbances of the test sample

Nitric oxide inhibitory activity

The inhibition of nitric oxide (NO) production in murine macrophage-like cell-line (RAW264.7) was evaluated by the method of Owolabi et al.[31,32] Cells were cultured in a culture flask at 37°C with a humidified atmosphere containing 5% CO2 using RPMI medium as culture medium. The RPMI medium was supplemented with 0.1% NaHCO3, 2 mM glutamine, penicillin G (100 units/mL), streptomycin (100 μg/mL), and 10% Fetal Bovine Serum (FBS). Cells (1×105 cells/well) were seeded in 96-well plates and adhered to the bottom of the well in the incubator for 1 hr. Then, the medium was replaced with samples, and 25 μg/mL of lipopolysaccharide (LPS) and incubated for 24 hr. The supernatant (100 μL) was collected and reacted with Griess reagent (100 μL) to measure the nitrite accumulation in the supernatant. The reaction mixture was measured at wavelength 570 nm. Indomethacin was used as a standard control. Each sample was conducted in triplicate. The % inhibition was calculated based on the following equation.

Inhibition (%)=[(A-B)/(A-C)]×100

WhereA = the absorbances of LPS solution,

B = the absorbances of the sample with LPS, and

C = the absorbances of the sample without LPS

Cytotoxicity

Cytotoxicity was also determined using the MTT colorimetric method.[33] The cytotoxicity was used to determine the test samples in fibroblast (L929) proliferation activity. L929 cells (5×103 cells/well) were seeded in 96-well plates and incubated for 24 hr. After that, the test samples were replaced in each well and incubated for 24 hr. Then the supernatant solution was removed and added 10 µL of MTT solution (5 mg/mL in PBS) then incubated for 2 hr. After that, the medium was removed and added DMSO to dissolve the formazan production in the cells. The formazan solution was measured with a microplate reader at 570 nm. The test samples were considered cytotoxic when the optical density of the sample-treated group was less than 80% of that in the control (vehicle-treated) group. While the percentage was more than 100% indicated that this sample could promote fibroblast proliferation.

Evaluation of Hydnophytum formicarum Jack.

Purification of DPP-4 inhibitory activities of HFT fractions

Dry powder of HFT was macerated with EtOH (950 g × 1.3 L) for 72 hr. The ethanolic extract was evaporated at 45°C by using a rotary vacuum evaporator. The crude ethanolic extract (25 g, % yield = 2.92%w/w) was dissolved in 10% methanol:H2O (3 L) to obtain a clear solution prior to fractionation by using a solvent partition method with hexane (2 L), ethyl acetate (EtOAc; 4 L), n-butanol (n-BuOH; 1.5 L), and water, respectively. Each partition fraction was evaporated by a rotary evaporator or freeze-drying. Finally, four fractions were obtained including HH (2.64 g, %yield = 9.52%w/w), HE (12.79 g, %yield = 46.13% w/w), HB (3.25 g, %yield = 11.75% w/w) and HW (7.27 g, %yield = 26.26% w/w), respectively.

Each fraction was analyzed for DPP-4 inhibitory activities, as the method described in section 2.6. The fraction that gave the highest DPP-4 inhibitory activity was further fractionated using bioassay-guided isolation of chromatographic peaks using silica gel, Sephadex LH-20, and Diaion HP-20 with various mobile phases such as hexane, EtOAc, MeOH, and CHCl3. In the purification process using each chromatographic technique, fractions were collected. The purity of the fractions was determined by using silica gel TLC, detected by UV-lamp at 254 and 356 nm, and by spraying p-anisaldehyde-sulfuric acid solution. The fractions that gave the same TLC patterns were combined, and the purity was checked again by TLC after combination.

Structure elucidation

Spectroscopic methods including infrared (IR), Ultraviolet (UV), nuclear magnetic resonance (NMR), and mass spectra (MS) were used to characterize the chemical structures of the isolated compounds. Mass spectrometry and nuclear magnetic resonance spectroscopy (1H and 13C) were used to give structural information about the pure isolated compounds. The compounds isolated from H. formicarum tuber extracts were well-characterized, and their DPP-4 inhibitory activity was confirmed to find their IC50 values.

Statistical analysis

The IC50 values were calculated from dose-response curves using Microsoft Excel. All results were shown as mean ± S.D. For statistical analysis, the data were statistically compared by one-way analysis of variance (ANOVA) at 95% significant level using SPSS (version 12) for windows. All bioassay experiments were performed in triplicate.

RESULTS

Phytochemical screening

Results on phytochemical screening of 26 selected plants on phenolic, flavonoid, terpenoid, anthocyanin, and alkaloid were indicated in Table 2. All plants contained detectable phenolic, flavonoid, and terpenoid components. S. corbularia (SCR), T. arjuna (TAF), and H. formicarum (HFT) showed the highest total phenolic content. H. formicarum (HFT) yielded the highest terpenoid content compared to the other plants.

Table 2: Phytochemical screening of the 26 selected plants.

Plant Phytochemical contents
Phenolic1 Flavonoid2 Terpenoid3 Anthocyanin4 Alkaloid5
AHW 22.54 ± 1.43 61.02 ± 0.27 1089.85 ± 1.59 9.83 ± 2.34 ND
AEW 54.25 ± 0.85 58.73 ± 0.61 901.94 ± 2.75 17.66 ± 4.03 8.31 ± 0.28
AMH 62.02 ± 1.72 70.99 ± 0.80 688.37 ± 1.59 5.31 ± 2.02 ND
APL 35.81 ± 1.56 82.95 ± 0.38 2346.51 ± 1.59 12.95 ± 2.66 1.59 ± 0.07
CMH 166.69 ± 2.18 31.59 ± 0.51 864.36 ± 4.20 10.22 ± 1.88 0.41 ± 0.05
CMT 34.20 ± 0.66 28.71 ± 0.70 967.94 ± 2.75 5.12 ± 2.87 ND
CRR 101.92 ± 1.82 67.97 ± 0.83 1235.59 ± 4.20 13.56 ± 1.14 ND
HPR 132.95 ± 1.03 66.87 ± 1.42 1613.23± 1.59 5.75 ± 3.96 ND
HAS 21.79 ± 1.19 58.49 ± 0.93 578.38 ± 1.59 19.69 ± 6.10 ND
HFT 246.56 ± 1.24 15.21 ± 0.22 9413.56 ± 1.59 10.83 ± 5.10 ND
ICR 14.25 ± 1.03 61.56 ± 0.22 1381.33 ± 1.59 4.09 ± 0.35 ND
LSL 211.83 ± 2.60 65.39 ± 0.41 2157.69 ± 1.59 15.64 ± 8.55 6.00 ± 0.22
OAW 93.96 ± 1.61 52.33 ± 0.81 1384.99 ± 1.59 23.55 ± 5.83 ND
POS 22.34 ± 0.38 11.94 ± 0.67 398.72 ± 2.75 13.27 ± 4.15 0.04 ± 0.05
RNW 34.17 ± 0.54 50.99 ± 0.44 1835.96 ± 1.59 23.69 ± 5.84 0.49 ± 0.06
SCH 143.10 ± 2.53 7.17 ± 0.48 2411.59 ± 2.75 6.86 ± 3.24 ND
SCR 388.22 ± 1.92 33.72 ± 1.18 1176.01 ± 1.59 3.04 ± 0.75 ND
SGR 30.83 ± 1.18 21.12 ± 0.54 1690.22 ± 4.20 17.04 ± 5.39 ND
SIF 6.26 ± 0.42 75.91 ± 1.61 945.94 ± 2.75 29.46 ± 7.68 ND
TAF 383.37 ± 4.45 76.85 ± 1.71 833.20 ± 2.75 1.94 ± 0.54 0.17 ± 0.41
TBF 43.05 ± 2.36 27.87 ± 0.62 1187.92 ± 2.75 21.21 ± 3.56 ND
TCF 152.44 ± 3.52 14.37 ± 0.11 832.28 ± 1.59 12.09 ± 4.62 ND
TCV 51.08 ± 0.94 38.98 ± 0.80 925.77 ± 1.59 22.14 ± 3.21 0.14 ± 0.04
TTW 29.71 ± 1.85 32.43 ± 0.86 1493.15± 2.75 ND ND
UMV 340.65 ± 6.74 30.99 ± 0.69 2173.27 ± 4.20 10.85 ± 0.64 ND
URV 95.93 ± 2.81 6.58 ± 0.27 1386.83 ± 4.20 10.04 ± 2.64 ND

The values represent as mean ± S.D. (n = 4). ND = not detect

1 Gallic acid equivalents (µg/g dry mass).

2 Quercetin equivalents (mg/g dry mass).

3 Linalool equivalents (g/g dry mass).

4 Cyanidin-3-glucoside equivalents (mg) for 100 g of sample.

5 Atropine equivalents (mg/g dry mass).

Anti-α-glucosidase activity

The inhibition of α-glucosidase activity of all extracts were illustrated in Figure 1. Most of all extracts exhibited very effective anti-α-glucosidase activity compared with the standard acarbose (18.59% ± 0.45 at 25 µg/mL). The range of anti-α-glucosidase activity in all tested samples was from 84.84% ± 0.56 to 4.46% ± 2.81 at a concentration of 25 µg/mL. The ethanolic extract showed inhibition (84.84% ± 0.56) that was 17 times higher than the aqueous extract of the recipe (4.46% ± 2.81). The most potent plants at inhibiting α-glucosidase (> 50%) were S. chinensis (SCH) (69.06% ± 0.78), L. speciosa (LSL) (67.05% ± 0.25), U. minutiflora (UMV) (63.53% ± 0.72), I. cylindrica (ICR) (61.74% ± 1.16), H. formicarum (HFT) (60.60% ± 1.49), and S. glabra (SGR) (59.91% ± 2.99).

Figure 1: Anti-α-glucosidase, and anti-α-amylase activities of the extracts from 26 selected plants in this study. The % inhibition was observed at the concentration 25 µg/mL for anti-α-glucosidase and anti-α-amylase activities are shown. Sample codes are referred to Table 1. AqEx and EtEx are the aqueous and ethanolic extracts of the recipe. The acarbose and sample were chosen the concentration at 25 µg/mL for compare the activity in the same concentration.

Anti-α-amylase activity

α-Amylase inhibitory activities of all extracts were shown in Figure 2. All tested samples at a concentration of 25 µg/mL inhibited α-amylase in different levels. The aqueous extract of the combined recipe exhibited slightly less inhibition (35.67% ± 1.89) than the ethanolic extract of the recipe (45.14% ± 0.45) and the same potency with acarbose (33.14% ± 0.79 at 25 µg/mL). More than 17 plants demonstrated effective inhibition (more than 50%). The plants that showed the highest inhibition were O. aristatus (OAW) (81.49% ± 12.80), T. arjuna (TAF) (79.61% ± 0.37), C. mitis (CMT) (78.10% ± 3.52), C. rotundus (CRR) (77.87% ± 4.71), and T. terrestris (TTW) (77.52% ± 1.57).

Figure 2: Anti-DPP-4 activities of the extracts from 26 selected plants in this study. The % inhibition was determined at the concentration of 50 µg/mL. Positive control diprotiin A was tested at the concentration of 50 µg/mL.[18] Sample codes are referred to Table 1. AqEx and EtEx are the aqueous and ethanolic extracts of the recipe.

Anti-DPP-4 activity

All samples showed DPP-4 inhibitory activity at a concentration of 50 µg/mL in the range of 9.81% - 82.75% (Figure 2). Diprotin A (as positive control) showed 90.07% inhibition at the same concentration. The aqueous extract of the recipe inhibited DPP-4 at moderate levels (41.43% ± 0.53), which was approximately two-fold higher than the ethanolic extract of the recipe (24.71% ± 0.50). For single plants, H. formicarum (HFT) exhibited the most DPP-4 inhibitory activity with the inhibition 82.75% ± 0.83 was displayed by other plants that exhibited DPP-4 inhibition at a high level (> 50%) included U. minutiflora (UMV) (71.89% ± 0.34), L. speciose (LSL) (71.07% ± 0.07), and T. arjuna (TAF) (60.11% ± 0.22).

Antioxidant activity

DPPH radical scavenging assay, ferric reducing anti-oxidation power assay, and hydroxyl radical scavenging assays were performed to investigate the inhibition of different kinds of reactive radical species. The antioxidant activity results were shown in Table 3. T. arjuna (TAF), U. minutiflora (UMV), T. bellirica (TBF), C. micracantha (CMH), and S. corbularia (SCR) showed good scavenging DPPH radicals with IC50 3.77, 6.42, 7.02, 9.17 and 9.24 µg/mL, respectively, compared with the standards ascorbic acid and BHT (4.28 and 4.82 µg/mL, respectively). Only TAF could neutralize DPPH radical equipotent to ascorbic acid and BHT. In FRAP assay, T. arjuna, L. speciosa, T. bellirica, C. micracantha, and U. minutiflora exhibited ferric-reducing effects equivalent to quercetin standard curve, 136, 629, 594, 448, and 444 mg/g dry mass, respectively. Meanwhile, T. arjuna, S. chinensis, A. ebracteatus, C. micracantha, and A. hirtum indicated a hydroxyl radical formation quite a bit better than the standard quercetin with percent inhibitions of 50.86, 34.94, 32.27, 27.36, and 25.28, respectively. Overall, T. arjuna very effectively inhibited all reactive species assessed in this study.

Table 3: The antioxidant activities of the twenty-six selected plants in this study. For DPPH assay the plant extracts and ascorbic acid tested at concentration of 0.15-75 µg/mL (Brand-Williams et al., 1995). For FRAP assay the plant extracts tested at 200 µg/mL compared with the standard curve of quercetin (Yıldırım et al., 2001). For HRSA assay the plant extracts and positive control tested at concentration of 30 µg/mL (Tian et al., 2009).

Plants DPPH1 FRAP2 HRSA3
Ascorbic acid 4.28 ± 0.02 NT NT
BHT 4.82 ± 0.02 NT NT
Quercetin NT NT 5.67 ± 6.05
AHW NA 0.02 ± 0.02 25.28 ± 1.98
AEW 59.45 ± 1.79 0.03 ± 0.01 32.27 ± 3.22
AMH NA 0.07 ± 0.01 20.59 ± 10.21
APL NA NA 3.27 ± 6.18
CMH 9.17 ± 0.68 0.45 ± 0.01 27.36 ± 7.79
CMT 109.21 ± 6.16 0.08 ± 0.01 7.14 ± 13.61
CRR 39.96 ± 0.59 0.13 ± 0.01 11.30 ± 2.52
HPR 20.33 ± 0.28 0.23 ± 0.07 NA
HAS NA 0.09 ± 0.01 11.38 ± 2.51
HFT 10.06 ± 1.04 0.39 ± 0.01 14.05 ± 5.22
ICR NA 0.01 ± 0.01 NA
LSL 10.25 ± 0.23 0.63 ± 0.01 17.47 ± 1.98
OAW 39.26 ± 1.21 0.21 ± 0.01 NA
POS NA NA NA
RNW NA 0.11 ± 0.01 NA
SCH 21.83 ± 0.40 0.13 ± 0.01 34.94 ± 12.48
SCR 9.24 ± 1.71 0.26 ± 0.01 4.61 ± 5.39
SGR 132.54 ± 3.43 0.02 ± 0.01 NA
SIF NA 0.04 ± 0.01 10.48 ± 2.06
TAF 3.77 ± 0.17 1.14 ± 0.06 50.86 ± 8.21
TBF 7.02 ± 0.08 0.59 ± 0.04 25.80 ± 3.59
TCF 9.59 ± 0.40 0.33 ± 0.04 4.16 ± 3.34
TCV 106.46 ± 9.44 NA 0.45 ± 1.88
TTW NA NA NA
UMV 6.42 ± 0.24 0.44 ± 0.02 NA
URV 28.17 ± 4.82 0.07 ± 0.01 8.18 ± 3.87

* NA = Not active

1 The half-maximal inhibitory concentration (IC50) (µg/mL).

2 Quercetin equivalents (mg/g dry mass).

3 % hydroxyl radical scavenging activity (%HRSA).

Anti-inflammatory activity

The ability to reduce nitric oxide production from RAW264.7 cells was used to assess inhibition of the inflammation process (Table 4). Due to their toxicity to RAW264.7 (data of % viability of RAW264.7 was showed in Table 4), the nitric oxide inhibition of A. myriophylla and T. terrestris could not be compared with others. A. paniculata, A. hirtum, R. nasutus, A. ebracteatus, and H. perforata effectively inhibited NO production with percent inhibition of 90.1% ± 2.4, 73.5% ± 2.8, 61.9% ± 1.9, 60.8% ± 3.6, and 54.0% ± 1.1, respectively. All of which were higher than the standard indomethacin (34.3% ± 2.4) at the concentration of 50 µg/mL. The ethanolic extract of the recipe also showed satisfactory activity (44.1% ± 2.3), slightly more potent than indomethacin.

Table 4: The percent inhibition of NO production and percent cell viability using RAW264.7 cells when treated with sample or positive control at a concentration of 50 µg/mL.

Plants % Inhibition of NO % Viability
AHW 73.5±2.8* 95.0±1.1
AEW 60.8±3.6 101.5±3.8
AMH 100.9±0.7* 20.4±1.5a
APL 90.1±2.4* 143.2±8.7
CMH 33.9±0.6 134.5±3.1
CMT 15.8±1.2 130.7±2.7
CRR 30.5±1.2 173.7±3.4
HPR 54.0±1.1 134.0±1.2
HAS 32.3±1.4 114.3±2.6
HFT 20.8±1.0 148.1±4.3
ICR 27.9±2.2 112.8±2.0
LSL 7.5±1.0 104.4±1.4
OAW 42.5±1.5 129.1±3.7
POS 26.5±0.5 123.8±1.5
RNW 61.9±1.9 95.6±2.6
SCH. 26.2±2.3 121.1±2.6
SCR. 6.2±1.8 140.7±4.4
SGR 16.2±0.8 145.5±3.3
SIF 17.3±0.8 133.6±3.3
TAF 21.2±1.7 128.5±4.0
TBF NA 107.1±5.4
TCF 3.9±1.1 135.0±1.3
TCV 20.0±2.6 126.0±3.2
TTW 40.8±2.7 71.8±1.1a
U.M.V. NA 125.5±3.6
URV 12.6±1.9 120.6±5.8
Aqueous extract 28.3±1.9 106.6±3.3
Ethanolic extract 44.1±2.3 152.4±7.8
Indomethacin 34.3±2.4 117.0±2.2

Value represents mean ± SEM (n = 4); NA = no activity; a cytotoxic effect was observed (% cell viability less than 80%)

a Cell viability less than 80%

*Significantly different higher activity than obtained with indomethacin, 50 µg/mL (The concentration of indomethacin and samples was used at 50 µg/mL for screen to establish the % inhibition).[32]

Cytotoxicity

The cell proliferation and viability of L929 fibroblast cells were performed with all plants using the concentration at 0, 3, 10, 30, 100 µg/mL as shown in Table 5.[33] U. minutiflora, T. terrestris, A. hirtum, A. myriophylla, S. chinensis, and T. arjuna can promote the L292 cell proliferation at all concentrations (more than 100%), whereas A. ebracteatus can promote cell proliferation only at low concentrations (3 µg/mL). Most of the samples showed lower % cell proliferation when treated with a high concentration of the sample (100 µg/mL). All extracts were not toxic to the cells and classified as safe because the cell viability remained higher than 80% of the control after being tested with the sample.[34] The promotion of fibroblast proliferation of the sample could promote wound healing in the proliferation phase.[33]

Table 5: The percent cell proliferation of L929 cells treated with DM plant extracts at a concentration of 3, 10, 30, and 100 µg/mL.

Plants % cell proliferation
3 µg/mL 10 µg/mL 30 µg/mL 100 µg/mL
AHW 120.58±6.4 114.09±11.6 107.38±6.3 105.12±7.9
AEW 122.23±12.4 107.58±1.7 105.88±3.8 84.26±3.2
AMH 121.68±14.8 115.67±7.5 115.67±7.5 115.60±0.9
APL 112.12±17.8 104.95±14.62 107.41±21.0 79.34±13.0
CMH 97.95±11.0 100.95±13.8 93.97±12.0 92.62±12.8
CMT 100.9±6.0 95.85±10.5 96.22±10.2 114.02±3.2
CRR 110.7±10.7 101.81±13.0 112.76±19.9 95.93±29.9
HPR 115.18±14.5 106.56±18.5 93.04±10.5 97.95±3.9
HAS 98.71±18.1 103.06±12.2 101.37±8.0 114.53±2.1
HFT 132.66±35.4 99.45±15.1 102.14±3.5 105.89±4.1
ICR 121.22±14.6 106.88±14.5 98.39±5.8 83.25±7.2
LSL 96.18±2.4 107.59±14.3 91.93±3.7 85.78±4.6
OAW 123.14±2.3 112.53±2.9 106.46±4.6 90.50±8.6
POS 110.49±9.8 99.42±11.31 90.48±3.3 80.42±5.1
RNW 116.09±9.8 108.86±11.8 96.93±13.2 93.92±10.74
SCH 140.27±6.6 128.95±5.2 123.10±8.3 113.71±4.1
SCR 122.07±2.3 124.45±6.5 118.04±7.2 96.40±6.8
SGR 116.73±15.2 106.20±12.3 116.95±7.5 90.49±10.4
SIF 105.19±8.8 102.72±13.7 92.96±9.7 97.02±8.8
TAF 139.42±11.3 134.93±11.78 120.19±6.9 111.16±3.48
TBF 120.71±15.3 103.23±15.3 101.80±13.9 96.77±6.9
TCF 104.02±14.1 92.44±16.3 88.27±8.9 79.85±18.0
TCV 116.23±6.4 109.39±8.3 98.58±5.7 91.18±7.1
TTW 128.83±12.6 122.8±7.6 135.06±18.4 132.289±11.1
UMV 143.43±20.9 146.75±18.0 152.58±12.9 136.91±13.3
URV 99.89±9.3 88.62±9.3 96.87±10.05 72.16±10.4

Evaluation of the most potential antidiabetic plants (Hydnophytum formicarum Jack.)

After screening the antidiabetic plants, it was found that H. formicarum tuber showed the highest DPP-4 inhibitory activity (82.75±0.83 % inhibition at the concentration of 50 µg/mL) as well as moderately high in anti-α-glucosidase and antioxidant activities. At the same time, it showed no toxicity to L929 cells. Thus, H. formicarum tuber was studied further to identify possible chemical components that contribute to its biological activities.

Purification and assessment of anti-DPP-4 activities of HFT fractions

The ethanol crude extract of H. formicarum (HFT extract) was subjected to further partition using different solvents resulting in four fractions, including HH, HE, HB, and HW. The physical appearances and % yield of all extracts are summarized in Table 6. All fractions were obtained as a dark brown-off red viscous liquid. The isolated yields and percentage yields of HH, HE, HB, and HW extract were 2.64 g (9.52% w/w), 12.79 g (46.13% w/w), 3.25 g (11.75% w/w), and 27.01 g (21.43% w/w), respectively, when compared to the crude ethanolic extract. The anti-DPP-4 activity was used for bioassay-guided isolation of purified active compounds. The four fractions were analyzed for anti-DPP-4 activity and compared with diprotin A, the positive standard. 50 µg/mL of diprotin A showed significant inhibitory activity of 90.07 ± 0.39%. The most inhibitory HFT fractions were the HB fraction (78.99 ± 0.40 %), the HW fraction (71.46 ± 0.61 %) and the HE fraction (67.59 ± 0.48 %). However, the inhibitory activity of these fractions was less than the crude extract (80.75 ± 0.83 %). The HH fraction did not give good DPP-4 inhibitory activity (1.78 ± 0.20 %). Thus, the extracts that yielded efficient inhibitory activity were from high polar solvents. (Table 7).

Table 6: The percentage yields and physical appearance of the extracts obtained from partition of H. formicarium.

Extract Physical appearance % Yield (%w/w)
Crude extract (HFT) Dark brown off red viscous liquid -
Hexane extract (HH) Dark brown off red viscous liquid 9.52
EtOAc extract (HE) Dark brown off red viscous liquid 46.13
n-BuOH extract (HB) Dark brown off red viscous liquid 11.75
H2O extract (HW) Dark brown off red viscous liquid 26.26

Table 7: The DPP-4 inhibitory activity (% inhibition) of 50 µg/mL of the extracts.

Samples % Inhibition of DPP-4 (at conc. 50 µg/mL)
Diprotin A 90.07 ± 0.39
Crude extract (HFT) 80.75 ± 0.83
Hexane extract (HH) 1.78 ± 0.20
EtOAc extract (HE) 67.59 ± 0.48
n-BuOH extract (HB) 78.99 ± 0.40
H2O extract (HW) 71.46 ± 0.61

Structure elucidation of pure compounds

Bioassay-guided isolation was used to obtain pure compounds. When the HH fraction (1,000 mg) was subjected to silica gel column chromatography using a sequential of hexane, EtOAc, and MeOH, it yielded 10 fractions: HH 1 (461.40 mg), HH 2 (20.90 mg), HH 3 (10.00 mg), HH 4 (46.00 mg), HH 5 (22.40 mg), HH 6 (7.70 mg), HH 7 (40.30 mg), HH 8 (53.90 mg), HH 9 (187.00 mg), HH 10 (515.10 mg). HH 1 (461.40 mg) fraction was selected to be further purified by a silica gel column chromatography using hexane and EtOAc in a gradient mode to give 6 sub-fractions: HH 1.1 (203.70 mg), HH 1.2 (301.40 mg), HH 1.3 (12.40 mg), HH 1.4 (46.50 mg), HH 1.5 (58.70 mg) and HH 1.6 (88.10 mg). Of these, two fractions, HH 1.2 and HH 1.4 were further purified by silica gel column chromatography with gradient mode using sequential hexane and EtOAc. HH 1.2 was isolated to give 8 fractions: HH 1.2.1 (15.30 mg), HH 1.2.2 (26.20 mg), HH 1.2.3 (71.10 mg), HH 1.2.4 (7.40 mg), HH 1.2.5 (47.60 mg), HH 1.2.6 (8.50 mg), HH 1.2.7 (1.20 mg) and HH 1.2.8 (19.10). HH 1.2.3 (71.10 mg) was further purified by Sephadex LH-20 using chloroform to give 6 fractions: HH 1.2.3.1 (480 mg), HH 1.2.3.2 (1.30mg), HH 1.2.3.3 (54 mg), HH 1.2.3.4 (5.30 mg), HH 1.2.3.5 (2.20 mg) and HH 1.2.3.6 (2.50 mg). Among these obtained fractions, fraction HH 1.2.3.4 provided white solid residue after drying by the evaporation process as shown the structure in Figure 3(1).

Figure 3: Structures of isolated compounds of Hydnophytum formicarum Jack.

HH 1.4 was also isolated to give 9 fractions: HH 1.4.1 (3.40 mg), HH 1.4.2 (3.60 mg), HH 1.4.3 (9.80 mg), HH 1.4.4 (3.70 mg), HH 1.4.5 (1.80 mg), HH 1.4.6 (0.90 mg), HH 1.4.7 (0.90 mg), HH 1.4.8 (4.70 mg) and HH 1.4.9 (10.30 mg). HH 1.4.3 was also further purified by Sephadex LH-20 column using the mixture of MeOH in EtOAc (1:1) to give 4 fractions: HH 1.4.3.1 (4.40 mg), HH 1.4.3.2 (5.90 mg), HH 1.4.3.3 (10 mg) and HH 1.4.3.4 (1.30 mg). HH 1.4.3.2 (5.90 mg) showed a clear pattern with an off-white solid after drying from this separation fraction as shown the structure in Figure 3(2) and (3).

The purified residues were further characterized by H1-NMR, FT-IR, UV, and mass spectroscopy. The spectral data were utilized for comparison with known compounds. HH 1.2.3.4 Yielded spectral and mass data that matched palmitic acid, as previously reported,[35] (Supplement 2). The HH 1.4.3 solid was also characterized by H1-NMR, FT-IR, and EIMS techniques and matched a report by Chaturvedula and Prakash,[36] which is a mixture of stigmasterol and ß-sitosterol (Supplement 3).

Assessment of anti-DPP-4 activities of isolated compounds

HH 1.2.3.4 and HH 1.4.3 were tested to quantify their inhibition activities against DPP-4 activity using a similar procedure to that used with the extracts. Diprotin A was used as a positive control in each experiment. The results demonstrated that palmitic acid (1) and the mixture of stigmasterol (2) and ß-sitosterol (3) gave more inhibition than the sugar from fraction HW, but they showed much less activity than a positive standard, diprotin A. We, therefore, suggest that these components of HFT could be useful as marker compounds for HFT, rather than as DPP-4 inhibitors in their own right. Their IC50 values are summarized in Table 8. The compounds with high anti-DPP-4 activity may be considered biomarkers for quality control of the HFT standardized extract preparation for antidiabetic activity.

Table 8: IC50 of pure compounds for DPP-4 inhibitory activities.

Compounds IC50 ± SD (µg/mL)
Palmitic acid (1) 73.82 ± 2.64
Stigmasterol (2) and ß-sitosterol (3) 78.58 ± 0.92
Sugar fraction 624.98 ± 11.99
Diprotin A 2.07 ± 0.01

DISCUSSION

Herbal antidiabetic medicines are extensively accepted as alternatives for DM patients in lowering blood sugar. In this study, a traditional antidiabetic recipe, which is being commonly prescribed at the Thai-traditional medicine department in Wang-Nam-Yen hospital, Thailand, was investigated for potentially beneficial DM-related activities. These included anti-DPP-4, anti-α-glucosidase, and anti-α-amylase, anti-inflammation, antioxidant, and fibroblast proliferation activity. Our previous report showed that some plants in this recipe, such as S. chinensis, L. speciosa, U minutiflora, I. cylindrica, and H. formicarum showed promising anti-α-glucosidase, and anti-α-amylase activities.[19] In this study, additional potential antidiabetic mechanisms were evaluated to support the broader clinical utilization of this remedy. Wang-Nam-Yen hospital formulation is administered via the oral route. Firstly, the aqueous extract of the recipe was tested for the inhibitory activities against DPP-4, α-glucosidase, and α-amylase enzymes. The results showed that the aqueous extract inhibited α-amylase and DPP-4 enzymes at a moderate level but only slightly inhibited α-glucosidase. This suggests that anti-α-glucosidase activities may not be a major mechanism of this extract. The ethanolic extract, compared with the aqueous extract, provided better inhibition of α-glucosidase and α-amylase enzymes, suggesting that ethanol extraction might improve the remedy’s targeting of these enzymes.

A number of medicinal plants with DPP-4 inhibitory activity have been previously reported, such as Castanospermum austral,[37,38] Mangiferia indica,[39] Withania somnifera,[40] Trigonella foenum-graecum,[40] Urena lobate,[41] and Berberis aristata.[42] H. formicarum is also used as the constituent in Thai traditional medicine, and some of its biological activities have been described previously.[43-45] Sinapinic acid has been reported to be an active compound isolated from the rhizome of H. formicarum, which can inhibit the growth of HeLa and HT29 cells via histone deacetylase inhibition.[46] In this study, H. formicarum showed the most potent DPP-4 inhibitory activity among all screened plants with 82.72% inhibition, nearly an equally potent to diprotin A. Jeli and Makiyah,[47] reported H. formicarum was able to increase the size of Langerhans islet, and the number of β-cells; they concluded that H. formicarum could minimize the damage of pancreases of alloxan-induced diabetic rats. Our results could support the use of H. formicarum as herbal medicine for antidiabetic.

Triterpenoids have been reported as anti-diabetes activities by several targets, such as α-glucosidase, α-amylase, aldose reductase, protein tyrosine phosphatase 1B, glycogen phosphorylase.[48] Due to the high contents of terpenoids, both α-glucosidase and α-amylase were inhibited in most of the plants tested. In addition, another study suggested flavonoids, particularly luteolin, apigenin, and resveratrol, could act as natural DPP-4 inhibitors.[49] This is consistent with our results because most of the plants we studied contained high levels of terpenoids and flavonoids, and most of them displayed an inhibition of all tested enzymes at a significant level. This work shows that H. formicarum, L. speciose, T. arjuna, and U.minutiflora provided superior inhibition profiles against DPP-4, α-glucosidase, and α-amylaseand are good candidates for further development in antidiabetic formulations.

As noted above, increased oxidative stress and inflammation was associated with the development and progression of diabetes and its complications. Both aqueous and ethanolic extracts of Wang-Nam-Yen and its component plants gave significant inhibition of DPPH radical and NO production. Our results agreed with other studies that propose that various phenolics and flavonoids could attenuate the oxidative radicals and NO.[20,31,32] T. arjura was the most active of the plants tested in this regard and gave the superior inhibition in all anti-oxidation assays, while A. hirtum, A. myriophylla, and A. paniculata exhibited considerable significant reductions in NO production.

H. formicarum was selected for further study of chemical components because it exhibited the best anti-DPP-4 activity. Successes in isolation of purified chemical constituents were obtained from hexane partitioned fractions. The two compounds identified in the hexane fraction were palmitic acid and a mixture of stigmasterol and ß-sitosterol. These constituents have been reported in previous studies of other plants.[45,50,51]

Unfortunately, neither palmitic acid nor the mixture of stigmasterol and ß-sitosterol showed low activity against DPP-4 compared to diprotin A. However, previous studies have shown sterols like stigmasterol and β-sitosterol to reduce blood glucose level, cholesterol absorption, and body weight in STZ induced diabetic mice.[52] Moreover, β-sitosterol was shown to normalize the altered levels of blood glucose in the diabetic rat by induced β-cells regeneration, glucose uptake, reduced toxicity to the β-cells by inhibition of ROS, reduction of β -cells apoptosis, activation of insulin receptor (IR), and glucose transporter 4 (GLUT4) proteins in the adipose tissue, and to increased insulin release.[53-56] Furthermore, β-sitosterol reduced blood glucose through various signaling pathways, including the reduction of PTP1B (Protein tyrosine phosphate 1B), which increases leptin and insulin signaling, reduction of glucose production by inhibition of phosphoenolpyruvate carboxykinase (PEPCK), inhibition of alpha-glucosidase and amylase enzyme activities, activation of AMPK pathway through AMPK, GSK-3, and ACC phosphorylation, and increasing of glycogen synthesis.[54,55]

Our results do not confirm the inhibition of DPP-4 enzyme by β-sitosterol and stigmasterol suggested in the report of Purnomo et al.[41] using in silico techniques.

Palmitic acid also exhibited antidiabetic activity by acute stimulation of glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells.[57] We suggested that palmitic acid, stigmasterol, and ß-sitosterol may also serve as useful biomarkers of H. formicarum, for quality control purposes.

CONCLUSION

Many of the plants in the Wang-Nam-Yen traditional anti-diabetes recipe displayed potent inhibition not only against α-glucosidase and α-amylase. Some also exhibited potent activity against DPP-4, anti-oxidation and anti-inflammation assays. The terpenoid and flavonoid contents of the constituent plants were quantified and are possibly related to the reported beneficial DM-related activities. H. formicarum was chosen for chemical evaluation, and the purified compounds. Palmitic acid, stigmasterol, and β-sitosterol were extracted from the hexane fraction. These chemicals have reported anti-DM activity but showed only weak activity in the specific DPP-4 inhibition assay. The potentially beneficial anti-DM assay results obtained from the constituent plants of Wang-Nam-Yen are consistent with its traditional use and argue for wider dissemination of its use in the clinical setting.

ACKNOWLEDGEMENT

The authors acknowledge the cooperation from all staff in the Department of Pharmacognosy and Pharmaceutical Botany, and Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University. The authors would like to thank Mr. Patarachai Pornpanyanukul from Pharmacy Division, Wangnamyen hospital, Sakeao Province, Thailand, for the material and data support. Thank you to the Prince of Songkla University research grant (Grant Number PHA6000985) for financial support. Moreover, the research was supported from Prince of Songkla University and Ministry of Higher Education, Science, Research, and Innovation under the Reinventing University Project (Grant Number REV64035).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ABBREVIATIONS

BCG: bromocresol green; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene; n-BuOH: n-butanol; DMEM: Dulbecco’s Modified Eagle Medium; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EtOAC: ethyl acetate; EtOH: Ethanol; FRAP: Ferric reducing antioxidant power; GP-pNA: gly-pro-p-nitroanilide; HB: n-butanol extract; HE: ethyl acetate extract; HH: Hexane extract; HRSA: hydroxyl radical scavenging activity; HW: aqueous extract; IR: infrared; LPS: lipopolysaccharide from Escherichia coli; MeOH: methanol; MS: mass spectra; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NMR: nuclear magnetic resonance; NO: nitric oxide; RAW264.7: murine macrophage-like cell-line; UV: Ultraviolet.

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GRAPHICAL ABSTRACT

images

SUMMARY

The 26 plants selected from Wang-Nam-Yen hospital receipt, Thai traditional antidiabetic receipt used for diabetic treatment, were evaluated for their DPP-4, α-glucosidase, and α-amylase inhibitory activity. Most of them showed DPP-4, α-glucosidase, and α-amylase inhibitory activity in the good range, however, in different extent.

The ethanolic extract of H. formicarum tubers was identified as the highest potential antidiabetic effect by inhibition of DPP-4 enzymes and showed high potential for inhibit enzyme α-glucosidase.

Palmitic acid and the mixture of stigmasterol and β-sitosterol were extracted from the hexane fraction. These chemicals have reported antidiabitic activity in the specific DPP-4 inhibition assay.

Cite this article: Rachpirom M, Barrows LR, Thengyai S, Ovatlarnporn C, Sontimuang C, Thiantongin P, et al. Antidiabetic Activities of Medicinal Plants in Wang Nam Yen Hospital Recipes and Candidate Antidiabetic Compounds from Hydnophytum formicarum Jack. Tubers. Pharmacog Res. 2022;14(1):89-99.