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

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.


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 is a result of a disturbance in glucose metabolism and presents as an elevation of fasting blood sugar.[3][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 Pharmacognosy Research, Vol 14, Issue 1, Jan-Mar, 2022 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][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][18][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.

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.

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 Folin-Ciocalteu 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 Na 2 CO 3 solution (0.8 mL).An aliquot of each mix (200 µL) was transferred into 96-well microplate and incubated at room temperature 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,

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.H 2 SO 4 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 CaCl 2 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; × 100 Where A control = the absorbance of blank A sample = 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 Na 2 CO 3 solution.The absorbance of the final solution was measured at a wavelength of 405 nm (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;

the absorbance of blank (without sample)
A sample = 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 Tris-HCl 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;

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;

the absorbance of blank (without sample)
A sample = 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 50°C 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 H 2 O 2 .After incubation at 37°C for 60 min, the absorbance of the reaction mixture was measured at wavelength 536 nm.Each sample was conducted in triplicate.The percentage of HRSA was calculated by the following equation; Where Ab = the absorbances of blank, A n = 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 macrophagelike 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% CO 2 using RPMI medium as culture medium.The RPMI medium was supplemented with 0.1% NaHCO 3 , 2 mM glutamine, penicillin G (100 units/mL), streptomycin (100 μg/mL), and 10% Fetal Bovine Serum (FBS).Cells (1×10 5 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
Where A = 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]he cytotoxicity was used to determine the test samples in fibroblast (L929) proliferation activity.L929 cells (5×10 3 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 (vehicletreated) group.While the percentage was more than 100% indicated that this sample could promote fibroblast proliferation.

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:H 2 O (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 bioassayguided isolation of chromatographic peaks using silica gel, Sephadex LH-20, and Diaion HP-20 with various mobile phases such as hexane, EtOAc, MeOH, and CHCl 3 .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 ( 1 H and 13 C) 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 IC 50 values.

Statistical analysis
The IC 50 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.

Phytochemical screening
Results on phytochemical screening of 26 selected plants on phenolic, flavonoid, terpenoid, anthocyanin, and alkaloid were indicated in Table 2.

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.

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, 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). 3Linalool equivalents (g/g dry mass). 4Cyanidin-3-glucoside equivalents (mg) for 100 g of sample. 5Atropine equivalents (mg/g dry mass).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]he promotion of fibroblast proliferation of the sample could promote wound healing in the proliferation phase. [33] 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

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.

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 Positive control diprotiin A was tested at the concentration of 50 µg/mL. [18]ample codes are referred to Table 1.AqEx and EtEx are the aqueous and ethanolic extracts of the recipe.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, 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]ncluding 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).

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 (461.40mg), HH 2 (20.90 mg), HH 3 (10.00mg), 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 1.2.3.4Yielded 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.

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-αamylase, 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-αamylase activities. [19]In this study, additional potential antidiabetic mechanisms were evaluated to support the broader clinical utilization of this remedy.[55][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]ur 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]e 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, antioxidation 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.

%
Inhibition = [(A control -A sample ) / A control ] × 100 Where A control = the absorbance of blank A sample = the absorbance of the sample

Figure 1 :
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.

Figure 2 :
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 Table1.AqEx and EtEx are the aqueous and ethanolic extracts of the recipe.