α-Amylase and α-glucosidase inhibitors from the aerial parts of Chamaecrista pumila (Lam.) K. Larsen

Abstract One new compound, 4,7-dihydroxy-2-hydroxymethyl-5,6-dimethoxyanthraquinone (1), along with eight known compounds (2–9) were isolated from the methanol extracts of the aerial parts of Chamaecrista pumila (Lam.) K. Larsen. Their chemical structure was determined based on spectroscopic data interpretation and comparison with the reported data. The inhibitory effects of them on α-amylase and α-glucosidase were performed. The results showed that compounds 4, 6, 8, and 9 against potent α-glucosidase with the inhibition values of 98.14 ± 0.05, 98.19 ± 0.04, 97.01 ± 0.49, 84.43 ± 0.6% at 50 µM, respectively. Compounds 4 and 6 displayed significance against α-amylase at 200 µM with inhibition values of 22.35 ± 1.10 and 60.47 ± 0.91%. Graphical Abstract


Introduction
Diabetes mellitus is known as a health-threatening concomitant disease throughout the world.It is a chronic metabolic disease from a deficiency in insulin secretion or action.One of the therapeutic approaches to reduce postprandial hyperglycemia is to retard digestion and absorption of dietary carbohydrates by inhibiting digesting enzymes (Ch'ng et al. 2019).a-Amylase and a-glucosidase are played as the two key enzymes involved in the carbohydrate hydrolysis process (Dona et al. 2010).Pancreatic a-amylase will break down dietary carbohydrates into monosaccharides in the digestive system and then be degraded by a-glucosidases to glucose which on absorption, enters the bloodstream.Therefore, the inhibition of a-amylase and a-glucosidase has considered an important strategy in the treatment of diabetes disease, since inhibitors of these enzymes can suppress carbohydrate digestion, delay glucose uptake and reduce blood sugar levels (Kajaria et al. 2013).Previous investigations reported that plant constituents such as coumarin, flavonoids, and iridoids as potential inhibitors for the treatment of type 2 diabetes mellitus (Leo et al. 2016).
The genus Chamaecrista is the most predominant of the family Fabaceae and comprises approximately 430 species (Theplantlist 2022).Chamaecrista species are mainly distributed throughout tropical and subtropical regions, including Africa, Asia, and North-South America.Plants of this genus have been used as folk medicines history in South American and Asian countries.They present several therapeutic properties as laxatives (C.biensis, C. cathartica, and C. lateriticola), wound and ulcer treatments (C.absus and C. nigricans), and show anti-ophidic effects against snake and scorpion poisons (C.apoucouita).Most medicinal plants are prepared as infusions or unguents (Lewis et al. 2005;Plants 2022).Chamaecrista pumila (Lam.)K. Larsen (synonym Cassia pumila Lam.), one of the Chamaecrista species, grows wild in many places such as coastal sandy beaches, roadsides, dikes, wastelands, and forests in Viet Nam (Ho 1999).C. pumila has been used in traditional medicine for treating liver diseases.The phytochemistry of this plant has not been exploited.Most of the works are centered on the phylogeny and taxonomical classification of plants from the genus.A few reports were found to indicate an interesting antimicrobial activity for this plant, caused apparently by a high content of flavonoids and phenolics (Liao et al. 2020).
In our search for antidiabetic agents of natural origins, an ethanolic extract from the aerial parts of C. pumila was found to show significant inhibitory activity against a-amylase (70.6 ± 1.30%) and a-glucosidase (66.4% ± 0.84%) at a concentration of 200 lg/mL.Therefore, the present study reports the isolation and structural elucidation of nine compounds (1-9, Figure 1) from the aerial parts of C. pumila.Their inhibition effects on a-glucosidase and a-amylase are investigated.

Results and discussion
Compound 1 was purified as a yellow, amorphous powder.Its molecular formula was found to be C 17 H 14 O 7 (11 indices of hydrogen deficiency) as inferred from the HR-ESI-MS ion peak at m/z 331.0813 331.0817) which was supported by the corresponding 1D nuclear magnetic resonance (NMR) data.The 1 H NMR spectrum of 1 displayed resonance signals for a tetrasubstituted aromatic ring [d H 7.66 (1H, d, J ¼ 2.5 Hz, H-1) and 7.24 (1H, d, J ¼ 2.5 Hz, H-3)] and an aromatic singlet [d H 7.51 (s, H-8)] suggested the presence of another pensubstituted aromatic ring system in the molecule of 1.Additionally, an oxymethylene proton [d H 4.60 (2H, s, H-15)] and two aromatic methoxy groups [d H 3.97 (3H, s, 5-OCH 3 ) and 3.99 (3H, 6-OCH 3 )], were also detected (Figure S2).A characteristic feature of the 13 C NMR spectrum was the apparent twinning of each carbon resonance, indicating substitution patterns in compound 1.Thus, with the aid of the heteronuclear single-quantum correlation (HSQC) spectrum, it was observed that the molecule contained 17 carbon atoms attributable to two carbonyl carbons [d C 183.2 (C-9) and 188.7 (C-10)], two methoxys [d C 61.9 (5-OCH 3 ) and 61.5 (6-OCH 3 )], one oxymethylene group [d C 64.1 (C-15)], and 12 aromatic carbons (d C 116.9-163.7 ppm of rings B and C, nine non-protonated sp 2 carbons).Among these, two downfield shifted carbon resonances were part of two ketone carbonyl functionalities adjacent to phenolic moiety [d C 183.2 (C-9) and 188.7 (C-10)] (Figure S3 and Table S1).The presence of two carbonyl carbons together with two benzenoid moieties suggested that this compound has an anthraquinone skeleton with one tetrasubstituted and one pensubstituted benzene ring, which was confirmed by HSQC and HMBC data (Figure S7).The five substituents of 1 are inclusive of one oxymethylene, two hydroxyls, and two methoxy groups which could be deduced based on the determined molecular formula.Compound 1 resembled the structure of 4,7-dihydruxy-2-hydroxymethyl-l,5,6-trimethoxyanthraquinone (Barba et al. 1994), except that one methoxy group (at C-1) in 4,7-dihydruxy-2-hydroxymethyl-l,5,6-trimethoxyanthraquinone was absent in 1, showing instead an aromatic carbon (d C 117.5, C-1).
The substituent pattern of 1 was determined by careful examination of the heteronuclear multiple-bond correlations (HMBC) of 1 confirmed the characteristic of a 9,10-  S7) and NOESY interactions of H-15 (d H 4.60) with H-1 and H-3.The attachment of the methoxy group to the carbon at C-5 was confirmed by HMBC correlations observed between d H 3.97 (5-OCH 3 ) with C-5 (d C 157.0), while the position of a secondary methoxy at C-6 could be deduced from its HMBC correlation d H 3.99 (6-OCH 3 ) with C-6 (d C 148.5), respectively.The carbon resonance for C-5 appeared at a relatively high field due to two electron donating groups in the ortho positions, which could be observed in the cases of trioxygenated anthraquinones.Similarly, HMBC correlations were used to reveal the locations of the remaining two hydroxy substituent groups at C-4 and C-7 (Figure S7), which was supported by the HR-ESI-MS data.Through the HMBC correlation of the remaining aromatic methine proton at d H 7.51 (H-8) with the carbonyl carbon at d C 183.2 (C-9), together with the carbon chemical shift at d C 158.5 (C-7), one more hydroxy group was assumed to be attached to C-7.Based on this cumulative analysis, the structure of 1 was established as depicted in Figure 1.As a new natural product, 1 was named 4,7-dihydroxy-2-hydroxymethyl-5,6dimethoxyanthraquinone.23.45 ± 0.32 88.37 ± 0.68 a Data represent the mean ± SD of at least three independent experiments performed in triplicates.b Acarbose (100 and 500 mM) was used as a positive control.NA: not activive.
Continuously, all isolates were screened for in vitro inhibitory activities against the a-amylase and a-glucosidase enzymes with acarbose used as a positive control.The best known a-amylase and a-glucosidase inhibitors are acarbose and miglitol; the former is a natural product isolated initially from an Actinoplanes strain, and the second is the N-hydroxyethyl analogue of 1-deoxynojirimycin, isolated from Morus spp.The results showed that compounds 4, 6, 8, and 9 against potent a-glucosidase with the inhibition percent values of 98.14 ± 0.05, 98.19 ± 0.04, 97.01 ± 0.49, and 84.43 ± 0.6% at 50 mM, respectively, comparison with the positive control (37.71 ± 1.26% at 100 mM).At the same concentration, compounds 5 and 7 exhibited strong activity against a-glucosidase with 57.29 ± 1.09 and 62.8 ± 1.06%, respectively.Other compounds showed significant activities with inhibition values from 22.09 ± 2.44 to 37.00 ± 1.43%.Increasing the concentration to 200 mM, compound 3 inhibited a-glucosidase up to 55.92 ± 1.69%, while compounds 5 and 7 exhibited potent activity with the inhibition percent values of 99.33 ± 0.52 and 94.2 ± 0.53%, respectively.The effect of compounds 4, 6, 8, and 9 showed slight differences at this concentration (Figure 2).
The structure of isolated compounds was suggested that one compound 1 is the anthraquinone, five compounds (2, 3, 4, 5, and 7) are flavonoids, and three compounds (6, 8, and 9) have a stilbene skeleton.Among them, flavonoids and stilbenes showed good activity against a-glucosidase at test concentrations.All stilbene compounds possessed a-glucosidase more than 50% at a concentration of 50 mM and increased up to nearly 100% at a concentration of 200 mM.Previous studies demonstrated that resveratrol and stilbene compounds, derived from them, are potent inhibitors of a-glucosidase (Lam et al. 2008).Two compounds, trans-scirpusin A (6) and piceatannol (8) inhibited a-glucosidase with IC 50 values of 34.32 and 1.13 mM, respectively (Wan et al. 2011).
Numerous studies have reported that flavonoids have protective effects in the development of diabetes as well as a mitigation effect of diabetic complications (Je et al. 2002;Nagasawa et al. 2003).In this research, five flavonoids displayed significant activity with an inhibition percent more than 50% at a concentration of 500 mM.Among them, a biflavonoid (7,4 0 -dihydroxyflavan-(4a!8)-epiafzelechin) and a chaconne [1-propanone, 3-(3,4-dihydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)], a flavone (luteolin) showed the strong inhibition activity against a-glucosidase.Luteolin is present in some vegetables, fruits, and medicinal herbs such as celery, green pepper, and carrot (Ando et al. 2009), and has recently been reported to act as an inhibitor of a-glucosidase (Matsui et al. 2002;Kuroda et al. 2012).According to the tested results of Kim et al. have compared the a-glucosidase inhibiting activities of twenty-one naturally occurring flavonoids and found that luteolin was the strongest inhibitor (Kim et al. 2000).This evidences suggested that luteolin could be a promising a-glucosidase inhibitor for further animal studies or clinical trials.
Although flavonoids have been shown to inhibit a-glucosidase, to our knowledge, biflavonoids have rarely been reported for a-glucosidase inhibitory activity.One biflavonoid, 5,7,4 0 ,3 0 ',5 0 ',7 0 ',4 0 ''-heptahydroxy-3,8-biflavanone from Garcinia kola stem bark, known as GB1, possessed a-glucosidase with an IC 50 value of 0.90 ± 0.01 mM, comparison with the standard drug 1-deoxynojirimycin (IC 50 ¼ 0.28 ± 0.02 mM) (Antia et al. 2010).CC Jia et al. have reported that paucinervin K exhibited strong a-glycosidase inhibition with an IC 50 value of 12.48 ± 4.60 mM (Jia et al. 2017).Other study, three biflavonoids of the flavanone-chalcone type, rotundaflavanochalcone, iso-rotundaflavanochalcone, and de-O-methyl rotundaflavanochalcone were reported from the roots of Boesenbergia rotunda, displayed significant activity with IC 50 values of 2.4 ± 0.4, 3.4 ± 0.9, and 1.3 ± 0.2 mM, respectively (Chatsumpun et al. 2017).The result of a biflavonoid 4 against a-glucosidase in this paper also is similar to the previous report and this is the first report about the a-glucosidase inhibition activity of compound 4.These studies suggested that biflavonoid may be a potential dietary supplement or phytomedicine for the prevention of type 2 diabetes mellitus.

General experimental procedures
The 1 H and 13 C NMR spectra were recorded in deuterated solvents on an AVANCE III HD 500 spectrometer (MA, USA) operating at 125 MHz for 13 C and 500 MHz for 1 H in CD 3 OD, with tetramethylsilane (TMS) as the internal standard.The HRESIMS were acquired on an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Emeryville, CA, USA).Silica gel (70-230, 230-400 mesh, Merck, Whitehouse Station, New Jersey), reverse-phase (RP)-18 (75 mm, Fuji Silysia Chemical Ltd., Kasugai, Japan), Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used as absorbents in the column chromatography (CC).Thin layer-chromatography (TLC) plates (silica gel 60 F 254 and RP-18 F 254S , 0.25 mm, Merck) were purchased from Merck KGaA (Darmstadt, Germany).Spots were detected under UV radiation (254 and 365 nm) and by spraying the plates with 10% H 2 SO 4 followed by heating with a heat gun.p-Nitrophenyl-a-D-glucopyranoside (p-NPG), 1-deoxynojirimycin, and genistein were from Sigma (Sigma-Aldrich, St. Louis, MO).Other reagents and solvents used were of analytical grade and were purchased from reliable commercial sources.

Plant material
The aerial parts, including the leaves, twigs, stems, and inflorescence of Chamaecrista pumila (Lam.)K. Larsen were collected at Lac Son district, Hoa Binh province, Vietnam by Nghiem Duc Trong in December 2017, who also identified this plant.A voucher specimen (collection number HNIP/18031/17) has been deposited at the Department of Botany, Hanoi University of Pharmacy.

Extraction and isolation
The dried aerial parts of Chamacrista pumila (3.0 kg) were macerated with 10 L ethanol (EtOH) three times.Evaporation of the solvent under reduced pressure gave EtOH extract (400 g).The EtOH extract was suspended in water and successively separated with dichloromethane (CH 2 Cl 2 ) and ethyl acetate (EtOAc) to yield CH 2 Cl 2 (150 g), EtOAc (116 g) extracts, and water layer, respectively.

Assay for a-amylase inhibition
The a-amylase (A8220, Sigma-Aldrich, St. Louis, MO) enzyme inhibitory activity was measured using the method reported by Kusano et al. (Kusano et al. 2011) with slight modifications.The substrate was prepared by boiling 100 mg potato starch in 5 mL phosphate buffer (pH 7.0) for 5 min, then cooling to room temperature.The sample (2 mL dissolved in DMSO 1%) and substrate (50 mL) were mixed in 30 mL of 0.1 M phosphate buffer (pH 7.0).After 5 min pre-incubation, 5 mg/mL a-amylase solution (20 mL) was added, and the solution was incubated at 37 C for 15 min.The reaction was stopped by adding 50 mL 1 M HCl and then 50 mL iodine solution was added.The absorbances were measured at 650 nm by a microplate reader.Acarbose was used as a positive control.

Assay for a-glucosidase inhibition
The a-glucosidase (G0660-750UN, Sigma-Aldrich, St. Louis, MO) enzyme inhibition assay was performed according to the previously described method (Ali et al. 2002).The sample solution (2 mL dissolved in dimethyl sulfoxide; DMSO 1%) and 0.5 U/ml a-glucosidase (40 ml) were mixed in 120 mL of 0.1 M phosphate buffer (pH 7.0).After 5 min pre-incubation, 5 mM p-nitrophenyl-a-D-glucopyranoside (p-NPG) solution (40 mL) was added, and the solution was incubated at 37 C for 30 min.The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices, Sunnyvale, CA).Acarbose was used as a positive control.

Statistical analysis
All data represent the mean ± SD of at least three independent experiments performed in triplicates.Statistical significance is determined by one-way ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
In summary, phytochemical fractionation of the ethanol extract of C. pumila aerial parts led to the isolation of 4,7-dihydroxy-2-hydroxymethyl-5,6-dimethoxyanthraquinone (1), together with eight known compounds (2-9).Compounds 4 and 6 showed inhibitory effects against both a-amylase and a-glucosidase activities.Especially, compounds 4, 6, 8, and 9 possessed potent a-glucosidase with an inhibition value of more than 95% at 50 mM.These results suggest that the aerial parts of C. pumila could be useful as sources of natural a-glucosidase inhibitor agents in the functional food and pharmaceutical industries.Besides, to the best of our knowledge, this represents the first report of chemical constituents and a-amylase and a-glucosidase activities from C. pumila.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Figure 2 .
Figure 2. Effect of isolated compounds (1-9) against a-glucosidase.Data represent the mean ± S.D. of at least three independent experiments performed in triplicates.Acarbose (100 mM and 500 mM) was used as a positive control.

Table 1 .
Inhibition effects of selected compounds against a-amylase.