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. 2021 Apr 28;7(4):e06904. doi: 10.1016/j.heliyon.2021.e06904

Antidiabetic and antimicrobial flavonoids from the twigs and roots of Erythrina subumbrans (Hassk.) Merr.

Piyaporn Phukhatmuen a,1, Pornphimol Meesakul a,1, Virayu Suthiphasilp a, Rawiwan Charoensup b,c, Tharakorn Maneerat a,c, Sarot Cheenpracha d, Thunwadee Limtharakul e, Stephen G Pyne f, Surat Laphookhieo a,c,∗
PMCID: PMC8102414  PMID: 33997418

Abstract

The phytochemical investigation of the twig and root extracts of Erythrina subumbrans (Hassk.) Merr. (Fabaceae) resulted in the isolation and identification of a new pterocarpan, erythrinocarpan (1), along with 27 known compounds (2–28). All isolated compounds were evaluated for their antidiabetic, antimicrobial, and anti-inflammatory properties. Compounds 3, 8, 9, and 22 had α-glucosidase inhibitory activity with IC50 values of 13.4 ± 0.05, 24.5 ± 0.13, 29.0 ± 0.05, and 12.8 ± 0.14 μM, respectively, while compound 2 inhibited α-amylase activity with an IC50 value of 67.6 ± 1.12 μM. Compounds 22 and 24 inhibited glycation activity with the IC50 values of 36.9 ± 0.62 and 40.5 ± 0.37 μM, respectively. From cell-based assays, compound 27 showed the highest ability to induce glucose consumption (IC50 29.1 ± 0.86 μM) and glucose uptake (2.8-fold), and to inhibit nitric oxide (NO) production (IC50 52.5 ± 0.56 μM) without cell toxicity. Furthermore, compound 9 showed antimicrobial activities against Gram-positive bacteria and fungi with MIC values ranging from 2–4 μg/mL.

Keywords: Erythrina subumbrans, Pterocarpan, Flavonoids, Antidiabetic activity, Antimicrobial activity

Erythrina subumbrans; Pterocarpan; Flavonoids; Antidiabetic activity; Antimicrobial activity.

1. Introduction

Diabetes mellitus (DM) is a chronic metabolic syndrome associated with insulin production disorder in the pancreas leading to hyperglycemia (ADA, 2009). This disease has emerged as a major health problem worldwide, with the number of DM patients having increased by 50% over the past decade. Type 2 DM (non-insulin-dependent) is found among 90–95 % of patients or approximately 451 million people from a 2017 report (Cho et al., 2018). Consistently high blood glucose levels can bring about serious health complications. DM, when not well controlled, can result in nerve damage (neuropathy), kidney failure, and blindness (retinopathy), and raises the risk of foot ulcers and inflammation which can result in a need for lower limb amputation (WHO, 2016). However, despite several DM medication reports, there is a wide range of side effects, including weight gain, hypoglycemia, and coronary heart disease (Pandey et al., 2011). Accordingly, identifying chemical entities that have the potential to become antidiabetic drugs is of importance.

One important class of natural compounds that have potential as new antidiabetic agents is flavonoids (Chen et al., 2015), which are phenolic compounds found in various species of plants (Panche et al., 2016). This group of natural products possess a diverse range of biological activities, and many of them are used as therapeutic agents to treat disease, and play an important role in the pharmaceutical industry (Brodowska, 2017). For antidiabetic activity, flavonoids show several mechanistic pathways such as the regulation of the metabolism of carbohydrates, insulin secretion and glucose uptake, the enhancement of the proliferation of β-cells, the mitigation of apoptosis, and the reduction of hyperglycemia by manipulating the liver's metabolism of glucose. Moreover, some of these compounds can reduce the risk of complications from DM (AL-Ishaq et al., 2019).

Erythrina subumbrans (Hassk.) Merr. (Fabaceae) or known as 'Thong Lang Pa' in Thailand has been reported to be a source of a large number of flavonoids, including flavanones, isoflavone, and pterocarpans (Rukachaisirikul et al., 2007a, 2008, 2014). This plant is a deciduous tree with spreading branches and stout prickles which is distributed in tropical and subtropical region such as, China, India, Sri Lanka, Myanmar, Thailand, Laos, Vietnam, Malaysia, Indonesia and the Philippines (Pillay et al., 2001). In Thailand, the leaves of this plant have been used as an ingredient in Thai cuisines. In contrast, the bark has been used in traditional medicinal remedies to treat microbial infections and nausea (Pillay et al., 2001) and a number of pterocarpans and flavonoids have been isolated that have antibacterial properties (Rukachaisirikul et al., 2007b). Although the isolated flavonoids from this plant show many biological activities (Rashid et al., 2019), the curative effect of the isolated compounds from E. subumbrans on diabetes and their complications has not been reported. In the current study, the twig and root extracts of E. subumbrans were used to investigate their chemical components and to evaluate their antidiabetic, anti-inflammatory, and antimicrobial activities. The project aimed to discover primary bioactive compounds for developing antidiabetic therapeutic drugs in the future.

2. Materials and methods

2.1. General procedure

The general information on the instrumentation used and chemicals were the same as in previous reports (Phukhatmuen et al., 2020; Raksat et al., 2020; Tantapakul et al., 2016).

2.2. Plant material

The roots and twigs of E. subumbrans (Fabaceae) were collected in 2019 from plants growing in Doi Tung, Chiang Rai Province, Thailand (20°20′ 27.00″ N and 99° 50′ 2.39″ E). The plant was authenticated by Mr. Martin Van de Bult, a botanist at Doi Tung Development Project, Chiang Rai, Thailand. A herbarium specimen number MFU-NPR0188 has been deposited at the Natural Products Research Laboratory, Mae Fah Luang University.

2.3. Extraction and isolation

Air-dried roots (2.7 kg) and twigs (4.1 kg) of E. subumbrans were extracted with ethyl acetate (EtOAc) (3 × 5 L) over a period of 3 days at room temperature. Removal of the solvent under reduced pressure yielded the EtOAc extract of the roots (49.3 g) and the twigs (128.7 g), respectively. The root extract (49.3 g) was fractionated by quick column chromatography (QCC) on silica gel eluting with a gradient of hexanes-acetone to give seven fractions (RES1-RES7). Compounds 2 (35.7 mg), 3 (3.0 mg), 7 (6.2 mg), 10 (16.4 mg), 12 (3.7 mg), 13 (2.7 mg), 14 (2.5 mg) and 22 (1.7 mg) were isolated from RES4 (2.73 g). Compounds 1 (2.9 mg), 4 (4.1 mg), 6 (3.7 mg), 8 (2.3 mg), 9 (2.2 mg), 11 (15.8 mg), 19 (7.1 mg), 20 (9.9 mg), and 21 (5.4 mg) were obtained from RES6 (6.3 g). For a flowchart of the isolation procedure, see Figure 1.

Figure 1.

Figure 1

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Flowchart of the isolation procedure from the root extract of E. subumbrans.

The twig extract (128.7 g) was subjected to QCC on silica gel eluting with a gradient of hexanes-EtOAc to obtain eight fractions (TES1-TES8). Compounds 5 (7.3 mg), 7 (2.6 mg), 19 (3.1 mg) and 27 (2.4 mg) were isolated from TES3 (6.3 g). Fraction TES4 (10.8 g) was subjected to CC on Sephadex LH-20 using 100% MeOH as eluent to afford six fractions (TES4.1-TSE4.6). Compounds 16 (2.8 mg), 23 (32.4 mg), and 26 (3.2 mg) were isolated from TES4.2 (433.7 mg) while compound 22 (3.4 mg) was obtained from TES4.3 (254.2 mg). Fraction TES4.5 (632.8 mg) was further purified by CC on silica gel yielding compounds 6 (4.6 mg), 24 (3.8 mg), and 25 (3.6 mg). Compounds 1 (1.0 mg), 17 (3.4 mg), and 28 (2.9 mg) were obtained from fraction TSE5 (4.1 g) whereas compounds 15 (12.1 mg) and 18 (5.2 mg) were obtained from TSE6 (9.2 g). For a flowchart of the isolation procedure, see Figure 2.

Figure 2.

Figure 2

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Flowchart of the isolation procedure from the twig extract of E. subumbrans.

Erythrinocarpan (1). Light yellow viscous oil. UV λ max (log ε): 246 (2.10), 286 (3.19) nm; IR (KBr) υ max: 3467, 1613, 1491, 1169 cm−1; 1H and 13C-NMR spectral data, see Table 1; HRESITOFMS m/z 443.1829, [M + Na]+ (calcd for C26H28NaO5, 443.1835).

Table 1.

NMR Spectroscopic data of compound 1 (500 MHz in CDCl3).

position δC δH (J in Hz) COSY (1H–1H) HMBC (1H →13C)
1 153.2 - - -
2 114.3 - - -
3 156.3 - - -
4 101.2 6.30 (s) - C-1, C-2, C-3, C-4a, C-11b
4a 154.0 - - -
6 65.0 5.44 (s) - C-4a, C-6a, C-6b, C-11a
6a 107.3 - - -
6b 118.7 - - -
7 116.1 6.80 (d, 8.2) H-8 C-6b, C-8, C-9
8 112.8 7.09 (d, 8.2) H-7 C-6a, C-6b, C-9, C-10, C-10a
9 152.1 - - -
10 110.0 - - -
10a 154.7 - - -
11a 145.9 - - -
11b 104.7 - - -
1′ 22.5 3.42 (d, 7.2) H-2′, H-4′, H-5′ C-1, C-2, C-3, C-1′, C-2′, C-3′
2′ 122.2 5.25 (t, 7.2) H-1′, H-4′, H-5′ -
3′ 135.2 - - -
4′ 18.0 1.84 (s) H-1′, H-2′ C-2′, C-3′, C-5′
5′ 25.9 1.76 (s) H-1′, H-2′ C-2′, C-3′
1″ 23.4 3.73 (d, 7.1) H-2″, H-4″, H-5″ C-9, C-10, C-10a, C-2″, C-3″
2″ 121.2 5.41 (t, 7.1) H-1″, H-4″, H-5″ -
3″ 135.7 - - -
4″ 18.0 1.87 (s) H-1″, H-2″ C-2″, C-3″, C-5″
5″ 25.9 1.77 (s) H-1″, H-2″ C-2″, C-4″
OMe-1 62.5 3.89 (s) - C-1
OH-3 5.46 (s) C-2, C-3, C-4
OH-9 5.32 (s) C-8, C-9, C-10
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2.4. α-Glucosidase inhibition activity

The α-glucosidase inhibitory assay was performed using the same method as described in our previous reports (Phukhatmuen et al., 2020; Raksat et al., 2020). The positive controls were acarbose, voglibose, and quercetin.

2.5. α-Amylase inhibition activity

The α-amylase inhibitory assay was modified from a previous report (Kusano et al., 2011). Acarbose, voglibose, and quercetin were used as positive controls.

2.6. Glycation inhibition activity

The procedure for the glycation inhibition assay was performed using the same procedure as the previous report (Justino et al., 2016). The standard control was quercetin.

2.7. Glucose uptake activity

The glucose uptake assay was conducted using the same procedure as the previous report with slight alteration (Sharma et al., 2019), and metformin was used as the standard control.

2.8. Glucose consumption activity

The glucose consumption assay was performed using the same procedure as our previous report (Phukhatmuen et al., 2020), and metformin was used as standard control. Cell viability was carried out by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, as previously described (Ahmed et al., 1994).

2.9. Nitric oxide production inhibition activity

The nitric oxide production (NO) inhibition assay was performed using the same procedure as previously reported (Dong et al., 2017), and aminoguanidine was used as a positive control and 0.1% DMSO as a negative control. Cell viability studies were evaluated by the MTT assay (Ahmed et al., 1994).

2.10. Antimicrobial activity

Bacterial strains including Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Micrococcus luteus DMST 15503, methicillin resistant Staphylococcus aureus NPRC 001R, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC, 19615), Gram-negative bacteria (Shigella flexneri DMST 4423, Pseudomonas aeruginosa ATCC 10145, Salmonella typhi DMST 22842, Salmonella typhimurium DMST 562, Escherichia coli TISTR 780) and fungi (Candida albicans ATCC 10231) used in these experiments were obtained from the Microbiological Resources Center, Thailand. A 2-fold serial dilution method (Nutrient broth) was used for the antibacterial assays and the determination of minimum inhibitory concentrations (MICs) (Wikler et al., 2006; Tantapakul et al., 2016). The antimicrobial assays were tested in triplicate, and the standard compounds were vancomycin and gentamycin.

3. Results and discussion

3.1. Isolation and structure elucidation

The EtOAc extracts of the twigs and roots of E. subumbrans were purified by repeated chromatographic techniques to obtain 28 compounds (Figure 3) including 14 pterocarpans (1–14), five flavanones (15 and 22–25), three flavones (16–18), three isoflavones (19–21) and three phenolic derivatives (26–28). The known compounds were identified as eryvarin E (2), eryvarin D (3) (Tanaka et al., 2001a), erycristagallin (4) (Hegde et al., 1997), erythribyssin N (5) (Nguyen et al., 2010), coumestrol (6) (Yuk et al., 2011), erycristin (7) (Mitscher et al., 1988), 1-methoxyerythrabyssin II (8) (Rukachaisirikul et al., 2008), erythrabyssin II (9) (Tanaka et al., 1998), sandwicensin (10) (Mckee et al., 1997), phaseollidin (11) (Dagne et al., 1993), erypoejin J (12) (Tanaka et al., 2003), orientanol C (13) (Tanaka et al., 1998), gangetin (14) (Purushothaman et al., 1986), pinocembrin (15) (Ramirez et al., 2013), 3,6,4′-trihydroxyflavone (16), 3,6-dihydroxyflavone (17) (Park et al., 2006), 5-methoxy-7-hydroxyflavone (18) (Dao et al., 2003), lupalbigenin (19) (Pistelli et al., 1996), erysubin F (20) (Tanaka et al., 2001b), genistein (21) (Ma et al., 2013), 5-hydroxysophoranone (22) (Matsuura et al., 1994), 7,2′,4′-trihydroxy-8,3′,5′-(3″-methyl- but-2″-enyl)flavanone (23) (Xia et al., 2019), 4′-hydroxy-6,3′,5′-triprenylisoflavanone (24), abyssinone V (25) (Hegde et al., 1997), xanthoxyletin (26) (Wu and Furukawa, 1983), 2,2-dimethyl-2H-chromene-6-carboxaldehyde (27) (Smith et al., 2003) and 2-hydroxy-4-methoxy-cinnamic acid (28) (Hofer et al., 1986) by comparisons with the literature reported spectroscopic data.

Figure 3.

Figure 3

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Compounds isolated from twigs and roots of E. subumbrans.

Compound (1) was obtained as a light yellow viscous oil. The molecular formula of C26H28O5 was determined from its HRESITOFMS, which showed an apparent molecular ion at m/z 443.1829 [M + Na]+ (calcd for C26H28NaO5, 443.1835). The IR spectrum displayed the hydroxy group at 3467 cm−1, while the UV spectrum showed maximum absorptions at λmax 246 and 286 nm. The 13C NMR and DEPT spectroscopic data (Table 1) displayed resonances for 26 carbons including five methyls (δC 18.0, 18.0, 25.9, 25.9, and 62.5), three methylenes (δC 65.0, 23.4, and 22.5), five methines (δC 122.2, 121.2, 116.1, 112.8, and 101.2), and 13 quaternary carbons (δC 156.3, 154.7, 154.0, 153.2, 152.1, 145.9, 135.7, 135.2, 118.7, 114.3, 110.0, 107.3, and 104.7). The 1H NMR spectroscopic data (Table 1) revealed resonances for three aromatic protons [δH 7.09 (1H, d, J = 8.2 Hz, H-7), 6.80 (1H, d, J = 8.2 Hz, H-8), and 6.30 (1H, s, H-4)], one methylene [δH 5.44 (2H, s, H-6)]. The NMR spectroscopic data suggested that the structure of 1 had a pterocarpan core structure, which was closely related in structure to that of erycristagallin (4) (Hegde et al., 1997). The difference between compounds 1 and 4 was that compound 1 displayed one extra a methoxy group at C-1 [δH 3.89 (3H, s, 1-OMe)]. In addition, two sets of isoprenyl units [δH 5.25 (1H, t, J = 7.2 Hz, H-2′), 3.42 (2H, d, J = 7.2 Hz, H-1′), 1.84 (3H, s, H-4′), and 1.76 (3H, s, H-5′) and 5.41 (1H, t, J = 7.1 Hz, H-2″), 3.73 (2H, d, J = 7.1 Hz, H-1″), 1.87 (3H, s, H-4″), and 1.77 (3H, s, H-5″)] were identified. The following HMBC correlations (Figure 4) established the attachments of a methoxy, a hydroxy, and an isoprenyl unit on the A ring at C-1, C-3, and C-2, respectively: MeO-1 (δH 3.89) with C-1 (δC 153.2); HO-3 (δH 5.46) with C-2 (δC 114.3), C-3 (156.3), and C-4 (δC 101.2); H2-1′ (δH 3.42) with C-1 (153.2), C-2 (δC 114.3), and C-3 (δC 156.3); H-4 (δH 6.30) with C-2, C-3, C-4a (δC 154.0), and C-11b (δC 104.7). In addition, the HMBC correlations between H2-1′′ (δH 3.73) with C-9 (δC 152.1), C-10 (δC 110.0), and C-10a (δC 154.7) and HO-9 (δH 5.32) with C-8 (δC 112.8), C-9 (δC 152.1), and C-10 (δC 110.0) confirmed the other isoprenyl unit and hydroxy group where on the D ring at C-10 and C-9, respectively. Therefore, the structure of 1 was characterized as erythrinocarpan.

Figure 4.

Figure 4

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1H–1H COSY and HMBC key correlations of erythrinocarpan (1).

3.2. Antidiabetic activities (enzymatic based assays)

Various natural products directly or indirectly affect diabetes pathways as enzyme inhibitors (Alam et al., 2019). Several enzymes are involved in the pathogenesis of the disease. In this study, the properties of α-glucosidase inhibition, α-amylase inhibition, and glycation inhibition of all isolated compounds (1–28) from E. subumbrans were evaluated (Table 2).

Table 2.

α-Glucosidase, α-amylase and glycation inhibition activities of compounds 1–28 isolated from E. subumbrans roots and twigs.

Compound α-Glucosidase inhibition (IC50, μM) α-Amylase inhibition (IC50, μM) Glycation inhibition (IC50, μM)
1 60.8 ± 0.25 Inactive Inactive
2 Inactive 67.6 ± 1.12 Inactive
3 13.4 ± 0.05 Inactive Inactive
4 90.8 ± 2.86 Inactive Inactive
5 99.9 ± 0.38 Inactive Inactive
6 Inactive Inactive Inactive
7 Inactive Inactive Inactive
8 24.5 ± 0.13 Inactive 53.1 ± 1.65
9 29.0 ± 0.05 Inactive Inactive
10 Inactive Inactive Inactive
11 Inactive Inactive Inactive
12 Inactive Inactive Inactive
13 Inactive Inactive Inactive
14 Inactive Inactive 95.6 ± 1.02
15 Inactive Inactive 83.1 ± 0.94
16 Inactive Inactive Inactive
17 Inactive Inactive Inactive
18 Inactive Inactive Inactive
19 Inactive Inactive Inactive
20 Inactive Inactive Inactive
21 Inactive Inactive Inactive
22 12.8 ± 0.14 Inactive 36.9 ± 0.62
23 Inactive Inactive Inactive
24 Inactive Inactive 40.5 ± 0.37
25 Inactive Inactive Inactive
26 Inactive Inactive Inactive
27 Inactive Inactive Inactive
28 Inactive Inactive Inactive
Acarbose 76.7 ± 1.42 103.4 ± 0.89 NT
Voglibose 134.2 ± 0.10 201.2 ± 1.94 NT
Quercetin 28.0 ± 0.18 174.3 ± 0.68 59.1 ± 0.78
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Inactive at >100 μM.

In the α-glucosidase inhibition assay, seven compounds including six pterocarpans (1, 3–5, 8 and 9), and one flavanone (22), showed α-glucosidase inhibition activities with IC50 values ranging from 12.8 ± 0.14–99.9 ± 0.38 μM. Of these compounds, compounds 22 (IC50 of 12.8 ± 0.14 μM), 3 (IC50 of 13.4 ± 0.05 μM), and 8 (IC50 of 24.5 ± 0.13 μM), showed inhibitory activity greater than that of the positive controls [acarbose (IC50 of 76.7 ± 1.42 μM), voglibose (IC50 of 134.2 ± 0.10 μM), and quercetin (IC50 of 28.0 ± 0.18 μM)] (Table 2). Compound 9 (IC50 of 29.0 ± 0.05 μM) also showed inhibitory activity better than that of acarbose and voglibose but was less active than quercetin. Compound 22 was the only flavanone that revealed significant α-glucosidase inhibitory activity. It is interesting to note that the structures of compounds 1 and 8 only differ by the degree of unsaturation at the B/C ring junction (Δ6a(11a)). The more unsaturated compound (1) showed significantly less α-glucosidase inhibition activity than its more saturated counterpart (8) (Table 2). Similarly, the presence of the prenyl unit at C-2 in compound 2 (IC50 > 100 μM) is crucially important to reduce the α-glucosidase inhibition activity when compared to compound 3 (IC50 of 13.4 μM) containing a proton.

In the case of α-amylase inhibitory activity, only pterocarpan 2 showed inhibitory activity with an IC50 value of 62.6 ± 1.12 μM, which was stronger than that of the positive controls [acarbose (IC50 103.4 ± 0.89 μM), voglibose (IC50 201.2 ± 1.94 μM), and quercetin (IC50 174.3 ± 0.68 μM)] (Table 2). The other compounds had IC50 values of >100 μM. It should be noted that the structure of pterocarpan 2 differed from pterocarpans 3, 4 and 7 only at C-2 (R2 = H), C-9 (R4 = OH), and the B/C ring junction (Δ6a(11a)), respectively. These differences have a much greater effect on reducing the α-amylase inhibitory activity of pterocarpans 3, 4 and 7.

Glycation inhibition assay was evaluated by advanced glycation end products (AGEs) analysis by emulating the conditions of proteins under hyperglycaemic conditions. This process is significantly related to diabetes long-term complications such as diabetic nephropathy, inflammatory conditions, and fibrosis (Uribarri et al., 2010). Among the tested compounds, flavanones 22 and 24 showed the best glycation inhibitory activities with IC50 values of 36.9 ± 0.62 and 40.5 ± 0.37 μM, respectively, which were better than the positive control (quercetin, IC50 value of 59.1 ± 0.78 μM, Table 2). Interestingly, flavanones 22 and 24 in which the A-ring prenyl unit is at C-8 and C-6, respectively, have very similar glycation inhibition activities.

3.3. Antidiabetic activities (cell-based assays)

Previous studies indicated that some natural products, especially flavonoids, can enhance glucose uptake activity in L-6 cell lines (Narmatha and Maneemegalai, 2019). In this study, cell-based assays on the antidiabetic properties of the isolated compounds via glucose consumption and glucose uptake assays were determined and the results are shown in Table 3. Only two compounds, compounds 26 and 27, illustrated the ability to induce glucose consumption into 3T3-L1 adipocyte cells with IC50 values of 33.4 ± 0.34 and 29.1 ± 0.86 μM, respectively without toxicity to the cells. To confirm the activity of promoting glucose consumption, these compounds were further evaluated for glucose uptake induced by L6 myotube cells. The results point out the relationship between glucose consumption with a ratio of glucose uptake at 1.3-fold and 2.8-fold compared with metformin positive control (Table 3). The outcome means that these compounds can transport glucose into cells to supply cells with energy in the form of adenosine triphosphate (ATP) and play an important role in many other cellular functions (Busik et al., 2002).

Table 3.

Glucose consumption, glucose uptake and NO inhibition of compounds 1–28 isolated from E.subumbrans roots and twigs.

Compound Glucose consumption (IC50, μM) Glucose uptake (fold) NO Inhibition (IC50,
μM)		 Cell viability of 3T3-L1 cells (%) Cell viability raw 264.7 cells (%)
1 Inactive Inactive Inactive 54.1 ± 0.9 99.4 ± 0.6
2 Inactive Inactive Inactive 76.5 ± 1.1 56.1 ± 2.4
3 Inactive Inactive Inactive 84.3 ± 1.6 13.2 ± 0.7
4 Inactive Inactive Inactive 94.4 ± 0.5 97.3 ± 0.3
5 Inactive Inactive Inactive 34.2 ± 0.9 98.6 ± 1.6
6 Inactive Inactive Inactive 65.5 ± 1.2 3.7 ± 0.2
7 Inactive Inactive Inactive 85.2 ± 1.8 3.1 ± 0.9
8 Inactive Inactive Inactive 40.5 ± 0.9 2.9 ± 0.6
9 Inactive Inactive Inactive 78.3 ± 0.7 3.3 ± 0.2
10 Inactive Inactive Inactive 64.2 ± 0.3 3.0 ± 0.1
11 Inactive Inactive Inactive 43.9 ± 0.9 18.9 ± 0.3
12 Inactive Inactive Inactive 56.5 ± 1.3 95.1 ± 0.7
13 Inactive Inactive Inactive 36.9 ± 0.8 3.4 ± 0.1
14 Inactive Inactive Inactive 43.1 ± 0.4 2.8 ± 0.1
15 Inactive Inactive Inactive 77.3 ± 1.4 92.9 ± 0.8
16 Inactive Inactive 96.2 ± 1.61 39.7 ± 1.9 98.4 ± 0.5
17 Inactive Inactive Inactive 43.6 ± 1.1 97.7 ± 0.6
18 Inactive Inactive Inactive 41.6 ± 1.8 5.7 ± 1.1
19 Inactive Inactive Inactive 79.6 ± 1.8 3.8 ± 0.1
20 Inactive Inactive Inactive 84.7 ± 0.9 93.7 ± 0.7
21 Inactive Inactive Inactive 74.5 ± 1.4 81.3 ± 0.5
22 Inactive Inactive Inactive 80.8 ± 1.2 3.67 ± 0.9
23 Inactive Inactive Inactive 26.9 ± 0.9 12.1 ± 1.1
24 Inactive Inactive Inactive 39.7 ± 0.7 3.7 ± 0.1
25 Inactive Inactive Inactive 62.4 ± 1.4 3.4 ± 0.2
26 33.4 ± 0.34 1.3 ± 0.21 Inactive 97.5 ± 0.3 21.6 ± 1.5
27 29.1 ± 0.86 2.8 ± 0.47 52.5 ± 0.56 98.2 ± 1.7 97.9 ± 1.2
28 Inactive Inactive Inactive 40.6 ± 1.1 97.1 ± 0.2
Metformin 47.2 ± 1.17 3.7 ± 0.96 NT 96.1 ± 0.9 99.4 ± 0.6
Aminoguanidine NT NT 22.4 ± 0.78 NT 97.9 ± 0.8
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Inactive at >100 μM.

3.4. Nitric oxide production inhibitory activity

Nitric oxide (NO) is a general signaling molecule considered a pro-inflammatory mediator that plays a part in inflammation pathogenesis (Luiking et al., 2010). This signaling induces inflammation due to over-production in abnormal situations (Sharma et al., 2007). Prolonged DM is one of the factors that is positively associated with inflammation by activating pathways for the production of pro-inflammatory cytokines (NO) (Kim et al., 2008). Only compounds 16 and 27 showed nitric oxide production inhibitory activity with IC50 values of 96.2 ± 1.61 and 52.5 ± 0.56 μM, respectively, which were less active than the standard control (aminoguanidine, IC50 value of 22.4 ± 0.78 μM, Table 3) Compared with prior studies, compounds in the same group of active compounds, including flavones, showed the greatest potential to inhibit nitric oxide production with IC50 values of 3.8–25.1 μM (Menini et al., 2020). The compounds in groups of phenylpropanoid derivatives similar to compound 27 have also been reported to inhibit nitric oxide production with IC50 values in the range of 26.3–31.6 μM (Cai et al., 2020). Our data can infer that compounds 16 and 27 are only moderately active inhibitors of LPS-stimulated NO production in RAW 264.7 cells.

3.5. Antimicrobial activity

The most severe complications of diabetes are foot ulcer infections, which can ultimately lead to lower limb amputations (Ramirez-Acuña et al., 2019). Therefore, compounds 1–28 were also evaluated for their antimicrobial activities against Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Micrococcus luteus DMST 15503, methicillin resistant Staphylococcus aureus NPRC 001R, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC, 19615), Gram-negative bacteria (Shigella flexneri DMST 4423, Pseudomonas aeruginosa ATCC 10145, Salmonella typhi DMST 22842, Salmonella typhimurium DMST 562, Escherichia coli TISTR 780) and fungi (Candida albicans ATCC 10231) using the broth microdilution assay. Compound 9 showed the best activities against Gram-positive bacterial and fungi with MIC values ranging from 2-4 μg/mL, whereas compound 10 was less active with MIC values ranging from 8-64 μg/mL (Table 4). Antibacterial activities against S. aureus and MRSA of pterocarpans 9 and 10 have been reported with MIC values in the range of 0.78–1.56 (Rukachaisirikul et al., 2007b) and 8–64 μg/mL (Innok et al., 2010), respectively. In this study, the MIC values against S. aureus and MRSA of pterocarpans 9 and 10 were 2–4 and 8–64 μg/mL, respectively, similar to that found in the previous study (Rukachaisirikul et al., 2007b; Innok et al., 2010). All remaining compounds were found to have weak activities or were inactive against all strains of bacteria and fungi. It should be noted that compounds 9 and 10 shared the same core structure but have a different number of hydroxy and isoprenyl groups. Compound 9 contains two hydroxy groups (C-3 and C-9) and two isoprenyl groups (C-2 and C-10) while compound 10 comprises a hydroxy (C-3), a methoxy (C-10), and an isoprenyl (C-10) group. From this information we might conclude that compound 9 has better antimicrobial activity than 10, due to the extra hydroxy and isoprenyl groups in the former compound.

Table 4.

Antimicrobial activity of compounds 1–28 isolated from E. subumbrans roots and twigs.

Compound MIC (μg/mL)
Gram-positive bacteria
 Gram-negative bacteria
 Fungi
EF MT MRSA SA SP SF PA ST STR EC CA
1 Inactive Inactive Inactive Inactive Inactive Inactive 128 128 128 128 Inactive
2 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive Inactive 128 Inactive
3 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive 128 128 Inactive
4 Inactive Inactive Inactive Inactive Inactive Inactive 128 128 Inactive 128 Inactive
5 Inactive Inactive Inactive Inactive Inactive Inactive 128 128 Inactive 128 Inactive
6 128 Inactive 32 Inactive Inactive 128 128 128 Inactive 128 128
7 128 128 128 64 128 128 128 128 Inactive 128 128
8 128 128 128 64 128 Inactive 128 128 Inactive 128 128
9 2 2 4 2 2 Inactive 128 128 Inactive Inactive 4
10 8 8 8 64 8 128 128 Inactive 128 Inactive 16
11 128 128 64 64 64 Inactive 128 128 Inactive Inactive 128
12 Inactive Inactive Inactive 128 Inactive Inactive 128 128 Inactive Inactive Inactive
13 Inactive Inactive Inactive 128 Inactive Inactive 128 128 Inactive 128 Inactive
14 128 128 64 32 64 Inactive 128 128 Inactive 128 128
15 Inactive Inactive Inactive 128 Inactive 128 128 Inactive Inactive 128 Inactive
16 Inactive Inactive Inactive 128 Inactive Inactive 128 Inactive Inactive 128 Inactive
17 Inactive Inactive Inactive 128 Inactive Inactive 128 Inactive Inactive Inactive Inactive
18 Inactive Inactive Inactive 128 Inactive Inactive 128 128 Inactive 128 Inactive
19 Inactive Inactive 128 128 64 Inactive 128 Inactive Inactive 128 128
20 128 Inactive Inactive 16 Inactive Inactive Inactive Inactive Inactive Inactive 16
21 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive Inactive Inactive Inactive
22 Inactive Inactive Inactive 128 128 128 128 Inactive Inactive 128 Inactive
23 Inactive Inactive Inactive 128 Inactive Inactive 128 Inactive Inactive 128 Inactive
24 Inactive Inactive 128 128 128 Inactive 128 128 Inactive 128 128
25 Inactive 128 Inactive 128 128 128 128 128 128 Inactive Inactive
26 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive Inactive Inactive Inactive
27 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive Inactive Inactive Inactive
28 Inactive Inactive Inactive Inactive Inactive Inactive 128 Inactive Inactive Inactive Inactive
Vancomycin 2 2 1 0.5 2 - - - - - 4
Gentamicin - - - - - 4 4 1 0.5 4 -
Open in a new tab

Inactive at >128 μg/mL. EF = Enterococcus faecalis (ATCC 29212); MT = Micrococcus luteus (DMST 15503); MRSA = Methicillin Resistant Staphylococcus aureus (NPRC 001R); SA = Staphylococcus aureus (ATCC 25923); SP = Streptococcus pyogenes (ATCC, 19615); SF = Shigella flexneri (DMST 4423); PSA = Pseudomonas aeruginosa (ATCC 10145); STP = Salmonella typhi (DMST 22842); STM = Salmonella typhimurium (DMST 562); EC = Escherichia coli (TISTR 780); CA = Candida albicans (ATCC 10231).

4. Conclusion

In conclusion, a new pterocarpan (1), was isolated together with 27 known compounds (2–28) from the root and twig extracts of E. subumbrans. The compounds were identified as 14 pterocarpans (1–14), five flavanones (15 and 22–25), three flavones (16–18), three isoflavones (19–21) and three phenolic derivatives (26–28). Five compounds (1, 6, 7, 19, and 22) were obtained from both extracts, whereas 12 compounds (2–4, 8–14, 20, and 22) and 11 compounds (5, 15–18, and 23–28) were obtained only from the root and twig extracts, respectively. Compounds 5, 6, 7, 12, 13, 15, 16, 17, 18, 21, 23, 24, 25, 26, 27, and 28 were isolated for the first time from this plant. All isolated compounds were evaluated for their potential antidiabetic activities (enzyme and cells-based assays) along with anti-inflammatory effects through nitric oxide production inhibitory activities and their antimicrobial activities. Compounds 22, 3, 8, and 9 displayed promising α-glucosidase inhibition activity, while compound 2 showed the best α-amylase inhibition activity. Furthermore, compounds 22 and 24 were effective to prevent AGEs. Compound 27 showed the best ability to induce glucose consumption and to enhance glucose uptake. It also had NO production inhibitory activity without causing toxicity to cells. These bioactive compounds might be considered as a group of antidiabetic agents with potential for further development as new drugs to treat diabetes mellitus and complications. Compounds 9 and 10 were identified as the most promising compounds showing potent activity against all Gram-positive strains and fungi.

Declarations

Author contribution statement

Piyaporn Phukhatmuen, Pornphimol Meesakul: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Virayu Suthiphasilp: Analyzed and interpreted the data; Wrote the paper.

Rawiwan Charoensup, Tharakorn Maneera, Sarot Cheenpracha: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Thunwadee Limtharakul, Stephen G. Pyne: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Surat Laphookhieo: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by Thailand Science Research and Innovation (DBG6280007 & DBG6180029), National Research Council of Thailand (NRCT5-RGJ63013-147), Mae Fah Luang University, Pornphimol Meesakul is supported by the Postdoctoral Fellowship from Mae Fah Luang University.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Acknowledgements

We would like to thanks Mr. Martin Van de Bult, Doi Tung Development Project, Chiang Rai, Thailand, for plant identification. Mae Fah Luang University and Chiang Mai University are also acknowledged for laboratory facilities.

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