Phenolic acids from Balanophora involucrata and their bioactivities

Fitoterapia 121 (2017) 129–135

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Phenolic acids from Balanophora involucrata and their bioactivities a,b

b

b

b

b

MARK b

Jiangchun Wei , Xiaokui Huo , Zhenlong Yu , Xiangge Tian , Sa Deng , Chengpeng Sun , Lei Fengb,⁎, Chao Wangb, Xiaochi Mab, Jingming Jiaa,⁎ a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China College of Pharmacy, Academy of Integrative Medicine, Liaoning Engineering Technology Centre of Target-based Nature Products for Prevention and Treatment of Ageingrelated Neurodegeneration, Dalian Medical University, Dalian 116044, PR China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Balanophora involucrata Phenolic acid α-Glucosidase Anti-oxidant GlmU

The bioactive substance investigation of Balanophora involucrata obtained 15 phenolic acids, including 5 new compounds (1–3, 8, 9), which were determined by various spectroscopic data analyses. Most isolated compounds displayed inhibitory effects on α-Glucosidase in vitro. For the potential inhibitors 8 (1.95 μM) and 10 (9.02 μM), the inhibition kinetics have been studied, which gave the Ki values as 0.68, 3.15 μM respectively. And, in silico docking analyses have been performed to investigate the inhibitory mechanism of compounds 8 and 10. Additionally, most isolated compounds showed anti-oxidant activities in the DPPH scavenging assay. New compound 8 also could inhibit the acetyl transfer activity of GlmU moderately with the IC50 value of 18.21 μM, which was a new antibacterial target.

1. Introduction Balanophora involucrata Hook. f., a parasitic plant growing on the roots of leguminous plants, is distributed mainly in the southwest of China, India, and Nepal. The whole plant is a folk medicine for the treatment of irregular menstruation, cough, hemoptysis, traumatic injury and bleeding, dizziness, and gastralgia [1,2]. Chemical investigations revealed the existences of flavanone glucosides, dihydrochalcones, cyanogenic glycosides, tannins, alkane glycosides, lignans, triterpenoids and steroids [3–8]. And, the isolated compounds displayed various biological activities, such as anti-inflammatory, BACE inhibitory, anti-oxidation, and analgesic effects [4,5,9]. It is obvious that the chemical substance of B. involucrata displayed the characteristic of chemical diversity, especially the phenolic acids. The present work investigated the chemical constituents of B. involucrata as well as their biological activities continually. As a result, fifteen compounds (1–15) were obtained from the whole plant of B. involucrata using various chromatographic techniques. Their structures were determined on the basis of widely spectroscopic data, including 1D NMR, 2D NMR, HRESIMS, and ECD. (Fig. 1) The inhibitory effects of isolated compounds against α-Glucosidase were evaluated in vitro as well as the inhibition kinetics of potential inhibitors were studied. In silico docking analyses were performed to investigate the inhibition mechanism. For the phenolic acids, the anti-oxidant effects of 1–15 were also evaluated using the DPPH assay. Additionally, the inhibitory

⁎

Corresponding authors. E-mail addresses: [email protected] (L. Feng), [email protected] (J. Jia).

http://dx.doi.org/10.1016/j.fitote.2017.07.003 Received 17 June 2017; Received in revised form 1 July 2017; Accepted 6 July 2017 Available online 08 July 2017 0367-326X/ © 2017 Published by Elsevier B.V.

effects of 1–15 on M. tb. GlmU were investigated in vitro, which was a new target for the treatment of tuberculosis. 2. Experimental 2.1. General methods Optical rotations were measured with on a JASCO P2000 automatic polarimeter. UV spectra were recorded by a JASCO V-650 UV spectrophotometer. NMR spectra were measured with tetramethylsilane (TMS) on Burker-500, -600 NMR spectrometers. ESI-MS and HRESIMS data were measured using an API 3200 mass spectrometer (AB SCIEX, Framingham, MA, USA) and an Agilent 1290 infinity 6540 UHD accurate Q-TOF mass spectrometer respectively. Analytical HPLC data were collected on an UltiMate 3000 instrument (Thermo Scientific dionex) equipped with a diode array detector (DAD). Preparative HPLC was performed by an Agel instrument with UV detector and a YMC C18 column (250 × 20 mm, 5 μm). Column chromatographic separations were carried out on silica gel (200–300 mesh Qingdao Marine Chemical Group Corporation, Qingdao, China), Waters Semi-Preparative to Preparative-Scale HPLC Purification (Waters, USA). GF254 silica gel TLC plates (Yantai Chemical Industrial Institute, Yantai, China) were used for analytical TLC. Acetonitrile and Methanol were used of chromatographic grade purchasing from Sigma-Aldrich in American. All other solvents were used of chemical grade (Kermel Chemical Co. Ltd.,

Fitoterapia 121 (2017) 129–135

J. Wei et al.

Fig. 1. Compounds 1–15 obtained from Balanophora involucrate.

(3.1 mg, 0.03% TFA, 30% CH3OH aqueous, tR = 24.98 min), 13 (6.9 mg, 0.03% TFA, 40% CH3OH aqueous, tR = 22.78 min). Fraction 4 (8.12 g) were subjected to a MPLC (ODS) eluted with CH3OH − H2O (0.03% TFA, 30%–60% CH3OH aqueous) to get 18 fractions. Subfractions 7, 10 and 13 were dealt with a fully automatic preparative HPLC with CH3OH − H2O (30%–60% CH3OH aqueous), and then purified by preparative HPLC (detected at 203 nm and 254 nm,) to afford compounds 1 (12.6 mg, 0.03% TFA, 35%, tR = 29.98 min), 2 (4.2 mg, 0.03% TFA, 35%, tR = 25.74 min), 6 (4.3 mg, 0.03% TFA, 35% CH3OH aqueous, tR = 28.20 min), 7 (1.7 mg, 0.03% TFA, 20% CH3OH aqueous, tR = 22.66 min), 8 (8.5 mg, 0.03% TFA, 30% CH3OH aqueous, tR = 34.02 min), 14 (2.3 mg, 0.03% TFA, 25% CH3OH aqueous, tR = 22.46 min), 15 (3.1 mg, 0.03% TFA, 35% CH3OH aqueous, tR = 30.22 min).

Tianjin, China). 2.2. Plant material The whole plant of Balanophora involucrate were collected from Enshi city of Hubei province in China and identified by Prof. Qing-Shan Yang, Anhui University of Chinese Medicine (2015). A voucher specimen was deposited in the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University (No. K-3262). 2.3. Extraction and isolation The powdered whole plant of Balanophora involucrate (2.5 kg) were extracted by 95% ethanol aqueous solution refluxed (1.5 h × 3) and then extracted with petroleum ether, ethyl acetate, n-Butanol successively. The ethyl acetate extracts (316 g) was dealt with a silica gel column by elution with CHCl3-CH3OH (50:1–1:1) in sequence to give fractions 1–32. Fraction 3 (5.23 g) was subjected to a MPLC (ODS) eluted with CH3OH − H2O (0.03% TFA, 30%–70%) to get 29 fractions. Sub-fractions 11, 14, 16 and 18 were dealt with a fully automatic preparative HPLC with CH3OH − H2O (30%–50%), and then purified by preparative HPLC (detected at 203 nm and 254 nm) to afford compounds 3 (12.6 mg, 0.03% TFA, 40% CH3OH aqueous, tR = 40.56 min), 4 (4.2 mg, 0.03% TFA, 30% CH3OH aqueous, tR = 50.34 min), 5 (4.3 mg, 0.03% TFA, 45% CH3OH aqueous, Rt = 24.24 min), 9 (1.7 mg, 0.03% TFA, 35% CH3OH aqueous, tR = 35.82 min), 10 (8.5 mg, 0.03% TFA, 35% CH3OH aqueous, tR = 22.56 min), 11(2.3 mg, 0.03% TFA, 25% CH3OH aqueous, tR = 24.10 min), 12

2.3.1. Inovonoid A (1) White amorphous powder; [α]D25 − 18.4 (c 0.1 Methanol); UV λmax 201.7 (4.18), 218.6 (3.10), 275.8 (2.46); ECD (Methanol) 224 (Δ ε − 3.49); 1H NMR (DMSO‑d6, 600 MHz), see Table 1; 13C NMR (DMSO‑d6, 150 MHz), see Table 2; HRESIMS m/z 347.0772 (calcd C17H15O8, 347.0768). 2.3.2. Inovonoid B (2) White amorphous powder; [α]D25 − 8.5 (c 0.1 Methanol); UV λmax 200.5 (4.31), 231.5 (3.15), 289.5 (3.08); ECD (Methanol) 255 (Δ ε + 1.18); 1H NMR (DMSO‑d6, 600 MHz), see Table 1; 13C NMR (DMSO‑d6, 150 MHz), see Table 2; HRESIMS m/z 357.0955 (calcd C19H18O7, 357.0974). 130

Fitoterapia 121 (2017) 129–135

J. Wei et al.

(DMSO‑d6, 600 MHz), see Table 1; 13C NMR (DMSO‑d6, 150 MHz), see Table 2; HRESIMS m/z 309.0562 (calcd C12H14O8Na, 309.0586).

Table 1 1 H NMR spectroscopic data of new compounds (600 MHz, DMSO‑d6, δ in ppm, J in Hz). No.

1

2

3

8

9

2

7.02 d (1.2)

6.94 d (1.8)

6.89 d (1.2)

2.58 dd (15.0, 4.8) 2.45 dd (15.0, 8.4) 4.16 m 4.09 m

7.67 d (1.2)

6.81 d (7.8) 6.86 dd (7.8,1.2) 4.9 d (7.2) 4.19 m

6.75 d (7.8) 6.79 dd (7.8, 1.8) 5.52 d (6.6) 3.47 m

3.53 brd (12.6) 3.34 dd (12.6, 4.8) 6.98 d (1.8)

3.72 m 3.62 m

6.72 d (7.8) 6.76 dd (8.4) 4.3d (6.6) 2.81 dd (3.72) 4.04 d (9.0)

3 4 5 6 7 8 9

2′

6.95 d (1.8)

4′ 5′ 6′

2.4. Yeast α-glucosidase inhibitory activity 6.40 dd (1.8)

Compounds 1–15 were tested for their ability to inhibit α-glucosidase. This assay was carried out as previously described [10].

4.22 d (5.4)

2.5. DPPH scavenging assay 6.97 s

The free radical scavenging rates of these samples were detected by DPPH assay as the previous and acknowledged method, which was widely used in the literature [11]. Meanwhile, vitamin C was used as a positive control.

6.74 d (1.8)

6.74 s

7.04 d (1.8)

7′ 8′

7.11 brd

6.88 s

7.44 d (16.2) 6.22 d (16.2)

4.75 d (6.0)

9′ OCH3

6.74 s

2.3.5. Inovonoid E (9) White amorphous powder; [α]D25 − 15.5 (c 0.1 Methanol); UV λmax 200.4 (4.57), 287.4 (3.08); 1H NMR (DMSO‑d6, 600 MHz), see Table 1; 13 C NMR (DMSO‑d6, 150 MHz), see Table 2; HRESIMS m/z303.0871 (calcd C16H15O6, 303.0869).

3.78 s

3.75 s

6.80 d (7.8) 6.58 dd (7.8, 1.8)

6.97 s

2.6. Inhibitory effects on mycobacterial tuberculosis GlmU GlmU acetyltransferase assay were performed by 5,5′-dithio-bis-(2nitrobenzoic acid) (DTNB) colorimetric assay using the purified M. tb GlmU protein [12].

3.34 m 3.72 m 3.08 t 3.76 s 3.76 s

3.59 s

2.7. Molecular docking

3.84 s 3.73 s

The stable configurations of compounds 8 and 10 were subjected to molecular docking studies based on the comparison of the geometrical parameters between calculated model and crystal structure. The preparation of the pdbqt files was done by standard procedure using AutoDock Tools 1.5.6 [13]. The docking procedures were performed in AutoDock Vina using the default scorning function. Exhaustiveness was set to 100, and number of output conformations was set to 20 [14]. The searching seed was random. The calculated geometries were ranked in terms of free energy of binding and the best poses were selected for further analysis [15].

Table 2 13 C NMR spectroscopic data of new compounds (150 MHz, DMSO‑d6, δ in ppm). No.

1

2

3

8

9

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1-OCH3 3-OCH3 7-OCH3 3′-OCH3 5′-OCH3

127.7 112.3 148.1 147.5 115.7 121.1 76.4 78.2 60.6 122.9 109.4 144.1 137.0 146.6 109.7 167.5

132.6 110.9 148.0 146.9 115.8 119.2 88.0 53.4 63.3 128.0 116.6 141.8 132.6 132.6 116.5 145.0 116.0 168.3

132.3 110.2 147.3 146.0 115.1 118.6 87.0 53.9 70.2 129.6 117.9 145.3 115.2 147.5 109.7 81.4 49.4 68.9

170.7 38.4 64.8 66.7

127.9 117.8 141.7 131.3 153.9 113.3 165.8 60.1

56.2

56.1

55.7

118.6 108.1 144.9 137.9 144.9 108.1 165.1

3. Results and discussion Compound 1 obtained as a white amorphous powder, had the molecular formula C17H16O8 determined by HRESIMS (−) m/z 347.0772 (calcd [M-H]− 347.0767), which indicated the unsaturation degree of 10. The UV spectrum displayed the absorption of aromatic moieties at λmax 201, 218.6, 275.8 nm. The 1H NMR spectrum indicated the existences of two oxygenated methines (δH 4.90, 4.19), one oxygenated methene (δH 3.53, 3.34), one methoxyl (δH 3.78), one aromatic ABX coupling system (δH 7.02 d, J = 1.2 Hz; 6.81 d, J = 7.8 Hz; 6.86 dd, J = 7.8, 1.2 Hz), two aromatic protons with meta-position (δH 6.98 d, J = 1.8 Hz; 7.04 d, J = 1.8 Hz). The 13C NMR spectrum gave 17 carbon signals, which indicated the presences of 12 aromatic carbons, one carboxylic carbon, three oxygenated carbons, and one methoxyl. Combination of the 1H and 13C NMR spectra, suggested the existences of one tri-substituted aromatic ring and one tetra-substituted aromatic ring. On the basis of the spectroscopic data, compound 1 was deduced to be a nor-lignan derivative similar to arteminorin D [16], possessing C7-O-C4′, C8-O-C3′ ether bonds. In the HMBC spectrum, the phenolic hydroxyl proton (δH 9.18) correlated with C-3 (δC 148.1), C-5 (δC 115.7), and the methoxyl protons (δH 3.78) correlated with C-3 (δC 148.1), which determined the 3-methoxyl-4-hydroxy-trisubstituted phenyl ring (Fig. 2a). The HMBC correlations from the oxygenated methine δH 4.90 to C-2, C-6, C-8, C-9 established the C6-C3 moiety for compound 1. For the other phenyl ring, the long range correlations of phenolic hydroxyl (δH 9.60)/C-5′, C-4′, H-2′ (δH 6.98)/C-7′, H-6′ (δH

125.6 113.1 146.5 145.0 114.5 121.5

50.7 51.4 55.0 55.7

2.3.3. Inovonoid C (3) White amorphous powder; [α]D25 − 10.3 (c 0.1 Methanol); UV λmax 200.5 (4.50), 229.4 (3.02), 280.7 (2.28); ECD (Methanol) 276 (Δ ε − 0.37); 1H NMR (DMSO‑d6, 600 MHz), see Table 1; 13C NMR (DMSO‑d6, 150 MHz), see Table 2; HRESIMS m/z 381.1313 (calcd C20H22O6Na, 381.1314). 2.3.4. Inovonoid D (8) White amorphous powder; [α]D25 − 720. (c 0.1 Methanol); UV λmax 215.9 (4.04), 274.9 (3.15); ECD (Methanol) 355 (Δ ε + 0.42); 1H NMR 131

Fitoterapia 121 (2017) 129–135

J. Wei et al.

Fig. 2. Key HMBC correlations (a) and ECD spectrum (b) of compound 1.

substituted phenyl (δH 6.74 s; 6.88 s; 6.74 s) groups was determined. The coupling constants of H-7/H-8, H-7′/H-8′ were J = 6.6 Hz, J = 6.0 Hz, respectively, which indicated the trans positions of H-7/H8, H-7′/H-8′. Therefore, the structure of compound 3 was elucidated as shown in Fig. 1, named Invonoid C. Compound 8 with the molecular formula C12H14O8 determined by HRESIMS displayed the aromatic absorptions at 215.9, 274.9 nm in the UV spectrum. Two aromatic protons δH 6.99 s (2H) was observed the 1H NMR spectrum. Combined with the 13C NMR spectrum, a galloyl group was established for the structure of 8. Additionally, one oxygenated methene δH 4.09 (2H), one oxygenated methine δH 4.16 (1H), one methoxyl δH 3.59 (3H), one methene δH 2.58, 2.45, as well as a carboxyl group (δC 170.7) were observed from the NMR data. In the HMBC spectrum, the long range correlations of H-2/C-4, C-3, C-1, H-3/C-1, OCH3/C-1, and H-4/C-7′ established a 3,4-dihydroxy-methyl butyrate moiety, which was attached to the C-7′ of galloyl group by C-4. The absolute configuration of 3-OH was determined by Rh2(OCOCF3)4 induced ECD experiment [22]. As a result, a positive Cotton effect at 360 nm indicated a 3S configuration for compound 8. Thus, compound 8 was determined to be Invonoid D. Compound 9 was also deduced to be a phenolic acid derivative on the basis of the spectroscopic data. The 1H NMR spectrum of 9 displayed the presences of an aromatic ABX system (δH 6.58 dd, J = 7.8, 1.8 Hz; 6.80 dd, J = 7.8 Hz; 6.74 d, J = 1.8 Hz), two aromatic protons (δH 7.67 d, J = 1.2 Hz; 7.40 d, J = 1.2 Hz), together with two phenolic hydroxyls (δH 9.48 s, 9.00, s) and two methoxyls (δH 3.73, 3.84, 3H, s respectively). The 13C NMR data confirmed the abovementioned moieties and indicated the carboxyl carbon (δC 165.8). Analysis of the spectroscopic data suggested that compound 9 was a biphenyl derivative. The HMBC correlations of H-2 (δH 7.67)/C-7 (δC 165.8), C-8 (δC 60.1), OCH3 (δH 3.84)/C-7, OH (δH 9.48)/C-5 established a 3,4,5-trisubstituted benzoate group. Similarly, a 1,3,4-trisubstituted phenyl group was established. Additionally, the long range correlations of H-2′ (δH 6.74)/C-4 (δC 131.3), H-6′ (δH 6.58)/C-4 linked two aromatic rings. On the basis of spectroscopic data, compound 9 was elucidated to be Inovonoid E. The other known compounds were identified to be (+)-5-hydroxypinoresinol (4) [23], (+)-pinoresinol (5) [23], isolariciresinol(6) [24], burselignan (7) [25], brevifolin (10) [26], brevifolin carboxylic acid (11) [26], methyl brevifolin carboxylate (19) [26], ethyl brevifolin carboxylate (13) [27], phyllanthusiin E (14) [28], phyllanthusiin E methyl ester (15) [28] on the basis of their spectroscopic data and literatures. The inhibitory effects of compounds 1–15 against α-Glucosidase have been evaluated in vitro with acarbose as the positive control. Most of the isolated compounds could inhibitory α-Glucosidase with the IC50 < 100 μM, as well as the IC50 of acarbose was 505.79 μM (Table 3). Lignans 1–6 displayed moderate inhibitory effects, which was weaker than phenolic acids 8 (1.95 μM) and 10 (9.02 μM). For the

7.04)/C-7′ established the tetra-substituted phenyl ring with a carboxylic carbon for compound 1. The ether bond linkage of C7-O-C4′, C8O-C3′ could also be determined by the weak long range correlations between H-7/C-4′, H-8/C-3′. Therefore, compound 1 was established to be nor-lignane. The coupling constant J = 7.2 Hz between H-7 and H-8 suggested the trans configuration [16]. The absolute configuration of 1 was determined using the electronic circular dichroism (ECD) exciton chirality method [17]. The ECD spectrum of 1 showed a negative Cotton effect at 224 nm due to the transition interaction between two different benzene moieties in the structure. The above information demonstrated a negative chirality for 1, and the two aforementioned chromophores should be oriented counterclockwise in space (Fig. 2b). Thus, the absolute configuration of 1 was determined as (7S, 8S). Thus, compound 1 was elucidated to be Invonoid A. Compound 2 with the molecular formula C19H18O7 determined by HRESIMS and 13C NMR data was also deduced to be a lignane derivative. The 1H NMR spectroscopic data indicated the existences of two methines (δH 5.52, 3.47), one oxygenated methene (δH 3.72, 3.62), one methoxyl (δH 3.75), and one E-olefinic bond (δH 6.22 d, J = 16.2 Hz; 7.44 d, J = 16.2 Hz), one ABX-system (δH 6.75 d, J = 7.8 Hz; 6.79 d, J = 7.8, 1.8 Hz; 6.94 d, J = 1.8 Hz), two meta aromatic protons (δH 7.11 d, J = 1.8 Hz; 6.95 d, J = 1.8 Hz). The 13C NMR spectrum of 2 gave 19 carbon signals, which confirmed the abovementioned moieties. Analysis of the spectroscopic data suggested that compound 2 possessed one tri-substituted phenylpropanoid and one tetra-substituted phenylpropionic acid substructures, which was similar to spicatolignan B, except for the phenolic hydroxyl group of the aromatic ring [18]. In the 1 H-1H COSY spectrum, H-7/H-8/H-9 spin-spin system was established by the correlations. And, the propyl group was located to the tri-substituted phenyl ring on the basis of long range correlations of H-7/C-1, C-2, C-6. The trans propionic acid moiety was assigned at the tetrasubstituted phenyl ring by the HMBC correlations H-7′/C-1′, C-2′. The furan ring was also established on the basis of HMBC correlations H-7/ C-4′, H-8/C-5′. Therefore, the planar structure of compound 2 was established. The coupling constant J = 6.6 Hz indicated the trans H-7/H8. The ECD spectrum displayed a positive Cotton effect at 281.5 nm, which indicated the 7S, 8R configuration for compound 2 on the basis of (−)-(7R,8S,7′E)-3,4,5,5′-tetramethoxy-4′,7-epoxy-8,3′-neolign-7′ene-9,9′-diol, and (+)-(7S,8R,7′E)-4-hydroxy-3,5′-dimethoxy-4′,7epoxy-8,3′-neolign-7′-ene-9,9′-diol 9′-ethyl ether [19].Therefore, compound 2 was determined to be Invonoid B. Analysis of the spectroscopic data indicated that compound 3 was a lignan possessed the framework of the pinoresinol-type with two benzene moieties, which was similar to lignans 4-(4-(3,5-dimethoxyphenyl) hexahydrofuro[3,4c]furan-1-yl)- 2-methoxy-phenol, (+)-pinoresinol, pandanusphenol A [20,21], except for the substituents of phenyl groups and the configuration. On the basis of the 1D-, and 2D–NMR spectroscopic data, one 1,3,4-tri-substituted phenyl (δH 6.76 dd, J = 7.8 Hz; 6.72 dd, J = 7.8, 1.2 Hz; 6.89 d, J = 1.2 Hz) and one 1,3,5-tri132

Fitoterapia 121 (2017) 129–135

J. Wei et al.

hydroxyls in the structures, antioxidant activities of 1–15 were evaluated using DPPH scavenging assay. As shown in Table 4, most compounds displayed inhibitory effects on DPPH scavenging, which suggested the antioxidant effects with the positive control vitamin C. Especially, compounds 10, 11 and 15 displayed significant antioxidant activities with IC50 8.74, 8.59, 7.46 μM compared with vitamin C. The GlmU protein widely existed in the bacteria is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities, which are mainly responsible for catalyzing the formation of UDP-N-acetylglucosamine (UDP-GlcNAc) from glucosamine-1-P (GlcN-1-P), UTP, and acetyl-CoA (Ac-CoA). And the final product UDP-GlcNAc is essential for two important biosynthetic pathways of the cell wall: lipopolysaccharide and peptidoglycan synthesis. Therefore, the GlmU is regarded as a novel vital target for the antibacteria substance development. In the present paper, the inhibitory effects of compounds 1–15 against GlmU were evaluated in vitro. Most compounds displayed weak inhibitory effects at the concentration 100 μM (Fig. 6). However, compound 8 could inhibit the acetyl transfer activity of GlmU significantly with the IC50 value of 18.21 μM.

Table 3 The inhibitory effects of compounds 1–15 on α-Glucosidase. Compounds

IC50 (μM)

Compounds

IC50 (μM)

1 2 3 4 5 6 7 8

70.34 92.63 69.25 83.43 89.36 87.52 > 100 1.95

9 10 11 12 13 14 15 Acarbose

86.53 9.02 45.64 68.32 66.05 > 100 85.66 505.79

potential inhibitors 8 and 10, the inhibition kinetics have been studied in vitro. On the basis of the inhibition kinetics and the intersection point in the horizontal axis in the Lineweaver–Burk plots, the inhibition type of compound 8 towards α-Glucosidase was best fit for the uncompetitive inhibition type. The inhibition kinetic parameter (Ki) was determined to be 0.68 μM (Fig. 3). And, compound 10 displayed a noncompetitive inhibition type on the basis of the inhibition kinetics and Lineweaver–Burk plots with IC50 value as 3.15 μM (Fig. 4). In silico docking analyses have been performed to investigate the molecular mechanism between compounds 8, 10 and α-Glucosidase. The docking experiment revealed Vander waals interaction between the molecular 8 and the residues Arg315, Arg442, Asp352. Meanwhile, hydrophobic interactions were observed between molecular 8 and the residues Phe178, Phe159, Tyr158. His280, Phe303 of α-Glucosidase. (Fig. 5a, b) As shown in Fig. 5c, d, hydrogen bonds were formed between the lactone and ketone oxygen atoms of 10 and Gln279 (3.0, 3.2 Å), His280 (3.0 Å). Additionally, hydrophobic interactions were observed between the molecular 10 and Tyr158, Phe159, Arg315, Tyr316, Phe314, Phe303. So, various Vander waals, hydrogen bonds and the hydrophobic interactions improved the interaction between compounds 8, 10 and α-Glucosidase, respectively. In consideration that most isolated compounds had phenolic

4. Conclusions The present bioactive substance investigation of Balanophora involucrata obtained 15 phenolic acids, including 5 new compounds (1–3, 8, 9). The bioactivities of isolated compounds were evaluated in vitro using various bioassays. The new compound 8 and known compound 10 could inhibit α-Glucosidase significantly with the IC50 values of 1.95, 9.02 μM respectively. In the inhibition kinetics experiment, the Ki values were determined to be 0.68, 3.15 μM respectively. And, in silico docking analyses observed various Vander waals, hydrogen bonds and the hydrophobic interactions between the molecular 8, 10 and αGlucosidase. Additionally, most isolated compounds showed anti-oxidant activities in the DPPH scavenging assay. New compound 8 also could inhibit the acetyl transfer activity of GlmU moderately with the IC50 value of 18.21 μM, which was a new antibacterial target.

Fig. 3. The inhibition kinetics of compound 8 towards α-Glucosidase. (a) Dose-dependent inhibition behavior of compound 8 towards the activity of α-Glucosidase; (b) Inhibition kinetics of compound 8 towards α-Glucosidase; (c) Lineweaver–Burk plot of 8′s inhibition on α-Glucosidase; (d) Dixon plot of 8′s inhibition on α-Glucosidase.

133

Fitoterapia 121 (2017) 129–135

J. Wei et al.

Fig. 4. The inhibition kinetics of compound 10 towards α-Glucosidase. (a) Dose-dependent inhibition behavior of compound 10 towards the activity of α-Glucosidase; (b) Inhibition kinetics of compound 10 towards α-Glucosidase; (c) Lineweaver–Burk plot of 10′s inhibition on α-Glucosidase; (d) Determination of inhibition kinetic parameter (Ki) using the slopes from Lineweaver–Burk plot towards the concentration of 10. The data points represent the mean value of duplicateexperiments.

Conflict of interest The authors have declared that there is no conflict of interest. Jiangchun Wei, Xiaokui Huo, Zhenlong Yu, Xiangge Tian, Sa Deng, Chengpeng Sun, Lei Feng, Chao Wang, Xiaochi Ma, Jingming Jia.

Natural Science Foundation of China (No. 81473423, 81503201, 81622047), and the Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University. We appreciate the help of Prof. Qing-Shan Yang at Anhui University of Chinese Medicine.

Acknowledgments

Appendix A. Supplementary data

This research program is financially supported by the National

1

H NMR,

13

C NMR, 2D NMR, and HRESIMS of new compounds Fig. 5. Docking analysis between compounds 8 (a, b) and 10 (c, d) and α-Glucosidase. (a, c) α-Glucosidase (surface) ̶ ligand (stick) complex. (b, d) Binding mode of both 8, 10 (yellow carbons) with αGlucosidase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

134

Fitoterapia 121 (2017) 129–135

J. Wei et al.

[7] B. Luo, K. Zou, Z.Y. Guo, F.J. Dan, J.Z. Wang, H. Wang, Balanoinvolin, a new steroid derivative from Balanophora involucrate, Chem. Nat. Compd. 45 (2009) 371. [8] H.L. Ruan, J. Li, X.Y. Zhao, Y.H. Zhang, M. Xiang, J.Z. Wu, Studies on anti- inflammatory and analgesic effects of Balanophora involucrata, Chin. Arch. Tradit. Chin. Med. 21 (2003) 910. [9] Z.Z. He, J.F. Yan, Z.J. Song, F. Ye, X. Liao, S.L. Peng, L.S. Ding, Chemical constituents from the aerial parts of Artemisia minor, J. Nat. Prod. 72 (2009) 1198–1201. [10] S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell, Y.T. Trinh, Q. Zhang, I.L. Urbatsch, G. Chang, Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding, Science 323 (2009) (1718–1722). [11] R. Apak, M. Ozyurek, K. Guçlu, E. Çapanog, Antioxidant activity/capacity measurement 1. Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays, J. Agric. Food Chem. 64 (2016) 997–1027. [12] W.L. Zhang, V.C. Jones, M.S. Scherman, S. Mahapatra, D. Crick, S. Bhamidi, Y. Xin, R. McNeil, Y.F. Ma, Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase, Int. J. Biochem. Cell Biol. 40 (2008) 2560–2571. [13] W. Luo, Y. Chen, T. Wang, C. Hong, L.P. Chang, C.C. Chang, Y.C. Yang, S.Q. Xie, C. Wang, Design, synthesis and evaluation of novel 7-aminoalkyl-substituted flavonoid derivatives with improved cholinesterase, Bioorg. Med. Chem. 24 (2016) 672–680. [14] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31 (2010) 455–461. [15] W. Xu, J. Huang, B. Shao, X. Xu, R. Jiang, M. Yuan, Design, synthesis, crystal structure, biological evaluation and molecular docking studies of carbazole-arylpiperazine, Bioorg. Med. Chem. 24 (2016) 5565–5572. [16] P.H. Nguyen, J.L. Yang, M.N. Uddin, S.L. Park, S. Lim, D.W. Jung, D.R. Williams, W.K. Oh, Protein tyrosine phosphatase 1B (PTP1B) inhibitors from Morinda citrifolia (Noni) and their insulin mimetic activity, J. Nat. Prod. 76 (2013) 2080–2087. [17] L. Xiong, C.G. Zhu, Y.R. Li, Y. Tian, S. Lin, S.P. Yuan, J. Hu, Q. Hou, N.H. Chen, Y. Yang, J.G. Shi, Lignans and neolignans from Sinocalamus affinis and their absolute configurations, J. Nat. Prod. 74 (2011) 1188–1200. [18] Y.N. Yang, H. Zhu, Z. Chen, F. Liu, Y.W. An, Z.M. Feng, J.S. Jiang, P.C. Zhang, NMR spectroscopic method for the assignment of 3,5-dioxygenated aromatic rings in natural products, J. Nat. Prod. 78 (2015) 705–711. [19] G.K. Wang, B.B. Lin, R. Rao, K. Zhu, X.Y. Qin, G.Y. Xie, M.J. Qin, A new lignan with anti-HBV activity from the roots of Bombax ceiba, Nat. Prod. Res. 27 (2013) 1348–1352. [20] X.P. Zhang, H.F. Wu, C.M. Wu, P. Guo, X.D. Xu, M.H. Yang, Pandanusphenol A and B: two new phenolic compounds from the fruits of Pandanus tectorius Soland, Rec. Nat. Prod. 7 (2013) 359–362. [21] H. Liu, C.J. Li, J.Z. Yang, N. Ning, Y.K. Si, L. Li, N.H. Chen, Q. Zhao, D.M. Zhang, Carbazole alkaloids from the stems of Clausena lansium, J. Nat. Prod. 75 (2012) 677–682. [22] M. Mitsuo, K. Hiroyuki, K. Hiromu, Biotransformation of lignans: a specific microbial oxidation of (+)-eudesmin and (+)-magnolin by Aspergillus niger, Phytochemistry 34 (1993) 1501–1507. [23] X.Y. Chen, J.Y. Liang, Chemical constituents of planted taxus chinensis var. mairei, Chin. J. Nat. Med. 4 (2006) 101–103. [24] A. Jutiviboonsuk, H.J. Zhang, G.T. Tan, C.Y. Ma, N.V. Hung, N.M. Cuong, N. Bunyapraphatsara, D.D. Soejarto, H.H.S. Fong, Bioactive constituents from roots of Bursera tonkinensis, Phytochemistry 66 (2005) 2745–2751. [25] M.A.M. Nawwar, S.A.M. Hussein, I. Merfort, NMR spectral analysis of polyphenols from Punica granatum, Phytochemistry 36 (1994) 793–798. [26] M. Wolniak, M. Tomczyk, J. Gudej, I. Wawer, Structural studies of methyl brevifolin carboxylate in solid state by means of NMR spectroscopy and DFT calculations, J. Mol. Struct. 825 (2006) 26–31. [27] H.E. Gottlieb, S. Kumar, M. Sahai, A.B. Ray, Ethyl brevifolin carboxylate from Flueggea microcarpa, Phytochemistry 30 (1991) 2435–2438. [28] K.P. Latte, H.K. Pelargoniins, New ellagitannins from Pelargonium reniforme, Phytochemistry 54 (2000) 701–708.

Table 4 The antioxidant effects of compounds 1–15 for the DPPH scavenging. Compounds

IC50 (μM)

Compounds

IC50 (μM)

1 2 3 4 5 6 7 8

> 50 96.47 24.98 23.91 38.56 45.26 63.84 12.09

9 10 11 12 13 14 15 Vitamin Cb

> 50 8.74 8.59 9.52 10.58 9.87 7.46 8.94

Inhibition rate of Glmu (%)

100 ** *

80 60 40 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0 Compounds Fig. 6. The GlmU inhibitory effects of compounds 1–15 (100 μM).

associated with this article can be found online. Supplementary data associated with this article can be found in the online version, at http:// dx.doi.org/10.1016/j.fitote.2017.07.003. References [1] X.L. Shen, Y.J. Hu, Y.M. Shen, Q.Z. Mu, Study on chemical constituents of Balanophora involucrate Hook. f, Chin. Tradit. Herb. Drugs 27 (1996) 259–260. [2] X.Z. Xia, H.X. Han, P.F. Tu, Studies on triterpenoids and steroids from Balanophora involucrate, Chin. Tradit. Herb. Drugs 32 (2001) 6–9. [3] J.Y. Pan, S. Zhang, L.S. Yan, J.D. Tai, Q. Xiao, K. Zou, Y. Zou, J. Wu, Separation of flavanone enantiomers and flavanone glucoside diastereomers from Balanophora involucrate. Hook. f. by capillary electrophoresis and reversed-phase high-performance liquid chromatography on a C18 column, J. Chromatogr. A 1185 (2008) 117–129. [4] J.Y. Tao, J. Zhao, Y. Zhao, Y.M. Cui, W.S. Fang, BACE inhibitory flavanones from Balanophora involucrate Hook. f, Fitoterapia 83 (2012) 1386–1390. [5] G.M. She, Y.J. Zhang, C.R. Yang, A new phenolic constituent and a ayanogenic glycoside from Balanophora involucrata (Balanophoraceae), Chem. Biodivers. 10 (2013) 1081–1087. [6] J.Y. Pan, Y. Zhou, K. Zou, J. Wu, Q.X. Li, S. Zhang, Chemical constituents of Balanophora involucrate, Chin. Tradit. Herb. Drugs 39 (2008) 327.

135