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Purification and characterization of four benzophenone derivatives from Mangifera indica L. leaves and their antioxidant, immunosuppressive and α-glucosidase inhibitory activities
Journal of Functional Foods 52 (2019) 709–714
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Purification and characterization of four benzophenone derivatives from Mangifera indica L. leaves and their antioxidant, immunosuppressive and α-glucosidase inhibitory activities
T
Chengzhen Gua,b, Meilian Yanga, Zhihong Zhouc, Afsar Khand, Jianxin Caoa, , Guiguang Chenga, ⁎
⁎
a
Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming 650500, People’s Republic of China College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, People's Republic of China c Institute of Traditional Chinese Medicine Research, Yunnan University of Traditional Chinese Medicine, Kunming 650504, People's Republic of China d Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Pakistan b
ARTICLE INFO
ABSTRACT
Keywords: Mangifera indica Phytochemicals ROS Immunosuppressive activity α-Glucosidase inhibition
The aim of the present study was to isolate phytochemicals from Mangifera indica L. leaves and to screen their bioactivities. Four benzophenone derivatives were isolated and characterized, two of which were new compounds, namely manindicins A and B. All the four compounds significantly inhibited the cellular ROS amount of H2O2-induced HepG2 cells. Stimulation index of spleen cells showed that manindicins A and B had a good and similar immunosuppressive activity, while mangiferin and norathyriol exhibited relatively weaker activity. Norathyriol showed the strongest inhibitory effect on α-glucosidase with an IC50 of 4.22 ± 0.19 μg/mL, which was lower than that of acarbose (16.28 ± 1.22 μg/mL). However, the manindicins A and B had weak inhibition of α-glucosidase. The present work suggests that M. indica leaves can be used potentially as functional-food supplement for improving human health.
1. Introduction The genus Mangifera (Anacardiaceae) has more than 70 species and 1000 varieties, and is widely distributed in the tropical and subtropical areas (Ni & Xie, 2009). The Mangifera indica L. fruit is one of the most important fruit-crops with extensive acceptance due to its succulence, exotic flavour, and nutritional value. In addition, the fruit is a good source of carotenoids, lupeol, mangiferin, and phenolic acids with a wide range of pharmacological properties for human health. Apart from consumption as ripe fruit, these are usually processed into juices, jam, and beverages. The rising demand for nutritional supplement of consumers resulted in a constant growth of mango fruits production. A large number of by-products (i.e. leaves, peels, and kernels) are discarded as industrial waste. Indeed, it is well known that mango byproducts contain significant amounts of health-enhancing substances including mangiferin, gallic acid, ferulic acid, epicatechin, gallotannins, phenolics, flavonoids, benzophenones, and so on (Burtonfreeman, Sandhu, & Edirisinghe, 2017; Fang et al., 2018; Fernández-Ponce, Medina-Ruiz, Casas, Mantell, & Martínez de la Ossa-Fernández, 2018; Kim et al., 2018; Matkowski, Kus, Goralska, & Wozniak, 2013; Pan, Yi, Zhang, et al., 2018). These bioactive compounds exhibited various
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biological activities, such as antioxidant (Amaya-Cruz et al., 2015; Gondi, Basha, Bhaskar, Salimath, & Prasada Rao, 2015), anti-inflammatory (Natal et al., 2017; Pan, Yi, Wang, Chen, & He, 2018), antidiabetic (Rodríguez-González et al., 2017), hepatoprotective (Medina Ramírez et al., 2018), cholesterol-lowering (Gururaja et al., 2017), and anti-obesogenic activities (Fang et al., 2018). The leaves of M. indica are a kind of crop waste. However, the leaves are consumed traditionally as folk tea and medicine to reduce the risk of respiratory diseases in China (Deng & Zeng, 2003). Pharmacological studies showed that the extracts of leaves have antioxidant, antibacterial, antiviral, antidiabetic, anti-inflammatory, and anti-tumor activities (Ahmed, 2007; Ajila, 2008; Liu, Wang, Zhou, Gong, & Deng, 2017; Rajendran & Ekambaram, 2008; Yoshimi & Matsunaga, 2001; Zheng & Lu, 1990). Previous studies on mango leaves reported different kinds of secondary metabolites, including flavonoids, terpenoids, phytosterols, coumarins, and lignans, of which phenolics are the characteristic constituents (Anjaneyulu, 1985; Rodeiro & Cancino, 2006). Among them, the benzophenone mangiferin contributed to the majority of phenolics. Since tea plays an important role in human life and mango leaves contain a high amount of bioactive phenolic compounds, the investigation of chemical constituents in mango leaves is thus
Corresponding authors. E-mail addresses: [email protected] (J. Cao), [email protected] (G. Cheng).
https://doi.org/10.1016/j.jff.2018.11.045 Received 5 August 2018; Received in revised form 13 November 2018; Accepted 28 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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necessary. A phytochemical investigation of the leaves of M. indica resulted in the isolation of four benzophenone derivatives. Manindicins A and B, benzophenone glycosides, were identified by extensive spectroscopic techniques, while two known compounds were characterized as mangiferin and norathyriol. Some of these compounds exhibited significant antioxidant, immunosuppressive, and α-glucosidase inhibitory activities. The study suggested that mango leaves can be served as dietary supplement for a functional food.
serum (FBS) and 1% antibiotic mixture of 100 U/mL penicillin/streptomycin under a humidified 5% CO2 atmosphere. Cytotoxicity assay of HepG2 cells was performed by MTT method (Mosmann, 1983) with some modifications. Briefly, cells were centrifuged at a density of at 4.0 × 105 cells/well and kept for 24 h. Different concentrations of the test compounds were added in serum-free medium. After 20 h of treatment, the cells were incubated with 150 μL MTT reagent (0.5 mg/ mL) for 4 h. Subsequently, the MTT reagent was removed, and 150 μL dimethyl sulfoxide (DMSO) was added for dissolving the formazan crystals. Then, HepG2 cells were washed carefully once with PBS, and maintained in serum-free DMEM with 100 μL of 0.5 mg/mL 0.05% MTT for 4 h. Then, 150 μL of DMSO was added per well for the dissolution of formazan. The spectrophotometric absorbance at 570 nm was measured by utilizing a microplate reader. The cell viability results showed that all samples under the test concentrations had no cytotoxicity to HepG2 cells by comparison with the control group.
2. Materials and methods 2.1. General experimental procedures Optical rotation values were recorded on a JASCO P-1020 digital polarimeter. IR data were recorded by a Bruker Tensor 27 spectrometer with KBr pellets; NMR spectra were performed on a Bruker DRX400 MHz spectrometer with TMS as an internal standard. High resolution electrospray ionization mass spectrometry (HRESIMS) in positive mode was obtained on a Thermo high resolution Q-exactive focus mass spectrometer. D101 (Canzhou Bon Chemical Co., Ltd. Hebei, China) macroporous resin, MCI-gel CHP-20P (75–150 μm, Mitsubishi, Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden) were used for column chromatography. Thin-layer chromatography (TLC) was performed on silica gel 60 G254 coated glass plates (Qingdao Ocean Chemical Co., Ltd, Qingdao, China), and the spots were observed either by UV light or by spraying the plates with 10% H2SO4.
2.5. Measurement of intracellular reactive oxygen species levels The intracellular ROS oxidative stress in oxidized HepG2 cells was determined using a 2′,7′-dichlorodihydrofluorescin diacetate (DCFHDA) ROS assay (Zhuang, Ma, Guo, & Sun, 2017). Briefly, HepG2 cells were exposed to the test compounds under different concentrations (12.5, 25, and 50 μM). After washing with PBS twice, the collected cells were resuspended in serum-free medium with 10 mM DCFH-DA at 37 °C in the dark for 20 min. After that, the supernatant was discarded and the cells were washed three times with PBS. The intracellular ROS generation was immediately recorded at excitation wavelength of 485 nm and emission wavelength of 535 nm by using a flow cytometry (Guava easy Cyte 6-2L; Millipore, Billerica, MA, US).
2.2. Plant material M. indica leaves were collected in June 2014 in Yuanjiang county, Yunnan province, China, and identified by Prof. Z. H. Zhou (Yunnan University of TCM, China). The voucher sample was deposited at the Functional Food Laboratory, Yunnan Institute of Food Safety, Kunming University of Science and Technology.
2.6. Splenocyte proliferation assay The immunosuppressive activity was carried out using the CCK-8 method as reported previously (Gao et al., 2017). Briefly, spleens were isolated from male BALB/c mice. The spleens were gently crushed and filtered through a 200 μm mesh screen, and washed repeatedly with cold PBS. Cells were collected by centrifugation. Recovered splenocytes were transferred into a sterile centrifuge tube, mixed with 3 mL TrisNH4Cl (Biosharp, China) for lysing the remaining erythrocytes. The cells were washed twice with PBS and re-suspended in RPMI 1640 complete medium (Gibco, USA) with 10% fetal bovine serum, and 1% penicillin–streptomycin. Cells were seeded into 96-well flat-bottom microtiter plates at a density of 1 × 106 cell/mL, and treated with concanavalin A (Con A, 10 μg/mL) in the presence and absence of the test compound (10, 20, and 40 μM) using dexamethasone (DXM) as a positive control. Splenocytes without Con A-treated were served as negative control. After incubation for 72 h at 37 °C in a humidified atmosphere, 10 μL of CCK-8 was added and incubated for another 4 h. The tests were conducted for three independent replicates, and the absorbance at 450 nm was measured on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The stimulation index (SI) was calculated based on the following formula: SI = the absorbance value for mitogen-cultures/the absorbance value for non-stimulated cultures.
2.3. Extraction and isolation The air-dried leaves of M. indica (25 kg) were smashed, and extracted with distilled water for three times (each time for four hours) under reflux. The filtrates were combined and evaporated in vacuum to yield a crude extract. The extract (1.0 kg) was dissolved in water and subjected to a Diaion101 column (MeOH-H2O, 10:90–100:0) to yield seven fractions (Fr-1–Fr-7). Fr-5 (723 mg) was applied to CHP-20P column (MeOH-H2O, 40:60–100:0) which afforded three subfractions (Fr-5-1–Fr-5-3). Fr-5-1 (211 mg) and Fr-5-2 (186 mg) were chromatographed on Sephadex LH-20 column, eluted with MeOH-H2O (20:80), to give compounds 1 (103 mg) and 2 (96 mg). Fr-3 (632 mg) was purified on an MCI-gel followed by CHP-20P CC (MeOH-H2O, 70:30), to afford compound 4 (18 mg). Fr-4 (332 mg) was purified by CHP-20P column (MeOH-H2O, 40:60) to yield compound 3 (121 mg). Manindicin A (1): yellow crystals; [α]25 D+139.9 (c 1.0, CH3OH), UV (CH3OH) λmax (log ε) 203, 305 nm; IR (KBr) νmax 3548, 3362, 1635, 1592, 1550, and 1507 cm−1; positive HR-ESI-MS m/z 405.1185 [M +H]+ (calcd for C20H21O9, 405.1180, Δ 1.2 ppm). Manindicin B (2): yellow crystals; [α]25 D+37.5 (c 1.0, CH3OH), UV (CH3OH) λmax (log ε) 204, 307 nm; IR (KBr) νmax 3424, 1634, 1605, 1574, and 1509 cm−1; positive HR-ESI-MS m/z 405.1185 [M+H]+ (calcd for C20H21O9, 405.1180, Δ 1.2 ppm).
2.7. α-Glucosidase inhibitory assay A 20 μL (0.2 U/mL) of α-glucosidase (Sigma, 129 k1426) and 20 μL of samples were added to 112 μL KH2PO4/K2HPO4 buffer (pH 6.8); the mixture was held at 37 °C for 15 min. A 20 μL (2.5 mmol) of 4-nitrophenyl-α-D-glucopyranoside (α-PNPG, Sigma) was put into the mixture and kept at 37 °C for 15 min. Finally, a 80 μL (0.2 mol/L) of Na2CO3 was added and the values of optical density (OD) were scaled at 405 nm by Multiskan MK3 Microplate Reader (Thermo Electron). There were four groups of the experiments [a: negative control
2.4. Cytoprotective effect on HepG2 against hydrogen peroxide-induced injury HepG2 cells were supplied from the cell bank of Kunming Institute of Zoology, CAS (Kunming, China), and then cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine 710
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Fig. 1. Structures of compounds 1–4.
(buffer + enzyme solution + substrate), b: blank control (buffer), c: sample group (samples + enzyme solution + substrate), d: sample control (sample + buffer)]; Triplicates of each experiment were performed. The inhibition capacity was calculated by the formula as follows: % inhibition = [1- (ODc-ODd)/(ODa-ODb)] × 100%.
Table 1 NMR spectroscopic data of compounds 1 and 2 (δ in ppm and J in Hz). No.
1
2
δH
3. Results and discussion
1 2 3 4 5 6 7 6-OH 4-OCH3 1′ 2′,6′ 3′,5′ 4′ 4′-OH 1″
3.1. Phytochemical investigation The extract from the leaves of Mangifera indica L. was chromatographed over various columns using D-101 macroporous resin, CHP20P, and Sephadex LH-20 to yield two new benzophenone derivatives (1–2) and two known xanthones (Fig. 1). The two known compounds were elucidated as mangiferin (3) (Qin, Deng, Feng, & Feng, 2008) and norathyriol (4) (Guo, Zheng, & Zheng, 2005). Compound 1 was obtained as yellow crystals. Its molecular formula was elucidated as C20H20O9 according to its HRESIMS (m/z 405.1185 [M+H]+), which indicated 11 degrees of unsaturation. The IR absorption bands revealed the presence of hydroxy groups (3548 and 3362 cm−1), aromatic ring (1592, 1550, and 1507 cm−1), and a conjugated ketone (1635 cm−1). An AA′BB′ spin-system at δH 7.62 (2H, d, J = 8.7 Hz) and 6.82 (2H, d, J = 8.7 Hz), and a penta-substituted benzene ring at δH 6.14 (1H, s) were observed in the 1H NMR spectrum. Moreover, the 1H NMR spectrum indicated the presence of a methoxy group (δH 3.80, 3H, s) and two phenolic-hydroxy groups (δH 10.42 and 10.25). The NMR data (Table 1) revealed that there were 20 carbon signals, six of them were attributed to a glycosyl moiety [δC 32.1 (C-1″), 119.9 (C-2″), 80.8 (C-3″), 75.7 (C-4″), 81.9 (C-5″), and 60.5 (C-6″)] according to 1H-1H COSY and HMBC correlations. The remaining 14 carbon-signals were assigned to one methoxyl (δC 55.4), five methines, and eight quaternary carbons (including one carbonyl [δC192.0]) (Table 1). The protons at δH 3.32 (1H, d, J = 16.4 Hz) and 2.68 (1H, d, J = 16.4 Hz) were connected to δC 32.1 (C-1″) on the basis of HSQC spectrum. The HMBC cross-peaks of δH 3.32 (Ha-1″) and 2.68 (Hb-1″) with δC 103.3 (C-3), 157.0 (C-2), and 157.7 (C-4) indicated that the methylene C-1″ was attached to C-3. The carbonyl resonance at δC 192.0 was readily assigned to C-7 according to the HMBC cross-peaks of δH 7.62 (H-2′/H-6′) with C-7. The NMR data of 1 were identical to those of aquilarinoside A, a benzophenone mono-glycoside (Qi, Lu, Liu, & Yu, 2009) with an unknown absolute configuration of glycoside moiety, except for one more methoxy group in 1. The HMBC correlation between δH 3.80 (CH3O) and δC 157.7 (C-4) indicated that the methoxy was assigned to C-4.
2″ 3″ 4″ 5″ 6″
δC
6.14 (1H, s) 10.42 (1H, s) 3.80 (3H, s) 7.62 (2H, d,8.7) 6.82 (2H, d,8.7) 10.25 (1H, s) 3.32 (1H, d,16.4) 2.68 (1H, d,16.4) 3.92 3.64 3.58 3.38 3.55
(1H, (1H, (1H, (1H, (1H,
t,11.2) m) m) m) m)
δH
103.1 157.0 103.3 157.7 92.1 158.6 192.0
6.13 (1H, s) 10.96 (1H, s) 3.81 (3H, s)
55.4 129.2 132.0 114.8 161.9
7.64 (2H, d,8.4) 6.83 (2H, d, 8.4) 10.31 (1H, s) 3.14 (1H, d,16.2) 2.90 (1H, d,16.2)
32.1 119.9 80.8 75.7 81.9 60.5
Recorded at 400 MHz for 1H and 100 MHz for
δC
3.83 3.45 3.66 3.01 3.27 13
(1H, (1H, (1H, (1H, (1H,
t,12.7) m) m) m) m)
102.9 157.9 102.8 158.3 91.9 160.2 193.2 55.5 129.8 131.9 114.5 161.5 33.1 116.9 79.4 73.8 83.8 63.7
C in DMSO‑d6.
The correlations between protons at δH 3.92 (t, H-3″) and 5.31 (d, HO-4″), 3.58 (m, H-5″), 3.32 (d, Ha-1″); 5.80 (d, HO-3″) and 3.64 (1H, m, H-4″), 3.32 (d, Ha-1″); 3.64 (1H, m, H-4″) and 2.68 (d, Hb-1″), 5.80 (d, HO-3″); 3.38 (1H, m, H-6″) and 3.64 (1H, m, H-4″) were observed in the ROESY spectrum, which suggested a trans configurations of H-3″/H4″ and H-4″/H-5″. Thus, the glycoside moiety was identified as α-Dfructofuranose which made a spiro-bicyclic system with benzophenone. The relative configuration of compound 1 was determined according to single crystal X-ray diffraction analysis (Fig. 2). Due to the above evidences, the final structure of compound 1 was identified and trivially named as manindicin A. Compound 2 had the same molecular formula of C20H20O9 as that of compound 1, indicated by the quasi-molecular ion HRESIMS peak at m/ z 405.1185 [M+H]+. The NMR data of 2 and 1 were similar, except for the upfield shift of C-2″, C-3″, and C-4″ by 3.0, 1.4, and 1.9 ppm, and downfield shift of C-1″, C-5″, and C-6″ by 1.0, 1.9, and 3.2 ppm, respectively, which revealed that compound 2 was a diastereomer of 1 at 711
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Fig. 3. The cell viabilities of HepG2 cells treated by four isolated compounds with MTT assay at different concentrations. The values were presented as mean ± SD of triplicate replicates. Fig. 2. ORTEP drawing of compound 1.
immunosuppressants characterized by high efficiency and low toxicity become more and more important because of the current immunosuppressants with toxic and side effects. The immunosuppressive results of the four compounds isolated from mango leaves are presented in Fig. 5. As shown in Fig. 5, all the compounds exhibited good inhibition on Con A-induced spleen cell proliferation in a concentrationdependent manner ranging from 10 to 40 μM. When tested at a concentration of 40 μM, the relative inhibition ratios of manindicin A, manindicin B, mangiferin and norathyiol were 31.92 ± 3.84%, 31.67 ± 3.43%, 12.09 ± 2.62% and 24.28 ± 2.89%, indicating that manindicin A and manindicin B had a similar (p > 0.05) and the strongest (p < 0.05) immunosuppressive activity, while mangiferin possessed the weakest activity (p < 0.05). However, the stimulation index of DXM (positive control) at each tested concentration was significantly lower than all those of four compounds at the same concentration (p < 0.05), which suggested that DXM had a better immunosuppressive activity. The relative inhibition ratio of DXM at 40 μM was 56.99 ± 4.22%. To our knowledge, this is the first report on immunosuppressive properties of M. indica metabolites in Con A-induced spleen cells. A previous study has also reported that the aqueous extract of M. indica bark and mangiferin exhibited inhibitory effects on macrophage activity, which suggested the extract and mangiferin possessed immunosuppressive effect and was partly consistent with the findings of the present work (Garcia, Delgado, Ubeira, & Leiro, 2002). Another study has also reported that mangiferin is the predominant component of M. indica L. bark for immunosuppressive anti-tumor effects in MDAMB231 breast cancer cells.
glycosyl moiety. The differences of the chemical shifts of the glycoside moieties in compounds 1 and 2 were identical to the differences between α-D-fructose and β-D-fructose (Pawank, 1992). The ROESY correlations between protons at δH 3.83 (t, H-3″) and 3.14 (d, Ha-1″), 5.33 (d, HO-4″); 3.45 (m, H-4″) and 3.27 (m, Hb-6″) indicated that the relative configurations of H-3″/H-4″ and H-4″/H-5″ were trans, which were same as 1. The ROESY correlations were observed between H-5″ (3.66, m) and Hb-1″ (2.90, d) in 2, which were absent in 1, indicating that the glycoside moiety in 2 was β-D-fructofuranose. Thus, compound 2 was elucidated and trivially named as manindicin B. Compounds 1 and 2 were the diastereomers, formed by the combination of benzophenone and α/β-D-fructose, respectively. 3.2. Cytoprotective effect on H2O2-treated HepG2 cells The cellular antioxidant activity was developed as a common model for quantifying the antioxidant capacity of phytochemicals in HepG2 cells, and especially in the assessment of their intracellular ROS scavenging activity. The reactive oxygen species (ROS), such as superoxide anion radical, hydroxy radical, and hydrogen peroxide are produced in the body and are beneficial as signal transducers and growth regulators for human beings. However, excessive ROS result in oxidative stress, which may lead to some chronic diseases (Wang et al., 2016). Therefore, the antioxidants are required to balance the level of ROS. The MTT result showed that all the compounds did not exhibit cytotoxicity to HepG2 cells at the tested concentration ranging from 12.5 to 50 μM (Fig. 3). The cellular ROS inhibition result of those four compounds is illustrated in Fig. 4. As shown in Fig. 4, the cellular ROS amount of HepG2 cells increased significantly when treated by H2O2. All compounds possessed good ROS inhibition effects with a concentration-dependent manner when compared to H2O2 treated group (p < 0.05). Among those four compounds, norathyriol showed the weakest inhibitory effect on ROS production at each tested concentration ranged from 12.5 to 50 μM (p < 0.05). However, manindicin A, manindicin B and mangiferin exhibited a similar inhibition against ROS generation at 25 and 50 μM (p > 0.05). When tested at 12.5 μM, manindicin A had lower ROS inhibitory activity (p < 0.05), while manindicin B and mangiferin shared a similar activity (p > 0.05).
3.4. α-Glucosidase inhibitory activity The α-glucosidase inhibition is a common and effective way for the treatment of hyperglycaemia in managing type II diabetes (Tewari et al., 2003). Previous studies showed that the ethanol extract of mango peels inhibited α-glucosidase activities with IC50 value of 3.5 μg/mL (Gondi & Prasada Rao, 2015). Another research on streptozotocin-induced diabetic rats exhibited the extract of mango by-product reduced serum glucose and triacylglycerides and was associated to its high content of phenolics (Rodríguez-González et al., 2017). However, the αglucosidase inhibitory effects of the chemical constituents of M. indica are hardly ever reported. Hence, the α-glucosidase inhibitory activity was tested for all the isolated compounds. As shown in Table 2, norathyriol exhibited an excellent α-glucosidase inhibition activity with an IC50 of 4.22 ± 0.19 μg/mL, which was about 4-fold lower than that of commercial inhibitor (acarbose, 16.28 ± 1.22 μg/mL). Subsequently, mangiferin has a moderate α-glucosidase inhibitory activity
3.3. Immunosuppressive activity Autoimmune diseases are mostly chronic or progressive diseases, which require long-term medication. Therefore, the requirement of 712
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Fig. 4. Intracellular ROS production of four isolated compounds on H2O2-induced HepG2 cells. The values were presented as mean ± SD of triplicate replicates. Different letters indicate significant differences (p < 0.05).
Table 2 α-Glucosidase inhibitory activities of the isolates. Compound
IC50 (μg/mL)
Acarbose Manindicin A Manindicins B Mangiferin Norathyriol
16.28 ± 1.22 > 300 > 300 32.11 ± 2.01 4.22 ± 0.19
(IC50 = 32.11 ± 2.01 μg/mL), half the efficacy of acarbose. However, manindicins A and B had weak inhibition of α-glucosidase with IC50 of both being more than 300 μg/mL (Table 2). The decreased α-glucosidase inhibitory effect of 3 may due to the increasing molecular size and polarity. After the replacement of H-3 by a glucose moiety, the increased steric hindrance may weaken the binding interaction between flavonoids and the enzyme. A previous study found luteolin had a better inhibitory effect on α-glucosidase than its C-glycosides, orientin and isoorientin, which is in part in agreement with our findings (Li et al., 2009). In addition, the O-glycosylation of flavonoid could also decrease the α-glucosidase inhibitory activity. Li and coworkers (Li, Zhou, Gao, Bian, & Shan, 2009) compared α-glucosidase inhibitory potential of
Fig. 5. Effect of the isolated compounds on Con A-stimulated splenocyte proliferation in vitro. DXM was used as the positive control. The values were presented as mean ± SD of triplicate replicates. Statically significant difference was determined by ANOVA and Tukey test (*p < 0.05, compared with the control group).
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(2017). Bioactive compounds of the Ubá mango juices decrease inflammation and hepatic steatosis in obese Wistar rats. Journal of Functional Foods, 32, 409–418. Ni, Z. G., & Xie, D. H. (2009). Present situation and suggestions of mango industry in Yunnan. Chinese Journal of Tropical Agriculture, 29, 52–56. Pan, J., Yi, X., Zhang, S., Cheng, J., Wang, Y., Liu, C., & He, X. (2018). Bioactive phenolics from mango leaves (Mangifera indica L.). Industrial Crops and Products, 111, 00–406. Pan, J., Yi, X., Wang, Y., Chen, G., & He, X. (2018). Benzophenones from mango leaves exhibit α-glucosidase and NO inhibitory activities. Journal of Agricultural and Food Chemistry, 64, 7475–7480. Pawank, A. (1992). NMR spectroscopy in the structure elucidation of oligosaccharides and glycosides. Phytochemistry, 31, 3307–3330. Qi, J., Lu, J. J., Liu, J. H., & Yu, B. Y. (2009). Flavonoid and a rare benzophenone glycoside from the leaves of Aquilaria sinensis. Chemical Pharmaceutical Bulletin, 57(2), 134–137. Qin, J. P., Deng, J. G., Feng, Y. Q., & Feng, X. (2008). Applications of organic mass spectrometry in structure identification of an impurity compound in prepared mangiferin extracted from Mangifera indica L. leaves. Journal of Chinese Mass Spectrometry Society, 29(4), 219–225. Rajendran, P., & Ekambaram, G. (2008). Chemopreventive efficacy of mangiferin against benzo-(a)pyrene induced lung carcinogenesis in experimental animals. Environmental Toxicology and Pharmacology, 26, 278–282. Rodeiro, I., & Cancino, L. (2006). Evaluation of the genotoxic potential of Mangifera indica L. extract (vimang), a new natural product with antioxidant activity. Sciences, 44, 1707–1713. Rodríguez-González, S., Gutiérrez-Ruíz, I. M., Pérez-Ramírez, I. F., Mora, O., RamosGomez, M., & Reynoso-Camacho, R. (2017). Mechanisms related to the anti-diabetic properties of mango (Mangifera indica L.) juice by-product. Journal of Functional Foods, 37, 190–199. Tewari, N., Tiwari, V. K., Mishra, R. C., Tripathi, R. P., Srivastava, A. K., Ahmad, R., ... Srivastava, B. S. (2003). Synthesis and bioevaluation of glycosyl ureas as αGlucosidase inhibitors and their effect on Mycobacterium. Bioorganic & Medicinal Chemistry, 11, 2911–2922. Wang, L., Ding, L., Yu, Z., Zhang, T., Ma, S., & Liu, J. (2016). Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells. Food Research International, 90, 33–41. Yoshimi, N., & Matsunaga, K. (2001). The inhibitory effects of mangiferin, anaturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats. Cancer Letters, 163(2), 163–170. Zheng, M. S., & Lu, Z. Y. (1990). Antiviral effect of mangiferin and isomangiferin on herpes si- mplex virus. Chinese Medical Sciences Journal, 103, 160–165. Zhuang, Y. L., Ma, Q. Y., Guo, Y., & Sun, L. P. (2017). Protective effects of rambutan (Nephelium lappaceum) peel phenolics on H2O2-induced oxidative damages in HepG2 cells and D-galactose-induced aging mice. Food & Chemical Toxicology, 108, 554–562.
4. Conclusion In this study, four benzophenone derivatives were isolated and characterized from M. indica L. leaves, two of which were the new compounds, namely manindicins A and B. It is found that the four compounds could inhibit the cellular ROS amount of H2O2-induced HepG2 cells. Meanwhile, spleen cells proliferation assay showed that manindicins A and B had a good and similar immunosuppressive activity, while mangiferin and norathyriol exhibited a relative weaker activity. Norathyriol showed the strongest inhibitory effect on α-glucosidase activity with an IC50 of 4.22 ± 0.19 μg/mL, which was lower than that of acarbose (16.28 ± 1.22 μg/mL), followed by mangiferin. However, the manindicins A and B had weak inhibition of α-glucosidase with an IC50 of both being more than 300 μg/mL. Declarations of interest None. Acknowledgements This work was supported by the National Natural Science Foundation of China [Grant No. 31600274 and 31460083], Applied Basic Research Project of Yunnan Province (Grant No. 2018FB036), and the Key Laboratory Training Program in Yunnan (Grant No. 2017DG006). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2018.11.045. References Ahmed, E. M. (2007). Egyptian mango by-product 2: Antioxidant and antimicrobial activities of extract and oil from mango seed kernel. Food Chemistry, 103, 1141–1152. Ajila, C. M. (2008). Protection against hydrogen peroxide induced oxidative damage in rat er- ythrocytes by Mangifera indica L. peel extract. Food and Chemical Toxicology, 46, 303–309. Amaya-Cruz, D. M., Rodríguez-González, S., Pérez-Ramírez, I. F., Loarca-Piña, G., AmayaLlano, S., Gallegos-Corona, M. A., & Reynoso-Camacho, R. (2015). Juice by-products as a source of dietary fibre and antioxidants and their effect on hepatic steatosis. Journal of Functional Foods, 17, 93–102. Anjaneyulu, V. (1985). Tritepenoids from Mangifera indica. Phytochemistry, 24, 2359–2367. Burtonfreeman, B. M., Sandhu, A. K., & Edirisinghe, I. (2017). Mangos and their bioactive components: Adding variety to the fruit plate for health. Food & Function, 8, 3010–3012. Deng, J. G., & Zeng, C. H. (2003). Thirty years Research progress of Mangifera indica L. and mangiferin in recent thirty years. Journal of Guangxi Traditional Chinese Medical University, 6, 44–47. Fang, C., Kim, H., Noratto, G., Sun, Y., Talcott, S. T., & Mertens-Talcott, S. U. (2018). Gallotannin derivatives from mango (Mangifera indica L.) suppress adipogenesis and increase thermogenesis in 3T3-L1 adipocytes in part through the AMPK pathway. Journal of Functional Foods, 46, 101–109. Fernández-Ponce, M. T., Medina-Ruiz, E., Casas, L., Mantell, C., & Martínez de la OssaFernández, E. J. (2018). Development of cotton fabric impregnated with antioxidant mango polyphenols by means of supercritical fluids. The Journal of Supercritical Fluids, 140, 310–319. Gao, F., Yao, Y. C., Cai, S. B., Zhao, T. R., Yang, X. Y., Fan, J., ... Cheng, G. G. (2017). Novel immunosuppressive pregnane glycosides from the leaves of Epigynum auritum. Fitoterapia, 118, 107–111. Garcia, D., Delgado, R., Ubeira, F. M., & Leiro, J. (2002). Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin. International Immunopharmacology, 2, 797–806. Gondi, M., Basha, S. A., Bhaskar, J. J., Salimath, P. V., & Prasada Rao, U. J. S. (2015).
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Purification and characterization of four benzophenone derivatives from Mangifera indica L. leaves and their antioxidant, immunosuppressive and α-glucosidase inhibitory activities
T
Chengzhen Gua,b, Meilian Yanga, Zhihong Zhouc, Afsar Khand, Jianxin Caoa, , Guiguang Chenga, ⁎
⁎
a
Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming 650500, People’s Republic of China College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, People's Republic of China c Institute of Traditional Chinese Medicine Research, Yunnan University of Traditional Chinese Medicine, Kunming 650504, People's Republic of China d Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Pakistan b
ARTICLE INFO
ABSTRACT
Keywords: Mangifera indica Phytochemicals ROS Immunosuppressive activity α-Glucosidase inhibition
The aim of the present study was to isolate phytochemicals from Mangifera indica L. leaves and to screen their bioactivities. Four benzophenone derivatives were isolated and characterized, two of which were new compounds, namely manindicins A and B. All the four compounds significantly inhibited the cellular ROS amount of H2O2-induced HepG2 cells. Stimulation index of spleen cells showed that manindicins A and B had a good and similar immunosuppressive activity, while mangiferin and norathyriol exhibited relatively weaker activity. Norathyriol showed the strongest inhibitory effect on α-glucosidase with an IC50 of 4.22 ± 0.19 μg/mL, which was lower than that of acarbose (16.28 ± 1.22 μg/mL). However, the manindicins A and B had weak inhibition of α-glucosidase. The present work suggests that M. indica leaves can be used potentially as functional-food supplement for improving human health.
1. Introduction The genus Mangifera (Anacardiaceae) has more than 70 species and 1000 varieties, and is widely distributed in the tropical and subtropical areas (Ni & Xie, 2009). The Mangifera indica L. fruit is one of the most important fruit-crops with extensive acceptance due to its succulence, exotic flavour, and nutritional value. In addition, the fruit is a good source of carotenoids, lupeol, mangiferin, and phenolic acids with a wide range of pharmacological properties for human health. Apart from consumption as ripe fruit, these are usually processed into juices, jam, and beverages. The rising demand for nutritional supplement of consumers resulted in a constant growth of mango fruits production. A large number of by-products (i.e. leaves, peels, and kernels) are discarded as industrial waste. Indeed, it is well known that mango byproducts contain significant amounts of health-enhancing substances including mangiferin, gallic acid, ferulic acid, epicatechin, gallotannins, phenolics, flavonoids, benzophenones, and so on (Burtonfreeman, Sandhu, & Edirisinghe, 2017; Fang et al., 2018; Fernández-Ponce, Medina-Ruiz, Casas, Mantell, & Martínez de la Ossa-Fernández, 2018; Kim et al., 2018; Matkowski, Kus, Goralska, & Wozniak, 2013; Pan, Yi, Zhang, et al., 2018). These bioactive compounds exhibited various
⁎
biological activities, such as antioxidant (Amaya-Cruz et al., 2015; Gondi, Basha, Bhaskar, Salimath, & Prasada Rao, 2015), anti-inflammatory (Natal et al., 2017; Pan, Yi, Wang, Chen, & He, 2018), antidiabetic (Rodríguez-González et al., 2017), hepatoprotective (Medina Ramírez et al., 2018), cholesterol-lowering (Gururaja et al., 2017), and anti-obesogenic activities (Fang et al., 2018). The leaves of M. indica are a kind of crop waste. However, the leaves are consumed traditionally as folk tea and medicine to reduce the risk of respiratory diseases in China (Deng & Zeng, 2003). Pharmacological studies showed that the extracts of leaves have antioxidant, antibacterial, antiviral, antidiabetic, anti-inflammatory, and anti-tumor activities (Ahmed, 2007; Ajila, 2008; Liu, Wang, Zhou, Gong, & Deng, 2017; Rajendran & Ekambaram, 2008; Yoshimi & Matsunaga, 2001; Zheng & Lu, 1990). Previous studies on mango leaves reported different kinds of secondary metabolites, including flavonoids, terpenoids, phytosterols, coumarins, and lignans, of which phenolics are the characteristic constituents (Anjaneyulu, 1985; Rodeiro & Cancino, 2006). Among them, the benzophenone mangiferin contributed to the majority of phenolics. Since tea plays an important role in human life and mango leaves contain a high amount of bioactive phenolic compounds, the investigation of chemical constituents in mango leaves is thus
Corresponding authors. E-mail addresses: [email protected] (J. Cao), [email protected] (G. Cheng).
https://doi.org/10.1016/j.jff.2018.11.045 Received 5 August 2018; Received in revised form 13 November 2018; Accepted 28 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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necessary. A phytochemical investigation of the leaves of M. indica resulted in the isolation of four benzophenone derivatives. Manindicins A and B, benzophenone glycosides, were identified by extensive spectroscopic techniques, while two known compounds were characterized as mangiferin and norathyriol. Some of these compounds exhibited significant antioxidant, immunosuppressive, and α-glucosidase inhibitory activities. The study suggested that mango leaves can be served as dietary supplement for a functional food.
serum (FBS) and 1% antibiotic mixture of 100 U/mL penicillin/streptomycin under a humidified 5% CO2 atmosphere. Cytotoxicity assay of HepG2 cells was performed by MTT method (Mosmann, 1983) with some modifications. Briefly, cells were centrifuged at a density of at 4.0 × 105 cells/well and kept for 24 h. Different concentrations of the test compounds were added in serum-free medium. After 20 h of treatment, the cells were incubated with 150 μL MTT reagent (0.5 mg/ mL) for 4 h. Subsequently, the MTT reagent was removed, and 150 μL dimethyl sulfoxide (DMSO) was added for dissolving the formazan crystals. Then, HepG2 cells were washed carefully once with PBS, and maintained in serum-free DMEM with 100 μL of 0.5 mg/mL 0.05% MTT for 4 h. Then, 150 μL of DMSO was added per well for the dissolution of formazan. The spectrophotometric absorbance at 570 nm was measured by utilizing a microplate reader. The cell viability results showed that all samples under the test concentrations had no cytotoxicity to HepG2 cells by comparison with the control group.
2. Materials and methods 2.1. General experimental procedures Optical rotation values were recorded on a JASCO P-1020 digital polarimeter. IR data were recorded by a Bruker Tensor 27 spectrometer with KBr pellets; NMR spectra were performed on a Bruker DRX400 MHz spectrometer with TMS as an internal standard. High resolution electrospray ionization mass spectrometry (HRESIMS) in positive mode was obtained on a Thermo high resolution Q-exactive focus mass spectrometer. D101 (Canzhou Bon Chemical Co., Ltd. Hebei, China) macroporous resin, MCI-gel CHP-20P (75–150 μm, Mitsubishi, Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden) were used for column chromatography. Thin-layer chromatography (TLC) was performed on silica gel 60 G254 coated glass plates (Qingdao Ocean Chemical Co., Ltd, Qingdao, China), and the spots were observed either by UV light or by spraying the plates with 10% H2SO4.
2.5. Measurement of intracellular reactive oxygen species levels The intracellular ROS oxidative stress in oxidized HepG2 cells was determined using a 2′,7′-dichlorodihydrofluorescin diacetate (DCFHDA) ROS assay (Zhuang, Ma, Guo, & Sun, 2017). Briefly, HepG2 cells were exposed to the test compounds under different concentrations (12.5, 25, and 50 μM). After washing with PBS twice, the collected cells were resuspended in serum-free medium with 10 mM DCFH-DA at 37 °C in the dark for 20 min. After that, the supernatant was discarded and the cells were washed three times with PBS. The intracellular ROS generation was immediately recorded at excitation wavelength of 485 nm and emission wavelength of 535 nm by using a flow cytometry (Guava easy Cyte 6-2L; Millipore, Billerica, MA, US).
2.2. Plant material M. indica leaves were collected in June 2014 in Yuanjiang county, Yunnan province, China, and identified by Prof. Z. H. Zhou (Yunnan University of TCM, China). The voucher sample was deposited at the Functional Food Laboratory, Yunnan Institute of Food Safety, Kunming University of Science and Technology.
2.6. Splenocyte proliferation assay The immunosuppressive activity was carried out using the CCK-8 method as reported previously (Gao et al., 2017). Briefly, spleens were isolated from male BALB/c mice. The spleens were gently crushed and filtered through a 200 μm mesh screen, and washed repeatedly with cold PBS. Cells were collected by centrifugation. Recovered splenocytes were transferred into a sterile centrifuge tube, mixed with 3 mL TrisNH4Cl (Biosharp, China) for lysing the remaining erythrocytes. The cells were washed twice with PBS and re-suspended in RPMI 1640 complete medium (Gibco, USA) with 10% fetal bovine serum, and 1% penicillin–streptomycin. Cells were seeded into 96-well flat-bottom microtiter plates at a density of 1 × 106 cell/mL, and treated with concanavalin A (Con A, 10 μg/mL) in the presence and absence of the test compound (10, 20, and 40 μM) using dexamethasone (DXM) as a positive control. Splenocytes without Con A-treated were served as negative control. After incubation for 72 h at 37 °C in a humidified atmosphere, 10 μL of CCK-8 was added and incubated for another 4 h. The tests were conducted for three independent replicates, and the absorbance at 450 nm was measured on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The stimulation index (SI) was calculated based on the following formula: SI = the absorbance value for mitogen-cultures/the absorbance value for non-stimulated cultures.
2.3. Extraction and isolation The air-dried leaves of M. indica (25 kg) were smashed, and extracted with distilled water for three times (each time for four hours) under reflux. The filtrates were combined and evaporated in vacuum to yield a crude extract. The extract (1.0 kg) was dissolved in water and subjected to a Diaion101 column (MeOH-H2O, 10:90–100:0) to yield seven fractions (Fr-1–Fr-7). Fr-5 (723 mg) was applied to CHP-20P column (MeOH-H2O, 40:60–100:0) which afforded three subfractions (Fr-5-1–Fr-5-3). Fr-5-1 (211 mg) and Fr-5-2 (186 mg) were chromatographed on Sephadex LH-20 column, eluted with MeOH-H2O (20:80), to give compounds 1 (103 mg) and 2 (96 mg). Fr-3 (632 mg) was purified on an MCI-gel followed by CHP-20P CC (MeOH-H2O, 70:30), to afford compound 4 (18 mg). Fr-4 (332 mg) was purified by CHP-20P column (MeOH-H2O, 40:60) to yield compound 3 (121 mg). Manindicin A (1): yellow crystals; [α]25 D+139.9 (c 1.0, CH3OH), UV (CH3OH) λmax (log ε) 203, 305 nm; IR (KBr) νmax 3548, 3362, 1635, 1592, 1550, and 1507 cm−1; positive HR-ESI-MS m/z 405.1185 [M +H]+ (calcd for C20H21O9, 405.1180, Δ 1.2 ppm). Manindicin B (2): yellow crystals; [α]25 D+37.5 (c 1.0, CH3OH), UV (CH3OH) λmax (log ε) 204, 307 nm; IR (KBr) νmax 3424, 1634, 1605, 1574, and 1509 cm−1; positive HR-ESI-MS m/z 405.1185 [M+H]+ (calcd for C20H21O9, 405.1180, Δ 1.2 ppm).
2.7. α-Glucosidase inhibitory assay A 20 μL (0.2 U/mL) of α-glucosidase (Sigma, 129 k1426) and 20 μL of samples were added to 112 μL KH2PO4/K2HPO4 buffer (pH 6.8); the mixture was held at 37 °C for 15 min. A 20 μL (2.5 mmol) of 4-nitrophenyl-α-D-glucopyranoside (α-PNPG, Sigma) was put into the mixture and kept at 37 °C for 15 min. Finally, a 80 μL (0.2 mol/L) of Na2CO3 was added and the values of optical density (OD) were scaled at 405 nm by Multiskan MK3 Microplate Reader (Thermo Electron). There were four groups of the experiments [a: negative control
2.4. Cytoprotective effect on HepG2 against hydrogen peroxide-induced injury HepG2 cells were supplied from the cell bank of Kunming Institute of Zoology, CAS (Kunming, China), and then cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine 710
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Fig. 1. Structures of compounds 1–4.
(buffer + enzyme solution + substrate), b: blank control (buffer), c: sample group (samples + enzyme solution + substrate), d: sample control (sample + buffer)]; Triplicates of each experiment were performed. The inhibition capacity was calculated by the formula as follows: % inhibition = [1- (ODc-ODd)/(ODa-ODb)] × 100%.
Table 1 NMR spectroscopic data of compounds 1 and 2 (δ in ppm and J in Hz). No.
1
2
δH
3. Results and discussion
1 2 3 4 5 6 7 6-OH 4-OCH3 1′ 2′,6′ 3′,5′ 4′ 4′-OH 1″
3.1. Phytochemical investigation The extract from the leaves of Mangifera indica L. was chromatographed over various columns using D-101 macroporous resin, CHP20P, and Sephadex LH-20 to yield two new benzophenone derivatives (1–2) and two known xanthones (Fig. 1). The two known compounds were elucidated as mangiferin (3) (Qin, Deng, Feng, & Feng, 2008) and norathyriol (4) (Guo, Zheng, & Zheng, 2005). Compound 1 was obtained as yellow crystals. Its molecular formula was elucidated as C20H20O9 according to its HRESIMS (m/z 405.1185 [M+H]+), which indicated 11 degrees of unsaturation. The IR absorption bands revealed the presence of hydroxy groups (3548 and 3362 cm−1), aromatic ring (1592, 1550, and 1507 cm−1), and a conjugated ketone (1635 cm−1). An AA′BB′ spin-system at δH 7.62 (2H, d, J = 8.7 Hz) and 6.82 (2H, d, J = 8.7 Hz), and a penta-substituted benzene ring at δH 6.14 (1H, s) were observed in the 1H NMR spectrum. Moreover, the 1H NMR spectrum indicated the presence of a methoxy group (δH 3.80, 3H, s) and two phenolic-hydroxy groups (δH 10.42 and 10.25). The NMR data (Table 1) revealed that there were 20 carbon signals, six of them were attributed to a glycosyl moiety [δC 32.1 (C-1″), 119.9 (C-2″), 80.8 (C-3″), 75.7 (C-4″), 81.9 (C-5″), and 60.5 (C-6″)] according to 1H-1H COSY and HMBC correlations. The remaining 14 carbon-signals were assigned to one methoxyl (δC 55.4), five methines, and eight quaternary carbons (including one carbonyl [δC192.0]) (Table 1). The protons at δH 3.32 (1H, d, J = 16.4 Hz) and 2.68 (1H, d, J = 16.4 Hz) were connected to δC 32.1 (C-1″) on the basis of HSQC spectrum. The HMBC cross-peaks of δH 3.32 (Ha-1″) and 2.68 (Hb-1″) with δC 103.3 (C-3), 157.0 (C-2), and 157.7 (C-4) indicated that the methylene C-1″ was attached to C-3. The carbonyl resonance at δC 192.0 was readily assigned to C-7 according to the HMBC cross-peaks of δH 7.62 (H-2′/H-6′) with C-7. The NMR data of 1 were identical to those of aquilarinoside A, a benzophenone mono-glycoside (Qi, Lu, Liu, & Yu, 2009) with an unknown absolute configuration of glycoside moiety, except for one more methoxy group in 1. The HMBC correlation between δH 3.80 (CH3O) and δC 157.7 (C-4) indicated that the methoxy was assigned to C-4.
2″ 3″ 4″ 5″ 6″
δC
6.14 (1H, s) 10.42 (1H, s) 3.80 (3H, s) 7.62 (2H, d,8.7) 6.82 (2H, d,8.7) 10.25 (1H, s) 3.32 (1H, d,16.4) 2.68 (1H, d,16.4) 3.92 3.64 3.58 3.38 3.55
(1H, (1H, (1H, (1H, (1H,
t,11.2) m) m) m) m)
δH
103.1 157.0 103.3 157.7 92.1 158.6 192.0
6.13 (1H, s) 10.96 (1H, s) 3.81 (3H, s)
55.4 129.2 132.0 114.8 161.9
7.64 (2H, d,8.4) 6.83 (2H, d, 8.4) 10.31 (1H, s) 3.14 (1H, d,16.2) 2.90 (1H, d,16.2)
32.1 119.9 80.8 75.7 81.9 60.5
Recorded at 400 MHz for 1H and 100 MHz for
δC
3.83 3.45 3.66 3.01 3.27 13
(1H, (1H, (1H, (1H, (1H,
t,12.7) m) m) m) m)
102.9 157.9 102.8 158.3 91.9 160.2 193.2 55.5 129.8 131.9 114.5 161.5 33.1 116.9 79.4 73.8 83.8 63.7
C in DMSO‑d6.
The correlations between protons at δH 3.92 (t, H-3″) and 5.31 (d, HO-4″), 3.58 (m, H-5″), 3.32 (d, Ha-1″); 5.80 (d, HO-3″) and 3.64 (1H, m, H-4″), 3.32 (d, Ha-1″); 3.64 (1H, m, H-4″) and 2.68 (d, Hb-1″), 5.80 (d, HO-3″); 3.38 (1H, m, H-6″) and 3.64 (1H, m, H-4″) were observed in the ROESY spectrum, which suggested a trans configurations of H-3″/H4″ and H-4″/H-5″. Thus, the glycoside moiety was identified as α-Dfructofuranose which made a spiro-bicyclic system with benzophenone. The relative configuration of compound 1 was determined according to single crystal X-ray diffraction analysis (Fig. 2). Due to the above evidences, the final structure of compound 1 was identified and trivially named as manindicin A. Compound 2 had the same molecular formula of C20H20O9 as that of compound 1, indicated by the quasi-molecular ion HRESIMS peak at m/ z 405.1185 [M+H]+. The NMR data of 2 and 1 were similar, except for the upfield shift of C-2″, C-3″, and C-4″ by 3.0, 1.4, and 1.9 ppm, and downfield shift of C-1″, C-5″, and C-6″ by 1.0, 1.9, and 3.2 ppm, respectively, which revealed that compound 2 was a diastereomer of 1 at 711
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Fig. 3. The cell viabilities of HepG2 cells treated by four isolated compounds with MTT assay at different concentrations. The values were presented as mean ± SD of triplicate replicates. Fig. 2. ORTEP drawing of compound 1.
immunosuppressants characterized by high efficiency and low toxicity become more and more important because of the current immunosuppressants with toxic and side effects. The immunosuppressive results of the four compounds isolated from mango leaves are presented in Fig. 5. As shown in Fig. 5, all the compounds exhibited good inhibition on Con A-induced spleen cell proliferation in a concentrationdependent manner ranging from 10 to 40 μM. When tested at a concentration of 40 μM, the relative inhibition ratios of manindicin A, manindicin B, mangiferin and norathyiol were 31.92 ± 3.84%, 31.67 ± 3.43%, 12.09 ± 2.62% and 24.28 ± 2.89%, indicating that manindicin A and manindicin B had a similar (p > 0.05) and the strongest (p < 0.05) immunosuppressive activity, while mangiferin possessed the weakest activity (p < 0.05). However, the stimulation index of DXM (positive control) at each tested concentration was significantly lower than all those of four compounds at the same concentration (p < 0.05), which suggested that DXM had a better immunosuppressive activity. The relative inhibition ratio of DXM at 40 μM was 56.99 ± 4.22%. To our knowledge, this is the first report on immunosuppressive properties of M. indica metabolites in Con A-induced spleen cells. A previous study has also reported that the aqueous extract of M. indica bark and mangiferin exhibited inhibitory effects on macrophage activity, which suggested the extract and mangiferin possessed immunosuppressive effect and was partly consistent with the findings of the present work (Garcia, Delgado, Ubeira, & Leiro, 2002). Another study has also reported that mangiferin is the predominant component of M. indica L. bark for immunosuppressive anti-tumor effects in MDAMB231 breast cancer cells.
glycosyl moiety. The differences of the chemical shifts of the glycoside moieties in compounds 1 and 2 were identical to the differences between α-D-fructose and β-D-fructose (Pawank, 1992). The ROESY correlations between protons at δH 3.83 (t, H-3″) and 3.14 (d, Ha-1″), 5.33 (d, HO-4″); 3.45 (m, H-4″) and 3.27 (m, Hb-6″) indicated that the relative configurations of H-3″/H-4″ and H-4″/H-5″ were trans, which were same as 1. The ROESY correlations were observed between H-5″ (3.66, m) and Hb-1″ (2.90, d) in 2, which were absent in 1, indicating that the glycoside moiety in 2 was β-D-fructofuranose. Thus, compound 2 was elucidated and trivially named as manindicin B. Compounds 1 and 2 were the diastereomers, formed by the combination of benzophenone and α/β-D-fructose, respectively. 3.2. Cytoprotective effect on H2O2-treated HepG2 cells The cellular antioxidant activity was developed as a common model for quantifying the antioxidant capacity of phytochemicals in HepG2 cells, and especially in the assessment of their intracellular ROS scavenging activity. The reactive oxygen species (ROS), such as superoxide anion radical, hydroxy radical, and hydrogen peroxide are produced in the body and are beneficial as signal transducers and growth regulators for human beings. However, excessive ROS result in oxidative stress, which may lead to some chronic diseases (Wang et al., 2016). Therefore, the antioxidants are required to balance the level of ROS. The MTT result showed that all the compounds did not exhibit cytotoxicity to HepG2 cells at the tested concentration ranging from 12.5 to 50 μM (Fig. 3). The cellular ROS inhibition result of those four compounds is illustrated in Fig. 4. As shown in Fig. 4, the cellular ROS amount of HepG2 cells increased significantly when treated by H2O2. All compounds possessed good ROS inhibition effects with a concentration-dependent manner when compared to H2O2 treated group (p < 0.05). Among those four compounds, norathyriol showed the weakest inhibitory effect on ROS production at each tested concentration ranged from 12.5 to 50 μM (p < 0.05). However, manindicin A, manindicin B and mangiferin exhibited a similar inhibition against ROS generation at 25 and 50 μM (p > 0.05). When tested at 12.5 μM, manindicin A had lower ROS inhibitory activity (p < 0.05), while manindicin B and mangiferin shared a similar activity (p > 0.05).
3.4. α-Glucosidase inhibitory activity The α-glucosidase inhibition is a common and effective way for the treatment of hyperglycaemia in managing type II diabetes (Tewari et al., 2003). Previous studies showed that the ethanol extract of mango peels inhibited α-glucosidase activities with IC50 value of 3.5 μg/mL (Gondi & Prasada Rao, 2015). Another research on streptozotocin-induced diabetic rats exhibited the extract of mango by-product reduced serum glucose and triacylglycerides and was associated to its high content of phenolics (Rodríguez-González et al., 2017). However, the αglucosidase inhibitory effects of the chemical constituents of M. indica are hardly ever reported. Hence, the α-glucosidase inhibitory activity was tested for all the isolated compounds. As shown in Table 2, norathyriol exhibited an excellent α-glucosidase inhibition activity with an IC50 of 4.22 ± 0.19 μg/mL, which was about 4-fold lower than that of commercial inhibitor (acarbose, 16.28 ± 1.22 μg/mL). Subsequently, mangiferin has a moderate α-glucosidase inhibitory activity
3.3. Immunosuppressive activity Autoimmune diseases are mostly chronic or progressive diseases, which require long-term medication. Therefore, the requirement of 712
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Fig. 4. Intracellular ROS production of four isolated compounds on H2O2-induced HepG2 cells. The values were presented as mean ± SD of triplicate replicates. Different letters indicate significant differences (p < 0.05).
Table 2 α-Glucosidase inhibitory activities of the isolates. Compound
IC50 (μg/mL)
Acarbose Manindicin A Manindicins B Mangiferin Norathyriol
16.28 ± 1.22 > 300 > 300 32.11 ± 2.01 4.22 ± 0.19
(IC50 = 32.11 ± 2.01 μg/mL), half the efficacy of acarbose. However, manindicins A and B had weak inhibition of α-glucosidase with IC50 of both being more than 300 μg/mL (Table 2). The decreased α-glucosidase inhibitory effect of 3 may due to the increasing molecular size and polarity. After the replacement of H-3 by a glucose moiety, the increased steric hindrance may weaken the binding interaction between flavonoids and the enzyme. A previous study found luteolin had a better inhibitory effect on α-glucosidase than its C-glycosides, orientin and isoorientin, which is in part in agreement with our findings (Li et al., 2009). In addition, the O-glycosylation of flavonoid could also decrease the α-glucosidase inhibitory activity. Li and coworkers (Li, Zhou, Gao, Bian, & Shan, 2009) compared α-glucosidase inhibitory potential of
Fig. 5. Effect of the isolated compounds on Con A-stimulated splenocyte proliferation in vitro. DXM was used as the positive control. The values were presented as mean ± SD of triplicate replicates. Statically significant difference was determined by ANOVA and Tukey test (*p < 0.05, compared with the control group).
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4. Conclusion In this study, four benzophenone derivatives were isolated and characterized from M. indica L. leaves, two of which were the new compounds, namely manindicins A and B. It is found that the four compounds could inhibit the cellular ROS amount of H2O2-induced HepG2 cells. Meanwhile, spleen cells proliferation assay showed that manindicins A and B had a good and similar immunosuppressive activity, while mangiferin and norathyriol exhibited a relative weaker activity. Norathyriol showed the strongest inhibitory effect on α-glucosidase activity with an IC50 of 4.22 ± 0.19 μg/mL, which was lower than that of acarbose (16.28 ± 1.22 μg/mL), followed by mangiferin. However, the manindicins A and B had weak inhibition of α-glucosidase with an IC50 of both being more than 300 μg/mL. Declarations of interest None. Acknowledgements This work was supported by the National Natural Science Foundation of China [Grant No. 31600274 and 31460083], Applied Basic Research Project of Yunnan Province (Grant No. 2018FB036), and the Key Laboratory Training Program in Yunnan (Grant No. 2017DG006). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2018.11.045. References Ahmed, E. M. (2007). Egyptian mango by-product 2: Antioxidant and antimicrobial activities of extract and oil from mango seed kernel. Food Chemistry, 103, 1141–1152. Ajila, C. M. (2008). Protection against hydrogen peroxide induced oxidative damage in rat er- ythrocytes by Mangifera indica L. peel extract. Food and Chemical Toxicology, 46, 303–309. Amaya-Cruz, D. M., Rodríguez-González, S., Pérez-Ramírez, I. F., Loarca-Piña, G., AmayaLlano, S., Gallegos-Corona, M. A., & Reynoso-Camacho, R. (2015). Juice by-products as a source of dietary fibre and antioxidants and their effect on hepatic steatosis. Journal of Functional Foods, 17, 93–102. Anjaneyulu, V. (1985). Tritepenoids from Mangifera indica. Phytochemistry, 24, 2359–2367. Burtonfreeman, B. M., Sandhu, A. K., & Edirisinghe, I. (2017). Mangos and their bioactive components: Adding variety to the fruit plate for health. Food & Function, 8, 3010–3012. Deng, J. G., & Zeng, C. H. (2003). Thirty years Research progress of Mangifera indica L. and mangiferin in recent thirty years. Journal of Guangxi Traditional Chinese Medical University, 6, 44–47. Fang, C., Kim, H., Noratto, G., Sun, Y., Talcott, S. T., & Mertens-Talcott, S. U. (2018). Gallotannin derivatives from mango (Mangifera indica L.) suppress adipogenesis and increase thermogenesis in 3T3-L1 adipocytes in part through the AMPK pathway. Journal of Functional Foods, 46, 101–109. Fernández-Ponce, M. T., Medina-Ruiz, E., Casas, L., Mantell, C., & Martínez de la OssaFernández, E. J. (2018). Development of cotton fabric impregnated with antioxidant mango polyphenols by means of supercritical fluids. The Journal of Supercritical Fluids, 140, 310–319. Gao, F., Yao, Y. C., Cai, S. B., Zhao, T. R., Yang, X. Y., Fan, J., ... Cheng, G. G. (2017). Novel immunosuppressive pregnane glycosides from the leaves of Epigynum auritum. Fitoterapia, 118, 107–111. Garcia, D., Delgado, R., Ubeira, F. M., & Leiro, J. (2002). Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin. International Immunopharmacology, 2, 797–806. Gondi, M., Basha, S. A., Bhaskar, J. J., Salimath, P. V., & Prasada Rao, U. J. S. (2015).
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