Recently, a number of new species of the Magnoliaceae family have been discovered and published such as Magnolia tiepii, M. lamdongensis, M. bidoupensis. Phytochemical study of the leaves of M. lamdongensis, an endemic species of Lam Dong province, Vietnam, resulted in the isolation of four compounds, which were named stigmast-5-en-3β-ol-3-O-β-D-glucopyranoside (1), stigmast-5-en-3β-ol (2), quercetin (3), and palmitic acid (4). Their structural elucidations were confirmed by 1 D and 2 D NMR experiments and a comparison with those reported in the literature. These metabolites isolated for the first time from this species.
Keywords: Magnolia lamdongensis; stigmast-5-en-3β-ol-3-O-β-D-glucopyranoside; stigmast-5-en-3β-ol; quercetin; palmitic acid
Magnolia (Magnoliaceae), a genus of trees or shrubs, has about 250 species that grow in temperate and tropical climates, primarily in India, Malaysia, Japan, China, Vietnam and America [1]. Many species in this genus are of great value and have been used in traditional medicines to treat a wide range of diseases. Because of its antiinflammatory activity, M. fargesii has been widely used in traditional medicine to treat empyema, nasal congestion, sinusitis, and allergic rhinitis [2]. M. grandiflora flowers and leaves have long been used to treat headaches, hypertension, fever, diarrhoea, and rheumatism [3]. The trunk bark of M.
ovata has been used to treat fever, and the leaves are thought to be useful in the treatment of diabetes [4]. Previous chemical studies on Magnolia species revealed the presence of lignans [5, 6], alkaloids [7], and sesquiterpenoids [8]. The biological activities of compounds isolated from Magnolia genus have also been thoroughly investigated. Some of the most notable biological activities in this genus are cytotoxic activity [9], antioxidant activity [10], and anti-inflammatory activity [6]. Among them, sesquiterpenoids have been shown to have coronary vasodilator activity [11] as well as cytotoxicity [12], anti-inflammatory, and antihyperalgesic properties [13].
Magnolia lamdongensis belonging to the genus Magnolia were collected in Lam Dong province. In the previous report, three flavonoids (astragalin, quercetin 3- neohesperidoside, and quercetin 3-O-α-L-rhamnopyranosyl(1→2)--D-galactopyranoside) and a sterol (stigmasterol) were isolated from the leaves of this species [14]. Continuing our research, four compounds 1-4 have been isolated for the first time from the leaves of M. lamdongensis. This is the first report about isolated compounds from this plant.
Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany) and reversed-phase silica gel (ODS- A, 12 nm S-150 mm, YMC Co., Ltd., Japan) resins. TLC used pre-coated silica gel 60 F254 (1.05554.0001, Merck) and RP-18 F254S plates (1.15685.0001, Merck), and compounds were visualized by spraying with aqueous 10% 1 H2SO4 and heating for 3–5 min. The H NMR (500 MHz) 13 and C NMR (125 MHz) spectra were recorded on an AVANCE III HD 500 (Bruker, Germany) FT-NMR spectrometer with tetramethylsilane (TMS) was used as an internal standard. ESI mass spectra were collected on Agilent 1100 LC/MS systems. The IR spectra were recorded on a JASCO FT/IR 4100 FT-IR in KBr.
Magnolia lamdongensis samples were collected in September 2020 at the Phu Son slope in Lamdong, Vietnam, and identified by Dr. Nong Van Duy of the Tay Nguyen Institute for Scientific Research, VAST. A voucher specimen (TN3/163) was deposited at the Tay Nguyen Institute for Scientific Research, VAST.
The air-dried and powdered leaves of M. lamdongensis (2.0 kg) were extracted three times with methanol (10L/time) at room temperature. The methanol solutions were filtered, combined, and concentrated under reduced pressure to obtain methanol residue (267 g). This was suspended in water (2L) and partitioned in turn with n-hexane, chloroform, and ethyl acetate to give the corresponding extracts: n-hexane (H, 15.0 g), CHCl3 (C, 20.8 g), EtOAc (E, 19.9 g), and water layer (W, 2L).
The extract C (20.8 g) was separated on silica gel CC with stepwise gradient elution of CHCl3/MeOH (1:0-0:1, v/v) to yield thirteen fractions, C1-C11. Fraction C11 (1.94 g) was fractionated by Sephadex LH-20 CC with stepwise gradient elution MeOH/H2O (9:1-1:0, v/v) to yield five subfractions, C11A-C11E. Subfraction C11C (234 mg) was subjected to a silica gel CC eluted with CHCl3/MeOH/H2O (5:1:0.1, v/v/v) to yield five subfractions, C11C1-C11C5. Subfraction C11C2 (104 mg) was purified by the RP-18 column eluted with MeOH/H2O (4:1, v/v) to yield compound 1 (15 mg). Fraction C10 (1.4 g) was further separated by sephadex LH- 20 CC with MeOH/H2O (1:1-1:0, v/v) to give four subfractions C10A-C10D. Subfraction C10D (72 mg) was separated by silica gel CC eluting with CHCl3/MeOH/H2O (3:1:0.1, v/v/v) and purified by RP-18 column using MeOH/H2O (1:1-1:0, v/v) as elution to yield compound 3 (10 mg).
The extract H (15.0 g) was separated on silica gel CC with stepwise gradient elution of hexane/EtOAc (1:0-0:1, v/v) to yield seven fractions, H1-H7. Fraction H4 (1.8 g) was fractionated by Sephadex LH-20 CC with stepwise gradient elution MeOH/H2O (9:1-1:0, v/v) to yield six subfractions, H4A-H4F. Subfraction H4F (89 mg) was purified by the silica gel column eluted with n-hexane:CH2Cl2 (10:1, v/v) to yield compound 2 (12 mg). Fraction H3 (4.8 g) was further separated by column chromatography on silica gel CC using a mixture of n-hexane:CH2Cl2 (20:1, v/v) to afford seven subfractions, H3A-H3G. Subfraction H3B (1.1 g) was fractionated by RP-18 CC with MeOH/H2O (9:1, v/v) to yield three subfractions, H3B1-H3B3. Subfraction H3B3 (82 mg) was subjected to chromatography on the silica gel column eluted with CH2Cl2:acetone (30:1, v/v) to yield compound 4 (22 mg).
1-4
White needle; molecular formula C35H60O6; ESI-MS m/z + -1 -1 577.43 [M+H] ; IR (KBr): 3433 cm (OH), 1639 cm -1 -1 (C=C), 1461 cm and 1380 cm (gem-dimethyl group), -1 1 13 1053 cm (C-O). H NMR (500 MHz, DMSO) and C NMR (125 MHz, DMSO):see table 1.
+ -1 C H O; ESI-MS m/z 415.12 [M+H] ; IR (KBr): 3424 cm 29 50 -1 3 -1 (OH), 2937 and 2870 cm (C-H sp ), 1641 cm (C=C), -1 -1 1 1379 cm (gem-dimethyl groups), 1056 cm (C-O). H 13 NMR (500 MHz, CDCl ) and C NMR (125 MHz, CDCl ):
3 3 see table 1.
Quercetin (3): Yellow powder; molecular formula + C H O ; ESI-MS: m/z 303.23 [M+H] . IR (KBr): 3406 15 10 7 -1 -1 -1 1 cm (OH), 1666 cm (C=O), 1610 cm (C=C, aren). H NMR (500 MHz, CD3OD)H: 7.75 (1H, br s, H-2), 7.65 (1H, d, J = 8.5 Hz, H-6), 6.91 (1H, d, J = 8.5 Hz, H-5), 6.41 13 (1H, br s, H-8), 6.21 (1H, br s, H-6); CNMR (125 MHz, CD3OD) C: 148.03 (C-2), 137.20 (C-3), 177.53 (C-4), 162.51 (C-5), 99.25 (C-6), 165.57 (C-7), 94.42 (C-8), 158.25 (C-9), 104.53 (C-10).
Palmitic acid (4): White amorphous powder; molecular + formula C16H32O2; ESI-MS m/z 257.35 [M+H] ; IR (KBr):
-1 -1 1 1702 cm (C=O), 3321 cm (OH). H NMR (600 MHz, 13 CDCl3) and C NMR (150 MHz, CDCl3):see table 1.
Compound 1 was isolated as a white needle with the molecular formula C35H60O6 determined by ESI-MS m/z + 577.43 [M+H] . The IR spectrum suggested the presence of -1 hydroxyl groups (3433 cm ), gem-dimethyl group (1461 -1 -1 and 1380 cm ), olefinic carbons (1639 cm ), and C-O bond
-1 1 (1053 cm ). The H-NMR spectra revealed the signals of six methyl groups including two tert-methyl groups at δ 0.95 H (s) and 0.65 (s), three secondary methyl groups at δ 0.90 (d, H methines, one oxygenated methine (C 76.75, C-3), and 3 three quaternary sp carbons. These signal were indicative of the presence of stigmastane-type steroid skeleton (aglycone) and one sugar moiety. The chemical shifts of sugar moiety is revealed by HSQC (Heteronuclear Single Quantum Coherence) spectra analysis [C100.79 (CH, C-1ʹ), 73.5 (CH, C-2ʹ), 76.97 (CH, C-3ʹ), 70.15 (CH, C-4ʹ), 76.78 (CH, C-5ʹ), 61.13 (CH , C-6ʹ)/ 4.22 (1H, d, J = 8.0 Hz, H-1ʹ), 2.90 J = 6.5 Hz), 0.80 (d, J = 5.5 Hz), and 0.78 (d, J = 5.5 Hz), and one primary methyl group at δ 0.82 (t, J = 7.5 Hz).
H confirm a double bond (C5=C6). Also, the correlation between anomeric proton H-1ʹ and carbon C-3 in the HMBC spectrum confirmed that the sugar moiety was attached to the O-atom of the aglycone. Compound 1 was identified as stigmast-5-en-3β-ol-3-O-β-D-glucopyranoside (daucosterol) (Figure 1) based on spectroscopic evidence and comparison with reported values in the literature [15].
Compound 2 was obtained as a white needle. The molecular formula C29H50O was deduced from ESI-MS m/z 415.12 + [M+H] . The IR spectra showed absorption peaks at 3424 -1 -1 3 -1 cm (OH), 2937 and 2870 cm (CH sp ), 1641 cm (C=C), -1 -1 1379 cm (gem-dimethyl groups), and 1056 cm (C-O).
1 13 Comparison of the H and C NMR data of 2 with those of 1 indicated that the structures of both compounds were similar, except for the replacement of the O-β-D- glucopyranoside in 1 with a hydroxylgroup (OH) in 2.
13 Detailed analysis of the CNMR and DEPT spectra revealed the presence of 29 carbon signals, including six 3 methyls, eleven methylenes, eight sp methines, one 3 oxygenated methine, and three quaternary sp carbons. The 1 H-NMR spectra of 2 showed signals for six methyl groups at δ 1.00 (s, H-18), 0.68 (s, H-19), 0.92 (d, J = 6.2 Hz, H- H 21), 0.83 (d, J = 6.5 Hz, H-26), 0.79 (d, J = 5.2 Hz, H-27), 0.84 (t, J = 5.2 Hz, H-29). The presence of a signal at δ H 3.52 indicated an oxymethine proton and one trisubstituted olefin at δH 5.35 (t, J = 5.2 Hz, H-6). By comparison of the NMR data of 2 with those of the published data [16], 2 was identified as stigmast-5-en-3β-ol (-sitosterol).
Compound 3 was isolated as a yellow powder with molecular formula C H O , which was deduced from the 15 10 7 + ESI-MS m/z: 303.23 [M+H] . The IR spectrum suggested -1 the presence of hydroxyl groups 3406 cm , carbonyl group -1 -1 1 1666 cm , doupble bonds 1610 cm (aren). The H NMR spectrashowedan ABX system at δH 7.75 (brs, H-2), 7.65 (d, J = 8.5, H-6), and 6.91 (d, J = 8.5 Hz, H-5) of B ring and a meta-coupled pattern for H-6 and H-8 protons (δ 6.21 H 13 and 6.41, br s). The C NMR spectra showed the presence 26 27 25 23 21 29 20 28 18 12 17 H 19 HO 1 H 9 14 15 10 8 H H 7 3 5 RO (1) R = -D-glucopyranoside (2) R = H of 15 carbon signals in the flavonoid skeleton. Based on these data,compound 3 was identified as quercetin [17]. Compound 4 was isolated as a white amorphous powder. The IR spectrum suggested the presence of carbonyl group -1 -1 at 1701 cm (C=O), hydroxyl group at 3321 cm (OH). Based on the ESI-MS molecular ion peak at m/z 257.35 + [M+H] , the spectral data of compound 4 indicated that it is palmitic acid, confirming the molecular formula of 13 C16H32O2. The C NMR spectrum revealed the presence of one methyl signal at δC 14.11 was assigned to a terminal methyl (C-16), one quaternary carbon signal at δ 179.83 C was assigned to the carboxylic acid (C-1). The signals at δC 22.70, 24.70, 31.94, and 34.02 were assigned to methylene carbons C-15, C-3, C-14, and C-2, respectively. The remaining methylene carbon signals from δC 29.08 to 1 29.25were assigned to carbons from C-4 to C-13. In the H NMR spectra of compound 4 showed the following signals, one methyl triplet at δH 0.88 (t, J = 7.2 Hz, H-16), a multiplet at δH 1.63 (m, H-3), a triplet at δH 2.34 (t, J = 7.2 Hz, H-2), and a singlet at δ 7.26 (OH) which could be H assigned to carbocylic group (COOH). In addition, the proton signals from δ 1.26 to 1.34 (24H, m) were H determined to be protons from H -4 to H -14. Based on the 2 2 spectroscopic evidences and comparison with the reported values in the literature [18], compound 4 was identified as palmitic acid (n-hexadecanoic acid).
These results contribute to additional data on the chemical composition of a new species of the genus Magnolia.In addition to the previously reported flavonoid glycosides, terpenoids, flavonoid aglycone, and fatty compound were isolated from the leaves of M. lamdongensis.
OH 3' OH 4' O 7 9 1' 2 HO 1 16 4 3 5 OH 3 11 OH O O (3) (4) -4.
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According to the results above, four compounds including stigmast-5-en-3β-ol-3-O-β-D-glucopyranoside, stigmast-5- en-3β-ol, kaempferol, and palmitic acid were isolated from the leaves extract of Magnolia lamdongensis for the first time. Their structures were identified by nuclear magnetic resonance (NMR), fourier transform-infrared spectroscopy (FT-IR), electrospay ionization mass spectrum (ESI-MS) as well as comparison with published data.
This work was financially supported by Vietnam national project of the Tay Nguyen Program in the period 2016-2020 (code: TN18/C09).
The authors declare that there is no conflict of interests.
All the authors have contributed equally in designing, drafting the manuscript as per the journal submission format. All authors read and approved the final manuscript.