Bioactive polar compounds from stem bark of Magnolia officinalis

Fitoterapia 83 (2012) 356–361

Contents lists available at SciVerse ScienceDirect

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

Bioactive polar compounds from stem bark of Magnolia officinalis Sheng-xian Yu a, b, Ren-yi Yan a, Ri-xin Liang a, Wei Wang a, Bin Yang a,⁎ a b

Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 16 Dongzhimengneinanxiao Street, Beijing, 100700, PR China BeiJing Double-crane Pharmaceutical Co., Ltd., No. 1, Li Ze East 2nd Road, Beijing, 100102, PR China

a r t i c l e

i n f o

Article history: Received 19 October 2011 Accepted in revised form 20 November 2011 Available online 4 December 2011 Keywords: Magnolia officinalis Hydrophilic compounds Anti-spasmodic Antioxidant activity

a b s t r a c t Two new phenylethanoid glycosides magnoloside D (1) and E (2), together with nine known compounds, were isolated from the polar part of methanol extract of the stem bark of Magnolia officinalis. The structures of the new compounds were established on the basis of spectral analysis. Anti-spasmodic activity of four major constituents (3, 4, 9 and 11) was tested in isolated colon of rat, compounds 3, 4, and 9 showed inhibition against acetylcholine, with the effect similar to that of magnolol and honokiol. At the same time, antioxidant activity of the isolated compounds was investigated using a DPPH and an ORAC assay. All of the compounds, except compound 8 showed potent antioxidant capacity in the ORAC assay, while compounds 1–5 and 11 exhibited potent antioxidant activity in the DPPH assay. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The bark of Magnolia officinalis (Magnoliaceae), entitled as Houpo in Chinese, has been used in traditional Chinese medicine (TCM) for the treatment of abdominal distention and pains, dyspepsia, and asthmatic cough [1]. In China, a number of prescriptions containing Houpo are still in use in modern clinical practice. Previous chemical studies resulted in isolation of a variety of neolignans, lignans, alkaloids and sesquiterpenes [2–5]. Magnolol and honokiol are the two major and attractive constituents of Houpo. Many studies showed that they have a wide variety of pharmaceutical properties, such as, anti-spasmodic, antioxidant, anti-cancer and antidepressant activities, etc. [6]. Also, total content of magnolol and honokiol is regarded as an important parameter to evaluate the quality of Houpo [7]. Traditionally, before Houpo is used in clinic, it will be processed. In our previous work we found that the content of some chemical ingredients of Houpo, especially those unknown polar compounds, changed

after Houpo is processed [8]. Subsequently, phytochemical investigation was focused on the polar fraction and resulted in the isolation of two new phenylethanoid glycosides, named magnoloside D (1) and E (2), together with 9 known compounds. The known compounds, magnoloside A (3) [9], magnoloside B (4) [10], manglieside D (5) [11], saikolignanoside A (6) [12], 1,1′-dibenzene-6′,8′,9′- trihydroxy-3-allyl-4-O-β-Dglucopyranoside (7) [13], syringaresinol-4,4′-O-bis-β-D-glucoside (8) [14], syringin (9) [15], caffeic acid methyl ester (10) [16], magnoflorine (11) [17] were identified by comparing their spectroscopic data with those reported in the literature (Fig. 1). Herein, we reported the isolation and structural elucidation of these polar compounds from methanol extraction of M. officinalis, as well as their anti-spasmodic and antioxidant effects. 2. Experimental 2.1. General experimental procedures

⁎ Corresponding author. Tel.: + 86 10 64014411 2848; fax: + 86 10 64013996. E-mail address: [email protected] (B. Yang). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.11.020

UV spectra were obtained in MeOH on a Jasco V-650 spectrophotometer. IR spectra were recorded in KBr pellets on a Thermo Nicolet 5700 infrared spectrometer. HR-ESIMS were obtained on an Agilent 6520 Accurate-Mass Q-TOF LC-MS. NMR spectra were taken on Varian Mercury-400 or Inova

S. Yu et al. / Fitoterapia 83 (2012) 356–361

357

Fig. 1. Chemical structures of compounds 1–11.

500 spectrometer with solvent peak as references. Macroporous resin D101 was a product of Chemical Plant of Nan Kai University (Tianjin, China). RP-C18 (YMC, 40–60 μm) and Sephadex LH-20 (Pharmacia, Sweden) were used for column chromatographic separation. MPLC was performed on an EZ Purifier II flash chromatography system (Shanghai Li Sui ETech CO. Ltd). Analytical HPLC was conducted on a Waters 2695 pumping system equipped with a Waters 2996 photodiode array detector. The semipreparative HPLC was performed using a Waters 600 pump, a Waters 2487 detector, and a C18 column (250 mm × 20 mm, 5 μm; YMC). DPPH and ORAC assays were performed on a Varioskan Flash Multimode Reader (Thermo scientific, Finland). GC was carried out on an Agilent 7890 GC system.

2.2. Plant material M. officinalis was collected from Enshi city, Hubei Province of China, in May 2009, and identified by Prof. Bin Yang. A voucher specimen (NO. 20090518) was deposited at Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

2.3. Extraction and isolation The dried and powdered M. officinalis (8 kg) was extracted with methanol (32 × 3 L) by ultrasonication. The MeOH solutions were combined and concentrated to yield a dried extract (1.5 kg). The MeOH extract (1.5 kg) was suspended in distilled water (3 L) and then partitioned with CHCl3 (3 L × 3). The water-soluble portion (650 g) was chromatographed on D101 macroporous resin, eluted with a gradient of EtOH-H2O (0:100 to 95:5), to obtain five fractions (Fr.1–Fr.5). Fr.2 (250 g, 20% EtOH elute) was subjected to MPLC on an ODS column (30 mL/min, 265 nm) eluted with a gradient of MeOH-H2O (5:95 to 100:0) to yield seven fractions (Fr.2.1–Fr.2.7). Fr.2.1 (10 g), eluted with a gradient of MeOH-H2O (15:85), was purified by MPLC on ODS (15 mL/min) to yield compound 11 (60 mg). Fr.2.2 (40 g), eluted with a gradient of MeOH-H2O (20:80). Compound 9 (600 mg) was crystallized from MeOH. Fr.2.3 (55 g), eluted with a gradient of MeOH-H2O (25:75), then chromatographed on Sephadex LH-20 (MeOH-H2O, 25:75) to obtain compound 4 (1 g). Fr.2.4 (60 g), eluted with a gradient of MeOH-H2O (30:70), then rechromatographed on Sephadex LH-20 (MeOH-H2O, 30:70) to give compound 3 (2 g). Fr.2.5

358

S. Yu et al. / Fitoterapia 83 (2012) 356–361

(50 g), eluted with a gradient of MeOH-H2O (35:65), then chromatographed on Sephadex LH-20 (MeOH-H2O, 35:65) to furnish three fractions (Fr.2.5.1–Fr.2.5.3). Compound 8 (35 mg) and compound 5 (10 mg) were crystallized from Fr.2.5.1 and Fr.2.5.2 using MeOH, respectively. Fr.2.6 (14 g) was purified by semipreparative RP-C18 HPLC, eluted with MeOH-H2O (30:70) to afford three compounds, compound 10 (6 mg), compound 7 (10 mg), compound 6 (30 mg). Fr.2.7 (20 g) was repeatedly chromatographed by MPLC on ODS (MeOH-H2O, 35:65) to afford compound 1 (760 mg) and compound 2 (46 mg) (Fig. 2). Magnoloside D (1): light yellow amorphous powder; [α]D20-63.8 (c 0.09, MeOH); UV (MeOH) λmax 294, 328 nm; IR (KBr) νmax 3335, 2931, 1689, 1631, 1605, 1520, 1282, 1043 cm − 1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Table 1; (−)-ESIMS m/z 623.0 [M-H]−; HR-ESIMS m/z 623.1951 [M-H] − (calcd. for C29H35O15, 623.1976). Magnoloside E (2): light yellow amorphous powder; [α]D20-28.8 (c 0.08, MeOH); UV (MeOH) λmax 290, 328 nm; IR (KBr) νmax 3320, 2933, 1693, 1631, 1604, 1517, 1282, 1044 cm − 1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data see Table 1; (−)-ESIMS m/z 609.0 [M-H]−; HR-ESIMS m/z 609.1817 [M-H] − (calcd. for C28H33O15, 609.1819).

neutralized with 10 mM HCl (1 mL) and extracted with EtOAc (3 × 1 mL). Then 1 M HCl (1 mL) was added to the water layer and refluxed at 90 °C for 4 h. After cooling, the solution was extracted with EtOAc (3 × 1 mL). The water layer was dried by a stream of N2. Anhydrous pyridine (1 mL) and L-cysteine methyl ester hydrochloride (1 mg) were added and reacted at 70 °C for 2 h. Then the mixture was dried by a stream of N2. Trimethylsilyl imidazole (0.20 mL) was added, and the mixture was reacted at 70 °C for 30 min. Then the mixture was partitioned between H2O (2 mL) and n-hexane (3 × 1 mL). The n-hexane layer was concentrated to 1 mL and filtered, and analyzed by GC using a HB-5 column (30 m × 0.32 mm, 0.25 μm). Temperatures of the injector and detector were 250 and 280 °C, respectively. The temperature of oven was at 100 °C during the first 2 min. Subsequently, the temperature changed from 100 °C to 250 °C in the next 15 min, and then the temperature was kept at 250 °C in the following 10 min. Peaks of the hydrolysate of 1 and 2 were detected by comparison with retention times of authentic samples of L-rhamnose (tR 15.084), Dallose (tR 16.375), L-allose (tR 16.463), and D-apiose (tR 14.207) after being treated simultaneously with the same manner.

2.4. Sugar analysis of Compounds 1–2

2.5. Anti-spasmodic assay

Compounds 1–2 (each 5.0 mg) were hydrolyzed with 10 mM NaOH (1 mL) at 60 °C for 2 h. The solution was

Male wistar rats (230–270 g) were fasted for 24 h, and then sacrificed by cervical dislocation, after that, colons

Fig. 2. Key HMBC correlations of compounds 1 and 2.

S. Yu et al. / Fitoterapia 83 (2012) 356–361 Table 1 1 H NMR and

13

359

C NMR data of compounds 1 (CD3OD) and 2 (C5D5N). 1

Position Aglycone 1 2 3 4 5 6 α β Allose 1′ 2′ 3′ 4′ 5′ 6′ Rhamnose 1" 2" 3" 4" 5" 6" Caffeoyl 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′ α′′′ β′′′ CO

2

δH

δC

6.62, 1H, d (2.0)

6.58, 1H, 6.48, 1H, 3.63, 1H, 2.71, 2H,

d (8.0) dd (2.0, 8.0) m; 3.91, 1H, m t (7.6)

4.70, 1H, 3.44, 1H, 4.18, 1H, 3.49, 1H, 3.91, 1H, 4.26, 1H, 4.41, 1H,

d (8.4) dd (2.8, t (2.8) dd (2.8, m dd (6.0, dd (2.0,

4.84, 1H, 3.86, 1H, 3.67, 1H, 3.37, 1H, 3.92, 1H, 1.19, 3H,

s dd (1.2, 3.6) dd (3.6, 9.6) t (9.6) m d (6.0)

8.4) 10.0) 12 .0) 12.0)

6.99, 1H, d (2.0)

6.71, 1H, 6.84, 1H, 6.24, 1H, 7.50, 1H,

d (8.0) dd (2.0, 8.0) d (15.6) d (15.6)

131.5 117.1 146.0 144.6 116.4 121.3 72.2 36.8 100.6 74.8 68.8 68.9 72.8 65.1

97.8 72.4 72.3 73.9 69.9 17.9 127.7 115.1 146.8 149.6 116.5 123.1 114.9 147.2 169.3

were isolated and flushed out using Tyrode's solution (composition 8.0 g NaCl, 0.2 g KCl, 0.2 g CaCl2, 1.0 g NaHCO3, 0.1 g MgCl2, 0.05 g NaH2PO4, 1.0 g glucose, and 1 L distilled water, pH 7.4). Colons of 1.5 cm in length were longitudinally mounted in a 20 mL organ bath (ALC-M, Tissue-Organ Bath system) with an initial tension of 2.0 g. One edge of colon strip was fixed to the bottom of organ bath using suture silk, and the other edge was connected to the force displacement transducer (ALC-M, Chengdu instrument factory). Tension signals were recorded by multi-channel physiological recorder (MP 150, BIOPAC). Acetylcholine (15 μg/mL) (Beijing Dingguo Biotech Co. Ltd, Batch NO. 9BE10120, 99% purity) was applied to produce contraction. Then different samples dissolved in 1% DMSO were added. Atropine (5 μg/mL) (98%, Tianjin Jinyao Amino Acid CO., Ltd, batch NO. 1004112) was employed as a positive control, and 1% DMSO was employed as a blank. 2.6. DPPH assay and ORAC assay See ref [18]. 3. Result and discussion Compound 1 was isolated as a light yellow amorphous powder. The molecular formula, C29H36O15, was deduced

Position Aglycone 1 2 3 4 5 6 α β Allose 1′ 2′ 3′ 4′ 5′ 6′ Apiose 1" 2" 3" 4" 5" Caffeoyl 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′ α′′′ β′′′ CO

δH

δC

7.20 overlap

7.16, 6.81, 3.94, 3.09,

1H, 1H, 1H, 2H,

d (8.0) dd (2.0, 8.0) m; 4.34, 1H, m m

5.44, 4.20, 4.91, 4.06, 4.60, 4.86, 5.05,

1H, 1H, 1H, 1H, 1H, 1H, 1H,

d (8.0) dd (3.0, 8.0) t (3.0) t (3.0, 10.0) m dd (6.0, 12.0) dd (2.0, 12.0)

5.97, 1H, d (1.5) 4.76, 1H, d (1.5) 4.34, 1H, d (9.5) 4.68, 1H, d (9.5) 4.31, 2H, s

7.59 overlap

7.22 overlap 7.13, 1H, d (2.0, 8.5) 6.69, 1H, d (16.0) 7.97, 1H, d (16.0)

131.5 117.4 147.8 146.3 118.4 121.5 72.2 37.4 101.3 75.2 69.5 69.6 73.7 65.8

107.3 78.8 81.7 76.5 67.1 127.8 116.6 148.4 150.2 117.6 123.1 116.0 146.8 168.6

from HRESIMS on the basis of the molecular ion peak at m/z 623.1951 [M–H] −. The UV spectrum showed absorptions maxima at 294 and 328 nm. The IR spectrum displayed absorption bands of hydroxyl group (3334 cm − 1), conjugated carbonyl group (1689 and 1631 cm − 1) and aromatic ring functionalities (1605 cm − 1). The 1H NMR spectrum of 1 (Table 1) exhibited the characteristic signals belonging to (E)-caffeic acid and 3,4-dihydroxyphenylethanol moieties: two sets of ABX-type aromatic signals at δH 6.62 (1H, d, J = 2.0 Hz), 6.58 (1H, d, J = 8.0 Hz), and 6.48 (1H, dd, J = 2.0, 8.0 Hz), and at δH 6.98 (1H, d, J = 2.0 Hz), 6.72 (1H, d, J = 8.0 Hz), and 6.85 (1H, dd, J = 8.0, 2.0 Hz), two trans-olefinic protons at δH 7.50 (1H, d, J = 15.6 Hz), 6.24 (1H, d, J = 15.6 Hz), β-methylene at δH 2.71 (2H, t, J = 7.6) of the side-chain of the aglycon moiety. Additionally, two anomeric proton resonances appeared at δH 4.70 (d, J = 8.4 Hz) and 4.84 (br s) which correlated, respectively, with signals at δC 100.6 and 97.8 in the HMQC spectrum. The methyl signal at δH 1.19 (d, J = 6.0 Hz) and δC 17.9 indicated the existence of rhamnose moiety. A series of signals in 1H NMR at δH 4.70 (1H, d, J = 8.4 Hz), 3.44 (1H, dd, J = 2.8, 8.4 Hz), 4.18 (1H, t, J = 2.8 Hz), 3.49 (1H, dd, J = 2.8, 10.0 Hz), 4.26 (1H, dd, J = 6.0, 12.0 Hz), 4.41 (1H, dd, J = 2.0, 12.0 Hz) and 3.91 (1H, m) indicated a rarely occurred β-allopyranosyl unit in the structures of 1. Also, it was confirmed by comparison NMR features of those of

360

S. Yu et al. / Fitoterapia 83 (2012) 356–361

Table 2 Antioxidant capacity of isolated compounds in DPPH and ORAC assaysa.

1 2 3 4 5 6 7 9 10 11 Magnolol Honokiol

DPPHb

ORACc

9.11 ± 0.16 9.41 ± 0.96 9.74 ± 0.36 7.00 ± 0.13 25.61 ± 1.78 –d – – – 21.51 ± 1.99 19.61 ± 4.25 12.79 ± 1.06

9.17 ± 0.93 12.93 ± 1.92 8.11 ± 0.82 9.83 ± 2.22 4.95 ± 0.90 3.13 ± 1.07 6.23 ± 0.55 1.10 ± 0.17 0.06 ± 0.01 5.75 ± 0.56 5.39 ± 0.79 4.94 ± 0.24

a All values were measured with thee replicates and are presented as means ± SD. b Concentration necessary for 50% inhibition (IC50, μg/mL). c μM Trolox/μM. d IC50 > 200 μg/mL.

magnoloside A [9], B and C [10] isolated from M. obovata, which contained allopyranose. Alkaline and acidic hydrolysis afforded monosaccharide components identified as D-allose and L-rhamnose by the GC analysis. An unambiguous determination of the sequence and linkage sites was obtained from the HMBC correlations. Thus, cross-peaks were observed between the anomeric proton of allose and C-α (δC 72.2) of the phenylethyl alcohol moiety, H-6′ (δH 4.26) of allose and carbonyl carbon (δC 169.3) of caffeoyl group, and H-1′′ (δH 4.84) of rhamnose and C-2′ (74.8) of allose. Therefore, the structure of compound 1 was established as 2-(3,4-dihydroxyphenyl)ethanol 1-O-[6-O-caffeoyl]-[α-Lrhamnopyranosyl-(1 → 2)-β-D-allopyranoside], named magnoloside D. Compound 2 was obtained as a light yellow amorphous powder. The HRESIMS of 2 gave a pseudomolecular ion peak at m/z 609.1819 [M–H] −, indicating a molecular formula of C28H34O15 and in good agreement with the observation of 5 methylenes, 15 methines, and 8 quaternary carbons in its DEPT and HSQC spectra. The UV spectrum showed absorptions maxima at 290 and 328 nm. The IR spectrum displayed absorption bands of hydroxyl group (3319 cm − 1),

conjugated carbonyl group (1693 and 1631 cm − 1) and aromatic ring functionalities (1604 cm − 1). Analysis of the 1D and 2D NMR spectra of compound 2 in comparison to that of 1 showed that the difference between the two compounds should be confined to the terminal sugar portion. The rhamnose in compound 1 characterized by upfield methyl signals was replaced by an apiose. The apiose moiety was confirmed by the observation of the anomeric proton at δH 5.97 (d, J = 1.5 Hz) couple with vicinal protons at δH 4.76 (d, J = 1.5 Hz), and an ABq signals at δH 4.68 (d, J = 9.5 Hz) and 4.34 (d, J = 9.5 Hz), and a methene at δH 4.31 (2H, s, H-5′′) in the 1H NMR. Meanwhile, in 13C NMR the signals for an apiose unit at δC 107.3 (d), 78.8 (d), 81.7 (s), 76.5 (t), 67.1(t) were consistent with the reported data [19]. Alkaline and acidic hydrolysis of 2 resulted in the liberation of D-allose and D-apiose, identified by GC. Finally, all connectivities within 2 were established by an HMBC experiment, where correlations between H-1′ (δH 5.44, d, J = 8.0 Hz) of the allose and the α-C atom (δC 72.2) of the phenylethyl alcohol moiety, H-1′′ (δH 5.97, d, J = 1.5 Hz) of the apiose and C-2′ (δC 75.2) of the allose, and H-6′ (δH 4.86, dd, J = 6.0, 12.0 Hz) of the allose and the carbonyl carbon (δC 168.6) of the caffeoyl moiety were observed. On the basis of the above evidence, compound 2 was elucidated as 2-(3,4dihydroxyphenyl)ethanol 1-O-[6-O-caffeoyl]-[β-D-apiofuranosyl-(1 → 2)-β-D-allopyranoside], named magnoloside E. Among the compounds isolated, 3–8, 10 and 11 were isolated from M. officinalis for the first time. In the DPPH assay, compound 1–4 exhibited more powerful free-radical scavenging capability than that of magnolol and honokiol, with IC50 values of 9.11, 9.41, 9.74, and 7.00 μg/mL, respectively. In the ORAC assay, all isolated compounds showed antioxidant activities, except for compound 8 (Table 2). Magnolol and honokiol were considered to be the main ingredients of M. officinalis to exert therapeutic effects on gastrointestinal diseases, which significantly inhibited the contraction of isolated gastrointestinal smooth muscles induced by multiple factors [6]. In this research, three (3, 4 and 9) of four major compounds in water-soluble portion showed the similar inhibition against acetylcholine induced colon contraction in rat. It means that the polar fraction plays an unnegligible role in the antispasmodic effects of M. officinalis extracts (Fig. 3).

Fig. 3. Inhibitory effects on acetylcholine (15 μg/mL) induced contraction in the colon of rat. Data are shown as means ± SD (n = 8), ⁎P b 0.05, ⁎⁎P b 0.01 and ⁎⁎⁎P b 0.001 compared to the corresponding values of blank contractility (t-test).

S. Yu et al. / Fitoterapia 83 (2012) 356–361

Acknowledgements This project was supported by the National Natural Science Foundation of China (No. 81073042), the Special Fund of National Bureau of Traditional Chinese Medicine (No. 200807020) and the Research Fund of China Academy of Chinese Medical Sciences (No. ZZ20090108). References [1] Jiangsu New Medical College. Chinese Drug Dictionary. Shanghai: Shanghai People Publishing House; 1977. p. 1628–30. [2] Sarker SD, Latif Z, Stewart M, Nabar L. Phytochemistry of the genus Magnolia. In: Sarker SD, Maruyama Y, editors. The genus Magnolia. CRC Press; 2002. p. 32–85. [3] Youn UJ, Chen QC, Jin WY, Lee IS, Kim HJ, Lee JP, et al. Cytotoxic lignans from the stem bark of Magnolia officinalis. J Nat Prod 2007;70:1687–9. [4] Shen CC, Ni CL, Shen YC, Huang YL, Kuo CH, Wu TS, et al. Phenolic constituents from the stem bark of Magnolia officinalis. J Nat Prod 2009;72: 168–71. [5] Guo ZF, Wang XB, Luo JG, Luo J, Wang JS, Kong LY. A novel aporphine alkaloid from Magnolia officinalis. Fitoterapia 2011;82:637–41. [6] Lee YJ, Lee YM, Lee CK, Jung JK, Han SB, Hong JT. Therapeutic applications of compounds in the Magnolia family. Pharmacol Ther 2011;130:157–76. [7] Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia I. Beijing: Chemical Industry Press; 2010. p. 235. [8] Yu SX, Zhang CX, Chen CY, Yan RY, Yang B, Liao CL, et al. Effects of primary processing on quality of cortex Magnolia officinalis. China J Chin Mater Med 2010;35:1831–5.

361

[9] Hasegawa T, Fukuyama Y, Yamada T, Nakagawa K. Isolation and structure of Magnoloside A, a new phenylpropanoid glycoside from Magnolia obovata Thunb. Chem Lett 1988;17:163–6. [10] Hasegawa T, Fukuyama Y, Yamada T, Nakagawa K. Structures of magnoloside B and C. Novel phenylpropanoid glycosides with allopyranose as core the sugar unit. Chem Pharm Bull 1988;36:1245–8. [11] Kiem PV, Tri MD, Tuong LVD, Tung NH, Hanh NN, Quang TH, et al. Chemical constituents from the leaves of Manglietia phuthoensis and their effects on osteoblastic MC3T3-E1 Cells. Chem Pharm Bull 2008;56:1270–5. [12] Tan L, Wang B, Zhao YY. A lignan glucoside from Bupleurum scorzonerifolium. Chin Chem Lett 2004;15:1053–6. [13] Deng SM, Dai HF, Zhou J. A new glycoside from Magnolia rostrata. Acta Bot Yunnanica 2002;24:397–400. [14] Jolad SD, Hoffmann JJ, Cole JR, Tempesta MS, Bates RB. Cytotoxic agent from Penstemon deustus (Scrophulariaceae): isolation and stereochemistry of liriodendrin, a symmetrically substituted furofuranoid lignan diglucoside. J Org Chem 1980;45:1327–9. [15] Munkombwe NM, Galebotswe P, Modibesane K, Morebodi N. Phenylpropanoid glycosides of Gnidia polycephala. Phytochemistry 2003;64: 1401–4. [16] Sugama K, Hayashi K, Nakagawa T, Mitsuhashi H, Yoshida N. Sesquiterpenoids from Petasites fragrans. Phytochemistry 1983;22: 1619–22. [17] Ishii H, Imai M, Johji S, Tan S, Chen IS, Ishikawa T. Studies on the chemical constituents of Xanthoxylum nitidum (Roxb.) D.C. (Fagara nitida Roxb.).II. Examination of the chemical constituents by membrane filtration: identification of magnoflorine, a water-soluble quaternary aporphine alkaloid. Chem. Pharm Bull 1994;42:108–11. [18] Yan RY, Cao YY, Chen CY, Dai HQ, Yu SX, Wei JL, et al. Antioxidant flavonoids from the seed of Oroxylum indicum. Fitoterapia 2011;82:841–8. [19] Kanchanapoom T, Kasai R, Yamasaki K. Phenolic glycosides from Markhamia stipulata. Phytochemistry 2002;59:557–63.