VIETNAM JOURNAL OF CHEMISTRY VOL. 51(3) 358-362 JUNE 2013
PHYTOCHEMICAL STUDY ON THE PLANTS OF THE ANTIDRUG MEDICATION HEANTOS 4
Part 3. Homolsoflavonold, flavonoid and phenolic compounds
Nguyen Thi Hoang Anh', Trinh Thi Thuy', Tran Van Sung'*, Tran Khuong D a n \ Nguyen Ba Chinh^
Nguyen QuangS Katrin Franke', Norbert Arnold\ and Ludger Wessjohann ' Institute of Chemistry, Vietnam Academy of Science and Technology
^Heantos group
^Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle/S., Germany Received 12 February 2013
Abstract
Fifty eight homoisoflavonoids, flavonoids and phenolic compounds were isolated from the n-hexane, ethyl acetate and n-butanol extracts of the Heantos 4 plants. Their structures have been elucidated by mass, NMR spectroscopy and comparison with published data. Among them, five homoisoflavonoids are new compounds.
Keywords: Heantos 4, homoisoflavonoid, flavonoid, phenolic compounds, antidrug medication.
1. INTRODUCTION
The antidrug medication Heantos 4 is a Vietnamese folk remedy used for the treatment of opiate addiction. It contains twelve herbal medicinal compositions. We carried out the study on phytochemistry of this remedy to learn more about its constituents as well as the relationship between the chemical structures and the biological activities. In the other papers [1, 2] we reported the isolation of alkaloid and nitrogen-containing compounds as well as terpenoids and steroids from Heantos 4 plants. In continuation of our phychemical study, this communication describe the isolation of homoisoflavonoids, flavonoids and phenolic compounds from Heantos plants and their biological activities.
2. EXPERIMENTAL 2.1. Instruments and Chemicals
NMR: Varian Unity 300; MS: AMD 402; for analytical purposes: Merck TLC aluminium sheets silica gel 60 F2S4 (layer thickness 0.2 mm) were used. Silica gel Merck 60 (0.040-0.063 mm) is used for column chromatography.
2.2. Extraction and Isolation method
The Heantos plants were collected and scientific
names determined. The plant materials were dried, ground and then exhaustively extracted with EtOH:H20 (90:10) at room temperature. The organic solvent was evaporated under vacuum and aq. soln. was partitioned with /i-hexane, EtOAc and n-BuOH, successively giving n-hexane; EtOAc- and n-BuOH-extracts.
Each obtained extract was purified over a silicagel column, washing gradient with appropriate solvent systems to give fractions. Rechromatography of these fractions on different chromatographic methods such as normal, flash silicagel, sephadex LH-20. reversed phase RP-18 columns eluting with convenient solvents to yield pure compounds. In some cases preparative thin layer chromatography or recrystallize methods have been used.
3. RESULTS AND DISCUSSION
The obtained compounds from Heantos plants contain homoisoflavonoids (1-13), flavonoids (14, 15), flavonoid glycosides (16-23), a chalcone glucoside (24) and phenolic compounds (25-58).
Among phenolic compounds, five are phenylethyl alcohol glycosides (54-58). Five of thirteen isolated homoisoflavonoids are new compounds. The structures of new compounds were determined by spectroscopic analysis as 5,7-dihydroxy-8-methoxy- 6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)- chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6- methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-
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one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy- 3'-methoxybenzyl)chroman-4-one (3), 2,5.7- trihydroxy-6,8-dimethyl-3-(3',4'-methylene- dioxy- ben!yl)chroman-4-one (4) and 2,5,7-frihydroxy-6,8- dimethyl-3-(4'-methoxybenzyl) chroman-4-one (5) [3]. The isolated homoisoflavonoids belong to the 3- benzyI-4-chromanone class of homoisoflavonoid.
This class consists of a sixteen carbon skeleton with a chromanone moiety, which attached a benzyl group at the 3-position. This is the commonest class of homoisoflavonoid. The 'H-, '^C-NMR and COSY spectra of compounds 1-3 revealed the presence of a homoisoflavonoid skeleton substituted with one chelated hydroxy group, one or two aromatic methyl groups at A ring as well as hydroxy and methoxy groups at A and B rings. The location of the functional groups and the assignment of the NMR signals were achieved by analysis of the direct (HMQC) and long-range (HMBC) 'H-'^C- conelations as well as NOESY spectrum. The ' H - ISMR spectrum of compound 4 showed two sets of signals with mostly identical coupling patterns but with different chemical shifts, supporting the assumption, that compound 4 existed as a mixture of two isomers in the ratio of about 1:0.87. The EIMS spectrum of 4 indicated a molecular ion peak at Jtdz 358 and a base peak at m/z 135 due to B-ring moiety and two fragment peaks due to A-ring at 181 and 223. High-resolution mass specfrometry indicated the elemental composition of 4 to be C^HigO? The comparison of ' H - N M R spectra of compounds 1 and 4 suggested that 4 also had a homoisoflavonoidal skeleton. But, instead of the oxy-methylene proton signals in compound 1, one hydroxylated methin signal at ^5.47 appeared in the
Tran Van Sung, et al.
' H - N M R spectrum of 4. Based on the fragmentation in MS spectrum and the proton signals it was concluded that the molecule of 4 has a hydroxyl group connected to C-2, resulting in a hemiacetal function. This function is the reason that the compound formed two isomers. In addition, this result was supported by the ' H - ' H - C O S Y spectrum, which showed the correlations for H-3/H-2, H- 1 l/H-3 and H-l l/H-2. The HMBC specfrum showed the couplings between both protons H-ll and C-2, C-3, C-4 as well as C-1', C-2', C-6'; between the protons of the 6-Me group and C-5, C-7, C-10;
between the protons of the 8-Me group and C-7, C- 9; between the protons of methylenedioxy group and C-3', C-4'. Consequently, the structure of 4 was determined as 2,5.7-trihydroxy-6,8-dimethyl-3- (3',4'-methylene-dioxybenzyI)chronian-4-one.
The EIMS specfra of compound 5 showed a molecular ion peak at m/z 344 [M]* and base peak at m/z 121. The elemental composition of 5 was C19H20O6 according to the high-resolution mass spectrometry. From the ' H - N M R spectra it was concluded to be a mixture of two isomers in a ratio of about 1:0.79. The ' H - N M R spectra of 4 and 5 were very similar. The signals for the nng A corresponded to each other, but the ring B was a para disubstitued benzene ring (<56.86 and 7.\8,J-
8.8 Hz) with a methoxy substituent {S 3.80 / 3.79) in 5. Thus, 5 was supported to be 2,5,7- tnhydroxy- 6,8-dimethyl -3 -(4'-methoxybenzyl)chroman-4-one.
This was confirmed by the analysis of the COSY, HMQC and HMBC experiments. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2 [3].
4 R, + R2 = -O-CH2-0 5 Ri=H,R2 = 0Me
Figure 1: Five new homoisoflavonoids
Homoisoflavonoids, a special class of flavonoids, are mainly distributed in the Liliaceae family. Up to 2008, about 110 homoisoflavonoids were isolated from natural materials, which showed many bioactivities like anti-oxidation, anti- mflammatory, esfrogenicy, antiestrogenic.
anticancer and angioprotective,...[4]. Some studies on their biological activities were described as below. A new homoisoflavonoid, named cambodianol isolated from the stems of Dracaena cambodiana exhibited significant cytotoxic activities against K562 and SGC-7901 cell line with the IC50
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values of 1.4 and 2.9 ug/ml, respectively [5]. Four naturally occurring homoisoflavonoids and eight analogs have been synthesized and their antioxidant activity determined by superoxide (NBT) and DPPH free radical scavenging methods. The study showed that 7-hydroxy-3-[(3,4,5-tri hydroxy phenyl) methyiene]chroman-4-one displayed excellent activity followed by sappanone A in both methods and were many times more potent than the commercial antioxidants like BHA, BHT, etc. These compounds were evaluated in vitro for their inhibitory activities against 5-lipoxygenase (5-LOX) enzyme [6]. Compound 7-hydroxy-3-[(N,N- dimethylaminophenyl)methylene] chroman-4-one was found to possess potent inhibitory activity and
Phytochemical study on the plants..., parti was comparable to that of the standard, nordihydroguiaretic acid. The studied results suggest that these homoisoflavonoids, with their potent antioxidant and 5-LOX inhibitory activities, may have useful applications as antioxidants and lead compounds for asthma and inflammatory diseases [6]. T. M. Hung et al reported the anti-inflammatory activities of three new homoisoflavonoids isolated from the roots of Ophiopogon japonicus (Liliaceae) investigating by their effects on the release of the inflammatory chemokine eotaxin, stimulated by IL-4 and the combination of IL-4 and TNF-alpha in BEAS-2B cells, which mimics the in vivo conditions in bronchial allergic asthma [7].
Table 1: The homoisoflavonoids. flavonoids and phenolic compounds from Heantos 4 plants Nr 1 Compounds
llitmoisoflavonoid 1
2 3 4 5 6 7 8 9 10
n
12 135,7-dihydroxy-8-methoxy-6-melhy!-3-{2'- hvdroxy-4'-inethoxybenzyl)chroman-4-one 7-hydroxy-5,8-dimethDxy-6-methyl-3-(2'- hydroxy-4'-methoxybenzyl)chroman-4-one 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'- methoxybenzyI)cliroman-4-one 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'- methylenedioxybenzyl)chroinaii.4-one 2,5,7-trihydroxy-6,8-dimethyl-3-(4'- methoxybenzyl)chroman-4-one
5,7-trihydroxy-6,8-dimethyl-3-(2'-hydroxy-3',4'- methylenedioxybenzyl)chromone Methylophiopogonone A Ophiopogonanone A Methylophiopogonanone B Methylophiopogonanone A 6-aldehydoisoophiopoganone A 5-hydroxy-7,8-dimethoxy-6-methyl-3-{3',4'- dihydroxyb€nzyl)chronian-4-one 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydrDxy-3'- methoxybenzyl)chroman.4K)ne Flavonoid, flavonoid glycoside 14
15 16 17 18 19 20 21 22 23 24
5,7-dimethoxy-3',4'-methylendioxy-flavan-3-ol 4-hydroxy-5,7,3'-trimethoxy-flavan-3-ol
!sovitexin-2"-0-;^D-glucopyranoside Spinosin
Spinosin A Spinosin B Spinosin C Ononin Rutin Liquiritin Isoliquiritin Phenolic compounds 25
26 27 28
Paradol Vanillic acid
6-Gingerol » Gingerdione
Nr 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
Compounds Dehydro-gingerdione Shogaol 6-GingerdioI glucoside Hexahydrocurcumin
1,5-epoxy-3-hydroxy-1 -(3,4-dihydroxy-5- methoxy-phenyI)-7-(4-hydroxy-3-methoxy- phenyl)- heptane
Methylisoeugenol Coumarin Cinnamaldehyde Cinnamic acid 2-hydroxy-c innamaldehyde 2-methoxy-c innamaldehyde 3-<2-hydroxyphenyl)-propanol 3-Phenyl-2-propen-1 -ol 3-Phenyl-2-propan-I -ol Peru lic aldehyde Syringaldehyde Syringin Vanillin
4,4'-Dihydroxy-3,3'-dimethoxy-stilben Coniferylsulfat
Ferulic acid glucoside Coniferylferulat Decursidat Ferulic acid trimer
2-hydroxy-3-methoxy-benzoic acid glucosyl ester Acteoside
Isoacteoside Martynoside Leucosceptoside A JionosideBl
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Five of isolated flavonoid glycosides ( 1 7 - 2 1 ) are flavone-C-glycosides, w h i c h w e r e isolated previously from the s e e d s o f Ziziphus jujuba Mill var. spinosa Hu [8]. It is reported that, spinosin (17) and its derivatives possess significant sedative activity [9].
Liquiritin and isoliquiritin, t h e main constituents of Glycyrrhiza uralensis, significantly reduced at doses of 10, 20 and 4 0 m g / k g t h e immobility time in the FST (Forced S w i m m i n g T e s t ) a n d T S T (Tail Suspension Test) in m i c e 3 0 m i n after treatment.
They increased the c o n c e n t r a t i o n s o f the main neurotransmitters 5-HT and N E in the h i p p o c a m p u s , hypothalamus and cortex. B o t h c o m p o u n d s also reduced the ratio of 5 - H I A A / 5 - H T in the hippocampus, h y p o t h a l a m u s and cortex, s l o w i n g down 5-HT metabolism c o m p a r e d with mice treated with vehicle+stress. Therefore, they produced significant antidepressant-like effects, and their mechanism of action may b e d u e to increased 5-HT and NE in the mouse h i p p o c a m p u s , h y p o t h a l a m u s and cortex [10].
Phenolic c o m p o u n d s are bioactive substances widely distributed in t h e vegetable k i n g d o m . They act as natural antioxidants and contribute to the color, flavor and aroma of food. T h i s group is composed of one or m o r e aromatic b e n z e n e rings with one or more hydroxyl groups and their redox properties are related with their chemical structure characteristics [ I I ] . C o u m a r i n s and phenylethyl alcohol glycosides are of the most active representatives from t h e isolated phenolic compounds of Heantos plants.
Coumarins have s h o w n m a n y biological activities, include antioxidant, anti-tumor, antimicrobial, antiviral, vasorelaxant, anti- inflammatory and enzymatic inhibitors. Some coumarins are n o w commercially available as medicines [ I I ] .
Acteoside, isoacteoside, martynoside, leucosceptoside A and j ionoside B1 are phenylpropanoid glycosides extracted from the
\e&ves of Rehmannia glutinosa. F o u r o f them display various biological activities. N a m e l y , acteoside and martynoside exhibited anticancer, cytotoxic and antimetastatic activities. T h e i r estrogenic/
antiestrogenic effects w e r e assessed in breast cancer cells (MCF7), endometrial c a n c e r cells (Ishikawa) and osteoblasts ( K S 4 8 3 ) , by m e a s u r i n g I G F B P 3 levels, cell viability and n u m b e r of mineralized nodules, respectively. Both c o m p o u n d s antagonized ERalpha and ERbeta (p < 0.001), w h e r e a s they reversed the effect of E ( 2 ) mainly via E R a l p h a (p<0.001). Martynoside w a s a potent antiestrogen in MCF-7 cells. In osteoblasts, m a r t y n o s i d e induced nodule mineralization. A c t e o s i d e w a s an antiestrogen in breast c a n c e r cells and osteoblasts.
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without a n y effect on endometrial cells [12]. Besides these, leucosceptoside A also display significant antioxidant activity in the D P P H assay [13].
A c k n o w l e d g e m e n t s : We thank the Bundesministerium fuer Bildung und Forschung (BMBF), Bonn. Germany for financial support. Dr.
A. Porzel and Dr. J. Schmidt, Institute for Plant Biochemistry Halle/S., Germany for their discussion on NMR and MS-spectra.
R E F E R E N C E S
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2. Nguyen Thi Hoang Anh, Trinh Thi Thuy, Tran Van Sung, Tran Khuong Dan, Nguyen Ba Chinh, Nguyen Quang, Katrin Franke, Norbert Arnold and Ludger Wessjohann. Phytochemical study on the plants of the antidrug medication Heantos 4. Part II Terpenoid- and steroid compounds, Vietnam Journal ofChemistry, 51(2) (2013).
3. Nguyen Thi Hoang Anh, Tran Van Sung, Andrea Porzel, Katrin Franke, Ludger A. Wessjohann.
Homoisoflavonoids from Ophiopogon japonicus Ker- Gawler, Phytochemistry, 62, 1153-1158 (2003).
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5. J, Liu, W. L. Mei, J. Wu, M. Peng, H. F. Dai, A new cytotoxic homoisoflavonoid from Dracaena cambodiana. Journal of Asian Natural Products Research, 11(2), 192-195 (2009).
6. V Siddaiah, C. V. Rao, S. Venkateswariu, A. V.
Krishnaraju, G. V. Subbaraju. Synthesis, stereochemical assignments and biological activities of homoisoflavonoids, Bioorganic & Medicinal Chemistry, 14(8), 2545-2551 (2006).
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Corresponding author: Tran Van Sung
Institute ofChemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
Tel. 0084^-37564794; Fax. 0084^-8361283 E-mail; [email protected].
