Chia-Chuan Chang, Pennaka Hari Kishore, Shoei-Sheng Lee*

Transcription

1 Chemical investigation of Phyllanthus maderaspatensis by hyphenation of liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy Chia-Chuan Chang, Pennaka Hari Kishore, Shoei-Sheng Lee* School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan, R..C Abstract Phyllanthus maderaspatensis, a traditional Indian herb (Euphorbiaceae), was investigated by liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR). Twelve compounds were characterized and identified from n-buh and CHCl 3 soluble s. EtH extract of P. maderaspatensis was triturated with H 2, n-buh, n-hexane and CHCl 3. The n-buh and CHCl 3 soluble s were roughly purified by centrifugal partition chromatography and silica gel column chromatography, respectively. Two flavonoids, rutin (1) and quercitrin-3--rhamnoside (2), one phenolic compound, gallic acid methyl ester 4--dimer (3), were isolated and from n-buh soluble subs, and nine cinnamoyl sucrose acetates (4-12) were from CHCl 3 soluble subs. Elucidation of their structures was based on 1D, 2D NMR and MS spectral analysis. This work was supported by the National Science Council, Taiwan, R..C. under the grant NSC B Key Words Phyllanthus maderaspatensis, solid phase extraction, LC-NMR 1. Introduction LC-SPE-NMR, the technique that applying SPE as a sample concentrating and manipulating interface of LC-NMR, has been developed in recent years. 1-3 Chromatographic separation can be done with cheap non-deuterated solvents or and even with additives which are not compatible with NMR spectroscopy. Aqueous eluents are removed by airflow without tedious concentration processes. Cartridges containing the adsorbed compounds are flushed by the deuterium solvents and pumped into the NMR flow probe for data acquisition. The deuterated solvent, which is used for the elution and transfer, is independent of the chromatographic conditions and can be selected to improve spectral quality and make exchangeable protons observable in NMR spectra. Several experiment results revealed that LC-SPE-NMR is a super powerful tool for natural product chemistry. Phyllanthus maderaspatensis (Euphorbiaceae), is an annual shrub distributed in south Guangdong (China), Africa, Australia, India, Indonesia and Sri Lanka. The alcoholic extract was reported to demonstrate preventive and suppressive effects on duck hepatitis B virus. 4-5 An infusion of the leaves is

2 used in headache, and the seeds possess laxative, carminative and diuretic properties. The crude EtH extract of P. maderaspatensis was triturated by with H 2, n-buh, n-hexane and CHCl 3 to give corresponding soluble s. After repeat column chromatographies on the CHCl 3 soluble, niruriside (4), 6 a cinnamoyl sucrose acetate, and several s of cinnamoyl sucrose acetates analogues were identified. This paper reported efficient use of LC-SPE-NMR on both n-buh and CHCl 3 soluble s for investigation of chemical constituents. 2. Experimental The LC system consisted of an Agilent 1100 pump (Waldbronn, Germany), an Agilent manual injector valve, a Bruker photo diarray detector (Bruker, Rheinstetten, Germany) and a Bruker BSFU connected to a Spark Prospekt II (Spark, AJ Emmen, The Netherlands). The SPE cartridge trays contain Hysphere C18 HD and Resin GP high-pressure SPE cartridges in a 96-well format purchased from Spark. A Merck LiChrocart and a Purospher STAR RP-18e (5µm) HPLC column ( mm, Merck, Darmstadt, Germany) was used with H 2 /CH 3 CN as eluents. Flow rate of auxiliary H 2 is 1.0 ml/min. Each peak was trapped under manual control, and the drying time for each cartridge is 120 min. The control unit consists of a computer with Windows 2000 operation system for use with Hhyster 2.3 software for hyphenated experiments. 3. Results and Discussions To the alcoholic extract P. maderaspatensis (1.2 kg) was added H 2 (1 L) and stirred continuously for 1 h, centrifugation. The residue was subjected to the same process for four times to obtain H 2 soluble and the residue. H 2 soluble was partitioned with n-buh (2 L 4) to obtain the n-buh soluble (169 g) and H 2 soluble (172 g). To the H 2 insoluble residue was added hexane (1 L) and stirred continuously for 1 h. The residue was subjected to the same process for four times and centrifugation to obtain n-hexane soluble (99 g) and the residue (980 g). To the n-hexane insoluble was added CHCl 3 (1 L) and stirred continuously for 1 hour, filtered. The residue was subjected to the same process for four times to give the CHCl 3 soluble (26 g) and residue (830 g) (Scheme 1). The n-buh soluble (0.4 g) was subjected to centrifugal partition chromatography (CPC) [Solvent system: CHCl 3 /MeH/n-BuH/H 2 (10/10/1/6); mobile phase: lower layer; stationary phase: upper layer; flow rate: 1.0 ml/min; rotational speed: 1,000 rpm; pressure: 4.4 mpa] to give six subs (A-F). The six s were further separated and analyzed by LC-SPE-NMR (Column: Merck LiChrospher 100 RP-18e 5µm HPLC column; detection wavelength: 265 nm; flow rate: 0.5 ml/min; mobile phase: 10/90 (MeCN/H 2 ); gradient: 90/10 (MeCN/H 2 ) at 15 min; injection volume 20

3 µl; SPE cartridges: Hysphere Resin GP; collection time: 0.3 min) to obtain 2 flavonoids and 1 phenolic compound. rutin (1) from sub B. Analysis of sub D by LC-SPE-NMR obtained quercitrin-3--rhamnoside (2), and gallic acid methyl ester 4--dimer (3) from sub E (Scheme 2). The CHCl 3 soluble (10 g) was separated by a silica gel column ( mesh, 5-80 % (acetone/toluene) to give subs 1-7. Sub 4 was subjected to a silica gel column ( mesh, 20 % acetone/toluene) to give niruriside (5.2 g). Sub 6 was subjected to a silica gel column ( mesh, 10 % acetone/toluene) to obtain subs 1-3 (645, 169 and 51 mg, respectively). Tube (22 mg) of sub 7 was separated and analyzed by LC-SPE-NMR [LC condition: Merck LiChrospher RP-18e (5µm) tube column; detection wavelength: 285 nm; flow rate: 1.0 ml/min; mobile phase: MeCN/H 2 (35/65); gradient: MeCN/H 2 (55/45) at 65 min; sample preparation: 22 mg in 1 ml MeH (22.0 mg/ml); injection volume: 20 µl (440 µg); SPE condition: C18 HD (7 µm), collection time 0.3 min] to obtain 5 cinnamoyl sucrose acetates (4-8). Tube (193 mg) of sub 7 was separated and characterized by LC-SPE-NMR [Column: Merck LiChrospher RP-18e (5µm) HPLC column; detection wavelength: 285 nm; flow rate: 0.2 ml/min; mobile phase: MeCN/H 2 (58/42); gradient: MeCN/H 2 (90/10) at 40 min; injection volume: 20 µl; SPE cartridges: Hysphere C18 HD (7 µm) and Resin GP (10-12 µm)] to obtain 4 cinnamoyl sucrose acetates (9-12)(Table 1). 2D heteronuclear experiments (HMBC and HSQC) were performed by automation measurement for compounds 1-3. The structures of the eight cinnamoyl sucrose acetates (5-12) were elucidated mainly by comparing their 1 H NMR spectra (in CD 3 CN) with that of niruriside (4), a major cinnamoyl sucrose acetate compound in the plant. 4. Conclusions The implementation of SPE between LC and NMR demonstrates high efficiency and versatile analytical application for rapid chemical screening of natural products, as shown here for a class of closely related sugar derivatives. With in a few days of work, an overview of the composition in parts of extract was obtained, starting from the crude alcoholic extract while only with the initial partition works and rapid pretreatments were needed. Furthermore, the small scale of isolation work speed up the whole screening progress and largely facilitates the identification of unknown compounds of natural products. Relative quantitative data obtained by LC-SPE-NMR also provide representative information on the compositions of an unknown sample. References 1. Johnson, S.; Morgan, E. D.; Wilson, I. D.; Spraul, M.;Hofmann, M. J. Chem. Soc., Perkin Trans., 1994,

4 1, Albert, K.; Schlotterbeck, G.; Braumann, U.; Handel, H.; Spraul, M.; Krack, G. Angew. Chem. Int. Ed. Engl. 1995, 34, Corcoran,.; Wilkinson, P. S.; Godejohann, M.; Braumann, U.; Hofmann, M.; Spraul, M. Am. Lab. 2002, 34, Munshi, A.; Mehrotra, R.; Panda, S. K. J. Med. Virology 1993, 40, Munshi, A.; Mehrotra, R.; Ramesh R.; Panda, S. K. J. Med. Virology 1993, 41, Qian.-Cutrone, J.; Huang, S.; Trimble, J.; Li, H.; Lin, P.-F.; Alam, M.; Klohr, S. E.; Kadow, K. F. J. Nat. Prod. 1996, 59,

5 Table 1. Structures of cinnamoyl sucrose acetate Compound R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 4 Ac C 6 H 5 (CH) 2 C H C 6 H 5 (CH) 2 C Ac H Ac Ac R 8 6' CH 2 5 Ac 3,4-DHC H 4-HC H H Ac 3,4-DHC 6 H 3,4-DHC H 4-HC Ac H Ac Ac 5' 4' R 6 3' R 7 1' 2' 2 R R CH 2 R 1 H 2C R R 3 7 Ac 3,4-DHC H 3-HC Ac H Ac Ac 8 Ac C 6 H 5 (CH) 2 C H C 6 H 5 (CH) 2 C H H Ac 3,4-DHC 9 Ac C 6 H 5 (CH) 2 C H H Ac H Ac Ac 10 H 3-HC H 3-HC Ac H Ac Ac 11 Ac 3-HC H 3-HC Ac H Ac Ac 12 H 3,4,5-THC H 3,4,5-THC H Ac H Ac 3,4-DHC = 3,4-dihydroxycinnamoyl (caffeyl); 4-HC = 4-hydroxycinnamoyl (p-coumaryl); 3-HC = 3-hydroxycinnamoyl (m-coumaryl); 3,4,5-THC = 3,4,5-trihydroxycinnamoyl

6 MeH extract of Phyllanthus maderaspatensis (1200 g) 1. H 2 4L 2. stir 4 h 3. centrifugation n-buh soluble (169 g) H 2 soluble n-buh 2L x 4 H 2 soluble (172 g) Hexane insoluble (980 g) H 2 insoluble residue (1080 g) 1. Hexane 4L 2. stir 4 h 3. centrifugation Hexane soluble (99 g) Scheme 1. Partitionation scheme of Phyllanthus maderaspatensis

7 P. maderaspatensis BuH extract 0.4 g CPC Fr. A 86 mg Fr. B 44 mg Fr. C 18 mg Fr. D 6 mg Fr. E 8 mg Fr. F LC-SPE-NMR 0.5 ml/min ACN:H 2 (1:9 to 9:1) LC-SPE-NMR 0.5 ml/min ACN:H 2 (1:9 to 9:1) LC-SPE-NMR 0.5 ml/min ACN:H 2 (1:9 to 9:1) H H CMe H H H H H 1 -Glu-Rha H 2 -Rha H H H H CMe 3 Scheme 2. Separation of n-buh soluble of P. maderaspatensis by LC-SPE-NMR