DOI 10.

1007/s10600-014-1136-0
Chemistry of Natural Compounds, Vol. 50, No. 5, November, 2014 [Russian original No. 5, SeptemberOctober, 2014]

ISOLATION OF ALKALOIDS FROM Kursi caper BY COUNTERCURRENT

CHROMATOGRAPHY WITH pH-DEPENDENT DISTRIBUTION

Z. Talat, Y. Ali, and H. A. Aisa*

Kursi caper consists of extracts from four plants and is used in Uighur traditional medicine to treat rheumatism,
arthritis, and gout. Our previous studies proved that Kursi caper is effective against arthritis [1]. Some of its active constituents
are the total alkaloids [2]. The currently used methods for separating the alkaloids are inconvenient and ineffective. In
continuation of this research, we isolated from Kursi caper the three alkaloids harmine (1), harmaline (2), and J-harmine (3) by
countercurrent chromatography with pH-dependent distribution:
Plant, g

Principal active constituents

Capparis spinosa L., 200

Peganum harmala L., 100
Fomes officinalis (Vill. ex. Fr.), 100
Operculina turpethum, 100

Isocodonocarpin, glucocapperin [3]

Harmine, harmaline [4]
Officinalic acid [5]
Operculinoside A [6].

Countercurrent chromatography with pH-dependent distribution is a special method for isolating ionized compounds
[7]. Herein we communicate an effective separation by this method of harmine and J-harmine together with the E-carboline
alkaloid harmaline and their structures.
Harmine and J-harmine are known to be analogs of E- and J-carboline alkaloids. Several E-carbolines possess strong
and varied CNS and antitumor activity whereas J-carbolines were studied as antitumor agents [8]. The synthesis of E- and
J-carbolines is interesting because of their biological and pharmaceutical significance [9].
Countercurrent chromatography with pH-dependent distribution requires finding a suitable distribution coefficient
(Kacid and Kbase). The samples must be very soluble in the solvent system [10]. The optimum solvent system turned out to be
methyl-tert-butyl ether (MtBE)MeCNH2O (2:2:3), which was used before [11]. The sample preparation was reported before [12].
Compounds 1 (304 mg), 2 (41 mg), and 3 (197 mg) of t95% purity were isolated from the total extract (1 g).
The compounds were identified by comparing their spectral data (UV, NMR, mass) with those in the literature.
Countercurrent chromatography with pH-dependent distribution used a column consisting of a multi-layer coil
(100 m, 1.8 mm ID, Model TBE-300A, Shanghai Tauto Biotech, Shanghai, PRC) and total volume 290 mL. The value
(E = r/R, where r is the rotation radius or distance from the coil to the axle holder and R, the circular rotation radius or distance
between the holder axis and centrifuge center axis) varied from 0.57 (inner) to 0.76 (outer). The solvent was pumped onto
the column at a constant flow rate by a TBE-300A pump (Shanghai Tauto Biotech). The sample was manually injected onto
the column using a 20-mL syringe. The pH values of the fractions were measured using an RHS-3C pH-meter (Shanghai
HongYi Instruments, Shanghai, PRC). The purity of the alkaloids was checked using an HPLC (Waters, USA). NMR spectra
were recorded in DMSO-d6 on a VNMRS-600 spectrometer (600 MHz for 1H) (Varian, USA). ESI-MS analysis was performed
on a QSTAR Elite System (AB-Sciex, USA). HPLC grade solvents were used for countercurrent chromatography and HPLC.
Crude extract was produced by wetting raw material with distilled H2O in a 1:10 ratio for 1 h, after which the mixture
was refluxed twice at 100C for 1.5 h. The resulting solution was decanted and precipitated with 50% EtOH. The liquid was
separated. The solvent was evaporated. The resulting extract was dried in vacuo to a dry powder. Dry extract (50 g) was
dissolved in HCl (300 mL, 2%). The extracts were combined and filtered. The filtrate was made basic with NH4OH to pH 9.5
and precipitated for 24 h. The liquid part and solvents were evaporated. The resulting extract was dried in vacuo to a powder
(2.4 g) of total alkaloids.
The Key Laboratory of Plant Resources and Chemistry of Arid Zone, State Key Laboratory Basis of Xinjiang Indigenous
Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
830011, Urumqi, P. R. China, e-mail: haji@ms.xjb.ac.cn. Translated from Khimiya Prirodnykh Soedinenii, No. 5, September
October, 2014, pp. 835836. Original article submitted March 14, 2014.
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0009-3130/14/5005-0968

2014

Springer Science+Business Media New York

A biphasic solvent system and sample solution were prepared using the biphasic solvent system MtBEMeCNH2O
(2:2:3). The two phases were separated before use. The upper organic phase was made basic with TEA (10 mM) and used as
the stationary phase. The lower aqueous phase was acidified by adding HCl (5 mM) and used as the mobile phase.
Samples were separated by injecting onto a column packed with the organic phase. The aqueous phase was pumped
onto the column at flow rate 2 mL/min in head-to-tail mode. The column was rotated at 850 rpm. The effluent was continuously
monitored using a UV detector at 254 nm. Fractions were collected every 5 min. The pH values of the fractions were
measured manually. After the separation, the column contents were collected in a graduated cylinder using compressed N2.
The retention of the stationary phase to the total column capacity was calculated. Fractions were evaporated to dryness and
analyzed by HPLC.
Fractions were analyzed by countercurrent chromatography with pH-dependent distribution on an HPLC over Waters
Sunfire C18 columns (250 mm u 4.6 mm ID) at 330 nm and 30C. The mobile phase was MeCNNH4OAc (20:80) buffer at
flow rate 1 mL/min.
Harmine (1) [13]. Yellow crystals, mp 251253q. UV spectrum (MeOH, Omax, nm): 240, 338. (+) ESI-MS, m/z 213
+
>M + H] . 1H NMR spectrum (400 MHz, DMSO-d6, G, ppm, J/Hz): 11.43 (1H, s, H-1), 8.16 (1H, d, J = 5.6, H-5), 8.03 (1H, d,
J = 9.2, H-9), 7.78 (1H, d, J = 4.8, H-6), 7.03 (1H, d, J = 1.6, H-12), 6.84 (1H, dd, J = 2.0, 8.0, H-10), 3.87 (3H, s, CH3O), 2.74
(3H, s, CH3). 13C NMR spectrum (100 MHz, DMSO-d6, G, ppm): 160.1 (C-11), 141.9 (C-3), 141.3 (C-2), 137.8 (C-5), 134.6
(C-13), 127.2 (C-8), 122.6 (C-9), 114.9 (C-7), 111.9 (C-6), 109.0 (C-10), 94.6 (C-12), 55.3 (CH3O), 20.3 (CH3).
Harmaline (2) [14]. Yellow crystals, mp 226228q. UV spectrum (MeOH, Omax, nm): 224, 338. (+) ESI-MS,
m/z 215 >M + H]+. 1H NMR spectrum (600 MHz, DMSO-d6, G, ppm, J/Hz): 11.16 (1H, s), 7.42 (1H, d, J = 8.4), 6.85 (1H, d,
J = 1.8), 6.70 (1H, dd, J = 2.4, 6.0), 3.78 (3H, s), 3.64 (2H, dd, J = 9.0, 7.8), 2.68 (2H, dd, J = 9.0, 7.8), 2.24 (3H, s).
13C NMR spectrum (150 MHz, DMSO-d , G, ppm): 157.1, 156.7, 137.5, 128.5, 120.3, 119.4, 114.5, 110.2, 94.6, 55.1, 47.6,
6
22.0, 19.1.
J-Harmine (3) [13]. Yellow crystals, mp 238240q. UV spectrum (MeOH, Omax, nm): 240, 323. (+) ESI-MS,
m/z 213 >M + H]+. 1H NMR spectrum (600 MHz, DMSO-d6, G, ppm, J/Hz): 12.70 (1H, s, H-1), 8.40 (1H, d, J = 6.0, H-3), 8.33
(1H, d, J = 6.6, H-4), 8.31 (1H, d, J = 9.0, H-9), 7.13 (1H, d, J = 1.2, H-12), 7.02 (1H, d, J = 9.0, H-10), 3.93 (3H, s, CH3O),
3.02 (3H, s, CH3). 13C NMR spectrum (150 MHz, DMSO-d6, G, ppm): 162.4 (C-11), 145.1 (C-13), 137.6 (C-6), 133.8 (C-7),
131.6 (C-2), 129.4 (C-4), 124.4 (C-9), 113.8 (C-3), 113.7 (C-8), 112.1 (C-10), 94.3 (C-12), 55.7 (CH3O), 16.2 (CH3).
ACKNOWLEDGMENT
The work was supported by the Foundation for International Collaboration and Exchange of the Chinese National
Foundation for Natural Sciences (No. 31110103908) and the Foundation for International Scientific-Technical Collaboration
of Xinjiang-Uighur Autonomous Region (No. 20126023).

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