Two bioactive flavonol glucosides, hyperoside and quercitrin, were successfully isolated in one step from the phytochemically unknown medicinal plant Osteomeles schwerinae by high-speed counter-current chromatography using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (0.5:5.5:1.5:4.5 v/v). From 160 mg of crude extract, 2.1 mg of hyperoside (98.6% purity, 83.3% recovery) and 4.5 mg of quercitrin (99.2% purity, 81.7% recovery) were separated and then their chemical structures were identified by (1)H and (13)C NMR analysis. In addition, the isolates were subjected to an in vitro bioassay to evaluate their inhibitory activity on rat lens aldose reductase. Both quercitrin and hyperoside showed excellent inhibitory activities toward rat lens aldose reductase with the IC(50) values of 0.16 and 4.33 muM, respectively, as compared with positive control, 3,3-tetramethyleneglutaric acid (28.7 muM). So far, chemical constituents and biological activities of O. schwerinae have never been reported. This is the first report on the chemical constituents and biological activity of this plant using semi-preparative high-speed counter-current chromatography separation technique.

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of flavonoid glycosides from the leaves of Nelumbo nucifera (Lotus) by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (HPLC). A total of 4.6 mg of isoquercitrin, 9.1 mg of hyperoside and 3.0 mg of astragalin with the purity of 95.8%, 97.5% and 98.3%, respectively, were obtained in one-step separation and less than 6 h from 80 mg of crude extract from the leaves of N. nucifera. The chemical structures of all the three compounds were identified by MS,(1)H NMR,(13)C NMR. Astragalin was obtained from N. nucifera for the first time.

An ethanol extract of air-dried flowers of Edgeworthia chrysantha Lindl. was partitioned between water and petroleum, ethyl acetate, and n-butanol. The n-butanol extraction was initially purified by silica gel column chromatography to give a partially purified sample. The bioactive compound rutin, along with nicotiflorin, were successfully separated from the partially purified sample by high-speed counter-current chromatography. The two compounds were isolated from the plant of Edgeworthia genus for the first time. The two-phase solvent system used was composed of ethyl acetate-n-butanol-water at an optimized ratio of 4:1:5 (v/v/v). High-speed counter-current chromatography yielded, from 108 mg of the partially purified extract, 53 mg rutin and 32 mg nicotiflorin with 92.5% and 92.2% recovery, with each at over 96.5% purity by high-performance liquid chromatography analysis. Their structures were identified by 1H NMR and 13C NMR.

Separation techniques with high efficiency and sensitive detection have been widely used for quality control of traditional Chinese medicines (TCMs). High-performance liquid chromatography, gas chromatography, and capillary electrophoresis are commonly used to separate various components in TCMs. Ultraviolet detection, fluorescence detection, evaporative light-scattering detection, mass spectrometry and nuclear magnetic resonance can be applied to separation techniques for qualitative and quantitative analysis of TCMs. The development of quality control for TCMs based on quantitative and qualitative analysis from 2000 to 2007 are reviewed; the fingerprint technique is also discussed due to its broad application in the quality control of TCMs. Prospects for further research based on our primary results are also discussed.

Since the 1990s, interest in natural product research has increased considerably. Following several outstanding developments in the areas of separation methods, spectroscopic techniques, and sensitive bioassays, natural product research has gained new attention for providing novel chemical entities. This updated review deals with sample preparation and purification, recent extraction techniques used for natural product separation, liquid-solid and liquid-liquid isolation techniques, as well as multi-step chromatographic operations. It covers examples of papers published since the NPR review 'Modern separation methods' by Marston and Hostettmann,1 with major emphasis on methods developed and the research undertaken since 2000.

A two-dimensional counter-current chromatographic system (2D-CCC) for preparative isolation and purification of three prenylflavonoids from Artocarpus altilis is presented. An upright CCC instrument (CCC1, total capacity: 1600 ml) was used as the first dimension. Effluent of interest from CCC1 was collected on-line into a 30 ml sample loop by a laboratory-prepared column-switching interface and introduced into a high-speed CCC instrument (CCC2, total capacity: 210 ml) for the second dimension separation. With this 2D-CCC system and a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:7:3 and 5:5:6.5:3.5, v/v/v/v), which had been selected by high-speed CCC, about a 500 mg amount of the crude extract was separated, yielding 9 mg of compound 1, 28 mg of compound 2 and 78 mg of compound 3. The purities of the three prenylflavonoids were 98.7 (1), 98.3 (2) and 97.2%(3), respectively, as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization MS,(1)H NMR and (13)C NMR.

Hydrophilic antioxidant constituents in the fruits of the vegetable Luffa cylindrica (L.) Roem (sponge gourds) were separated by an antioxidant-guided assay to yield eight compounds: p-coumaric acid (1), 1-O-feruloyl-beta-D-glucose (2), 1-O-p-coumaroyl-beta-D-glucose (3), 1-O-caffeoyl-beta-D-glucose (4), 1-O-(4-hydroxybenzoyl)glucose (5), diosmetin-7-O-beta-D-glucuronide methyl ester (6), apigenin-7-O-beta-D-glucuronide methyl ester (7), and luteolin-7-O-beta-D-glucuronide methyl ester (8). The eight compounds were isolated by high-speed countercurrent chromatography and identified by electrospray ionization-mass spectrometry and NMR analysis, and the antioxidant activity was evaluated by the radical scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical. High-performance liquid chromatography analysis showed that a total amount of the eight compounds in the dried gourds without skin was about 1%. The results demonstrate that the consumption of sponge gourds can supply some antioxidant constituents to human body.

Counter-current chromatography is a very versatile separation technique which does not require a solid stationary phase. It relies simply on the partition of a sample between the two phases of an immiscible solvent system. Some of the more recent applications of the method to the separation of plant-derived natural products are described here. Crude plant extracts and semi-pure fractions can be chromatographed, with sample loads ranging from milligrams to grams. Aqueous and non-aqueous solvent systems are used and the separation of compounds with a wide range of polarities is possible. The technique is complementary to other chromatographic methods and is compatible with gradient systems. The possibilities for solvent selection are almost limitless but some guidelines for the choice of successful systems are presented.

Several polymethoxylated flavones including nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, tangeretin and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone were separated from Tangerine peel (Juhong in Chinese) by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.8:1:1, v/v). Then, 26 mg of nobiletin, 6 mg of 3,5,6,7,8,3',4'-heptamethoxyflavone, 35 mg of tangeretin and 11 mg of 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone could be obtained from 150 mg crude extracts and their purities were 98.6%, 95.9%, 99.8% and 96.8%, respectively. All these constituents were identified by EI-MS and 1H NMR.

Flavan-3-ol phloroglucinol adducts were synthesised through acid catalysed degradation of a procyanidins-rich grape seed extract in the presence of phloroglucinol. The reaction mixture (3.3 g) was fractionated without further sample preparation using the all-liquid chromatographic technique of high-speed counter-current chromatography (HSCCC). Selected solvent systems were hexane-ethyl acetate-methanol-water (0.1:5:0.1:5, v/v/v/v) and (1.5:10:1.5:10, v/v/v/v). The fractions obtained were found to contain almost pure compounds, in some cases final purification was achieved by preparative HPLC. The so-obtained pure standards of (+)catechin-(4alpha-->2)-phloroglucinol,(-)epicatechin-(4beta-->2)-phloroglucinol,(+)catechine,(-)epicatechin-3-O-galloyl-(4beta-->2)-phloroglucinol,(-)epicatechin, and (-)epicatechin gallate are required for quantification of acid-catalysed phloroglucinol degradation products of procyanidins.

A lignan, sisymbrifolin (1) found in the fruits of Solanum sisymbriflolium has been isolated from the bark extract of Salix alba (Salicaceae). Its structure was elucidated by its direct spectrum data of ESI-MS and one- and two-dimensional NMR spectroscopy for the first time.

Hydrophilic antioxidant constituents in the fruits of the vegetable Luffa cylindrica (L.) Roem (sponge gourds) were separated by an antioxidant-guided assay to yield eight compounds: p-coumaric acid (1), 1-O-feruloyl-beta-D-glucose (2), 1-O-p-coumaroyl-beta-D-glucose (3), 1-O-caffeoyl-beta-D-glucose (4), 1-O-(4-hydroxybenzoyl)glucose (5), diosmetin-7-O-beta-D-glucuronide methyl ester (6), apigenin-7-O-beta-D-glucuronide methyl ester (7), and luteolin-7-O-beta-D-glucuronide methyl ester (8). The eight compounds were isolated by high-speed countercurrent chromatography and identified by electrospray ionization-mass spectrometry and NMR analysis, and the antioxidant activity was evaluated by the radical scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical. High-performance liquid chromatography analysis showed that a total amount of the eight compounds in the dried gourds without skin was about 1%. The results demonstrate that the consumption of sponge gourds can supply some antioxidant constituents to human body.

High-speed counter-current chromatography (HSCCC) was used for the separation of anthocyanins on a preparative scale from bilberry fruit crude extract (Vaccinium myrtillus, Ericaceae) with a biphasic solvent system composed of methyl tert-butyl ether-n-butanol-acetonitrile-water-trifluoroacetic acid (1:4:1:5:0.01, v/v). Each injection of 500 mg crude extract yielded 130 mg of pure delphinidin-3-O-sambubioside, and 77 mg of pure cyanidin-3-O-sambubioside. The two anthocyanin disaccharides with a glucose (2 --> 1) xylose unit are novel compounds for bilberry fruit, and were elucidated by means of electrospray ionization MS-MS, 1H, 13C, distortion less enhancement by polarization transfer NMR, and two-dimensional HC-correlation experiments (heteronuclear multiple bond correlation and heteronuclear multiple quantum correlation).

The Chinese phytomedicinal formulation Sanqi Zongdai Pian, traditionally prepared from crude extracts from roots of Panax notoginseng (Araliaceae), contains highly polar dammarane saponins which were separated at a preparative scale using high-speed counter-current chromatography (HSCCC). In each operation, 283 mg methanolic extract of five tablets was separated and yielded pure 157, 17, 13 and 56 mg of ginsenoside-Rb1, notoginsenoside-R1, ginsenoside-Re and ginsenoside-Rg1, respectively, n-hexane-n-butanol-water (3:4:7, v/v/v) was used for the two-phase solvent system of the HSCCC separation. The chemical structures of three ginsenosides and one notoginsenoside were elaborated by means of electrospray ionization MS-MS and NMR analysis.

An activity-guided isolation of bioactive stilbenes has been carried out with the grapevine shoot extract Vineatrol® 30. After hexane precipitation of the polymeric constituents, the stilbene mixture was separated on a preparative scale using low speed rotary countercurrent chromatography (LSRCCC). The antiproliferative activity of the separated LSRCCC fractions was then screened in the human cancer cell line A-431 and trans-resveratrol, trans--viniferin, r-2 viniferin, hopeaphenol, and miyabenol C were identified as active principles. In addition, a new class of stilbene derivatives, which exhibit a -lactam ring system and exert a weak growth-inhibiting activity in A-431 cells, has been identified.

In this study, preparative ion-pair high-speed countercurrent chromatography was directly coupled to an electrospray ionization mass-spectrometry device (IP-HSCCC/ESI-MS-MS) for target-guided fractionation of high molecular weight acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra (Nyctaginaceae). The direct identification of six principal acyl-oligosaccharide linked betacyanins in the mass range between m/z 859 and m/z 1359 was achieved by positive ESI-MS ionization and gave access to the genuine pigment profile already during the proceeding of the preparative separation. Inclusively, all MS/MS-fragmentation data were provided during the chromatographic run for a complete analysis of substitution pattern. On-line purity evaluation of the recovered fractions is of high value in target-guided screening procedures and for immediate decisions about suitable fractions used for further structural analysis. The applied preparative hyphenation was shown to be a versatile screening method for on-line monitoring of countercurrent chromatographic separations of polar crude pigment extracts and also traced some minor concentrated compounds. For the separation of 760mg crude pigment extract the biphasic solvent system tert.-butylmethylether/n-butanol/acetonitrile/water 2:2:1:5 (v/v/v/v) was used with addition of ion-pair forming reagent trifluoroacetic acid. The preparative HSCCC-eluate had to be modified by post-column addition of a make-up solvent stream containing formic acid to reduce ion-suppression caused by trifluoroacetic acid and later significantly maximized response of ESI-MS/MS detection of target substances. A variable low-pressure split-unit guided a micro-eluate to the ESI-MS-interface for sensitive and direct on-line detection, and the major volume of the effluent stream was directed to the fraction collector for preparative sample recovery. The applied make-up solvent mixture significantly improved smoothness of the continuously measured IP-HSCCC-ESI-MS base peak ion trace in the experimental range of m/z 50-2200 by masking stationary phase bleeding and generating a stable single solvent phase for ESI-MS/MS detection. Immediate structural data were retrieved throughout the countercurrent chromatography run containing complete MS/MS-fragmentation pattern of the separated acyl-substituted betanidin oligoglycosides. Single ion monitoring indicated clearly the base-line separation of higher concentrated acylated betacyanin components.

The natural pigment composition of purple bracts of Bougainvillea glabra (Nyctaginaceae) consists of a highly complex mixture of betacyanins solely differing by the substitution with a variety of acyl-oligoglycoside units. This study was focused on a two-dimensional chromatography approach, a combination of preparative high-speed countercurrent chromatography (HSCCC) and analytical C18-HPLC with ESI-DAD-MS/MS detection which finally enabled a more detailed view into the pigment profile and elucidated the existence of an overwhelming amount of varying betacyanin structures occurring in Bougainvillea bracts. The detected molecular weights of the pigments reached so far unknown high values and ranged up to maximum values of 1653 and 1683 Da for the largest molecules due to oligosaccharide linkage and multiple acyl substitutions. The preparative IP-HSCCC separation yielded 15 complex fractions containing betacyanins of enhanced polarity as well as structures with highly increased lipophilicity. Betacyanin structures extended by large oligosaccharide chains with bigger number of glycoside units and also carrying a reduced number of hydroxycinnamic acid substitutions were characteristic for polar pigments occurring mainly in the early eluting CCC fractions. IP-HSCCC was proven to be extremely effective for fractionating this complex crude betalain pigment extract into more defined 'polarity-windows'. Structural analysis by analytical LC-ESI-MS/MS in the positive ionization mode detected a total sum of 146 different betacyanin pigments in the CCC fractions of reduced complexity.

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution-extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical 'head-to-tail' mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol-acetonitrile-aqueous TFA 0.7%(5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether-1-butanol-acetonitrile-aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K(D) ratios and selectivity factors alpha around 1.0-1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization-MS using also authentic reference materials.

Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid-liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid-liquid partitioning with ethyl acetate-methanol-water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether-n-butanol-acetonitrile-water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC-high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.

The first preparative fractionation of betalain pigments by means of ion-pair high-speed counter-current chromatography (IP-HSCCC) from berry extracts of Phytolacca americana (Phytolaccaceae) is presented. A novel HSCCC solvent system consisting of 1-butanol-acetonitrile-water (5:1:6, v/v/v) was applied using ion-pair forming trifluoroacetic acid at low concentration (0.7%, v/v). Affinity of polar betacyanins and betaxanthins to the organic stationary phase of the biphasic HSCCC solvent mixture was considerably improved. Partitioning coefficient values and influence of increasing trifluoroacetic acid additions to the biphasic solvent mixture were measured for all identified betacyanins and betaxanthins. Gentle separation by IP-HSCCC of the injected pigment extract (900 mg) yielded sufficient amounts of the principal pigments 15S-betanin/15R-isobetanin. The pure epimers separated by C18-HPLC were immediately studied by one- and two-dimensional NMR. In the recovered fractions, minor concentrated betacyanins and betaxanthins were significantly enriched by IP-HSCCC and were detected for the first time in the extracts of P. americana. IP-HSCCC and C18-HPLC were shown to be complementary techniques in the isolation procedure of recovering minor concentrated, highly polar and chemically instable betacyanins and betaxanthin from complex plant matrices. Altogether, identification of 17 betalains was achieved by HPLC-diode array detection-electrospray ionization MS/MS in the HSCCC fractions with their respective isomers, also resulting in the tentative elucidation of betacyanins with novel salicylic acid substitution pattern in the berry extracts of P. americana.

A new flavonoid, 6-(2-hydroxy-5-carboxyphenyl)-apigenin (1), together with two new natural products, 3-(4-hydroxyphenyl)-6,7-dihydroxy coumarin (2), 1-methoxy-3-methylanthraquinone (3) and four known compounds, were isolated from Selaginella tamariscina (BEAUV.) SPRING. The structures of the new isolated compounds were elucidated on the basis of 1D and 2D NMR as well as ESI-HR-MS spectroscopic analysis.

A new glycocerebroside (1), along with one reported one (2), was isolated from the ethanol extract of Sagina japonica (Caryophyllaceae) and was fully characterized. The structures of two compounds were identified as (2S, 3S, 4R, 8E)-1-(beta-D-glucopyranosyl-3, 4-dihydroxy-2-[(R)-2'- hydroxypalmitoyl]amino-8-heptadecaene (1) and (2S, 3R, 8E)-1-(beta-D-glucopyranosyl-3-hydroxy-2-[(R)-2'-hydroxypalmitoyl]amino-8-octadecaene (2) by using spectroscopic methods ((1)H,(13)C, and 2D NMR, MS) and chemical degradation.

Two new guaiane-type sesquiterpene glycosides, 11-O-acetyl-torilolone 8-O-beta-D-glucopyranoside (1) and 1beta-hydroxytorilolone 11-O-beta-D-glucopyranoside (2), were isolated from the fruits of Daucus carota L. Their chemical structures were elucidated on the basis of MS, NMR spectroscopic analyses coupled with chemical degradation.

The essential oil from leaves of Guarea guidonia was subjected to chromatographic separation procedures to afford nine sesquiterpenes; two of them are new eudesmane derivatives. The chemical structures of the obtained compounds were characterised by spectrometric analysis, mainly mass spectrometry and NMR.

A preparative high-speed counter-current chromatography method for separation and purification of liensinine, isoliensinine and neferine from seed embryo of Nelumbo nucifera GAERTN was successfully established by using n-hexane-ethyl acetate-methanol-water (5:8:4:5, v/v, containing 0.5% NH(4)OH) as the two-phase solvent system. From 200 mg of crude extract, 18.4 mg of liensinine, 19.6 mg of isoliensinine and 58.4 mg of neferine were obtained with the purity of 96.8, 95.9, and 98.6%, respectively. The identification of the three alkaloids was performed with (1)H NMR and (13)C NMR.

Two new C(13) nor-isoprene glycosides,(6S,9S)-6,9-dihydroxymegastiman-4-en-9-O-beta-D-glucopyranoside (1) and (6S,9S)-6,9-dihydroxymegastiman-4-en-9-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (2) were isolated from the leaves of Casearia sylvestris, along with icariside B(5)(3), byzantionoside B (4), blumenol B (5), blumenol C (6) and loliolide (7). The structures of these compounds were determined on the basis of 1D and 2D NMR, MS and circular dichroism (CD) spectroscopic analyses, chemical methods and comparison with the literature data.

To study the chemical constituents of the Maackia amurensis, the constituents were isolated by various chromatographies and the structures were elucidated on the basis of chemical and spectroscopic data (ESI-MS, 1D and 2D NMR). Thirteen isoflavone glycosides were isolated from the n-BuOH-soluble fraction of the 70% ethanol extract and identified as 7-hydroxy-4',6-dimethoxyisoflavone-7-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1), dalsympathetin (2), ononin (3), glycitin (4), genestin (5), saikoisoflavonoside A (6), afrormosin-7-O-beta-D-glucopyranoside (7), gehuain (8), kushenol O (9), 7-hydroxy-4',6-dimethoxyisoflavone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (10), tectoridin (11), biochanin A 7-O-beta-D-gentiobioside (12) and 7-hydroxy-4'-methoxyisoflavone-7-O-beta-D-apiofuranosy l-(1-->6)-beta-D-glucopyranoside (13). Compound 1 is a new isoflavone glycoside, named as maackiaisoflavonoside, compounds 2, 8, 9, 10, 12 and 13 were isolated from this genus for the first time.

Two compounds were isolated from the leaves of Panax ginseng. Their structures were identified as 3beta,6alpha,12beta-triol-22,23,24,25,26,27-hexanordammaran-20-one, and dammar-20(22),24-diene-3beta,6alpha,12beta-triol by spectral and chemical methods. The complete signal assignments of the two compounds were carried out by means of 2D NMR spectral analysis.

The fungus, Tritirachium sp. HKI 0317, was isolated from the Antarctic lichen Neuropogon sp. Fermentation of this strain, extraction of the culture broth, and preparative separation of produced compounds furnished 4-carboxy-5,5'-dihydroxy-3,3'-dimethyldiphenylether (1), macrosphelide A (2), and macrosphelide J (3). The structures were elucidated on the basis of MS and NMR measurements, and the previously published data for this compounds.

Phytochemical investigation of chemical constituents of the CHCl3 fraction of 75% alcohol extract from the leaves of Cyclocarya paliurus has resulted in the isolation of five compounds, alpha-boswellic (I), beta-boswellic (II), Oleanolic acid (III), 4-hydroxyl-3-methoxyl-benzoic acid (IV) and 3,6,3',5'-tetramenthox-5,7,4'-trihydroxy-flavonol (V). Their structures were elucidated on the basis of spectroscopic analyses, especially NMR. Compunds I, II, IV and V were obtained from this plant for the first time.