Chromatographic determination of plant saponins—An update (2002–2005)

Journal of Chromatography A, 1112 (2006) 78–91

Review

Chromatographic determination of plant saponins—An update (2002–2005) W. Oleszek ∗ , Z. Bialy Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland Available online 31 January 2006

Abstract The developments during 2002–2005 in the methods used for saponin analyses in plant material are presented. There were number of papers published on isolation and identification of new saponins by chromatographic techniques. Some new developments can be found in separation techniques or solid and mobiles phases used. Separation of individual saponins is still complicated and time consuming. This is due to the fact that in most of the plant species saponins occur as a multi-component mixture of compounds of very similar polarities. Thus, to isolate single compound for structure elucidation or biological activity testing, a combination of different chromatographic techniques has to be used, e.g. first separation of the mixture to simpler sub-fractions on reversed phase C18 has to be followed by further purification on normal phase Silica gel column. Especially difficult is determination of saponins in plant material as these compounds do not posses chromophores and their profiles cannot be registered in UV. Most HPLC methods apply not only specific registration at 200–210 nm, but these methods are not applicable for determination of many saponins in plant material at levels lower than 200–300 mg/kg. Some new or improved techniques for quantification of saponins in plant material were published in reviewed period. These include further progress in the application of evaporative light scattering detection (ELSD) for saponin profiling and quantification, which is also not only specific but also more sensitive in comparison to 200–210 nm detection. Some progress in development of new applications for liquid chromatography-electrospray mass spectrometry (LC/ESI/MS) for saponin determination has also been done. This method gives highest sensitivity and on line identification of separated saponins and should be recommended for specialized analyses of extracts and pharmaceutical formulas like the validation of a new assay. From non-chromatographic techniques for saponin determination, a sensitive and compound specific ELISA tests for some saponins were developed. © 2006 Elsevier B.V. All rights reserved. Keywords: Reviews; Saponins; Triterpene glycosides; Steroidal glycosides; TLC; Low pressure column chromatography; HPLC; LC-PAD-ESI-MS

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-chromatographic techniques of saponin analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thin-layer chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low-pressure column chromatography (LPCC) for isolation of saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . High-performance liquid chromatography (HPLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid chromatography-electrospray ionization mass spectrometry (LC/ESIMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Saponins are the group of secondary metabolites which are found in great number of plant species and in some marine

∗

Corresponding author. Tel.: +48 81 8863421x205; fax: +48 81 8864547. E-mail address: [email protected] (W. Oleszek).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.01.037

78 80 80 80 85 87 89 89 89

organisms. They are characterized by the surfactant properties and give in most cases stable, soap-like foams in aqueous solutions. Most of the available literature on saponin occurrence in plant kingdom was based on this test. In the Orient, these compounds were used as soap and many trivial names of saponin-rich species are derived from this feature, e.g. soapwort (Saponaria officinalis), soaproot (Chlorogalum pomeridianum), soapbark (Quillaja saponaria), soapberry (Sapindus saponaria) or soap-

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79

Fig. 1. Basic sapogenin skeletons—steroidal (left column) and triterpene (right column).

nut (Sapindus mukurossi) [1]. Some of them find a commercial application as drugs and medicines, adjuvants, taste modifiers, emulsifiers, precursors of hormone synthesis and sweeteners [1–3]. Saponins are complex molecules consisting of non-sugar aglycone coupled to sugar chain units. The aglycones can be divided into the groups of saponins containing triterpene or steroidal aglycones—sapogenins (Fig. 1). Both triterpene and steroidal aglycones have a number of different substituents ( H, COOH, CH3 ). The sugars can be attached to the aglycone either as one, two or three side chains. Thus, number of constituents and different possibilities of sugar chain composition and attachment causes great natural diversity of saponin structures. Even within one plants species different parts may harbor saponins with different structures. Due to the fact that saponins usually occur in plants as a mixture of structurally related forms with very similar polarities, their separation still remains a challenge. It is a usual practice in isolation of these compounds that a number of different separation techniques (TLC, column chromatography, flash chromatography, Sephadex chromatography and HPLC) should be used to obtain pure compounds for the structure and biologi-

cal activity determination. Early work on saponins included hot extraction of plant material with alcohol–water solutions followed by evaporation of alcohol and extraction of saponins into butanol (liquid–liquid extraction). However, hot extraction may disintegrate some labile functions (acylated forms) and produce artifacts rather than genuine saponins. Besides, extraction with methanol in some cases, especially for steroidal saponins may result in formation of methyl derivatives, not found originally in plants. Thus, for obtaining real composition of saponins, cold extraction with ethanol–water solutions should be rather recommended. In liquid–liquid extraction, some highly polar saponins (bidesmosides, tridesmosides) can be lost or extraction may not be quantitative. The alternative for liquid–liquid extraction is selective solid phase extraction (SPE) on number of sorbents (C18, C8). In SPE method, saponin extract (aqueous 10–20% methanol) can be loaded on preconditioned sorbent and washed with methanol–water. Ratio of methanol–water has to be optimized individually in preliminary tests for different classes of saponins on ready to use 1–2 cm3 cartridges. The procedure is very convenient for preparation of highly purified saponin mixtures for column separations of for biological activity test.

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Similar problems may occur also with their determination in plant material, for which both semi-quantitative methods (gravimetry, spectrophotometry, Trichoderma viride, Tribolium castaneum, seed germination tests) and analytical techniques (TLC, GC, HPLC, LC/MS, CE) were developed. The general principles of these techniques have been described in previous review [4]. The present paper reviews progress in saponin analysis that was done predominantly during 2002–2005.

Densitometric technique was applied to measure saponin content in some legumes [40]. The principle of this method was based on precise purification of saponins from non-saponin components and spotting such samples in rows on TLC plates along with different concentration soyasaponin standards. Plates were sprayed without developing with a solvent with sulphuric acid and heated. Intensity of spots was measured with densitometer and was compared to the intensity of a standard samples (range of 1.25–10 ␮g of saponins applied).

2. Non-chromatographic techniques of saponin analysis Immunoassays which use monoclonal antibodies (MAbs) against drugs and low molecular weight natural bioactive compounds are becoming an important tool in saponin analyses. They show high specificity and sensitivity for receptor binding analyses, enzyme assays and qualitative and quantitative analytical techniques. They find an application in new Eastern blotting and in immuno-affinity column chromatography, for diagnosis, therapy and drug monitoring. Enzyme-linked immunosorbent essay (ELISA) based on MAbs are in many cases more sensitive than conventional HPLC methods. Number of MAbs were also developed for saponins. These include ginsenoside Rb1 [5] (measuring range of ELISA assay from 20 to 400 ng/ml), ginsenoside Rg1 [6] (measuring range 0.3–10 ␮g/ml), glycyrrhizin [7] (measuring range 20–200 ng/ml), saikosaponin [8] measuring range 26 ng/ml to 1.5 ␮g/ml), oleanolic acid [9] (lowest concentration 100–200 nmol/␮l) and solamargine [10]. The antibodies of cell line 10F10 competed with triterpenoid saponins from leaves of Betula pendula Roth, leaves of Hedera helix L., fruits of Aesculus hippocastanum L., roots of Glycyrrhiza glabra L., bark of Quillaja saponaria Molina, bark of Gypsophila paniculata L., roots of Polygala senega L., but there was no competition observed with steroidal saponins of Smilex aristolochiifolia Mill [9]. 3. Thin-layer chromatography (TLC) Thin-layer chromatography is becoming rather a supporting technique in analysis of saponin fractions obtained from column chromatography. This has been also used for confirmation of purity and identity of isolated compounds. Such an application of TLC over a period covered by present review was very often reported and the data are presented in Table 1. Some trials of using TLC for qualitative and quantitative analysis of multicomponent saponin mixtures was also reported. The TLC separation of 18 saponins of Medicago sativa was developed [52]. The technique was based on two-dimentional (2D) TLC with a sorbent gradient. First development was performed on RP-18 plate and in the perpendicular direction on silica gel plates. Since the use of RP-18 and silica gel as a bilayer is rather complicated due to possibility of modification of stationary phases by first solvent system, two single layer TLC plates were used. The way of connection of two plates was described in detail in separate publication [53]. Such a sorbent gradient 2D TLC gave substantially better separation of saponins as compared to normal 2D TLC on one sorbent.

4. Low-pressure column chromatography (LPCC) for isolation of saponins As documented in a number of publications, a preparative separation of individual saponins for structure determination and biological activity evaluation was performed by combinations of low-pressure liquid chromatography (Table 2). For this purpose proper careful selection of stationary and mobile phases was essential for successful work. There were still a limited number of possibilities as regard to stationary phases. Most frequently, the first step of crude extract separation employed Sephadex LH-20 molecular filtration, which allowed preliminary separation of complex matrix of the extract into saponins and into accompanying impurities [35]. In some cases during this preliminary separation, simultaneous partition of mixtures of saponins into simpler subfractions could also be achieved. Similar goal could also be accomplished by passing water solution of plant crude extract trough a porous polymer gel column Diaion HP-20 (synthetic polyaromatic adsorbent with pore volume 1.3 ml/g, surface area 500 m2 /g, pore radius >200) [45]. Washing this column with water removed some impurities and adsorbed saponins which were eluted with MeOH. Plant extract could also be separated to different classes of phytochemicals by selective solid phase extraction/fractionation on RP-18 short column [109]. Next step of saponin isolation includes column chromatography (CC) on silica gel with mobile phase composed of different combinations of CHCl3 –MeOH–H2 O and EtOAc– MeOH–H2 O, or on reversed phase silica gel RP-18, with mobile phase composed of MeOH–H2 O or MeCN–H2 O and their acidified (HOAc, TFA) modifications. In most cases, one column separation was not efficient enough and combination of normal and reversed phase separation were needed. Final purification could also be accomplished using HPLC systems with normal or reversed phase columns, but this procedure was rather tedious and did not allow to obtain bigger amounts of pure saponins. Higher quantities of separated compounds could be obtained from high-speed counter-current chromatography, the system working without any solid support, with separation based on fast partitioning effects of the analytes between two immiscible liquid phases. With this technique irreversible absorption effects and artefact formation was minimal. This was successfully applied for the high yield separation of ginsenosides Rb1, R1, Re and Rg1 from Chinese phytomedicinal formulation Sanqi Zongdai Pian [41]. The n-hexane–n-BuOH–H2 O (3:4:7, v/v/v) was used for two-phase solvent system.

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Table 1 Analysis of saponins by thin layer chromatography (TLC) Sample

Sorbent

Solvent system

Reference

Abizia sap.

Silica gel 60 F

CHCl3 –MeOH–HOAc–H2 O (15:8:3:2) CHCl3 –MeOH–H2 O (65:40:1)

[11]

Albiziasaponins A–E

Silica gel 60 F RP-18 WF

CHCl3 –MeOH–H2 O (15:3:1) MeOH–H2 O (7:3)

[12]

Alium sap. Argania sap. Arnica sap.

Silica gel Silica gel Silica gel 60 RP-18

n-BuOH–HOAc–H2 O (60:15:25) n-BuOH–HOAc–H2 O (12:3:5) CH2 Cl2 –EtOAc (9:1) MeOH

[13] [14] [15]

Astragalus sap. Azadirachta sap.

RP-18 DC-cards Silica gel GF60

MeOH–H2 O (8.5:1:5) Petrol–EtOAc (7:3) Petroleum ether–EtOAc (6.5:3.5)

[16] [17]

Capsicoside E–G Carpolobia sap.

Silica gel Silica gel 60 F

n-BuOH–HOAc–H2 O (12:3:5) CHCl3 –MeOH–HOAc–H2 O (15:8:3:2) CH2 Cl2 –MeOH (19:1)

[18] [19]

Chelioclinium sap. Chenopodium sap. Cussonia sap. Cussosaponins A–E Dammarane sap. Dioscorea sap.

Silica gel Silica gel Silica gel 60 F Silica gel Silica gel 60 F Silica gel

[20] [21] [22] [23] [24] [25]

Draconins A–C

Silica gel 60 F

Hexane–EtOAc (4:1) with 0.05% HOAc n-BuOH–HOAc–H2 O (60:15:25) MeOH–CH2 Cl2 –HOAc–H2 O (40:55:3:2) CH2 Cl2 –MeOH–H2 O (17:6:1) CHCl3 –MeOH–H2 O (65:35:6) CHCl3 –MeOH–H2 O (13:7:1) CHCl3 –MeOH (9:1) n-Hexane–EtOAc (20:1, 7:3) Toluene–PrOH (20:1) CHCl3 –MeOH (10:1)

Eranthisaponins A and B

Silica gel 60 F RP-18

CHCl3 –MeOH–H2 O (14:8:1) MeCN–H2 O (2:5, 4:1, 1:3)

[27]

Eucalyptus sap. Fomefficinic acid A–E Gambeya sap. Ginsenosides

Silica gel 60 F Silica gel Silica gel Silica gel 60 Silica gel 60 F RP-18

n-Hexane–CH2 Cl2 –EtOAc (16:16:1) CHCl3 –MeOH (95:5) CHCl3 –MeOH–H2 O (8:5:1) CHCl3 –MeOH–H2 O (13:7:1) CHCl3 –MeOH–H2 O (10:3:1) MeOH–H2 O (7:3)

[28] [29] [30] [41] [42]

Glinus Sap.

Silica gel 60 F RP-18 Silica gel Sil G-100

MeOH–H2 O (4:1), CHCl3 –MeOH–H2 O (70:50:4) MeOH–H2 O (4:1) CHCl3 –MeOH–H2 O (70:50:4)

[31]

Harpulia sap.

Silica gel 60 F

CHCl3 –MeOH–H2 O (12:8:1) CHCl3 –MeOH–HCOOH (65:35:1)

[32]

Ilex sap.

Silica gel

n-BuOH–HOAc–H2 O (65:15:25) CHCl3 –MeOH–H2 O (70:30:3) CHCl3 –MeOH–n-PrOH–H2 O (5:6:5:1:4) upper phase

[33]

Lotoidesides A–F Lupinus sap. Lyciantosides A–C

Silica gel 60 F Silica gel Silica gel

CHCl3 -MeOH-H2 O (13:7:1, 28:12:1) Hexane–Me2 CO (75:25, 60:40, 40:60, 10:90) n-BuOH–HOAc–H2 O (60:15:25) CHCl3 –MeOH–H2 O (7:3:0.3)

[34] [35] [36]

Maytenus sap. Medicago sap. Meryta sap. Soyasaponin Pastuchoside A–E

Silica gel Silica gel Silica gel 60 F Silica gel Silica gel

100% MeCN EtOAc–HOAc–H2 O (7:2:2) CHCl3 –MeOH–H2 O-EtOAc (28:35:5:32) CHCl3 –MeOH–H2 O (65:35:10) lover phase CHCl3 –MeOH–H2 O (26:14:3) n-BuOH–HOAc–H2 O (4:1:5) CHCl3 –MeOH (20:1) CH2 Cl2 –MeOH–H2 O (50:25:5)

[37] [38] [39] [40] [43]

Pittoviridoside Pittoviridoside

Silica gel C-18 Silica gel

n-BuOH–EtOH–H2 O (5:1:4) 70% MeOH EtOAc–EtOH–H2 O (7:2:1, 7:10:1)

[44] [45]

Silene sap. Solanum sap. Symplocos sap. Tribulus sap.

Silica gel C-18 Silica gel 60 F Silica gel 60 F

CHCl3 –MeOH–HOAc–H2 O (15:8:3:2) MeCN–H2 O (7:3) CHCl3 –MeOH (9:1) n-BuOH–HOAc–H2 O (60:15:25) CHCl3 –MeOH–H2 O (40:9:1)

[46] [47] [48] [49]

Tripterygium sap.

Silica gel 60 F

Hexane–EtOH (2:1) CHCl3 –hexane (8:2)

[50]

Tuberosides N-U

Silica gel

n-BuOH–HOAc–H2 O (4:5:1) CHCl3 –MeOH–H2 O (7:3:0.5)

[51]

[26]

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Table 2 Low pressure column chromatographic conditions for saponin separation Sample

Column

Solvent system

Refs

Acaciosides

C-18 Silica gel RP-18

Gradient 50 → 100% MeOH Gradient CHCl3 –MeOH Gradient MeOH–H2 O

[45] [54]

Acutangulosides A–F

C-18

(0–100% MeOH in H2 O) 70–100% MeOH; MeOH–1% TFA 40% MeCN–1% TFA 45% MeCN

[55]

Aesculus sap.

D-101 macroreticular column Silica gel Silica gel(BW-200)

H2 O; 30% EtOH; 70% EtOH 95% EtOH Petrol–EtOAc (20:1 → 80:1) CHCl3 –MeOH–H2 O (15:3:1 → 6:4:1) MeOH–H2 O (70:30) MeCN–1% aqueous HOAc (35:75 → 80:20)

[56]

Agrostophyllum sap. Albiziasaponin A–E

ODS A YMC Pack Allium sap.

[57] [42]

RP-18 Diaion HP-20 Silica gel RP-18 Sephadex LH-20 Sephadex LH-20 Silica gel

Gradient MeOH–H2 O Gradient EtOH–H2 O CH2 Cl2 –MeOH–H2 O (5:1:0.15 → 1:1: 0.3) 65–80% MeOH MeOH MeOH CH2 Cl2 ; CH2 Cl2 –EtOAc (9:1, 7:3, 2:3) EtOAc–MeOH (9:1, 3:1, 1:1) Cyclohexane–CH2 Cl2 (3:2)

Asterosaponins

Silica gel Sephadex LH-20 RP-18 lichroprep

CHCl3 –n-BuOH (satur. H2 O)–MeOH (2:1:0 → 0:6:1) lower phase MeOH–H2 O (2:1) MeOH–H2 O (46:54)

[59]

Azadirachta sap. Bacopaside

Silica gel 60 GF Sephadex LH-20 Diaion HP-20 MCI-gel CHP20p C-18 Comosil

CHCl3 , CHCl3 –MeOH (9.9:0.1 → 9.85:0.15) EtOH H2 O–MeOH H2 O–MeOH (1:0 → 0:1) H2 O–MeOH (1:0 → 1:1)

[17] [60]

Barringtogenol Cangorosin sap.

RP 18 Silica gel

MeOH–H2 O (4:6 → 6:4) Gradient CH2 Cl2 –EtOAc Gradient n-hexane–EtOAc

[61] [62]

Celtis sap.

Silica gel

CHCl3 –MeOH (100:0 → 2:1) CHCl3 –Me2 CO (100:0 → 5:1) EtOH–Me2 CO (50:1 → 2:1)

[63]

Certonardoside

ODS A YMC Pack Silica gel PF254

Gradient MeOH:H2 O (33–0%) MeOH–CHCl3 (17–100%)

[64]

Certonardosides A–J

ODS-A YMC Silica gel 60 C-18 5E Shodex

Gradient H2 O–MeOH MeOH–CHCl3 (0–50%) 80% MeOH

[65]

Certonardoa sap.

ODS Silica gel 60 ODS YMC Pack C-18-5E Shodex YMC-Pack NH2 C-8 YMC-Pack

Gradient H2 O–MeOH MeOH–CH2 Cl2 (0–70%) 75% MeOH 75% MeOH 90% MeCN 75% MeOH

[66]

Cheiloclinium sap. Chenopodium sap.

Silica gel C-18 Silica gel Diaion HP-20 Sephadex LH-20

Gradient hexane–EtOAc MeOH–H2 O (1:2) EtOAc–MeOH–H2 O–hexane (22.5:1:0.8:0.8) H2 O–EtOH (3:7 → 1:9) Me2 CO 90% MeOH

[20] [67]

Clematis sap.

Diaion HP-20 Silica gel

MeOH (30,60, 80,100%); EtOH, EtOAc Gradient CHCl3 –MeOH (4:1 → 0:1) CHCl3 –MeOH–H2 O (40:10:1 → 7:4:1) MeOH–H2 O (8:5); MeCN–H2 O (5:8)

[68]

ODS

[58] [51]

[13] [15]

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Table 2 (Continued ) Sample

Column

Solvent system

Refs

Combretaceae sap.

Sephadex LH-20 Diaion HP-20

TCM (trichloromethane) → MeOH 10% Me2 CO in MeOH

[69]

Conyzasaponins I–Q Cucumariosides

Silica gel 60 Silica gel

MeOH–H2 O (1:9 → 10:0) CHCl3 –MeOH–H2 O (4:8:1) CHCl3 –PrOH–H2 O (10:30:4) CHCl3 –EtOH (5:2)

[70] [71]

Cussonia sap.

Silica gel

Hexane–EtOAc (9:1; 3:2; 3;7) CH2 Cl2 –MeOH (4:1 → 3:2)

[22]

Cussosaponins A–E

Silica gel RP-18

CH2 Cl2 –MeOH–H2 O (17:4:0.5 → 17; 8:2) MeOH–H2 O (2:8 → 10:0)

[23]

Cycloartane sap.

MCI gel

H2 O–MeOH (5:6 → 0:10) MeOH–CH2 Cl2 (10:0 → 8:2)

[16]

Silica gel

Hexane–EtOAc (9:1 → 0:10)

Dioscorea sap.

C-18 Silica gel

MeOH–H2 O (5:5 → 4:1) CHCl3 –MeOH–H2 O (13:7:2) lower phase

[25]

Diosgenin Diploclisia sap.

Amberlite XAD-2 Silica gel Sephadex LH-20

MeOH–H2 O (6:4 → 10:0) 20 → 40% MeOH in EtOAc MeOH

[72] [73]

Dizygotheca sap.

Diaion HP-20 Silica gel Sephadex LH-20 Silica gel

H2 O; 50, 70; 100% MeOH CHCl3 –MeOH (65:35) MeOH CHCl3 –MeOH–H2 O (80:12:2 → 61:33:6)

[74]

Draconins A–C

Silica gel

[26]

Sephadex LH-20 Diaion HP-20

CHCl3 –MeOH (8:2) n-Hexane–CH2 Cl2 –MeOH (2:1:1) n-Hexane–CH2 Cl2 –MeOH (2:1:1) Gradient MeOH–H2 O

Eranthisaponins A and B

Silica gel Diaion HP-20

CHCl3 –MeOH–H2 O (14:8:1) 30 → 100% MeOH; EtOH; EtOAC

[27]

Euphorbia sap. Ficus sap. Gambeya sap.

Silica gel Silica gel Silica gel

Gradient n-hexane-EtOAc Gradient hexane-EtOAc Hexane-EtOAc (100:0 → 0:100) EtOAc-MeOH (100:0 → 0:100)

[75] [76] [30]

Silica gel 60 H Silica gel 60 C

Hexane–EtOAc (5:5) CH2 Cl2 –MeOH (9:5 → 90:10)

RP-18 Silica gel

Gradient H2 O–MeOH CH2 Cl2 –MeOH–H2 O (80:20:5 → 65:35:5) MeOH–H2 O (8:2 → 6:4)

Sephadex LH-20 Sephadex LH-20 Silica gel Silica gel BW-200 ODS DM 1020 Diaion HP-20

MeOH–H2 O (1:1) MeOH Gradient CHCl3 –CH3 OH–H2 O (10:1:0 → 6:4:1) CHCl3 –MeOH–H2 O (10:3:1 → 6:4:1) MeOH–H2 O (25:75 → 70:30) H2 O; 50%, 100% MeOH

Sephadex LH-20 Silica gel

MeOH CHCl3 –MeOH–H2 O (13:7:2) CHCl3 –MeOH–H2 O (8:5:1)

RP 18 Lichroprep

50% MeOH

Hydrocotylosides I–VII Harpullia sap. Helleborus sap. Hookerosides A and B

Diaion HP-20 Silica gel Sephadex LH-20 Silica gel ODS

50, 60, 70, 100% MeOH CHCl3 –MeOH–H2 O (8:2:0 → 15:10:) MeOH 80% MeOH 80% MeOH

[80] [32] [49] [81]

Hopane saponins

Sephadex LH-20 RP-18

MeOH 15% MeCN

[31]

Ilex sap.

LH-20

MeOH

[33]

Ginsenosides

Glandulosides A–D

[77]

[78] [41]

[79]

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Table 2 (Continued ) Sample

Column

Solvent system

Refs

Jenisseensosid

Sephadex LH-20 Silica gel 60 RP-18

MeOH CHCl3 –MeOH–H2 O (15:7:2 → 8:5:1) MeOH–H2 O (linear gradient 50 → 70%)

[46]

Jujubogenin Justiciosides A–D

Sephadex LH-20 Silica gel

MeOH EtOAc–MeOH (9:1) EtOAc–MeOH–H2 O (40:10:1) Gradient H2 O → MeOH

[82] [83]

Lanostane sap.

Silica gel LPCL

CHCl3 –MeOH (19:1 → 8:2) Petroleum ether–EtOAc (10:0 → 8:2) CHCl3 –MeOH (97:3 → 90:10)

[29]

Lotoideside A–F Lupane triterpene Lupinus sap.

Silica gel Silica gel Sephadex LH-20 RP-18

CHCl3 –MeOH (1:0 → 3:2) Gradient n-hexane–EtOAc, MeOH MeOH MeOH–0.15% TFA in water (70:30 → 100:0)

[34] [84] [35]

Lycianthosides Maesopsis sap.

Sephadex LH-20 Silica gel

MeOH CHCl3 -MeOH (9:1) Gradient CH2 Cl2 -MeOH

[36] [85]

Maytenus sap.

Silica gel ODS MPLC Silica gel MPLC

CH2 Cl2 –EtOAc (1:0 → 0:1) CH3 C–H2 O (8:2 → 1:0) Gradient n-hexane–EtOAc

[62]

Mimengosides C–G

Diaion HP-20 Silica gel

20 → 100% MeOH 60% MeOH

[86]

Nepheliosides I–VI

Silica gel RP-18

Gradient hexane–Me2 CO–MeOH (8:1:0.1 → 2:1:0.1) 75% MeOH

[87]

Ocotilone

Silica gel ODS

CHCl3 –MeOH MeOH–H2 O (30:70 → 70:30) n-Hexane–CHCl3 –EtOAc (16:16:1 → 8:1:1)

[88]

Oleanone sap. Oleanane sap.

Sephadex LH-20 Diaion HP-20 Silica gel

MeOH MeOH CHCl3 –MeOH–H2 O (65:33:2)

[21] [89]

Pastuchoside A-E

Silica gel C-18

CHCl3 –MeOH–H2 O (26:14:3) MeOH–H2 O (2:8 → 8:2)

[42]

Pentacyclic triterpene

Silica gel 60

Gradient n-hexane–EtOAc n-Hexane–CHCl3 –EtOAc (16:16:1 → 8:1:1)

[28]

Pentandrosides A–G

C-18 Backer C-18 Lichroprep Silica gel

Gradient H2 O → MeOH 20 → 25% MeCN CHCl3 –MeOH (9:1)

[90]

Pittoviridosides Polygonatosides A-D

C-18 Silica gel RP-18

MeOH–H2 O (5:5 → 9:1) CHCl3 –MeOH–H2 O (7:2.5:0.4) Gradient MeOH–H2 O

[43]

Silica gel 60 Sephadex Silica gel Diaion HP-20 Diaion HP-20

CHCl3 –MeOH–H2 O (8:5:1) LH-20 80% MeOH CHCl3 –MeOH–EtOAc–H2 O (2:1:4:1) 40% MeOH H2 O; 20, 40, 60, 80, 100% MeOH 40, 60, 80,100% Me2 CO

[92] [93]

ODS

Presenegenin Pulsatilla sap. Saniculasaponin I–XI Sapindaceae

[91]

[94] [95]

Silica gel

CHCl3 –MeOH (21:1 → 2:1)

Sapotaceae

Diaion HP-20 Sephadex LH-20

H2 O, 50% MeOH, MeOH MeOH

[14]

Scabiosaponins A–K Schefflera sap.

Diaion HP-20 Silica gel Sephadex LH-20

40, 60, 80, 100% MeOH CHCl3 –MeOH (85:15 and 83:17) Gradient MeOH–H2 O

[81] [96]

Sophora sap.

Polymer MCI RP-18 Silica gel

20, 50, 100% MeOH MeOH–H2 O (7:3) EtOAc (satur. H2 O)

[97]

W. Oleszek, Z. Bialy / J. Chromatogr. A 1112 (2006) 78–91

85

Table 2 (Continued ) Sample

Column

Solvent system

RP-18 Silica gel Sephadex LH-20

80% MeOH CHCl3 –MeOH–H2 O (7:3:1) MeOH

Soyasaponins

Silica gel C-18 Sep-Pac ODS C-18 C-18 Nova Pac

CHCl3 –MeOH–H2 O–HOAc (65:35:9.8:0.2) 100% MeOH MeCN-1–PrOH–H2 O–OHAc (80:6:13.9:0.1) A: 4.0% OHAc, B: 100% MeOH

[98] [99]

Spirostane sap. .

Silica gel VCL silica gel Silica gel Diaion HP-20 Silica gel

CHCl3 –MeOH (95:5 → 9:1) n-Hexane–CH2 Cl2 –MeOH mix. Gradient CH2 Cl2 –MeOH (95:5) 30 → 50% MeOH; MeOH; EtOH; EtOAc Gradient CHCl3 → MeOH

[100] [47]

Symplocososides A–F

Silica gel RP-18

CH2 Cl2 –MeOH–H2 O MeOH–H2 O (79:21)

[102]

Symplocososides

Silica gel RP-18

Gradient CHCl3 → MeOH MeOH–H2 O (6:4 → 8:2)

[48]

Synallactosides

Silica gel Sephadex DEAE

CHCl3 –EtOH (6:1 → 4:1) 55% EtOH

Diaion HP-20 Silica gel Silica gel Silica gel Sephadex LH-20 Toyopearl HW-40

MeOH–H2 O (1:1) Gradient CHCl3 –MeOH CH2 Cl2 –MeOH–H2 O (7:3:1) lower phase CHCl3 ; CHCl3 –MeOH (97:3 → 9:1) MeOH CHCl3 –MeOH (2:1)

[104]

RP-18 Silica gel Sephadex LH-20 Silica gel

Gradient H2 O → MeOH MeOH–CHCl3 –HOAc (10:88:2) MeOH CHCl3 –MeOH–H2 O (10:1:0 → 6:4:1)

[106] [107] [108]

RP 18

MeOH–H2 O (4:6 → 9:1)

Taverniera sap. Ternstroemiasides A–F Tripterigium sap.

Triquetrosides Vaccinium sap. Yesanchinosides

5. High-performance liquid chromatography (HPLC) The absence of a chromophore in saponins hampers their detection in ultraviolet light and allows non-specific detection at 200–210 nm. Thus, most of published data are based on recording HPLC profiles at 200–210 nm (Table 3). But at this wavelength other than saponin components of the analyte may overlap with saponins making determination difficult. Only for 2,3-dihydro-2,5-dihydroxy-6-methyl-4-pyrone (DDMP) conjugated soyasaponins, which have an UV absorption maximum at 295 nm, glycyrrhetinic acid glycosides and cucurbitacins detection with UV–vis detectors could be successful. To overcome these problems and to be able to develop validated analytical methods for quality control of some products, several trials were performed to apply evaporative light scattering detection (ELSD) for detection of saponins. This detector was successfully applied for measuring soyasapogenols A and B, separated on C18 column with MeCN–PrOH–H2 O–HOAc (80:6:13.9:0.1) in soybean [99]. Validated HPLC method with ELSD was also developed for determination of major ginsenosides in samples of Chinese traditional medicine [125]. Saponins were successfully separated on Spherisorb ODS2, C18 column in MeCN–H2 O gradient and quantified using calibration curves, with detection limits of 50 ng.

Refs

[101]

[103]

[105] [50]

Similar validated method using ELSD was developed for saponin determination in Flos Lonicerae (dried buds of several species of the genus Lonicera) used as a herb in traditional Chinese medicine [111]. Seven saponins, including the macranthoidins A and B, macranthosides A and B, dipsacoside B and two hederagenin glycosides were successfully separated on C18 column using MeCN–H2 O–HOAc gradient and quantified. But still ELSD is not much in use for the routine quality control of saponin containing herbal products and LC with UV detection remains the method of choice. For Ginseng products, an interlaboratory studies were performed to evaluate repeatability, reproducibility and recovery [126]. Twelve collaborating laboratories assayed four products namely Panax ginseng, Panax quinquefolius, and two ginseng products, for six ginsenosides: Rb1 , Rb2 , Rc , Rd , Re and Rg1 . Analyses were performed on methanol extracts using C18 (5 ␮m particle size, 250 mm × 4.6 mm) column and UV detection at 203 nm. Obtained results agreed reasonably well among participating laboratories suggesting that the method worked well. This allowed detection of individual gnisenosides if their concentration in plant material was higher than 200 mg/kg. Only in case of Rg1 and Re for which separation was rather poor concentration in plant material could not be lower than 300 mg/kg. It was concluded that for routine analyses polar reversed-phase columns would be desired; they would reduce time of analyses

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Table 3 The high performance liquid chromatographic separation of saponins Saponin

Column

Solvent system detection

Reference

Acutangulosides A–F Aesculiside A Albiziasaponins A–E Asterosaponins Avicins D and G

RP-18, phenyl ODS YGW C18 YMC-Pack ODS-A RP-18 Intersil RP-18 Flurosep-RP-phenyl C-18 201SP510 YMC-Pack ODS-AQ RP-18 ␮-Bondapak C-18 YMC J’sphere ODS-H80 YMC-Pack ODS YMC-Pak ODS C18-5E Shodex YMS-Pak C-8 Pegasil ODS-II

MeOH–H2 O MeCN–H2 O–HOAc MeCN–H2 O–HOAc MeOH–H2 O MeOH–H2 O MeCN–H2 O MeCN–H2 O–TFA MeOH–H2 O MeCN MeOH–H2 O MeCN–H2 O MeOH–H2 O MeOH–H2 O MeOH–H2 O MeOH–H2 O MeOH–H2 O MeCN–H2 O

[55] [56] [12] [59] [110]

Chenopodium sap. Cucumariosides Dioscorea sap. Echinocystic acid glc. Elburzensosides Ficus triterp. Flos sap. Ginsenosides

␮-Bondapak C-18 Silasorb C-18 Cosmogel C-18 ODS ␮-Bondapak C-18 Silica gel Zorbax SB-C18 YMC-Pack ODS-AQ YMC-Pack ODS-AQ303 Synergi Hydro-RP

MeOH–H2 O CH3 COCH3 –H2 O MeOH–H2 O MeCN–H2 O–TFA MeOH–H2 O EtOAc–hexane MeCN–H2 O–OHAc MeCN–H2 O MeCN–10 mM K-phosphate buffer MeCN–H2 O

[21] [71] [72] [74] [58] [111] [112] [113] [114] [77]

Glinus sap. Hederagenin sap.

RP-18 Silica gel YMC R&D ODS ␮-Bondapak C-18 ODS Capcell Pak Ph PrepLC ␮-Bondapak C-18 Lichrospher RP-18 ␮-Bondapak RP-18 YMC ODS X-Terra C-18 ␮-Bondapak C-18 Kromasil Sil Reliasil C-18 Hypersil BDS C18 XTerra RP-18 RP-18 RP-18

MeCN–H2 O CHCl3 –MeOH MeOH–H2 O–TFA MeOH–H2 O MeCN–H2 O MeCN–H2 O–TFA MeCN–H2 O–TFA MeOH–H2 O MeCN–H2 O–TFA MeOH–H2 O MeCN–H2 O MeOH–H2 O–TFA MeOH–H2 O Cyclohexane–EtOAc MeCN–H2 O–TFA NH4 OAc–MeOH–MeCN–H2 O MeCN–MeOH–H2 O MeCN–H2 O–HOAc MeCN–H2 O–HOAc

[31] [95] [115] [49] [80]

Mimengosides C–G Morolic acid Nephelioside I–VI Notoginsenosides Oenothera triterpenoids Oleanolic acid sap.

Pegasil OGS-II RP-18 201SP ODS-AQ YMC-Pak ODS-A Lichrosorb Diol Develosil PhA RP-18 201SP YMC R&D ODS

MeOH–H2 O MeCN–H2 O–TFA MeOH–H2 O MeCN–H2 O–HOAc n-Hexane–EtOAc MeCN–H2 O–TFA MeCN–H2 O–TFA MeOH–H2 O–TFA

[86] [119] [87] [42] [120] [89] [119] [74]

Pachyelasides A–D Phytolaccagenic acid glc. Pittoviridoside Protoaescigenin sap. Protobassic acid sap. Pulsatilla sap. Quinonemethide triter. Saniculasaponins I–XI

Asahipack GS-320 STR Prep-ODS 20 YMC ODS-A C-18 201SP510 ODS Zorbax Spherisorb ODS 2 RP-18 ODS

MeOH MeOH–H2 O MeOH–tetrahydrofuran–H2 O–HOAc MeCN–H2 O–TFA MeCN–H2 O MeCN–H2 O MeOH–H2 O–H3 PO4 MeCN–H2 O

[121] [73] [122] [32] [14] [93] [20] [94]

Barringtogenol C Barringtonia sap. Boswellic acid Capsicosides E–G Celtis triterpenes Certonardosides A–J

Conyzasaponins I–Q

Helleborus sap. Hydrocotylosides I–VII Hydroxyimberic acid sap. Ilex sap. Jenisseensosides A–C Jujubogenins Justicosides A–D Lotoidesides A–F Lycianosides A–C Lupanes Lupinus sap. Maesa sap. Medicagenic acid sap. Medicago sap.

[32] [44] [84] [18] [63] [65] [66]

[70]

[69] [33] [79] [82] [83] [34] [36] [54] [35] [116] [38] [117] [118]

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87

Table 3 (Continued ) Saponin

Column

Solvent system detection

Reference

Scabiosaponins A–K Serianic acid sap. Soyasapogenol A and B Soyasaponins

Pegasil ODS STR Prep-ODS 20 ODS RP-18 Lichroprep RP-18 ␮-Bondapak RP-18 Zorbax Eclipse XDB-C18 Aquasil RP-18

MeOH–H2 O MeOH–H2 O MeCN–PrOH–H2 O–HOAc MeCN–H2 O–TFA MeOH–isoPrOH–H2 O–HCOOH MeCN–H2 O MeCN–H2 O–HOAc

[81] [73] [99] [123] [98] [124] [125]

Steroidal sap.

Lichrospher C-18 Lichrospher C-18

MeCN–H2 O MeCN–H2 O

[100] [26]

Symplocososides

YMC-Pack ODS-A

MeOH–H2 O MeCN–H2 O

[102]

Synallactosides Ternstroemiasides A–F Tripterygium sap. Triquetrosides Tropeosides A and B Ursolic acid Zafaral

Silasorb C-18 YMC ODS-H80 YMC-Park SIL-06 RP-18 RP-18 Nova-Pak RP-18 RP-18

EtOH–H2 O MeOH–H2 O Hexane–EtOAc MeOH–H2 O MeOH–H2 O MeOH–H2 O–HOAc MeCN–H2 O

[103] [105] [50] [106] [106] [107] [17]

(60 min in cited studies) and also improve separation of Rg1 and Re . 6. Liquid chromatography-electrospray ionization mass spectrometry (LC/ESIMS) Difficulties in detection of saponins by the LC/UV methods encouraged development of hyphenated techniques combining liquid chromatography and mass spectrometry. Applying MS to structural and analytical problems has become increasingly common over last few years, and MS has been extensively used to characterize, to confirm and to determine saponins in plant extracts. Most extensive work was performed on commercially important plants, e.g. soyasaponins in soybean products (Glycine max) [98,122,123] and black bean (Vigna mungo L. Hepper) [127], ginseng (Panax notoginseng) saponins [77,128] and recently on Medicago saponins [116,117,129] due to their importance to metabolomics and functional genomics of Fabaceae. Soybean saponins can be divided into two classes namely A and B. Saponins of class A possess soyasapogenol A in an aglycone part and have bidesmosidic character (3 and 22O substituted with up to four sugars). Additionally, saponins of this group contain three or four acetyl groups in their genuine form. Saponins of B class are monodesmosidic structures possessing soyasapogenol B or E in an aglycone part. Likewise, the soyasaponins belonging to group B occur as both DDMP and non-DDMP forms. Both acetyl groups in A type and DDMP in B type saponins are very labile substitutions and can be partially lost during sample preparation, which additionally increase number of saponins (artifacts) in a matrix. This diversity hinders the separation and determination of individual compounds and in fact no method determining all genuine soybean saponins is available. To reduce complexity and to increase stability of soybean saponins, partial alkaline degradation cleaving acetyl and DDMP groups prior to LC/MS analysis

was proposed [98]. Such deacetylated and non-DDMP saponins could be easily quantified using selective ions of their [M − H]− ions. Some improvement to this procedure allowing fingerprinting of soybean saponins was developed [123]. This was based on monitoring of the soyasaponin specific protonated aglycones and dehydrated aglycone ions. For series A saponins these ions of soyasapogenol A (m/z 475) corresponded to peaks at m/z 457 [A-H2 O]+ , 439 [A-2H2 O]+ , 421 [A-3H2 O]+ and for soyasapogenol B (m/z 457) corresponded to peaks 441 [B-H2 O]+ , 423 [B-2H2 O]+ , 405 [B-3H2 O]+ . Characteristic of continuously recorded positive ion in total ion chromatogram (m/z 200–1500) allowed identification of several groups A and B saponins and some isoflavones in one run and a quantification of the soyasaponin Bb based on available Bb standard. Combination of ELSD and ESI/MS was applied for developing procedure for separation and determination of group A soyasaponins with different degree of acetylation and of group B soyasaponins in both their DDMP-conjugated and nonconjugated forms occurring in soybean [124]. For ginseng saponins situation seem to be even more complex than for soybeans. Till recently 64 structures have been reported and the standards are available for only seven glycosides [128]. For such a complex mixture reliable methods for on line characterization is essential. As shown recently, ginsenosides under ESI/MS form stable adduct ions with small alkali and transition metals. Negative ion and MS/MS experiments allowed for the determination of molecular mass, type of triterpene core, type of sugars (pentose and hexose) and type of attachment points of the sugars to the core. MS analyses of ginseng root extracts [77] showed that the main ions observed were the sodiated molecular ions [M + Na]+ and to lesser extent [M + K]+ ions. MS/MS experiments showed that cleavage occurred predominantly at the glycosidic linkage at C-20. Barrel medic (Medicago truncatula), a close relative of alfalfa (Medicago sativa), has gained recently much attention as a model crop for the research of genomics of Fabaceae. Extensive

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Fig. 2. Direct injection ESI/MS of Medicago truncatula saponins [117] (Thermo Finnigan LCQ Advantage Max ion trap MS; syringe pump at flow rate 0.5 ␮l/min; spray voltage 4.2 kV; capillary offset voltage −60 V; capillary temperature 220 ◦ C; nitrogen flow rate 0.9 l/min; calibration mass range 400–2000 Da). Peak at m/z 1251 represents three compound, peaks at m/z 1367 and 1383 represents two saponins each.

work on possibility of metabolite profiling using LC/ESIMS/MS technique was performed on this plant. Research on comparison of saponin profiles in barrel medic and alfalfa showed that roots of barrel medic are richer source of saponins than

roots of alfalfa [130]. Thirty individual compounds were identified in both species based on observed LC/ESIMS profiles and LC/ESIMS/MS experiments. Some saponins were quantified in roots and aerial parts of barrel medic based on the same

Fig. 3. Total ion current (TIC) chromatogram (upper) of Medicago truncatula saponins [117] and selective ion chromatogram (SIC) for the mass of m/z 1251. Compounds: (2) 3-O-β-Glc-(1 → 3)-β-Glc,28-O-β-Api-(1 → 3)-α-Rha-(1 → 2)-α-Ara-zanhic acid; (4) 3-O-β-Glc-(1 → 3)-β-Glc,28-O-α-Ara-(1 → 3)-α-Rha-(1 → 2)α-Ara-zanhic acid; (7) 3-O-β-Glc-(1 → 3)-β-Glc,28-O-β-Xyl-(1 → 4)-α-Rha-(1 → 2)-α-Ara-zanhic acid. LC conditions: Finnigan Surveyor pump with gradient controller, autosampler and PDA detector; column 250 mm × 4 mm i.d., 5 ␮m, Eurospher 100 C18 ; mobile phase solvent B (0.05% acetic acid in water), solvent A (0.05% acetic acid in acetonitrile); flow rate 0.5 ml/min; run time 90 min; gradient program: from 18% A to 36% A in 55 min, from 36% A to 100% A in 20 min; injection volume 25 ␮L.

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techniques [116]. These trials, however, clearly show that for successful application of LC/ESIMS technique for quantification of saponins a set of original standards is essential. In the work on alfalfa roots fifteen saponins were proposed to be identified [129], but unfortunately all major medicagenic acid and zanhic acid glycosides previously isolated [130] or determined with HPLC [131] were missed. It was especially surprising that 3GlcA,28 AraRhaXyl medicagenic acid, the major medicagenic acid glycoside present in roots and aerial part of alfalfa was not recognized in LC/ESIMS/MS profile. Similar situation showing the weakness of LC/ESIMS data interpretation was evident from comparison of data on saponin composition and concentration in aerial parts of barrel medic. In the work of Huhman [116] about fifteen saponins were identified and quantified in aerial parts of barrel medic and again both composition and concentration was substantially different from the data published on the same species when appropriate standards were available [117]. As shown in our recent research on LC/ESIMS/MS of aerial parts of three Medicago truncatula subspecies they all possessed similar saponin profiles rich with medicagenic acid, zanhic acid and soyasapogenol saponins. Direct injection of solid phase extracted saponins (Fig. 2) showed 13 major peaks corresponding to [M − H]− ions of dominant saponins. Some peaks corresponded to two or three different compounds having identical m/z values and these were recognized based on the retention times and selected ion chromatograms in relation to original standards (Fig. 3). Without appropriate standards this differentiation would not be possible. The discussed examples of using LC/ESIMS/MS technique for identification/conformation and determination of saponins show that this technique is still a challenge and has several limitations. For the development of reliable routine method the availability of appropriate standards obtained by classical separation methods is essential. Even structural confirmation is not absolutely certain by this technique. As shown in the research on barrel medic, identical LC/ESIMS/MS sequencing of two compounds and identical retention times cannot distinguish between (1 → 2) and (1 → 3) sugar bounding, the difference which differentiates some saponins of barrel medic and alfalfa. 7. Conclusion Since saponins are polar compounds occurring in plant material in a multi-component mixtures their separation and determination still creates problems. Practically most of them have to be purified by the combination of several chromatographic techniques. A good example for difficulties in saponin separation is Ginseng. In spite of the fact that great number of papers have been published on this plant, and about 40 individual compounds were described, only six to seven standards are available. For future developments in saponin research, the development of a new reversed phase polar stationary phases is highly desired. None of actually existing chromatographic techniques provides full fingerprinting and quality assurance required for registration of herbal medicinal products. The LC method seems to be most often acceptable. However, the TLC methods are still recommended by U.S. Pharmacopeial Forum if further

89

confirmation of peak identity is required. Most reliable technique, giving possibility for both quantitation and identification is LC–MS/MS. But even in this technique, as discussed in this review, solid separation work on obtaining purified standards is essential for reliable conclusions. Structural information from MS/MS analyses is quite limited; type of sugar linkage, place of substitution cannot be concluded. Besides, separation of compounds with the same number of sugars attached, but differing in the type of sugars, is difficult or even impossible. Again new stationary phases for separation are highly desired. Acknowledgement Financial support was provided by Polish Ministry of Science, grant number 2 P06A 037 27. References [1] K.A. Hostettman, A. Marston, Saponins, Cambidge University Press, Cambridge, 1995. [2] G.R. Waller, K. Yamasaki, Saponins Used in Food and Agriculture, Plenum Press, New York, 1996. [3] W. Oleszek, A. Marston, Saponins in Food, in: Feedstuffs and Medicinal Plants, Kluwer Academic Publishers, Dordrecht, 2000. [4] W.A. Oleszek, J. Chromatogr. A 967 (2002) 147. [5] H. Tanaka, N. Fukuda, Y. Shoyama, Cytotechnology 29 (1999) 115. [6] N. Fukuda, H. Tanaka, Y. Shoyama, Cytotechnology 34 (2000) 197. [7] S. Shan, H. Tanaka, Y. Shoyama, Anal. Chem. 73 (2001) 5784. [8] S.-H. Zhu, S.-I. Shimokawa, H. Tanaka, Y. Shoyama, Biol. Pharm. Bull. 27 (2004) 66. [9] K.M.G. Brandt, C. Klein, I. Z¨udorf, T. Dingermann, W. Kn¨oss, Planta Med. 70 (2004) 986. [10] M. Ishiyama, Y. Shoyama, H. Murakami, H. Shinohara, Cytotechnology 18 (1996) 153. [11] M. Haddad, T. Miyomoto, V. Laurens, M.A. Lacaille-Dubois, J. Nat. Prod. 66 (2002) 376. [12] M. Yoshikawa, T. Morikawa, K. Nakano, Y. Pongpiriyadacha, T. Murakami, H. Matsuda, J. Nat. Prod. 65 (2002) 1641. [13] I. Dini, G.C. Tenore, E. Trimarco, A. Dini, Food Chem. 93 (2005) 206. [14] A. Alaoui, Z. Charrouf, M. Soufiaoui, V. Carbone, A. Malorni, C. Pizza, S. Piacente, J. Agric. Food Chem. 50 (2002) 4601. [15] T.J. Schmidt, J. von Raison, G. Willuhn, Planta Media 70 (2004) 968. [16] M.M. Radwan, A. Farooq, N.A. El-Sebakhy, A.M. Asaad, S.M. Toaima, D.G.I. Kingston, J. Nat. Prod. 67 (2004) 489. [17] B.S. Siddiqui, F. Afshan, T. Gulzar, M. Hanif, Phytochemistry 65 (2004) 2363. [18] M. Iorrizi, V. Lanzotti, G. Ranalli, S. De Marino, F. Zollo, J. Agric. Food Chem. 50 (2002) 4312. [19] A.C. Mitaine-Offer, T. Miyamoto, I.A. Khan, C. Delaude, M.A. Lacaille-Dubois, J. Nat. Prod. 65 (2002) 556. [20] A.H. Jeller, D.H.S. Silva, L.M. Liao, V.S. Bolzani, M. Furlan, Phytochemistry 65 (2004) 1977. [21] I. Dini, G.C. Tenore, A. Dini, J. Nat. Prod. 65 (2002) 1025. [22] L.A. Tapondjou, D. Lontsi, B.L. Sondengam, F. Shaheen, M.I. Choudhary, A. Rahman, F.R. van Heerden, H.J. Park, K.T. Lee, J. Nat. Prod. 66 (2003) 1268. [23] L. Harinantenaina, R. Kasai, K. Yamasaki, Chem. Pharm. Bull. 50 (2002) 1292. [24] A. Maciuk, C. Lavaud, P. Thepenier, M.J. Jacquier, K. Ghedira, M. Zeches-Hanrot, J. Nat. Prod. 67 (2004) 1642. [25] M. Sautour, A.C. Mitaine-Offer, T. Miyamoto, A. Dongmo, M.A. Lacaille-Dubois, Chem. Pharm. Bull. 52 (2004) 1355.

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