Saponins As Cytotoxic Agents: a Review

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Phytochem Rev (2010) 9:425–474 DOI 10.1007/s11101-010-9183-z

Saponins as cytotoxic agents: a review

Irma Podolak • Agnieszka Galanty • Danuta Sobolewska

Received: 13 January 2010 / Accepted: 29 April 2010 / Published online: 25 June 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Saponins are natural glycosides which Con A Concanavalin A possess a wide range of pharmacological properties ER Endoplasmic reticulum including cytotoxic activity. In this review, the recent ERK Extracellular signal-regulated studies (2005–2009) concerning the cytotoxic activity kinase of saponins have been summarized. The correlations GADD Growth arrest and DNA damage- between the structure and the cytotoxicity of both inducible gene steroid and triterpenoid saponins have been described GRP Glucose regulated protein as well as the most common mechanisms of action. hTERT Telomerase reverse transcriptase JAK Janus kinase Keywords Cytotoxic mechanisms MEK = MAPK Mitogen-activated protein kinase Glycosides Sar Steroid Triterpenoid MMP Matrix metalloproteinase mTOR Mammalian target of rapamycin Abbreviations NFjB Nuclear factor kappa-light-chain- AMPK AMP activated protein kinase enhancer of activated B cells BiP Binding protein NO Nitric oxide BrDU Bromodeoxyuridine PARP Poly ADP ribose polymerase CCAAT Cytidine-cytidine-adenosine- PCNA Proliferating cell nuclear antigen adenosine-thymidine PI3K Phosphoinositide-3-kinase CD Cluster of differentiation molecule PP Protein phosphatase CDK Cyclin-dependent kinase PPAR-c Peroxisome proliferator-activated CEBP CCAAT-enhancer-binding protein receptor c CHOP CEPB homology protein Raf Serine/threonine speciﬁc kinase STAT Signal transducer and activator of transcription TIMP Tissue inhibitor of metalloproteinase TSC Tuberous sclerosis complex VEGF Vascular endothelial growth factor & I. Podolak ( ) A. Galanty D. Sobolewska XIAP X-linked inhibitor of apoptosis Department of Pharmacognosy, Jagiellonian University, Medical College, Medyczna 9, 30-688 Cracow, Poland protein e-mail: [email protected] 123 426 Phytochem Rev (2010) 9:425–474

Introduction Steroidal sapogenins (27C) can have a 6-ring spirostane or a 5-ring furostane skeleton whereas in Saponins are secondary metabolites of glycosidic the case of triterpenoid sapogenins (30C), which are nature widely distributed in higher plants but also much more structurally diverse, the basic ring system found in some animal sources, like e.g. marine is most often made of five or, more seldom, of four invertebrates. Despite their fairly large structural units. Pentacyclic triterpenoid saponins belong, in a diversity these compounds share some unique bio- majority of cases, to oleanane-type, other skeleton logical properties like the ability to lyse erythrocytes types include ursane, lupane, hopane, germanicane, or to foam (Bruneton1995; Rao and Gurfinkel 2000; dammarane (Vincken et al. 2007). The presence in Francis et al. 2002). The latter contributed to naming the polycyclic sapogenin of different substituents, this group ‘‘saponins’’, which is derived from Latin such as for example hydroxyls, hydroxymethyls, sapo meaning soap. Haemolysis of red blood cells carboxyls and acyl groups, as well as differences in seems to result from saponin ability to form com- the composition, linkage and number of sugar chains plexes with cell membrane cholesterol leading in account for significant structural diversity of saponins consequence to pore formation and cell permeabili- and also their diverse bioactivity. zation, and also to cause alterations in the negatively Saponins exert a wide range of pharmacological charged carbohydrate portions on the cell surface activities including expectorant, antiinflammatory, (Abe et al. 1981; Melzig et al. 2001; Gauthier et al. vasoprotective, hypocholesterolemic, immunomodu- 2009a). It should be mentioned however that the latory, hypoglycaemic, molluscicidal, antifungal, exact mechanism of haemolytic activity of saponins antiparasitic and many others (Sparg et al. 2004; is not clearly understood and is the subject of Sahu et al. 2008). Plants rich in saponins, like Panax discussions within the scientific community. ginseng or Glycyrrhiza glabra, have been used for Surface activity responsible for foaming proper- medicinal purposes since ancient times (Fiore et al. ties, as well as some other biological functions 2005) and to date continue to play a significant role including haemolytic activity, are attributed to char- not only in medicine but also in food and cosmetic acteristic structural features of saponins and their industry, where they are utilized as emulsifiers or amphiphilic nature which results from the presence of sweeteners (Gu¨çlu¨-U¨ stu¨ndag˘ and Mazza 2007). a hydrophilic sugar moiety and a hydrophobic genin Another important application of saponins is their (called sapogenin). Saponins can possess from one to use as adjuvants in the production of vaccines (Sun three straight or branched sugar chains, most often et al. 2009b). Steroidal sapogenins since many years composed of D-glucose, L-rhamnose, D-galactose, have served as economically important raw materials D-glucuronic acid, L-arabinose, D-xylose or D-fucose. for the pharmaceutical industry in the production of The sugar chain can contain from one to several steroidal hormones (Blunden et al. 1975;Gu¨çlu¨- monosaccharide residues, and is usually attached at U¨ stu¨ndag˘ and Mazza 2007). C-3 (Vincken et al. 2007). Many reviews summarized different aspects The aglycone may have steroid or triterpenoid related to saponins, some of the more recent tackled structure according to which saponins are usually biosynthesis (Haralampidis et al. 2002; Kalinowska classified. Steroidal compounds are less common and et al. 2005), distribution (Henry 2005; Vincken et al. usually found among the Liliopsida (former Mono- 2007), structures (Vincken et al. 2007; Sahu et al. cotyledones) in members of such families as Lilia- 2008), biological and pharmacological activities of ceae, Dioscoreaceae, Agavaceae, while triterpenoid saponins (Sparg et al. 2004; Yang et al. 2006; Wang saponins are more widely distributed and typical of et al. 2007; Sun et al. 2009b), application and the Magnoliopsida families (former Dicotyledones), processing (Gu¨çlu¨-U¨ stu¨ndag˘ and Mazza 2007), meth- e.g. Primulaceae, Sapotaceae, Caryophyllaceae and ods employed in their analysis (Oleszek and Bialy others. In rare cases both types of saponins may 2006; Sahu et al. 2008) and chemical synthesis of accumulate in a plant, like for example in Avena sp. saponins (Yu et al. 2007; Yu and Sun 2009; Gauthier (monocotyledonous Poaceae) (Osbourn 2003)or et al. 2009b). Lysimachia paridiformis (dicotyledonous Myrsina- Cytotoxicity and the chemopreventive role of ceae) (Xu et al. 2007b). saponins was also discussed in a number of review 123 Phytochem Rev (2010) 9:425–474 427 papers (Rao and Sung 1995; Konoshima 1996; As far as cytotoxicity data are concerned the Shibata 2001; Kerwin 2004;Kim2008; Bachran following observations can be made. Most studies et al. 2008; Fuchs et al. 2009). While most of these were performed on a relatively narrow range of cell focus on triterpenoids, and especially on ginseng and lines, usually from one to five, sometimes up to ten. soy saponins, chemopreventive and cancer control The most notable exceptions in this respect are the activity of a steroidal sapogenin—diosgenin has been reports, in which isolated compounds were assayed by recently summarized (Raju and Mehta 2009). Cyto- National Cancer Institute (NCI) in anticancer drug toxicity of dioscins was also discussed in a review on discovery screen. For example, Zhang (Zhang and Li bioactive steroid saponins from the Dioscorea genus 2007) tested triterpenoid saponins isolated from (Sautour and Mitaine-Offer 2007). Bachran et al. Aesculus pavia against a panel of 59 cell lines from (Bachran et al. 2008) in their review of saponins nine different human cancers such as leukemia, non- playing a role in tumor therapy focused on certain small cell lung, colon, CNS, melanoma, ovarian, renal, groups of compounds, such as dioscins, saikosapo- prostate and breast. Similarly, cytotoxicity of seven nins, julibrosides, avicins, soy and ginseng saponins, steroid saponins, isolated from Dioscorea collettii var. and on combinations of saponins and conventional hypoglauca, was compared on such a wide panel of anti-tumorigenic drugs. cancer cell lines (Sautour and Mitaine-Offer 2007). This review focuses on the studies from the last One of the saponins, furostanol protoneodioscin 5 years referring to saponins for which cytotoxic (NSC-698789) isolated from the rhizomes of this activity in vitro against human cancer cell lines was plant, showed high activity against leukemia lines reported. Some data from animal models are also (CCRF-CEM, K562 and MOLT-4), colon cancer lines presented. (HCT-15, KM12), CNS cancer line (SNB-75), mela- Due to enormous number of reports that appear noma line (M14), renal cancer line (CAKI-1), prostate worldwide in which some reference to cytotoxicity is cancer (DU-145) and breast cancer line (MDA-MB- -1 made we had to set some limits. Thus, literature 435) with GI50 B 2.0 lmol l . Leukemia, CNS can- searches were conducted in the following electronic cer, and prostate cancer appeared the most sensitive databases: MEDLINE/PubMed, SCOPUS/Elsevier, subpanels to protoneodioscin. A comparison with Springer/ICM, INSPEC/ICM, SCI-Ex/ICM. The other compounds in the NCI’s database indicated that search terms used were: saponins, cancer, cytotoxicity. protoneodioscin had a novel mechanism of anticancer In this review special emphasis is given to studies action (Hu and Yao 2002). Also, the corresponding on structure-activity relationship and a summary of methyl artifacts such as e.g. methyl protodioscin mechanisms of action is also provided. (Fig. 1), have shown cytotoxic activity at concentrations below 2 lmol l-1 in most studies performed so far (Hu and Yao 2003a; Wang et al. 2006). Cytotoxic saponins (2005–2009) Generally, in most papers cited here, the choice of cell lines seems to be random, most studies with a In the past 5 years a relatively large number of wider range of lines use leukemia and solid tumors publications devoted to various aspects of phyto- cells.Very few studies compared cells from the same chemical analysis of saponins contained also some tumor but differing in malignancy or other charac- references to their biological activity including cyto- teristics. For example, Tapandjou et al. and Note´ toxicity. These data are summarized in Tables 1 et al. evaluated cytotoxicity of isolated triterpene and 2. saponins against two colon cancer cell lines, HCT- In the majority of cases, while structure elucida- 116 and HT-29 (Tapondjou et al. 2006; Note´ et al. tion process was the main focus of the research, 2009). There were slight differences in the potency of cytotoxic activity tests were performed as additional tested compounds, however none of them seemed to part of experimental studies. However, there was also be selective. In a study by Einbond et al., saponins a considerable number of reports in which the from Cimicifuga sp. were tested in two human breast mechanisms of action or structure-activity relation- cancer lines, MDA-MB-453 (ER negative, Her2 ships were tackled in more detail and these are overexpressing) and MCF-7 (ER positive, Her2 reviewed in the following sections of this paper. low). The latter cells were found much more resistant 123 428 123 Table 1 Cytotoxic triterpene saponins (2005–2009) Source Saponoside Cell line Concentration Assay/effect References

21 Aesculus pavia L. 5 saponins (21, 22, 26, 27, and 28) 59 cell lines from nine different human cancers IC50 lmol l SRB; cytotoxic Zhang and Li including leukemia, non-small cell lung, (2007) colon, CNS, melanoma, ovarian, renal, prostate, and breast (Hippocastanceae) Compounds 26– 28 0.175–8.71 21 Albizia chinensis Albizosides A-C, most potent: IC50 lmol l MTT; cytotoxic Liu et al. (2009b) (Osb.) Merr., Albizoside C HCT-8 0.4 (Fabaceae/ Mimosoidae) Bel-7402 0.4 BGC-823 1.7 A549 0.01 A2780 0.3 Control: camptothecin HCT-8 3.2 Bel-7402 12.5 BGC-823 9.7 A549 3.1 A2780 0.3 21 Albizia coriaria IC50 [lmol l ] MTT; cytotoxic Note´ et al. (2009) Welw. ex Oliv. (Fabaceae/

Mimosoidae) 9:425–474 (2010) Rev Phytochem Coriarioside A HCT 116 4.2 HT-29 2.7 Gummiferaoside C HCT 116 6.7 HT-29 7.9 Control: paclitaxel HCT 116 3.5 9 10-3 HT-29 3.4 9 10-3 htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

21 Albizia gummifera IC50 [lmol l ] CellTiter Glo Cao et al. (2007) luminescent; antiproliferative (J. F. Gmel.) C. A. Gummiferaoside A A2780 0.37 Sm. var. gummifera (Fabaceae/ Mimosoidae) MDA-MB-435 0.84 HT-29 6.61 H522-T1 [10 U937 0.19 Gummiferaoside B A2780 0.70 MDA-MB-435 ND HT-29 ND H522-T1 ND U937 ND Gummiferaoside C A2780 0.26 MDA-MB-435 0.48 HT-29 0.61 H522-T1 0.64 U937 0.29 Control: actinomycin D, vinblastine— data not given Albizia julibrissin Julibroside J21 Bel-7402 10 lgml-1 SRB; cytotoxic Zou et al. DURAZZ. (80.8% inhib.) (2006b) (Fabaceae/ Mimosoidae) 21 Albizia procera 3-O-[b-D-Xyl-(1 ? 2)-a-L-Ara-(1 ? 6)-2- HEPG2 IC50 [lgmll ] SRB; cytotoxic Melek et al. (Fabaceae/ acetamido-2-deoxy-b-D-Glc] echinocystic 9.13 (2007) Mimosoidae) acid 123 3-O-[a-L-Ara-(1 ? 2)-a-L-Ara-(1 ? 6)-2- 10.0 acetamido-2-deoxy-b-D-Glc] echinocystic acid 429 430 123 Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

21 Ardisia japonica 11 ardisianosides A-K together with 10 known IC50 [lmol l ] MTT; cytotoxic Chang et al. (Thunb.) Bl. saponins; most potent: HL-60 1.9 (2007) (Myrsinaceae) 3b-O-b-D-Glc-(1 ? 2)-[a-L-Rha-(1 ? 2)-b-D- KATO-III 0.4 Glc-(1 ? 4)]-a-L-Ara-13b,28-epoxy-16a- hydroxyoleanane A549 3.7 Control: etoposide HL-60 0.3 KATO-III 0.1 A549 20.9 21 Ardisia pusilla A. 5 saponins, most potent: IC50 [lmol l ] MTT; cytotoxic Tian et al. (2009) DC (Myrsinaceae) 3b-O-{b-D-Glc-(1 ? 2)-{b-D-Xyl-(1 ? 2)-[b- U251MG 2.57 D-Glc-(1 ? 3)-b-D-Glc-(1 ? 3)]-b-D-Glc- (1 ? 4)}-a-L-Ara}-16a-hydroxy-13b,28- epoxy-oleanan-30-al Human astrocytes [100 Control: nimustine hydrochloride U251MG 0.98 Human astrocytes [100 21 Bacopa monnieri 15 saponins, most potent: Brine shrimp IC50 [lgml ] Brine shrimp Sivaramakrishna lethality assay et al. (2005) (Linn.) Pennel Bacopasaponin C 3.9 9:425–474 (2010) Rev Phytochem (Scrophulariaceae) Control: podophyllotoxin 4.5 21 Caryocar Caryocaroside II-7 Human keratinocytes LC50 [lmol l ] XTT; cytotoxic Magid et al. 23.0 (2006) Villosum (Aubl.) Caryocaroside II-22 20.9 Pers. (Caryocaraceae) htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

21 Cephalaria 11 saponins, most potent: IC50 [lmol l ] MTT; cytotoxic Tabatadze et al. gigantea (2007) (LEDEB.) BOBROV (Dipsacaceae) Giganteoside D MEL-5 7.50 HL-60 3.15 Giganteoside E MEL-5 7.50 HL-60 6.80 Control: camptothecin MEL-5 0.40 HL-60 0.05 21 Cimicifuga sp. Series of triterpene glycosides, most potent: IC50 [lmol l ] MTT; coulter Einbond et al. (Ranunculaceae) counter; (2008) antiproliferative 25-acetyl-7,8-didehydrocimigeno 3-O-b-D-Xyl MDAMB-453 3.2 Cucumaria frondosa 3 saponins, most potent: HL-60 2 lmol l-1 NRR; cytotoxic Jin et al. (2009) (Cucumariidae) Frondoside A 21 Dianthus versicolor 9 compounds, most potent: IC50 [lgmll ] MTT; cytotoxic Ma et al. (2009) Fisch. ex Link. (Caryophyllaceae) dianversicoside C HFL-I 3.2 EVC-304 3.6 BGC-803 3.1 MCF-7 [10 Hep G2 [10 Control: cisplatin HFL-I 0.11 EVC-304 0.49 BGC-803 6.8

123 MCF-7 3.9 Hep G2 7.7 431 432 123 Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

21 Duranta repens LC50 [lgmll ] SRB; cytotoxic Ahmed et al. (Verbenaceae) (2009) Durantanin IV HepG2 1.68 Durantanin V 1.07 21 Eclipta prostrata IC50 [lgml ] MTT; cytotoxic Khanna and (Asteraceae) Kannabiran (2009) Dasyscyphin C HeLa 50.0 Control: 5- ﬂuorouracil 36 21 Glinus lotoides L. Lotoidosides A-G, most potent: IC50 [lmol l ] MTT; Hamed et al. (Molluginaceae) antiproliferative (2005) Lotoidoside E J774.A1 0.018 HEK-293 0.02 WEHI-164 0.03 Control: 6- mercaptopurine J774.A1 0.003 HEK-293 0.007 WEHI-164 0.017 21 Glochidion J. R. IC50 [lmol l ] MTT; Kiem et al. FORST. & G. antiproliferative (2009) FORST. (Euphorbiaceae)

Glochierioside A HL-60 5.5 9:425–474 (2010) Rev Phytochem HT-29 6.8 MCF-7 29.1 SK-OV-3 22.7 Glochierioside B HL-60 6.6 HT-29 18.6 MCF-7 36.1 SK-OV-3 16.0 Control: mitoxantrone htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

HL-60 7.2 HT-29 8.4 MCF-7 10.3 SK-OV-3 12.1 21 Gordonia Gordonosides, most potent: IC50 [lmol l ] MTT; cytotoxic Yu et al. (2009) chrysandra Cowan (Theaceae) Gordonoside C HCT-8 1.2 Bel-7402 0.7 BGC-823 2.5 A549 1.8 A2780 0.4 Control: paclitaxel HCT-8 3.6 Bel-7402 6.3 BGC-823 0.04 A549 1.0 9 10-3 A2780 0.9 21 Gymnema sylvestre Gymnemagenol HeLa IC50 [lmol l ] MTT; cytotoxic Khanna and (Asclepiadaceae) 37.0 Kannabiran (2009) Control: 5- ﬂuorouracil 36.0 21 Hydrocotyle Several saponins, most potent: ED50 [lmol l ] MTT; cytotoxic Huang et al. sibthorpioides (2008b) (Apiaceae) Hydrocosisaponin E KB 8.54 Daoy 5.21 WiDr 5.06

123 KB 14.03 Daoy 4.89

WiDr 4.24 433 434 123 Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

Control: mitomycin-C Hydrocotyloside VII KB 0.04 Daoy 0.07 WiDr 0.05 21 Impatiens siculifer 19 saponins, most potent: IC50 [lmol l ] MTT; cytotoxic Li et al. 2009 (Balsaminaceae) Impatienoside G HL-60 21.8 KATO-III 36.7 A549 24.8 Control: etoposide HL-60 0.3 KATO-III 0.1 A549 20.6 21 Lonicera Macranthoside B IC50 [lmol l ] MTT; Wang et al. macranthoides antiproliferative (2009) (Caprifoliaceae) HepG2 10.1 MGC-803, 16.8 MCF-7 12.61 SW1116 14.55 U251 18.26 C6 14.35 B16F1 15.30

B16F10 16.74 9:425–474 (2010) Rev Phytochem 21 Lysimachia Capilliposide D A2780 IC50 [lgmll ] No data; Tian et al. (2006) capillipes Hemsl 0.2 lgml-1 cytotoxic (Myrsinaceae/ Primulaceae) Lysimachia 25 lgml-1 Trypan blue Galanty et al. thyrsiﬂora L. exclusion; (2008) (Myrsinaceae/ cytotoxic Primulaceae) htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

LTS-4 HTB-140 (50.1% dead HSF cells) (68.3% dead cells) -1 Panax ginseng C. A. Ginsenoside Rk1 HepG2 75 lmol l Cell counting kit- Kim et al. MEYER (55% dead cells) 8 (tetrazolium (2008a, b) (Araliaceae) salt); cytotoxic 21 Panax 10 saponins, most potent: IC50 [lgmll ] MTT; cytotoxic Qiu et al. (2009) quinquefolium (Araliaceae)

Ginsenoside Rk1 MCF-7 20.0 21 Physena sessiliﬂora LD50 [lgmll ] Brine shrimp Inoue et al. Tul. lethality assay; (2009) (Capparidaceae) cytotoxic Physenoside S7 Brine shrimp 8.5 Physenoside S8 22.1 Control: etoposide phosphate 3.5 21 Physospermum 4 saponins, most potent: buddlejasaponin IV IC50 [lmol l ] SRB; cytotoxic Tundis et al. verticillatum (2009) (Apiaceae) ACHN 7.9 A375 14.6 C32 26.9 MCF-7 [50 LNCaP [50 A549 38.3 Huh-7D12 [50 COR-L23 0.4 CaCo-2 40.7 142BR [50 123 Control for MCF- 7: TaxolÒ

0.09 435 436 123 Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

21 Pseudocolochirus IC50 [lmol l ] SRB; cytotoxic Zhang et al. violaceus (2007) (Cucumariidae) 3-O-{6-O-sodiumsulfate-3-O-methyl-b-D-Glc- MKN-45 0.442 (1 ? 3)-b-D-Xyl-(1 ? 4)-[b-D-Xyl-(1 ? 2)]-b -D-Qui-(1 ? 2)-4-O-sodiumsulfate-b-D- Xyl}-16b-acetoxy-holosta-7, 24-diene-3b-ol HCT-116 0.052 Intercedenside B MKN-45 0.378 HCT-116 0.066 Control: 10-hydroxycamptothecin MKN-45 0.164 HCT-116 0.135 21 Pulsatilla koreana 17 saponosides; most potent: oleanolic acid ED50 [lmol l ] SRB; Bang et al. N. 3-O-a-L-Rha-(1 ? 2)-[b-D-Glc-(1 ? 4)]-a- antiproliferative (2005) (Ranunculaceae) L-Ara A-549 2.56 SK-OV-3, 2.31 SK-MEL-2 1.57 HCT15 8.36 Control: doxorubicin A-549 0.017

SK-OV-3, 0.094 9:425–474 (2010) Rev Phytochem SK-MEL-2 0.036 HCT15 0.792 21 Sapindus mukorossi 13 saponins most potent: sapinmusaponin K ED50 [lmol l ] MTT; cytotoxic Huang et al. Gaertn. (2008a) (Sapindaceae) Hela 18.10 WiDr 19.31 KB 5.49 Daoy 7.09 htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

Hepa59T/VGH 11.07 Control: mitomycin-C Hela 0.52 WiDr 0.38 KB 0.70 Daoy 0.38 Hepa59T/VGH 0.35 21 Schefﬂera 5 saponins, most potent: IC50 [lgmll ] MTT; cytotoxic Tapondjou et al. abyssinica (2006) (Hochst. Ex A. Rich.)Harms (Araliaceae) Fatsiaside C1 HCT 116 \4 Guaianin N HT-29 \4 Control: paclitaxel HCT 116 9.9 9 10-3 HT-29 14.4 9 10-3 21 Sideroxylon IC50 [lgmll ] MTT; cytotoxic Sa´nchez-Medina foetidissimum et al. (2009) subsp. gaumeri (Sapotaceae) A 5:4 mixture comprising 3-O-(b-D-Glc)-28-O- RAW 264.7 11.9 (a-L-Rha-(1 ? 3)-b-D-Xyl-(1 ? 4)[b-D-Api- (1 ? 3)]-a-L-Rha-(1 ? 2)-a-L-Ara)-16a- hydroxy-protobassic acid and Control: podophyllotoxin -1 3-O-(b-D-Api-(1 ? 3)-b-D-Glc)-28-O-(a-L- 29.0 nmol l Rha-(1 ? 3)[b-D-Xyl-(1 ? 4)]-b-D-Xyl- (1 ? 4)-a-L-Rha-(1 ? 2)-a-L-Ara)-16a- hydroxy-protobassic acid 123 437 438 123 Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

Silphium radula (urs-12-ene-3b,6b,6b-triol-3-O-b-Gal- MDA-MB-231 25 lgml-1 (80% PI; Calabria et al. Nutt. (Asteraceae) (1 ? 2)-b-Glc inhib.) antiproliferative (2008) 21 Synapta maculata Synaptoside A HeLa IC50 [lgmll ] MTS; cytotoxic Avilov et al. (Synaptida, 8.6 (2008) Apodida) 21 Synthetic betulinic 16 compounds; most potent: 1-O-[3-b-acetoxy- IC50 [lmol l ] Calcein AM Cmoch et al. acid derivatives lup-20(29)-ene-28-oyl]-a-D-Mann Fluorescent (2008) viability; cytotoxic CEM 10.4 MCF 7 22.7 A-549 43.3 HeLa 34.7 BJ-H-tert 38.7 RPMI 8226 19.4 G 361 22.7 Control: betulinic acid CEM 40 MCF 7 [50 A-549 [50 HeLa 47.6 BJ-H-tert [50 RPMI 8226 34.6 G 361 [50 htce e 21)9:425–474 (2010) Rev Phytochem 21 Xanthoceras 6 saponins: most potent xanifolia-Y3 IC50 [lmol l ] MTT; Chan et al. sorbifolia Bunge antiproliferative (2008) (Sapindaceae) OVCAR3 4.0 htce e 21)9:425–474 (2010) Rev Phytochem Table 1 continued Source Saponoside Cell line Concentration Assay/effect References

Control: paclitaxel 1.0 10-3 9 10-3 Cancer cell lines: Prostate: LNCaP, PC-3, DU-145; Leukemia: CEM, K562, HL60, *L1210, P388; Ovarian: SK-OV-3, A2780, OVCAR3, HO-8910; Breast: MDA-MB-231, MCF-7, MDA-MB-453, MDA-MB-435; Lung: A-549, H522-T1, COR-L23, NCI-H460, LTEP-a-2, *LA795, SPC-A-1; Melanoma: G361, MEL-5, A375, C32, *B16F1, *B16F10, SK-MEL-2, HTB-140; Colon: HCT-15, HT-29, WiDr, HCT116, CaCo-2, SW1116, HCT-8, DLD-1, LoVo, KM20L2; Lymphoma: U937; Pharynx: CNE, KB, Eca-109; Liver: HepG2, Huh-7D12, Bel-7402, Hepa59T/VGH, SMMC7721; Stomach: MGC-803, MKN-45, BGC-803, BGC823, KATO-III, SGC7901, AGS; Pancreas: Bx-PC3; CNS: U251, SF268, *C6, U251MG, U373, Daoy, XF498; Renal: ACHN; Bone: 1547, *WEHI-164, *XC, RPMI 8226; Uterus: HeLa Normal cell lines: Fibroblasts: HSF, BJ-H-tert, 142BR, HFL-I, WS1; Macrophages: *RAW 264.7, *J774.A1; Epithelium/endothelium: EVC-304, MCF-10A; Kidney embryonic: HEK-293. * Murine origin

Table 2 Cytotoxic steroid saponins (2005–2009) Source Saponoside Cell line Concentration Assay References

-1 Agave utahensis IC50 [lgmll ] MTT; Yokosuka (Agavaceae) atiproliferative and Mimaki 2009 (25R)-2b-hydroxy-5a-spirostan-3b-yl HL-60 12.3 O-b-D-Xyl-(1 ? 2)- O-b-D-Glc- (1 ? 4)-b-D-Gal (25R)-5a-spirostan-3b-yl O-b-D-Glc- 9.4 (1 ? 2)-O-[b-D-Glc-(1 ? 3)]- O-b- D-Glc-(1 ? 4)-b-D-Gal (25R)-2a-hydroxy-5a-spirostan-3b-yl 5.5 O-b-D-Glc-(1 ? 2)-O-[b-D-Glc- (1 ? 3)]- O-b-D-Glc-(1 ? 4)-b-D- Gal (25R)-2a-hydroxy-5a-spirostan-3b-yl 11.3 O-b-D-Glc-(1 ? 2)-O-b-D-Glc- (1 ? 4)-b-D-Gal Control: etoposide 0.20 (25R-5b-spirostan-3b-yl O-b-D-Glc– 4.90 Yokosuka (1?4)-b-D-Gal et al. (2009) -1 Allium leucanthum IC50 [lmol l ] Resazurin Mskhiladze (Alliaceae) reduction; et al. antiproliferative (2008) Yayoisaponin A549 3.7 DLD-1 5.6 WS1 3.0 Eruboside B A549 5.3 DLD-1 8.2 WS1 3.6 Aginoside A549 5.8 DLD-1 7.9 WS1 3.6 (25R)-5a-spirostane-3b,6b-diol 3-O- A549 9.0 b-D-Glc-(1 ? 2)-[b-D-Xyl- DLD-1 13.0 (1 ? 3)]-b-D-Glc-(1 ? 4)-b-D-Gal WS1 3.1 Leucospiroside A A549 5.0 DLD-1 7.2 WS1 4.55 (25R)-5a-spirostane-2a,3b,6b-triol A549 22.0 3-O-b-D-Glc-(1 ? 2)-b-D-Glc- DLD-1 22.0 (1 ? 4)-b-D-Gal WS1 14.5 (25R)-5a-spirostane-3b,6b-diol 3-O- A549 7.8 b-D-Glc-(1 ? 3)-b-D-Glc-(1 ? 2)- DLD-1 8.9 [b-D-Glc-(1 ? 3)]-b-D-Glc- WS1 7.7 (1 ? 4)-b-D-Gal Control: etoposide

123 Phytochem Rev (2010) 9:425–474 441

Table 2 continued Source Saponoside Cell line Concentration Assay References

A549 1.1 DLD-1 4.8 WS1 Not determined Control: 5-fluorouracil A549 48.0 DLD-1 11.0 WS1 20.01 -1 Allium IC50 [lmol l ] MTT; cytotoxic Chen et al. macrostemon (2009) (Alliaceae) 26-O-b-D-Glc-5a-furost-25(27)-ene- SF-268 35.2 3b,12b,22,26-tetraol 3-O-b-D-Glc- (1 ? 2)-[b-D-Glc-(1 ? 3)]-b-D- Glc-(1 ? 4)-b-D-Gal 26-O-b-D-Glc-5b-furost-20(22)- NCI-H460 25.7 25(27)-dien-3b,12b,26-triol 3-O-b- D-Glc-(1 ? 2)-b-D-Gal SF-268 35.4 Allium ursinum A mixture of: 2 lgml-1 Trypan Blue Sobolewska (Alliaceae) et al. (2006) (25R)-spirost-5-en-3b-ol 3-O-a-L-Rha- B16 (100% dead cells) Exclusion; (1 ? 4)-a-L-Rha-(1 ? 4)-[a-L-Rha- (1 ? 2)]-b-D-Glc and XC (100% dead cells) cytotoxic (25R)-spirost-5,25(27)-dien-3b-ol 3-O-a-L-Rha-(1 ? 4)-a-L-Rha- (1 ? 4)-[a-L-Rha-(1 ? 2)]-b-D-Glc 21 Anemarrhena IC50 [lgml ] MTT; cytotoxic Bao et al. asphodelodes (2007) (Liliaceae) Sarsapogenin HepG2 42.4 (48 h) 25.0 (72 h) 21 Asparagus filicinus IC50 [lgml ] SRB; cytotoxic Zhou et al. (Asparagaceae) (2007) Filiasparoside A A549 13.3 MCF-7 11.2 Filiasparoside B A549 16.4 MCF-7 16.8 Filiasparoside C A549 2.3 MCF-7 3.0 Filiasparoside D A549 2.4 MCF-7 10.3 Aspafilioside A A549 9.4 MCF-7 [20 Aspafilioside B A549 7.6 MCF-7 10.4

123 442 Phytochem Rev (2010) 9:425–474

Table 2 continued Source Saponoside Cell line Concentration Assay References

A549 Control: etoposide 0.8 MCF-7 2.8 -1 Asparagus Asparanin A HepG2 IC50 [lmol l ] MTT; cytotoxic Liu et al. ofﬁcinalis (2009a) (Asparagaceae) 6.20 (24 h) 4.11 (48 h) 2.90 (72 h) -1 Asparagus IC50 [lmol l ] MTT; cytotoxic Huang et al. ofﬁcinalis (2008c) (Asparagaceae) (25S)-5b-spirostan-3b-ol 3-O-b-D- A2780 10.57 Glc-(1 ? 2)-b-D-6-O-acetyl-Glc MGC-803 8.39 CNE 5.50 Asparagoside A A2780 10.22 HO-8910 5.04 (25R)-5b-spirostan-3b-ol 3-O-b-D-Glc HO-8910 24.83 L1210 12.33 Sarsasapogenin A2780 6.09 (25S)-neospirost-4-en-3-one A2780 18.85 Eca-109 2.91 (25S)-5b-spirostan-3b-ol 3-O-b-D- LTEP-a-2 2.85 Glc-(1 ? 2)-[b-D-Xyl-(1 ? 4)]- b-D-Glc KB 2.66 L1210 1.46 (25S)-5b-spirostan-3b-ol 3-O-b-D- Eca-109 4.03 Glc-(1 ? 2)-b-D-Glc MGC-803 3.72 LTEP-a-2 3.16 KB 1.38 L1210 4.32 (25S)-5b-spirostan-3b-ol 3-O-a-L- Eca-109 10.15 Rha-(1 ? 2)-[a-L-Rha-(1 ? 4)- b-D-Glc] CNE 12.88 Control: doxorubicin A2780 0.15 HO-8910 1.45 Eca-109 0.36 MGC-803 0.72 CNE 0.58 LTEP-a-2 1.33 KB 0.43

123 Phytochem Rev (2010) 9:425–474 443

Table 2 continued Source Saponoside Cell line Concentration Assay References

L1210 0.26 21 Asparagus IC50 [lgml ] SRB; cytotoxic Kim et al. oligoclonos (2005) (Asparagaceae) Asparanin A A549 2.05 SK-OV-3 2.43 SK-MEL-2 2.41 XF498 2.56 HCT15 2.47 Aspaoligonin A A549 2.25 SK-OV-3 2.66 SK-MEL-2 2.53 XF498 2.84 HCT15 2.59 Aspaoligonin B A549 2.55 SK-OV-3 2.73 SK-MEL-2 2.59 XF498 2.51 HCT15 2.57 Control: adriamycin A549 0.004 SK-OV-3 0.18 SK-MEL-2 0.03 XF498 0.012 HCT15 0.08 -1 Balanites IC50 [lmol l ] MTT; cytotoxic Gnoula et al. aegyptiaca (2008) (Balanitaceae) A mixture of balanitin-6 and A549 0.3 balanitin-7 U373 0.5 PC-3 0.9 Bx-PC3 1.2 LoVo 1.5 MCF-7 2.6 Control: oxaliplatin A549 3.9 U373 3.3 PC-3 9.2 Bx-PC3 [10.0 LoVo 4.7 MCF-7 8.9 Control: etoposide A549 3.1 U373 1.4 PC-3 [10.0 123 444 Phytochem Rev (2010) 9:425–474

Table 2 continued Source Saponoside Cell line Concentration Assay References

Bx-PC3 [10.0 LoVo 4.9 MCF-7 [10.0 Control: 7-ethyl-10- hydroxycamptothecin A549 0.04 U373 0.001 PC-3 0.2 Bx-PC3 0.07 LoVo 0.4 MCF-7 0.03 Control: TaxolÒ A549 0.009 U373 0.001 PC-3 0.002 Bx-PC3 0.003 LoVo 0.005 MCF-7 0.001 -1 Chlorophytum Borivilianoside H IC50 [lmol l ] MTT; cytotoxic Acharya borivilianum et al. (Liliaceae) (2009) HT-29 2.6 HCT 116 0.38 Control: paclitaxel HT-29 3.6 9 10-3 HCT 116 1.1 9 10-3 -1 Dioscorea Pennogenin-3-O-a-L-Rha-(1 ? 3)-[a- Bel-7402 10 lmol l (99.1% MTT; Liu et al. bulbifera L-Rha-(1 ? 2)]-b-D-Glc inhib.) antiproliferative (2009a) (Dioscoreaceae) -1 SMMC7721 IC50 4.54 lmol l -1 Pennogenin-3-O-a-L-Rha-(1 ? 4)-[a- Bel-7402 10 lmol l (92.6% L-Rha-(1 ? 2)]-b-D-Glc inhib.) -1 SMMC7721 IC50 4.85 lmol l -1 Dioscorea villosa IC50 [lmol l ] MTT; Hu et al. (Dioscoreaceae) antiproliferative (2007) (25R)-spirost-5-en-3b-ol 3-O-a-L-Rha- HepG2 9.02 (1 ? 2)-O-[b-D-Glc-(1 ? 4)]-b-D- Glc HEK293 13.21 MCF7 16.74 -1 Paris polyphylla IC50 [lmol l ] MTT; cytotoxic Siu et al. (Liliaceae) (2008) Polyphyllin D NCI-H460 3.0 SF-268 3.0 Hep G2 3.5 HeLa 3.7 MCF-7 3.7

123 Phytochem Rev (2010) 9:425–474 445

Table 2 continued Source Saponoside Cell line Concentration Assay References

HEK-293 11.0 Control: cisplatin NCI-H460 25 SF-268 15.6 Hep G2 20.2 HeLa 11.6 MCF-7 19 HEK-293 [100 Paris polyphylla 2.5 lgml-1 MTT; cytotoxic Yun et al. var. chinensis (2007) (Liliaceae) Diosgenin-3-O-a-L-Rha-(1 ? 2)-O- HepG2 60.59% dead cells b-D-Glc SGC7901 70.14% dead cells BxPC3 76.52% dead cells Pennogenin-3-O-a-L-Rha-(1 ? 2)- HepG2 44.22% dead cells O-b-D-Glc SGC7901 50.13% dead cells BxPC3 73.32% dead cells Diosgenin-3-O-a-L-Rha-(1 ? 2)- HepG2 83.76% dead cells [a-L-Ara-(1 ? 4)]-b-D-Glc SGC7901 88.19% dead cells BxPC3 87.06% dead cells Pennogenin-3-O-a-L-Rha-(1 ? 2)- HepG2 65.22% dead cells [a-L-Ara-(1 ? 4)]-b-D-Glc SGC7901 70.39% dead cells BxPC3 79.64% dead cells Diosgenin-3-O-a-L-Rha-(1 ? 2)- HepG2 68.35% dead cells [b-D-Glc-(1 ? 3)]-b-D-Glc SGC7901 77.34% dead cells BxPC3 76.63% dead cells Diosgenin-3-O-a-L-Rha-(1 ? 4)- a-L- HepG2 61.70% dead cells Rha-(1 ? 4)-[a-L-Rha-(1 ? 2)]-b- D-Glc SGC7901 75.81% dead cells BxPC3 82.92% dead cells Pennogenin-3-O-a-L-Rha-(1 ? 4)- a- HepG2 35.75% dead cells L-Rha-(1 ? 4)-[a-L-Rha-(1 ? 2)]- b-D-Glc SGC7901 47.58% dead cells BxPC3 81.77% dead cells Control: epirubicin- HCl HepG2 78.89% dead cells SGC7901 70.73% dead cells BxPC3 65.78% dead cells

123 446 Phytochem Rev (2010) 9:425–474

Table 2 continued Source Saponoside Cell line Concentration Assay References

-1 Paris polyphylla IC50 [lmol l ] MTT; cytotoxic Yan et al. var. yunnanensis (2009) (Liliaceae) Diosgenin-3-O-a-L-Ara-(1 ? 4)- LA795 1.85 [a-L-Rha-(1 ? 2)]-b-D-Glc Pennogenin-3-O-a-L-Ara-(1 ? 4)- 5.14 [a-L-Rha-(1 ? 2)]-b-D-Glc Diosgenin-3-O-a-L-Rha-(1 ? 4)- 3.06 [a-L-Rha-(1 ? 2)]-b-D-Glc Pennogenin-3-O-a-L-Rha-(1 ? 4)- 2.26 [a-L-Rha-(1 ? 2)]-b-D-Glc Diosgenin-3-O-a-L-Rha-(1 ? 4)- 1.35 [a-L-Rha-(1 ? 4)]-[a-L-Rha- (1 ? 2)]-b-D-Glc 21 Paris polyphylla IC50 [lgml ] MTT; cytotoxic Zhao et al. var. yunnanensis (2009) (Liliaceae) Pennogenin-3-O-a-L-Ara-(1 ? 4)- HL-60 0.51 [a-L-Rha-(1 ? 2)]-b-D-Glc Tg 0.30 Tb 1.72 Pennogenin-3-O- b-D-Glc-(1 ? 3)- 3.75 a-L-Rha-(1 ? 2)]-b-D-Glc Pa 0.18 Pb 0.14 Gracillin 0.53 Prosapogenin A of dioscin 0.38 Reclinatoside 0.52 Loureiroside 0.72 Control: cis- diamminedichloro- platinum 0.038 21 Polygonatum IC50 [lgml ] MTT; cytotoxic Yang and punctatum Yang (Liliaceae) (2006) Polypunctoside A HeLa 39.8 Polypunctoside B 3.2 Polypunctoside C 2.5 Polypunctoside D 2.6 Dioscin 20.4 Control: cisplatin 0.75 21 Sansevieria GI50 [lgml ] SRB; Pettit et al. ehrenbergii antiproliferative (2005) (Agavaceae) Sansevistatin 1 P388 1.6 BXPC-3 1.1

123 Phytochem Rev (2010) 9:425–474 447

Table 2 continued Source Saponoside Cell line Concentration Assay References

MCF-7 1.1 SF-268 1.3 NCI-H460 0.43 KM20L2 0.47 DU-145 1.0 Sansevistatin 2 P388 1.7 BXPC-3 0.93 MCF-7 0.62 SF-268 0.68 NCI-H460 0.26 KM20L2 0.22 DU-145 0.42 Dioscin P388 1.5 BXPC-3 1.1 MCF-7 1.6 SF-268 1.2 NCI-H460 1.6 KM20L2 1.6 DU-145 1.6 3b-O-[a-L-Rha-(1 ? 2)]-b-D-Glc- P388 1.5 (25R)-spirost-5-ene BXPC-3 1.8 MCF-7 2 SF-268 1.8 NCI-H460 1.8 KM20L2 1.7 DU-145 1.6 3b-O-[a-L-Rha-(1 ? 2)]-[b-D-Xyl- P388 2.6 (1 ? 4)]- b-D-Glc-(25R)-spirost- 5-ene BXPC-3 1.7 MCF-7 1.4 SF-268 1.3 NCI-H460 1.2 KM20L2 0.5 DU-145 1.1 -1 Solanum nigrum IC50 [lmol l ] MTT; cytotoxic Zhou et al. (Solanaceae) (2006) Degalactotigonin HepG2 0.25 NCI-H460 4.49 MCF-7 1.57 SF-268 3.19 Control: 10-hydroxy- camptothecine HepG2 6.49 NCI-H460 38.55 123 448 Phytochem Rev (2010) 9:425–474

Table 2 continued Source Saponoside Cell line Concentration Assay References

MCF-7 19.12 SF-268 29.88 21 Solanum torvum IC50 [lgml ] MTT; cytotoxic Lu et al. (Solanaceae) (2009) Yamogenin-3-O- b-D-Glc-(1 ? 6)- MGC-803 25.2 b-D-Glc HepG2 32.7 AS49 34.2 MCF-7 29.4 Neochlorogenin-3-O- b-D-Glc- MGC-803 57.5 (1 ? 6)-b-D-Glc HepG2 65.5 AS49 58.3 MCF-7 48.4 Control: cis-diammine- dichloroplatinum MGC-803 14.4 HepG2 13.2 AS49 14.1 MCF-7 15.3 -1 Tribulus IC50 [lmol l ] Acid phosphatase Perrone parvispinus assay; et al. (Zygophyllaceae) (2005) (25R)-26-O-b-D-Glc-5a-furostan- U937 0.5 3b,22a,26-triol 3-O-{b-D-Gal- (1 ? 2)-O-[b-D-Xyl-(1 ? 3)]-O- b-D-Glc-(1 ? 4)-b-D-Gal} Gitonin U937 0.1 WST1; cytotoxic MCF-7 3.0 HepG2 3.0 Control: resveratrol U937 17.0 MCF-7 100 HepG2 50 Tupistra chinensis 10 lgml-1 MTT; Zou et al. (Liliaceae) antiproliferative (2006a) Fraction containing: HeLa (81% inhib.) (25S)-26-O-(b-D-Glc)-furost-5-en- HL-60 (92% inhib.) 3b,22a,26-triol 3-O- b-D-Glc- (1 ? 2)-b-D-Glc-(1 ? 4)-b-D-Glc and (25R)-26-O-(b-D-Glc)-furost-5-en- 3b,22a,26-triol 3-O-b-D-Glc- (1 ? 2)-b-D-Glc-(1 ? 4)-b-D-Glc 21 Ypsilandra IC50 [lgml ] MTT; cytotoxic Xie et al. thibetica (2009) (Liliaceae) Ypsilandroside G K562 4.4

123 Phytochem Rev (2010) 9:425–474 449

Table 2 continued Source Saponoside Cell line Concentration Assay References

Pennogenin-3-O-a-L-Rha-(1 ? 2)-[a- SPC-A-1 6.3 L-Rha-(1 ? 4)- a-L-Rha-(1 ? 4)]- b-D-Glc BGC-823 6.9 Eca-109 8.0 AGS 8.9 K562 4.7 SPC-A-1 6.3 BGC-823 6.9 Eca-109 8.0 AGS 8.9 Control: cisplatin— data not given Cancer cell lines: Prostate: LNCaP, PC-3, DU-145; Leukemia: CEM, K562, HL60, *L1210, P388; Ovarian: SK-OV-3, A2780, OVCAR3, HO-8910; Breast: MDA-MB-231, MCF-7, MDA-MB-453, MDA-MB-435; Lung: A-549, H522-T1, COR-L23, NCI- H460, LTEP-a-2, *LA795, SPC-A-1; Melanoma: G361, MEL-5, A375, C32, *B16F1, *B16F10, SK-MEL-2, HTB-140; Colon: HCT-15, HT-29, WiDr, HCT116, CaCo-2, SW1116, HCT-8, DLD-1, LoVo, KM20L2; Lymphoma: U937; Pharynx: CNE, KB, Eca- 109; Liver: HepG2, Huh-7D12, Bel-7402, Hepa59T/VGH, SMMC7721; Stomach: MGC-803, MKN-45, BGC-803, BGC823, KATO- III, SGC7901, AGS; Pancreas: Bx-PC3; CNS: U251, SF268, *C6, U251MG, U373, Daoy, XF498; Renal: ACHN; Bone: 1547, *WEHI-164, *XC, RPMI 8226; Uterus: HeLa Normal cell lines: Fibroblasts: HSF, BJ-H-tert, 142BR, HFL-I, WS1; Macrophages: *RAW 264.7, *J774.A1; Epithelium/ endothelium: EVC-304, MCF-10A; Kidney embryonic: HEK-293; * Murine origin

Concentration: IC50 half maximal inhibitory concentration; ED50 effective dose; GI50 growth inhibition; LC50 lethal concentration; LD50 lethal dose Assays: MTT—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; XTT—2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino) carbonyl]-2H-tetrazolium hydroxide; MTS—3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium; WST-1—2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium, SRB—sulphorhodamine. PI—propidium iodide. NRR—neutral red release to saponin treatment (Einbond et al. 2008). Another are provided. One of worth noting recent examples is interesting example is a study on the effect of avicin the study by Tian et al. who tested triterpenoid saponins D from Acacia victoriae on apoptosis (Zhang et al. isolated from Ardisia crispa against human glioblas- 2008). It was observed on three lymphoma cell lines toma U251MG cell line (Tian et al. 2009). While all the (MJ, Hut78, HH) and on cells from patients with four compounds exhibited signiﬁcant cytotoxicity -1 Se´zary syndrome. Moreover, cells from healthy against cancer cells (IC50 2.57–12.20 lmol l ) donors and two normal T-cell lines (CD4? and they did not affect the growth of primary cultured activated CD4?) were used as comparative material. human astrocytes at concentrations as high as [100 Apoptosis was seen in all lymphoma cell lines and in lmol l-1. Se´zary syndrome patient cells whereas only slight The most preferred methods used for cytotoxicity apoptotic changes were detected in normal cells. screening were MTT (formazan based assay) and SRB The study by Zhang et al. was well designed also in (sulphorhodamine B assay), however in some studies another aspect, because the major drawback of cyto- other techniques were also applied, like eg. neutral red toxicity tests summarized in this review, is that only release assay. In rare cases, brine shrimp lethality test few compare the effect on cancer cells with the effect is still applied even though it is rather unspeciﬁc. on normal cells. Thus, it is very hard to draw any According to Houghton et al. the SRB assay is more conclusions on the future potential of these compounds sensitive and rapid than MTT, which was more as anticancer drugs because no data on their selectivity popular among authors of studies cited in Tables 1

123 450 Phytochem Rev (2010) 9:425–474

Fig. 1 Chemical structures O of polyphyllin D (1), methyl protodioscin (2), albizoside O C(3) and saponin PE (4)

O HO O O OH O HO HO O OH

HO O

HO OH

1. OH O OH O OH OMe OH

HO O

O O HO O HO O HO O HO OH HO OH

O O OH HO O

OH O OH O O O O OH O O HO HO O O O O HO HO HO O O HO OH O HO OH O HO O OH OH O HO OH O HO OH HO O

HO O HO OH OH

OH OH 3.

O O CH2

O O O Na+SO3O- HO O O O O HO HO OH OH O HO O

HO MeO OH

123 Phytochem Rev (2010) 9:425–474 451

and 2 (Houghton et al. 2007). There may be some IC50 values between control compounds, for example -1 minute differences in IC50 values obtained with these IC50 of etoposide against A549 was 1.1 lmol l methods (Dongre et al. 2007). whereas IC50 of 5-fluorouracil against the same cell -1 In the majority of cases IC50 values were com- line was as high as 48 lmol l (Mskhiladze et al. pared with a control. Usually, the reference substance 2008). Gnoula et al. in their study of cytotoxic was a known, clinically used anticancer agent, like activity of a mixture of balanitin-6 and balanitin-7 etoposide, paclitaxel, doxorubicin, adriamycin, cis- against a panel of six cell lines (A549, U373, PC-3, platin, in some cases however, compounds still under Bx-PC3, LoVo, MCF-7) compared IC50 values with investigation like betulinic acid (Cmoch et al. 2008) as much as four reference substances: oxaliplatin, or resveratrol (Perrone et al. 2005) were applied. etoposide, 7-ethyl-10-hydroxycamptothecin and pac- Sometimes, information on the use of a positive litaxel (Gnoula et al. 2008). The tested saponin control was not accompanied by IC50 data (Xie et al. mixture was significantly more active (IC50 from 0.3 -1 2009; Cao et al. 2007). to 2.6 lmol l ) than oxaliplatin (IC50 from 3.3 to -1 As can be seen from comparison of IC50 values [10 lmol l ) but significantly less active than -1 (Tables 1, 2) most saponins, both triterpenoid and paclitaxel (IC50 from 0.001 to 0.009 lmol l ). Thus, steroid, were less active than the reference com- a choice of reference may lead to various conclusions pounds. In some cases, however, their cytotoxicity with respect to the potency of cytotoxic compounds. was comparable or even higher. Among triterpenoid Most often, IC50 values for paclitaxel are of saponins, acylated oleanane-type glochierioside A, nmol l-1 order whereas for saponins in the same cell was cytotoxic against HL-60, MCF-7, SK-OV-3 and line—lmol l-1. It is interesting to note that one of

HT-29 cell lines with IC50 values similar to mitoxan- triterpene saponins isolated from Gordonia chrysan- trone (Kiem et al. 2009). One of the structurally dra (Theaceae)—gordonoside C, was significantly related acylated oleanane-type albizosides, albizoside more potent than paclitaxel against Bel-7402 (IC50 C (Fig. 1) isolated from Albizia chinensis, was more 0.7 and 6.3 lmol l-1, respectively) (Yu et al. 2009). potent than camptothecin on five cell lines, the Another issue worth mentioning with respect to difference was especially notable against lung car- cytotoxicity values presented in Tables 1 and 2 is that -1 cinoma A549, with IC50 0.01 lmol l vs. there is often no obvious correlation between the in 3.1 lmol l-1 (Liu et al. 2009b). Other examples vitro and in vivo effects, therefore compounds of worth noting are holostane saponins from a sea lesser activity might prove more valuable when tested cucumber Pseudocolochirus violaceus, which exhib- in animal models. A good example is provided by the ited distinctly higher cytotoxicity than 10-hydroxy- study of Bang et al. An oleanolic acid saponin which camptothecin used as a positive control against was most active in vitro had an inferior in vivo colon cancer HCT-116 (IC50 0.052–0.066 and antitumor activity, contrary to a hederagenin glyco- 0.135 lmol l-1, respectively) (Zhang et al. 2007). side (Bang et al. 2005). Among steroid saponins, the activity of filiasparoside Bioavailability would be an important aspect to C(Asparagus filicinus) against MCF-7 was compa- discuss in this respect and more studies are certainly -1 rable to etoposide (IC50 3.0 and 2.8 lgml , respec- needed as only limited data are available in the tively) (Zhou et al. 2007). Also, similar levels of cell literature with regard to the pharmacokinetics and growth inhibition were observed for diosgenin-3-O-a- bioavailability of saponins both triterpene and steroi- L-Rhap-(1 ? 2)-[a-L-Araf-(1 ? 4)]-b-D-Glcp and dal. Most of the available data indicate that these control epirubicin-HCl against HepG2, SGC7901 compounds are very poorly absorbed following oral and BxPC3 (Yun et al. 2007). Degalactotigonin from administration to animals and humans. As these Solanum nigrum exhibited much higher cytotoxicity issues are outside the scope of the present review, than 10-hydroxycamptothecin on a panel of four cell only examples of reports which refer to saponins with lines (HepG2, MCF-7, SF-268, NCI-H460) with cytotoxic or anti-tumor activities are presented.

IC50 values ranging from 0.25–4.49 and 6.49– In the study by Li et al., it was shown that the 38.55 lmol l-1, respectively (Zhou et al. 2006). absolute bioavailability of dioscin after oral admin- Comparison of cytotoxicity level is, however, only istration to rats was very low (0.2%). Dioscin an approximation as there are notable differences in underwent a prolonged absorption from the 123 452 Phytochem Rev (2010) 9:425–474 intestines, its level in the intestine remained quite 1,961.72 ± 686.60 lgml-1 h compared to 7.87 ± high even after 120 h after administration (Li et al. 2.85 and 148.58 ± 36.73 lgml-1 h). (Xiong et al. 2005). Similar results were obtained in the study by 2008). Ren et al. The 3H labeled total saponins of Dioscorea Another interesting example that shows how by nipponica (including dioscin, pseudoprotodioscin, modifying a drug’s formulation its bioavailability may protodioscin and methyl protodioscin) were orally be enhanced is a study of Gu et al. on beagle dogs. The administered (80 mg kg-1) to rats. The compounds authors compared oral bioavailability of anti-tumor were found to have a long half-life in rats. The 20(R)-ginsenoside Rh2 and its micronized formulation. highest emission of radioactivity level at the 120th h The results obtained indicated that the bioavailability was measured in the liver, adrenal gland, and walls of of oral micronized 20(R)-Rh2 was almost two-fold the gastrointestinal tract. The blood concentration of higher than that of native ginsenoside (AUC0-µ 3H labeled compounds was very low (Ren et al. 2375.9 ± 882.5 and 1,215.7 ± 598.6 ng ml-1 h,

2008). respectively; Cmax 730.0 ± 325.5 and 371 ± 199.6 -1 He et al. concluded that oral bioavailability of ng ml , respectively; Tmax 1.8 ± 0.7 and 2.6 ± -1 methyl protodioscin in rat (80 mg kg ) was good. 1.3 h, respectively). The authors concluded that Rh2 Ten metabolites of the parent compound, including in the micro-particle formulation seems to be better dioscin, were identified in the urine. Some of them dissolved in the intestinal juice and transported across showed potent antiproliferative activities towards the intestinal epithelial cells (Gu et al. 2006). HepG2, NCI-H460, MCF-7 and HeLa human cell lines, but their cytotoxic effects were much weaker in comparison to methyl protodioscin activity (He et al. 2006). Correlation between the structure and cytotoxicity Amongst triterpene saponins, tubeimoside I, iso- of saponins lated from the tubers of Bolbostemma paniculatum which has gained much attention due to its antitumor Observations from numerous studies confirm that the and antitumor-promoting effects, has been examined biological activity of saponins is influenced both by in order to assess pharmacokinetics and bioavailabil- the aglycone and the sugar moiety. Most existing ity after intravenous and oral administration to rats. information of the structure-activity relationships Its oral bioavailability (50 mg kg-1) was only 0.23% comes from research on the haemolytic action of which indicated poor absorption or acid-induced saponins. As was shown for triterpenoid and steroid degradation (Liang et al. 2007). compounds there are certain structural features which However, there were also reports which proposed seem to influence the strength of the lysis of new strategies to increase absorption of such amphi- erythrocytes, however, it is hardly possible to estab- philic compounds as saponins. In studies on ginseng lish any uninamous correlation. In the case of saponins it was suggested that worse permeability triterpene saponins for example, acylation seems to from gastrointestinal tract may be due to surface be beneficial in terms of their haemolytic potential activity of saponins which might self-assemble as (Melzig et al. 2001; Chwalek et al. 2004), for steroid micelles in solution. Such micellar aggregates would saponins most studies conclude that furostanols are in have an increased molecular size and hydrophyllic general inactive. Similarly, the nature of carbohy- property and thus result in poor permeability. To prove drate constituents influences haemolytic action of that this suggestion holds true Xiong et al. designed a both types of saponins but this effect is not repeatable lipid-based formulation that was expected to prevent with the same sugar moiety and a different aglycone. ginsenosides from aggregation and then compared the The lysis of erythrocytes is one of the most relative bioavailability of ginsenosides Rg1 and Rb1 characteristic biological features of saponins and is from an aqueous solution and this new formulation. considered as a major drawback in their clinical After intraduodenal administration of the new formu- development. As was mentioned earlier, it is believed lation ginsenoside bioavailability in rats was remark- to result mainly from the ability of these compounds ably enhanced relative to the aqueous solution to interact with membrane cholesterol leading to

(AUC0-µ of Rg1 and Rb1 were 207.52 ± 53.95 and membrane destabilization but not only (Francis et al. 123 Phytochem Rev (2010) 9:425–474 453

2002; Melzig et al. 2001). The same mechanism is and natural triterpenoid saponins (Gauthier et al. also involved in the cytotoxicity of saponins how- 2009a). In their previous work (Gauthier et al. 2006), ever, as was shown in many studies, a large number the authors investigated only lupane-type compounds, of compounds act via different modes of action, this extended study encompassed compounds with including apoptotic and non-apoptotic cell death lupane and oleanane skeleton The authors subdivided inducement, which are also reviewed in this paper. the tested compounds into five groups: free triterp- Comparison of the steroidal saponins haemolytic enoids such as betulin, betulinic acid, hederagenin activity with their cytotoxicity revealed that these two and oleanolic acid; lupane monodesmosides (with activities were not correlated because of different a-L-Rhap, a-L-Araf, a-D-Manp, b-D-Glcp, b-D-Galp, mechanisms of action (Wang et al. 2007). It was b-D-Xylp); lupane bidesmosides (with a-L-Rhap and observed however, that some highly haemolytic b-D-Glcp or a-L-Rhap); lupane saponins containing compounds were highly cytotoxic as well, and a-L-arabinopyranose (mono and bidesmosides with similarly some weakly toxic steroidal saponins had either a-L-Rhap or/and b-D-Glcp); oleanane saponins weak haemolytic activity. At the same time, several (a-hederin, b-hederin, d-hederin, hederacolchiside A glycosides showing similar cytotoxicity had disparate and hederacolchiside A1). Human cell lines assessed haemolytic activity and opposite, some compounds in this study included lung carcinoma A549, colo- possessing comparable haemolysis potency had dis- rectal adenocarcinoma DLD-1 as well as normal skin parate cytotoxicity. Therefore, there is no clear fibroblasts WS1. correlation between the haemolytic activity and Based on the obtained results the authors con- cytotoxicity of steroidal saponins and similarly the cluded, that in the case of oleanane type saponins SAR data referring to haemolysis cannot be directly those having oleanolic acid as an aglycone were more extrapolated to relationships crucial for cytotoxic potent than hederagenin saponins irrespective of the activity. sugar moiety. Again, this study confirms that as far as While most studies in which cytotoxic activity of sugar part is concerned it seems that the presence at saponins is reported focus on structure elucidation the C-3 position of the aglycone of an a-L-Rhap- there are sparse data, if any, on SAR relevant to (1 ? 2)-a-L-Arap moiety is a structural requirement cytotoxicity results, which are often published as essential for potent cytotoxic activity of oleanane additional information accompanying structure eluci- saponins. dation procedures. Some information is usually pro- What is worth noting is the fact, that the same vided by authors who isolate, from a given plant sugar sequence has no effect on the cytotoxicity of source, a greater variety of structures. However, there lupane-type saponins, such compounds were inactive is still a fairly limited number of comparative studies even at the highest concentrations tested in this study -1 which directly concentrate on establishment of struc- (IC50 [ 100 lmol l ) whereas the presence of the ture-activity relationships relevant to cytotoxicity. sole a-L-Arap moiety had a significantly positive Several studies which explored the correlation effect. The cytotoxicity of betulin and betulinic acid between haemolytic activity and cytotoxicity of derivatives significantly varied according to the saponins have been published, the most recent are nature of monosaccharides linked to C-3 and C-28. the reports by Gauthier et al. on triterpenoid glyco- In monodesmosidic compounds the most active were sides and by Wang et al. on steroid saponins a-L-rhamnopyranosides and in bidesmosidic saponins (Gauthier et al. 2009a; Wang et al. 2007). Most those containing two rhamnose moieties. In fact, crucial structure-cytotoxic activity relationships are betulin bis-3,28-a-L-rhamnopyranoside was the most summarized in Table 3. potent inhibitor of cell lines of all compounds tested, -1 with IC50 ranging from 1.3 to 1.9 lmol l . How- Correlation between the structure and cytotoxicity ever, this last saponoside was not selective towards of triterpenoid saponins cancer cells. In this respect, the most promising was betulinic acid a-L-rhamnopyranoside, which exerted

The most recent SAR study by Gauthier et al. strong cytotoxicity on cancer lines with IC50 from 2.6 -1 compared the effect on haemolysis, cytotoxicity and to 3.9 lmol l while IC50 for human ﬁbroblasts was membrane cell permeabilization of 35 semi-synthetic as high as 31 lmol l-1. 123 454 Phytochem Rev (2010) 9:425–474

Table 3 Structure-cytotoxic activity relationships of saponins Structural feature Enhancement of activity References

Type of triterpene aglycone Oleanane [ lupane Bang et al. (2005) Oleanane [ hopane Hamed et al. (2005) Lupane [ germanicane Thibeault et al. (2007) Oleanolic acid [ hederagenin in vitro Gauthier et al. (2009a), Bang et al. (2005) Hederagenin [ oleanolic acid in vivo Bang et al. (2005) Betulinic acid [ betulin Thibeault et al. (2007) Oleanane-type aglycone Free carboxyl at C-28 Chwalek et al. (2006), Bang et al. (2005) Acylation at C-21 and C-22 Zhang and Li (2007), Chan (2007), Chan et al. (2008), Yu et al. (2009) Oleanane-type aglycone acylated Methyl at C-24 [ hydroxymethyl at Chan et al. (2008) at C-21 and C-22 C-24 Free hydroxyl at C-16 Yu et al. (2009) 13,28-epoxyoleanane-type Aldehyde/methyl at C-30 [ carboxyl/ Tian et al. (2009) aglycone hydroxymethyl at C-30 Free hydroxyl at C-16 Chang et al. (2007) Acacic acid-type aglycone Oligosaccharide ester at C-28 Cao et al. (2007) Free hydroxyl at C-16 Cao et al. (2007) Acylation at C-21 (monoterpene- Cao et al. (2007), Note´ et al. (2009), Liu et al. (2009b) quinovopyranosyl moiety) Oleanane-type saponins Monodesmosides [ bidesmosides Calabria et al. (2008), Bang et al. (2005), Gauthier Ursane-type saponins et al. (2009a) Lupane-type saponins Acacic acid-type saponins Bidesmosides [ monodesmosides Zou et al. (2006b), Cao et al. (2007) Oleanane-type saponins a-L-Arap at C-3 Jung et al. (2004), Chang et al. (2007) a-L-Rhap-(1 ? 2)-a-L-Arap moiety at Chwalek et al. (2006), Bang et al. (2005), Gauthier C-3 et al. (2009a) Branched trisaccharide at Bang et al. (2005) C-3 [ disaccharide at C-3 Straight chain length ? contradictory Jung et al. (2004), Ahmed et al. (2009), Huang et al. data (2008b), Tundis et al. (2009) Lupane-type saponins a-L-Rhap at C-3 Gauthier et al. (2009a) Betulin saponins a-D-Manp at C-3 Thibeault et al. (2007) Monomannosides [ bimannosides Cmoch et al. (2008) Diosgenyl saponins a-L-Rhap-(1 ? 2)-b-D-Glcp at C-3 Mimaki et al. (2001), Herna´ndez et al. (2008) Elongation of sugar chain Yan et al. (2009) Diosgenin and pennogenin Terminal Rhap linked to C-2 of the Zhao et al. (2009) saponins inner Glcp

With regard to the other monosaccharides evalu- rhamnose facilitates this process by its interaction ated in this study, it seems that the presence of a b-D- with lectins and thus, saponins bearing this sugar-unit Glcp either at C-3 or C-28 has a detrimental effect on more easily enter the cell exerting anticancer action. the cytotoxicity of lupane-type saponins. The possi- The presence of a rhamnose residue in the sugar ble explanation of this phenomenon may be the high chain attached at C-3 as a feature responsible for polarity of the glucose moiety which might hinder the cytotoxicity was also pointed out by other reports diffusion of glucose bearing saponins into the cyto- (Chang et al. 1998; Cham and Daunter 1990), and plasm. On the other hand, it was postulated that more recently by Tabatadze et al. who studied the 123 Phytochem Rev (2010) 9:425–474 455 effect of giganteosides D and E on leukemia HL-60 (1 ? 4)]-a-L-arabino-pyranoside) gained much inter- and MEL-5 cell lines (Tabatadze et al. 2007). est as it showed only moderate activity against cancer Another observation from the study of Gauthier cells in vitro whereas had a potent antitumor action et al. (2009a) is that haemolytic activity, membrane on a solid tumor in vivo (IR, 82%), which was higher cell permeabilization and cytotoxicity of oleanane- than adriamycin (IR, 64%) (Kim et al. 2004). type saponins seem to be directly correlated whereas The results from a SAR study by Bang et al. the cytotoxic action of lupane saponins does not (2005) indicate that generally, the cytotoxicities of result from inducement of membrane permeabiliza- the lupane-type saponins were much weaker than tion and is due to another mechanisms. those of oleananes, when an a-L-Rhap-(1 ? 2)-a-L- In another SAR study by the same group with Arap sequence was present in a branched sugar synthetic triterpenoid glycosides of germanicane- and moiety at C-3 of the aglycone. These results were in lupane-type (Thibeault et al. 2007) the authors agreement with those obtained by Gauthier (Gauthier concluded that: (1) germanicane-type saponins are et al. 2009a) however sugar moieties in most weaker anticancer agents than lupane-type com- compounds evaluated by Bang et al. were composed pounds, (2) betulinic acid saponins generally exert of more monosaccharide units. Another observation higher in vitro cytotoxicity than those derived from concerning aglycone part, that the existence of a free betulin, with the exception of betulin 3b-O-a-D- carboxyl at C-28 is important for antitumor activity, mannopyranoside, (3) other sugars linked to the C3 of conformed with some reports (Chwalek et al. 2006) betulin (D-glucose, L-rhamnose, D-arabinose, D-gal- but was contradictory to other (Gauthier et al. 2009a) actose, D-xylose) supress anticancer activity. who showed that some bidesmosidic betulinic acid Based on reports that mono- and trimannosyl saponins glycosylated at C-28 were highly cytotoxic. residues may be regarded a convenient means of Moreover, comparison of activities exerted by sapo- delivering drugs into human dendritic cells or mac- nins derived from oleanolic acid and hederagenin rophages, Cmoch et al. synthesized a number of such made the authors postulate that the hydroxyl group at mannosylated derivatives of lupeol, betulin and C-23 has a negative effect on cytotoxicity. betulinic acid and compared their cytotoxic activity The comparison of the relationship between the (Cmoch et al. 2008). Cytotoxicity was assayed on structure of the sugar moiety and cytotoxic activity of normal human fibroblasts (BJ-H-tert) and a panel of oleanane saponins was in agreement with other cancer cells: T-lymphoblastic leukemia (CEM), reports (Gauthier et al. 2009a)—again the presence breast carcinoma (MCF-7), lung carcinoma (A-549), of an a-L-Rhap-(1 ? 2)-a-L-Arap sequence at C-3 multiple myeloma (RPMI 8226), cervical carcinoma produced higher cytotoxicity. Moreover, trisaccha- (HeLa), malignant melanoma (G361). The data rides with a third sugar linked at C-40 of arabinose or obtained from this study showed that the tested C-30 of rhamnose exhibited stronger cytotoxicities lupane-type compounds selectively inhibit the growth than disaccharides. of cancer cells without affecting normal cells. With A very interesting observation comes from in vivo regard to the sugar moiety, monomannosyl deriva- experiments, as no direct correlation was seen with in tives of lupeol, betulinic acid and betulin were vitro cytotoxicity. The main difference was in strongly cytotoxic whereas trisaccharide analogues oleanane aglycones—oleanolic acid derivatives were inactive. showing much stronger cytotoxicities in vitro than Similar groups of saponins, that is of lupane-type those of hederagenin, had an inferior in vivo antitu- (eight compounds) and oleanane-type (nine com- mor activity. This may be due to the differences in pounds) were evaluated by Bang et al. against human bioavailability of these structures, so the presence of solid tumors cell lines A-549, SK-OV-3, SK-MEL-2 a hydroxyl at C-23 seems to be more effective for in and HCT-15 and also subjected to in vivo test (Bang vivo antitumor activity. With regard to sugar moiety, et al. 2005). The compounds were isolated from one both the number of glucopyranosides and their plant species, namely Pulsatilla koreana roots. Pre- binding position to the active O-a-L-Rhap-(1 ? 2)- viously, one of the saponins from this plant species, a-L-Arap sequence is of importance for in vivo so called Pulsatilla saponin D (hederagenin 3-O-a-L- effects. The authors postulate that this may be related rhamnopyranosyl-(1 ? 2)-[b-D-glucopyranosyl- to the rate of glucose hydrolysis in the cell, the more 123 456 Phytochem Rev (2010) 9:425–474 glucose molecules in the chain the lower hydrolysis saponin was a-hederin. This fact provided further rate. Moreover, the third glucopyranoside attached at evidence that the a-L-Rhap-(1 ? 2)-a-L-Arap osidic C-400 is more resistant to glucose-hydrolase than at sequence is essential for the cytotoxic activity of C-200 and thus, such a saponin can more effectively hederagenin glycosides. reach the target site. So, it seems that a trisaccharide As far as aglycone is concerned, esterification in chain O-a-L-Rhap-(1 ? 2)-[b-D-Glcp-(1 ? 4)]-a-L- position 28 generally decreased cytotoxicity. It is Arap is responsible for the improvement of the interesting to note that haemolytic activity was bioavailability in tumor tissue in vivo. In a later study increased in the majority of cases what supports by Bang (Bang et al. 2007), a method for simple previous observations that it is impossible to correlate synthesis of this trisaccharide moiety was described. haemolytic and cytotoxic activities of saponins. Chwalek et al. examined structure-activity rela- In another SAR study (Jung et al. 2004) oleanane tionships of oleanane glycosides with cytotoxicity, disaccharides isolated from Akebia quinata were apoptosis induction and haemolysis (Chwalek et al. evaluated against the same human cell lines as those 2006). The tested saponins were 25 synthetic heder- used in the study by Bang et al., that is lung cancer agenin derivatives including six methyl esters. The A549, ovarian cancer SK-OV-3, melanoma SK- authors focused mainly on determination of the MEL-2, colon cancer HCT-15 and in addition brain influence of the monosaccharide composition and tumor XF498. Cytotoxicity against normal human interglycosidic linkages within the sugar moieties cells was not evaluated. The study included a more because they aimed at verification of the importance limited number of compounds—six saponins with of sugar chain. However, a comparison was also oleanolic acid or hederagenin as aglycones and with made between compounds with a free or esterified a-L-Arap as the first sugar attached to the sapogenin. carboxyl at C-28. The compounds were divided into The authors suggested that cytotoxicity of saponins is two groups of hederagenin saponins, those containing predominantly due to the sugar moiety as the effects an a-L-arabinose or b-D-glucose as the first sugar as of the two compounds with the same sugar sequence well as their methyl esters. Moreover, saponins were very similar. Moreover, comparison of saponins having a non-naturally occurring b configuration with a different linkage position between monosac- between the aglycone and L-arabinose were also charides, namely 1 ? 2 and 1 ? 3, showed, that investigated. In the arabinose series, positional these linkages are not crucial for cytotoxicity. disaccharide isomers bearing a-L-rhamnose, b-D- However, the nature and linkage of the second sugar xylose or b-D-glucose attached to positions 2, 3 or 4 is of importance, rhamnose manifested more potent were included in the study, whereas in the glucose activity than glucose, and axially attached 40-O-b-D- series, saponins with the following dissaccharide glucosyl was the least effective. Also, elongation residues were considered: cellobiosyl, lactosyl, of the sugar chain had a detrimental effect on maltosyl, gentiobiosyl, isomaltosyl and melibiosyl. cytotoxicity. Cytotoxicity was tested on KB cells with SN 38 as a This last observation seems to need further con- positive control. firmation as there are also reports with contradictory As far as the sugar moiety is concerned, the data. For example, in another study on hederagenin following observations were made. Saponins having a saponins (isolated from Duranta repens) those with non-natural anomeric configuration of b-L-ora-D- four sugar units exhibited greater cytotoxicity than were inactive or only slightly active. Glucose at the those possessing three sugars (Ahmed et al. 2009), first position of the disaccharide chain is unfavorable however these differences were seen on only one cell for cytotoxic activity when compared to arabinose. line—HepG2. Same effects were noted when com- Neither the nature of the second sugar nor the paring cytotoxic activity of seven oleanane-type interglycosidic linkages are in themselves determi- saponins (hydrocosisaponins A-F and hydrocotylo- nant as each sugar can affect cytotoxicity when at a side VII) from a Chinese folk medicinal plant specific position. Thus, for an a-L-rhamnose the Hydrocotyle sibthorpioides (Huang et al. 2008b). following order of activity was established Compounds with four sugar units, three D-glucoses (1 ? 2) [ (1 ? 3) [ (1 ? 4) and for b-D-xylose and one L-arabinose, were much more active than (1 ? 2) [ (1 ? 4) [ (1 ? 3). The most potent those depleted of a terminal glucose. 123 Phytochem Rev (2010) 9:425–474 457

On the other hand, cytotoxicity assay performed seems that the presence of two acyl groups is on a wider panel of human cell lines (malignant important for cytotoxicity as non-acylated saponins melanoma A375, amelanotic melanoma C32, Cauca- showed no or only weak activity (Zhang and Li sian lung carcinoma A549, hepatocellular carcinoma 2007). Huh-7D12, Caucasian lung large cell carcinoma This observation conformed with other reports, for COR-L23, Caucasian colon adenocarcinoma CaCo- example on saponins isolated from Xanthoceras 2, prostate carcinoma LNCaP, renal adenocarcinoma sorbifolia (Chan 2007). Structure-activity relationship ACHN, breast cancer MCF-7, skin fibroblast 142BR) study was performed on an oleanane-type compound of two saponins sharing the same aglycon, buddleja- Xantifolia-Y, the characteristic features of which are saponin IV and songarosaponin D but differing in the diangeloyl groups at C-21 and C-22 and a trisaccharide sugar chain length (two glucoses ? fucose; three chain at C-3 composed of (-[b-D-galactopyranosyl- glucoses ? fucose, respectively) does not indicated (1 ? 2)]-a-L-arabinofuranosyl-(1 ? 3)-b-D-glucur- any general influence of this structural feature on the onopyranosyl). For the purpose of the SAR study, cytotoxicity. Compound with three monosaccharides angeloyl groups or sugar moiety were separately was much more active than saponin bearing a four- removed and thus obtained derivatives together with -1 sugar chain against ACHN cells (IC50 7.9 lmol l b-escin and xantifolia-X (saponin with one angelolyl at vs. 37.2 lmol l-1) whereas it was much weaker C-22) were comparatively assayed against ovarian -1 against CaCo-2 cells (IC50 40.7 lmol l vs. cancer cells OVCAR3. Moreover, effect on haemol- 16.7 lmol l-1) (Tundis et al. 2009). ysis was also evaluated. Based on the obtained results it Calabria et al., in their study on the effects on was concluded that while removal of the sugar moiety cancer cells proliferation of nine oleanene- and significantly decreases both cytotoxicity and haemo- ursene-type saponins from Silphium radula, also lytic activity the removal of angelolyls at C-21 and observed that the length and linkage of the sugar C-22 completely eliminates these activities. It seems chain influences cytotoxicity. Moreover, the authors also that acylation with angelolyl group increases suggested that a monodesmosidic disaccharide chain cytotoxicity when compared to acetyl group. containing a galactopyranosyl residue produces In this study by Chan a very limited number of higher activity than bidesmosidic or monodesmosidic compounds were compared versus only one cell line, single sugar moiety (Calabria et al. 2008). however in a later report (Chan et al. 2008) six more In a cytotoxicity assay of dammarane saponins on saponins were evaluated. Again it was confirmed that human breast cancer MCF-7 cell line it was shown acylation at both C-21 and C-22 increases cytotox- that the number of glycosyls affects the activity of icity, and moreover it was postulated that analogues ginsenosides, those with one glycosyl are more potent with a C-24 methyl group (instead of CH2OH) and a than those bearing three or four glycosyls (Qiu et al. galactose unit (instead of glucose) in the trisacharide

2009). In this study ginsenoside Rh2 was used as a moiety at C-3 exhibit higher potency. -1 positive control (IC50 20 lmol l ). In another study Seven structurally related oleanane-type saponins on dammarane-type saponins, it was concluded that with diangeloyl groups at C-21 and C-22, additional the number of hydroxyl groups in the side-chain plays oxygenated substituents at C-15 and/or C-16, and a a crucial role in their cytotoxicity, two hydroxyls disaccharide chain at C-3 isolated from Gordonia being more beneficial than one or three (Huang et al. chrysandra were evaluated against a wider panel of 2008a). human cancer cell lines (HCT-8, Bel-7402, BGC- Zhang and Li screened five pre-selected saponins 823, A549 and A2780) (Yu et al. 2009). The obtained isolated from Aesculus pavia against a panel of 59 results indicated that a free hydroxyl at C-16 may be cell lines from nine human cancers: leukemia, non- of importance in mediating cytotoxicity. Its acetyla- small cell lung, colon, CNS, melanoma, ovarian, tion and the presence of a hydroxyl at C-15 had a renal, prostate and breast. These saponins were detrimental effect. polyhydroxyoleanenes with or without acyl groups However, in studies on triterpene glycosides at C-21 and/or C-22 and a sugar moiety composed of isolated from sea cucumbers Cucumaria sp. (Jin a-L-Araf, b-D-GlcAp, b-D-Galp. From the results et al. 2009) against various leukemia cells, acetyl obtained with a large array of cancer cell lines it group at C-16 (as opposed to keto group) was 123 458 Phytochem Rev (2010) 9:425–474 selected as the structural feature responsible for moiety at C-16, (2) glucopyranosyl moiety at C-2 of increased cytotoxicity of these compounds. a-L-arabinopyranose. Acetylation at another position, namely C-25, Although many studies reported that while mono- proved to be important for the growth inhibitory desmoside saponins are cytotoxic, bidesmosides are activity of cimigenol derivatives on the Her2 over- often inactive in the same cell lines it was noteworthy expressing MDA-MB-453 human breast cancer cells that a mixture of two bidesmosides from Sideroxylon (Einbond et al. 2008). The most potent saponin was foetidissimum subsp. gaumeri exerted cytotoxic -1 25-acetyl-7,8-didehydrocimigenol 3-O-b-D-xylopy- activity (IC50 11.9 lgml ) against the murine -1 ranoside with an IC50 5 lmol l . macrophage-like cell line RAW 264.7 (Sańchez- Interesting observations were made in a compar- Medina et al. 2009). Moderate activity against human ison of cytotoxic activity against glioblastoma leukemia HL-60, stomach adenocarcinoma KATO-III U251MG cells of saponins isolated from Ardisia and lung adenocarcinoma A549 cells, was also pulsilla (Tian et al. 2009). Five tested compounds reported for echinocystic acid bidesmoside, impatie- -1 were all monodesmosides and belonged to two noside G (IC50 21.8–36.7 lmol l ) (Li et al. 2009). closely related aglycone types, those with olean-12- However, other compounds tested in this study, ene or a 13,28-epoxyoleanane skeleton. The saponins including mono- and bidesmosides of soyasapogenol differed in a constituent at C-30 and in the length of A were inactive at concentrations 50 lmol l-1. the branched sugar chain (tetra- or hexasaccharide). It seems therefore, that the cytotoxicity of bides- Based on the results from this study, as well as earlier mosidic saponins varies dependent of the aglycone. reports on similar compounds, it was concluded that The presence of an oligosaccharide ester at C-28 was both the aglycone and the sugar moiety play an postulated to be an important structural feature essen- important role in their cytotoxicity. It seems that the tial for growth inhibitory activity of many cytotoxic most important structural feature is the presence of acacic acid-type saponins, such as for example avicins the aldehyde or methyl group at C-30, a replacement from Acacia victoriae or julibrosides from Albizia of which by a carboxyl or a hydroxymethyl results in julibrissin. Other structural features essential for their loss of activity. Moreover, the results from this study action include a monoterpene-quinovopyranosyl moi- were also interesting in terms of the used cell line ety at C-21 and a hydroxyl group at C-16 (Cao et al. because glioblastoma multiforme is very resistant to 2007). The crucial role in mediating cytotoxicity is chemotherapy. The most active saponin (3b-O-{b-D- attributed to an ester substituent at C-21 (Note´ et al. glucopyranosyl-(1 ? 2)-{b-D-xylopyranosyl-(1 ? 2)- 2009) and for the most potent activity, two mono- [b-D-glucopyranosyl-(1 ? 3)-b-D-glucopyranosyl- terpene-quinovopyranosyl groups acylated at C-21 of (1 ? 3)]-b-D-glucopyranosyl-(1 ? 4)}-a-L-arabino- the aglycon are required (Liu et al. 2009b). pyranosyl}-16a-hydroxy-13b,28-epoxy-oleanan-30-al) Structurally related compounds isolated from -1 had IC50 of 2.57 lmol l and at the same time did not Albizia gummifera had significant antiproliferative -1 affect the growth of normal human astrocytes at effects (IC50 0.1–6.6 lmol l ) on the histiocytic concentration as high as 100 lmol l-1. lymphoma U937, human ovarian A2780, colon HT- Another species of genus Ardisia, A. japonica, 29, nonsmall-cell lung H522-T1, and breast MDA- afforded 21 saponins which were evaluated against MB-435cancer cell lines (Cao et al. 2007). Also, three human cell lines, HL-60 (myeloid leukemia), bidesmosidic julibrozide J21, with acacic acid agly- KATO-III (stomach adenocarcinoma) and A549 cone, showed a marked inhibitory action against (lung adenocarcinoma) (Chang et al. 2007). All human liver cancer cell line Bel-7402 (Zou et al. -1 cytotoxic saponins (IC50 from 1.9–37.2 lmol l ) 2006b). From a study of Melek et al. it was concluded were derivatives of either cyclamiretin A or proto- that saponins containing acacic acid lactone as the primulagenin A, that is they shared a common 13,28- triterpene moiety are inactive (Melek et al. 2007). epoxyoleanane skeleton and possessed a branched Some of fairly early SAR experiments were sugar chain with a-L-arabinopyranose as the first performed on saponins with previously reported sugar attached at C-3 of the aglycon. Comparison of antitumor activity, namely tubeimosides I–III from

IC50 values indicated that the following structural Bolbostemma paniculatum (Yu et al. 2001). These features seem to improve the activity: (1) hydroxyl compounds are oleananes belonging to a group of 123 Phytochem Rev (2010) 9:425–474 459 rare cyclic bidesmosides with a 3-hydroxy-3-methyl- Similarly to Mimaki’s studies, diosgenyl a-L-rhamno- glutarate bridge joining two sugar chains. Tubeimo- pyranosyl-(1 ? 2)-b-D-glucopyranoside displayed sides I–III differ in the aglycone part by the presence strong cytotoxic activity with an IC50 of about or absence of a hydroxyl at C-16 and in the linkage 5-7 lmol l-1. Glycosides containing other sugar position of the bridge between two sugar chains. instead of rhamnose were, in general, less cytotoxic Antitumor activities were compared in vivo on in this study. Selective acetylation of a hydroxyl group 0 transplantable mouse S180 in BALB/c mice. The at C-6 of glucose of diosgenyl a-L-rhamnopyranosyl- obtained data indicated that C-16 hydroxyl group is (1 ? 2)-b-D-glucopyranoside did not change signif- important for the activity and at the same time for the icantly the cytotoxic activity when compared to decrease in toxicity, whereas sugar chain linkage deacetylated compound. It was concluded that free such as in tubeimoside III, elevates antitumor effect hydroxyl group at C-60 of glucose may be of no but also the toxicity of the compound. So, tube- importance for this effect. Additional acetylation of imoside II seemed to be the most promising agent the hydroxyl group at C-400 of rhamnose significantly for cancer chemotherapy among saponins of decreased the cytotoxic activity. The reduction of B. paniculatum. cytotoxic effect was observed when a second rhamnose was attached to C-400 of rhamnose. A significant decrease in growth inhibitory activity was observed Correlation between the structure and cytotoxicity on SK-MEL-1 in response to the compounds having of steroid saponins other than rhamnose moiety attached to glucose -1 residue (IC50 [ 100 lmol l ). These results showed Influence of sugar chain the importance of rhamnose for diosgenyl glycosides’ cytotoxicity. In 2001, Mimaki et al. reported some structure- Diglycosides with mannose or fucose moieties activity relationships of steroidal saponins. The induced significant G1 arrest at the expense of S- and compounds were mainly isolated from the Liliaceae G2/M-phase cell population. Diosgenyl a-L-rhamn- plants and tested against HL-60 human promyelo- pyranosyl-(1 ? 2)-b-D-glucopyranoside produced cytic leukemia cells. Diosgenin b-D-glucoside was alterations in cell cycle progression, induced G2-M -1 not active (IC50 [ 20 lgml ), however an addi- arrest at the expense of G1 phase cell number. No tional a-L-rhamnopyranosyl moiety at C-2 of the evidence of changes in the cell cycle phases was glucose considerably increased the activity (IC50 observed in response to compound containing a 1.8 lgml-1). Among different diosgenin rhamno- galactosyl group (Hernańdez et al. 2008). pyranosyl-glucosides only a-L-rhamnopyranosyl- In Mimaki’s studies saponins with glycosyl groups (1 ? 2)-b-D-glucopyranoside of diosgenin was at C-1 of aglycone showed no activity, the only active. Compounds with a-L-Rhap moiety at C-3 or exceptions were compounds possessing some acyl C-4 of the glucose showed no cytotoxic activity. groups at the glycosyl moiety. Also, a (25S)-spirost-5- Further attachment of a-L-arabinofuranosyl, b-D-glu- ene-1b,3b-diol with a sugar chain containing D-fucose copyranosyl or a-L-rhamnopyranosyl groups to C-3 and L-rhamnose was cytotoxic. The authors suggested or C-4 of the inner glucosyl group did not influ- that cytotoxic activity of such saponins was connected ence activity or caused only slight increase with a certain degree of lipophilicity (Mimaki et al. (0.5–3.3 lgml-1). The authors concluded that the 2001). number and the sequence of sugar moieties seem to be The importance of the number of sugar moieties in important for cytotoxic activity (Mimaki et al. 2001). a sugar chain and the structure of aglycone was also Some diosgenyl a-L-rhamnopyranosyl-(1 ? 2)- confirmed by the studies on saponins isolated from b-D-glucopyranoside analogues with different mono- Agave fourcroydes. Chlorogenin, hecogenin and saccharides (D-Glc, D-Gal, D-Man, L-Ara, D-Xyl, and tigogenin hexasaccharides (with the same sequence D-Fuc) in the place of the rhamnose moiety were of sugar chains) showed cytotoxic activity against -1 synthesized (Hernańdez et al. 2008). Their effects on HeLa cells (IC50 13.1, 5.2, and 4.8 lgml , respec- the growth of human myeloid (HL-60 and U937) and tively), while diglucosides were inactive (Ohtsuki melanoma (SK-MEL-1) cell lines were evaluated. et al. 2004). 123 460 Phytochem Rev (2010) 9:425–474

The correlation between the cytotoxic activity and cycle in G2/M (52.6% compared to 20.3% for the number of sugar residues in the sugar chain was control), while smilagenin blocked cell cycle in observed in cytotoxicity test of saponins isolated G0/G1 phase. Sarsasapogenin significantly enhanced from Paris polyphylla var. yunnanensis. Diosgenin DNA fragmentation (6.9-fold compared to control). tetrasaccharide showed stronger cytotoxic activity Results from this study provide evidence for the than compounds containing three or two sugar resi- importance of the conformation at C-25 (Trouillas dues. Two diosgenin and pennogenin disaccharides et al. 2005). exhibited the lowest inhibtion properties. Diosgenin- The R/S configuration at C-25 was essential for 3-O-a-L-arabinofuranosyl-(1 ? 4)-[a-L-rhamnopyr- selectivity in leukemia cells observed between methyl anosyl-(1 ? 2)]-b-D-glycopyranoside showed higher protoneogracillin (C-25 S configuration) and methyl cytotoxicity rate than diosgenin-3-O-a-L-rhamnopyr- protogracillin (C-25 R configuration) isolated from the anosyl-(1 ? 4)-[a-L-rhamnopyranosyl-(1 ? 2)]-b-D- rhizomes of Dioscorea collettii var. hypoglauca.A glyco-pyranoside (1.85 and 3.06 lmol l-1, respectively). 6–11 fold difference was observed on three leukemia Although the number of monosaccharides in the cell lines (CCRF-CEM, MOLT-4 and PRMT-8226), sugar moiety were the same they varied in compo- GI50 for methyl protoneogracillin was 1.79, 4.59, and sition (Yan et al. 2009). 1.57 lmol l-1, respectively whereas for methyl protogracillin 20.2, 25.6, and 12.0 lmol l-1, respectively. Influence of aglycone However, no difference in selectivity against solid tumors was observed for these two compounds, A series of steroidal sapogenins (diosgenin, tigoge- indicating that R or S configuration at C-25 was not nin, sarsasapogenin, smilagenin and others) has been critical for the activity against solid tumors (Hu and tested for the influence on cell proliferation, cell Yao 2003b). cycle distribution and apoptosis in 1,547 human In vitro studies on steroidal saponins of Paris osteosarcoma cells. To study the role of a 5,6-double polyphylla var. yunnanensis displayed that glycosides bond in the cytotoxic effect, the activities of of diosgenin were more active against LA 795 cells diosgenin (possessing a double bond), smilagenin than those of pennogenin-type (which belongs to the and tigogenin (5b and 5a stereoisomers respectively; 17-hydroxy-steroidal sapogenins) (Yan et al. 2009). no 5,6-double bonds) were compared. In the studies on cytotoxic activity of steroidal Diosgenin induced apoptosis in 1,547 cells (5.6- saponins isolated from Liliaceae plants the introduc- fold compared to control) whereas smilagenin and tion of C-17 hydroxyl group to diosgenyl glycoside tigogenin showed weak apoptotic activity. Diosgenin reduced the cytotoxicity slightly whereas additional and smilagenin induced strong inhibition of cell C-27 hydroxyl group considerably decreased the proliferation while tigogenin inhibited it only mod- cytotoxic activity (Mimaki et al. 2001). erately. After 24 h treatment diosgenin and smilage- The cytotoxic activity of steroidal saponins isolated nin blocked cell cycle in G0/G1 phase in 54.1 and from the fresh whole plants of Agave utahensis against 54.9% respectively compared to 39.6% for control HL-60 human promyelotic leukemia cells was studied. while tigogenin had no effect. The results from this Two smilagenin diglycosides and one trisaccharide -1 study indicate that the difference in the spatial showed high cytotoxicity with IC50 4.9–7 lgml . conformation of the A- and B-rings and the presence The additional C-2 hydroxyl group or C-12 car- or lack of 5,6-double bond do not affect the mode of bonyl group in the 5b-spirostanol structure signifi- action on the cell cycle (diosgenin and smilagenin cantly reduced cytotoxic activity (Yokosuka et al. arrested the cell cycle in the same phase) (Trouillas 2009). et al. 2005). Methyl protodioscin (a furostanol compound) and The importance of stereoisomery at C-25 on cell dioscin (a spirostanol glycoside) isolated from the proliferation with regard to apoptosis and cell cycle root of Polygonatum zanlanscianense were tested data were studied using sarsasapogenin and smilage- against human leukemia HL-60 cells in vitro. The nin (the 25-isomer of smilagenin). Sarsasapogenin spirostanol glycoside induced differentiation, apop- showed strong apoptotic effect whereas smilagenin tosis and cytotoxicity in the HL-60 cells, while the induced weak apoptosis. Sarsasapogenin arrested cell other had no effect. It was concluded that the 123 Phytochem Rev (2010) 9:425–474 461 spirostanol structure must be critical in the effects on surface, which are stimulated by specific molecules the HL-60 cells (Wang et al. 2001). known as pro-apoptotic ligands (Apo2L/TRAIL and Some spirostanol diosgenin derivatives were inves- CD95L/FasL). Among the reviewed saponins, only tigated for cytotoxic effect against human colorectal saponin fraction isolated from Bupleurum kaoi HCT 116 carcinoma cells. Compounds which bear S induced the extrinsic pathway of apoptosis (Hsu and Se atoms on the diosgenin F ring were synthesized. et al. 2004). Oppenauer oxidation of diosgenin, solasodin (a nitro- The influence of triterpene saponin-enriched frac- gen analogue of diosgenin) and the synthesized tion (SEF) from Bupleurum kaoi on human non-small heterospirostanols gave corresponding a,b-unsatu- cell lung cancer A549 was investigated by Hsu rated ketones. Amongst compounds studied solasodine et al. The antiproliferative effect of SEF was and its a,b-unsaturated ketone derivative exhibited observed, in a dose-dependent manner, with IC50 -1 -1 high activity (IC50 = 6 and 14 lmol l , respec- values 196 lgml . Inhibition of proliferation of the tively). The activities of other compounds were lower. cells was through the apoptotic pathway, with the The results of this study suggested that N-atom on the F observed enhancement in Fas and its two ligand ring was important for cytotoxic activity against HCT forms, membrane-bound Fas ligand and soluble Fas 116 cells. The activity of N-spirostanol compound was ligand. In cells treated with caspase-8 inhibitor, higher when there was no conjugated system in the A growth inhibition and apoptosis were significantly ring (Quan et al. 2002). decreased, which also proves an important role of Fas/FasL system in SEF-mediated apoptosis of A549 cells (Hsu et al. 2004). Cytotoxic mechanisms of saponins The intrinsic pathway of apoptosis begins usually in response to different types of cell stress: cellular Saponins are potential anticancer agents, with differ- signals resulting from DNA damage, a defective cell ent mechanisms of action. Their cytotoxic effects cycle, detachment from the extracellular matrix, may be due to either apoptosis inducement or non- hypoxia, loss of cell survival factors. This pathway apoptotic cell death stimulation. There is a number of involves the release of pro-apoptotic proteins that well-known processes which lead to the same activate caspase enzymes from the mitochondria, but effect—cell death—but involve different mechanisms also depends on the balance between the pro- and of action, like stimulation of autophagic cell death, anti-apoptotic proteins from Bcl-2 superfamily (Xu decrease in NO production in cells, or cytoskeleton et al. 2009). integrity disassembly. The immunosuppressive activity of a triterpene The cytotoxic effects of saponins, discussed in this saikosaponin a from Bupleurum falcatum on the paper, were studied mainly in vitro, but also some in concanavalin A (Con A) stimulated mouse CD3? T vivo experiments were conducted. The most impor- cells, isolated from the lymph node of BALB/c mice, tant mechanisms are summarized in Table 4. was examined, as well as the underlying mechanism of the action. The compound had potent antiproliferative effect, and also significantly inhibited the In vitro experiments activation of the cells, in a concentration-dependent manner. Moreover, saikosaponin a potently sup- Apoptosis—programmed cell death—is one of the pressed the production of IL-2, IFN-c and TNF-a in most important processes involved in the regulation T cells stimulated by Con A. The G0/G1 arrest of the of tissue development and homeostasis. It is also well activated T cells by saikosaponin a was also known that research of novel anticancer agents in observed, and the effect was due to down-regulating recent years focuses on their potential pro-apoptotic of CDK6 and cyclin D3 protein level and up- properties. The process may be initiated either at regulating of p27kip protein level. The apoptosis of plasma membrane (extrinsic pathway) or within the the activated T cells, in comparison to the non- cell (intrinsic pathway) (Bachran et al. 2008). activated cells, was induced by the compound in The extrinsic pathway is triggered by the activa- dose-dependent manner, probably via mitochondrial tion of specific pro-apoptotic receptors on the cell pathway—a significant collapse of the mitochondrial 123 462 Phytochem Rev (2010) 9:425–474

Table 4 Mechanisms of cytotoxic activity of saponins Process Mode of action References

Apoptosis Cytochrome c release Sun et al. (2009a), Li et al. (2008), Xu et al. (2009), Wang et al. (2008), Jin intrinsic et al. (2009), Zhang et al. (2008), Haridas et al. (2009), Herna´ndez et al. pathway (2004), Siu et al. (2008), Liu et al. (2005) Mitochondria membrane depolimerization GADD153/CHOP transcription factor expression increase Caspase 3 and 9 activation PARP-1 cleavage MAPK cascade activation p-STAT-3 decrease Bcl-2, c-myc, survivin downregulation VEGF downregulation p53 stimulation NF-jB inhibition JAKs dephosphorylation Protein phosphatase-1 activation Intracellular Ca2? homeostasis disruption Apoptosis Fas enhancement Hsu et al. (2004) extrinsic pathway Fas ligand enhancement Apoptosis non- Telomerase activity inhibition Kim et al. (2008a) speciﬁed Cell cycle arrest CDK6 downregulation Sun et al. (2009a, b), Ellington et al. (2005), Raju and Mehta (2009), Srinivasan et al. (2009), Liu et al. (2005) Cyclin D3 downregulation Cyclin D1 downregulation p27kip upregulation CDK2 inhibition Autophagy Microtubule-associated protein Ellington et al. (2005), Xu et al. (2007a) light chain-3 increase Autophagic vacuoles number increase AMPK-TSC2 regulation Cellular ATP decrease AMPK activation Angiogenesis Thrombospondin-1 expression Wang et al. (2008), Tian et al. (2005) inhibition increase PKC expression decrease ERK activation P38MAPK activation Endothelial cell proliferation inhibition Tube-formation inhibition

123 Phytochem Rev (2010) 9:425–474 463

Table 4 continued Process Mode of action References

Cytoskeleton Microtubule dissasembly Li et al. (2008), Ma et al. (2008), Einbond et al. (2008), Galanty et al. (2008), disintegration Gnoula et al. (2008) Tubulin polymerization inhibition Actin ﬁlaments disruption Intracellular ATP decrease Nucleus morphology changes Metastasis Cell motility and migration Tian et al. (2005), Galanty et al. (2008), Man et al. (2009) inhibition inhibition Cell adhesion inhibition MMP-2, MMP-9 downregulation TIMP-2 upregulation

membrane potential and release of cytochrome c endothelial cells HUVEC and ECV304 was studied. from mitochondria to cytosol was observed (Sun The compound inhibited both endothelial cell lines et al. 2009a). proliferation in dose-dependent manner, also causing The anticancer properties of ardisiacrispin (A ? B), apoptosis of the cells. Moreover, b-escin sodium a mixture (fixed proportion 2:1) of triterpene saponins suppressed endothelial cells migration and motility. ardisiacrispin A and B from Ardisia crenata, against a The possible mechanism of action of the compound number of human cancer cell lines were evaluated. The may be through increasing expression of thrombo- highest antiproliferative effect of the tested substance spondin-1 and decreasing expression of protein kinase was observed on human hepatoma Bel-7402 cells. C, an intracellular messenger necessary for endothelial Moreover, the substance induced apoptosis in cells proliferation and migration. Additionally, the Bel-7402 cells, which was observed by the changes compound activates p44/42 mitogen-activated protein of the mitochondrial membrane depolarization, mem- kinase (ERK) and p38 mitogen-activated protein brane permeability enhancement and nuclear conden- kinase (p38MAPK), which are involved in MAPK sation, and the effects were dose-dependent (Li et al. signaling cascade, whose activation is linked to cell 2008). proliferation, motility and apoptosis. The obtained data Cytotoxic effect of a triterpene saponin tubeimo- indicate the potential anti-angiogenic activity of side 1 (TBMS1) from Bolbostemma paniculatum was b-escin sodium due to its direct effects on endothelial tested on HeLa cells. In the cells treated with the cells (Wang et al. 2008). compound, profound protein alterations were The effects of two triterpene glycosides: frondo- observed, suggesting apoptotic process, with the side A and cucumaroside A2-2, isolated from sea involvement of mitochondria and endoplasmic retic- cucumber Cucumaria frondosa, on human leukemia ulum. TBMS1 induced the depletion of mitochondrial cells death-inducing capability were determined and transmembrane potential, causing the activation of compared in this study. Both compounds strongly caspase-dependent apoptotic cell death. Moreover, induced apoptosis of leukemic cells, but the process the increased expression of GADD153/CHOP tran- was more rapid and potent in cells treated with scription factor, associated with growth arrest and frondoside A, in comparison to cucumaroside A2-2. apoptosis in the event of prolonged ER stress, was The obtained data indicate that only apoptosis detected. The results of the study suggest the induced by cucumaroside A2-2 was caspase-depen- cytotoxic effect of TBMS1 through the mitochon- dent (Jin et al. 2009). drial, but also ER stress cell death pathways (Xu et al. The influence of avicin D and G, triterpene 2009). saponins obtained from seedpods of Acacia victoriae, The effect of b-escin sodium on proliferation, on metabolic state of isolated rat mitochondria was migration and apoptosis of human umbilical vein studied by Lemeshko et al. Following avicin 123 464 Phytochem Rev (2010) 9:425–474 treatment, a significant decrease in oxygen consump- expression of Stat-3-regulated proteins—c-myc, tion rate was observed. This process was reversible cyclin D1, Bcl2, survivin, VEGF—was observed. by the addition of exogenous cytochrome c, which Downregulation of Stat-3 activity and inhibitory suggests that avicin-induced inhibition of respiration effect on expression of pro-survival proteins contrib- may be due to the release of cytochrome c from uted to the induction of apoptotic process in tumor mitochondria. Moreover, avicins increased the rote- cells treated with avicin D. The results of the study none-intensive oxidation of external NADH in the suggest that avicin D, by the inhibition of Stat-3 presence of exogenous cytochrome c, which may activity, suppressed also proinflammatory and pro- indicate that the compounds increase permeabiliza- oxidant stromal environment of tumors. Moreover, tion of mitochondrial membrane to cytochrome c. avicin D activated PP-1, a cellular economizer, which The results explain selective induction of apoptosis in can lead to redirecting metabolism of tumor cells tumor cells by avicins, especially avicin G (Leme- from growth promoting to energy sparing pathways shko et al. 2006). (Haridas et al. 2009). The influence of avicin D, also isolated from Steroid sapogenin icogenin, isolated from Dra- A. victoriae, on different cutaneous T-cell lympho- caena draco, was active against the myeloid leuke- -1 mas cell lines (CTCL): MJ, Hut78, HH and also on mia cell line HL-60 (IC50 2.6 lmol l at 72 h) and cells obtained from patients with Se´zary syndrome inhibited cell growth by induction of apoptosis. and healthy donors, with normal CD4? and activated Nuclear changes and the fragmentation of poly CD4? T-cells was investigated by Zhang et al. (ADP-ribose) polymerase-1, an enzyme involved in Avicin D induced apoptosis in three CTCL cell lines, DNA repairing and in apoptosis process, were in time- and dose-dependent manner. Apoptotic cell observed (Hernańdez et al. 2004). death was more notable in avicin D-treated cells from Polyphyllin D (PD), a steroid saponin (Fig. 1) the patients with Se´zary syndrome than in cells from isolated from Paris polyphylla, was identified as a healthy donors. The compound activated caspase-3 in potential ER stress inducer on human non-small cell CTCL cell lines and cells from Se´zary syndrome lung cancer NCI-H460 cell line. PD treatment led to patients, but not in cells from healthy donors. Avicin upregulation of typical ER stress-related proteins/ D did not influence the expression of signal trans- genes including glucose-regulated protein 78 (BiP/ ducer and activator of transcription-3 (STAT-3) but it GRP78) and protein disulfide isomerase (PDI). In decreased phosphor-signal transducer and activator of particular, elevated expression of C/EBP homologous transcription-3 (p-STAT-3) protein level in CTCL transcription factor (chop) and activation of caspase-4 cell lines and cells from the Se´zary syndrome occurred at early time point (8 h) of PD treatment, patients. The compound also selectively inhibited suggesting an initial ER stress-mediated apoptosis. the expression of antiapoptotic protein bcl-2, while Induction of tumor suppressor p53, disruption of did not influence the expression of proapoptotic mitochondrial membrane, activation of caspase-9 and protein bax in CTCL cells and cells from the patients caspase-3 were also detected upon prolonged PD with Se´zary syndrome. In conclusion, avicin D treatment. Collectively, these data revealed that PD selectively induced apoptosis, which was probably induced the cytotoxic effect through a mechanism dependent on caspase-3 and downregulation of initiated by ER stress followed by mitochondrial p-STAT-3 and bcl-2 proteins (Zhang et al. 2008). apoptotic pathway (Siu et al. 2008). Haridas et al. investigated the effect of avicin D on Diosgenin, a naturally occurring steroidal sapoge- phosphorylation/dephosphorylation of Stat-3 process nin, has been suggested to play an important role in in a number of human tumor cell lines: U266, RPMI- anticancer protection. Cytotoxic activity of the com- 8226, SKO-007, MM1, A431, OVCAR3, HepG2. pound is mainly due to its inhibition of cell cycle

Avicin D stimulated dephosphorylation of Stat-3 in signaling and induction of apoptosis. It induced G1 all cell lines, what lead to a decrease in transcription phase cell cycle arrest in 1,547 osteosarcoma cells rate of Stat-3. This process may be due to avicin D and induced apoptotic demise. The activation of p53 inﬂuence on dephosphorylation of JAKs (Janus and binding of NFjB to DNA independent of PPAR- kinases) and activation of protein phosphatase-1. c was demonstrated (Raju and Mehta 2009). In other

Moreover, in avicin D-treated cells the reduction of experiment, diosgenin induced cell cycle G2/M arrest 123 Phytochem Rev (2010) 9:425–474 465 and apoptosis. The disruption of intracellular Ca2? tumor growth and malignant transformation of cells, homeostasis, generation of reactive oxygen species which is an interesting alternative, especially in and caspase activation were observed (Liu et al. apoptosis-resistant cancer cells (Xu et al. 2007a). 2005). Ellington et al. evaluated the effect of a purified Srinivasan et al. examined the effect of diosgenin soybean B-group saponin extract on human HCT-15 on Akt signaling in estrogen receptor positive and colon adenocarcinoma cells. The extract significantly negative breast cancer cells. Diosgenin inhibited decreased the viability of the cells, in dose- and time- pAkt expression and Akt kinase activity, but it did dependent manner and the cells were accumulated in not affect the levels of PI3K, which lead to the phase S of the cell cycle, due to the reduction of inhibition of Akt downstream targets, NF-jB, Bcl-2, cyclin-dependent kinase (CDK2) activity. The exam- survivin and XIAP and the Raf/MEK/ERK pathway ination of the cells treated with the extract under the in ER?, but not in ER- BCa cells. In both ER? and transmission electronic microscope indicated the non- - ER BCa cells diosgenin caused G1 cell cycle arrest apoptotic programmed cell death morphology by downregulating cyclin D1, cdk-2 and cdk-4 changes—a number of autophagic vacuoles, suggest- expression what resulted in cell proliferation inhibi- ing autophagic cell death. Moreover, the increased tion and induced apoptosis. Diosgenin was not level of microtubule-associated protein light-chain 3, significantly toxic towards the normal breast epithe- a specific marker of macroautophagy, following lial cells MCF-10A (Srinivasan et al. 2009). soyasaponin treatment was observed (Ellington Platycodin D, a triterpenoid bidesmoside isolated et al. 2005). from the root of Platycodon grandiflorum, was The effect of avicin D and avicin G, isolated from examined as a potential antitumor compound on the root of Acacia victoriae, on the autophagy different human leukemia cell lines: U937, THP-1 induction in a number of human tumor cells: A549, and K562. The compound exerted a dose-dependent SKOV3, PC3, U2OS, MDA-MB-231 and T47D was cytotoxic effect, and also inhibited the activity of examined. Cells treated with an apoptosis inhibitor telomerase via down-regulation of hTERT (telome- (benzyloxycarbonylvalyl-alanyl-aspartic acid (O- rase reverse transcriptase) expression, which can be methyl)-fluoro-methylketone) still could undergo cell associated with non-specified apoptotic process. In death under the influence of avicin D, what indicated platycodin D treated cells the decrease of c-Myc and non-apoptotic cell death. Moreover, this process was Sp1 (regulators of hTERT transcription) protein not due to the deregulation of amino acids but based levels and reducement in their DNA binding activity on the obtained data it seems that avicin D induced were observed. The compound also down-regulated disruption of cellular bioenergetics, what in turn the activation of Akt, and thus reduced phosphory- suggested autophagy. Avicin D decreased cellular lation and nuclear translocation of hTERT. The ATP levels and stimulated the activation of AMP- results indicate that cytotoxic effect of platycodin D activated protein kinase (AMPK), which suggests the on human leukemia cells may be due to telomerase possible mechanism of avicin D-induced autophagic, activity suppression through transcriptional and post- caspase-independent cell death, via regulation of translational suppression of hTERT (Kim et al. AMPK-TSC2 (tuberous sclerosis complex 2)-mTOR 2008b). (mammalian target of rapamycin) pathway (Xu et al. Anti-angiogenic and antitumor properties of PE 2007). (Fig. 1), a sulfated triterpene saponin isolated from The excessive production of nitric oxide (NO) in the sea cucumber Pentacta quadrangularis, were the cell may be due to viral or bacterial infections and examined. The compound inhibited proliferation, can promote pathogenesis by promoting oxidative migration, adhesion and tube-formation in two types stress, tissue injury and even cancer. There is some of human endothelial cells: HMECs and HUVECs. evidence that NO production inhibitory activity of Moreover, non-specified apoptosis in the cells treated saponins is correlated with their cytotoxic effect with PE was also observed (Tian et al. 2005). (Tundis et al. 2009). Autophagy is a process used by stressed cells to The cytotoxic activity of four triterpene sapo- catabolize their own contents (proteins, organelles) to nins—saikosaponin a, buddlejasaponin IV, song- provide cellular energy. The process may also inhibit arosaponin D and saikosapogenin a (obtained by 123 466 Phytochem Rev (2010) 9:425–474 hydrolysis of sugar unit from saikosaponin a)—from influenced the cytoskeleton of Bel-7402 cells, caus- the roots of Physospermum verticillatum, against seven ing microtubules dissasembly (Li et al. 2008). cancer cell lines—ACHN, C32, Caco-2, COR-L23, In our own investigations, the effect of LTS-4, a A375, A549 and Huh-7D12—and skin fibroblasts triterpene saponoside from Lysimachia thyrsiflora, 142BR was examined. All the saponins exhibited upon the viability, proliferation, morphology and cell strong cytotoxic activity against COR-L23 line, motility of human melanoma HTB-140 cells and whereas none of the tested compounds was active human skin fibroblasts (HSFs) was tested. The against the fibroblasts. Buddlejasaponin IV and song- compound decreased the viability and cell growth arosaponin D inhibited NO production in lipopolysac- of both cell types in time- and dose-dependent charide-stimulated murine monocytic RAW 264.7 manner, and was cytotoxic against cancer cells at macrophages (Tundis et al. 2009). significantly lower concentrations in comparison to Normal cell division, cellular motility, intracellu- fibroblasts. LTS-4 affected also the morphology of lar transport and proper cell shape are all dependent examined cells, causing vacuolization and cell shape on cytoskeleton integrity and stability. Saponins may changes. The compound caused actin cytoskeleton stimulate disintegration of microtubular network or disintegration—disappearance of stress fibres and actin filaments of cancer cells, which can lead to random distribution of actin in the cytoplasm. further processes of apoptotic or non-apoptotic cell Moreover, LTS-4 exerted an inhibitory effect on death (Ma et al. 2008; Einbond et al. 2008). tumour cells motility (Galanty et al. 2008). Anti-microtubule role of tubeimoside I (TBMS1), The mixture of steroid glycosides balanitin-6 and isolated from Bolbostemma paniculatum, on human balanitin-7 (referred to as bal6/7), isolated from the nasopharyngeal carcinoma CNE-2Z cell line was kernels of African plant Balanites aegyptiaca, dis- investigated by Ma et al. The dose- and time- played high cytotoxic activity against human cancer dependent inhibition of cell growth treated with lines: A549 non-small cell lung cancer and U373 TBMS1 was significant. In the cells treated with glioblastoma. Its activity was higher than etoposide 25 lmol l-1 of TBMS1 for 3 h the disruption of and oxaliplatine, but markedly lower than paclitaxel. microtubular network was observed, and the analysis It was suggested that its antitumor activity resulted showed the increase of the cytosolic tubulin level in from the decrease of intracellular ATP, mediated by the treated cells, which suggest the inhibition of bal6/7, which induced in turn major disorganization tubulin polymerization. The effect was weak in of actin cytoskeleton. The experiment indicated that comparison to colchicine. Moreover, the tested the cytotoxic effect was not mediated by apoptotic compound inhibited colchicine binding to tubuline, mechanism (Gnoula et al. 2008). in contrast to vinblastine binding, which indicates the competitive binding of TBMS1 to tubulin at colchi- In vivo experiments cine binding site (Ma et al. 2008). The growth inhibitory effect of extracts and isolated Steroidal saponins of Paris polyphylla var. yunnan- compounds from Cimicifuga species were investigated ensis (e.g. polyphyllin D, dioscin, formosanin C, on two human breast cancer cells: MDA-MB-453 (ER methylprotogracillin, and trillin) have been found to negative, Her2 overexpressing) and MCF7 (ER possess strong antitumor activity. The experiment positive, Her2 low). The results indicated that the was performed on T739 bearing LA795 mice which growth inhibitory activity of these extracts was related received the saponin extract by intragastric adminis- to triterpene glycoside composition. Actein, a purified tration. The observed antitumor and antimetastatic isolated compound, influenced the composition of effects were due to inducing apoptosis and upregu- actin cytoskeleton by alteration of distribution of actin lating the expression of TIMP-2 and down-regulating filaments in both type of cells—actin was dispersed in the level of MMP-2 and MMP-9 (Man et al. 2009). the cytoplasm and aggregated around the nucleus. Oral administration of diosgenin-3-O-a-L-arabino- Moreover, the nuclei changed their shape in a manner furanosyl-(1 ? 4)-[a-L-rhamnopyranosyl-(1 ? 2)]- typical of apoptosis (Einbond et al. 2008). b-D-glucopyranoside, isolated from the rhizome of Ardisiacrispin A and B from Ardisia crenata, Paris polyphylla var. yunnanensis, to T739 mice mentioned above due to their apoptotic activity, also bearing LA795 lung adenocarcinoma significantly 123 Phytochem Rev (2010) 9:425–474 467 inhibited tumor growth (by 29.44%). The increase of PE, a triterpene saponin isolated from the sea apoptosis rate in tumor cells in comparison to cells in cucumber Pentacta quadrangularis, suppressed spon- control mice was observed (Yan et al. 2009). taneous angiogenesis in chorioallantoic membrane of Diosgenin significantly inhibited tumor growth in the chicken embryo at very low concentrations vivo in MCF-7 and MDA-231 (breast cancer) xeno- (5 nmol l-1 per egg). Moreover, in two animal tumor grafts in nude mice. Both in vivo and in vitro results models: sarcoma 180 and hepatoma 22, PE on (diosgenin inhibited pAkt expression and Akt kinase intravenous administration significantly inhibited activity in vitro) led authors to conclude that dios- tumor growth by triggering apoptosis. It was also genin may be a potential chemotherapeutic agent for shown that PE suppressed the active forms of the treatment of breast cancer, however no data on VEGFR2 (vascular endothelial growth factor), Akt, the route of administration were given (Srinivasan ERK (extracellular signal-regulated kinase), FAK et al. 2009). (focal adhesion kinase) and paxillin, which mediate Diosgenin when administrated with diet to the angiogenesis of endothelial cells and apoptosis azoxymethane-induced colon carcinogenesis rats (Tian et al. 2005). suppressed both invasive and non-invasive colon Avicin D from Acacia victoriae inhibited the level tumor incidence (up to 60%). Experimental diet of VEGF (vascular endothelial growth factor) and containing 0.1% of diosgenin significantly reduced CD31, an endothelial cell marker indicating the colon tumor multiplicity (adenocarcinomas/rat) in microvascular networks within the cells, in mouse rats. A significant increase in BrdU labeling and skin carcinogenesis model. The compound was decrease in PCNA labeling in colonic crypts and applied to the shaved skin of animals. The results tumors was detected (Malisetty et al. 2005). suggest its antiangiogenic potential (Haridas et al. The mixture of steroidal saponins balanitin-6 and 2009). balanitin-7 (bal6/7) isolated from Balanites aegyptiaca led to increase of the survival time of mice bearing murine L1210 leukemia grafts to the same Saponins-toxin cytotoxicity enhancement extent as vincristine, when administered i.p. (Gnoula et al. 2008). The combined application of saponins with other Fifteen triterpene saponins from Pulsatilla kore- antitumor compounds may increase cytotoxic activity ana were administered intraperitoneally to BDF1 of the latter, which is an interesting new possibility in mice bearing Lewis lung carcinoma. Saponins con- cancer treatment research. One of the first experi- taining an ester group at C-28 of the aglycone were ments was done by Hebestreit (Hebestreit and Melzig inactive while those with a-L-rhamnopyranosyl- 2003) on the saponins from Agrostemma githago (1 ? 2)-a-L-arabinopyranosyl moiety at C-3 and a seeds. The agrostemmasaponins significantly free carboxylic acid at C-28 of the oleanane skeleton increased cytotoxicity of agrostin, a toxic protein of had the highest antitumor potency. The authors the seeds. The effect was probably due to the claimed that the oleanane-type saponins were more enhanced penetration of agrostin, when applicated active in vivo than the lupane-type saponins. The with the saponins, through the cell membrane. most potent antitumor compound was hederagenin Although no summarizing review has been published 3-O-a-L-rhamnopyranosyl-(1 ? 2)-[b-D-glucopyr- so far, a number of experiments were conducted in anosyl-(1 ? 4)]-a-L-arabinopyranoside, the activity recent years to examine this promising option in of which was higher than standard chemotherapeutic tumor therapy. agent—paclitaxel (Bang et al. 2005). In the study by Wang et al, hederagenin saponin In vitro experiments macranthoside B, isolated from the flower buds of Lonicera macranthoides, was injected intravenously Bachran et al. analyzed the influence of seven to BALB/cA nude mice. The compound significantly saponins: saponinum album (from Gypsophila pan- supressed the tumor growth and the effect was similar iculata L.), b-aescin (from Aesculus hippocastanum to cyclophosphamide treated control group (Wang L.), quillajasaponin (from Quillaja saponaria Mol.), et al. 2009). hederasaponin C (from Hedera helix L.), ginsenoside 123 468 Phytochem Rev (2010) 9:425–474

Rd (from Panax ginseng C.A. Mey.), glycyrrhizic acid active in comparison to their previous experiments (from Glycyrrhiza glabra L.) and helianthoside 2 with unlabeled compound, which was also observed (from Helianthus annuus L.), on the cytotoxicity in the case of combined 3H-labeled saponinum album enhancement of a targeted chimeric toxin, composed and chimeric toxin (Bachran et al. 2010). of saporin and human epidermal growth factor, against Enhancement of cytotoxicity of lectins and pep- untransfected Swiss mouse embryo NIH-3T3 cells and tidic toxins—agrostin, microcystin-LR, nigrin b, HER14 cells (NIH-3T3 cells transfected with human saporin, his-saporin, diphtheria toxin, diphtheria toxin epidermal growth factor receptor). The compounds A-chain and ricin A-chain—against ECV-304 cells differed in their structures mainly by a formyl group at was tested in the presence of saponinum album by C-4 or a branched sugar chain at C-3. Cytotoxicity of Hebestreit et al. The significant effect was observed the saponins alone was also determined against for lectins, but not for other peptidic toxins, with the HER14 and NIH-3T3 cells, resulting in 90% cell highest enhancement for agrostin and saporin, which survival at the concentration of 1.5 lgml-1.This suggest the specific interaction between the lectins concentration was chosen for further experiments with and saponins. The increase in cytotoxicity of lectins saponin-toxin combination. Two of the tested sapo- was due to the enhancement of their penetration nins—quillajasaponin and saponinum album— through the cell membrane. The effect was attenuated enhanced the cytotoxicity of the chimeric toxin by by latruculin (inhibitor of endocytosis) and bafilo- more than 1,000-fold, the enhancement factors of the mycin (inhibitor of transport from early to late other saponins were about tenfold only. For saponi- endosomes and lysosomes) but was neither influ- num album, the effect was receptor specific, in enced by brefeldin A, which causes dissolution of the contrast to quillajasaponin and the saponins with low Golgi stacks nor by the addition of monosaccharides, enhancement factor. The compound decreased IC50 which support the probable mechanism of the value from 2.4 nmol l-1 to 0.18 pmol l-1. The enhancement as receptor-mediated endocytosis. The authors conclude that the overall structure of the effect might be associated with an enhanced capacity triterpenoid aglycone cannot be solely responsible for to form vesicles, induced by saponins, which trans- the enhancement, also the branched sugar chain at C-3 port the proteins via endocytosis into the cell may play a role (Bachran et al. 2006). This statement (Hebestreit et al. 2006). conforms to observations of Melzig et al. (2005), who Heisler et al. investigated the effect of the studied agrostin cytotoxicity enhancement by a num- combined application of saponinum album and ber of different saponins. As far as aglycone structure chimeric toxin (consisting of saporin-3, epidermal is concerned, the authors emphasize the importance of growth factor and a molecular adapter) on the formyl group at C-4 (Bachran et al. 2006). This feature untransfected Swiss mouse embryo NIH-3T3 cells, was also pointed our by Sołtysik et al. (1995), who as control, HER 14 cells (NIH-3T3 cells transfected suggested that it may be of importance in the adjuvant with human epidermal growth factor receptor) and function of saponins. However, these conclusions human breast adenocarcinoma cell line MCF-7 as need further verification and more SAR studies are targeted cell lines. Pre-applied saponin enhanced the certainly needed. cytotoxic effect of the chimeric toxin against the In 2009, the same authors described the effect of targeted cell lines, whereas no effect was observed on the combined application of saponinum album and the control cells at the same concentration. The epidermal growth factor receptor (EGFR)-targeted application of the chimeric toxin or the saponin alone chimeric toxins (SA2E) against murine mammary at the optimal concentration did not exhibit cytotoxic adenocarcinoma cells (TSA), transfected with EGFR. effect, which indicates the synergistic effect of the The 20,000-fold enhancement of cytotoxicity of combined application of both compounds (Heisler SA2E was observed with low concentrations of et al. 2005). saponinum album, in a synergistic manner (Bachran et al. 2009). In vivo experiments Cytotoxicity of 3H-labeled saponinum album alone and in combination with chimeric toxin was analysed In the study by Heisler et al. saponinum album and on HER 14 cells. 3H-labeled saponin was much less chimeric toxin were applied subcutaneously to 123 Phytochem Rev (2010) 9:425–474 469

BALB/c mice in combination or alone. Only the their cytotoxic activity. This review has summarized combined treatment gave good therapeutic effects, saponins with such an activity, which were reported with only slight side effects. The probable mecha- in the last 5 years and their most common mechanism suggests the saponin-mediated elongation of the nisms of action. Correlations between structure and retention period of endocytosed proteins inside the cytotoxicity of both triterpenoid and steroid the endosome and thus, a greater chance of crossing saponins were also described. the endosomal membrane by the toxins (Heisler et al. In the majority of cases these were in vitro studies, 2005). only a limited number was performed in animal Bachran et al. investigated the effects of applica- models. The most preferred assays used were formation of saponinum album to mice bearing tumor cells. zan based (MTT) and sulphorhodamine B (SRB). The In their ﬁrst study, saponinum album and SA2E were choice of cell lines (usually from one to ﬁve) in most applicated subcutaneously to BALB/c mice with solid reports cited in this review seemed to be random and tumor (TSA cells transfected with EGFR). The very few compared cells from the same tumor but application resulted in 94% tumor volume reduction, differing, e.g. in malignancy, or activity against and the effect was obtained with 50-fold lower normal cells. Reference compounds were used in chimeric toxin concentration in comparison to pure most of studies cited here and in general saponins, SA2E treatment. Only moderate and reversible side both triterpenoid and steroid, were less active. effects were observed during the application (Bach- Comparison of cytotoxicity level is however only ran et al. 2009). an approximation as there were notable differences in

In another experiment of this group, the distribu- IC50 values between control compounds themselves. tion of labeled saponinum album in BALB/c mice As far as structure-activity relationship is con- with tumor was measured by radioactivity analysis in cerned, some information was usually given by samples of organs, blood, urine and tumor tissues. No authors who isolated, from a plant source, a greater preferential accumulation in tumor tissues or other variety of structures. In recent years some wider organs was observed and the highest radioactivity comparative studies have appeared, which directly level in the urine was after 30 min. Different concentrated on establishment of SARs relevant to pretreatment times were measured in tumor-bearing cytotoxicity. From these data some general conclu- mice, using sequential combination of saponin (given sions can be drawn, however with such an enormous first) and chimeric toxin. Promising antitumor effi- structural diversity of saponins and still fairly limited cacy was observed after 60 min pretreatment with sets of compounds that were evaluated in this regard, saponin, but no significant changes after 10 min this is very difficult and may be subject to bias. pretreatment with the compound. No information was Nevertheless, it seems that for all saponins both provided on the metabolism of this labeled saponin aglycone and sugar part play an important role for mixture and no control group which would receive cytotoxic activity. The main structural features non-labeled, acetylated saponinum album was used responsible for enhancement of cytotoxicity of ole- for comparison of the results (Bachran et al. 2010). In ananes are: free carboxyl at C-28, free hydroxyl at another experiment of the same group, the authors C-16 and acylation at C-21 and C-22. As far as sugar 3 claimed that the H-acetyl labeling of the sugar chain moiety is concerned the presence of a-L-Arap as the did not influence the activity of the saponinum album first sugar attached at C-3 in oleananes (and espe- in vitro (Weng et al. 2009). However, data on saponin cially an a-L-Rhap-(1 ? 2)-a-L-Arap sequence) and labeling and its effect on the activity are still very a-L-Rhap in lupanes was pointed out. scarce. With respect to steroidal saponins, similarly to triterpenes, the number and the sequence of sugar residues in a carbohydrate chain, and the position of Summary and conclusions sugar attachment to the aglycone were important for cytotoxicity. For diosgenyl saponins the presence of In recent years more and more researchers who an a-L-Rhap-(1 ? 2)-b-D-Glcp sequence at C-3 was isolated new saponins from natural sources, along considered beneficial for this activity. Also, elonga- with structure elucidation process, reported also on tion of the sugar chain resulted in an increase in 123 470 Phytochem Rev (2010) 9:425–474 cytotoxicity. However, in order to obtain a better Ahmed WS, Mohamed MA, El-Dib RA et al (2009) New tri- insight into SAR relevant to cytotoxicity there is still terpene saponins from Duranta repens Linn. and their cytotoxic activity. Molecules 14:1952–1965 a need for a greater number of studies which would Avilov SA, Silchenko AS, Antonov AS et al (2008) Synaptosides compare a wider array of compounds, ideally deriv- A and A1, triterpene glycosides from the sea cucumber atives of a single sapogenin with systematic varia- Synapta maculata containing 3-O-methylglucuronic acid tions of the sugar chains(s) or a group of different and their cytotoxic activity against tumor cells. J Nat Prod 71:525–531 sapogenins bearing identical sugar chain(s). 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