Bioactive saponins from Astragalus suberi L. growing in Yemen

From the aerial parts of Astragalus suberi L., Fabaceae, seven saponins were isolated. Based on spectral data (IR, 1H and 13C NMR and HR-FABMS), the structures were established as 3-O-(beta-D-glucopyranosyl)-soyasapogenol B (1); 3-O-(beta-D-glucuronopyranosyl)-soyasapogenol B (2); 3-O-[beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-soyasapogenol B (3); 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-soyasapogenol B (4); 3-O-[beta-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucopyranosyl]-11-hydroxy-soyasapogenol B (5); 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucuronopyranosyl]-soyasapogenol B (6) and 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-beta-D-glucuronopyranosyl]-complogenin (7). The isolated saponins exhibited antibacterial activities against Gram-positive and Gram-negative bacteria with minimum inhibitory concentration values >100 microg/ml, antifungal activity against all the strains tested with minimum fungicidal concentration values between 25 and 50 microg/ml and inhibited the growth of Hep-2 (human carcinoma of larynx), with IC50 values between 50 microg/ml (compounds 5-7) and 100 microg/ml (compounds 1-4), and Hela (human carcinoma of cervix) cell lines in culture with different IC50 values [74 (compound 7), 98 (compound 5) and 180 microg/ml (compounds 1-4 and 6)].     

Synthesis and cytotoxic activity of new azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolin-12-ones

A series of azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolin-12-ones (3a–f), that were conformationally restricted analogs of lead compound 2, were designed as potential cytotoxic compounds and synthesized using a radical oxidative aromatic substitution reaction as the key step. Compounds 3a–f were tested on five tumor cell lines to determine the conformational requirements for biological activity of compound 2. The results show that conformational restrictions on compound 2, generating the derivatives 3a–f, do not appreciably reduce the cytotoxic activity of 2, although compound 3d (R = Br) showed good activity against U-251 cells. Preliminary structure–activity relationship studies with these compounds revealed the importance of halogens bonded to the isoquinoline moiety. Additionally, derivatives 3f (R = NO₂) and 3b (R = F) were cytotoxic to PC-3 and K-562 cells. However, none of the azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolinones inhibited the enzymatic activity of CDK1/cyclin B, CDK5/p25, or GSK-3.     

A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557

A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 μg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC₅₀ = 16.7 ± 0.34 μg/ml). This was the first report of spatozoate isolation from bacterial sources.     

Recombination of diterpenoid structure units: Synthesis of antitumor amides bearing functionalized bicyclo[3.2.1]octane ring

In this work, 23 new amides (14–36) bearing a representative diterpenoid structure unit, the functionalized bicyclo[3.2.1]octane ring, have been synthesized and its antitumor potential is studied. In vitro studies demonstrate that a number of amides with the bicyclo[3.2.1]oct-3-en-2-one subunit are active against HL-60, SMMC-7721, A-549, SK-BR-3, and PANC-1 tumor cell lines. The hybrid derivative, compound 20, was found to be the most potent compound (IC₅₀ = 1.05 μM against HL-60) and more active than cisplatin (DDP), the positive control. Additionally, compound 20 exhibited broad spectrum in vitro anticancer activity with IC₅₀ values of 1.1–4.3 μM against the five tested cancer cell lines.     

Structure of tyrolobibenzyl D and biological activity of tyrolobibenzyls from Scorzonera humilis

A novel tyrolobibenzyl derivative, 1-->beta-D-apiosyl-beta-D-glucopyranosyl 4-[2-(4-hydroxyphenyl)ethyl]benzofuran-2-carboxylate 3 (tyrolobibenzyl D) was isolated from Scorzonera humilis L. and its structure established by mass spectrometry and 1D- and 2D-NMR spectroscopy. The biological activities of the new compound and related tyrolobibenzyls A-C (1-2, 4) and the semi-synthetic peracetyl derivatives of tyrolobibenzyls B (2a) and C (4a) were assessed. The results revealed no cytotoxic activity against P388 cells and neither anti-bacterial activity against Bacillus subtilis nor antifungal activity against Candida albicans and Trichophyton mentagrophytes for any of the investigated compounds. An evaluation of potential chemopreventive activity of 2, 2a, 4, and 4a also revealed no pronounced activity in any of the employed assaying systems.     

Three new triterpenoids from Salacia hainanensis Chun et How showed effective anti-α-glucosidase activity

To identify the chemical constituents with hypoglycemic activity in Salacia hainanensis Chun et How, this study was carried out on the roots of S. hainanensis. By means of a bioassay-guided method, three new triterpenoids (2β,3β-dihydroxylup-20(29)-ene [compound 1], 30-hydroxy-D:A-friedo-olean-1-en-3-one [compound 2], and 24,25,26-trihydroxytirucall-7-en-3-one [compound 3]) along with three known compounds (olibanumol J [compound 4], 21α-hydroxy-D:A-friedo-olean-3-one [compound 5], and 29-hydroxy-D:A-friedo-olean-3-one [compound 6]) were isolated from the EtOAc part and were shown to have effective α-glucosidase inhibitory activity. Their structures were established on the basis of spectral analysis, especially according to the data obtained by two dimensional nuclear magnetic resonance spectroscopic and high-resolution mass spectra experiments. All compounds with the exception of compound 2 showed much stronger inhibitory activity against α-glucosidase than did the positive control (acarbose, IC₅₀ 1.02 μM).     

Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of Mycobacterium tuberculosis InhA

A series of twenty seven substituted 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives were designed based on our earlier reported Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein reductase (InhA) lead. Compounds were evaluated for MTB InhA inhibition study, in vitro activity against drug-sensitive and -resistant MTB strains, and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30) was found to be the most promising compound with IC₅₀ of 5.12 ± 0.44 μM against MTB InhA, inhibited drug sensitive MTB with MIC 17.11 μM and was non-cytotoxic at 100 μM. The interaction with protein and enhancement of protein stability in complex with compound 30 was further confirmed biophysically by differential scanning fluorimetry.     

Protective effects of steroid saponins from Paris polyphylla var. yunnanensis on ethanol- or indomethacin-induced gastric mucosal lesions in rats: structural requirement for activity and mode of action

The methanolic extract from the rhizomes of Paris polyphylla SM. var. yunnanensis (FR.) H-M. was found to potently inhibit ethanol-induced gastric lesions in rats. Through bioassay-guided separation, four known spirostanol-type steroid saponins, pennogenin 3-O-alpha-L-rhamnopyranosyl(1-->2)-[alpha-L-arabinofuranosyl(1-->4)]-beta-D-glucopyranoside (1), pennogenin 3-O-alpha-L-rhamnopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (2), diosgenin 3-O-alpha-L-rhamnopyranosyl(1-->2)-[alpha-L-arabinofuranosyl(1-->4)]-beta-D-glucopyranoside (3), and diosgenin 3-O-alpha-L-rhamnopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (4), and a new furostanol-type steroid saponin, parisaponin I (5), together with two known furostanol-type steroid saponins, trigofoenoside A (6) and protogracillin (7), were isolated from the active fraction. Compounds 1-4 (1.25-10 mg/kg, po) strongly inhibited gastric lesions induced by ethanol and indomethacin. With regard to structural requirement of steroid saponins, the 3-O-glycoside moiety and spirostanol structure were found to be essential for the activity and the 17-hydroxyl group in the aglycon part enhanced the protective effects against ethanol-induced gastric lesions. The protective effects of 1 and 3 against ethanol-induced gastric lesions were attenuated by pretreatment with indomethacin and N-ethylmaleimide. Compounds 1 and 3 weakly inhibited acid secretions in pylorus-ligated rats. These findings suggested that endogenous prostaglandins and sulfhydryl compounds were involved in the protective activity.     

Novel 4-N-substituted aryl but-3-ene-1,2-dione derivatives of piperazinyloxazolidinones as antibacterial agents

Novel (5S)-N-[3-(3-fluoro-4-{4-[2-oxo-4-(substituted aryl)-but-3-enoyl]-piperazin-1-yl}-phenyl)-2-oxo-oxazolidin-5-ylmethyl]-acetamide 3a–j analogues were synthesized and their in vitro antibacterial activity was evaluated. Most of the compounds of series showed superior in vitro activity against Gram-positive resistant strains than linezolid. Compound 3f is the most potent compound in the series with 0.04–0.39 μg/mL MIC.     

Triterpenoids in Gypsophila trichotoma Wend.

A new triterpeniod saponin 3-O-β-arabinopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (1), together with the known saponin 3-O-β-xylopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (2), and three known triterpenes gypsogenic acid (3), quillaic acid (4) and gypsogenin (5) were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Cytotoxic activity of compounds 1 and 2 were tested against seven human cancer cell lines. Compound 1 showed cytotoxic activity against all of them, while compound 2 only against two cell lines.     

Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2-carboxanilides

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 28.4 μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 14.2 μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC = 46.7 μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC₅₀ value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3 μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure–activity relationships are discussed.     

The synthesis of 2- and 3-aryl indoles and 1,3,4,5-tetrahydropyrano[4,3-b]indoles and their antibacterial and antifungal activity

A series of 2- and 3-aryl substituted indoles and two 1,3,4,5-tetrahydropyrano[4,3-b]indoles were synthesized from indole and 5-methoxyindole. The 2-aryl indoles were synthesized from the 1-(phenylsulfonyl)indole derivatives using magnesiation followed by iodination. The 2-iodinated compounds were then subjected to Suzuki–Miyaura reactions. In addition, the 3-aryl indoles were made from the corresponding 3-bromoindoles using Suzuki–Miyaura reactions. The 1,3,4,5-tetrahydropyrano[4,3-b]indoles were also synthesized from 1-(phenylsulfonyl)indole by magnesiation followed by treatment with allylbromide. The product was then converted into [2-allyl-1-(phenylsulfonyl)-1H-indol-3-yl]methanol which upon exposure to Hg(OAc)₂ and NaBH₄ afforded tetrahydropyrano[4,3-b]indoles. A number of the 2- and 3-aryl indoles displayed noteworthy antimicrobial activity, with compound 13a displaying the most significant activity (3.9 μg/mL) against the Gram-positive micro-organism Bacillus cereus.     

Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ⩽0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC₅₀ = 961 μg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC₅₀ = 269 μg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.     

Characterisation of new oligoglycosidic compounds in two Chinese medicinal herbs

A series of caffeic acid derivatives (3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and 4,5-dicaffeoyl-quinic acid), and the new compound beta,3,4-trihydroxyphenethyl-O-[beta-apiofuranosyl-(1-->4)-alpha- rhamnopyranosyl-(1-->3)]-(4-O-caffeoyl)-beta-glucopyranoside (wedelosin), as well as three known flavonoid glycosides (quercetin 3-O-beta-glucoside, kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside, and astragalin or kaempferol 3-O-beta-glucoside) were isolated from the Chinese medicinal herb Wedelia chinensis. Wedelosin showed an inhibitory activity on both the classical and the alternative activation pathway of the complement system. Another Chinese medicinal herb, Kyllinga brevifolia, yielded two known flavonoid glycosides [kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside and isorhamnetin 3-O-beta-apiosyl-(1-2)-beta-glucoside], and a new quercetin triglycoside [quercetin 3-O-beta-apiofuranosyl-(1-->2)-beta-glucopyranoside 7-O-alpha-rhamnopyranoside]. The latter compound showed a moderate anti-viral activity.     

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6–16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R¹, R², and R³). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC₅₀ = 420 nM) and 9 (IC₅₀ = 930 nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.     

Cytotoxic cholestane and pregnane glycosides from Tribulus macropterus

The methanol extract of the whole parts of Tribulus macropterus Boiss. (family Zygophyllaceae) showed cytotoxic activity against a human tumour cell line (hepatocyte generation 2, HepG2) (IC50 = 2.9 microg/ml). The n-butanolic fraction obtained from successive fractionation of the methanolic extract exhibited activity against HepG2 (IC50 = 2.6 microg/ml). Therefore, this fraction was subjected to separation using different chromatographic techniques. Five compounds, 1-5, were isolated and identified as: (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranoside (1), (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranoside (2), sucrose (3), D-pinitol (4) and 3beta-hydroxy-5a-pregn-16(17)en-20-one-3-O-beta-D-xylopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-ga-lactopyranoside (5) on the basis of spectroscopic and chemical data. The three steroidal compounds 1, 2 and 5 were also tested against the same cell line HepG2 and their IC50 values were 2.4, 2.2 and 1.1 microg/ml, respectively.     

A facile synthesis and biological activity of novel tetrahydrobenzo[4′,5′]thieno[3′,2′:5,6]pyrido[4,3-d]pyrimidin-4(3H)-ones

The synthesis and biological activity of 2-substituted-8,9,10,11-tetrahydrobenzo[4′,5′]thieno[3′,2′:5,6] pyrido[4,3-d]pyrimidin-4(3H)-ones are described. Bioassay results indicated that these compounds have antifungal activity against Botrytis cinerea at a concentration of 50 mg/L. In addition, compounds 5m and 5n were effective to both KB cells and their parent multidrug resistant KBv200 cells with the overexpression of ABCB1. For example, compound 5m showed the best inhibition against KB and KBv200 cells with IC₅₀ values of 17.4 and 25.4 μM, respectively.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Structure–activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids,and their tricyclic and bicyclic analogues

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-β-carboline, showed the best in vitro activity, with an IC₅₀ value of 0.45 μM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI > 1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure–activity relationships are discussed and compared with related naturally occurring compounds.     

Haemolytic acylated triterpenoid saponins from Harpullia austro-caledonica

Eight new acylated triterpenoid saponins were isolated from the stem bark of Harpullia austro-caledonica along with the known harpuloside (9). Their structures were established using 1D and 2D NMR and mass spectrometry as 3-O-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylbarringtogenol C (1), 3-O-alpha-L-rhamnopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloyl barringtogenol C (2), 3-O-alpha-L-arabinofuranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylbarringtogenol C (3), 3-O-alpha-L-arabinofuranosyl-(1-->2)-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylprotoaescigenin (4), 3-O-alpha-L-arabinofuranosyl-(1-->3)-[alpha-L-arabinofuranosyl-(1-->2)]-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloyl protoaescigenin (5), 3-O-alpha-L-arabinofuranosyl-(1-->3)-[beta-D-xylopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylprotoaescigenin (6), 3-O-alpha-L-arabinofuranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylprotoaescigenin (7), 3-O-beta-D-xylopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21 beta, 22 alpha-di-O-angeloylprotoaescigenin (8). The EtOH extract of the stem bark showed in vitro cytotoxic activity against KB cells (90% at 10 microg/ml). At a concentration of 5 microg/ml, the saponin mixture showed haemolytic activity and caused 100% haemolysis of a 10% suspension of sheep erythrocytes.