Challenges and pitfalls in antioxidant research.

Over the last decade, much research has focused on the potential health benefits of antioxidants and indeed many synthetic and natural compounds have been evaluated for their antioxidant profile. However, in several studies only a limited number of assays, often poorly validated, are used and the techniques available frequently lack specificity. These limitations may incorrectly influence the results. This review will therefore focus on several pitfalls that may emerge in vitro and in vivo antioxidant research. First, different in vitro techniques to determine antioxidant potential are discussed, including radical scavenging assays and fingerprinting methods. As a rule, a panel of different assays is indispensable to characterize and establish in vitro antioxidant activity. Furthermore, as problems of absorption, distribution, metabolism and excretion are only accounted for by in vivo studies, the need for in vivo antioxidant research is pointed out. Several methods to characterize the in vivo activity of antioxidants, including major drawbacks and pitfalls of some assays, have been discussed. The availability of both a representative "oxidative stress" animal model and a battery of well-validated assays to assess the broad diversity of oxidative damage and antioxidative defence parameters, are crucial for antioxidant research in vivo.

Proanthocyanidins in health care: current and new trends.

Polyphenolic compounds are widely distributed in higher plants and are an integral part of the human diet. Recent interest in these substances has been stimulated by their potential health benefits, which are believed to arise mainly from their antioxidant activity. In the past years, the antioxidant activity of flavonoids has been studied in detail. An important but often overlooked group of polyphenols is that of the proanthocyanidins. Therefore, the present review is focused mainly on the antioxidant activity of proanthocyanidins and its relevancy in vivo. The three most important mechanisms of their antioxidant action will be discussed, i.e. free radical scavenging activity, chelation of transition metals, and inhibition of enzymes. In addition, the protective role of proanthocyanidins against lipid peroxidation and peroxynitrite, as well as their antimicrobial properties will be discussed. To study the in vivo relevancy of the proanthocyanidin activities, the knowledge of their pharmacokinetic parameters is crucial. Although bioavailability and metabolism data on polyphenols in general and proanthocyanidins in particular are still largely unavailable, the first reports indicate that at least monomers and smaller oligomeric procyanidins are absorbed. There is also considerable scientific and public interest in the important role that antioxidants may play in health care, e.g. by acting as cancer chemopreventive and anti-inflammatory agents and by reducing risk of cardiovascular mortality. Each of these aspects will be discussed, with special attention to the role of proanthocyanidins on apoptosis, gene expression and transcription factors, such as NF-kappa B.

New acylated triterpenoid saponins from Maesa lanceolata.

Ten new acylated triterpenoid saponins were isolated from the leaves of Maesa lanceolata. For their structure elucidation extensive use was made of homo- and heteronuclear 2D NMR techniques such as COSY, NOESY, HSQC and HMBC. All saponins identified contained the same tetraglycosidic side chain, but the triterpenoid moiety showed a variable esterification pattern. Monoester, diester and triester derivatives were present. Maesasaponin I was a 21-monoester derivative, i.e. ¿3 beta-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl- (1-->3)]-[beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl+ ++¿-21 beta-angeloyloxy-13 beta, 28-oxidoolean-16 alpha, 22 alpha, 28 alpha-triol. Maesasaponins III, IV3, V3 and VI2 had an additional acetyl, propanoyl, n-butanoyl and angeloyl substituent, respectively, in position 22. Maesasaponins II, IV2, V2, VI3 and VII1 were characterised as the 16-acetyl derivatives of maesasaponins I, III, IV3, V3 and VI2, respectively. Structures of saponins previously reported in M. lanceolata had to be revised.

Triterpenoid saponins from Maesa lanceolata.

Six new homologous triterpenoid saponins were isolated from the methanol extract of the leaves of Maesa lanceolata and characterized as 3 beta-O-[alpha-L-rhamnopyranosyl(1 --> 2)-beta-D-galactopyranosyl (1 --> 3)]-[beta-D-galactopyranosyl(1 --> 2)]-beta-D-glucuronopyranosides alpha-diol, 22 alpha-angeloyloxy-16 alpha-butanoyloxy-13 beta,28-oxydoolean-21 beta,28 alpha-diol, 16 alpha,22 alpha-diangeloyloxy-13 beta,28-oxydoolean-21 beta,-28 alpha-diol, 22 alpha-angeloyloxy-13 beta,28-oxydo-16 alpha-(2-methyl-butanoyloxy)-olean-21 beta,28 alpha-diol, 21 beta-acetoxy-22 alpha-angeloyloxy-13 beta,28-oxydo-16 alpha-propanoyloxyolean-28 alpha-ol, 21 beta-acetoxy-22 alpha-angeloyloxy-16 alpha-butanoyloxy-13 beta,28-oxydoolean-28 alpha-01. The structures were established on the basis of chemical and spectral evidence.

Oumarone, bissaone, and aissatone, unusual prenylated polyketides from harrisonia abyssinica

Fractionation of the n-hexane extract of the leaves of Harrisonia abyssinica, collected in Guinea, afforded three novel and unusual prenylated polyketides, which were named oumarone (1), bissaone (2), and aissatone (3). Their structures were elucidated by spectroscopic methods, mainly 1D and 2D NMR spectroscopy.

Isoquinoline derivatives isolated from the fruit of Annona muricata as 5-HTergic 5-HT1A receptor agonists in rats: unexploited antidepressive (lead) products.

The fruit and the leaves of Annona muricata (Annonaceae) are used in traditional medicine for their tranquillizing and sedative properties. Extracts of the plant have been shown to inhibit binding of [3H]rauwolscine to 5-HTergic 5-HT1A receptors in calf hippocampus, and three alkaloids, annonaine (1), nornuciferine (2) and asimilobine (3), isolated from the fruit have been shown to have IC50 values of 3 microM, 9 microM and 5 microM, respectively, although in ligand-binding studies it was not possible to determine whether interaction of these ligands with the receptor was agonistic or antagonistic. This paper presents the results of functional assays of the alkaloids. The inhibition of cAMP accumulation was tested in NIH-3T3 cells stably transfected with the 5-HT1A receptor from man. None of the alkaloids showed antagonistic properties towards the 5-HT1A receptors because in the antagonistic tests no influence on the forskolin-stimulated increase of cAMP level was detected. Full agonistic properties were measured for all three compounds; the inhibition constants (Ki) for 1, 2 and 3 were < 10 microM. Inhibition of the binding of the radioligand to the 5-HT1A receptor was observed in every ligand-binding assay performed with the alkaloids; the Ki values for 1, 2 and 3 were in the microM range. These results imply that the fruit of Annona muricata possesses anti-depressive effects, possibly induced by compounds 1, 2 and 3, and that in the past potent leads for the development of anti-depressive therapeutics have not been used.

Cirsimarin and cirsimaritin, flavonoids of Microtea debilis (Phytolaccaceae) with adenosine antagonistic properties in rats: leads for new therapeutics in acute renal failure.

In traditional medicine Microtea debilis is used against proteinuria. In ligand-binding studies extracts of Microtea debilis have been shown to inhibit the binding of [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) to adenosine-A1 receptors in rat forebrain membranes. Subsequently, cirsimarin, a flavonoid, was isolated as the active component and was shown to function as adenosine antagonist at the adenosine-A1 receptor in-vitro. In this study we have investigated the adenosine-A2 receptor activity of cirsimarin the in-vivo inhibition of the effects of adenosine by cirsimarin in rats, the absorption of cirsimarin and the inhibition of the binding of [3H]DPCPX to the adenosine-A1 receptor by urine samples obtained after oral administration of crude extract of Microtea debilis, cirsimarin or cirsimaritin to rats. Cirsimarin inhibited the binding of [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to adenosine-A2 receptors in rat striatum with an inhibition constant, Ki, of 6.5 +/- 0.3 microM. The decrease of heart rate and blood pressure induced by adenosine was significantly inhibited by cirsimarin. After oral administration of 8 and 80 mg kg-1 cirsimarin, the compound could not be detected in either plasma or urine, but the presence of cirsimaritin was established. By use of beta-glucuronidase, glucuronides of cirsimaritin were also detected in the urine. The concentrations of cirsimaritin in the plasma were 0.126 +/- 0.04, 0.138 +/- 0.015, and 0.120 +/- 0.022 microM, respectively, 2, 5 and 12 h after administration of 8 mg kg-1 cirsimarin. The concentrations of cirsimaritin in the urine at the same times after administration of the same dose were 2.05 +/- 1.86, 5.05 +/- 2.6 and 2.06 +/- 0.09 microM, respectively. The inhibition of the binding of [3H]DPCPX to the adenosine-A1 receptor by urine samples collected 2, 5 and 12 h after oral administration of 8 mg kg-1 cirsimarin or a crude extract of Microtea debilis containing approximately 8 mg kg-1 cirsimarin and 2.8 mg kg-1 cirsimaritin, or 6.8 mg kg-1 cirsimaritin, was not significantly different from that of urine samples collected from untreated rats, in contrast with urine samples collected 1 and 2 days after oral administration of 80 mg kg-1 cirsimarin. Approximately 3% of the cirsimarin was excreted in the urine as cirsimaritin. The results indicate that in the kidney and urinary tract the concentrations of cirsimaritin produced after ingestion of more than 8 mg kg-1 cirsimarin can be high enough to inhibit the interaction of adenosine with its receptors; this might explain the effectiveness of Microtea debilis preparations against proteinuria in traditional medicine.

Comparative study of eight well-known polyphenolic antioxidants.

Eight antioxidants from five different polyphenolic classes (cinnamic acids, benzoic acids, flavonoids, proanthocyanidins and stilbenes), and the water-soluble vitamin E derivative trolox were examined for their antioxidant activity in-vitro. In addition, the compounds were tested for their cytotoxicity on growing fibroblasts and their inhibition of the classical pathway of the complement system. Procyanidin C1 was shown to be a good scavenger of both DPPH(*) and HO(*), and a strong inhibitor of lipid peroxidation and the classical pathway of the complement system. Consequently, procyanidin C1 was classified as the most promising antioxidant in-vitro of all compounds tested. In contrast, genistein exhibited a very low antioxidant activity in both the lipid peroxidation and the DPPH(*) scavenging assay, a high cytotoxicity and a low complement-inhibiting activity.

Radical scavenging and xanthine oxidase inhibitory activity of phenolic compounds from Bridelia ferruginea stem bark.

Bridelia ferruginea Benth. (Euphorbiaceae) is a subtropical medicinal plant widely used in traditional African medicine against various diseases, including rheumatic pains. Seven of its constituents (3-O-methylquercetin (1), 3,7,3',4'-tetra-O-methylquercetin (rutisin, 2), myricetin (3), 3',4',5'-tri-O-methylmyricetin (ferrugin, 4), 3,3',4',5'-tetra-O-methylmyricetin (5), quercetin 3-O-glucoside (6), and a biflavanol gallocatechin-[4'-O-7]-epigallocatechin (7)) have been evaluated in-vitro in the xanthine-xanthine oxidase enzymatic system for inhibition of xanthine oxidase and radical scavenging activity. Results indicated that compounds 1, 3, 4 and 6 exhibited, at different levels, xanthine oxidase inhibiting and superoxide scavenging activity at micromolar concentrations, whereas compound 7 showed scavenging activity only. Compounds 2 and 5 were inactive in both cases. Study of the structure-activity relationship demonstrated that for flavonoids the xanthine oxidase inhibitory activity was reduced by methylation of the hydroxyl functionality at C-3 and in rings A and B. These results may partly explain and support the use of B. ferruginea stem bark for the treatment of rheumatic pains in traditional medicine.

Further evaluation of Rwandan medicinal plant extracts for their antimicrobial and antiviral activities.

A total of 45 Rwandan plant extracts, belonging to 37 different plant species out of 21 families, were investigated for their antibacterial, antifungal, and antiviral properties. The plants were selected on the base of their ethnomedicinal use against infections and autoimmune diseases. From all the plant extracts tested, only Clematis hirsuta (leaves) showed a pronounced antifungal activity against Candida albicans and the dermatophytes Trichophyton rubrum, Epidermophyton floccosum, and Microsporum canis. Seven plant extracts showed a high antiviral activity against the DNA-virus Herpes simplex type 1, while five and three plant extracts were highly active against the RNA-viruses Coxsackie and Polio, respectively. Only Macaranga kilimandscharica (leaves) showed an interesting anti-measles activity, whereas Eriosema montanum (leaves) and Entada abyssinica (leaves) were highly active against Semliki forest virus. Some plant extracts showed an antibacterial activity against Gram-positive bacteria and Mycobacterium fortuitum, but none of them were active against the Gram-negative bacteria tested.

Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo.

The chemical composition of essential oils from 15 aromatic medicinal plant species growing in the Democratic Republic of Congo have been studied. More than 15 constituents in an amount higher than 0.1% were identified in each essential oil. 1,8-cineole, alpha and beta-pinene, p-cymene, myrcene, gamma-terpinene, alpha-terpineol and limonene were prevalent constituents in almost more than 10 selected plant species. Results from the antibacterial testing by the diffusion method indicate that all essential oils (5 microl per disc) inhibited the growth of selected bacteria at different extents. The most active antibacterial essential oils were those of the leaves of Eucalyptus camadulensis and Eucalyptus terticornis (12-30 mm zone diameter of inhibition). They showed particularly a most potent inhibition of Pseudomonas aeruginosa growth (15-16 mm), followed by Eucalyptus robusta (12 mm). Essential oils from the leaves of Eucalyptus alba, Eucalyptus citriodora, Eucalyptus deglupta, Eucalyptus globulus, Eucalyptus saligna, Eucalyptus robusta, Aframomum stipulatum, Cymbopogon citratus, Ocimum americanum and that of the seeds of Monodora myristica showed also a good antibacterial activity (10-18 mm). Eucalyptus propinqua, Eucalyptus urophylla and Ocimum gratissimum essential oils were the less active samples against the selected bacteria. No correlation between the amount of major constituents such as 1,8-cineol, alpha-pinene, p-cymene, cryptone or thymol and the antibacterial activity was observed.

Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo.

Twenty extracts including ten EtOH and ten CH2Cl2 from different parts of nine African medicinal plants used in Congolese traditional medicine for the treatment of malaria, were submitted to a pharmacological test in order to evaluate their effect on P. falciparum growth in vitro. Of these plant species, 14 (70%) extracts including EtOH and CH2Cl2 from Cassia occidentalis leaves, Cryptolepis sanguinolenta root bark, Euphorbia hirta whole plant, Garcinia kola stem bark and seeds, Morinda lucida leaves and Phyllanthus niruri whole plant produced more than 60% inhibition of the parasite growth in vitro at a test concentration of 6 microg/ml. Extracts from E. hirta, C. sanguinolenta and M. morindoides showed a significant chemosuppression of parasitaemia in mice infected with P. berghei berghei at orally given doses of 100-400 mg/kg per day.

In vitro antiplasmodial activity of callus culture extracts and fractions from fresh apical stems of Phyllanthus niruri L. (Euphorbiaceae): part 2.

The ethanolic extracts from fresh apical stems of Phyllanthus niruri L. (Euphorbiaceae) cultured on Murashige and Skoog (MS) medium supplemented with IBA/BAP/Coco nucifera L. milk for 1, 2, 4 and 6 months were phytochemically and biologically investigated and compared with intact plant part and whole plant extracts. Results from the in vitro antiplasmodial testing indicated that the EtOH extract of a 1-month-old callus culture (IC(50) = 16.3 +/- 2.5 microg/ml) exhibited a higher activity than the ethanolic extracts of the fresh apical stem (IC(50) = 18.2 +/- 2.4 microg/ml) and callus cultures of 2-, 4- and 6-months-old (25 microg/ml < IC(50) < 40 microg/ml). These activities were however lower than that displayed by the ethanolic extract of the whole plant (IC(50) < 3 microg/ml). The EtOH extract of 1-month-old callus culture (the most active) was fractionated with solvents of different polarities. Its CH(2)Cl(2) fraction rich in terpenic constituents (IC(50) = 9.2 +/- 3.4 microg/ml) exhibited a higher antiplasmodial activity than its isoamylic alcohol fraction obtained at pH 2-3 (IC(50) = 25.6 +/- 2.3 microg/ml) rich in flavonoids. The activity of these two fractions was lower than that displayed by the same fractions from the whole plant (2 microg/ml < IC(50) < 3 microg/ml). Alkaloidic fractions from the whole plant and 1-month-old callus culture of fresh apical stem were considered as inactive (IC(50) > 100 microg/ml).

In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo.

The in vitro antiplasmodial activity of seven EtOH extracts and twenty fractions from the partition of the initial ethanolic extracts from seven African medicinal plants used in the Democratic Republic of Congo (DR Congo) for the treatment of malaria was evaluated. The most active EtOH extracts (IC50 < 3 microg/ml) were those from Cassia occidentalis leaves, Euphorbia hirta whole plant, Garcinia kola stem bark and Phyllanthus niruri whole plant. Their respective petroleum ether soluble fractions also exhibited an antiplasmodial activity with IC50 < 3 microg/ml. EtOH extracts from Vernonia amygdalina leaves (5 < IC50 < 10 microg/ml), Tetracera poggei leaves (10 < IC50 < 50 microg/ml) and Morinda morindoides leaves (50 < IC50 < 100 microg/ml) were less active, but their petroleum ether fractions exhibited a pronounced antiplasmodial activity (IC50 < 3 microg/ml). The same observation could also be made for the petroleum ether fraction from Cassia occidentalis, Euphorbia hirta, Garcinia kola and Phyllanthus niruri. Isoamyl alcohol fractions from Euphorbia hirta, Phyllanthus niruri and Vernonia amygdalina showed IC50) values less than 3 microg/ml, and from Cassia occidentalis, Garcinia kola, Morinda morindoides and Tetracera poggei between 10 and 50 microg/ml. The observed antiplasmodial activity may be related to the presence of terpenes, steroids, coumarins, flavonoids, phenolic acids, lignans, xanthones and anthraquinones.

Structure-activity relationship of flavonoids with superoxide scavenging activity.

The superoxide scavenging activities of 12 flavonoids were measured. The superoxide anions were generated by a hypoxanthine-xanthine oxidase system and measured by the nitrite method. The results showed that the scavenging ability enhanced with an increasing number of hydroxyl groups in ring B. Substitution at C3 position with a hydroxyl group increased the activity. Compared to a methoxyl group or a glycoside in this position, a free hydroxyl group showed the highest activity. A saturated C2-C3 bond showed a higher activity than a unsaturated bond. The absence of a carbonyl group at C4 position increased the activity.

Synthesis and antiviral activity of 7-O-(omega-substituted)-alkyl-3-O-methylquercetin derivatives.

3',4'-Di-O-benzyl-3-O-methylquercetin (2), the precursor in the synthesis of an important antivirally active flavone 3-O-methylquercetin (1), was regioselectively alkylated at the 7-OH position by a series of 1,omega-dihaloalkanes and omega-bromoalkanols. Dimerization of the flavone had to be avoided by applying strict reaction conditions. Subsequent debenzylation was carried out by catalytic transfer hydrogenolysis, affording quantitatively the 7-O-(omega-haloalkyl)-3-O-methylquercetin (11-14) and 7-O-(omega-hydroxyalkyl)-3-O-methylquercetin derivatives (15, 16). All compounds were tested for their antiviral activity against poliomyelitis- and HIV-viruses.

Conflict management style and marital satisfaction.

The aim of this study was to investigate whether there is one conflict management style that correlated more significantly with marital satisfaction than any other. In addition, spousal satisfaction with how marital conflict is managed was also examined, as were gender differences. Fifty-seven couples who had been married for at least 10 years took part in the study. Results showed that the collaborative conflict management style has the highest correlation with both marital satisfaction and spousal satisfaction with conflict management in the marriage. In contrast, where one or both of the spouses used the competitive conflict management style, the lowest marital satisfaction was reported. The results were also interpreted in terms of cultural and gender differences.

Isolation of a dihydrobenzofuran lignan from South American dragon's blood (Croton spp.) as an inhibitor of cell proliferation.

Dragon's blood is a red viscous latex extracted from the cortex of various Croton spp. (Euphorbiaceae), most commonly Croton lechleri, Croton draconoides (or Croton palanostigma), and Croton erythrochilus. It is used in South American popular medicine for several purposes, including wound healing. Bioassay-guided fractionation of dragon's blood, using an in vitro test system for the stimulation of human umbilical vein endothelial cells, has resulted in the isolation of a dihydrobenzofuran lignan, 3',4-O-dimethylcedrusin or 4-O-methyldihydrodehydrodiconiferyl alcohol [2-(3',4'-dimethoxyphenyl)-3-hydroxymethyl-2,3-dihydro-7-methoxybenzo furan-5- propan-1-ol] [1] as the biologically active principle. A related compound, 4-O-methylcedrusin [2-(3',4'-dimethoxyphenyl)-3-hydroxymethyl-2,3-dihydro-7-hydroxybenzo furan-5- propan-1-ol] [2], and the alkaloid taspine [3], also isolated from dragon's blood, were not active in the same assay. A cell proliferation assay, measuring the incorporation of tritiated thymidine in endothelial cells, showed that compound 1 did not stimulate cell proliferation, but rather inhibited thymidine incorporation, while protecting cells against degradation in a starvation medium.

In vitro and in vivo antiplasmodial activity of cryptolepine and related alkaloids from Cryptolepis sanguinolenta.

Three different extracts and four alkaloids from the root bark of Cryptolepis sanguinolenta have been assessed in vitro against Plasmodium falciparum D-6 (chloroquine-sensitive strain), K-1, and W-2 (chloroquine-resistant strains). Cryptolepine (1) and its hydrochloride (2), 11-hydroxycryptolepine (3), and neocryptolepine (5) showed a strong antiplasmodial activity against P. falciparum chloroquine-resistant strains. Quindoline (4) was less active. The highest activity was obtained with compound 1. In vivo tests on infected mice showed that cryptolepine (1), when tested as its hydrochloride (2), exhibited a significant chemosuppressive effect against Plasmodium berghei yoelii and Plasmodium berghei, berghei, while 1 had the same effect against P. berghei yoelii only. Compounds 3 and 4 did not show activity in this in vivo test system.

Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection.

Many compounds of plant origin have been identified that inhibit different stages in the replication cycle of human immunodeficiency virus (HIV): 1) virus adsorption: chromone alkaloids (schumannificine), isoquinoline alkaloids (michellamines), sulphated polysaccharides and polyphenolics, flavonoids, coumarins (glycocoumarin, licopyranocoumarin) phenolics (caffeic acid derivatives, galloyl acid derivatives, catechinic acid derivatives), tannins and triterpenes (glycyrrhizin and analogues, soyasaponin and analogues); 2) virus-cell fusion: lectins (mannose- and N-acetylglucosamine-specific) and triterpenes (betulinic acid and analogues); 3) reverse transcription; alkaloids (benzophenanthridines, protoberberines, isoquinolines, quinolines), coumarins (calanolides and analogues), flavonoids, phloroglucinols, lactones (protolichesterinic acid), tannins, iridoids (fulvoplumierin) and triterpenes; 4) integration: coumarins (3-substituted-4-hydroxycoumarins), depsidones, O-caffeoyl derivatives, lignans (arctigenin and analogues) and phenolics (curcumin); 5) translation: single chain ribosome inactivating proteins (SCRIP's); 6) proteolytic cleavage (protease inhibition): saponins (ursolic and maslinic acids), xanthones (mangostin and analogues) and coumarins; 7) glycosylation: alkaloids including indolizidines (castanospermine and analogues), piperidines (1-deoxynojirimicin and analogues) and pyrrolizidines (australine and analogues); 8) assembly/release: naphthodianthrones (hypericin and pseudohypericin), photosensitisers (terthiophenes and furoisocoumarins) and phospholipids. The target of action of several anti-HIV substances including alkaloids (O-demethyl-buchenavianine, papaverine), polysaccharides (acemannan), lignans (intheriotherins, schisantherin), phenolics (gossypol, lignins, catechol dimers such as peltatols, naphthoquinones such as conocurvone) and saponins (celasdin B, Gleditsia and Gymnocladus saponins), has not been elucidated or does not fit in the proposed scheme. Only a very few of these plant-derived anti-HIV products have been used in a limited number of patients suffering from AIDS viz. glycyrrhizin, papaverine, trichosanthin, castanospermine, N-butyl-1-deoxynojirimicin and acemannan.