Band 63, Heft 1-2

Chromatographic separation of a 70% aqueous methanol extract (AME) of Pongamia pinnata (Linn.) Pierre (Leguminosae) leaves has led to the isolation of two new isoflavonoid diglycosides, 4′-O-methyl-genistein 7-O-β- D -rutinoside (2) and 2′,5′-dimethoxy-genistein 7- O-β- D -apiofuranosyl-(1‴→ 6″)-O-β- D -glucopyranoside (6), and a new rotenoid, 12a-hydroxy-α-toxicarol (5), together with nine known metabolites, vecinin-2 (1), kaempferol 3-O-β-drutinoside (3), rutin (4), vitexin (7), isoquercitrin (8), kaempferol 3-O-β-d-glucopyranoside (9), 11,12a-dihydroxy-munduserone (10), kaempferol (11), and quercetin (12). Their structures were elucidated on the basis of chemical and spectroscopic analyses.

From a pyoverdin-negative mutant of Pseudomonas fluorescens AF76 a new lipopeptidic siderophore (ornicorrugatin) could be isolated. It is structurally related to the siderophore of Pseudomonas corrugata differing in the replacement of one Dab unit by Orn.

Phaleria macrocarpa (Scheff.) Boerl., a member of the Thymelaeaceae, is traditionally used in Indonesia as medicinal plant against cancer. In this context, we isolated the lignans pinoresinol, lariciresinol and matairesinol from different parts of this plant. The enantiomeric composition of these lignans was determined by chiral column analysis. Pinoresinol and lariciresinol were mixtures of both enantiomers with (79 ± 4)% and (55 ± 6)% enantiomeric excess for the (−)-enantiomers, respectively, whereas matairesinol was found as pure (+)-enantiomer.

Contents of total polyphenols, condensed tannins and proanthocyanidins, and their stability to various pH values and temperatures were studied in Mexican blueberry, cuautecomate fruit, garambullo fruit, aubergine, coffee pulp and residues of black grapes. Several aqueous extracts, obtained through a one-pass-extraction process, were analyzed using liquid chromatography in order to quantify the condensed tannin (proanthocyanidin) content responsible for their antioxidant activity and colour. All tested samples included high proanthocyanidin contents demonstrating that these Mexican fruits and vegetables are good sources of natural antioxidants, and they all could be considered as excellent functional foods due to their bioactivity measured as the condensed tannin level.

The antifungal activity of 40 coumarins was tested against the fungal strains: Candida albicans (ATCC 14053), Aspergillus fumigatus (ATCC 16913) and Fusarium solani (ATCC 36031), using the broth microdilution method. Osthenol showed the most effective antifungal activity among all the compounds tested, with a MIC value of 125 μg/ml for Fusarium solani and 250 μg/ml for Candida albicans and Aspergillus fumigatus. The antifungal potential of this prenylated coumarin can be related to the presence of an alkyl group at C-8 position.

Esters of 2-hydroxy-4-methoxy-6-n-pentylbenzoic acid (2−8) (methyl, ethyl, butyl, pentyl, isopropyl, sec-butyl and benzyl), olivetol (9), methyl, ethyl, butyl perlatolates (10−12), 2,4- dihydroxy-6-n-pentylbenzoic acid (15), and methyl and ethyl esters of (15) were prepared through structural modifications of perlatolic acid (1) with the aim to detect new antifungal and antibacterial substances and also to evaluate the toxicity by the brine shrimp lethality assay against Artemia salina. The antifungal assays were carried out against the fungus Cladosporium sphaerospermum through the bioautography method, and methyl 2,4-dihydroxy- 6-n-pentylbenzoate (13) showed the highest antifungal activity (2.5 μg). Olivetol (9) and 2,4- dihydroxy-6-n-pentylbenzoic acid (15) are also potent inhibitors of the growth of the fungus (5.0 μg). Except for methyl (10), the ethyl (11) and butyl (12) perlatolates were less active than perlatolic acid (1). The activities presented by methyl (2) and ethyl (3) 2-hydroxy-4- methoxy-6-n-pentylbenzoates and methyl (13) and ethyl (14) 2,4-dihydroxy-6-n-pentylbenzoates suggest that compounds with a free hydroxy group in the aromatic ring (C-4) have a more pronounced effect against C. sphaerospermum. Antibacterial activities were tested by the disc diffusion method using pathogenic strains of S. aureus and E. coli. The compounds were weakly active with inhibition zones between 9−15 mm. The 2-hydroxy-4-methoxy-6-npentylbenzoic esters 2−8 and alkyl perlatolates 10−12 were selective against E. coli. Perlatolic acid (1) and methyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (2) were the most active with LD 50 values of 24.1 μ M and 27.2 μ M , respectively. The other compounds were not toxic to Artemia salina larvae.

A new methylated flavonol, 5,7,2 ′,4 ′-tetrahydroxy-3-methoxyflavone (1), had been isolated from the methanol extract of the wood of Morus australis, along with nine known compounds, kuwanon C (2), morusin (3), morachalcone A (4), oxyresveratrol (5), 4 ′-(2-methyl-2-buten- 4-yl)oxyresveratrol (6), moracins M (7) and C (8), alboctalol (9), and macrourin B (10). The structures of these compounds were determined based on spectral evidence, including UV, IR, NMR, and mass spectra. Cytotoxic properties of compounds 1 →10 were evaluated against murine leukemia P-388 cells. The prenylated stilbene 6 and 2-arylbenzofuran 8, and morusin (3) were found to have strong cytotoxic effects with IC 50 values of 6.9, 8.7, and 10.1 μ M , respectively.

Baccharis dracunculifolia D.C. (Asteraceae) is the most important plant source of the Brazilian green propolis. Since propolis is known for its antimicrobial activity, the aim of this work was to evaluate the antimicrobial activities of B. dracunculifolia and some of its isolated compounds. The results showed that the leaves extract of B. dracunculifolia (BdE) presents antifungal and antibacterial activities, especially against Candida krusei and Cryptococcus neoformans, for which the BdE showed IC 50 values of 65 μg mL −1 and 40 μg mL −1 , respectively. In comparison to the BdE, it was observed that the green propolis extract (GPE) showed better antimicrobial activity, displaying an IC 50 value of 9 μg mL −1 against C. krusei. Also, a phytochemical study of the BdE was carried out, affording the isolation of ursolic acid (1), 2α-hydroxy-ursolic acid (2), isosakuranetin (3), aromadendrin-4′-methylether (4), baccharin (5), viscidone (6), hautriwaic acid lactone (7), and the clerodane diterpene 8. This is the first time that the presence of compounds 1, 2, and 8 in B. dracunculifolia has been reported. Among the isolated compounds, 1 and 2 showed antibacterial activity against methicillin- resistant Staphylococcus aureus, displaying IC 50 values of 5 μg mL −1 and 3 μg mL −1 , respectively. 3 was active against C. neoformans, showing an IC 50 value of 15 μg mL −1 and a MIC value of 40 μg mL −1 , while compounds 4→8 were inactive against all tested microorganisms. The results showed that the BdE, similar to the GPE, displays antimicrobial activity, which may be related to the effect of several compounds present in the crude extract.

Chromatographic separation of an ethyl acetate extract from Embelia schimperi led to the isolation of a new compound identified as 2,5-dihydroxy-3-methyl-1,4-benzoquinone (1) on the basis of spectroscopic and physical data. The plant’s crude extract and pure compound 1 were assayed for in vitro antimicrobial activity against clinical strains of Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus. Disc diffusion method was used and zones of inhibition, after respective incubation periods, were used to quantify antimicrobial activity. Standard antibiotics namely: augmentin, cotrimoxazole, gentamycin, tetracycline and lyncomycin were used as controls. The crude extract was inactive while the pure compound 1 showed significant activities against Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus with zones of inhibition ranging from 10−20 mm. The most sensitive microorganism was P. aeruginosa while C. neoformans was insensitive to both the crude extract and compound 1.

Five ingenane compounds, 1−5, kansuinins A and B, isolated from Euphorbia kansui, and their derivatives 7 and 9 were tested for termiticidal activity against the Japanese termite, Reticulitermes speratus. At 72 hours after treatment, the ingenane compounds 1 to 5 caused 100% mortality in R. speratus at 50, 25 and 12.5 μg/disk, respectively, except for compound 1, which gave a mortality rate of (93.06 ± 5.56)% at 12.5 μg/disk. At 36, 48 and 60 hours after treatment, compounds 1 to 5 showed more termiticidal activity than kansuinins A and B and their derivatives. The kansuinins showed no or only slight activity against termites in the filter paper bioassay under the conditions tested compared with a solvent control.

Under the bioassay-guided method, two diterpenes, 3-O-(2″,3″-dimethylbutanoyl)-13-Ododecanoylingenol (1) and 3-O-(2″,3″-dimethylbutanoyl)-13-O-decanoylingenol (2) isolated from Euphorbia kansui, showed a pronounced antinematodal activity against the nematode Bursaphelenchus xylophilus at the same minimum effective dose (MED) of 5 μg per cotton ball and still displayed antinematodal activity at a dose of 2.5 μg per cotton ball. Compounds 3 − 6 were obtained, and the structure of the new compound 6 was elucidated based on 1D-and 2D-NMR analyses and physicochemical data. Preliminary structure-biological activity relationships of ingenane-type compounds were deduced.

Flavonoids and organic acids were recommended in the literature as the main active constituents of Camptosorus sibiricus Rupr. Assay-guided fractionation led to the isolation of 9 flavonoids and 8 phenolic acids. All compounds were tested for DPPH scavenging activity, SOD-like and aldose reductase inhibition. Among them, compounds 1, 2, 3, 5, 6, 7, 8, 9, 11, 15 showed activities. The most active free radical scavenger and antioxidant was compound 8, while compound 1 exhibited strong inhibiting activity of aldose reductase. The structureactivity relation was dicussed briefly.

DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme’s activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC 50 ) of rhamnolipid PS-17 was 14.5 μg/ml against HSV-1 and 13 μg/ml against HSV-2. The IC 50 values of the complex were 435 μg/ml for HSV-1 and 482 μg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: ∆log CCID 50 of 1.84−2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 μg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 μg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.

Water supplemented with 10% or 20% (v/v) of Zarrouk medium was used to cultivate Spirulina platensis in closed and open bioreactors under controlled conditions (30 °C, 32.5 μmol m −2 s −1 , 12 h light/dark photoperiod) and in a greenhouse (9.4 to 46 °C, up to 2800 μmol m −2 s −1 , variable day length photoperiod) using different initial biomass concentrations (X 0 ) in the extreme south of Brazil (32.05° S, 52.11° W). Under controlled conditions the maximum specific growth rate (μ max ) was 0.102 d −1 , the biomass doubling time (t d ) was 6.8 d, the maximum dry biomass concentration (Xmax) was 1.94 g LD1 and the maximum productivity (P max ) was 0.059 g L −1 d −1 , while the corresponding values in the greenhouse experiments were μ max = 0.322 d −1 , t d = 2.2 d, X max = 1.73 g L −1 and P max = 0.112 g L −1 d −1 . Under controlled conditions the highest values for these parameters occurred when X 0 = 0.15 g L −1 , while in the greenhouse X 0 = 0.4 g L −1 produced the highest values. These results show that the cultivation of S. platensis in greenhouses in the extreme south of Brazil is technically viable and that the S. platensis inoculum and the concentration of Zarrouk medium can be combined in such a way as to obtain growth and productivity parameters comparable, or superior, to those occurring in bioreactors under controlled conditions of temperature, illuminance and photoperiod.

Quantitative composition of saponins (ginsenosides) in Panax quinquefolium originating from field cultivation in Poland and harvested at the beginning of the growing season, during the blossoming period and at the end of growth was determined. A colourimetric method gave lower values compared to results of HPLC analysis, but the tendency of change in the content of saponins was similar in both instances. Ginsenoside amounts in P. quinquefolium organs changed depending on the specific time during the vegetation season the samples were taken. This study found that the highest content of these metabolites −66 mg/g d. w. − occurred in the roots of the plant at the time of bloom. Two among the six metabolites examined in our study were dominant independently of the vegetation season. These were Rb 1 and Re, with values of 25.4−33.8 mg/g d. w. and 16.4−19.7 mg/g d. w., respectively.

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 ± 0.36) μg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 ± 0.23) μg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.

The amino acid proline is accumulated in plant tissues in response to a variety of stresses. The existence of two routes for its biosynthesis is well documented. However, little is known about the contribution of each pathway to the accumulation of free proline under stress conditions. In the present study young barley plants were subjected to osmotic stress by treating their roots with 25% polyethylene glycol. Prior to stress imposition roots were incubated for 24 h in nutrient solution containing proline or one of its metabolic precursors: glutamate and ornithine. Free proline quantity in the leaves was measured before and after stress. Relative water content (RWC) was used as a measure of the plant water status. Foliar proline levels showed a significant increase in ornithine- and proline-pretreated plants compared to the control. Nevertheless, no considerable changes in leaf RWC were observed. It was shown that before stress application only ornithine but not glutamate was immediately metabolized to proline. Under stress conditions, however, both precursors were converted into proline. The possible role of this amino acid in the processes of post stress recovery is discussed.

Aminopeptidase, preferring phenylalanine-p-nitroanilide as substrate, and proline iminopeptidase, highly-specific for proline-p-nitroanilide, were isolated from cabbage leaves (Brassica oleraceae var. capitata). As pH optima, 7.2−7.5 for aminopeptidase activity and 8.0−8.5 for proline iminopeptidase were determined. Both peptidases were strongly inhibited by p-chloromercuribenzoic acid, heavy metal ions and urea. The molecular weights were determined by gel filtration to be 56 and 204 kDa, respectively. The iminopeptidase was decomposed during SDS electrophoresis to four subunits of 50 kDa. Minor impurities of myrosinase- associated protein (~70 kDa) were found in both preparations. Preliminary data of their amino acid sequences showed similarities to those of aminopeptidases N (family M1) and proline iminopeptidases (family S33).

Plants, as well as fungi, use ambient sunlight as information to regulate photomorphogenetic processes. The photobiological control of this information showed that the development of photobiological greenhouse plastic covers simulates a photonic information that leads to a physiological enhancement of plant productivity and fungal disease control, thus minimizing the need for the use of agrochemicals. The main characteristics of these photobiological greenhouse plastic covers are the high transmission of photosynthetically active radiation (PAR, 400 − 700 nm) combined with an increase of the factor ζ =RL (655 −665 nm) /FRL (725 −735 nm) , which affects the cellular phytochromic equilibrium Φ = Pfr/(Pfr + Pr) and regulates the photosynthetic activity and therefore the plant productivity. Additionally, increase of the spectral ratios from the transmitted light: BL( 420-500 nm)/nearUV( 290-370 nm ) and BL (420 −500 nm) / FRL (725 −735 nm) , cause mainly the induction of biochemical, physiological and morphological responses, regulated by cryptochromes in plants (e. g. inflorescence and infructescence) and mycochrome in fungi (e. g. inhibition of sporulation). In the present work, comparative studies with randomly selected greenhouse plastics showed that small changes in the abovementioned “photobiological” parameters raise the productivity of tomato plants and inhibit the sporulation of several isolates of the fungal pathogen Botrytis cinerea. Thus, a model for the photoregulation of these two phenomena in greenhouses is proposed.

Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including β-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A 260 /A 230 and A 260 /A 280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g −1 fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.

Cell culture is an effective technology for taxol production. This paper discusses the effect of Taxus cell cultures on the 18S rRNA gene sequences based on the phylogenetic analysis of cultured T. chinensis cells and related species. The phylogenetic tree is reconstructed using the maximum parsimony method and the relative rate test to test the hypothesis of a molecular clock. The phylogenetic analysis indicates that cell culture changes the phylogenetic position of cultured T. chinensis cells. More than that, the 18S rRNA gene of cultured T. chinensis cells has a faster rate of substitution than that of T. chinensis. With T. media as reference, the divergence time of the cultured T. chinensis cells is 7 Ma (million years) more than that of the T. chinensis cells based on the 18S rRNA gene sequences.

In an attempt to estimate the occurrence of phenol hydroxylase-related gene sequences we performed a dot blot hybridization assay with DNA from phenol utilizing Trichosporon cutaneum R57 strain NBIMCC 2414 and microbial isolates from different wastewaters. The used oligonucletides were homologous to the 5 ′-end of TORPHD locus (NCBI)-coding phenol hydroxylase in Trichosporon cutaneum ATCC 46490 and to the 5 ′-end of TORCCMLE locus (NCBI)-coding cis,cis-muconate-lactonizing enzyme in Trichosporon cutaneum ATCC 58094. Two microbial strains, Escherichia coli JM 109 and Lactobacillus acidophilus ATCC 4356, incapable to degrade phenol were used as negative controls. We established the presence of hybridization with both used oligonucleotide probes in T. cutaneum R57 and T. cutaneum ATCC 46490 yeast strains. The experiments implemented with microbial isolates obtained from three industrialized areas in Bulgaria showed that 7 of them may carry sequences hybridizing with a phenol hydroxylase oligonucleotide probe. A subsequent hybridization test for the cis,cis-muconate-lactonizing enzyme showed that only 3 of them displayed a positive signal. Lactobacillus acidophilus ATCC 4356 and Escherichia coli JM 109 strains’ DNA used as negative controls in the experiments did not reveal any sequence similarity to the both applied oligonucleotides. The partial nucleotide sequences of 16S rDNAs of the isolated strains C1 and K1 obtained as PCR products were determined and sequenced. A comparison of these nucleotide sequences with similar sequences in NCBI Data Bank indicated that both C1 and K1 strains are closely related to the genera Acinetobacter and Burkholderia.

Four genes encoding parasporins, cytotoxins preferentially killing human cancer cells in vitro, were isolated from four Vietnamese strains of Bacillus thuringiensis. Nucleotide sequence analysis revealed that: (1) three genes fall into the two known classes, ps1Aa and ps1Ab, and (2) another one belongs to ps1Ac, a novel gene class established in this study. Upon proteolytic activation, parasporal protein of the organism with ps1Ac exhibited strong cytocidal activity against human cancer cells, HeLa and Hep G2, but not to non-cancer normal cells, UtSMC and HC.

A filamentous microorganism, morphologically similar to the cyanobacterium Arthrospira, was isolated from Mangueira Lagoon in Brazil, from which Arthrospira has not previously been isolated. Random amplified polymorphic DNA (RAPD) comparison with the standard Arthrospira platensis strains LEB 52 and Paracas indicated that the organism isolated was an Arthrospira isolate, which we denominated strain LEB 18. The RAPD analysis showed conserved sequences which indicated that the three strains belonged to the same genus, and were all Arthrospira species, but there were sufficient differences between them suggesting that they were separate strains. The strain LEB 18 was cultivated in undiluted Zarrouk medium and in 60% and 20% (v/v) Zarrouk medium diluted with sterilized Mangueira Lagoon water (MLW) using illuminance rates of 32.5, 45.5 and 58.5 μmol m −2 s −1 according to a complete 3 2 factorial design with a triplicate central point. The strains LEB 52 and Paracas were cultived in the conditions central point. Our new isolate produced the highest specific growth rate (μ max = 0.22 d −1 ) in 60% Zarrouk medium diluted with MLW and illuminated with 58.5 μmol m −2 s −1 and the highest protein content (86.0% w/w).

A Notable decrease of the peak intensity of the capillary electrophoregram due to the dimeric SOD molecule was observed when a solution containing copper(II) chloride and ascorbic acid was added to the SOD solution, indicating that the capillary electrophoresis method is useful to detect the dissociation of the dimeric SOD molecule in solution, and that dissociation of the dimeric SOD molecule is induced by the presence of hydrogen peroxide. The present results may give reasonable countermeasures towards the sporadic amyotrophic lateral sclerosis in future.

We have investigated the mechanism of manganese ion uptake by apo-transferrin using a capillary electrophoresis method, and obtained clear evidence that oxidation state +3 and the binuclear unit of a manganese chelate are essential factors for the facile uptake by apotransferrin, similar to that observed for Fe(III) chelates. These results may give valuable information to understand the pathogenesis of manganism and to develop new countermeasures for the neurotoxicity by manganese ions.

A method named “bag culture” was developed for coculturing of Linum persicum (section Syllinum) and L. austriacum (section Linum) hairy roots. For this propose L. austriacum and L. persicum hairy root cultures were established using Agrobacterium rhizogenes in McCown medium. L. persicum hairy roots in bags (1 mm 2 mesh) were successfully grown together with L. austriacum hairy roots. The amounts of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (MPTOX) produced by L. persicum hairy root cultures were detected using HPLC. The results indicated that the amounts of both lignans and growth indexes of the two hairy roots decreased, that may be partly due to a competition between the two types of culture in using precursors of biosynthetic metabolites and the amount of culture medium which is available for each hairy root. However, MPTOX (0.17 g/100 g DW) and PTOX (0.02 g/100 g DW) levels of the L. persicum single culture in bag were significantly higher than of the other cultures which may be due to the immobilization effect of the bag.