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Screening of some Siberian medicinal plants for antimicrobial activity
Journal of Ethnopharmacology 82 (2002) 51 /53 www.elsevier.com/locate/jethpharm
Short communication
Screening of some Siberian medicinal plants for antimicrobial activity L. Kokoska a,*, Z. Polesny a, V. Rada b, A. Nepovim c, T. Vanek c a
Department of Tropical and Subtropical Crops, Czech University of Agriculture, Kamycka 129, Prague 6-Suchdol, Czech Republic Department of Microbiology and Biotechnology, Czech University of Agriculture, Kamycka 129, Prague 6-Suchdol, Czech Republic c Department of Plant Tissue Cultures, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic b
Accepted 24 May 2002
Abstract The antimicrobial activity of crude ethanolic extracts of 16 Siberian medicinal plants was tested against five species of microorganisms: Bacillus cereus , Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , and Candida albicans . Of the 16 plants tested, 12 showed antimicrobial activity against one or more species of microorganisms. The most active antimicrobial plants were Bergenia crassifolia, Chelidonium majus, Rhaponticum carthamoides, Sanguisorba officinalis , and Tussilago farfara . # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antimicrobial activity; Medicinal plants; Crude extracts; Herbal medicine
1. Introduction In many parts of Russia, particularly in Siberia, there is a rich tradition in the use of herbal medicine for the treatment of various infectious diseases, inflammations and injuries (Gammerman and Grom, 1976). Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, much recent attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine (Essawi and Srour, 2000). Plant based antimicrobials represent a vast untapped source for medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Iwu et al., 1999). In this study ethanolic extracts of different parts of 16 plants, which had been described in herbal books and
* Corresponding author. Tel.: /420-2-2438-2180; fax: /420-22092-1363 E-mail address: [email protected] (L. Kokoska).
folklore medicine of Russia were screened for their antimicrobial activity.
2. Materials and methods 2.1. Plant materials The seeds and seedlings of tested plants were obtained from the Botanical Garden of All-Russian Research Institute of Medicinal and Aromatic Plants (Moscow, Russia). They were grown in the experimental field of the Institute of Tropical and Subtropical Agriculture of the Czech University of Agriculture in Prague (ITSA CUA in Prague). The plant parts were collected during the months of May/September of 2001. Voucher specimens have been deposited at ITSA CUA in Prague. 2.2. Preparation of extract Dried plant material (15.0 g) was macerated with 80% ethanol (450 ml) for 5 days, filtered and the mare was exhaustively percolated with the same solvent. Filtrate and percolate were combined and evaporated to a thick residue at 40 8C. The residue was suspended or dissolved in 30.0 ml of Tris Buffer Saline (pH 7.6).
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 1 4 3 - 5
52
L. Kokoska et al. / Journal of Ethnopharmacology 82 (2002) 51 /53
for the bacteria and Amphotericin B (Sigma) was used for the yeast.
2.3. Microorganisms tested The following strains of bacteria were used: Escherichia coli ATCC 25922, Bacillus cereus ATCC 11778, Pseudomonas aeruginosa ATTC 27853, and Salmonella enteritidis ATCC 13076 (Oxoid, England). The yeast strain used in this study was Candida albicans ATCC 10231 (Oxoid). Microorganisms were grown overnight at 37 8C in Mueller-Hinton Broth (Oxoid) at pH 7.4. Their sensitivity to reference antibiotics has been checked (Table 2). Erythromycin and Gentamicin (Sigma, USA) were used
2.4. Antibacterial testing Antimicrobial activity of the crude ethanolic extracts of different plants was determined by the liquid dilution method (Vanden Berghe and Vlietinck, 1991). Fourfold dilutions (three) of tested extract sterilized by filtration through a 0.23 membrane filter were carried out starting from a dilution of 1/2. The tubes were inoculated with a microorganism suspension at a density of 105 CFU/ml.
Table 1 Antibacterial activity of ethanol crude extracts of some Siberian medicinal plants Species (Family) and voucher specimen number
Folk medicine use
Part tested Microorganisma/MICb
Achillea millefolium L. (Compositae) 0145
Anti-haemorrhage; antiphlogistic
Aerial part Rhizome
Arctium lappa L. (Compositae) 0133
Diuretic; diaphoretic; anti-ulcer
Aerial part Root
Bergenia crassifolia (L.) Fritsch (Saxifragaceae) 0142 Antidote
Aerial part Rhizome
Chelidonium majus L. (Papaveraceae) 0134
Aerial part Root
Cholagogue; spasmolytic; analgetic
Cichorium intybus L. (Compositae) 0141
Cholagogue; digestive; hypoglycaemic Aerial part Root
Glycyrrhiza uralensis Fischer (Leguminosae) 0112
Antiphlogistic; antitussive; expectorant
Bc
Ca
Ec
Pa
Sa
62.50
n.a.
n.a.
n.a.
62.50
n.a.
n.a.
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
n.a.
62.50
n.a.
n.a.
n.a.
62.50
62.50
250.00 62.50
15.63
62.50
15.63
15.63
15.63
62.50
62.50
n.a.
n.a.
n.a.
n.a.
n.a.
15.63
62.50
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
250.00
250.00 n.a.
n.a.
n.a.
250.00
Aerial part Root Fruit
250.00 n.a.
n.a.
n.a.
250.00
250.00 n.a. n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
Hippophae rhamnoides L. (Elaeagnaceae) 0125
Antiphlogistic, vulnerary
Leaf Root Fruit
250.00 n.a. 250.00 n.a. 62.50 n.a.
n.a. 62.50 62.50
n.a. n.a. 250.00 n.a. 62.50 62.50
Lamium album L. (Labiatae) 0146
Anti-haemorrhage, tonic
Aerial part Rhizome
n.a.
n.a.
n.a.
n.a.
n.a.
250.00 n.a.
n.a.
n.a.
250.00
15.63
n.a.
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
15.63
62.50
n.a.
250.00 250.00 62.50
15.63
250.00 62.50
62.50
250.00
Rhaponticum carthamoides (Willd.) Iljin (Compositae) Stimulant 0110
Aerial part Root
Sanguisorba officinalis L. (Rosaceae) 0135
Aerial part Rhizome
Anti-haemorrhage; antiphlogistic
Tussilago farfara L. (Compositae) 0117
Antiseptic; antiphlogistic
Aerial part Rhizome
15.63
n.a.
n.a.
n.a.
62.50
62.50
n.a.
n.a.
n.a.
62.50
Vaccinium myrtillus L. (Ericaceae) 0128
Astringent; antiphlogistic; hypoglycaemic
Leaf
n.a.
n.a.
250.00 250.00 n.a.
n.a.: not active. a Microorganisms: Bc , Bacillus cereus ; Ca , Candida albicans ; Ec , Escherichia coli ; Sa , Staphylococcus aureus ; Pa , Pseudomonas aeruginosa . b MIC, Minimum inhibitory concentration (mg of dry plant material/ml).
L. Kokoska et al. / Journal of Ethnopharmacology 82 (2002) 51 /53
Table 2 Antimicrobial reference standards Antibiotic
Amphotericin B Erythromycin Gentamicin
Microorganisma/MICb Bc
Ca
Ec
Pa
Sa
/ 0.78 /
0.39 / /
/ / 1.56
/ / 3.13
/ 1.56 /
( /): not determined. a Microorganisms: Bc, Bacillus cereus; Ca, Candida albicans; Ec, Escherichia coli; Sa, Staphylococcus aureus; Pa, Pseudomonas aeruginosa. b MIC: Minimum Inhibitory Concentration (mg/ml).
The tubes were incubated at 37 8C for 24 h (or 48 h for the yeast) and then observed for the minimum inhibitory concentration (MIC). The growth of organisms was observed as turbidity determined by the spectrophotometer (Ultrospec III, Pharmacia LKB, UK) at 620 nm. Control tubes without tested extracts were assayed simultaneously. All samples were tested in triplicate.
3. Results and discussion A total of 33 ethanolic extracts from 16 different plant species were investigated. Determination of the MIC by means of the liquid dilution method (Table 1) showed that 22 plant extracts tested exhibited an antimicrobial effect against some of the five tested microorganisms. Results showed that extracts from Achillea millefolium , Bergenia crassifolia , Sanguisorba officinalis , Tussilago farfara (aerial part; rhizome), Arctium lappa , Cichorium intybus , Glycyrrhiza uralensis , Rhaponticum carthamoides (aerial part; root), Chelidonium majus (root), Hippophae rhamnoides (leaf; root; fruit), Lamium album (rhizome), and Vaccinium myrtillus (leaf) possessed antimicrobial activity. Although the plants differ significantly in their activities against the tested microorganisms, most of the extracts showed antimicrobial activity against B. cereus and Staphylococcus aureus then against E. coli and P. aeruginosa . Only extracts from B. crassifolia shown significant inhibitory activity against C. albicans . According to the liquid dilution screening method for antimicrobial activity of higher plants reported by Vanden Berghe and Vlietinck (1991) a prominent antibacterial effect, worthy of further investigation, is obtained if not only the 1/2, but also the 1/8 and 1/32 dilutions show inhibitory activities. An inhibition only for the 1/2 dilution is less promising for further investigation. From this study we can conclude that B. crassifolia, C. majus, R. carthamoides, S. officinalis and T. farfara were the most active antimicrobial plants. All of these
53
species are perennial herbs widely distributed in Siberia from the Altai Region to Lake Baical (Gammerman and Grom, 1976). Antiviral activities of extracts isolated from S. officinalis and the antimicrobial activity of extracts from leaves B. crassifolia have been previously reported (Kim et al., 2001; Fedoseyeva et al., 2000). Lectin with potent antimicrobial properties against multiresistant enterococci and staphylococci has been isolated from C. majus (Fik et al., 2001); antiviral and antifugal activity of alkaloids isolated from this plant have also been reported (Kery et al., 1987; Ma et al., 2000). Despite many published reports dealing with bioactivity of isolated compounds from T. farfara and R. carthamoides, little was known about their antimicrobial activity prior to our investigation. We are continuing our investigations of the antibacterial principles from these plants.
Acknowledgements This research was financially supported by the Grant Agency of the Czech Republic (Project No. 525/02/ 0257).
References Essawi, T., Srour, M., 2000. Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology 70, 343 /349. Fedoseyeva, L.M., Kerasheva, S.I., Karabasova, E.V., 2000. Antimicrobial activity of dry extract from leaves of Bergenia crassifolia (L.) Fritsch. with respect to pathogenes of some suppurative inflammatory diseases. Rastitelnye Resursy 36, 153 /157. Fik, E., Wolun-Cholewa, M., Kistowska, M., Warchol, J.B., Gozdzicka-Jozefiak, A., 2001. Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture. Folia Histochemica et Cytobiologica 39, 215 /216. Gammerman, A.F., Grom, I.I., 1976. Wild growing medicinal plants of USSR. Medicine, Moscow, p. 8, 95, 118, 264, 275. Iwu, M.W., Duncan, A.R., Okunji, C.O., 1999. New antimicrobials of plant origin. In: Janick, J. (Ed.), Perspectives on New Crops and New Uses. ASHS Press, Alexandria, VA, pp. 457 /462. Kery, A., Horvath, J., Nasz, I., Verzar-Petri, G., Kulcsar, G., Dan, P., 1987. Antiviral alkaloid in Chelidonium majus L. Acta Biologica Hungarica 57, 19 /25. Kim, T.G., Kang, S.Y., Jung, K.K., Kang, J.H., Lee, E., Han, H.M., Kim, S.H., 2001. Antiviral activities of extracts isolated from Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. against hepatitis B virus. Phytotheraphy Research 15, 718 /720. Ma, W.G., Fukushi, Y., Tahara, S., Osawa, T., 2000. Fungitoxic alkaloids from Hokkaido Papaveraceae . Fitoterapia 71, 527 /534. Vanden Berghe, D.A., Vlietinck, A.J., 1991. Screening methods for antibacterial and antiviral agents from higher plants. In: Dey, P.M., Harborne, J.B., Hostettman, K. (Eds.), Methods in Plant Biochemistry. Assays for Bioactivity, vol. 6. Academic Press, London, pp. 47 /69.
Short communication
Screening of some Siberian medicinal plants for antimicrobial activity L. Kokoska a,*, Z. Polesny a, V. Rada b, A. Nepovim c, T. Vanek c a
Department of Tropical and Subtropical Crops, Czech University of Agriculture, Kamycka 129, Prague 6-Suchdol, Czech Republic Department of Microbiology and Biotechnology, Czech University of Agriculture, Kamycka 129, Prague 6-Suchdol, Czech Republic c Department of Plant Tissue Cultures, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic b
Accepted 24 May 2002
Abstract The antimicrobial activity of crude ethanolic extracts of 16 Siberian medicinal plants was tested against five species of microorganisms: Bacillus cereus , Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , and Candida albicans . Of the 16 plants tested, 12 showed antimicrobial activity against one or more species of microorganisms. The most active antimicrobial plants were Bergenia crassifolia, Chelidonium majus, Rhaponticum carthamoides, Sanguisorba officinalis , and Tussilago farfara . # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antimicrobial activity; Medicinal plants; Crude extracts; Herbal medicine
1. Introduction In many parts of Russia, particularly in Siberia, there is a rich tradition in the use of herbal medicine for the treatment of various infectious diseases, inflammations and injuries (Gammerman and Grom, 1976). Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, much recent attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine (Essawi and Srour, 2000). Plant based antimicrobials represent a vast untapped source for medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Iwu et al., 1999). In this study ethanolic extracts of different parts of 16 plants, which had been described in herbal books and
* Corresponding author. Tel.: /420-2-2438-2180; fax: /420-22092-1363 E-mail address: [email protected] (L. Kokoska).
folklore medicine of Russia were screened for their antimicrobial activity.
2. Materials and methods 2.1. Plant materials The seeds and seedlings of tested plants were obtained from the Botanical Garden of All-Russian Research Institute of Medicinal and Aromatic Plants (Moscow, Russia). They were grown in the experimental field of the Institute of Tropical and Subtropical Agriculture of the Czech University of Agriculture in Prague (ITSA CUA in Prague). The plant parts were collected during the months of May/September of 2001. Voucher specimens have been deposited at ITSA CUA in Prague. 2.2. Preparation of extract Dried plant material (15.0 g) was macerated with 80% ethanol (450 ml) for 5 days, filtered and the mare was exhaustively percolated with the same solvent. Filtrate and percolate were combined and evaporated to a thick residue at 40 8C. The residue was suspended or dissolved in 30.0 ml of Tris Buffer Saline (pH 7.6).
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 1 4 3 - 5
52
L. Kokoska et al. / Journal of Ethnopharmacology 82 (2002) 51 /53
for the bacteria and Amphotericin B (Sigma) was used for the yeast.
2.3. Microorganisms tested The following strains of bacteria were used: Escherichia coli ATCC 25922, Bacillus cereus ATCC 11778, Pseudomonas aeruginosa ATTC 27853, and Salmonella enteritidis ATCC 13076 (Oxoid, England). The yeast strain used in this study was Candida albicans ATCC 10231 (Oxoid). Microorganisms were grown overnight at 37 8C in Mueller-Hinton Broth (Oxoid) at pH 7.4. Their sensitivity to reference antibiotics has been checked (Table 2). Erythromycin and Gentamicin (Sigma, USA) were used
2.4. Antibacterial testing Antimicrobial activity of the crude ethanolic extracts of different plants was determined by the liquid dilution method (Vanden Berghe and Vlietinck, 1991). Fourfold dilutions (three) of tested extract sterilized by filtration through a 0.23 membrane filter were carried out starting from a dilution of 1/2. The tubes were inoculated with a microorganism suspension at a density of 105 CFU/ml.
Table 1 Antibacterial activity of ethanol crude extracts of some Siberian medicinal plants Species (Family) and voucher specimen number
Folk medicine use
Part tested Microorganisma/MICb
Achillea millefolium L. (Compositae) 0145
Anti-haemorrhage; antiphlogistic
Aerial part Rhizome
Arctium lappa L. (Compositae) 0133
Diuretic; diaphoretic; anti-ulcer
Aerial part Root
Bergenia crassifolia (L.) Fritsch (Saxifragaceae) 0142 Antidote
Aerial part Rhizome
Chelidonium majus L. (Papaveraceae) 0134
Aerial part Root
Cholagogue; spasmolytic; analgetic
Cichorium intybus L. (Compositae) 0141
Cholagogue; digestive; hypoglycaemic Aerial part Root
Glycyrrhiza uralensis Fischer (Leguminosae) 0112
Antiphlogistic; antitussive; expectorant
Bc
Ca
Ec
Pa
Sa
62.50
n.a.
n.a.
n.a.
62.50
n.a.
n.a.
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
n.a.
62.50
n.a.
n.a.
n.a.
62.50
62.50
250.00 62.50
15.63
62.50
15.63
15.63
15.63
62.50
62.50
n.a.
n.a.
n.a.
n.a.
n.a.
15.63
62.50
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
250.00
250.00 n.a.
n.a.
n.a.
250.00
Aerial part Root Fruit
250.00 n.a.
n.a.
n.a.
250.00
250.00 n.a. n.a. n.a.
n.a. n.a.
n.a. n.a.
n.a. n.a.
Hippophae rhamnoides L. (Elaeagnaceae) 0125
Antiphlogistic, vulnerary
Leaf Root Fruit
250.00 n.a. 250.00 n.a. 62.50 n.a.
n.a. 62.50 62.50
n.a. n.a. 250.00 n.a. 62.50 62.50
Lamium album L. (Labiatae) 0146
Anti-haemorrhage, tonic
Aerial part Rhizome
n.a.
n.a.
n.a.
n.a.
n.a.
250.00 n.a.
n.a.
n.a.
250.00
15.63
n.a.
n.a.
n.a.
62.50
250.00 n.a.
n.a.
n.a.
15.63
62.50
n.a.
250.00 250.00 62.50
15.63
250.00 62.50
62.50
250.00
Rhaponticum carthamoides (Willd.) Iljin (Compositae) Stimulant 0110
Aerial part Root
Sanguisorba officinalis L. (Rosaceae) 0135
Aerial part Rhizome
Anti-haemorrhage; antiphlogistic
Tussilago farfara L. (Compositae) 0117
Antiseptic; antiphlogistic
Aerial part Rhizome
15.63
n.a.
n.a.
n.a.
62.50
62.50
n.a.
n.a.
n.a.
62.50
Vaccinium myrtillus L. (Ericaceae) 0128
Astringent; antiphlogistic; hypoglycaemic
Leaf
n.a.
n.a.
250.00 250.00 n.a.
n.a.: not active. a Microorganisms: Bc , Bacillus cereus ; Ca , Candida albicans ; Ec , Escherichia coli ; Sa , Staphylococcus aureus ; Pa , Pseudomonas aeruginosa . b MIC, Minimum inhibitory concentration (mg of dry plant material/ml).
L. Kokoska et al. / Journal of Ethnopharmacology 82 (2002) 51 /53
Table 2 Antimicrobial reference standards Antibiotic
Amphotericin B Erythromycin Gentamicin
Microorganisma/MICb Bc
Ca
Ec
Pa
Sa
/ 0.78 /
0.39 / /
/ / 1.56
/ / 3.13
/ 1.56 /
( /): not determined. a Microorganisms: Bc, Bacillus cereus; Ca, Candida albicans; Ec, Escherichia coli; Sa, Staphylococcus aureus; Pa, Pseudomonas aeruginosa. b MIC: Minimum Inhibitory Concentration (mg/ml).
The tubes were incubated at 37 8C for 24 h (or 48 h for the yeast) and then observed for the minimum inhibitory concentration (MIC). The growth of organisms was observed as turbidity determined by the spectrophotometer (Ultrospec III, Pharmacia LKB, UK) at 620 nm. Control tubes without tested extracts were assayed simultaneously. All samples were tested in triplicate.
3. Results and discussion A total of 33 ethanolic extracts from 16 different plant species were investigated. Determination of the MIC by means of the liquid dilution method (Table 1) showed that 22 plant extracts tested exhibited an antimicrobial effect against some of the five tested microorganisms. Results showed that extracts from Achillea millefolium , Bergenia crassifolia , Sanguisorba officinalis , Tussilago farfara (aerial part; rhizome), Arctium lappa , Cichorium intybus , Glycyrrhiza uralensis , Rhaponticum carthamoides (aerial part; root), Chelidonium majus (root), Hippophae rhamnoides (leaf; root; fruit), Lamium album (rhizome), and Vaccinium myrtillus (leaf) possessed antimicrobial activity. Although the plants differ significantly in their activities against the tested microorganisms, most of the extracts showed antimicrobial activity against B. cereus and Staphylococcus aureus then against E. coli and P. aeruginosa . Only extracts from B. crassifolia shown significant inhibitory activity against C. albicans . According to the liquid dilution screening method for antimicrobial activity of higher plants reported by Vanden Berghe and Vlietinck (1991) a prominent antibacterial effect, worthy of further investigation, is obtained if not only the 1/2, but also the 1/8 and 1/32 dilutions show inhibitory activities. An inhibition only for the 1/2 dilution is less promising for further investigation. From this study we can conclude that B. crassifolia, C. majus, R. carthamoides, S. officinalis and T. farfara were the most active antimicrobial plants. All of these
53
species are perennial herbs widely distributed in Siberia from the Altai Region to Lake Baical (Gammerman and Grom, 1976). Antiviral activities of extracts isolated from S. officinalis and the antimicrobial activity of extracts from leaves B. crassifolia have been previously reported (Kim et al., 2001; Fedoseyeva et al., 2000). Lectin with potent antimicrobial properties against multiresistant enterococci and staphylococci has been isolated from C. majus (Fik et al., 2001); antiviral and antifugal activity of alkaloids isolated from this plant have also been reported (Kery et al., 1987; Ma et al., 2000). Despite many published reports dealing with bioactivity of isolated compounds from T. farfara and R. carthamoides, little was known about their antimicrobial activity prior to our investigation. We are continuing our investigations of the antibacterial principles from these plants.
Acknowledgements This research was financially supported by the Grant Agency of the Czech Republic (Project No. 525/02/ 0257).
References Essawi, T., Srour, M., 2000. Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology 70, 343 /349. Fedoseyeva, L.M., Kerasheva, S.I., Karabasova, E.V., 2000. Antimicrobial activity of dry extract from leaves of Bergenia crassifolia (L.) Fritsch. with respect to pathogenes of some suppurative inflammatory diseases. Rastitelnye Resursy 36, 153 /157. Fik, E., Wolun-Cholewa, M., Kistowska, M., Warchol, J.B., Gozdzicka-Jozefiak, A., 2001. Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture. Folia Histochemica et Cytobiologica 39, 215 /216. Gammerman, A.F., Grom, I.I., 1976. Wild growing medicinal plants of USSR. Medicine, Moscow, p. 8, 95, 118, 264, 275. Iwu, M.W., Duncan, A.R., Okunji, C.O., 1999. New antimicrobials of plant origin. In: Janick, J. (Ed.), Perspectives on New Crops and New Uses. ASHS Press, Alexandria, VA, pp. 457 /462. Kery, A., Horvath, J., Nasz, I., Verzar-Petri, G., Kulcsar, G., Dan, P., 1987. Antiviral alkaloid in Chelidonium majus L. Acta Biologica Hungarica 57, 19 /25. Kim, T.G., Kang, S.Y., Jung, K.K., Kang, J.H., Lee, E., Han, H.M., Kim, S.H., 2001. Antiviral activities of extracts isolated from Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. against hepatitis B virus. Phytotheraphy Research 15, 718 /720. Ma, W.G., Fukushi, Y., Tahara, S., Osawa, T., 2000. Fungitoxic alkaloids from Hokkaido Papaveraceae . Fitoterapia 71, 527 /534. Vanden Berghe, D.A., Vlietinck, A.J., 1991. Screening methods for antibacterial and antiviral agents from higher plants. In: Dey, P.M., Harborne, J.B., Hostettman, K. (Eds.), Methods in Plant Biochemistry. Assays for Bioactivity, vol. 6. Academic Press, London, pp. 47 /69.