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Screening of Echinops ellenbeckii and Echinops longisetus for biological activities and chemical constituents.

Abstract

Members of the genus Echinops in the family of Asteraceae are widely used in Ethiopian herbal medicine for the treatment of various diseases and illness such as migraine, diarrhea, heart pain, different forms of infections, intestinal worm infestation and hemorrhoid. Hydroalcoholic extracts of the root, flower head, leaf and stem of Echinops ellenbeckii O. Hoffm, and Echinops longisetus A. Rich were investigated for their chemical constituents and biological activities. The presence of alkaloids, saponins, phytosterols, polyphenols and carotenoids in the different parts of the plants was observed whilst anthraquinones were not detected. The leaf extracts of both plants and stem extract of E. longisetus showed strong inhibitory activity against cultures of Staphylococcus aureus. None of the extracts were found to be active against Gram-positive organisms. The flower extract of E. ellenbeckii showed strong inhibitory activity against Candida albicans. Root and flower extracts of the plants investigated showed lethal activity against earthworms. Moreover, the extracts of the roots of both plants showed molluscicidal activity against schistosome-transmitting snail hosts. The biological activities observed were dose dependent.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Echinops ellenbeckii; Echinops longisetus; Biological screening; Molluscicidal; Antimicrobial; Antifungal; Anthelmintic

Introduction

The genus Echinops, belongs to the family Asteraceae and comprises over 120 species, of which 12 are known to occur in Ethiopia. Among the 12 species occurring in Ethiopia, Echinops ellenbeckii O. Hoffm, Echinops longisetus. A. Rich, E. kebericho Mesfin and E. buhaitensis Mesfin are confined to the highlands of the country. E. ellenbeckii is known from a few localities in Ethiopia (Arsi and Harerge provinces) between 7-9[degrees]30'N and 38[degrees]45'E and at altitudes between 2200m and 2900m whereas E. longisetus has a much wider distribution in Ethiopian highlands (Tadesse and Abegaz, 1990).

Chemical constituents of members of the genus have been studied to some extent. E. grijsii and E. giganteus are reported to contain tricyclic sesquiterpenes (e.g. [alpha]- and [beta]-caryophyllene; [alpha]- and [beta]-bisabolene, [alpha]- and [beta]-santalene, guaiene, etc.), sesquiterpene alcohols (e.g. (-)-nopsan-4-ol and (+)-prenopsan-8-ol, silphiperfol-6-ene) and sesquiterpene lactones (e.g. [alpha]- and [beta]-caryophyllene epoxide) in their volatile fractions (Weyerstahl et al., 1998; Menut et al., 1997; Guo et al., 1994). Monoterpenoids have also been reported from E. ritro and E. grijsii (Guo et al., 1994; Dopke and Fritsch, 1969). The genus Echinops is one of the taxa with well-characterized alkaloids within the Asteraceae family. The occurrence of simple quinoline alkaloids in the aerial and/or underground parts of more than 14 species including E. echinatus, E. ritro and E. sphaerocephalus has been reported (Chaudhuri, 1992; Dopke and Fritsch, 1969; Schroeder and Luckner, 1968). Members of the genus Echinops are also reported to contain flavonoids, thiophene acetylene compounds and fatty acids (Liu et al., 2002; Hymete and Afifi, 1997; Singh and Pandey, 1994).

Some species of the genus Echinops are known to have use in Ethiopian herbal medicine. E. kebericho Mesfin is claimed to be useful in the treatment of migraine, diarrhea, heart pain and other ailments. The root and flower heads of E. ellenbeckii are useful in the herbal treatment of hemorrhoid (Dawit and Ahadu, 1993). Alcoholic extract of the roots of E. kebericho has been shown to have a very strong lethal activity against earthworm. Worms kept in a cabinet together with the powdered roots of E. kebericho were found dead after a few hours suggesting that the volatile constituents could be responsible for the lethal activity (Hymete and Kidane, 1991). Some other species of this genus are also claimed to exhibit vermicidal activities.

The phytochemical screening and biological activity testing of E. ellenbeckii and E. longisetus, have to the authors' present knowledge not been carried out before. The aim of this study was to determine the chemical groups and biological activities of the extracts of the roots, stems, leaves and flower heads of these plants to elaborate and evaluate their potential medicinal uses.

Experimental

Plant material and extraction

The whole plants of E. ellenbeckii and E. longisetus were collected from Kofele (South Ethiopia) and Toke (West Ethiopia) respectively, in February 2003. Professor Sebsebie Demissew of the National Herbarium, Department of Biology, Addis Ababa University, confirmed the plants collected. Voucher specimens were conserved at the National Herbarium, Addis Ababa University, under the accession numbers Ariaya H. 5 and Ariaya H.2, respectively. The leaves, flower heads, stems and roots were separated and dried in the shade. The plant parts were ground into a fine powder using an electric grinder. Powdered plant parts were extracted with 80% MeOH in a soxhlet apparatus until the last portion of the extract became colorless. The resultant extract was evaporated to dryness in vacuo.

Microorganisms

The cultures of microorganisms used for the in vivo antibacterial tests in this study were Staphylococcus aureus ATCC 6538 (Gram-positive), Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 (Gram-negative) for the antibacterial tests. Candida albicans (yeast, clinical isolate), Aspergillus niger ATCC 10535 (mold) and Trichophyton mentagrophytes ATCC 18748 (dermatophyte) were used for the antifungal tests. The organisms were obtained from the Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia. The nutritive media used during the investigation included Nutrient agar (LB medium) (Sigma-Aldrich) for bacteria and Sabouraud medium Batch no. 251385 (Oxoid Ltd.) for the fungi.

Antimicrobial sensitivity test

Three different concentrations (2.5, 5.0 and 10 [micro]g/[micro]l) of the dried 80% MeOH extracts were prepared by dissolving in distilled water. Tests were performed by the hole plate diffusion method on agar plates in triplicates following standard procedures (Desta, 1993).

Seeded agar was punched with sterile cork borer (10mm diameter) and 100 [micro]l of the test solutions were placed in each hole. The plates were then incubated at 37[degrees]C for 24 h for the antibacterial tests. For the antifungal tests the plates were incubated at 25[degrees]C for 48 h in the case of C. albicans and A. niger. Incubation of 7 days was done for T. mentagrophytes. Antibacterial or antifungal activities were then measured as indicated by the clear zones of inhibition. Gentamycin 0.1 [micro]g/[micro]l (in water) and ketoconazole 0.25 [micro]g/[micro]l were used as a positive control for the antibacterial and antifungal tests, respectively (Desta, 1993).

Anthelmintic activity test

The dried extracts (500 mg each) of the different morphological parts of E. ellenbeckii and E. longisetus were suspended in de-chlorinated tap water and volume adjusted to 10 ml. Test solutions with different concentration (1500, 1000, 500, 250, 125, 62.5 and 31.3 [micro]g/ml) were prepared by serial dilution. The anthelmintic activity was determined in triplicate by placing 10 earthworms in a petridish containing 50 ml of the test solution. Earthworm mortality was observed after 16 h of exposure to the test solutions. Niclosamide suspension was used as reference and de-chlorinated tap water as control (Hymete and Kidane, 1991).

Molluscicidal activity test

The 80% MeOH extract (1.0 g) [equivalent to 10.62 g of E. ellenbeckii root (EER) and 11.51 g E. longisetus root (ELR) powder] was suspended in 11 de-chlorinated tap water to give a 1.0 [micro]g/[micro]l solution. Test solutions with different concentrations (4.50, 9.00, 11.25, 13.50, 15.75, 18.00, 20.25, 27.00, 29.25, 31.50, 33.75, 36.00, 45.00 [micro]g/ml) were prepared by serial dilution. The extract suspension was vigorously shaken before preparing each dilution to obtain a uniform mixture. Biomphalaria pfeifferi (7-9 mm shell diameter) collected from Finchaa stream in western Ethiopia was used for molluscicidal tests. Ten snails were placed in 1000 ml capacity beakers for each dilution. Control groups were also placed in beakers of the same capacity containing de-chlorinated tap water only. After 24 h of exposure, test solutions were poured off and snails washed with water and then kept in de-chlorinated tap water for another 24 h for recovery. Snail mortality was observed and recorded after a 24 h recovery period (Erko et al., 1998).

Phytochemical tests

The 80% MeOH extract or the powdered plant material where ever required was used for testing the presence or absence of secondary metabolites such as phytosterols, carotenoids, polyphenols (tannins, flavonoids and coumarins), saponins, alkaloids and anthraquinones following standard procedures (Debela, 2002). Extracts equivalent to equal amounts of the dried plant materials was used in the tests.

Results

Yield of the 80% MeOH extracts (based on dry weight of plant material) for E. ellenbeckii were 16.13%, 17.71%, 9.37% and 9.42% for the leaves (EEL), flowers (EEF), stem (EES) and roots (EER), respectively. E. longisetus gave 5.43%, 15.33%, 9.6% and 8.69% extract for the leaves (ELL), flowers (ELF), stem (ELS) and roots (ELR), respectively.

The in vitro tests for antibacterial activity revealed that the 80% MeOH extracts of the leaf of E. ellenbeckii (EEL) and the leaf and stem of E. longisetus (ELL and ELS) inhibited the growth of S. aureus in a dose dependent manner. The antibacterial activity was detected at 5 [micro]g/[micro]l concentration for EEL and 2.5 [micro]g/[micro]l concentration for ELL and ELS (Table 1). No inhibitory activity was observed for EER, EEF, EES, ELF and ELR. None of the extracts showed inhibitory activity against the Gram-negative organisms (E. coli and P. aeruginosa). All the test organisms were sensitive to the positive control (gentamycine).

The flower extract of E. ellenbeckii (EEF) showed inhibitory effect against C. albicans. None of the other extracts examined inhibited growth of fungi tested in this study at the concentrations selected (Table 1). Ketoconazole at 0.25 [micro]g/[micro]l inhibited the growth of the test fungi, C. albicans (34.3 + 0.58 mm), and T. mentagrophytes (34.3 + 0.58). A higher concentration (0.40 [micro]g/[micro]l) of ketoconazole was required to get a measurable zone of inhibition in the case of A. niger (25.0 + 0.0).

The test for anthelmentic activity revealed that the 80% MeOH extract of the roots of E. ellenbeckii (EER) had a very strong activity (Table 2). The root extract of E. longisetus (ELR) was also shown to be active but at a relatively higher concentration compared to that of E. ellenbeckii. Flower extracts of both plants (EEF and ELF) showed a very weak activity at concentrations above 250 and 1500 [micro]g/ml, respectively. All the other extracts showed no activity to earthworms even at the highest concentration tested.

The root extract of E. ellenbeckii (EER) exhibited a relatively stronger molluscicidal activity compared to the other extracts (Table 3). The root extract of E. longisetus (ELR) also showed activity but a weaker one (Table 4). EER and ELR showed 100% molluscicidal activity at concentrations above 20.25 and 45 [micro]g/ml, respectively. On the other hand the leaf, stem and flower extracts of E. ellenbeckii and E. longisetus showed no molluscicidal activity even with the highest concentration (45 [micro]g/ml) studied.

The phytochemical screening revealed the presence of alkaloids, saponins, phytosterols and polyphenols in the roots of both plants investigated. The flowers of the plants investigated were found to contain phytosterols and polyphenols. In addition the flower of E. longisetus is proven to contain large amounts of alkaloids. The leaves of both plants contained carotenoids and polyphenols. It was possible to detect polyphenols and phytosterols in the stems of these plants. Anthraquinones were found to be absent in all morphological parts of the plants investigated (Table 5).

Discussion and conclusions

In Ethiopian traditional medicine some species of the genus Echinops have been used to treat migraine, diarrhea, heart pain, different forms of infection, worm infestation, hemorrhoid and other ailments (Dawit and Ahadu, 1993). The results from the present study have proved the scientific basis for traditional uses of the genus Echinops in the treatment of some ailments. Species of the genus Echinops are known to contain flavonoids and thiophene acetylene compounds (Singh and Pandey, 1994). Thiophene acetylene compounds are known to have anthelmintic activity. The strong anthelmintic activity observed in the roots and weak activity in the flowers of E. longisetus and E. ellenbeckii indicates the presence of such compounds in these morphological parts. The results from the previous study (Hymete and Kidane, 1991) also indicated the existence of scientific basis for the traditional uses of these two and probably other members of the genus Echinops in the treatment of intestinal worm infestation.

Flavonoid compounds are also reported to have antimicrobial property and these compounds could be the constituents of the leaves of the two plants and the stems of E. longisetus as they have shown strong inhibitory effect against Gram negative organisms. The zone of inhibition observed in the antimicrobial test for the active extracts is almost comparable to that of the positive control (0.1 [micro]g/[micro]l gentamycine). This further corroborates the usefulness of plant species of the genus in the treatment of some infectious ailments.

Molluscicidal activity (100%) was observed for EER and ELR at concentrations of 20.25 and 45 [micro]g/ml, respectively. The L[C.sub.100] for EER and ELR are relatively high when compared to that reported for endod (Phytolacca dodecandra). This plant contains triterpenoidal saponins, shows very strong molluscicidal activity and is now in use in the control of snails transmitting schistosomiasis in Ethiopia. The L[C.sub.100] for endod type 44 was 10 ppm (Lugt, 1981) and the L[C.sub.50] of endod type S was 4 mg/l (Lambert et al., 1991) against B. pfeifferi. Although the investigated root extracts of Echinops spp. have been shown to contain saponins (Table 5), the high dose level observed indicated that they might not be potential candidates for use in vector control of schistosomiasis. In this study, 80% MeOH was used as solvent of extraction to obtain an extract that could be used both for the chemical and biological screenings. It is thus recommended that water or butanol should especially be used as solvents for the extraction of saponins before a conclusion can be made. Also, other species of the diverse genus Echinops should be screened for their molluscicidal activities. Additionally, it is suggested that further investigations must be carried out to determine possible toxicities of the plants to ensure the safety of the extracts of these plants that are widely used in traditional medicine.

Acknowledgements

One of the authors (Ariaya Hymete) would like to acknowledge the Norwegian State Lanekassen for the financial assistance.

References

Chaudhuri, P.K., 1992. 7-hydroxyechinozolinone, a new alkaloid from the flowers of Echinops echinatus. J. Nat. Prod. 55 (2), 249-250.

Dawit, A., Ahadu, A., 1993. Medicinal Plants and Enigmatic Health Practices of Northern Ethiopia. Birhanena Selam, Addis Ababa (pp. 37-44, 83-89).

Debela, A., 2002. Manual for Phytochemical Screening of Medicinal Plants. Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia (pp. 35-47).

Desta, B., 1993. Ethiopian traditional herbal drugs. Part II: antimicrobial activity of 63 medicinal plants. J. Ethnopharmacol. 39, 129-139.

Dopke, W., Fritsch, G., 1969. Echinine a dihydroquinoline alkaloid from Echinops ritro seeds. Pharmazie 24 (12), 782.

Erko, B., Balcha, F., Kelbessa, E., 1998. Preliminary observations on molluscicidal activities of the inflorescences of Hagena abyssinisa (Bruce). J. F. Gmel. Ethiop. Pharm. J. 16, 49-52.

Guo, D., Lou, Z., Liu, Z., 1994. Chemical components of volatile oil from Echinops grijsii Hance. Zhongguo Zhongyao Zazhi 19 (2), 100-101.

Hymete, A., Afifi, M.S., 1997. Investigation of the fixed oil isolated from the roots of Echinops kebericho Mesfin. Mansoura. J. Pharm. Sci. 13 (1), 59-69.

Hymete, A., Kidane, A., 1991. Screening for anthelmintic activity in two Echinops spp. Ethiop. Pharm. J. 9 (1), 67-71.

Lambert, J.D.H., Temmink, J.H.M., Marquis, J., Parkhurst, R.M., Lugt, C.B., Lemmich, E., Wolde-Yohannes, L., de Savigny, D., 1991. Endod: safety evaluation of a plant molluscicide. Regulat. Toxicol. Pharmacol. 14 (2), 189-201.

Liu, Y., Ye, M., Guo, H.-Z., Zhao, Y.-Y., Guo, D.-A., 2002. New thiophenes from Echinops grijisii. J. Asian Nat. Prod. Res. 4 (3), 175-178.

Lugt, C.B., 1981. Phytolacca dodecandra Berries as a Means of Controlling Bilharzia-transmitting Snails. Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia (61pp.).

Menut, C., Lamty, G., Weyerstahl, P., Marschall, H., Seelmann, I., Amvam Zollo, P.H., 1997. Aromatic plants of Tropical Central Africa. Part XXXI. Tricyclic sesquiterpenes from the roots of Echinops giganteus var. lelyi C.D. Adams. Flav. Fragr. J. 12 (6), 415-421.

Schroeder, P., Luckner, M., 1968. Physiology of formation of the quinoline alkaloid echinorine in Echinops ritro. Planta Med. 16 (1), 99-108.

Singh, R.P., Pandey, V.B., 1994. Further flavonoids of Echinops niveus. Fitoterapia 65 (4), 374.

Tadesse, M., Abegaz, B., 1990. A revision of the genus Echinops (Composite, Craude) in Ethiopia, with notes on phytogeography and chemistry. Proceedings of the 12th Plenary Meeting of AETFAT, pp. 605-629.

Weyerstahl, P., Marchall, H., Seelmann, I., Jakupovic, J., 1998. Cameroonane, prenopsane and nopsane, three new tricyclic sesquiterpene skeletons. Eur. J. Org. Chem. 6, 1205-1212.

A. Hymete (a), T.-H. Iversen (a), J. Rohloff (a,*), B. Erko (b)

(a) Department of Biology, The Plant Biocentre, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway

(b) Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

Received 8 December 2003; accepted 16 January 2004

*Corresponding author. Department of Biochemistry, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knoell-Str. 8, Jena 07745, Germany. Tel.: +47 73 59 01 74; fax: + 47 73 59 01 77.

E-mail address: jens.rohloff@bio.ntnu.no (J. Rohloff).
Table 1. Results from the antimicrobial and antifungal tests for the
extracts of E. longisetus and E. ellenbeckii (mean values [+ or -] S.D.
(mm) of three separate experiments)

Organism type Extracts 2.5 [micro]g/[micro]l

S. aureus ELL 21.5 [+ or -] 1.30
 ELS 20.5 [+ or -] 1.00
 EEL --
 Gentamycine (0.1 24.7 [+ or -] 0.60
 [micro]g/[micro]l)
C. albicans EEF --
 Ketoconazole (0.25 34.3 [+ or -] 0.58
 [micro]g/[micro]l)

Organism type Extracts 5.0 [micro]g/[micro]l

S. aureus ELL 21.5 [+ or -] 1.00
 ELS 21.2 [+ or -] 0.80
 EEL 20.7 [+ or -] 1.80
 Gentamycine (0.1
 [micro]g/[micro]l)
C. albicans EEF --
 Ketoconazole (0.25
 [micro]g/[micro]l)

Organism type Extracts 10.0 [micro]g/[micro]l

S. aureus ELL 23.0 [+ or -] 0.50
 ELS 23.2 [+ or -] 0.30
 EEL 21.5 [+ or -] 1.00
 Gentamycine (0.1
 [micro]g/[micro]l)
C. albicans EEF 18.9 [+ or -] 0.17
 Ketoconazole (0.25
 [micro]g/[micro]l)

-- No inhibition zone.

Table 2. Mortality rate (%) observed for the anthelmintic activity tests
of the extracts of E. longisetus and E. ellenbeckii (mean values
[+ or -] S.D. (%) of three separate experiments)

Concentration ([micro]g/ml) EER EEF

 31.3 53.3 [+ or -] 11.5 --
 62.5 63.3 [+ or -] 5.8 --
 125.0 76.7 [+ or -] 5.8 --
 250.0 86.7 [+ or -] 11.5 6.7 [+ or -] 11.5
 500.0 100 [+ or -] 0.0 16.7 [+ or -] 5.8
1000.0 100 [+ or -] 0.0 23.3 [+ or -] 5.8
1500.0 100 [+ or -] 0.0 36.7 [+ or -] 5.8
Niclosamide L[D.sub.50] = 84.5
 [micro]g/ml
Water --

Concentration ([micro]g/ml) ELR ELF

 31.3 -- --
 62.5 -- --
 125.0 -- --
 250.0 56.7 [+ or -] 5.8 --
 500.0 100 [+ or -] 0.0 --
1000.0 100 [+ or -] 0.0 --
1500.0 100 [+ or -] 0.0 6.7 [+ or -] 11.5
Niclosamide
Water

-- No death recorded.

Table 3. Results of the molluscicidal activity test (mortality rate in
%) using root extracts of E. ellenbeckii (mean values [+ or -] S.D. (%)
of three separate experiments)

Concentration ([micro]g/ml) Mortality observed (%)

 4.50 --
 9.00 20.0 [+ or -] 0.0
11.25 26.7 [+ or -] 1.5
13.50 33.3 [+ or -] 1.5
15.75 60.0 [+ or -] 2.0
18.00 86.7 [+ or -] 1.5
20.25 100 [+ or -] 0.0
Control --

-- No death recorded.

Table 4. Results of the molluscicidal activity test (mortality rate in
%) using root extracts of E. longisetus (mean values [+ or -] S.D. (%)
of three separate experiments)

Concentration ([micro]g/ml) Mortality observed (%)

 9.00 --
18.00 26.7 [+ or -] 1.2
27.00 33.3 [+ or -] 2.3
29.25 40.0 [+ or -] 2.0
31.50 53.3 [+ or -] 2.3
33.75 60.0 [+ or -] 2.0
36.00 73.3 [+ or -] 3.1
45.00 100 [+ or -] 0.0
Control --

-- No death recorded.

Table 5. Results of the phytochemical screening of the extracts of E.
longisetus and E. ellenbeckii

Sample Alkaloids Saponins Phytosterols Carotenoids

ELL - - - + + +
ELF + + + - + + + -
ELS - - + -
ELR + + + + + + -
EEL - - - + + +
EEF - - + -
EES - - + -
EER + + + + + + -

Sample Polyphenols Anthraquinones

ELL + -
ELF + + + -
ELS + + + -
ELR + + + -
EEL + + + -
EEF + + -
EES + -
EER + + + -

+ Positive.
+ + Strong positive.
+ + + Very strong positive.
- Not detected.
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Author:Hymete, A.; Iversen, T.-H.; Rohloff, J.; Erko, B.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Sep 1, 2005
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