|
Screening of Echinops Ellenbeckii
9/1/2005
Phytomedicine: International
Journal of Phytotherapy & Phytopharmacology;
By B. Erko
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.
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.
|
Latest News 
