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Studies on antimicrobial activity of Inula helenium L Romanian cultivar

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Camelia Filofteia Diguta at University of Agricultural Sciences and Veterinary Medicine of Bucharest
Camelia Filofteia Diguta
Calina Petruta Cornea at University of Agricultural Sciences and Veterinary Medicine of Bucharest
Calina Petruta Cornea
L. Ioniţă
L. Ioniţă
L. Ioniţă
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Abstract and Figures

This study was carried out to evaluate the in vitro antimicrobial activities of dried roots of a Romanian indigenous population of Inula helenium L. The powdered dried root of the plant was extracted in ethanol (using 30%, 50% and 70% v/v). The antimicrobial activity has been tested on five potential pathogenic bacterial species (Bacillus subtilis, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus) and four fungal species (Candida albicans, C. parapsilosis, C. lipolytica and Aspergillus niger), all of veterinary interest. Antimicrobial activity was investigated by the drop-diffusion test method, measuring the inhibition zones. The preliminary results of our study indicated that ethanolic extracts (50% and 70%) from the roots of a Romanian cultivar of I. helenium L. showed significant antimicrobial activity against all tested microorganisms, except the pathogenic philamentous fungi A. niger. On the dermatophytic species (Candida sp.) the inhibitory effects of 50% and 70% extracts are very similar. Further investigation will be done to determine the minimal inhibitory concentrations for each susceptible microorganism. As final goal is the design of a new veterinary product with antimicrobial effects.
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Romanian Biotechnological Letters Vol. 19, No. 5,2014
Copyright © 2014 University of Bucharest Printed in Romania. All rights reserved
ORIGINAL PAPER
Romanian Biotechnological Letters, Vol. 19, No. 5, 2014 9699
Studies on antimicrobial activity of Inula helenium L Romanian cultivar
Received for publication, July 21, 2014
Accepted, September 22, 2014
DIGUȚĂ,C.1,2, CORNEA C.P.2, IONIȚĂ L.3, BRÎNDUȘE, E.4, FARCAȘ, N.5,
BOBIT D.6, MATEI F.1,2*
1 CBM BIOTEHGEN, 59 Mărăști Bld., Bucharest 011464, Romania
2 UASMV Bucharest, Faculty of Biotechnologies, 59, Mărăști Bld., 011464,
Bucharest 1, Romania
3 UASMV Bucharest, Faculty of Veterinary Medicine, 105, Splaiul Independenței
050097, Bucharest 5, Romania
4 ICDVV Valea Călugărească, Prahova, Romania
5 UASMV Bucharest, Faculty of Agriculture, 59, Mărăști Bld., 011464, Bucharest 1,
Romania
6 SC DACIA PLANT SRL, Hărmanului fn, Brasov, Romania
*corresponding author: florentina.matei@biotehnologii.usamv.ro
Abstract
This study was carried out to evaluate the in vitro antimicrobial activities of dried roots of a
Romanian indigenous population of Inula helenium L. The powdered dried root of the plant was
extracted in ethanol (using 30%, 50% and 70% v/v). The antimicrobial activity has been tested on
five potential pathogenic bacterial species (Bacillus subtilis, Bacillus cereus, Enterococcus faecalis,
Escherichia coli, Staphylococcus aureus) and four fungal species (Candida albicans, C.
parapsilosis, C. lipolytica and Aspergillus niger), all of veterinary interest. Antimicrobial activity
was investigated by the drop-diffusion test method, measuring the inhibition zones. The preliminary
results of our study indicated that ethanolic extracts (50% and 70%) from the roots of a Romanian
cultivar of I. helenium L. showed significant antimicrobial activity against all tested
microorganisms, except the pathogenic philamentous fungi A. niger. On the dermatophytic species
(Candida sp.) the inhibitory effects of 50% and 70% extracts are very similar. Further investigation
will be done to determine the minimal inhibitory concentrations for each susceptible
microorganism. As final goal is the design of a new veterinary product with antimicrobial effects.
Keywords: in vitro, Inula helenium, ethanolic extracts, antibacterial, antifungal
1. Introduction
Since the beginning of the twentieth century, the discovery and development of antibiotics
revolutionized human and veterinary medicine. However, due to excessive use of antibiotics
to treat the infectious diseases, many microorganisms developed several mechanisms to resist
conventional antibiotics, creating serious problems worldwide. For this reason, many research
have been directed towards finding alternatives such as new antimicrobial compounds from
natural sources in order to achieve the effective treatment. Since ancient times, medicinal and
aromatic plants, or spices have been used as food and feed additives, food supplements and
medicinal value. More recently, much attention has been paid to investigate weed plants as
sources of new bioactive compounds. The presence of antimicrobial and other biological
activities have been already demonstrated in extracts of Eupatorium spp. (L.-L. JI & al. [1];
N. ARVIND and S. AMIT [2]), Helleborus spp. (S. PUGLISI & al. [3]), Inula spp. (T.
KONISHI & al. [4]; A. STOJAKOWSKA & al. [5, 6, 7]; P.D. LOKHANDE & al. [8]; A.
DERIU & al. [9]; Z. STOJANOVIĆ-RADIĆ & al. [10]; H. YAN & al. [11]).
DIGUȚĂ,C., CORNEA C.P., IONIȚĂ L., BRÎNDUȘE, E., FARCAȘ, N., BOBIT D., MATEI F.
9700 Romanian Biotechnological Letters, Vol. 19, No. 5, 2014
The genus Inula (Asteraceae; tribe Inuleae) consists of approximately 100 species mainly
distributed in Europe, Africa and Asia. Recently, several species (Inula britannica L., I.
ensifolia I. helenium L, I. racemosa L., and I. viscosa L.) have been investigated for their
pharmacological benefit including antioxidant and anti-inflammatory activities,
hepatoprotective characteristics, cytotoxicity, and antimicrobial properties (Y.M. ZHAO &
al. [12]; W. H. TALIB & al. [13]; S. AMIN & al. [14]).
Among these species, Inula helenium L. also known as elecampane is a perennial herbaceous
plant, with tuberous, thick roots. Chemical analysis of the rhizome and roots showed that I.
helenium contains many bioactive compounds including polysaccharide inulin (up to 44%),
essential oil with eudesmane –type (up to 5%), sesquiterpene lactones (mainly alantolactone
and isoalantolactone), thymol derivatives, terpenes, and sterols (T. KONISHI & al. [4]; A.
STOJAKOWSKA & al. [5, 6]; A. DERIU & al. [9]; Z. STOJANOVIĆ-RADIĆ & al. [10]; H.
YAN & al. [11]; J. WANG & al. [15]). It is known that a number of these compounds are
responsible for its anti-inflammatory, anti-microbial and anti-helminthic properties.
Sesquiterpene lactones (Z. STOJANOVIĆ-RADIĆ & al. [10]) and thymol derivatives (A.
STOJAKOWSKA & al. [5, 6]) from elecampane have been demonstrated to be main
antimicrobial agents in the roots.
There are few reports concerning the antimicrobial activity of others members of the Inula
spp. against several microorganisms, including bacteria and fungi. P.D. LOKHANDE & al.
[8] reported that alantolactone isolated from root of the plant I. racemosa have been shown
maximum antibacterial activity as compared to other constituents and ethyl acetate extract of
the roots. Y.M. ZHAO & al. [12] isolated and identified five thymol derivatives from the
roots of I. hupehensis. One of these compounds was the most active, not only displaying
moderate antibacterial activities against S.aureus, Methicillin-resistant S. aureus and E. coli,
but also exhibiting inhibitory activities against three plant pathogenic fungi: Rhizoctonia
solani, Phytophthora melonis and P. litchi, and its content accounted for 0.033% of the roots
of I. hupehensis (Y.M. ZHAO & al. [12]). W. H. TALIB & al. [13] investigated the
antiproliferative and antimicrobial effects of thirteen compounds isolated from Inula viscosa
L. and demonstrated that two flavonoids (3-O-methylquercetin and 3,3′-di-O-
methylquercetin) inhibit Salmonella typhimurium (at MIC of 125 μg/mL) and Bacillus cereus
(at MIC of 125 μg/mL and 62.5 μg/mL, respectively).
In the present study, we have proceeded to a qualitative screening regarding the antimicrobial
activity of a Romanian cultivar of Inula helenium L root alcoholic extracts, in order to future
use of this plant as a source of new antimicrobial products for veterinary use. In Romania,
Inula helenium grows on plane and hills area, very close to the villages and it has been used as
old medicinal plant. As a general remark, it has been noticed that Romanian indigenous Inula
plants are shorter than the other Southern European cultivars
2. Materials and methods
Collection of plant material
Dried roots from a Romanian cultivar of Inula heleniumL. have been harvested from the
experimental field of Dacia Plant S.A., Bod, Romania. The plants have been cultivated under
certified ecological conditions. The cultivar has been introduced in the collection from a local
Inula population growing in South-Eastern side of Romania (Brăila) in a plane area.
Herbarium specimen was preserved at the manufacturer.
Preparation of plant extracts
Roots of Inula heleniumL. have been dried and chopped in small pieces of 0.2-1 cm and
extracted in ethanol with different concentrations: 30%v/v (I30o), 50% v/v (I50o) and 70%v/v
Studies on antimicrobial activity of Inula helenium L Romanian cultivar
Romanian Biotechnological Letters, Vol. 19, No. 5, 2014 9701
(I70
o
). After preliminary tests (data not shown) the optimal extraction time in hydro-alcoholic
solutions has been established at 6 hours at room temperature.
Test microorganisms
The Inula extracts have been tested on six potential pathogen bacterial strains and four fungal
strains which can affect the animals (Table 1).
Table 1 - Microorganisms tested for their susceptibility to Inula helenium alcoholic extracts
No. Strain Characteristics Origin
1. Bacillus subtilis ICCF 276 Gram-positive, catalase-positive Collection ICCF, Bucharest,
Romania
2. Bacillus cereus CP1 Gram-positive, beta hemolytic
bacterium
Collection of Faculty of
Biotechnologies, Bucharest,
Romania
3. Escherichia coli MI 57 Gram-negative, non-toxinogenic
serotype
Collection of Faculty of
Biotechnologies, Bucharest,
Romania
4. Escherichia coli CP2 Gram-negative, toxinogenic
serotype
Collection of Faculty of
Biotechnologies, Bucharest,
Romania
5. Staphylococcus aureus CP 3 Gram-positive, catalase-positive Collection of Faculty of
Biotechnologies, Bucharest,
Romania
6. Enterococcus faecalis CP 4 Gram-positive, sensible to β-
lactam-based antibiotics
Collection of Faculty of
Biotechnologies, Bucharest,
Romania
7. Candida albicans
ATCC10231
Serotype A, sensible to nystatin Collection of Microbial Genetics
and Biotechnology, Faculty of
Biology, Bucharest, Romania
8. Candida parapsilosis
CBS604
Sensible to amphotericin B,
nystatine, essential oils of
Coriander sativum
Collection of Microbial Genetics
and Biotechnology, Faculty of
Biology, Bucharest, Romania
9. Candida lypolytica MI 2 non-conventional yeast, lipase
production
Collection of Faculty of
Biotechnologies, Bucharest,
Romania
10. Aspergillus niger F2T Responsible for pulmonary
infections in dogs
Collection CBM Biotehgen
Bucharest, Romania
The bacterial strains were maintained and tested on nutrient agar (0.5% peptone; 0.3% yeast
extract; 1.5% agar; 0.5% NaCl) at 37
o
C, using 24 hours old inoculums. Yeasts and fungal
strains were maintained and tested on Yeast Extract Glucose Agar (1% yeast extract; 2%
peptone; 2% glucose; 2% agar) at 30
o
C, using 24 hours old inoculums for yeasts or mycelium
block (5 mm) for filamentous fungi.
Antimicrobial Analysis
In vitro, the antimicrobial activity was assessed by the "drop agar
diffusion" method (fig.1). The microorganisms were spread using
100μl of suspension containing 10
8
CFU/ml on nutrient agar or on
Yeast Extract Glucose Agar respectively.
Fig.1 - Aspects from the "drop agar diffusion" screening method
Concerning A. niger F2T, the mycelium block (5 mm) was placed in
DIGUȚĂ,C., CORNEA C.P., IONIȚĂ L., BRÎNDUȘE, E., FARCAȘ, N., BOBIT D., MATEI F.
9702 Romanian Biotechnological Letters, Vol. 19, No. 5, 2014
the centre of the medium in Petri dishes. After 1 hour, 10 μL of each sterile extract have been
added in the agar plates pre- inoculated with the tested microorganisms. Non diluted
ampicillin (for bacteria) and fluconazole (for fungi) have been used as positive control and the
Inula extracts solvent (hydro ethanolic solutions of 30%, 50% and 70%) have been used in the
master plates. All plates were incubated according to the itemed test microorganisms. The
diameter of growth inhibition zones (halos) was measured in millimetres on two axes and the
mean value reported. The results are the mean of two separate experiments with three
repetitions for each sample.
3. Results and discussion
On international levels main reports on antimicrobial activity of Inula sp. are linked to
extracts made of other species that Inula hellenium, like Inula racemosa (P.D. LOKHANDE
& al. [8]), Inula hupehensis (J. ZHAO & al [16]) or Inula viscosa (W. H. TALIB & al. [13]).
The present study was conducted on ethanol extracts (30%, 50%, 70%) from dried roots of a
Romanian cultivar of Inula helenium L. Antimicrobial activity was determined by drop agar
diffusion method, measuring the diameter of zone of inhibition. All the measurements have
been done in triplicate. As control, solutions of ethanol of same concentrations as the plant
extracts have been used. As general remark, the results indicated that the antimicrobial
activity increases while increasing the solvent concentration from 30% to 50%, while from
50% to 70% concentration no significant activity increase has been noticed (tables 2 and 3)
comparing to the control.
Fig.2 - Inula’ s extract
antimicrobial activity on
non-toxinogenic serotype
Escherichia coli MI 57 (a-
I30, b- I50, c- I70).
Against the bacterial strains, the ethanol extracts (I50˚ and I70˚) exhibited moderate to high
inhibitory activity, while the ethanol extract I30˚ proved not significant inhibitory activity
only on non-toxinogenic serotype Escherichia coli MI 57 (fig. 2, table 2), close to the control.
Table 2- Antibacterial activity of dried root extracts from I. helenium L.
Legend:
-: non inhibitory halo
+: low inhibitory activity
++: moderate inhibitory
activity
+++: high inhibitory activity
The susceptibility of the fungal strains inhibition is shown in Table 3. Ethanol extract (I50˚)
showed moderate antifungal activity against Candida albicans and good activity against
C.parapsilosis and C. lipolytica. No antifungal activity was found against A. niger (table 3). It
has been noticed that on the testes yeast (Candida spp.) the inhibitory effects of I50˚ and I70˚
are very similar.
Test bacteria
Inhibitory effect
Extract I30˚ Extract I50˚ Extract I70˚
Escherichia coli MI 57 + + + + + +
Escherichia coli CP2 - + + +
Bacillus spp. ICFF 276 - + + + +
Bacillus cereus CP1 - + + + +
Staphylococcus aureus CP3 - + + +
Enterococcus faecalis CP4 - + + + +
Studies on antimicrobial activity of Inula helenium L Romanian cultivar
Romanian Biotechnological Letters, Vol. 19, No. 5, 2014 9703
Table 3- Antifungal activity of dried root extracts from I. helenium L.
Legend:
-: no inhibitory halo
+: low inhibitory
activity
++: moderate
inhibitory activity
+++: high inhibitory
activity
These results are in agreement with the data reported by A. DERIU & al. [9], assaying root
essential oil of I. helenium against some Gram-positive and Gram-negative bacteria and
Candida species.
Regarding the Inula effects on the filamentous fungi, few reports have been issued. Some
recent data (TE ZHAO & [17]) reported the inhibitory effect of Inula britannica extracts on
most important plant pathogenic fungi (Fusarium, Phytophthora, Colletotrichum, etc) and the
potential to produce botanical fungicide to protect crops. In our study, the Inula helenium
ethanolic extracts did not show any inhibitory effect on animal pathogenic Aspergillus niger
which is the main responsible for pulmonary infections in dogs, while other authors (M.A.
NAN & [18]) reported some inhibitory activity on 50 mg/l (MIC).
4. Conclusions
The preliminary results of our study, evaluated by the diameter of the inhibition zone of
microbial growth, indicated that both ethanol extracts (I50˚ and I70˚) from the roots of a
Romanian cultivar of I. helenium L. have significant activity against the pathogenic bacteria
and dermatophytic fungi, while on filamentous fungi A. niger there is no inhibitory activity.
On Candida sp. the inhibitory effects of I50˚ and I70˚ are very similar. In order to develop a
veterinary product with anti-aspergillosis effect some other plant extract (Helleborus,
Eupatorium) should be taken into account.
Further research is required to identify and analyze the bioactive compounds present in the
extracts and to establish the minimal inhibitory concentration for each type of pathogenic
microorganisms. Considering the bioactive potential of roots extracts from indigenous
population of I. helenium could be of great interest for the development of new
pharmaceuticals for veterinary uses.
Acknowledgements
This work was supported by the Executive Agency for Higher Education, Research,
Development and Innovation Funding in the frame of Partnerships in S&T priority domains
Program, Project no. 134/2012, acronym PHYTOIMMUVET.
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This study aimed to investigate the antioxidant, antimicrobial, and cytotoxic potential of ethanolic extracts obtained from Gentiana asclepiadea L. and Inula helenium L. roots, in relation to their chemical composition. The total polyphenols, flavonoids, and phenolic acids were determined by spectrophotometric methods, while LC-MS analysis was used to evaluate the individual constituents. The antioxidant properties were tested using the FRAP and DPPH methods. The standard well diffusion and broth microdilution assays were carried out to establish in vitro antimicrobial efficacy and minimum inhibitory and bactericidal concentrations. The cytotoxicity was tested on rat intestinal epithelial cells using the MTT assay. The results pointed out important constituents such as secoiridoid glycoside (amarogentin), phenolic acids (caffeic acid, chlorogenic acid, trans-p-coumaric acid, salicylic acid), and flavonoids (apigenin, chrysin, luteolin, luteolin-7-O-glucoside, quercetin, rutoside, and naringenin) and promising antioxidant properties. The in vitro antimicrobial effect was noticed towards several pathogens (Bacillus cereus > Staphylococcus aureus > Enterococcus faecalis > Salmonella typhimurium and Salmonella enteritidis > Escherichia coli), with a pronounced bactericidal activity. Rat intestinal epithelial cell viability was not affected by the selected concentrations of these two extracts. These data support the ethnomedicinal recommendations of these species and highlight them as valuable sources of bioactive compounds.
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... It was determined that some species belonging to the genus Inula have antibacterial, antifungal, diuretic, antispasmodic, antihemorrhoidal, antiinflammatory, antitussive, bactericidal, antiproliferative, antidiabetic and hepatoprotective activities, and the wellknown chemical components of the genus include mono-, sesqui-and diterpenes, flavonoids and glycolipids (Akcın and Akcın, 2017;Paliwal et al., 2017;Gökbulut et al., 2013). In the chemical analysis of rhizomes and roots of I. helenium in particular, also known as elecampane among these species, many bioactive compounds, including polysaccharide inulin, eudesmane type essential oil, sesquiterpene lactones with various biological activities, thymol derivatives, terpenes and sterols were detected (Zheng et al., 2021;Diguță et al., 2014). Traditionally, I. helenium species is used in the treatment of arthritis, diabetes, rheumatism, pulmonary tuberculosis, and acute respiratory diseases as expectorant, antitussive and diaphoretic (Zhao et al., 2006;Zlatic et al., 2019). ...
Genetic Relationship of Seven Endemic Inula L. (Asteraceae) Species Grown in Turkey
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  • Apr 2022
In this study, genetic relationship of ISSR markers of seven endemic Inula species distributed in Turkey was carried out. Plant samples were collected from different regions of Turkey in 2013 and gDNA was obtained by DNA isolation from green leaves. Genetic relationship between species was determined using 12 ISSR primers. PCR products were run on agarose gel electrophoresis and visualized under UV light. All gel images were examined and the presence and absence of polymorphic bands were scored as 0 and 1. A total of 85 bands were obtained from the primers. Of these, 74 polymorphic and 11 monomorphic bands were obtained. The total polymorphism rate was found to be approximately 87.05%. The phylogenetic tree and genetic distances between species were calculated using the PAUP 0 4.0b10 analysis program. According to the distance matrix, the genetic distance was found between tthe closest Inula fragilis and Inula sarana (0.28571), while the farthest between Inula sarana and Inula macrocephala (0.56000) species. The phylogenetic tree was obtained using the UPGMA algorithm, and the tree consisted of two groups. The results were compared with the morphological, palynological nrDNA and cpDNA results of the past. Our findings supported previous studies.
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... The essential oil isolated from the roots of I. helenium exhibited a good antimicrobial activity against Staphylococcus aureus, by inducing cell membrane damage (increased permeability, followed by lysis of the exposed cells), the most active constituents being alantolactone, isoalantolactone and diplophyllin (Stojanović-Radić et al., 2012). The hydroalcoholic extract from roots showed a moderate to high antimicrobial activity against several bacterial strains (Escherichia coli, Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis) and fungi (Candida albicans, Candida parapsilosis and Candida lipolytica) (Diguta et al., 2014). I. helenium extract and isolated sesquiterpenes exhibited in vivo (animal model) antiinflammatory activity, and the oral intake alleviated the paw swelling and arthritic severity in rat arthritis models, these effects suggesting their possible use in the prevention and treatment of rheumatoid arthritis (Gao et al., 2017). ...
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A Review of Phytochemical and Pharmacological Studies of Inula Species
Article
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  • Feb 2019
Abstract: Background: Plants of the genus Inula are perennial herbs of the family Asteraceae. This genus includes more than 100 species, widely distributed throughout Europe, Africa and Asia including India. Many of them are indicated in traditional medicine, e.g., in Ayurveda. This review explores chemical constituents, medicinal uses and pharmacological actions of Inula species. Methods: Major databases and research and review articles retrieved through Scopus, Web of Science, and Medline were consulted to obtain information on the pharmacological activities of the genus Inula published from 1994 to 2017. Results: Inula species are used either alone or as an important ingredient of various formulations to cure dysfunctions of the cardiovascular system, respiratory system, urinary system, central nervous system and digestive system, and for the treatment of asthma, diabetes, cancers, skin disorders, hepatic disease, fungal and bacterial infections. A range of phytochemicals including alkaloids, essential and volatile oils, flavonoids, terpenes, and lactones has been isolated from herbs of the genus Inula, which might possibly explain traditional uses of these plants. Conclusion: The present review is focused on chemical constituents, medicinal uses and pharmacological actions of Inula species and provides valuable insight into its medicinal potential. Keywords: Inula species, Phytoconstituents, Sesquiterpenoid lactone, Traditional pharmacology
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Essential Oil from Inula britannica Extraction with SF-CO2 and Its Antifungal Activity
Article
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  • Oct 2013
The aim of this study was to determine the extraction technique of supercritical fluid carbon dioxide (SF-CO2) for the essential oil from Inula britannica flowers and its antifungal activities against plant pathogenic fungi for its potential application as botanical fungicide. The effects of factors, including extraction temperature, extraction pressure, SF-CO2 flow rate, flower powder size, and time on the essential oil yield were studied using the single factor experiment. An orthogonal experiment was conducted to determine the best operating conditions for the maximum extraction oil yield. Adopting the optimum conditions, the maximum yield reached 10.01% at 40°C temperature, 30 MPa pressure, 60 mesh flower powder size, 20 L h−1 SF-CO2 flow rate, and 90 min extraction time. The antifungal activities of I. britannica essential oil using the SF-CO2 against the most important plant pathogenic fungi were also examined through in vitro and in vivo tests. Sixteen plant pathogenic fungi were inhibited to varying degrees at 1 mg mL−1 concentration of the essential oil. The mycelial growth of Gaeumannomyces graminis var. tritici was completely inhibited. The radial growths of Phytophthora capsici and Fusarium monilifome were also inhibited by 83.76 and 64.69%, respectively. In addition, the essential oil can inhibit the spore germination of Fusarium oxysporum f. sp. Vasinfectum, Phytophthora capsici, Colletotrichum orbiculare, and Pyricularia grisea, and the corresponding inhibition rates were 98.26, 96.54, 87.89, and 87.35% respectively. The present study has demonstrated that the essential oil of I. britannica flowers extracted through the SF-CO2 technique is one potential and promising antifungal agent that can be used as botanical fungicide to protect crops.
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Antimicrobial Potential of Eupatorium adenophorum Spreng
Article
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  • Jan 2011
The antimicrobial activity of Petroleum-ether, Benzene, Chloroform, methanol and aqueous extracts of crude leaves of Eupatorium adenophorum Spreng were tested against the growth of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes and Pseudomonas aeruginosa by Paper disk diffusion technique. Nearly all the extracts were found to exhibit moderate to good antibacterial activity against the bacterial pathogens tested and the petroleum ether extract recorded largest zone of inhibition against B. subtilis. The positive results so obtained were compared with that of the reference standard antibiotic ciprofloxacin. Antifungal activity of the above extracts were tested on fungal strains Aspergilus niger, Aspergilus candidus and Candida albicans using Fluconazole as the standard drug. The result shows significant antimicrobial activity of extracts against tested fungi and bacteria.
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Chemical constituents of the roots of Inula helenium
Article
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UDC 547.918 Inula helenium L. (Compositae) is widely distributed in the north of China. Its roots have been used to treat pain of upper body, emesia, and diarrhea, and kill parasite in traditional Chinese medicine [1]. Moreover, in some European pharmacopoeias, the roots are officially listed as a diuretic, diaphoretic, expectorant, and anthelmintic [2]. Some studies on this plant show that it has some bioactivities [3]. Recently, our research showed that the chloroform fraction of the 95% ethanol extract of roots of the plant exhibited significant cytotoxicity activity in vitro. Its bioactivities prompted us to continue to investigate its chemical components. In the present work, the chemical investigation of the chloroform extract of the roots led to the isolation of ten compounds 1–10: 12(13)-en-betulinic acid methyl ester (1), alantolactone (2), isoalantolactone (3), 11DH,13-dihydroisoalantolactone (4), macrophyllilactone E (5), 5D,6D-epoxyalantolactone (6), 3-oxo-4(5),11-eudesmadien-8,12-olide (7), 4D-hydroxy-1E-guaia-11(13),10(14)-dien-12,8D-olide (8), E-sitosterol (9), and stigmasterol (10). Compound 1 was isolated as a white amorphous powder with the molecular formula C 31 H 48 O 3 , as determined on the basis of a molecular ion peak at m/z 468 [M] + in EI-MS. Its 1 H and 13 C NMR data were almost the same as the NMR data [4] of betulinic acid methyl ester with some minor differences. In comparison with the 1 H and 13 C NMR data of betulinic acid methyl ester, the chemical shifts of C-10 and C-14 of compound 1 are 5.7 and 2.5 ppm downfield, respectively. Meanwhile, a signal of the olefinic proton (G 6.27) can be found in the NMR spectra of compound 1 with classic olefinic carbon signals (C-12, G 124.4; C-13, 131.4) that are absent in the NMR spectra of the betulinic acid methyl ester. On the basis of the above observations, the structure of compound 1 was elucidated to be 12(13)-en-betulinic acid methyl ester. A search in the SciFinder and Reaxys database shows that there are only three references about compound 1 [5–7]. One of them [5] is about synthesis of compound 1 from methyl acetylbetulinate. Another one [6] is about the bioactivity of compound 1 prepared by the derivatization of betulinic acid. The third one [7] is about the biotransformation of compound 1, but there was an error in the NMR data stating that the compound is the betulinic acid methyl ester instead of the 12(13)-en-betulinic acid methyl ester. Thus, this is the first time that compound 1 was isolated from the natural source. The 13 C NMR data are shown in Table 1, and its 1 H NMR data are as follows: 1 H NMR (CDCl 3 , G, ppm): 0.8–0.9 (15H, m, H-23, 24, 25, 26, 27), 4.68 (1H, s, 2H-9a), 4.72 (1H, s, 2H-9b), 4.46 (1H, m, H-12). Alantolactone (2) [8]. White powder (MeOH), C 15 H 20 O 2 . EI-MS m/z 232 (M +). 1 H NMR (CDCl 3 , G, ppm, J/Hz): 1.40–1.82 (6H, m, H-1, 2, 3), 2.43 (1H, m, H-4), 5.13 (1H, d, J = 8, H-6), 3.56 (1H, m, H-7), 4.80 (1H, m, H-8), 2.09 (1H, dd, J = 6, 6, H-9a), 1.53 (1H, m, H-9b), 6.17 (1H, d, J = 4, H-13a), 5.60 (1H, d, J = 4, H-13b), 1.17 (3H, s, 14-CH 3), 1.07 (3H, s, 15-CH 3). 13 C NMR (CDCl 3 , G, ppm): 41.7 (C-1), 16.7 (C-2), 32.7 (C-3), 37.5 (C-4), 149.0 (C-5), 118.8 (C-6), 39.4 (C-7), 76.4 (C-8), 42.6 (C-9), 32.6 (C-10), 139.8 (C-11), 170.3 (C-12), 121.5 (C-13), 22.5 (C-14), 28.5 (C-15). Isoalantolactone (3) [8]. White powder (MeOH), C 15 H 20 O 2 . EI-MS m/z 232 (M +). 1 H NMR (CDCl 3 , G, ppm, J/Hz): 1.50–2.20 (6H, m, H-1, 2, 3), 2.34 (1H, m, H-5), 1.71 (1H, m, H-6a), 1.24 (1H, m, H-6b), 2.97 (1H, m, H-7), 4.48 (1H, m, H-8), 1.99 (1H, m, H-9a), 1.39 (1H, m, H-9b), 6.11 (1H, d, J = 2, H-13a), 5.57 (1H, d, J = 2, H-13b), 0.82 (3H, s, CH 3 -14), 4.76 (1H, d, J = 3, H-15a), 4.43 (1H, d, J = 3, H-15b). 13 C NMR (CDCl 3 , G, ppm): 42.. 11DH,13-Dihydroisoalantolactone (4) [9]. White powder (MeOH), C 15 H 22 O 2 . EI-MS m/z 234 (M +). 1 H NMR (CDCl 3 , G, ppm, J/Hz): 1.40–1.80 (6H, m, H-1, 2, 3), 2.34 (1H, m, H-5), 1.99 (1H, m, H-6a), 1.15 (1H, m, H-6b), 2.39 (1H, m, H-7), 4.46 (1H, m, H-8), 1.47 (1H, dd, J = 9, 9, H-9a), 2.15 (1H, dd, J = 4, 4, H-9b), 2.79 (1H, t, J = 7, H-11), 1.21 (3H, d, J = 15, H-13), 0.80 (3H, s, CH 3 -14), 4.77 (1H, d, J = 6, H-15a), 4.47 (1H, d, J = 3, H-15b). 13 C NMR (CDCl 3 , G, ppm): 41.
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Antiproliferative, Antimicrobial and Apoptosis Inducing Effects of Compounds Isolated from Inula viscosa
Article
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The antiproliferative and antimicrobial effects of thirteen compounds isolated from Inula viscosa (L.) were tested in this study. The antiproliferative activity was tested against three cell lines using the MTT assay. The microdilution method was used to study the antimicrobial activity against two Gram positive bacteria, two Gram negative bacteria and one fungus. The apoptotic activity was determined using a TUNEL colorimetric assay. Scanning electron microscopy was used to study the morphological changes in treated cancer cells and bacteria. Antiproliferative activity was observed in four flavonoids (nepetin, 3,3'-di-O-methylquercetin, hispidulin, and 3-O-methylquercetin). 3,3'-di-O-Methylquercetin and 3-O-methylquercetin showed selective antiproliferative activity against MCF-7 cells, with IC(50) values of 10.11 and 11.23 µg/mL, respectively. Both compounds exert their antiproliferative effect by inducing apoptosis as indicted by the presence of DNA fragmentation, nuclear condensation, and formation of apoptotic bodies in treated cancer cells. The antimicrobial effect of Inula viscosa were also noticed in 3,3'-di-O-methylquercetin and 3-O-methyquercetin that inhibited Bacillus cereus at MIC of 62.5 and 125 µg/mL, respectively. Salmonella typhimurium was inhibited by both compounds at MIC of 125 µg/mL. 3,3'-di-O-Methylquercetin induced damage in bacterial cell walls and cytoplasmic membranes. Methylated quercetins isolated from Inula viscosa have improved anticancer and antimicrobial properties compared with other flavonoids and are promising as potential anticancer and antimicrobial agents.
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Medicinal importance of genus Origanum: A review
Article
  • Jan 2013
Genus Origanum is important medicinally as it has antimicrobial, antifungal, antioxidant, antibacterial, antithrombin, antimutagenic, angiogenic, antiparasetic and antihyperglycaemic activities. Phytochemical investigations of the species of this genus have resulted in the extraction of a number of important bioactive compounds. This emphasizes on the need of extensive study for reporting the additional information on the medicinal importance of other unattended species of genus Origanum.
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Studies on the antimicrobial activities of extracts from Eupatorium lindleyanum DC against food spoilage and food-borne pathogens
Article
Antimicrobial activity of extracts from Eupatorium lindleyanum DC (EEL), a traditional Chinese medicine, was screened against eight selected food spoilage and food-borne pathogens. The EEL was observed to show effective antimicrobial effects with a remarkable dose–response relationship and broad antimicrobial spectrum on all test Gram-positive bacteria and Gram-negative species. Acid environment or medium helps enhance the antimicrobial activity. The activity remains rather stable under conventional food sterilization conditions and normal shelving temperature. The EEL obtained by water decoction exhibited the maximum inhibitory effect on test strains with its MIC (minimum inhibitory concentration) and/or MBC (minimum bactericidal concentration) value of 0.4–0.8mgml−1. When the EEL was used in commercial orange juice at a concentration of 0.4mgml−1, a similar antimicrobial effect to potassium sorbate at the level of 0.2mgml−1 was observed. In conclusion, in addition to use as a functional food ingredient, the EEL may be selected as an inhibitor to preserve food products where a natural preservative is desired.
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Ultrasound-assisted extraction of total flavonoids from Inula helenium
Article
  • Apr 2012
Inula helenium was a perennial herb belonging to composite family and the roots of I. helenium have been widely used in traditional Chinese medicine. In this study, I. helenium was used as an experimental matrix. Ultrasound-assisted extraction (UAE) of total flavonoids from I. helenium was studied with dual wavelength UV-VIS spectrophotometer. Effects of various factors including ratio of material to liquid, ultrasonic time, ethanol concentration and extraction times on extraction yield of total flavonoids were evaluated. Then, orthogonal design of four factors at three levels was applied for optimization the extraction yields of flavonoids from the root of I. helenium. The optimal extracting process of the total flavonoids from the root of the I. helenium was 1 g plant sample with 20 ml of 60% ethanol, extracting twice and each time for 20 min. Under these optimal conditions, the yield of total flavonoids was (17.36±0.94) mg/g. UAE was more efficient and time saving for the extraction of flavonoids from plant materials.
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Antimicrobial activities of some thymol derivatives from the roots of Inula hupehensis
Article
In a search for some naturally occurring antimicrobial compounds found in medicinal plants, herbs and spice extracts, four thymol derivatives were isolated from the roots of Inula hupehensis (a medicinal plant) and identified as the new compound 9-O-β-d-glucopyranosyl-9-hydroxythymol (1), together with 8,9-epoxy-10-isobutyryloxythymyl isobutyrate (2), 8-hydroxy-9, 10-diisobutyloxythymol (3) and 8,9,10-trihydroxythymol (4). These thymol derivatives, with a semi-synthetic compound 9,10-dihydroxy-8-methoxythymol (5), were tested for their antimicrobial activities against three bacteria and six plant pathogenic fungi. Within the series of thymol derivatives tested, compound 3 was the most active, particularly displaying moderate antibacterial activities against Staphylococcusaureus, Methicillin-resistant S. aureus and Escherichia coli with MICs of 62.3, 62.8, and 250 μg/ml. Moreover, this compound exhibited inhibitory activities against three plant pathogenic fungi: Rhizoctonia solani, Phytophthora melonis and Peronophythora litchi, with EC50 values of 157, 180 and 141 μg/ml. To our knowledge, this is the first report that these five thymol derivatives show inhibitory activity against plant pathogenic fungi.
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