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PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY OF LEAF

EXTRACTS OF ERYNGIUM FOETIDUM L. (APIACEAE)

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 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876

PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY OF LEAF

EXTRACTS OF ERYNGIUM FOETIDUM L. (APIACEAE)

D P Lingaraju1*, M S Sudarshana2, C Mahendra 2, K Poornachandra Rao3

1
A V Kanthamma College for Women, Hassan, Karnataka, India – 573201.
2
Department of Studies in Botany, University of Mysore, Mysoru, Karnataka – 570006.
3
Department of Microbiology, University of Mysore, Mysoru, Karnataka.

ARTICLE INFO ABSTRACT

Article history Eryngium foetidum L., belonging to Apiaceae is a biannual herb. It is used as an ethno-
Received 29/01/2016 medicinal plant for the treatment of various ailments. The leaf decoction is used against
Available online gastrointestinal disorders and leaf paste as wound healing agent by the ethnic communities in
29/02/2016 the Kodagu district of Karnataka. Hence a study was designed to examine the antibacterial
and antifungal activity of leaves of E. foetidum. The Soxhlet extraction was performed by
Keywords using organic solvents like petroleum ether, chloroform, ethyl acetate, methanol. Each extract
Eryngium Foetidum, was subjected to preliminary phytochemical tests. The results of phytochemical screening
Extraction, revealed the presence of some active principles like glycosides, flavonoids, terpenoids,
Phytochemicals, steroids and tannins. The antimicrobial activity of isolated extracts was performed with
Antimicrobial Activity. microorganisms like two Gram positive bacteria - Bacillus subtilis, Staphylococcus aureus,
two Gram negative bacteria - Escherichia coli, Pseudomonas aeruginosa and a fungus
Candida albicans by agar well diffusion method. The result of the antimicrobial activity
revealed that the ethylacetate extract of E. foetidum showed the strongest antimicrobial
activity among the five extracts against the four bacterial strains and C. albicans. This is also
revealed that inhibition of ethylacetate extract against P.aeruginosa (28mm ) and C. albicans
(18mm) was comparatively higher to that of the standard Gentamycin (18mm) and Nystatin
(14mm) respectively. The result of present study supports traditional usage of E. foetidum and
also suggests that ethyl acetate leaf extract possesses compounds with antimicrobial property
that can be used as antimicrobial agents in new drug for the therapy of infectious diseases
caused by pathogens.

Corresponding author
Lingaraju D P
Assistant Professor of Botany
AV Kanthamma College for Women, Hassan
Karnataka, India- 573201
rajuponnappa@yahoo.in
+919448996416 4339

Please cite this article in press as Lingaraju et al. Phytochemical Screening and Antimicrobial Activity of Leaf Extracts of
Eryngium Foetidum L. (Apiaceae. Indo American Journal of Pharmaceutical Research.2016:6(02).

Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
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Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Vol 6, Issue 02, 2016. Lingaraju et al. ISSN NO: 2231-6876

INTRODUCTION
Health-care, which was a part of the traditional culture of the people, has become a professional in the modern industrial
world. Synthetic drugs created by the affluent and influential pharmaceutical industries have given rise to effects which are more
dangerous than the diseases they claim to cure. Now microorganisms have become resistant to many antibiotics due to increased use
of drugs, which is decreasing efficiency of conventional medicines[1] . The development of multidrug resistance in the pathogenic
bacteria has created major clinical problems in the treatment of infectious diseases. So, it has become necessary to find out new
antimicrobial agents. Plants are rich reservoir of antimicrobials. Plant based antimicrobials have enormous therapeutic potential as
they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobials. Biomolecules of plant origin
appear to be one of the alternatives for the control of these antibiotic resistant human pathogens. In recent times, there has been
increasing interest in the study of bioactive compounds from leaves, flowers, seeds, and stem bark due to their antioxidative,
antimicrobial and other health promoting properties [2]. Now-a-days maximum number of plants is being screened for their possible
pharmacological value. The phytochemical research based on ethno-pharmacological information is generally considered an effective
approach in the discovery of new anti-infective agents from higher plants. So, research has gained momentum to establish a scientific
basis of folkloric use of medicinal plants and for the development of new effective drugs. Owing to the significance in the above
context, such preliminary phytochemical screening of plants is the need of the hour in order to discover and develop novel therapeutic
agents with improved efficacy. Several therapeutically important plants are listed and described in ancient literature. However,
majority of ethnomedicinal plants have not been systematically screened against various disease causing microorganisms. Considering
all these in mind, the present study is concentrated on the ethnomedicinal plant Eryngium foetidum L.
Eryngium foetidum L., (Fam: Apiaceae) is a biennial herb indigenous to Tropical America and West Indies [3]. The plant is
native to the Caribbean islands. Today, it is has been introduced to large parts of South East Asia. It is also cultivated as leafy
vegetable crops in Asia and Africa [4]. It is an endemic medicinal and aromatic herb found in diverse, specific geographic locations in
India [5]. It is also known as E. antihystericum. The specific name antihystericum reflects the fact that this plant has traditionally been
used for epilepsy. So it is known by the common names spirit weed and fit weed. It is used as an ethno-medicinal plant for the
treatment of a number of ailments such as fevers, chills, vomiting, burns, hypertension, headache, intestinal worms, earache,
stomachache, constipation, asthma, arthritis, snake bites, scorpion stings, diarrhea, malaria, pneumonia, flu, infertility complications
and epilepsy [6, 7]. Pharmacological investigations have demonstrated it as anthelmintic, anti-inflammatory, analgesic [8], anti-
convulsant, anti-carcinogenic, anti-diabetic [9, 10]. It is one of the plants used in a Japanese patent for having developed a skin-
whitening agent [11]. The leaves and roots are used in the treatment of gastrointestinal problems and leaf paste is used as an antiseptic
agent by the ethnic communities residing in the forests of Kodagu district (Karnataka).

Figure 1. Eryngium Foetidum.

Eryngium foetidum L, contains decanal and dodecanal as an important fragrance and flavor agents [12]. Leaves are an
excellent source of vitamin A, B1, B2 and C[13]. Earlier studies have shown that the main constituent of essential oil of the plant is
eryngial [14]. Many studies have done on this plant regarding to their chemical properties and volatile components, despite the
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widespread use of these herbs for food and as ethno medicinal agents. Most of the investigations were on the extraction and
identification of essential oil compounds. So there remains a lack of comprehensive, integral information on its antimicrobial activity.
Hence a study was designed to examine the antibacterial and antifungal activity of different organic extracts of leaves of E. foetidum.
The aim and objective of this study is to screen the aqueous and different organic solvent extracts of E. foetidum leaves for
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their biologically active principles, with a view to provide a scientific justification for ethnomedicinal use of this plant in the treatment
of various ailments. The study also aims at to determine the antimicrobial activities on some selected microorganisms.

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Vol 6, Issue 02, 2016. Lingaraju et al. ISSN NO: 2231-6876

MATERIALS AND METHODS

Collection of plant materials
Fresh leaves of Eryngium foetidum were collected from Pushpagiri hills of Somwarpet taluk, Kodagu district (Karnataka).
The plant was taxonomically identified by using Flora of Presidency of Madras [15]. A specimen of the plant was made into a
herbarium and it was kept in the herbarium of Department of Studies in Botany, University of Mysore, Mysoru.

Preparation of Solvent extracts

The leaves of E. foetidum were washed thoroughly 2-3 times with running water and once with sterile water, chopped into
small pieces, shade dried, coarse powdered in a mechanical grinder, sieved and used for extraction. The dried material (50gm) was
extracted with petroleum ether, chloroform, ethyl acetate, and methanol by using Soxhlet apparatus. The extract was filtered with
Whatman No. 1 filter paper and concentrated using rotary flash evaporator and preserved at 5 oC in air tight bottle until further use. All
the extracts were subjected to antimicrobial activity assay and preliminary phytochemical tests.

Test microorganisms
The test microorganisms used were Escherrichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus
and a fungus - Candida albicans. All these microorganisms were obtained from Herbal Drug Technology Laboratory, Department of
studies in Microbiology, University of Mysore. The bacteria were maintained on nutrient broth (NB) at 37°C and fungus were
maintained on Potato dextrose agar at 28°C.

Evaluation of antimicrobial activity by agar well diffusion method

The antimicrobial activities of different solvent extracts of leaves of E. foetidum were evaluated against pathogenic Gram
positive bacteria – Staphylococcus aureus, Bacillus subtilis and two Gram –negative bacteria- Pseudomonas aeruginosa, Escherrichia
coli and a fungus Candida albicans by agar well diffusion method with slight modifications as adapted by Singh et al [16]. All the
microbial cultures were adjusted to 0.5 McFarland standards, which is visually comparable to a microbial suspension of approximately
1.5 ×108 cfu/ml. 20 ml of agar media was poured into each Petri plate, allowed to solidify and plates were swabbed with 100 µl
inoculate of the test microorganisms. Using sterile cork borer of 6 mm diameter, wells were bored into the seeded agar plates and
these were loaded with a 50µl volume with concentration of 50 mg/ml of different solvent extract constituted in the respective
solvents used for extraction. All the plates were incubated at 37˚C for 24 h for the bacteria and 26 oC for 48 h for the fungus.
Antimicrobial activities of different solvent extracts were evaluated by measuring the zone of growth inhibition against the test
microorganisms. The well with pure solvents used for extraction was used as a negative control whereas Gentamycin (standard
antibacterial drug) and Nystatin (standard antifungal drug) were used as positive control at concentration of 10 µl/well from 1mg/ml of
stock solution to confirm that all the microorganisms tested were inhibited by the antibiotic. The experiments were performed in
triplicates. The diameter of the inhibition zone was measured in mm.

Determination of minimum inhibitory concentration

The MIC is defined as the lowest concentration of the compound at which the microorganism does not demonstrate visible
growth. The minimum inhibitory concentrations (MIC) were evaluated by the microbroth dilution method in a 96-well microtitre
plates by following the procedure adapted by Yakob et al., [17] with modifications. It was performed by using standard inocula
consisted of 2×106 cfu/ml for bacteria and 2×105 cfu/ml a for fungi. Two-fold serial dilutions of the test compounds, dissolved in
solvents used for extraction were prepared to final concentrations of 200 - 0.18 μg/ml in Mueller-Hinton Broth and Sabourauds
dextrose broth for bacteria and fungi respectively. To each well 10 μL of microbial inocula were added. In the tests,
triphenyltetrazoliumchloride and methylthioazyltetrazolim bromide (Aldrich Chemical Company Inc., USA) at concentration of
0.05ml was added to the culture medium as a growth indicator. The microbial growth was determined by the absorbance at 600 nm
using a universal microplate reader after incubation at 37 ˚C for 24 h for the bacteria, and at 26 ˚C for 48h for the fungus. The lowest
concentration of the extracts which inhibited microbial growth (no turbidity) was recorded as the minimum inhibitory concentration.

Preliminary phytochemical screening

The preliminary phytochemical tests were conducted to find the presence of the active chemical constituents such as
alkaloids, glycosides, flavonoids, triterpenes, sterols and tannins following the method adapted by Harbone [18] and Edeoga et al [19]. 4341
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Vol 6, Issue 02, 2016. Lingaraju et al. ISSN NO: 2231-6876

RESULTS AND DISCUSSION

Results of antibacterial activity
The petroleum ether, chloroform, ethylacetate, methanol and aqueous extracts of E. foetidum tested for antibacterial activity
against two Gram positive and two Gram negative bacteria and one fungus were presented in Table.1. The degree of activity was
varied with reference to different solvent extracts of the plant. The result of the antibacterial and antifungal activity revealed that the
ethylacetate extract of E. foetidum showed the strongest antimicrobial activity among the five extracts against the four bacterial strains
and a fungus C. albicans. This indicates that the active principles which inhibit the growth of susceptible bacteria and fungus may
dissolve better in ethylacetate than in the other solvents. This is also revealed that inhibition of ethylacetate extract against
P.aeruginosa (28mm ) was comparatively higher to that of the standard Gentamycin (18mm); and similar against S.aureus (25mm).
The inhibition against C. albicans (18mm) was higher when compared to that of standard Nystatin (14mm).
Petroleum ether extract showed inhibition against all bacteria tested, but no activity against C. albicans. Chloroform extract
showed inhibition only against Gram negative bacteria- B.subtilis (17mm ) and S.aureus ( 18mm). Methanol extract showed activity
against S. aureus (20mm), B. subtilis (16mm) and E. coli (15mm). Aqueous extract was found to be effective against Gram positive
bacteria- B.subtilis ( 14mm ) and S. aureus (18mm).
E.coli was sensitive to ethyl acetate extract followed by methanol and petroleum ether extracts, but insensitive to chloroform
and aqueous extracts. P.aeruginosa was found sensitive to petroleum ether and ethylacetate extracts. B.subtilis was sensitive to all
kinds of organic extracts used; it was maximum in ethylacetate followed by petroleum ether, chloroform, methanol and water.
S.aureus showed sensitivity to all kinds of solvent extracts; it was maximum in ethylacetate similar to standard Gentamycin.
C.albicans showed sensitivity only against ethylacetate and aqueous extracts. The ethyl acetate extract showed highest
inhibitory activity (18 mm) when compare to standard Nystatin (14mm).

Table 1: Results of Antmicrobial activity of different solvent extracts of leaves of E. foetidum by agar well diffusion method
(zone of inhibition in mm).
Compound E.coli P.aeruginosa B.subtilis S.aureus C. albicans
Nystatin for fungi NT NT NT NT 14
Gentamycin for bacteria 25 18 30 25 NT
Pet .ether extract 14 20 18 20 NS
Chloroform extract NS NS 17 18 NS
Ethyl acetate extract 17 28 20 25 18
Methanol extract 15 NS 16 20 NS
Aqueous extract NS NS 14 18 12
NT-Not tested, NS - Not sensitive.

The minimal inhibition concentration (MIC) was determined using five extracts of E.foetidum against a wide range of
bacterial strains and a fungus- C.albicans. The result of MIC assay is shown in Table-2. Ethylacetate extract exhibited the highest
antibacterial efficacy against P.aeruginosa at 3.12µg/ml concentration when compared to Gentamycin (12.5µg /ml. Ethyl acetate
extract showed the highest antifungal efficacy against C.albicans at 1.56 µg /ml concentration when compared to Nystatin (3.12 µg
/ml). In this study, P. aeruginosa and C. albicans were found to be sensitive to ethyl acetate extract.

Table 2: MIC (µg/ml) of solvent extracts of leaves of E. foetidum against bacteria and C.albicans.

Compound E.coli P.aeruginosa B.subtilis S.aureus C.albicans

Nystatin for fungi NT NT NT NT 3.12
Gentamycin for bacteria 3.12 12.5 0.39 1.56 NT
Pet .ether extract 100 12.5 3.12 3.12 NS
Chloroform extract NS NS 100 50 NS
Ethyl acetate extract 50 3.12 50 3.12 1.56
Methanol extract 200 NS 200 3.12 NS
Aqueous extract NS NS 100 50 12.5
†NT -Not tested, NS - Not sensitive.

Results of phytochemical screening

The phytochemical constituents are summarized in Table-3. The results revealed the presence of some active principles like
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glycosides, flavonoids, terpenoids, steroids, tannins but there were no alkaloids and saponins. The ethylacetate being solvent that has
low polarity was found to extract the maximum active principles.
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Vol 6, Issue 02, 2016. Lingaraju et al. ISSN NO: 2231-6876

Table 3. Qualitative preliminary phytochemical analysis of leaf extracts of E. foetidum .

Phytocontituents Tests PE CL EA MN AQ
Alkaloids Dragendroff’s test -- -- -- -- --
Wagner’s test -- -- -- -- --
Mayer’s test -- -- -- -- --
Glycosides Keller Killani test -- -- + + +
Molisch’s test -- -- + + --
Flavonoids Shinoda test + -- + + +
Ferric chloride test + -- + + +
Lead acetate test + - + + +
Triterpenes Libermann-Burchard test -- + + + --
Salkowski’s test -- + + + --
Sterols Libermann-Burchard test + + + + --
Salkowski’s test + + + + --
Tannins Gelatin test -- -- + + +
Ferric chloride test -- -- + + +
Saponins Foam test -- -- -- -- --
+ Present, -- Absent.
PE- Petroleum ether, CL- Chloroform, EA-Ethyl acetate, MN- Methanol, AQ- Aqueous

CONCLUSION
The solvent extracts of E. foetidum showed antibacterial and antifungal activity against both Gram positive and Gram
negative bacteria and a fungus tested. The result of the antimicrobial activity revealed that the ethylacetate extract of E. foetidum
showed the strongest antimicrobial activity among the five extracts against the four bacterial strains and C albicans. This indicates that
the active principles which inhibit the growth of microorganisms tested may dissolve better in ethylacetate than in the other solvents.
This is also revealed that inhibition of ethylacetate extract against P.aeruginosa, and C. albicans was comparatively higher to that of
the standard Gentamycin and Nystatin respectively.
The preliminary phytochemical screening of leaves E. foetidum revealed the presence of glycosides, flavonoids, terpenoids,
steroids and tannins. These active principles could be responsible for the antimicrobial properties. The extracts could inhibit both
Gram positive and Gram negative bacteria indicating that the active secondary metabolites are broad spectrum compounds. However,
it needs better thorough research to recognize the phytoconstituents before being used for the development of any drugs. Hence,
further research is required for the isolation and identification of the bioactive compounds.

ACKNOWLEDGEMENT
I extremely thankful to Chairman, Department of Botany, University of Mysore, for providing necessary facilities to carry
out present research work. I wish to thank the Chairman, Department of Microbiology, University of Mysore, for providing bacterial
and fungal strains. I would like to thank Principal and all my colleague of AVK College for women, Hassan, for their encouragement
and kind support.

Authors’ Statement
The authors declare no conflict of interest

REFERENCES
1. Amit Pandey, Parul Singh. Antibacterial activity of Syzygium aromaticum (clove) with metal ion effect against food borne
pathogens. Asian journal of plant science and research 2011;1(2):69-80.
2. Geetha and Padal SB. Antibacterial Activity in Extract of Bauhinia Vahlii. BioMed Research, BMR Microbiology 2014; 1(2): 1-
4.
3. Ramcharan C. Culantro: A much utilized, little understood herb. In: J. Janick (ed.), Perspectives on new crops and new uses.
ASHS Press: Alexandria: VA: 506–509; 1999.
4. Wang Ping, Su Zushang, Yuan, Wei, Deng, Guangrui., et al. Phytochemical constituents and pharmacological activities of
Eryngium L.(Apiaceae). Stephen F. Austin State University SFA ScholarWorks: Faculty Publications: Pharmaceutical Crops
2012; 99-120 .
5. Chandrika R, Komal Kumar J, Thara Saraswathi KJ, Deviprasad AG, FTIR spectroscopic studies and antimicrobial activity in
4343

populations of Eryngium foetidum L. International Journal of Pharmacy 2013; 3(4): 813-818.

6. Wong W. Some folk medicinal plants from Trinidad. Econ. Bot. 1976; 30:103–142.
7. Mahabir K. Medicinal and edible plants used by East Indians of Trinidad and Tobago. Chackra Publ. House: El Dorado: Trinidad:
W.I; 1991.
8. Sáenz MT, Fernández MA, García MD. Antiinflammatory and analgesic properties from leaves of Eryngium foetidum L.
Page

(Apiaceae). Phytotherapy Research 1997; 11 (5): 380-384.

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Vol 6, Issue 02, 2016. Lingaraju et al. ISSN NO: 2231-6876

9. Simon OR, Singh N. Demonstration of anticonvulsant properties of an aqueous extract of Spirit Weed (Eryngium foetidum L.).
The West Indian medical journal 1986; 35 (2): 121–5.
10. Honeychurch PN. Caribbean wild plants and their uses. Letchworth Press: Barbados: W.I; 1980.
11. Paula JHA, Seafortha CE, Tikasinghb T. Eryngium foetidum L.: a review. Fitoterapia 2011; 82 (3): 302-308.
12. Shavandi MA, Zahra Haddadian & Mohd Halim Shah Ismail. Eryngium foetidum L. Coriandrum sativum And Persicaria
odorata L.: A Review, Journal Of Asian Scientific Research 2012; 2(8): 410-426.
13. Bautista OK, Kosiyachinda S, Rahman ASA, Soenoeadji. Traditional vegetables of Asia. Asian Food Journal 1988; 4:47–58.
14. Yarnell A. Home Field Advantage, Chemical & Engineering News 2004; 82(23): 33.
15. Gamble JS. Flora of Presidency of Madras, vol. I-III. Bishan Singh Mahendra Pal Singh Publications: Dehra Dun: India; 1995.
16. Singh DP, Kumar K, Sharma C. Antimicrobial active macrocyclic complexes of Cr(III), Mn(III) and Fe(III) with their
spectroscopic approach. Eur. J. Med. Chem. 2009; 44: 3299-3304.
17. Yakob HK, Sulaiman S, and Uyub AM. Antioxidant and Antibacterial Activity of Ludwigia octovalvis on Escherichia coli
O157:H7 and Some Pathogenic Bacteria. World Applied Sciences Journal. 2012; 16(1): 22-29.
18. Harborne JB. Phytochemical Methods ; A Guide to Modern Techniques of Plants Analysis. 3 rd Edition. Chapman & Hall:
London: England; 1998.
19. Edeoga HO, Okwu DE, Mbaebre BO. Phytochemical constituent of some Nigerian Medicinal Plants. Afr .J. biotechnol 2005; 4(7)
: 685-688.

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