52 Chapter 3
3.1 Introduction
Plants contain numerous biologically active compounds,
many of which have exhibited antimicrobial properties (Perumal
Samy and Ignacimuthu, 1998). Over the past few years, strains of
many pathogenic species resistant to all widely available antibiotics
have emerged and proliferated (Tomasz, 1994). The antibiotics are
sometimes associated with adverse effects on hosts, which include
hypersensitivity, immunosuppression, allergic reactions and
depletion of beneficial gut and mucosal microbes (Idose et al.,
1968). As in human, reports of antibiotic resistant bacteria
emerging in animal populations are appearing with increasing
frequency (Bates et al., 1993). In many parts of the world plants are
used for antibacterial, antifungal and antiviral medicine. The
extracts were used as a source of medicinal agents to cure urinary
tract infections, cervicitis, vaginitis and gastrointestinal disorders
(Caceres et al., 1990).
In this study few locally available ferns were screened for
their antibacterial activity; some of them had been described as
ethnobotanically relevant. 80% ethanol extracts of different plants
were investigated for antibacterial activity towards selected
bacterial strains.
3.2 Materials and Methods
3.2.1 Procurement of plant materials
Fresh specimens were collected based on their richness in
the month of November from the Vagamon Hills and nearby areas
of Kottayam District of Kerala State, India. Plants of limited
population were avoided in order to conserve them. Voucher
specimens identified by Dr. P.V. Madhusoodanan (Pteridologist and
Antibacterial and Phytochemical Screening of Ferns 53
Professor of Botany, Calicut University Kerala-673635, India.) were
deposited at the herbarium of Calicut University Herbarium (CALI).
Table 3.1 List of ferns selected for antibacterial screening and
their ethnobotanical importance.
N0
Botanical Name and Family (Voucher
Specimen No)
Medicinal uses of plants/their genus
1. Angiopteris evecta (G.
Forst.) Hoffm.
Angiopteridaceae
(TT 2049)
Aromatic oil from large sized fern used for
perfuming coconut oil in south sea Islands. In
Central India the rhizomes are used against
scabies (Vasudeva, 1999). Treatment of Cancer/
Sarcoma (Defilpps et al., 1998)
2. Osmunda regalis L.
Osmundaceae
(TT 2050)
Cultivated as ornamental and local tribals of
Madhya Pradesh, India utilised its first frond of
the year to cure toothache (Vasudeva, 1999).
3. Lygodium Flexuosum
(L.) Sw.
Schizaceae
(TT 2051)
Syn. Ophioglossum
fluxuosum L.
The plant has been described as an expectorant
and its roots extract in mustard oil is considered
an effective remedy for the treatment of wounds
and enzema. Leaf paste also applied over the skin
to treat skin disease (Nayar, 1959).
4.
Acrostichum aureum L.
Pteridaceae
(TT 2052)
Plant is used as styptic and anthelmintic. Also
used as a worm remedy and as an astringent in
haemorrhage (Chopra et al., 1956). Used in
traditional medicine of Nicobarese tribes (Dagar,
1989)
5. Pteris quadriaurita Retz.
Pteridaceae
(TT 2053)
Prteis aquilinia L. used as antihelmintic (Nayar,
1959). Decoction of fresh rhizome and fronds are
given in chronic disorders arising from
obstructions of viscera and spleen. (Chopra et al.,
1956).
54 Chapter 3
6. Cheilanthes bullosa
Kunze.
Sinopteridaceae
(TT 2054)
Syn. Cheilanthes
farinosa Kaulf var
flaccida Bedd.
Cheilanthes farinosa Kaulf The plant juice filtered
and mixed with rice starch is given thrice a day for
the discharge of yellowish urine in the doses of 20-
30 ml (Lal et al., 1996).
7. Cheilanthes tenuifolia
(Burm.f.) Sw.
Sinopteridaceae
(TT 2055)
Syn. Trichomanes
tenuifolia Burm.f.
Used by tribal as a general tonic (Nayar, 1959).
8. Doryopteris concolor
(Langsd. & Fischer) Kuhn
in Decken
Sinopteridaceae (TT 2056)
Syn. Pteris concolor
Langsd. & Fischer
Prteis aquilinia L. used as antihelmintic (Nayar,
1959). Decoction of fresh rhizome and fronds are
given in chronic disorders arising from
obstructions of viscera and spleen. (Chopra et al.,
1956).
9. Parahemionitis cordata
(Roxb. ex Hook. & Grev.)
Fraser-Jenk.
Hemionitidaceae (TT
2057)
Syn. Hemionitis cordata
Roxb. ex Hook. & Grev.
The Rabbits Ear Fern used in the treatment of
earaches and as a vermifuge. The whole plant
parts are used. (Vasudeva, 1999).
10. Pityrogramma austro-
americana Domin
Hemionitidaceae (TT
2058)
Syn. Pityrogramma
calomelanos var.
aureoflava (Hook.)
Weath. ex Baily
The Golden fern cultivated in the botanical
gardens as ornamental plants due to their
splendid beauty and grace (Vasudeva, 1999).
Antibacterial and Phytochemical Screening of Ferns 55
11. Pityrogramma
calomelanos (L.) Link
Hemionitidaceae (TT
2059)
Syn. Acrostichum
calomelanos L.
The decoction of rhizomes used as an anthelmintic
(Vasudeva, 1999). Smoke the leaf for colds in head
and chests (Caius, 1935).
12. Adiantum caudatum L.
Adiantaceae (TT 2060) Used in cough and fever (Vasudeva, 1999).
13. Adiantum latifolium
Lam.
Adiantaceae (TT 2061)
Syn. Adiantum
denticulatum Sw.
The whole plant parts are used as medicinal. Plant
parts are mucilaginous, pectoral, expectorant and
emmenagogue. The leaves are used as a febrifuge
and catarrhal affections. Used popularly as a
cough medicine and for all throat infections
(Nayar, 1959). In France large quantities are used
to prepare the famous “Sirop de capillare”.
14.
Adiantum lunulatum
Burm.f.
Adiantaceae (TT 2062)
Syn. Adiantum
philippense L.
Rhizome used to cure glandular swelling
accompanied by fever. Juice of leaves used in
treatment of dysentery, diseases of blood, ulcers,
etc. In Ayurveda the plant is recommended to cure
for epilepsy. The spores are used in treatment of
leprosy and other skin diseases (Nayar, 1959).
Used as contraceptive agent to induce sterility
(Dhiman, 1998)
15. Adiantum raddianum C.
Presl.
Adiantaceae (TT 2063)
Syn. Adiantum
cuneatum Langsd. &
Fisch.
Adiantum lunulatum rhizome used to cure
glandular swelling accompanied by fever. Juice of
leaves used in treatment of dysentery, diseases of
blood, ulcers, etc. In Ayurveda the plant is
recommended to cure for epilepsy. The spores are
used in treatment of leprosy and other skin
diseases (Nayar, 1959).
56 Chapter 3
16. Microlepia speluncae
(L.) T. Moore
Dennstaedtiaceae (TT
2064)
Syn. Polypodium
speluncae L.
Microlepia scaberula used against eye disease
(McClatchey, 1993) Used in traditional medicine of
Nicobarese tribes (Dagar, 1989)
17. Lindsaea ensifolia Sw.
Lindsaeaceae (TT
2065)
Syn. Schizoloma
ensifolium (Sw.) J.Sm.
in Hook.
Fronds of Sphenomeris chinensis, Lindsaeaceae are
used to produce red dye. The plants are used
internally for chronic enteritis (Fosberg, 1942).
18. Odontosoria chinensis
(L.) J.Sm.
Lindsaeaceae (TT 2066)
Syn. Trichomanes
chinensis L.
Used in the treatment of chronic enteritis (Nayar,
1959).
19. Nephrolepis hirsutula (G.
Forst.) C.Presl.
Oleandraceae (TT 2067)
Syn. Polypodium
hirsutulum G. Forst.
In Garhwal a decoction of fresh tubers and fronds
is given to cure cough (Shah and Singh, 1990).
Commonly cultivated in the botanical gardens as
ornamentals and as potted plants for indoor
decoration (Kholia and Punetha, 2005)
20. Dicranopteris linearis
var. linearis Holttum
Gleicheniaceae (TT
2068)
Syn. Polypodium
linearis Burm. f.
Young fronds with cow’s milk given in woman’s
sterility. The rhizomes are used as antihelmintic
(Vasudeva, 1999). Externally onto wounds
(Futscher, 1959) Used in traditional medicine of
Nicobarese tribes (Dagar, 1989)
21. Alsophila gigantea Wall.
ex Hook.
Cyatheaceae (TT 2069)
Syn Cyathea gigantea
(Wall. ex Hook.)
Holttum
Pith from the trunk is used as a food product
(Lawrence, 1951) Cyathea albo-setacea used in
traditional medicine of Nicobarese tribes (Dagar,
1989)
Antibacterial and Phytochemical Screening of Ferns 57
22. Alsophila spinulosa
(Wall. ex Hook.) R.M.
Tryon
Cyatheaceae (TT 2070)
Syn Cyathea spinulosa
Wall. ex Hook.
Ornamental plant (Vasudeva, 1999). Cyathea albo-
setacea used in traditional medicine of Nicobarese
tribes (Dagar, 1989)
23. Christella dentata
(Forssk.)Brownsey &
Jermy (TT 2071)
Thelypteridaceae
Syn. Polypodium
dentatum Forssk.
Used as cusion for cattle by Raji tribes (Pande and
Pangtey, 1987).
24. Christella parasitica (L.)
H. Lev.
Thelypteridaceae (TT
2072)
Syn. Polypodium
parasiticum L.
Used against wounds (Gogoi, 2002)
25. Cyclosorus interruptus
(Willd.) H.Ito
Thelypteridaceae (TT
2073)
Syn. Pteris interrupta
Willd.
Pteris ensiformis used as astringent in Manipur
(Singh et al., 2001)
Boiled fronds of Cyclosorus used against boils,
ulcers and wounds (Powell, 1976).
26. Macrothelypteris
torresiana (Gaud.)
Ching
Thelypteridaceae (TT
2074)
Syn. Polystichum
torresianum Gaud.
Cultivated as ornamentals in the botanical
gardens. As they generally spread by creeping
rhizomes (Vasudeva, 1999).
27. Asplenium formoum
Willd.
Aspleniaceae (TT 2075)
Genus Asplinium with antihelminthic, used
against Jaundice (Vasudeva, 1999).
58 Chapter 3
28. Diplazium esculentum
(Retz.) Sw.
Athyriaceae (TT 2076)
Syn. Hemionitis
esculenta Retz.
Young fronds are cooked and eaten by tribal
people for maintaining health (Kaushik and
Dhiman, 1995) Diplazium cochleata used against
epilepsy / Seizures (Dhiman, 1998)
29.
Dryopteris cochleata
(D.Don) C.Chr.
Dryopteridaceae (TT
2077)
Syn. Nephrodium
cochleatum D.Don.
A small portion of the rhizome of the plant is
powdered and taken with water twice a day in
rheumatism, epilepsy & leprosy (Shah and Singh,
1990). The whole plant is crushed in a bowl and
their extract is given twice a day orally, in case of
snake bite besides a paste of the plant is also applied
on the bite wound to prevent infection (Verma et al.,
1995). Rhizome used in swellings and pain and
antifungal properties (Asolkar et al., 1992).
30. Dryopteris hirtipes
(Blume) Kuntze
Dryopteridaceae (TT
2078)
Syn. Aspidium hirtipes
Blume
The juice obtained from the leaves is given in
epilepsy. And also used as an antibiotic.
(Vasudeva, 1999).
31. Tectaria coadunata
(Wall. ex Hook. & Grev.)
C.Chr.
Dryopteridaceae (TT
2079)
Syn. Aspidium
coadunatum Wall. ex
Hook. & Grev.
Tectaria wightii decoction of the rhizomes is given
to children, in stomach-ache in kumaon (Pande
et al., 1994).
32. Tectaria wightii (C.B.
Clarke) Ching
Dryopteridaceae (TT
2080)
Syn. Nephrodium
wightii C.B.Clarke
The decoction of the rhizomes is given to children,
in stomach-ache in Kumaon (Pande et al., 1994).
The plant used in asthma, bronchitis and in
getting relief from bites and stings by insects and
centipedes (Vyas and Sharma, 1988).Plants used
as antihelmintic (Kaushik and Dhiman, 1995)
Antibacterial and Phytochemical Screening of Ferns 59
33. Bolbitis x prolifera
(Bory) C.Chr.
Lomariopsidaceae (TT
2081)
Syn. Polybotrya
prolifera Bory in Bel.
General unspecified use of Moorea, French
Polynesia
Bolbitis lonchophora- Moorea (Nicole Baltrushes,
2006)
34. Blechnum orientale L.
Blechnaceae (TT 2082)
Syn. Blechnum orientale
var. grande B.K. Nair &
Geev.
The plants are used as poultice for boils by the
tribal people of central India. Rhizomes are used
as antihelminthic. Plants used as medicine for
diarrhoea and stomach disorders (Vasudeva,
1999).
35. Stenochlaena palustris
(Burm.f.)Bedd.
Syn. Polypodium
palustre Burm.f.
Blechnaceae (TT 2083)
Stenochlaena palustris are eaten as food (Powell,
1976). Stenochlaena palustris used in traditional
medicine of Nicobarese tribes (Dagar, 1989)
36. Drynaria quercifolia (L.)
J. Sm.
Polypodiaceae (TT
2084)
Syn. Polypodium
quercifolium L.
Rhizome used in treatment of typhoid, hectic fever
dyspepsia and cough and phthesis (Nayar, 1959).
Fronds used against swelling (May, 1978).
37. Microsorum punctatum
(L.) Copel.
Polypodiaceae (TT 2085)
Syn. Acrostichum
punctatum L.
Microsorum punctatum used in traditional
medicine of Nicobarese tribes (Dagar, 1989)
Microsorum grossum reported to be antiinflamatory
and antibacterial (Whistler, 1992)
38. Phymatosorus
nigrescens (Blume)
Pic.Serm.
Polypodiaceae (TT
2086)
Syn. Polypodium
nigrescens Blume
Used against nasal infections, the smoke of a
species of Polypodium (Powell, 1976).
60 Chapter 3
39. Pyrrosia heterophylla
(L.) M.G.Price.
Polypodiaceae (TT
2087)
Plants used in urinary calculus and rheumatism
(Caius, 1935).
40. Pyrrosia lanceolata (L.)
Farwell
Polypodiaceae (TT
2088)
Syn. Acrostichum
lanceolatum L.
Pyrrosia heterophylla used in urinary calculus and
rheumatism (Caius, 1935).
41. Pyrrosia porosa
(C.Presl) Hovenkamp
Polypodiaceae (TT
2089)
Syn. Niphobolus
porosus C.Pres
Pyrrosia heterophylla used in urinary calculus and
rheumatism (Caius, 1935).
42. Marsilea minuta L.
Masileaceae (TT 2090)
Syn. Marsilea crenulata
Desv.
Decoction of leaves mixed with ginger is given in
bronchitis and cough by the tribes (Vasudeva,
1999). Used to cure eye disease (Dhiman, 1998)
43. Salvinia molesta D.S.
Mitch.
Salviniaceae (TT 2091)
Weed (Mitchel, 1979)
44. Azolla pinnata R.Br.
Azollaceae (TT 2092)
Azolla filiculoides as a biofertiliser (Ripley et al.,
2003)
3.2.2 Preparation of plant extract
The air-dried plant material was ground and used for
preparing extracts. 20 grams of powdered plant material was
extracted with 80% ethanol by maceration and kept it for a
period of 24 hours at room temperature. The resultant extract
could be called as cold extract. The extracts were filtered using
Antibacterial and Phytochemical Screening of Ferns 61
Whatman No. I filter papers and each extract was concentrated
in a rotary evaporator to remove alcohol. Each cold extract was
dissolved in sterile distilled water to obtain a sample
concentration of 100 mg/ml (Cos et al., 2002).
3.2.3 Bacterial strains
The test organisms were collected from the culture collection
of the Institute of Microbial Technology (IMTECH) Chandigarh.
These include Micrococcus luteus (MTCC 6164), Escherichia coli
(MTCC 443), Staphylococcus aureus subsp. aureus (MTCC 96), and
Pseudomonas aeruginosa (MTCC 741) and Serratia marcescens
(MTCC 97) (referred to as MTCC strains henceforth). The bacteria
were sub-cultured on nutrient agar slants (Monica, 1985)
incubated at 37oC for 24 hours and stored at 4oC in the refrigerator
to maintain stock culture.
3.2.4 Maintenance of bacterial strains
Organisms were maintained by periodic subculture in
nutrient agar slants.
3.2.4.1 Nutrient agar slants
Medium and preparation
Beef extract 1 g
Yeast extract 2 g
Peptone 5 g
Sodium chloride 5 g
Agar 2 g
Water 1 L
62 Chapter 3
All the ingredients except agar were dissolved in distilled
water, gently heated, cooled and pH was adjusted to 7.4. Agar was
then added and dissolved by heating. The medium was then
dispensed in 5 ml quantities in test tubes. Sterilisation was done
by autoclaving at 121°C for 15 minutes (Monica, 1985). The
medium in the tubes was then allowed to solidify as slants.
3.2.5. The Inoculum
The nutrient broth cultures of the organisms, grown at 37°C
for approximately 3 to 4 hours were used as inocula.
3.2.6. Mueller-Hinton agar
Mueller-Hinton agar medium was employed for disc diffusion
sensitivity testing. Dehydrated medium supplied by Hi-Media
Laboratories Ltd., Bombay was used. The medium contained per
litre, infusion from 300 g beef, casein hydrolysate 17.5 g, starch
1.5 g, and agar 17 g (Monica, 1985). The medium was prepared by
dissolving the dehydrated mixture of ingredients in distilled water.
After boiling, pH was adjusted to 7.4 and sterilised by autoclaving
at 121°C for 15 minutes. The medium was poured in Petri plates.
3.2.7 In vitro antibacterial assay
The disc diffusion method as illustrated by Bauer et al.,
(1966) was used to determine the growth inhibition of bacteria by
plant extracts. Sterile liquid Mueller Hinton Agar media (pH 7.4 ±
2) was poured into sterile Petri dish, after solidification the
bacteria (1 ml bacterial broth of approximately105 cells) were
swabbed with a sterile needle under aseptic conditions.
Commercially available blank sterile discs (Hi Media Laboratories
Antibacterial and Phytochemical Screening of Ferns 63
Pvt. Ltd, Bombay) of 6 mm diameter were used in the study. Each
disc was impregnated with 20 µl containing about 1 mg of plant
extract. A disc with solvent blank (80% ethanol) was used as
control. The discs (including control) were used after drying them
in an incubator at 60°C to remove any trace of solvent. Discs were
introduced onto the surface of the medium. The plates were
incubated at 37oC for 24 hours to obtain inhibition zones.
Experiments were conducted in six replicates and average
inhibitory zone diameter was determined.
Antibacterial evaluation was conducted in two different
levels in the initial phase. Single ethanolic extract was analysed
for antibacterial activity against MTCC strains. In the second
phase, plants that showed greater level of antibacterial activity
were evaluated against twelve strains of pathogenic bacteria. The
extracts were prepared in different solvents of increasing polarity.
Plants that showed higher potential of antibacterial activity
in the first phase of screening were Osmunda regalis, Acrostichum
aureum, Lygodium flexuosum, Pteris quadri-aurita, Cheilanthes
bullosa, Cheilanthes tenuifolia, Adiantum latifolium, Lindsaea
ensifolia, Odontosoria chinensis, Dicranopteris linearis, Christella
dentata, Dryopteris cochleata, Blechnum orientale and Drynaria
quercifolia.
3.2.8 Plant extracts for second level of evaluation
The air-dried plant material was ground and used for
preparing extracts. About 50 g of powdered plant material was
successively extracted using petroleum ether (60-80oC) (relative
polarity 0.117), acetone (relative polarity 0.355), methanol
64 Chapter 3
(relative polarity 0.762) and water (relative polarity 1.000) for a
period of 24 hrs (Eloff, 1998). The extracts were filtered using
Whatman No. I filter papers and each extract was concentrated in
vacuum using rotary evaporator at 40oC to remove the last trace
of solvent. The solid material was dissolved in the same solvent to
obtain a sample concentration of 50 mg/ml.
3.2.9 Bacterial strains for the second level of evaluation
The test organisms were collected from the culture collection
of the Institute of Microbial Technology (IMTECH) Chandigarh,
India as described in section 3.2.3. The Sree Chitra Tirunal
Institute of Medical Sciences and Technology, Thiruvananthapuram
Kerala, India supplied some clinical isolates like Staphylococcus
albus, Salmonella typhi, Salmonella paratyphi, Citrobacter freundii;
Klebsiella pneumoniae, Shigella sonnei and Shigella dysentriae.
3.2.10 Antibacterial evaluation for the second level of evaluation
The disc diffusion method as illustrated by Bauer et al.,
(1966) as described in section 3.2.7 was used to determine the
growth inhibition of bacteria by plant extracts.
3.2.11 Preliminary detection of phytochemicals
Alkaloids were detected by Iodine Potassium iodide reagent
(Stahl, 1969), Dragendroff reagent (Thies and Reuther, 1954, Tyihak,
1964) and Margui reagent (Harborne, 1973). Presence of flavonoids
was detected by spraying with 25% aqueous solution of basic lead
acetate yielded fluorescence in long wave U.V light (Horhammer
et al., 1964). Spraying 1% aluminium chloride solution in ethanol
yielded yellow fluorescence in long wave U.V light (Gage et al., 1951)
indicated the presence of flavonoids. Phenols were detected by
Antibacterial and Phytochemical Screening of Ferns 65
spraying TLC plates with saturated aqueous silver nitrate solution;
light pink to deep green spots were yielded after spraying (Burke et
al., 1960). Undiluted Folin ciocalteu reagent was sprayed in order to
find out phenols (Keith et al., 1958). Stannic chloride was used to
detect titerpenes, sterols and steroids, phenol and poly phenols
(Scheideggeer and Cherbuliez, 1955). Anisaldehyde-sulphuric acid
reagent was used to detect terpenes, steroid and sugars (Stahl and
Kaltenbach, 1961).
3.3 Results and discussion
The activity was evaluated by measuring the inhibition zone
diameter around the disc and was recorded as follows: no obvious
growth inhibition (-); zone of inhibition with diameter 7 mm-10.99
mm (+); 11 mm-14.99 mm as ++ and zone of inhibition with
diameter 15-21 mm (+++). Inhibition zones were identified by
means of the disc diffusion method. The results are displayed in
Table 3.2. The results showed that some of the extracts like that of
Osmunda regalis and Lygodium flexuosum exhibited an
antibacterial effect towards all tested organisms. Therefore the
extracts were active against both gram-positive and gram-negative
bacteria. Plant extracts of Pteris quadri-auriata showed maximum
antibacterial activity i.e. greater than 20 mm. towards
Pseudomonas aeruginosa, a gram-negative bacterium (Table 3.2).
Pteris quadri-auriata showed moderate level of inhibition towards
Staphylococcus aureus, Escheichia coli and Micrococcus luteus. The
plant extract showed little sensitivity towards Serratia marcescens.
66 Chapter 3
Table 3.2 Antibacterial activity of ethanol extracts of ferns
The meaning of symbols used is as follows.
(-) = no obvious growth inhibition
(+) = zone of inhibition with diameter 7.00 -10.99 mm
[++] = zone of inhibition with diameter 11.00 -14.99 mm
(+++) = zone of inhibition with diameter 15.00-21.00 mm
Inhibition zone developed towards various bacterial strains
Family & Name of the plant
Part of the
plant used
MTC
C 9
6
MTC
C 4
43
MTC
C 7
41
MT
CC
616
4
MTC
C 9
7
Angiopteridaceae
Angiopteris evecta Fronds + + - - -
Osmundaceae
Osmunda regalis
Whole plant + + ++ ++ +
Schizaceae
Lygodium flexuosum
Whole plant + + ++ ++ +
Pteridaceae
Acrostichum aureum
Stem & Fronds ++ + ++ - -
Pteris quadri-aurita
Whole plant + + +++ + -
Antibacterial and Phytochemical Screening of Ferns 67
Sinopteridaceae
Cheilanthes bullosa
Whole plant - - ++ ++ +
Cheilanthes tenuifolia
Whole plant - ++ ++ - -
Doryopteris concolor
Whole plant - + + - -
Hemionitidaceae
Parahemionitis cordata
Whole plant - - + + -
Pitrogramma austroamericana
Whole plant + - + - +
Pitrogramma calomelanos
Whole plant + + + + -
Adiantaceae
Adiantum caudatum
Whole plant + - - + -
Adiantum latifolium
Whole plant + - ++ ++ -
Adiantum lunulatum
Whole plant + + + - -
Adiantum raddianum
Whole plant
+ - - - -
Dennstaedtiaceae
Microlepia speluncae
Whole plant + - - + -
68 Chapter 3
Lindsaeaceae
Lindsaea ensifolia
Whole plant + + +++ - +
Odontosoria chinensis
Stem & Fronds ++ ++ + + -
Oleandraceae
Nephrolepis hirsutula
Stem & Fronds + + - + ++
Gleicheniaceae
Dicranopteris linearis Rhizome ++ ++ + - +
Cyatheaceae
Alsophila gigantea Fronds + + - ++ -
Alsophila spinulosa Fronds + + - + -
Thelypteridaceae
Christella dentata
Whole plant
- - +++ ++ -
Christella parasitica Fronds - - + - -
Cyclosorus interruptus Fronds - - + - -
Macrothelypteris torresiana
Fronds + + + + -
Aspleniaceae
Asplenium formoum
Whole plant - + + + +
Antibacterial and Phytochemical Screening of Ferns 69
Athyriaceae
Diplazium esculentum
Whole plant - - + + -
Dryopteridaceae
Dryopteris cochleata Whole
plant ++ + ++ + -
Dryopteris hirtipes Fronds + - - - -
Tectaria coadunata
Whole plant - - + - -
Tectaria wightii
Stem & Fronds + - + + -
Lomariopsidaceae
Bolbitis prolifera
Stem &
Fronds - + + + -
Blechnaceae
Blechnum orientale Rhizome - ++ + ++ +
Stenochlaena palustris
Whole plant
- + + - +
Polypodiaceae
Drynaria quercifolia Rhizome +++ + ++ ++ +
Microsorum punctatum
Whole plant - + + + ++
Phymatosorus nigrescens
Whole plant - + + + +
Pyrrosia heterophylla
Whole plant + + + + -
Pyrrosia lanceolata
Whole plant - - - - -
70 Chapter 3
Pyrrosia porosa
Whole plant - - - - -
Masileaceae
Marsilea minuta
Whole plant + + + + -
Salviniaceae
Salvinia molesta
Whole plant - - - - -
Azollaceae
Azolla pinnata
Whole plant - - - - -
Plants exhibited greater level of antibacterial activity were
Osmunda regalis, Lygodium flexuosum, Acrostichum aureum, Pteris
quadri-aurita, Cheilanthes bullosa, Cheilanthes tenuifolia, Adiantum
latifolium, Lindsaea ensifolia, Odontosoria chinensis, Dicranopteris
linearis, Christella dentata, Dryopteris cochleata, Blechnum orientale
and Drynaria quercifolia. Pseudomonas aeruginosa was found to be
the most sensitive organism to various extracts of ferns. Serratia
marcescens did not show any sensitivity to plant extracts.
The results of second level of screening of the plants for
antibacterial activities against various strains can be summarised
into following observations and assumptions.
Acetone and methanol extracts of most of the plants were more
active than petroleum ether and water extracts.
No antibacterial activity was recorded in the case of water
extracts of all plants. Water extraction was done at the end of
Antibacterial and Phytochemical Screening of Ferns 71
successive extractions and there was little possibility of finding
out active components in water extracts.
Petroleum ether extracts were less effective compared to
acetone and methanol extracts.
There is a general tendency that some medium-polar
compounds are involved in antibacterial activity.
Most of the extracts were active against Micrococcus luteus and
staphylococcus aureus.
Blechnum orientale L. showed broad range of antibacterial
activity against nine bacterial strains including gram-positive
and gram negative.
Maximum antibacterial activity was recorded for acetone
extracts derived from the rhizome extracts of Drynaria
quercifolia Dicranopteris linearis and Blechnum orientale.
The results of preliminary screening studies prompted us to
select Drynaria quercifolia for further studies.
Preliminary phytochemical tests demonstrated that the
occurrence of flavonoids, phenols and terpenes were more or less a
common feature of all plant extracts. However, plants differ
significantly in their activity against gram-positive and gram-
negative microorganisms. These differences may be attributed to the
fact that the cell wall in gram-positive bacteria consists of a single
72 Chapter 3
layer, whereas the gram-negative cell wall is a multi-layered
structure and quite complex (Madigan et al., 2000).
Experimental result of antibacterial activity of plant extracts
could be correlated with the ethnobotanical relevance of various
plants cited in Table 3.1. The presence of phenolics and flavonoids
was a general feature of different plant extracts (Table 3.3). While
alkaloid a more evolved compound generally observed in
Angiosperms was not observed as a common feature of Ferns.
Alkaloids are said to be more evolved in the sense that they are well
observed in highly evolved plant group, angiosperms. A few plants
like Microlepia speluncae, Nephrolepis hirsutula and Microsorum
punctatum showed the presence of alkaloids. Plants that showed
greater level of antibacterial activity exhibited the presence of both
flavonoid and terpenes. Both flavonoids and terpenes have
antibacterial properties (Vasange et al., 1997, Ramesh et al., 2001,
Shokeen et al., 2005). Since the medicinal plants studied appear to
have a broad antimicrobial activity spectrum, they could be useful in
antiseptic and disinfectant formulations as well as in chemotherapy
(Olukoya et al., 1993). Dicranopteris linearis is used traditionally
against wound infection (Futscher, 1959), the antibacterial potential
of the plant is confirmed by the present result especially towards
Staphylococcus aureus.
The antibacterial activity of Lindsaea ensifolia supported by
its ethnobotanical potential for chronic enteritis (Fosberg, 1942). The
optimal effectiveness of a medicinal plant may not be due to one
main active constituent, but to the combined action of different
compounds originallyin the plant (Bai, 1990). From this study we
conclude that promising plants exhibited broad spectrum of activity
against gram-positive and gram-negative bacteria and the results
Antibacterial and Phytochemical Screening of Ferns 73
confirm the use of these plants in traditional medicine for the
treatment of infections.
Table 3.3 Phytochemical contents of ethanolic extract of ferns
studied
The symbol [+] indicates present; and [-] indicates absence
Phytochemicals (+ present; - absent) Family and name of the
plant Alkaloids Flavonoids Terpenes Phenols
Angiopteridaceae
Angiopteris evecta - + + +
Osmundaceae
Osmunda regalis - + + +
Schizaceae
Lygodium flexuosum - + + +
Pteridaceae
Acrostichum aureum - + - +
Pteris quadri-aurita - + + +
Sinopteridaceae
Cheilanthes bullosa - - + +
Cheilanthes tenuifolia - + + +
Doryopteris concolor - + + +
74 Chapter 3
Hemionitidaceae
Parahemionitis cordata - + + +
Pitrogramma austroamericana
- + + +
Pitrogramma calomelanos - - - +
Adiantaceae
Adiantum lunulatum - + - +
Adiantum caudatum - + + +
Adiantum latifolium - + - +
Adiantum raddianum - + - +
Dennstaedtiaceae
Microlepia speluncae + + + +
Lindsaeaceae
Lindsaea ensifolia - + + +
Odontosoria chinensis - + + +
Oleandraceae
Nephrolepis hirsutula + + - +
Gleicheniaceae
Dicranopteris linearis - + + +
Antibacterial and Phytochemical Screening of Ferns 75
Cyatheaceae
Alsophila spinulosa - + + +
Alsophila gigantea - + + +
Thelypteridaceae
Christella dentata - + + +
Christella parasitica - + + +
Cyclosorus interruptus - + + +
Macrothelypteris torresiana - + + +
Aspleniaceae
Asplenium formoum - + + +
Athyriaceae
Diplazium esculentum - + + +
Dryopteridaceae
Dryopteris cochleata - + + +
Dryopteris hirtipes - + + +
Tectaria coadunata - + - +
Tectaria wightii - + - +
76 Chapter 3
Lomariopsidaceae
Bolbitis prolifera - + - +
Blechnaceae
Blechnum orientale - + + +
Stenochlaena palustris - + + +
Polypodiaceae
Drynaria quercifolia - + + +
Microsorum punctatum + + + +
Phymatosorus nigrescens - + + +
Pyrrosia heterophylla - - - +
Pyrrosia lanceolata - + + +
Masileaceae
Marsilea minuta - + + +
Salviniaceae
Salvinia molesta - - + +
Azollaceae
Azolla pinnata - - + +
77
Table 3.4 Detailed antibacterial activity of plants showed greater potential against pathogenic strains of bacteria.
The meaning of symbols used is as follows. (-) = no obvious growth inhibition; (+) = zone of inhibition with diameter 7.00-10.99 mm; (++) = 11.00-14.99 mm; (+++) = zone of inhibition with diameter 15.00-21.00 mm Abbreviations: PE - Petroleum ether Extract; AE - Acetone Extract; ME - Methanol Extract WE - Water Extract. S.al - Staphylococcus albus; S.au - Staphylococcus aureus; M.lu – Micrococcus luteus; S.ma - Serratia marcescens; S.ty - Salmonella typhi; S.pt - Salmonella paratyphi; C.fe - Citrobacter freundii; K.pn - Klebsiella pneumoniae; S.so - Shigella sonnei; S.dy - Shigella dysentriae; E. co - Escherichia coli; P.ae - Pseudomonas aeruginosa
Sl NO
Nam
e of
th
e pl
ant
and
Fam
ily
Part
u
sed
Ext
ra c
t u
sed
S.al
S.au
M.lu
S.m
a
S.ty
S.pt
C.f
r
K.p
n
S.so
S.dy
E.c
o
P.ae
PE - - - - - - - - - - - - AE + + - + - - - - - - - + ME - - - + - - + - - - - -
1. Osmunda regalis Osmundaceae
Whole plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - ++ ++ + - - + - - - - - ME - - - - - - - - - - - -
2. Lygodium flexuosum Schizaceae
Whole plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + - - - - - - + - - - ME - ++ ++ - - - + - - - - -
3. Acrostichum aureum Pteridaceae
Stem & Fronds
WA - - - - - - - - - - - -
Antibacterial and Phytochemical Screening of Ferns
77
PE - - - - - - - - - - - - AE - - + + - - + - - - + + ME + + + - + - - - + - ++ -
4. Pteris quadri-aurita Pteridaceae
Whole plant
WA - - - - - - - - - - - - PE + - - - - - - - - - - - AE - + - + + - - + + - + + ME + - + - - - - - - - - -
5. Cheilanthes bullosa Sinopteridaceae
Whole plant
WA - - - + - - - - - - - - PE - - - - - - - - - - - - AE + - - - + - + - - - - - ME + - - + - - - + - - + -
6. Cheilanthes tenuifolia Sinopteridaceae
Whole plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + - - - + - + + - - + ME + - + - - - - - - - - ++
7. Adiantum latifolium Adiantaceae
Whole plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - ++ - - - - - - - - - - ME + - ++ - - - - - - - - +
8. Lindsaea ensifolia Lindsaeaceae
Whole plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE + - ++ - - - - - - - + - ME - + - + - + - + - - + -
9. Odontosoria chinensis Lindsaeaceae
Stem & Fronds
WA - - - - - - - - - - - -
78
Chapter 3
79
PE - - - - - - - - - - - - AE - ++ + + - - ++ - - + - + ME + - - - - - - - + - + + 10.
Dicranopteris linearis Gleicheniaceae
Rhizome
WA - - - - - - - - - - - -
PE - - - - - - - - - - - AE + - + - - - + - - - + - ME - + - - - - - - + - + -
11.
Christella dentata Thelypteridaceae
Whole Plant
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE + - + - - - - - - - - - ME - + - - - - - - - - - -
12. Dryopteris cochleata Dryopteridaceae
Stem Fronds
WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + + - + - ++ - - - + ++ ME + - + - - - - + - + + -
13. Blechnum orientale Blechnaceae
Rhizome
WA - - - - - - - - - - - - PE - - + - - + ++ - - + - - AE - ++ + + - + ++ - - ++ - ++ ME - + - - + - + - - + - +
14.
Drynaria quercifolia Polypodiaceae
Rhizome
WA - - - - - - - - - - - -
Antibacterial and Phytochemical Screening of Ferns
79