Chapter 3

Antibacterial and Phytochemical Screening of Ferns

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.

Botanical Name and N0 Family (Voucher Specimen No) 1. Angiopteris evecta (G. Forst.) Hoffm. Angiopteridaceae (TT 2049) 2. Osmunda regalis L. Osmundaceae (TT 2050) 3. Lygodium Flexuosum (L.) Sw. Schizaceae (TT 2051) Syn. Ophioglossum fluxuosum L. 4. Acrostichum aureum L. Pteridaceae (TT 2052)

Medicinal uses of plants/their genus

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) Cultivated as ornamental and local tribals of Madhya Pradesh, India utilised its first frond of the year to cure toothache (Vasudeva, 1999). 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). 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 1956). in chronic disorders arising from obstructions of viscera and spleen. (Chopra et al.,

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 2030 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 1956). in chronic disorders arising from obstructions of viscera and spleen. (Chopra et al.,

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 austroamericana Domin Hemionitidaceae (TT 2058) Syn. Pityrogramma calomelanos var. aureoflava (Hook.) Weath. ex Baily The Golden fern as cultivated in the plants due botanical to their

gardens

ornamental

splendid beauty and grace (Vasudeva, 1999).

Antibacterial and Phytochemical Screening of Ferns

11. Pityrogramma calomelanos (L.) Link Hemionitidaceae (TT 2059) Syn. Acrostichum calomelanos L. 12. Adiantum caudatum L. Used in cough and fever (Vasudeva, 1999). Adiantaceae (TT 2060) 13. Adiantum latifolium Lam. Adiantaceae (TT 2061) Syn. Adiantum denticulatum Sw. 14. Adiantum lunulatum Burm.f. Adiantaceae (TT 2062) Syn. Adiantum philippense L.

55

The decoction of rhizomes used as an anthelmintic (Vasudeva, 1999). Smoke the leaf for colds in head and chests (Caius, 1935).

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. 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) Young fronds with cows milk given in womans sterility. The rhizomes are used as antihelmintic (Vasudeva, 1999). Externally onto wounds (Futscher, 1959) Used in traditional medicine of Nicobarese tribes (Dagar, 1989)

20.

Dicranopteris linearis var. linearis Holttum Gleicheniaceae (TT 2068) Syn. Polypodium linearis Burm. f.

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

22. Alsophila spinulosa (Wall. ex Hook.) R.M. Tryon Cyatheaceae (TT 2070) Syn Cyathea spinulosa Wall. ex Hook. 23. Christella dentata (Forssk.)Brownsey & Jermy (TT 2071) Thelypteridaceae Syn. Polypodium dentatum Forssk. 24. Christella parasitica (L.) H. Lev. Thelypteridaceae (TT 2072) Syn. Polypodium parasiticum L. 25. Cyclosorus interruptus (Willd.) H.Ito Thelypteridaceae (TT 2073) Syn. Pteris interrupta Willd. 26. Macrothelypteris torresiana (Gaud.) Ching Thelypteridaceae (TT 2074) Syn. Polystichum torresianum Gaud. 27. Asplenium formoum Willd. Aspleniaceae (TT 2075) Genus Asplinium with antihelminthic, Cultivated as ornamentals in the Used against wounds (Gogoi, 2002)

57

Ornamental plant (Vasudeva, 1999). Cyathea albosetacea used in traditional medicine of Nicobarese tribes (Dagar, 1989)

Used as cusion for cattle by Raji tribes (Pande and Pangtey, 1987).

Pteris ensiformis used as astringent in Manipur (Singh et al., 2001) Boiled fronds of Cyclosorus used against boils,

ulcers and wounds (Powell, 1976).

botanical

gardens. As they generally spread by creeping rhizomes (Vasudeva, 1999).

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) A small portion of the rhizome of the plant is

29. Dryopteris cochleata (D.Don) C.Chr. Dryopteridaceae (TT 2077) Syn. Nephrodium cochleatum D.Don.

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

33. Bolbitis x prolifera (Bory) C.Chr. Lomariopsidaceae (TT 2081) Syn. Polybotrya prolifera Bory in Bel. 34. Blechnum orientale L. Blechnaceae (TT 2082) Syn. Blechnum orientale var. grande B.K. Nair & Geev. 35. Stenochlaena palustris (Burm.f.)Bedd. Syn. Polypodium palustre Burm.f. Blechnaceae (TT 2083) 36. Drynaria quercifolia (L.) J. Sm. Polypodiaceae (TT 2084) Syn. Polypodium quercifolium L. 37. Microsorum punctatum (L.) Copel. Polypodiaceae (TT 2085) Syn. Acrostichum punctatum L. 38. Phymatosorus nigrescens (Blume) Pic.Serm. Polypodiaceae (TT 2086) Syn. Polypodium nigrescens Blume Microsorum medicine of punctatum Nicobarese used tribes in General Polynesia unspecified use of Moorea,

59

French

Bolbitis lonchophora- Moorea (Nicole Baltrushes, 2006)

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 1999). and stomach disorders (Vasudeva,

Stenochlaena palustris are eaten as food (Powell, 1976). Stenochlaena palustris used in traditional medicine of Nicobarese tribes (Dagar, 1989)

Rhizome used in treatment of typhoid, hectic fever dyspepsia and cough and phthesis (Nayar, 1959). Fronds used against swelling (May, 1978).

traditional 1989)

(Dagar,

Microsorum grossum reported to be antiinflamatory and antibacterial (Whistler, 1992)

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 Yeast extract Peptone Sodium chloride Agar Water 1g 2g 5g 5g 2g 1L

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 121C 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 37C 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 121C 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 l 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 60C 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 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 cochleata, Blechnum orientale and Drynaria

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

MTCC 6164 MTCC 443 MTCC 741 MTCC 96 MTCC 97

Family & Name of the plant

Part of the plant used

Angiopteridaceae Angiopteris evecta Osmundaceae Osmunda regalis Schizaceae Lygodium flexuosum Pteridaceae Acrostichum aureum Pteris quadriaurita Stem & Fronds Whole plant ++ + ++ Whole plant + + ++ ++ + Whole plant + + ++ ++ + Fronds + + -

+++

Antibacterial and Phytochemical Screening of Ferns

Sinopteridaceae Cheilanthes bullosa Cheilanthes tenuifolia Doryopteris concolor Hemionitidaceae Parahemioniti s cordata Pitrogramma austroameric ana Pitrogramma calomelanos Adiantaceae Adiantum caudatum Adiantum latifolium Adiantum lunulatum Adiantum raddianum Dennstaedtiaceae Microlepia speluncae Whole plant + + Whole plant Whole plant Whole plant Whole plant + + Whole plant Whole plant Whole plant + + Whole plant Whole plant Whole plant ++ ++ +

67

++

++

++

++

+ +

+ -

+ -

68

Chapter 3

Lindsaeaceae Lindsaea ensifolia Odontosoria chinensis Oleandraceae Nephrolepis hirsutula Gleicheniaceae Dicranopteris linearis Cyatheaceae Alsophila gigantea Alsophila spinulosa Thelypteridaceae Christella dentata Christella parasitica Cyclosorus interruptus Macrothelypt eris torresiana Aspleniaceae Asplenium formoum Whole plant + + + + Whole plant Fronds +++ ++ Fronds + + ++ Rhizome ++ ++ + + Stem & Fronds + + + ++ Whole plant Stem & Fronds + + +++ +

++

++

Fronds

Fronds

Fronds

Antibacterial and Phytochemical Screening of Ferns

Athyriaceae Diplazium esculentum Dryopteridaceae Dryopteris cochleata Whole plant Whole plant + + -

69

++

++

Dryopteris hirtipes Tectaria coadunata Tectaria wightii Lomariopsidaceae Bolbitis prolifera

Fronds Whole plant Stem & Fronds

Stem & Fronds + + + -

Blechnaceae Blechnum orientale Stenochlaena palustris Polypodiaceae Drynaria quercifolia Microsorum punctatum Phymatosoru s nigrescens Pyrrosia heterophylla Pyrrosia lanceolata Rhizome Whole plant Whole plant Whole plant Whole plant +++ + ++ ++ + Rhizome Whole plant ++ + + + ++ + +

++

70

Chapter 3

Pyrrosia porosa Masileaceae Marsilea minuta Salviniaceae Salvinia molesta Azollaceae Azolla pinnata

Whole plant

Whole plant

Whole plant

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 gramnegative 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
Family and name of the plant Angiopteridaceae Angiopteris evecta Osmundaceae Osmunda regalis Schizaceae Lygodium flexuosum Pteridaceae Acrostichum aureum Pteris quadriaurita Sinopteridaceae Cheilanthes bullosa Cheilanthes tenuifolia Doryopteris concolor + + + + + + + + + + + + + Phytochemicals
Alkaloids Flavonoids

(+ present; - absent)
Terpenes Phenols

74

Chapter 3

Hemionitidaceae Parahemioniti s cordata Pitrogramma austroameric ana 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

Cyatheaceae Alsophila spinulosa Alsophila gigantea Thelypteridaceae Christella dentata Christella parasitica Cyclosorus interruptus Macrothelypteri s torresiana Aspleniaceae Asplenium formoum Athyriaceae Diplazium esculentum Dryopteridaceae Dryopteris cochleata Dryopteris hirtipes Tectaria coadunata Tectaria wightii + + + + + + + + + + + + + + +

75

+ +

+ +

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

Antibacterial and Phytochemical Screening of Ferns

Name of the plant and Family

Extra ct used

S.ma

Part used

Sl NO

K.pn

M.lu

E.co

S.dy

S.au

1.

Osmunda regalis Osmundaceae Lygodium flexuosum Schizaceae Acrostichum aureum Pteridaceae

Whole plant

2. 3.

Whole plant

Stem & Fronds

PE AE ME WA PE AE ME WA PE AE ME WA

+ -

+ ++ + ++ -

++ ++ -

+ + + -

+ + + -

+ -

P.ae + -

S.so

S.pt

S.ty

C.fr

S.al

77

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4.

Pteris quadriaurita Pteridaceae Cheilanthes bullosa Sinopteridaceae Cheilanthes tenuifolia Sinopteridaceae Adiantum latifolium Adiantaceae Lindsaea ensifolia Lindsaeaceae Odontosoria chinensis Lindsaeaceae

Whole plant

5.

Whole plant

6.

Whole plant

7.

Whole plant

8. 9.

Whole plant

Stem & Fronds

PE AE ME WA PE AE ME WA PE AE ME WA PE AE ME WA PE AE ME WA PE AE ME WA

+ + + + + + + + -

+ + + ++ + -

+ + + + ++ ++ -

+ + + + + -

+ + + -

+ + -

+ + -

+ + + + -

+ + + -

+ ++ + + + + -

+ + + ++ + -

Chapter 3

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Antibacterial and Phytochemical Screening of Ferns

10.

Dicranopteris linearis Gleicheniaceae

Rhizome

PE AE ME WA PE AE ME WA PE AE ME WA PE AE ME WA PE AE ME WA

+ + + + -

++ + + + ++ + -

+ + + + + + + -

+ + -

+ + -

+ + -

++ + ++ ++ ++ + -

+ -

+ + -

+ + + ++ + -

+ + + + + -

+ + ++ ++ + -

11.

Christella dentata Thelypteridaceae Dryopteris cochleata Dryopteridaceae Blechnum orientale Blechnaceae Drynaria quercifolia Polypodiaceae

Whole Plant

12.

Stem Fronds

13.

Rhizome

14.

Rhizome

79