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CHAPTER 8

PRELIMINARY PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL

ACTIVITY OF SOME SELECTED MACROLICHENS

8.1 Introduction

Lichens are one of the well-known prolific sources and biologically active natural

products (Chauhan and Abraham, 2013). Lichens, therefore, has been used

traditionally in various fields such as perfumes, dyeing industry, as food supplements

(Llano, 1948) and especially as a source of natural drugs in pharmaceutical industry

(Müller, 2001; Ingolfsdottir, 2002; Choudhary et al., 2005; Stocker-Wörgötter, 2005).

Lichens produce a great number of primary (intracellular) as well as unique secondary

(extracellular) metabolites. The metabolites produced by lichens are often restricted to

a specific area in the thallus of the lichens (Feige and Lumbsch, 1995; Nybakken and

Gauslaa, 2007).

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Few primary metabolites that are essential for the lichen metabolism and structural

growth are amino acids, protein, vitamins and polysaccharides. Amongst the primary

metabolites, some few are either produced by the mycobiont or the photobiont whilst

other by the synergistic action of both the algal and the fungal partners. Almost all the

intracellular products isolated from lichens are not exceptional as they also occur in

free-living fungi, algae and higher plants (Hale, 1983). The secondary metabolites

which are exclusively present in the lichens are produced by none other than the

mycobionts (Elix, 1996). The secondary metabolites are found accumulated as

extracellular tiny crystals or as amorphous substances on the outer surface of the

hyphael cell wall. Barbatic acid, orcinol, β-orcinol, parietin, protolichesterinic acid,

salazinic acid, usnic acid and vulpinic acid are few common known secondary

metabolites produced by the fungal partner of the lichen.

The phenolic compounds, anthraquinones, dibenzofurans, depsides, depsidones and

depsones produced by the lichens have a wide range of being used in medicines since

time immemorial. Lichen species were said to have been used in various traditional

systems of medicines like the Traditional Chinese Medicine (TCM), Homeopathic and

Western Medical Herbals for effectively curing dyspepsia, bleeding piles, bronchitis,

scabies, stomach disorders and many disorders of blood and heart (Saklani and Upreti,

1992; Lal and Upreti, 1995; Negi and Kareem, 1996) and is still holding significant

attention as substitute for treatment of various diseases in various parts of the world.

In India too, lichen have been a household item since ancient times as medicines

(Traditional Indian Medicine, TIM) and in various cultural events. „Shipal' in

Atharveda (1500 B.C) was the first record of using lichens as medicine. The lichens

under the vernacular name „Charila' was generally applied to Parmotrema chinense,

P. perforatum and Everniastrum cirrhatum and was widely used in Ayurveda, an

ancient system of medicine in India (Kumar and Upreti, 2001). Besides, there were

records of using lichens in the preparation of indigenous perfume named “Otto” for

the past 800 years and also as a major ingredient of common condiments.

Lichen secondary metabolites can also function as allelopathic agents and affect the

development and growth of neighbouring lichens, fungi, mosses and vascular plants,

as well as other microorganisms (Lawrey, 1984, 1986; Macías et al., 2007).

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Studies revealed that the multitude utility of lichens as antibiotic, antibacterial,

antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic

properties is due to a range of secondary compounds produced by them (Boustie and

Grube, 2007). Lichens have around 1050 secondary compounds (Stocker-Wörgötter,

2008), of which 550 are unique and have been characterized. The dry weight of the

thallus amount between 0.1 and 10% or sometimes reaches up to 30% (Galun, 1988).

Dayan and Romagni, (2001) and Abdullah et al., (2007) reported that lichens develop

diverse biosynthetic pathways to produce such complex arrays of secondary

metabolites and further depicted that the polyketide biosynthetic pathway was

responsible for most of the classes of lichen compounds, whereas pulvinic acids are

shikimate derivatives, and the large quantity of di- and triterpenoids present in lichens

are formed via the mevalonate pathway.

Lichens have fascinated great attention to large number of investigators as new

sources of bioactive substances due to significant or marked antimicrobial activities

(Mitscher et al., 1987). Usnic acid and the thallus of lichens with usnic acid as

secondary metabolites are the most frequently investigated lichens for its

pharmaceutical application. Burkholder et al., (1944) reported that the antibacterial

properties of lichen extracts have been known for many years. This report has led to

an exponential increased in studies on identification of the antibiotic components of

lichens. Studies also revealed that the antibiotic effects of many lichens were found to

be significantly active in gram-positive bacteria but not much significant in gram

negative bacteria. However, only few lichen substances have been screened in detail

for their biological activity and therapeutic potential, due to extreme difficulties in

obtaining lichens in large quantities and purity so that it would be sufficient for

structural elucidation and pharmacological testing.

The growing population of drug-resistant microorganisms and the problem of treating

infectious diseases which are continuously emerging and re-emerging at a fast rate

have motivated for search for novel bioactive secondary metabolites as an alternative

antimicrobial drugs. With this aim, preliminary phytochemical screening and

antibacterial activity of five most abundant macrolichens (Cladonia verticillata

(Hoffm.) Shaer., Parmotrema austrosinense (Zahlbr.) Hale., Parmotrema tinctorum

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(Despr. ex Nyl.) Hale., Phaeophyscia hispidula (Ach.) Moberg and Ramalina

conduplicans (Vain.) from this region was evaluated and documented in this Chapter.

8.2 Methodology

8.2.1 Identification of the lichen sample

Five macrolichens viz C. verticillata (Hoffm.) Shaer., P. austrosinense (Zahlbr.) Hale.,

P. tinctorum (Despr. ex Nyl.) Hale., P. hispidula (Ach.) Moberg and R. conduplicans

(Vain.) collected from different regions of central part of Manipur were characterized

for studying the bioprospective capabilities. The morphological and anatomical details

of the specimens were studied as mentioned in Chapter 3. The specimens were then

identified by comparing the morphometric and biochemical test results with the

literature and identification keys provided by Awasthi (2007) and Divakar and Upreti

(2005). Representative specimens for the five identified species were kept in the

herbarium of Phycology and Lichenogical laboratory of Department of Ecology and

Environment, Assam University, Silchar (AUS).

8.2.2 Identification of the lichen acid

Lichen acid or the secondary metabolites of were identified by dissolving the

powdered lichen samples in acetone (10 mg/ml). The lichen extracts were then spotted

on silica gel thin layer chromatography (TLC) plates (silica gel 60 F254 aluminium

plates, Merck). The chromatograms were developed in solvent system A and treated

as mentioned in Chapter 3. The Rf values for each spot were calculated and compared

with the standard (Walker and James, 1980).

8.2.3 Phytochemical screening

Phytochemical analysis of 70% methanol extract of the lichen samples was carried out

using standard qualitative methods as described previously by Harborne (1995) and

Kokate et al., (1995). The components analysed for qualitative phytochemicals were

alkaloids, anthraquinones, cardiac glycosides, flavonoids, phenols, tannins, terpenoids,

saponins and steroids. The detail assay for qualitative phytochemical screening was

highlighted in Chapter 3.

Plate 8.1.The five macrolichen test samples for preliminary phytochemical screening

and antibacterial avtivity: (A) Cladonia verticillata (Hoffm.) Shaer., (B) Parmotrema

austrosinense (Zahlbr.) Hale, (C) Parmotrema tinctorum (Despr. ex Nyl.) Hale (D),

Phaeophyscia hispidula (Ach.) Moberg. (E) Ramalina conduplicans Vain.

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8.2.4 Antibacterial activity

The antibacterial activity of the crude lichen extracts against two test bacteria

Staphylococcus aureus, and Escherichia coli was determined using paper disk

diffusion assay (Kirby et al 1957; Bauer et al 1959, 1966; Larkin 1982; National

Committee for Clinical Laboratory Standards, 1993). Bacterial cell suspension was

procured at the Bacteriological Laboratory, Department of Microbiology, Assam

University, Silchar. The bacterial strains were inoculated and zone of inhibition for all

acetone and methanol extracts were determined as per assay mentioned in Chapter 3.

Bioactivity of the lichen substances were assessed using the following rating system

of Quinto and Santos (2005) after deducting the inhibition zone of the control: (1)

very active (> 19 mm zone of inhibition); (2) active (13-19 mm); (3) partially active

(10-12 mm); (4) inactive (<10 mm).

8.3 Results and Discussion

8.3.1 Phytochemical screening

Preliminary phytochemical analysis and thin layer chromatography (TLC) were

employed to detect various secondary metabolites in the lichen extracts. Five

macrolichens viz. Cladonia verticillata (Hoffm.) Shaer., Parmotrema austrosinense

(Zahlbr.) Hale, Parmotrema tinctorum (Despr. ex Nyl.) Hale, Phaeophyscia hispidula

(Ach.) Moberg. and Ramalina conduplicans (Vain.) were subjected for preliminary

qualitative phytochemical screening.

The preliminary phytochemical analysis (Table 8.1) and TLC (Table 8.2) revealed the

presence of fumarprotocetraric and protocetraric acid with a tinge of tannins,

terpenoids and saponins in C. verticillata, a dimorphic terricolous (soil) lichen. P.

tinctorum, a common corticolous lichen showed the presence of atranorin and

lecanoric acid in TLC while high level of alkaloids with moderate terpenoids and

cardiac glycosides was detected in phytochemical screening of P. tinctorum

(Plate 8.1). P. hispidula, pollution tolerant corticolous lichen showed a tinge of

flavanoid and saponin however no lichen acid was detected in TLC.

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Table 8.1 Phytochemicals constituents of C. verticillata, P. tinctorum, P. hispidula

and R. conduplicans

Phytochemical Species

C.

verticillata

P.

austrosinense

P.

tinctorum

P.

hispidula

R.

conduplicans

Alkaloids ‒ ‒ + ‒ +

Anthraquinones ‒ ‒ ‒ ‒ ‒

Cardiac glycosides ‒ + + ‒ +

Flavonoids ‒ ‒ ‒ ‒ ‒

Tannins + ‒ + ‒ +

Terpenoids + ‒ + ‒ +

Saponins + ‒ + + +

Steroids ‒ ‒ ‒ ‒ ‒

The preliminary phytochemical analysis of R. conduplicans, a bark inhibiting

(corticolous) fruticose lichen revealed the presence of strain of usnic acid, salazinic

acid whereas sekikaic acid that aggregates with phytochemicals like terpenoids,

tannin, cardiac glycosides and steroids was detected in TLC.

Table 8.2 Secondary metabolites detected in Thin Layer Chromatography (TLC)

Metabolites

Species

C.

verticillata

P.

austrosinense

P.

tinctorum

P.

hispidula

R.

conduplicans

Atranorin — + + — —

Fumarprotocetraric + — — — —

Lecanoric acid + + — —

Protocetraric acid + — — — —

Sekikaic acid — — — — +

Salazinic acid — — — — +

Usnic acid — — — — +

8.3.2 Antibacterial activity

The five macrolichens subjected for phytochemicals were again used for analysis of

therapeutic potential. In vitro antibacterial activity of the five macrolichens were

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checked using two test bacterial strains Staphylococcus aureus and Escherichia coli

using paper disk diffusion assay. The results were recorded as presence or absence of

zones of inhibition around the well. The inhibitory zone around the well indicated the

absence of growth and is reported as positive and absence of inhibition zone i.e.

presence of growth as negative (Table 8.3).

The level of the antimicrobial activity of the same lichen species may differ depending

on the solvent used in extraction. Yilmaz et al., (2004) reported that solvents with

medium polarity such as acetone and diethyl ether were the proper solvents for the

extraction of substances as the amount of residues is less when compared to solvents

with high polarity like ethanol, methanol and with very low polarity like petroleum

ether.

Table 8.3 Antibacterial property of the five macrolichens samples

*values are in arithmetic mean ± standard deviation (n=3)

Two solvent viz. acetone and methanol were used for extraction of the lichen sample

for the preliminary test. In vitro antibacterial activity of the acetone extracts of the five

macrolichens were checked using the two test bacteria, S. aureus (gram positive) and

E. coli (gram negative). Acetone extract was used as recommended by Huneck and

Yoshimura (1996) who stated that acetone was one of the most suitable solvent for

extraction of the lichen acid as most of the lichen substances were soluble in this

solvent. But not even a single species showed positive response against both the tested

bacteria. These may be due to the reason that the secondary metabolites present in the

five macrolichens selected for the study failed to express well in acetone extract or

Species Acetone Methanol

S. aureus E. coli S. aureus E. coli

C. verticillata — — — —

P. austrosinense — — — —

P. tinctorum — — 13 ± 0.14 —

P. hispidula — — — —

R. conduplicans — — 15 ± 0.14 —

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may be due to the fact that the acetone extract of the five lichens do not affect on the

growth of the two tested bacteria.

In vitro antibacterial potential of methanolic extracts of the same five macrolichens

were again tested using the same two test bacteria. Of the five methanolic extracts,

only the extracts of P. tinctorum and R. conduplicans exhibited positive response

against the gram-positive bacteria, (S. aureus) whereas none of the lichen test samples

showed zones of inhibition against the gram negative bacteria, E.coli (Plate 8.2).

Similar finding was reported by Kosanić and Ranković (2010) who stated that the

gram negative bacterium, E. coli were resistant to all the five lichen extracts

(Lecanora atra, L. muralis, Parmelia saxatilis, P. sulcata and Parmeliopsis ambigua)

they had tested.

According to the rating systems, as provided by Quinto and Santos (2005), the

bioactivity of the extract of R. conduplicans and P. tinctorum were both found to be

active (15 mm ± 0.14 and 13 mm ± 0.14 respectively) against S. aureus as the zone

of inhibition falls between 13-19 mm (Table 8.3). From the above results, we can also

predict that methanolic extracts of P. tinctorum and R. conduplicans can restrict the

growth of S. aureus while the acetone extract of the same lichen sample were

ineffective. Further, it could also be inferred that the gram positive bacteria are much

better inhibited by lichen acids in general (Yılmaz et al., 2004).

The findings is again consistent with the observation of Ranković et al., (2007b,

2007c) who reported that the strongest antimicrobial activity was exhibited in

methanolic extracts when compared to the extracts in other solvents. Kumar et al.,

(2010) also claimed that the antifungal activities of methanolic extract of Ramalina

hossei H. Magn and G. Awasthi against two fungal strains, Aspergillus niger and A.

fumigaus were found to be more pronounce than other extracts. The antibacterial

activity using different extracts of some lichens collected from Sikkim and Nepal was

evaluated and amongst all the extract, the methanolic extracts was found to be most

active against five bacterial strains viz. S. aureus, E. coli, Vibrio cholera, Shigella

dysenteriae and S. flexneri (Sinha and Biswas, 2011).

The results are however not always valid for all lichens species. Tiwari et al., (2011)

examined the in-vitro antifungal activity using different extract of P. tinctorum against

Plate 8.2. Phytochemical screening of the methanolic lichen extracts

Plate 8.3. Inhibition zone of the methanolic extract in Staphylococcus aureus

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ten plant pathogenic fungi and found out that the inhibition of the different solvent

extract of P. tinctorum differed on different test organism. The methanolic extract was

found to be more effective against some investigated plant pathogenic fungi while the

acetone extract showed maximum inhibition zone against Aspergillus flavus and the

chloroform extract had the strongest inhibition activity against A. fumigatus.

The effectiveness of the methanolic extracts of P. tinctorum against S. aureus may be

attributed to lichen substances atranorin and lecanoric acid in P. tinctorum

respectively. The better effectiveness of methanol and acetone extract of P. tinctorum

against some plant pathogenic fungi are due to the presence of lichen acids such as

lecanoric and orsellinic acid (Gomes et al., 2002; Tiwari et al., 2010). Tough the

present study noticed the presence of atranorin and lecanoric acid in TLC of P.

austrosinense which is same as that of P. tinctorum, no zone of inhibition was

observed in case of P. austrosinense against both the test bacteria. This could be due

to the presence of additional metabolites in P. tinctorum.

Studies have revealed that the higher the content of usnic acid in the species tested, the

stronger will be the antimicrobial activity. This statement holds true in case of R.

conduplicans. The effectiveness of the methanolic extracts of R. conduplicans against

S. aureus may be accredited mainly due to the presence of usnic acid in R.

conduplicans. The antibiotic action of usnic acid is due to the inhibition of oxidative

phosphorylation which inhibits oxygen consumption, electron transport chain, and

other key mitochondrial functions in the cells (Nash III, 1996; Frankos, 2005).

The secondary metabolites namely atranorin, lecanoric acid and usnic acid detected in

TLC of the tested lichen species are known to have relatively strong antimicrobial

effects against human, animal and plant pathogens, mycotoxin-producers and food-

spoilage organisms (Okeke et al., 2008).

8.4 Conclusion

The results presented herein indicate that the lichen extracts manifest mild

antimicrobial activity, which suggests that extracted components from various lichens

may prove useful in treating many diseases caused by microorganisms.

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The wide disparity of antimicrobial activity among different extracts of different

lichen species might be probably accredited to the difference in solubility of wide

variety of bioactive compounds, such as flavonoids, phenolics, tannins and terpenoids

present in the selected lichen specimens. Moreover, environment, extraction methods,

time of collecting samples and genetic differences between tested samples are factors

that can lead to significant differences in antibacterial activity (Shan et al., 2005).

The variation in sensitivity against the different lichen extracts can also be ascribed to

the morphological variations between the microorganisms and the differences in level

of permeability of the solvent in cell wall (Nostro et al., 2000).

As gram negative bacteria are the major pathogens of gastrointestinal diseases, a

further study is needed to improve the efficacy of lichen extracts against the microbes

tested.