2. Materials and Methods 2.1. Study area The study was conducted at Kalyani University Campus which is situated in the district of Nadia, West Bengal (Latitude 2304/N, Longitude 88052/E). This University campus has 400 acre field area and different types of plants. 2.1. Sampling The study was carried out during the period of March 2020. During which the average maximum and minimum temperature was 360C and 140C and the average value of relative humidity was 86%. The lichen sample collected from trees upto a height of about 10ft. Specimens were collected in paper bags and brought to the laboratory for analyses. Specimens are presented in the herbarium of Department of Botany, University of Kalyani, Kalyani, Nadia, WB. 2.2. Identification The lichen specimens were collected from all the available substrates (i.e. Tree bark, Twing, Leaf surface, Rock, Forest flore etc.) The collected specimens after their proper drying, were investigated morphologically, anatomically and chemically (Upreti DK, Divakar PK, Nayaka S (2005)). The color spot tests were performed with the usual reagents of K(5% Potassium hydroxide), C (Aqueous soln. of Calcium hypochlorite) and P (Paraphenylenediamine)( Rashmi S and Rajkumar HG. (2014) ). 2.3. Phytochemical Screening 2.3.1. Qualitative chemical evaluation The obtained different lichen extracts were qualitatively examined for the presence of various phytochemicals. 2.3.2. Test for Tannins Ferric chloride Test; 2 ml Crude extract was mixed with a few drops of 5% ferric chloride solution. Formation of a blue colour showed the presence of tannins. 2.3.3. Test for Alkaloids Dragondroff’s test; 2 ml of crude extract is added to 1% HCl, steam for 10 minutes. To this add 5 drops Dragondroff’s reagent; the reddish · brown precipitate indicates the presence of alkaloids. 2.3.4. Test for Saponins 2 ml of Crude lichen extract was mixed with 5 ml of distilled H2O in a test tube and it was shaken vigorously. The formation of a foam shows the presence of saponins. 2.3.5. Test for glycosides Keller-kilani test: 2 ml of lichen Crude extract was added with 2ml of glacial acetic acid and 1-2 drops of 2% solution of FeCl3. The mixture was then poured into an another test tube and added 2 ml of concentrated H2SO4. A brown coloured ring at the interphase showed the presence of cardiac glycosides. 2.3.6. Test for Flavonoids NaOH solution
test; 2 ml of crude extract is added to 2 ml of 10% NaOH solution. Formation of
Yellowish orange colour indicates the presence of flavanoids. 2.3.7. Test for Proteins Xanthoproteic
test: 2 ml of crude lichen extract is mixed with 2 ml of HNO3 and then boiled
in a water bath. Formation of Orange colour indicates the presence of proteins. 2.3.8. Test for Triterpenoids Salkowski
Test: 2 ml of crude lichen extract is shaken with 1 ml of chloroform and 5
drops of concentrated sulphuric acid were added along the side of test tube. A reddish-brown
colour formed at the interface showed the test as a positive for triterpenoids. 2.3.9. Test for carbohydrates Benedict’s
test: 2 ml of Crude lichen extract when mixed with 2 ml of Benedict’s reagent
and then boiled, a reddish-brown precipitate formed which showed the presence
of the carbohydrates 2.3.10. Test for Steroids Liebermann-Burchard reaction: 2 ml of crude lichen
extract is added with 2 ml acetic anhydride and a few drops of conc. H2SO4.
Blueish green coloured ring indicates the presence of steroids. 3. Preparation of lichen extract Before
extraction the plant materials were washed in tap water and then blot and dried
at room temperature (30+/-20C). Then the dried material were powdered in grinder, to prepare stalk solution 10 gm of
the powder were taken and then added to 100ml of solvents of methanol, ethanol
and distil water (W/V, 10gm/100ml) each extract was filtrated through whatman filter paper. 3.1. Test organism Bacteria
such as Micrococcus luteus; Shigella dysenteriae;
Shigella flexneri; Escherichia
coli; Staphylococcus aureus; Pseudomonas aeruginosa;
Bacillus subtilis; Vibrio cholerae were used as
test organism. All the bacterial culture were produce
from ID & BG, Hospitals, Kolkata. These bacterial strains culture maintained
on nutrient plate at 40C temp. 3.2. Screening of Antibacterial Antibacterial
test of selected microorganisms were carried out using
agar well diffusion method (Perez et al. 1990). The plates were incubated at 370C
for 24 hours during which activity also evidence by the presence of a zone of
inhibition surrounding the well. Each test was repeated three times and the
antibacterial activity was expressed as the mean of diameter of the inhibition
zone (mm). 4. Results and Discussion In
the presence study two lichen sample (LK1, Cryptothecia
striata and LK2,
Cryptothecia scripta) were tested for the presence of different phytochemicals.
The result was presented in Table 1. Both the lichens samples exhibited in the
presence of saponin, tannin, terpenoids, reducing sugar and alkaloids.
Flavonoids are absence in both the lichens. However, the lichens LK2 contained
carbohydrate. These biochemical test might be utilise
for the farther identification of lichens. The aqueous as well as methanolic and
ethanolic extract for both lichens thallus studied for their antibacterial and
it was presented in Table2 and Table3. All the extract showed antibacterial activities against all the
bacterial tests. The solvent extract shows maximum activity as compare to the water extract indicating the released of
bioactive component of lichens in the solvent in greater amount. The methanolic
extract of LK1 showed highest antibacterial activities and those of ethanolic
extract. Methanolic extract of LK1 exhibited maximum activity against Micrococcus luteus and minimum activity
against Staphylococcus aureus.On the other hand the ethanolic extract of LK1
showed maximum inhibition zone against Bacillus subtilis and
minimal inhibition zone against Pseudomonas aeruginosa . On the
other hand water extract of LK1 exhibited higher
activity against Pseudomonas aeruginosa and less activity against
E. Coli. The
methanolic extract of LK2 also exhibited considerable activities against all
the bacteria with highest activities recorded against E. Coli and
less activity against Pseudomonas aeruginosa. On the other hand the ethanolic extract of LK2 showed maximum inhibition
zone against Shigella flexneri and minimal inhibition
zone against Bacillus subtilis. On the other hand
water extract of LK2 exhibited higher activity against Micrococcus luteus
and less activity against Shigella flexneri.
In the present study both gram positive
and gram
negative
bacteria are inhibited by the lichen extract which indicate the presence of
broad spectrum antibacterial compound. 5. Conclussion The present work described the antibacterial efficiency and the presence of different phytochemicals of the lichens growing in Kalyani University Campus. The biochemical tests were done for the presence of Tannins, Alkaloids, Saponins, glycosides, Flavonoids, Proteins, Triterpenoids, Carbohydrates and Steroids. Lichens were screening antibacterial properties and it was found that Methanolic and Ethanolic extract shows maximum activity as compare to the water extract indicating the released of bioactive component of lichens in the solvent in greater amount. It is the first time study of antimicrobial activity of Cryptothecia striata and Cryptothecia scripta. However, further investigation on isolation and characterization of the active principle of this lichen species responsible for the antibacterial activity is necessary. REFERENCES Fleishner TL. (1994) Ecological costs of livestock grazing in western north America. Conservation Biology. 8(3) :633. Retrieved from https://doi.org/10.1046/j.1523-1739.1994.08030629.x Honegger R. Simon Schwendener (1829-1919) and the dual Hypothesis of Lichens. The Bryologist (2009).103(Summer 2000) :307-313. Retrieved from https://www.jstor.org/stable/3244159 Huneck S and Yoshimura I. (1996) Identification of Lichen Substances. DOI :10.1007/978-3-642-85243-5. Retrieved from https://link.springer.com/chapter/10.1007/978-3-642-85243-5_2 Rashmi S and Rajkumar HG. (2014) Preliminary phytochemical screening of different solvent extracts of lichens from Kodagu district, Karnataka. Journal of pharmacognosy and phytochemistry. 3(4) 209-212. Upreti DK, Divakar PK, Nayaka S (2005). Commercial and ethnic use of lichens in India. Economic Botany. 59(3) 269. Retrieved from https://link.springer.com/article/10.1663/0013-0001(2005)059[0269:CAEUOL]2.0.CO;2
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