Antibacterial activity of Crude extracts and Semi synthetic Hydrazone derivatives of Rimelia reticulata

 

R Meera1*, P Devi 2, B Madhumitha and B Kameswari3

1Department of Pharmaceutical Chemistry, 2Department of Pharmacognosy,

3Department of Biochemistry, K. M.College of Pharmacy, Madurai -625107 Tamilnadu, India.

*Corresponding Author E-mail: meeraharsa@yahoo.com

 

ABSTRACT

Anti microbial activity of Ether, Acetone Dichloromethane extract and Choloroform extracts and semi synthetic derivatives of Rimelia reticulata were tested against Gram(+) and Gram(-) bacteria strains using zone of inhibition by Agar disc diffusion method.. The bacteria used in test were Gram positive ( Staphylococus aureus, Bacillus subtilis and  Micrococcus luteus and Gram negative (Escherichia coli, Salmonella typhi and Pseudomonas aureginosa). Moderate antibacterial activity for Ether and Dichloromethane extract, and in Chloroform extract having more than 100% activity and Acetone extract having( 90%) activity in Gram (+) organism. In Gram(-) all the extracts having moderate activity (80%). In Semi synthetic derivatives of Rimelia reticulata the Gram (+) and Gram (-) organisms are used. Among the four derivatives  2,4 di nitro hydrazine derivative has more than (100%) activity and 4 nitro phenyl hydrazine, phenyl hydrazine having moderate (80%) activity against Gram (+). In Gram (-) Hydrazine hydrate has shown more than (100% ) and Phenyl hydrazine, 4 nitro phenyl hydrazine having (90%)  activity. The standard drug used in this activity is Lincomycin. Based on the current findings it can be concluded that the lichen Rimelia reticulata posses potent antibacterial activity.

 

KEYWORDS:  Rimelia reticulata, Antibacterial activity, Agar disc diffusion method, crude extracts, Semisyntheticderivatives.

 

 

INTRODUCTION:

The antimicrobial action of lichen substances is well known and was reviewed by Asahina, Bustinza1, Vatrio2, Kristin3. The most promising lichen substance is the yellow pigment usnicacid, a broad spectrum antibiotic. It strongly inhibits Mycobacterium and has found commercial use as the active ingredient in a salve called Usno4.This medication is more effective than penicillin salves in curing external burns and wounds. It is now widely available in Europe and is active against fungi and yeast. For semi synthetic approaches towards preparation of usnic acid derivatives where the reports of Kutney et.al5,6 who have prepared hydroxylamine derivatives-I and Iso usnic acid derivatives –II. Takahashi et al 7,8,9 have prepared a series of methyl dihydro usnic acid, anhydromethyl dihydro usnic acid , iso anhydro methyl usnic acid derivatives. So in semi synthetic derivative also the antimicrobial action is present.

 

Rimelia Reticulata is a lichen plant occurring in hill regions of Kanyakumari district.It is widely used in the treatment of fever, jaundice, lung diseases, skin infections and diabetes10,11 .

 

The objective of the present study was asses to anti bacterial activity of the extract. The lichen Rimelia reticulata  having aminoacids. The lichens are used in  rabies, expectorants, diabetes, jaundice and intermittent fevers and liverailments. Lecanoric acid, gyrophoric acid, apthosin and atranorin are the main constiuents of Rimelia reticulata.

 

Keeping this in view the present study was undertaken to investigate the antibacterial activity of crude extracts and semi synthetic derivatives of Rimelia reticulata  against various strains of bacteria.

 

MATERIALS AND METHODS:

Plant materials:

Rimelia reticulata a Lichen material were collected during the Mahendragiri hill regions of Kanyakumari. The lichen species were identified by Dr.D.K.Upreti, National Botanical Research Institute, Lichenology Laboratory, Lucknow. The voucher specimens KMCP-PM 83 and PM 84  have been submitted.

 

The lichen materials were dried under shade with occasional shifiting and then powdered with a mechanical grinder and stored in airtight container.  The powdered lichen material 250gm was placed into a extractor of a soxhlet  apparatus and by hot percolation method. The extraction was carried out with 2.5 liters of each solvent for a period of 72 hrs. The solvent was then removed under reduced pressure. The yield was respectively 2.5, 1.2, 1.002 gm. The dried extracts were dissolved in dimethyl sulfoxide and used for the present study. Qualitative analysis for all extracts was carried out of all extracts reveal the presence of glycosides, reducing sugars and aminoacids.

 

The antibacterial activity for semi synthetic derivatives of Rimelia reticulata was also studied. For that the hydrazine derivatives of usnicacid were prepared. As a part of the investigations, 4 hydrazone derivatives were phenyl hydrazine, 4 nitro phenyl hydrazine, 2,4 di nitro phenyl hydrazine and Hydrazine hydrate.

 

A mixture of usnicacid  (3.52gm,0.1M), absolute ethanol(100ml), Phenyl hydrazine (1.446gm,0.1M) were placed in a 250ml Round Bottom flask. It was refluxed over 4 hours with vigorous stirring. After 4 hours the reaction mixture was cooled and filtered. The crude product was recrystallised from absolute ethanol. The procedure was same for other 3 hydrazine derivatives.

 

Microorganisms and media:

Gram-positive bacteria: Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25922) and Micrococcus luteus (ATCC 10240). Gram-negative bacteria: Escherichia coli (ATCC 25922) Pseudomonas aureginosa ( ATCC 27853 ) and  Salmonella typhi   (ATCC  43579)

 

The bacterial stock cultures were maintained on Muller Hinton agar and Sabouraud-dextrose agar slants, respectively, which were stored at 4ŗC. Six microorganisms maintained on nutrient agar base were used to assess the anti bacterial activity of the plant extracts.

Antimicrobial screening:

Agar cultures of the test microorganisms were prepared as described by Mackeen et.al,199712. Three to five similar colonies were selected and transferred to 5ml broth with a loop and the broth cultures were incubated for 24 h at 37 ŗC. The crude extracts and semi synthetic derivatives were dissolved in dimethyl sulfoxide with a magnetic stirrer. For screening, sterile 6-mm filter paper discs were impregnated with 250µg of the extracts and derivatives and then placed in Muller Hinton agar medium. The inoculums for each organism was prepared from broth cultures. The concentration of cultures was 1×105 colony forming units/ml. The results were recorded by measuring the zones of growth inhibition surrounding the disc. Clear inhibition zones around the discs indicate the presence of antimicrobial activity. The antibacterial Lincomycin (10µg/ml)was used as reference standard as recommended by the National Committee for clinical laboratory standards.

 

RESULTS:

It was observed that the both extracts and semi synthetic derivatives showed antibacterial activity against all the tested organisms. The results are in Table 1 and Table 2. where there is moderate antibacterial activity  for  Ether and Dichloromethane extract, and in Chloroform extract having more than 100% activity and Acetone extract having( 90%) activity in Gram (+) organism. In Gram (-) all the extracts having moderate activity (80%) .The standard used is Lincomycin.

 

In Semi synthetic derivatives of Rimelia reticulata the Gram (+)  and Gram (-)  using Lincomycin as standard. Among the four derivatives  2,4 di nitro hydrazine derivative has more than (100%) activity and 4 nitro phenyl hydrazine, phenyl hydrazine having moderate (80%) activity against Gram (+).In Gram (-) Hydrazine hydrate has shown more than (100% ) and Phenyl hydrazine, 4 nitro phenyl hydrazine having (90%)  activity.

 

DISCUSSION:

Disc diffusion methods are used extensively to investigate the antibacterial activity of natural substances and plant extracts. These assays are based on the use of discs as reservoirs containing solutions of the substances to be examined.In the case of solutions with a low activity, a large concentration or volume is needed.

 

Most of the bacterial species were inhibited by the extracts of  Rimelia reticulata. In this study six different bacterial species were used to screen the possible antimicrobial activites of the extracts of  Rimelia reticulata. The extracts showed broad spectrum of activity against all the bacterial strains at the tested concentrations of 250µg/ml.Lincomycin used as positive control for bacteria, respectively.

.

Results of Antibacterial activites for extracts(Table1) and Semi Synthetic Derivatives (Table 2)of  Rimelia reticulata

Table 1

Microorganism

 

Inhibition Zones(mm)

 

 

Chloroform extract

Acetone extract

Ether extract

DCM extract

Positive standard

Bacteria

Gram( +)

Staphylococcus aureus

Bacillus subtilis

Micrococcus luteus

 

 

 

16

15

16

 

 

10

10

10

 

 

13

12

12

 

 

13

12

12

 

 

13

13

13

Gram(-)

Escherichia coli

Salmonella typhi

Pseudomonas aureginosa

 

12

12

12

 

12

11

12

 

12

11

11

 

12

12

11

 

15

15

14

 

Table 2

Microorganism

 

Inhibition Zones(mm)

 

 

Phenyl hydrazine

4-nitro

Phenyl hydrazine

2,4 dinitro

Phenyl hydrazine

Hydrazine

hydrate

Positive standard

Bacteria

Gram( +)

Staphylococcus aureus

Bacillus subtilis

Micrococcus luteus

 

 

 

7

6

6

 

 

7

6

7

 

 

8

9

6

 

 

6

7

7

 

 

6

6

7

Gram(-)

Escherichia coli

Salmonella typhi

Pseudomonas aureginosa

 

6

6

7

 

6

7

7

 

7

6

6

 

8

8

7

 

7

7

7

 

 

CONCLUSION:

As evident from the results antibacterial action of the extracts are more active on Gram(+)  than Gram(-) . Usnic acid one of the common lichen metabolites is a quite powerful antibiotic active against many Gram (+) bacteria13.The antibacterial activity of these plants may be due to the presence of active principles present in the lichens

 

ACKNOWLEDGEMENT:

Dr. D. K. Upreti Scientist and Group Leader of Lichenology Laboratory in National Botanical Research Instiute, Lucknow for providing the authentification of the Lichen Rimelia reticulata.

 

REFERENCES:

1.        Bustnza F. Endeavour.1951,10:161.

2.        Vartia k. Antibiotics in Lichens.  In: The Lichens (ed.V.Ahamadjan and Hale H.E.).1973; Page no : 547-561.

3.        Kristin I, Sally F.B, Peter J.H., Antimicrobial agents.Chemotherapy.1995; 289-92.

4.        Barry VC , Monographs and Reports, The Royal Institute of Chemistry .1952 ; Vol 2.

5.        James P. Kutney , Ignaci I.H , Sanchez and Trevor Yee.Studies in the Usnic Acid Series II. The Condensation of (+)-Usnic Acid with Hydroxylamine.Canadian Journal of Chemistry.1976;54:3713-371.

6.        James P. Kutney , Ignaci I.H , Sanchez and Trevor Yee. Studies in the Usnic Acid Series III. The Base Catalysed Usnic Acid- Isousnic Acid  Rearrangement . The Synthesis of (+)-Isousnic Acid . Canadian Journal of Chemistry.1976;54:3721-373.

7.        Kotaro Takahashi , Shuichi Miiyashita., Usnic Acid  I. Methyl Dihydrousnic acid Chem.Pharm.Bull.1962;10: 603-607.

8.        Kotaro Takahashi , Shuichi Miiyashita., Usnic Acid III. An hydro Methyl Dihydrousnic acid . Chem.Pharm.Bull.1963;11:209-213.

9.        Kotaro Takahashi , Shuichi Miiyashita, Yoshie Ueda., Usnic Acid IV. Isoanhydro Methyl Dihydrousnic acid . Chem.Pharm.Bull.1963;11: 472-478.

10.     Subramanian S.S and Ramakrishnan S., Curr.Sci.1964;33:522.

11.     Fuzikawa H., J.Pharm.Soc.(Japan).1939;59:615.

12.     Mackeen MM,Ali AM, EI-Sharkawy SH, Salleh MY , Lajis NH and Kawazu K, Antimicrobial and cytotoxic properties of some Malaysian traditional vegetables, Int J Pharmacogn, 1997;35:237-243.

13.     Stoll A, Brack A and Renz J., Experentia. 1947;3:115.

 

 

 

 

Received on 06.06.2009        Modified on 10.08.2009

Accepted on 16.09.2009        © AJRC All right reserved

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 445-447