Anti Tuberculous Activity and Various Enzyme Activities of Rimelia Reticulata

R Meera*, P Devi , B Madhumitha and B Kameswari

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

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

ABSTRACT

Bioassay guided fractionation of the Acetone, Benzene and Usnic acid (column ether extract) of Rimelia reticulata using drugs sensitive strain of Mycobacterium tuberculosis in vitro resulted in the Anti-TB activity. In enzyme activity the particular enzymes used are Amylase, Urease, Trypsin and Papain with standard inhibitors. For Amylase and Urease the Usnic acid and crude ether extracts shown 100% inhibition. For Trypsin and Papain alcohol extract shown 100% inhibition , in this study. The standard inhibitor for Amylase, Trypsin and Papain is Mercuric chloride. For Urease is P-chloro mercuric benzoate. Preliminary phytochemical screening was performed and different phytochemical constituents present in the extracts were identified.

KEY WORDS: Rimelia reticulata, Mycobacterium tuberculosis, Enzymes, Various extracts.

INTRODUCTION:

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 diabetes^{1,2,3 .}The objective of the present study was asses to anti inflammatory activity of the extract. The lichen Rimelia reticulata having aminoacids. The lichens are used in intermittent fever and liver ailments. Lecanoric acid, gyrophoric acid, apthosin and atranorin are the main constiuents of Rimelia reticulata.To our knowledge , there were no scientific reports on the antitubercular activity of Rimelia reticulata.In the present study the whole lichen was successively extracted with Ether, Dichloromethane, Chloroform, Benzene, Acetone, Ethylacetate and Alcohol by soxhlet hot extraction process. Preliminary phytochemical analysis was carried out for different extracts⁴. It was found that aminoacids and glycosides present in the extracts. The vacuum dried extracts were screened for antituberculous and enzyme activites. TLC studies were carried out using Hexane:ether:formicacid {5:4:1}. R_fvalues are calculated for the different extracts like 0.54, 0.55, 0.64 and 0.65.

Material and Methods :

The lichens were identified by NBRS {National Botanical Research Institute } Lucknow having No NBRI\LICH\DK-2004-95. After authentification lichens were collected in bulk from the Kanyakumari during early summer , washed,

shade dried and then milled into coarse powder by a mechanical grinder.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 which obtained the residue. Qualitative analysis for all extracts was carried out of all extracts reveal the presence of glycosides, reducing sugars and aminoacids.

Preliminary phytochemical screening was performed as per standardized procedure⁵and various phytoconstituents present in the extracts were identified.

The extracts were subjected to Antitubercular activity against Mycobacterium tuberculosios H₃₇ R_V strain by Minimum inhibitory concentration method using Rifampicin as the reference standard^6,7. For this method first prepare Antibiotics and synthesized compounds containing Lowenstein Jenson medium. 120 mg of accurately weighed quantity of Rifampicin and the synthesized compounds (Usnic acid, Acetone extract, Benzene extract) were dissolved individually with 30 ml of ethanol to produce 4000µg/ml of stock solution. Then 6.4 ml of stock solution was dissolved individually with 3.6ml of water to produced 2650 µg/ml of working solution. Then 5ml of working solution was dissolved individually with 5ml of water to produce 1280 µg /ml and preserved at 20° c. Final concentration of 5,10, 20 and 40 µg /ml were prepared. Finally 1ml of Rifampicin and the synthesized compounds (Usnic acid, Acetone extract, and Benzene extract) were

Table I: Effect of extract of Rimelia reticulata on Mycobacterium tuberculosis

S. No	Tested material	Drug concentration (µg/ml )
S. No	Tested material	5	10	20	40
1	Rifampicin (anti TB drug)	0.002	0.002	0.002	0.002
2	Usnic acid	0.03	0.03	0.02	0.01
3	Acetone extract	0.07	0.06	0.04	0.03
4	Benzene extract	0.07	0.07	0.06	0.05

Table II Amylase and Urease activities in extracts of Rimelia eticulata

Enzyme inhibition	Standard inhibitor	Extract ( % of inhibition)
Enzyme inhibition	Standard inhibitor	Standard inhibitor	Usnic acid	DCM extract	Acetone extract	Crude ether extract
Amylase	Mercuric chloride	100%	100%	Nil	60%	100%
Urease	Parachloro mercuric benzoate	100%	70%	Nil	60%	80%

Table III Papain and Trypsin activities in Rimelia reticulate on Mycobacterium tuberculosis

Enzyme inhibition	Standard Inhibitor	Extract ( % of inhibition)
Enzyme inhibition	Standard Inhibitor	Standard inhibitor	Alcohol extract	Benzene extract	Acetone Extract
Papain	Mercuric chloride	100%	100%	Nil	70%
Trypsin	Parachloro mercuric benzoate	100%	100%	Nil	70%

withdrawn and separately mixed with 9ml of L.J.medium using micropipette. Then prepare Dubos and Davis medium. This medium contains Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Sodium citrate, Magnesium sulphate, Tween -80, Casein hydrolysate, Distilled water and Bovine albumin solution. Dissolved each salt separately in portions of the water then mixed these solutions into the tween and casein hydrolysate dissolved in the remaining water. The medium should be at pH 7.2 distributed in cultured containers, autoclave at 115°c for 10 minutes and added Bovine albumin with sterile precautions. Approximately 4 mg moist weight of bacterial culture Mycobacterium tuberculosios was removed from a L.J.slope with 3mm external diameter (24 gauge wire) loop and transferred to Dubos and Davis medium to produce uniform suspension. One loop full of inoculum was inoculated on each slope using a 3mm external diameter (27 gauge wire) loop. The tubes were incubated at 37°c for 4 weeks.

For Amylase enzyme activity, the salivary amylase and crude inhibitor was prepared by the method of Buonocore⁸. Amylase activity was determined by the method of Bernfeld⁹. In this method, the absorbance was measured at 530nm in an Erma photoelectric colorimeter.

For assay of Papain the method was carried out by Arnon¹⁰ using casein as substrate. The soluble extracts after the enzymatic hydrolysis were measured at 280nm. For assay of Trypsin – a unit obtained from sodium dodecyl sulphate (SDS ) and was assayed according to the method of Birk¹¹using casein as substrate.

For assay of Urease enzyme was prepared from horse gram seeds. The assay of enzyme was carried out the method described by Malhothra¹² using Nesslers reagent. A standard

Was prepared using ammonium sulphate.Urease inhibition was compared with parachloromercuricbenzoate. Under

these conditions, the crude extract showed 100% of inhibition.

All the above enzyme activies, the absorbance were measured and the % inhibition was calculated.

Abs of control (no ihibitor) - Abs of sample

% inhibition = ------------------------------------------------×100

Abs of control

RESULTS AND DISCUSSIONS:

The present study reveals the presence of phytoconstituents such as aminoacids and tannins.The MICs of extracts Usnic acid ,Acetone extract ,Benzene extract were found to be 0.03, 0.04 and 0.06 mg/ml respectively against Mycobacterium tuberculosis. The ether extract showed maximum inhibition at 5 µg/ml. The Acetone and Benzene extracts have shown at 20µg/ml.

In the enzyme activity of Amylase and Urease, the Usnic acid and crude ether extract of Rimelia reticulata having superable (100%) inhibition and Acetone extract has moderate(60%) inhibiton .DCM extract has no inhibition activity. In Trypsin and Papain alcohol extract has shown superable (100%) inhibition, Acetone extract has shown moderate (60%) inhibition and Benzene extract has no inhibition activity.

ACKNOWLEDGEMENTS:

The authors are thankful to Mr.Dr.D.K.Upreti Scientist and Group Leader of Lichenology. Laboratory in National Botanical Research Institute, Lucknow for providing the authentification of the Lichen Rimelia reticulata.

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Received on 20.10.2008 Modified on 22.11.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 131-133