Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 RESEARCH ARTICLE Evaluation of Phytochemical Analysis and Antithrombolytic Activities of “Dual Organisms Lichens” – Parmotrema perlatum Rajha Viknesh Madheshwar1, Thiyagarajan perumal1*, Rubalakshmi G2 and Nirubama K2 1 2 Department of Microbiology, Bharathidasan University, Tiruchirappalli -620024,Tamilnadu, India GRD Bio clinical Research, Rasipuram, Namakkal, Tamilnadu, India. Received: 17 Feb 2017 Revised: 19 Mar 2017 Accepted: 25 Apr 2017 *Address for correspondence Dr.Thiyagarajan perumal Department of Microbiology, Bharathidasan University, Tiruchirappalli -24, Tamilnadu,India. Email: rajanphd2004@yahoo.com This is an Open Access Journal /article distributed under the terms of the Creative Commons Attribution License (CC BY-NC-ND 3.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. All rights reserved. ABSTRACT Lichens are known for their extraordinary secondary metabolites and thrombolytic activity. Parmotrema perlatum commonly called as Canary moss or Mangalore spices.This lichen is used as the spices in the food in the Indian tradition. The use of P.perlatum in medicine is based on the fact that they contain unique and varied biologically active substances, as natural Antioxidant, Antimicrobial and Anticoagulant. Since they are natural antibiotics, their metabolites exert a wide variety of biological actions including anti-mycotic, antiviral, anti-inflammatory, analgesic, antipyretic, anti-proliferative, and cytotoxic effects, they are considered as potential drugs. They contain a variety of secondary metabolites flavonoids, triterpenoids, saponins and phytosterols. It also acts as an anti-thrombolytic for the cardiovascular disease by preventing the clotting of blood in the veins and arteries by the action of P.perlatum. The lichens can be used as active ingredient for the preparation of drugs for its broad range of activity. The need for the control of cardiovascular diseases is in high rate. The use of synthetic drugs results in the adverse effect along with the temporary relief. But the implementation of traditional herbs on the cardiovascular patient’s results will be astonished. This paper is a tribute to the wealth of Indian traditional knowledge that exists about lichens. Keywords: Parmotrema perlatum, Secondary metabolites, Anti-coagulant, Thrombolysis. 12285 Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. INTRODUCTION Lichens are symbiotic organisms which are composed of fungi and algae. Lichens have been used in various fields, especially as a source of natural drugs in pharmaceutical industry and food supplement. Lichens are effective in the treatment of bronchitis, tuberculosis, and haemorrhoids. Lichens are important traditional medicines in many different cultures. This information has been made available to us from the contributions of hundreds of traditional knowledge holders in communities across the world. It is our responsibility to respect and value the knowledge that has been given to us. Lichens produce a wide range of organic compounds that can be divided into two groups, called primary metabolites and secondary metabolites. Primary metabolites are proteins, lipids, carbohydrates, and other organic compounds that are essential to the lichen’s metabolism and structure. Some of these metabolites are produced by the lichen’s fungal partner and algal or cyano bacterial partners. Secondary metabolites are produced by the fungus alone and secreted onto the surface of hyphae either in amorphous forms or as crystals. If these substances are only found in lichens, then they are called lichen substances [1]. Once such a lichens is Kalpasi (Parmotrema perlatum) which belongs to the family of parmoleaceae is a foliose lichens used as traditional medicines. Extracts and metabolites from this lichens exhibit pharmacological properties such as anti-inflammatory, antiulcer, anthelmintic, antibacterial, and free radical scavenging activity. Beside medicinal uses, this lichens has high economic value due to its spice properties and provides substantial livelihood support to local inhabitants. A wide range of chemical compounds including atranorin, chloroatranarin, salazinic acid, lecanoric acid, imbricaric acid, lecanora. Two terpenes, parmelanostene and permelabdone and usnic acid have been isolated from this species. The present study summarizes the information concerning the traditional uses, phytochemistry and thrombolytic activity of hydroalcoholic extract of Parmotrema perlatum [2].Parmotrema perlatum (parmelacieae), commonly known as kalpasi has a long, rich history in herbal medicine with a lengthy recorded indigenous use. It had also been found to be a promising new anti-tumor agent in numerous in vitro studies. Parmotrema perlatum is frequently utilized as a spice to improve the taste and flavour of the foodstuff and compound similar to usnic acid, 3-ketooleanane, tridecyl myristate, icosan-1-ol, azolitmin, erythrolein, orcin, spaniolitmint, atranorin and parmelanostene permelabdone were also present. Occurrences of phytochemical components in the traditional are responsible for healing of human syndrome and they are the primary and secondary metabolites [3]. An acidic phytochemical substance present in this lichen has also been exploited as an existing antibiotic for treating skin diseases in human beings [4]. The antibacterial properties of several lichens have been investigated in 1940s and 1950s targeting the invention of antibiotic penicillin by utilizing a fungus. Numerous secondary metabolites have been screened from lichens and usnic acid is an incredibly potent material employed in pharmaceutical treatment in relation to viruses along with analgesics and antipyretics [5]. In this present study preliminary phytochemical analysis and thrombolytic activity was carried out to find out the major chemical components available in this lichens and their potent thrombolytic activity. Lichens are utilized for detecting and examining of the phytochemical components that are beneficial for production of novel drugs and also significant in research organizations as well as pharmaceutical companies for the treatment of various diseases.The development of medicine from the traditional herbs and plants to prove the traditional spices has its efficiency in healing the disease without creating any side effects. There is no systematic work done it in this lichens and this is the first report of phytochemical and antithrombotic properties of hydroalcholic extract of Parmotrema perlatum. 12286 Indian Journal of Natural Sciences www.tnsroindia.org.in ©IJONS Vol.7 / Issue 42 / June 2017 International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. MATERIALS AND METHODS Collection and Identification Parmotrema perlatum used in the study was identified and the reference material has been kept under reference GRD/SC/05/16-17. Lichens of Parmotrema perlatum was collected randomly from the region of in around Mangalore forest, Karnataka. Preparation of Extract The shade dried coarsely powdered lichens of Parmotrema perlatum (25g) was extracted with 250 ml of 70% ethanol and 30% aqueous by soxhlet apparatus at room temperature for 72 hours. After extraction, the extract was filtered, concentrated to dryness in rota vapour under reduced pressure and controlled temperature. Dark yellowish brown colour residue was obtained and it was coded as Parmotrema perlatum. The residue was then stored in desiccators. The extractive value of hydro alcohol extract of Parmotrema perlatum was found to be 5g. Phytochemical Screening Analysis of Parmotrema perlatum Qualitative Phytochemical Analysis Preliminary phytochemicals analysis was carried out for all the extracts as per standard methods described by Brain and Turner 1975 and Evans 1996.Parmotrema perlatum extracts obtained by the above method was subjected to qualitative analysis for the presence of Phenolic groups, Glycosides, Alkaloids, Flavonoids, Tannins, Terpenoids, Saponins, Oils and gums as described by the method of and also as specified in the book of Practical Pharmacognosy [6]. Detection of Alkaloids Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were used to test the presence of alkaloids.   Mayer’s test: Filtrates were treated with Mayer’s reagent. Formation of a yellow cream precipitate indicates the presence of alkaloids. Wagner’s test: Filtrates were treated with Wagner’s reagent. Formation of brown/reddish brown precipitate indicates the presence of alkaloids. Detection of Flavonoids   Lead acetate test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids. H2SO4 test: Extracts were treated with few drops of H2SO4. Formation of orange colour indicates the presence of flavonoids. Detection of Steroids 2ml of acetic anhydride was added to 0.5g of the extracts, each with 2ml of H2 SO4. The colour changed from violet to blue or green in some samples indicate the presence of steroids. 12287 Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. Detection of Terpenoids  Salkowski’s test 0.2g of the extract of the whole plant sample was mixed with 2ml of chloroform and concentrated H2SO4 (3ml) was carefully added to form a layer. A reddish brown coloration of the inner face was indicates the presence of terpenoids. Detection of Anthraquinones  Borntrager’s test About 0.2g of the extract was boiled with 10% HCl for few minutes in a water bath. It was filtered and allowed to cool. Equal volume of CHCl3 was added to the filtrate. Few drops of 10% NH3 were added to the mixture and heated. Formation of pink color indicates the presence anthraquinones. Detection of Phenols   Ferric chloride test: Extracts were treated with few drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenol. Lead acetate test: Extract was treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of phenol. Detection of Saponins About 0.2g of the extract was shaken with 5ml of distilled water. Formation of frothing (appearance of creamy miss of small bubbles) shows the presence of saponins. Detection of Tannins A small quantity of extract was mixed with water and heated on water bath. The mixture was filtered and ferric chloride was added to the filtrate. A dark green color formation indicates the presence of tannins. Detection of Carbohydrates Extracts were dissolved individually in 5ml distilled water and filtered. The filtrate was used to test the presence of carbohydrates. Detection of Oils and Resins Test solution was applied on filter paper. It develops a transparent appearance on the filter paper. It indicates the presence of oils and resins. Thrombolytic Assay In vitro clot lysis activity of test drug was carried out according to the method of Prasad et al., 2006 with minor modifications. Briefly, venous blood drawn from the healthy volunteers was distributed in different pre weighed sterile micro centrifuge tube (0.5 ml/tube) and incubated at 37°C for 45 min. After clot formation, serum was completely removed without disturbing the clot and each tube having clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube –weight of tube alone). To each micro centrifuge tube containing 12288 Indian Journal of Natural Sciences www.tnsroindia.org.in ©IJONS Vol.7 / Issue 42 / June 2017 International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. pre-weighed clot, 100 μl of test drug was added separately. As a standard, 100 μl of Streptokinase (SK) and as a nonthrombolytic control, 100 μl of distilled water were separately added to the control tubes numbered I and II. All the tubes were then incubated at 37°C for 90 min and observed for clot lysis. After incubation, fluid released was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The experiment was repeated with the blood samples of the 5 volunteers. RESULTS AND DISCUSSION As a result further fractionation and scrutiny of the effective portions may be initiated along with the usage of these solvents (aqueous, hydroalcolhol, ethanol, petroleum ether and chloroform). In the preliminary phytochemical analysis the occurrence of phytochemical was confirmed in the extracts that are displayed in the Table 1. Phytochemical studies of P.perlatum have led to the isolation of various chemical constituents such as atranorin, chloroatranarin, salazinic acid [8] ,lecanoric acid, imbricaric acid [9], and lecanora.[10].Two terpenes, parmelanostene and permelabdone and usnic acid have also been isolated from this lichen.Results of the phytochemical screening of hydro alcoholic extract Parmotrema peraltum showed the flavonoids, saponins, triterpenoids, tannins, polyphenol and sterols were present and absence of and alkaloids, protein, carbohydrate and glycosides [11].The interaction between platelets and blood vessels is important in the development of thrombosis and cardiovascular diseases. The treatment and prevention of these cardiovascular diseases the inhibition of platelet aggregation is of fundamental importance [11]. The ability of P.peraltum to lysis of blood clot is recorded in this report.Medicinal plants contain different therapeutic agents which may have thrombolytic activity, cytotoxic effect etc. Working with different extract showed that they can lyses thrombus as streptokinase [12] Traditional drugs have a long history of utilization for the prevention and treatment of human illnesses. Today, numerous pharmaceuticals at present sanction by the Food and Drug Administration (FDA) have inceptions to natural sources. A major role for natural-derived compounds based on the reported immunemodulatory effects has emerged in recent times and has prompted the thorough experimental examination to focus viability and wellbeing.Nowadays phyto pharmacological investigation has created a new field to discovery spices derivative drugs, which are effective in remedial of certain diseases, and renewed the attention in herbal medicines. It is estimated that about 30% of the pharmaceuticals are prepared from plants derivatives. A failure of hemostasis and consequent formation of blood clots in the circulatory system can produce severe outcomes such as stroke and myocardial infraction. Pathological development of blood clots requires clinical intervention with fibrinolytic agents such as urokinase, tissue plasminogen activator and streptokinase. A number of research works have been conducted to discover the plants and natural food sources and their supplements having antithrombotic (anticoagulant and antiplatelet) effect and there is indication that consuming such food leads to prevention of coronary events and stroke. In the present study hydroalcholic extract of parmotrema perlatum showed significant thrombolytic activity this effect may be possibly due to phyto constituents present in the lichens extract affecting activation of plasminogen both by fibrin-dependent and fibrin-independent mechanisms similar to Streptokinase which causes extra production of plasmin which breaks down fibrin the major constituent of thrombi, to dissolve unwanted blood clots. CONCLUSION The present study may be useful to supplement the information with regard to its standardization and identification and in carrying out further research as a significant new source for novel bioactive substances. It can be concluded that Parmotrema perlatum has got the potential as a candidate for future thrombolytic agent. It can also be investigated 12289 Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. as a possible source of antithrombotic drugs. This is only a preliminary study and investigated be phytochemically and to exploit their medicinal and pharmaceutical potentials. Moreover, these Parmotrema perlatum lichen is also used as fodder for animals and in spices for human consumption where it could play major role for possible formulation of new drug to fight against pathogens. REFERENCES 1. Elix J. A. 1996, Biochemistry and Secondary Metabolites. In: Lichen Biology (Nash III, T. H., ed.). Cambridge University Press,Cambridge, pp. 154-181. 2. Thippeswamy, B., Sushma, NR., Naveenkumar, KJ., Antimicrobial property of bioactive factor isolated from Parmelia perlata . Internati Multidiscipli Resear J. 2012; 2(2): 01-05. 3. Krishnaiah, D., Sarbatly, R., Bono, A., phytochemical antioxidants for health and medicine: A move towards nature. Biotechnol Mol Biol Rev. 2007; 1: 97-104. 4. Momoh, MA., Adikwu, MU., Evaluation of the effect of colloidal silver on the antibacterial activity of ethanolic extract of the lichen Parmelia perlata. Afri J Pharm and Pharmacol. 2008; 2(6): 106-109. 5. Proksa, B., Proksova, A., Lichens metabolites. Usnic acid and its biological activity. Farm. Obz. 1999; 68: 139–143. 6. Brain KR, and Turner TD. Practical Evaluation of Phyto Pharmaceuticals, Wright –Science technica, Bristol, 1975; Vol. 144. 7. Ali, K., Ahmad, H., Khan, N. & Jury, S. 2014. Future of Abies pindrow in Swat district, northern Pakistan. Journal of Forestry Research, 25, 211-214. 8. G.L. Chopra, A text book of fungi, 14th ed., S.L. Jain, Fors Nagin and Co., Partab Road, Jullundur city, pp. 350354, 1979. 9. S.T. Abdulla, H. Hamid, M. Ali, S.H. Ansari, and M.S.Alam, “Two new terpenes from the lichen Parmelia perlata”, Indian Journal of Chemistry, Section B, Organic and Medicinal Chemistry, vol. 46(1), pp.173-176, 2007. 10. F. Chicita, Culbertson and William Louis Culbertson, Published by: American Bryological and Lichenological Society, Vol.69 (2), pp.192- 202, 1966. 11. Antiplatelet Trialist Collaboration. 1994. Collaborative overview of randomised trials of antiplatelet therapy 1: Prevention of death, myocardial infarction and stroke by prolonged Antiplatelet therapy in various categories of patients. Br Med J 308: 81–106. 12. Sweta P., Rajpal S.K., Jayant Y.D,. Hemant J.P., Gerhard M.T., and Hatim F.D.. Effect of Fagonia arabica (Dhamasa) on in vitro thrombolysis. BMC Complementary and Alternative Medicine. 2007; 7(36):1-6. Fig 1. Fresh Parmotrema perlatum Fig 2. Dried Parmotrema perlatum 12290 Indian Journal of Natural Sciences www.tnsroindia.org.in ©IJONS Vol.7 / Issue 42 / June 2017 International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. Table 1:Phytochemical Analysis of Different Extract of Parmotrema perlatum S.No. Aqueous Hydro alcohol 1 Alkaloid 2 Flavonoid 3 Triterpenoid 4 Phenolic compound 5 Protein ND 6 Carbohydrates ND - 7 Saponin ++ 8 Steroids 9 Ethanol Petroleum ether Chloroform - + ND ND +++ - + ND + ++ - ND + + ++ + + + - - ND ND - ND ND +++ - ND ++ ND ++ + + ND Glycosides + - - ND ND 10 Amino acid ND - - ND ND 11 Tannin ++ + + 12 Oil 13 Gums & Musilage 14 Chlorogenic compound + - ND - - ND ND + - - ND ND ND - - ND ND +++ = high; ++ = moderate; + = low; ND = not detectable. Table2 : Behaviour of drug powder of hydroalcoholic extract Parmotrema perlatum with various chemical reagents S.No Test for Reagents Reaction Observation 1. 2. Flavonoid Alkaloids Mg bits + HCl Mayer’s reagent Magenta colour Cream precipitate Dragendroff’s reagent (+)ve (-)ve (-)ve Reddish brown precipitate Hager’s reagent Wagner’s reagent 3. Saponin Water shake (-)ve Yellow precipitate Reddish brown precipitate Leather formation (-)ve (+)ve 12291 Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. 4. Tannins and Phenolic compound Ferric chloride Brownish green or blue-black (+)ve 5. Sterol Acetic anhydride + Sulphuric acid Bluish green (+)ve 6. 7. Chloroform + H2SO4 Ninhydrin Reddish brown No blue colour (+)ve (-)ve 8. Triterpenoids Protein and Amino acids Carbohydrates Glycosides Brick red precipitate Pink to red colour (-)ve 9. Fehling’s A and B solution Sodium nitroprusside solution (-)ve Table 3.Thrombolytic Activity of Hydroalcoholic Extract Parmotrema perlatum S.No. 01 02 03 04 05 Weight of empty tube A(g) 1.093 1.017 1.062 1.015 1.010 Weight of tube with clot B(g) 1.384 1.571 1.459 1.587 1.544 Weight of clot C (B-A) (g) 0.291 0.554 0.397 0.572 0.534 Weight of tube with clot after lysis D(g) 1.260 1.307 1.238 1.342 1.298 Weight of lysis E (B-D)(g) 0.124 0.264 0.221 0.245 0.246 % of clot lysis Average % of clot lysis 42.61 47.65 55.66 42.83 46.06 46.96 % of clot lysis = (wt of released clot /clot wt) × 100.Negative control: 2.048, Positive control: 56.12,Test drug: 46.96 Fig - 3: Percentage of Clot Lysis by Distilled Water, Streptokinase and Hydroalcoholi Extract Parmotrema Perlatum. 1. Control (sterile distilled water) 2. Standard drug-Streptokinase 3 to 7- Healthy volunteers treated with Hydroalcoholic extract parmotrema peraltum 12292 Indian Journal of Natural Sciences Vol.7 / Issue 42 / June 2017 www.tnsroindia.org.in ©IJONS International Bimonthly ISSN: 0976 – 0997 Rajha Viknesh Madheshwar et al. Fig – 4: Dissolved Clots after Treating with Hydroalcoholic Extract Parmotrema perlatum 12293