Context: Lichens have been used for various purposes such as dyes, perfumes and remedies in folk medicine indicating the pharmaceutical potential of lichens. Objective: Lichen growth in nature is very slow. To overcome this major drawback, we standardized the culture media to culture the lichen Usnea complanata (Müll.Arg.) Motyka (Parmeliaceae) for (
Keywords: Lichen culture; fermentation; enzyme inhibitory kinetics; antioxidants
Free radicals in relation to cardiovascular diseases and oxidative modification of circulating lipoproteins are important for the development of atherosclerosis. Particularly low-density lipoproteins, which are known to be a risk factor for cardiovascular diseases, may promote atherogenesis for several reasons. This eventually causes disruption of cell membranes, leading to the release of cell contents and death. Antioxidant therapy may inhibit atherosclerosis and thereby prevent the clinical complications of the disease such as coronary artery disease, including hypertension, hypercholesterolemia, postmenopausal state, thrombotic tendency and the risk of developing myocardial infraction. In healthy individuals, antioxidants protect components of the body against free radical damage ([
Uncontrolled platelet aggregation is critical in arterial thrombosis and may cause life-threatening disorders such as heart attacks, unstable angina and reocclusion after angioplasty ([
β-Hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), a rate-limiting enzyme in endogenous cholesterol synthesis, is a 97-kDa glycoprotein and catalyzes the reductive deacylation of HMG-CoA to mevalonate ([
Lichen-forming fungi are a large and diverse group of organisms that includes more than 13,000 described species. Lichens occur in all ecosystems on all continents and are the dominating organisms in extreme environments, including polar or alpine vegetations. They are able to grow on different substrates, including bare soil, rocks, barks of trees, wood, shells of barnacles and leaf surfaces. Past and current studies on biological activities of some lichen secondary metabolites of natural thallus exert a wide variety of biological actions including antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, plant growth inhibitory, antiherbivore, enzyme inhibitory, antiproliferative and cytotoxic effects, which are best supported by the available evidence ([
If the lichen metabolites having potential biological activities are to be used for modern pharmaceutical/nutraceutical applications, then the suitable in vitro culture conditions of the source organisms should be optimized ([
Recently, we have tested the antimicrobial, antioxidative and cardiovascular-protective activity of the natural thallus extract of a lichen Usnea complanata (Müll. Arg.) Motyka (Parmeliaceae) in our laboratory. The extract showed moderate-to-strong action on the above biological activities ([
Lichen growth in nature is very slow and for screening of the biological activities of a particular species huge amount of natural thallus is required for the extraction, which is not feasible in most of the cases ([
With this background, the present work is an extension to our previous work. In this work we have standardized the culture media to culture the lichen U. complanata and their growth optimization for in vitro synthesis of lichen substances usnic acid and psoromic acid, and determined the antioxidative and cardiovascular-protective activity of total extract of cultured lichen tissue and purified compound alone.
1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2-tert-butyl-4-methoxyphenol (BHA) and pyrocatechol were purchased from HiMedia, Merck. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR), HMG- CoA, pravastatin, angiotensin converting enzyme (ACE), hippuryl-l-histidyl-l-leucine (HHL), captopril and plasmin were purchased from Sigma-Aldrich Chemical Co., USA. All other routine chemicals used were of AR grade obtained from one of the following suppliers SRL, HiMedia, Merck.
Natural thallus of lichen species U. complanata was collected from Silver Oak trees in Mahabaleshwar (Satara-District, Maharashtra, India) in July 2008 and authenticated by the lichen taxonomist Dr. U.V. Makhija at Mycology Group, Agharakar Research Institute, Pune. A part of the specimen (Accession no: 70.31) has been deposited at Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune, India (Figure 1A).
Graph: Figure 1. A: Natural thallus of lichen Usnea complanata. B: 90-day-old culture cell derived from natural thallus fragments. C: Cells composed of blue colored fungal hyphae and dark green algal cells (micropreparation from the same culture). D: Culture in the conical flask containing liquid MY medium. E: Fermentation of cells derived from thallus.
Natural thallus of lichen species U. complanata was cultured following the methodology described by Yamamoto et al. ([
Graph: Figure 2. A: HPLC chromatogram showing production of usnic acid and psoromic acid in the three month old symbiont culture of U. complanata. B: HPLC chromatogram of purified usnic acid from in vitro cultured lichen U. complanata. C: HPLC chromatogram of purified psoromic acid from in vitro cultured lichen U. complanata.
After 30 days, a small portion of fresh cultured lichen material was collected from the Erlenmeyer flask and was then inoculated in the 5-L fermentor (BIOSTAT B plus-5L CC, Sartorius Stedim Biotech, Germany) containing 3 L of liquid medium of Malt-Yeast extract with pH 6.0 at stirrer 200 rpm (Figure 1E). Temperature was controlled between 18 and 20°C with an alternating photoperiod of 10 h light/14 h dark. The batch was run for a period of 30 days and then the lichen culture biomass was harvested. The lichen biomass was then weighed and further processed for extraction of biomass with different organic solvents and then processed for subsequent studies, i.e., quantification, isolation of lichen substances and to study their biological activities.
Cultured tissue of lichen species U. complanata was air-dried at room temperature and then extracted by various organic solvents such as ethanol, methanol, ethyl acetate and acetone using a Soxhlet extractor. The extract obtained was filtered using Whatman No. 1 filter paper and then evaporated in a water bath at 40°C until a solid powder was produced. This powder was weighed and further dissolved in respective solvents.
In order to obtain pure lichen substances, usnic acid and psoromic acid from the total extract collected from cultured tissue in bioreactor and stationary flask culture were vacuum freeze dried for 12 h. Dried extract (5–10 g) was further dissolved in acetone for 4 h at room temperature and then preparative TLC was performed with solvent system TDA. For usnic acid, Rf class was found to be 6.0 cm and for psoromic acid it was 3.0 cm, which was similar to the respective standards used. The two separated spots of usnic acid and psoromic acid on silica plate were scraped carefully and redissolved separately in acetone. The supernatant obtained after centrifugation at 8000 rpm containing the pure lichen compound was carefully decanted to other screw cap bottle and then acetone was evaporated in a water bath at temperature 40°C. Finally, the purity of the substance was further checked by HPLC. Lichen substances were identified by their peak symmetry and their retention time (retention time for usnic acid was 6.9 min and for psoromic acid was 3.3 min), by comparison with authentic substances made to the standard concentration (Figure 2B and C).
In order to determine the presence of antioxidant compounds in the crude lichen extracts, the extracts were analyzed by TLC using aluminium-backed TLC plates (Silica gel 60 F254) following the methodology of [
Graph: Figure 3. A: Yellow colored spot developed after spraying DPPH solution indicates presence of antioxidant compound in the extract of cultured lichen U. complanata. Spot 1: Ethanol extract, 2: acetone extract, 3: ethyl acetate extract. B: Identification type of phenolic group present in the cultured acetone extract of U. complanata symbiont.
Lichens mainly produce secondary metabolites are phenolic compounds along with other accessory pigments ([
Like other higher plants, lichens also produce polysaccharides and polyphenolic compounds. Thus, the polysaccharide and polyphenolic content in the culture extract were determined using the phenol-sulphuric acid method described by [
Antioxidant activity of the cultured lichen extract and the purified lichen compound usnic acid and psoromic acid was measured in terms of free radical scavenging activity (FRSA), nitric oxide radical scavenging activity (NORSA) and inhibition of lipid peroxidation. Details of the antioxidant assay procedure with slight modification have been described by us earlier ([
HMGR inhibitory activity was determined following the method described by [
The ACE inhibitory activity of the lichen purified compound usnic acid and psoromic acid was determined using the method of [
In order to study the ACE inhibitory kinetics, the substrate HHL concentrations 2.5, 5.0, 10, 15, 20 and 25 mM and inhibitor usnic acid and psoromic acid at concentrations of 0, 50 and 250 µg were used. Similarly for HMG-CoA reductase inhibitory kinetics, the substrate HMG-CoA concentrations 50, 100, 200, 250 and 300 mM and inhibitor usnic acid and psoromic acid at concentrations of 0, 50 and 250 µg were used. The mode of action of ACE and HMG-CoA reductase in the presence of inhibitor or without inhibitor was determined by using Lineweaver Burk plot analysis and their Km and Vmax values were recorded.
The purified lichen metabolites usnic acid and psoromic acid were incubated at 4°C for 1 month in the refrigerator and residual activity in terms of lipid peroxidation inhibition (LPI) was estimated. Further, in order to know the thermosensitivity of the purified lichen metabolites, usnic acid and psoromic acid were incubated at 40°C for 2 h and LPI activity was estimated.
In the present study, we have investigated the antioxidant and cardiovascular-protective activities of total extract and isolated compound usnic acid and psoromic acid alone of the lichen species U. complanata grown in stationary flask culture and in the fermentor.
Antioxidant activity in terms of FRSA, NORSA and LPI of the total extract and the purified compound usnic acid and psoromic acid alone were measured, and the results are presented in Tables 1–4. All the individual organic solvent extract of lichen tissue showed concentration-dependent FRSA, NORSA and LPI activity. The increasing ethanol extract concentration from 25 to 100 µg/mL in the assay mixture showed 55 to 71% FRSA comparable to other organic solvent extract for this activity. In the case of NORSA, methanol extract concentration from 25 to 100 µg/mL showed 25 to 61% activity, whereas other solvent extract with same concentration showed 29 to 35% activity. Furthermore, methanol extract concentration of 25 to 100 µg/mL inhibited 27 to 60% lipid peroxidation, other solvent extract showed 18.5 to 40% LPI. All the solvent extracts that showed antioxidant activity were found to be less than the positive control used as BHA a synthetic antioxidant at a concentration of 50 µg/mL that showed 78.9% FRSA, 62.3% NORSA and 67.3% LPI (Table 1).
Table 1. Antioxidant activity in terms of FRSA, NORSA and LPI with various concentrations of extracts of cultured lichen.
Extract concentration (μg/mL) FRSA (%)* NORSA (%)* LPI (%)* M E A EA M E A EA M E A EA 25 30.12 54.66 50.44 50.0 25.07 30.58 29.23 33.41 27.18 25.61 27.35 18.59 50 48.55 58.0 53.33 52.0 36.25 31.69 32.92 33.39 37.91 30.33 33.80 22.39 100 59.35 71.33 62.00 60.44 60.81 34.64 33.53 35.38 60.40 40.0 37.76 31.65 BHA (50 μg/mL) 78.89 62.28 67.26
10 M, methanol; E, ethanol; A, acetone; EA, ethyl acetate; BHA, butylated hydroxyl anisol (synthetic antioxidant). *Values presented are mean of five independent measurements.
As far as IC
Table 2. IC50 values in μg of the cultured lichen U. complanata extract for FRSA, NORSA and LPI.
Extract IC50 (μg/mL) FRSA* NORSA* LPI* Methanol 80 72.52 74.58 Ethanol 22.86 144.34 125 Acetone 25.0 149.12 132.41 Ethyl acetate 25.0 141.32 157.97 BHA (standard) 31.68 40.14 37.16
11 *Values presented are mean of five independent measurements.
In many reports, it has been found that in some cases the purified compound alone is bioactive, and in other cases extract is bioactive, suggesting a synergistic effect with other compounds in the extract ([
Antioxidant activity of purified usnic acid and psoromic acid was ascertained in terms of FRSA, NORSA and LPI. The results are presented in Table 3. The purified compound usnic acid and psoromic acid showed concentration-dependent FRSA, NORSA and LPI activity. Usnic acid concentration from 0.005 to 0.2 mg/mL showed FRSA 4.85 to 51.2%, NORSA 23.5 to 53.2% and LPI 24.4 to 46.6%, respectively. Psoromic acid at the same concentration showed FRSA 2.8 to 36.8%, NORSA 19.3 to 47.5% and LPI 27.8 to 57.3%, respectively. The positive control BHA a synthetic antioxidant at a concentration of 0.05 mg/mL showed FRSA 78.9%, NORSA 62.3% and LPI 67.3%, respectively. These results showed 50% of antioxidant activity by usnic acid with an IC
Table 3. Antioxidant activity in terms of FRSA, NORSA and LPI activities of lichen metabolites usnic acid and psoromic acid developed from lichen U. complanata in vitro culture.
Concentration (mg/mL) FRSA (%)* NORSA (%)* LPI (%)* Usnic acid Psoromic acid Usnic Acid Psoromic acid Usnic acid Psoromic acid 0.005 4.85 2.83 23.46 19.28 24.44 27.82 0.025 19.02 27.53 36.48 26.28 31.79 31.79 0.1 32.79 31.17 42.62 36.60 36.04 51.33 0.2 51.21 36.84 53.19 47.54 46.57 57.31 BHA (0.05) 78.89 62.28 67.26
12 *Values presented are mean of five independent measurements.
Table 4. IC50 value in mg/mL of purified lichen metabolites usnic acid and psoromic acid from in vitro cultured lichen U. complanata for their antioxidant activity in terms of FRSA, NORSA and LPI.
Antioxidant activity Lichen metabolite IC50 (mg/mL)* Standard IC50 (mg/mL)* Usnic acid Psoromic acid BHA FRSA 0.195 0.271 0.031 NORSA 0.188 0.21 0.040 LPI 0.214 0.174 0.037
13 *Values presented are mean of five Independent measurements.
Cardiovascular-protective activity of the solvent extract and the purified compound usnic acid and psoromic acid obtained from the cells of lichen U. complanata grown under submerged fermentation condition is presented in the Tables 5–8. This activity was measured in terms of HMGR and ACE inhibition and fibrinolytic activity. At the concentration of 60 µg/mL, methanol and ethyl acetate solvent extract showed 65.18 to 74.81% HMGR inhibition and was found higher than the ethanol and acetone extract which inhibited HMGR from 2.22 to 21.48% at the same concentration. However, HMGR inhibition showed by the extracts was found to be lower than the positive standard HMGR inhibitor pravastatin (95.55%) at concentration of 50 µg/mL (Table 5).
Table 5. HMGR inhibition by the solvent extract of in vitro cultured lichen U. complanata in the presence of 200 μM concentration of HMG-COA substrate.
Extract (60 μg/mL) Inhibition (%)* Ethanol 21.48 Methanol 65.18 Acetone 02.22 Ethyl acetate 74.81 Standard pravastatin (50 μg) 95.55
14 *Values presented are mean of five independent measurements.
As far as ACE inhibition by the solvent extract is concerned, the methanol extract at the concentration of 10 µg/mL inhibited ACE 43.47%, which is almost double than the ethanol and acetone extract 21.73% to 23.18% at the same concentration. No inhibition of ACE was found by the ethyl acetate extract at the same concentration (Table 6).
Table 6. ACE inhibition by different solvent extract of in vitro cultured lichen U. complanata in the presence of substrate HHL at a concentration 10 mM.
Extract (10 μg) Inhibition (%)* Ethanol 21.73 Methanol 43.47 Acetone 23.18 Ethyl acetate — Captopril (10 μg) 32.66
15 *Values presented are mean of five Independent measurements.
The mode of action of purified usnic acid and psoromic acid on the HMGR and ACE was studied by inhibition kinetics (Table 7 and Figure 4). In the case of HMGR inhibition, usnic acid used as inhibitor at the concentration of 50 and 250 µg with the substrate HMG-CoA concentration 50, 100, 200, 250 and 300 mM showed noncompetitive type of inhibition with a Vmax 1.4 and 1.16 U/mgP; Km 51.0 mM. Further, 50 and 250 µg psoromic acid used for the inhibition of HMGR showed competitive type of inhibition with Vmax 3.8 U/mgP and Km 100 to 125 mM. As far as inhibition kinetics of ACE is concerned by the inhibitor usnic acid and psoromic acid at the same concentration with the substrate concentration HHL 2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 mM, usnic acid showed uncompetitive type of inhibition with Vmax 0.50 and 0.38 U/mL; Km 3.78 and 2.55 mM and psoromic acid showed mixed type of inhibition with Vmax 0.38 and 0.34 U/mL; Km 10.86 and 16.06 mM, respectively.
Table 7. Enzyme kinetics for inhibition of ACE and HMG-CoA reductase enzyme by the lichen acids developed in vitro and their Vmax and Km values.
Inhibitor ACE inhibitory Type of inhibition HMG Co-A reductase inhibitory Type of inhibition Vmax (U/mL) Vmax (U/mgP) UA 50 μg 0.50 3.78 Uncompetitive 1.4 51.0 Noncompetitive UA 250 μg 0.38 2.55 1.16 51.0 PS 50 μg 0.38 10.86 Mixed 3.8 100.0 Competitive PS 250 μg 0.34 16.06 3.8 125.0
16 UA, usnic acid; PS, psoromic acid.
Graph: Figure 4. Lineweaver-Burk plots were drawn from assays using a range of lichen metabolite (inhibitor i.e. usnic and psoromic acid) at concentrations (0, 50, 250 µg/ml) with various substrate HMG-CoA concentrations (
Fibrinolytic activity in terms of zone of hydrolysis of fibrinogen in the presence of purified lichen compound usnic acid and psoromic acid was measured and the results are presented in Table 8. At 100 µg concentration of usnic acid or psoromic acid shown zone of hydrolysis 0.8 to 1.4 cm
Table 8. Fibrinolytic activity in terms of zone of hydrolysis of fibrinogen in the presence of lichen metabolites purified from the in vitro cultured lichen U. complanata.
Lichen metabolite Concentration (μg) Zone of hydrolysis (cm2) Usnic acid 100 1.4 Psoromic acid 100 0.8 Plasmin (control) 12.5 1.6
Since solvent extract had shown from moderate-to-strong antioxidative and cardiovascular-protective activity, in order to know apart from the lichen substances (usnic acid and psoromic acid) what other compounds might play a role in the observed biological activities, we measured total polyphenol and polysaccharide content in the solvent extract. The results are presented in Table 9. A concentration of 100 µg of solvent extracts showed total soluble polyphenol ranging from 17.5 to 24.5 µg and polysaccharide 7.76 to 16.45 µg.
Table 9. Polyphenol and polysaccharide content in the 100 μg of in vitro cultured U. complanata extract.
Extract Total polyphenol (μg/100 μg extract)* Polysaccharide (μg/100 μg extract)* Ethanol 17.5 12.42 Methanol 24.5 16.45 Acetone 20.0 7.76 Ethyl acetate 21.0 12.21
17 *Values presented are mean of five Independent measurements.
Stability and thermosensitivity of usnic acid and psoromic acid isolated from the cell grown from lichen U. complanata under submerged fermentation condition has been studied and the results are presented in Table 10. In this study, on day 1, we measured antioxidative activity in terms of LPI by adding usnic acid and psoromic acid individually at concentration of 100 µg/mL in the assay mixture and measured the LPI activity. Further, the usnic acid and psoromic acid that were preserved in the refrigerator for 20 days at 4°C were again tested for its potential for LPI activity. About 4.6% to 6.5% decrease in the LPI activity was found. Similarly, thermosensitivity of usnic acid and psoromic acid was also tested by keeping the bottle containing usnic acid and psoromic acid in a water bath at 40°C for 2 h. After 2 h of thermal incubation again LPI was measured. The concentrations of usnic acid and psoromic acid used in the assay were same as indicated above; 14.21% to 20.48% decrease in LPI activity was observed. This result suggests that the purified usnic acid and psoromic acid can retain the LPI potential for longer period, if they will be preserved at <4°C.
Table 10. Incubation stability of the lichen metabolites isolated and purified from in vitro cultured lichen U. complanata measured antioxidative activity in terms of LPI.
Compound Concentration (μg/mL) LPI activity first day/0 h After 20 days incubation at 4°C After 2 h incubation at 40°C LPI Activity % R.A. % Loss LPI Activity % R.A. % Loss Usnic acid 100 36.04 34.38 95.39 4.61 30.92 85.79 14.21 Psoromic acid 100 51.38 48.0 93.51 6.49 40.82 79.52 20.48
18 % R.A., Percentage residual activity.
Lichens are well-known for the diversity of secondary metabolites that they produce and display various biological activities. Throughout the ages, lichens have been used for various purposes as dyes, perfumes and remedies in folk medicines and also have potential healing/curing power. Even though the lichens, or the natural compounds they produce, have immense potential for the development of important therapeutic drugs, they have been long neglected by the modern pharmaceutical industries. The main reason for this is lichens grow very slow in nature and their axenic culture in laboratory is very difficult. For studying the biological activities of lichens, screening program has been the starting point in the drug research ([
The culture of lichen species U. complanata has been tried with various media, i.e., MY, LB, BB, Bischoff and Bold, and MS medium. Although initially observed very slow growth of mycelia in LB, BB, Bischoff and Bold, and MS medium till the period of 60 days, thereafter there was no growth of mycelia. When microscopically observed, the grown tissue in LB, BB, Bischoff and Bold, and MS medium, we found that there was no algal cells development. Further, we have checked the production of lichen substances usnic acid and psoromic acid by TLC method but we could not find even trace of lichen substances production except few unidentified pigments spots. However, MY medium favored the growth of symbionts for the production of usnic acid and psoromic acid in this lichen under laboratory conditions as evident by microscopical, TLC and HPLC testing. In the fermentor, we observed both myco- and photobionts aggregatively grown (evident from slide prepared from wet lichen culture) and produced usnic and psoromic acids. When this species has been cultured under stationary flask containing liquid broth of MY medium extract (Figure 1D), after 90 days of incubation afforded 0.705 g dry biomass/flask in that batch culture from 30 conical flasks was collected, total biomass obtained was 21.1 g, whereas under fermentation 11.1 g dry biomass was obtained after 30 days. Quantity of purified compounds obtained was usnic acid 31 mg and psoromic acid 27 mg from 15 g dried cultured biomass. Our results are in agreement with those reported that the chemical profile of the bionts is influenced and modulated qualitatively and quantitatively by varying osmotic conditions, composition of the nutrient medium, the physiological state or culture age, culture conditions, or the presence of photobiont ([
The lichen substances are unique as they are unknown in other plant sources. Lichens contain many characteristic aromatic compounds with known antiviral, antimicrobial, antiproliferative, antimitotic and antioxidant activities ([
With this information, in the present study we have evaluated the antioxidative, cardiovascular-protective potential of the total extract and two lichen substances isolated and purified from a lichen species that grow in stationary flask culture and in the fermentor. Antioxidative activity was measured in terms of FRSA, NORSA and LPI. All the extract showed concentration-dependent antioxidant activity, in which ethanol extract had 71% FRSA, methanol extract showed NORSA 61% and LPI 60% at a higher concentration of 100 µg/mL were found to be lower than the BHA a synthetic antioxidant (positive control) at a concentration of 50 µg/mL. As far as half-inhibiting ethanol, acetone and ethyl acetate extract concentration for FRSA is concerned, IC
Cardiovascular-protective effects of extract and purified lichen compound usnic acid and psoromic acid were evaluated in terms of their HMGR inhibition, ACE inhibition and fibrinolytic potential. Like antioxidative potential, cardiovascular-protective effects were also found by lichen extract and the purified compound in a dose-dependent manner. Methanol and ethyl acetate extract were strong HMGR inhibitors (65.18–74.81%) at a concentration of 60 µg/mL but methanol extract alone showed moderate inhibition of ACE (43.47%) at 10 µg/mL. When the mode of action of purified lichen compound usnic acid and psoromic acid on the inhibition of the HMGR and ACE was studied by inhibition kinetics, usnic acid showed a noncompetitive type and psoromic acid competitive type HMGR inhibition; further, usnic acid showed uncompetitive type and psoromic acid had mixed type of ACE inhibition. As far as fibrinolytic activity is concerned, even at higher concentration (100 µg/mL) of usnic acid or psoromic acid, there was no or very poor zone of hydrolysis of fibrinogen. Further phytochemical composition analysis of the extract showed the presence of total soluble polyphenol ranging from 17.5 to 24.5 µg and polysaccharide 7.76 to 16.45 µg at the concentration of 100 µg of solvent extract. The variation in the mode of action by the extract or purified compound toward the cardiovascular-protective effects could be attributed to several reasons. Secondary substances produced by the "tissue" cultures in many cases chemistry are usually different from the chemosyndrome of the corresponding natural lichen thalli (Yamamoto et al., [
As far as stability and thermosensitivity of the purified usnic acid and psoromic acid for the observed biological activity antioxidative potential is concerned, the experimental results indicated that both the compounds had slightly decreased in LPI potential preserved at 4°C for 20 days, whereas 14.21 to 20.48% decrease in LPI potential at 40°C incubated for 2 h. This decrease in LPI activity could be attributed to the decrease of the concentration of active compound(s) or to the decomposition of active compound(s) at higher temperature ([
In conclusion, we were able to culture lichen U. complanata in stationary flask and in fermentor under submerged condition with the production of lichen substances usnic acid and psoromic acid. The cultured lichen extracts and purified usnic acid and psoromic acid showed moderate-to-strong antioxidative inhibition of HMGR and ACE activities. The properties of lichen substances make them possible pharmaceutical applications. However, applications derived from the activities described for usnic acid and psoromic acid have to be thoroughly studied, particularly antioxidative, antihyperlipidemia and antihypertensive domains. With current culturing techniques and rapid growth of this lichen, fungi can be screened industrially for potentially useful natural products.
We are very grateful to the Department of Biotechnology, Government of India, New Delhi, for financial support. We are thankful to Dr. U.V. Makhija, Mycology Group, for helping in taxonomic identification of lichen species. We are also thankful to Dr. D.G. Naik, Chemistry Group, Agharkar Research Institute, for providing the HPLC facility.
The Authors report no conflicts of interest.
By Bhaskar C. Behera; Nutan Mahadik and Mangesh Morey
Reported by Author; Author; Author