Exploring Lichen Derived Compounds for Therapeutic Potential in Diabetes Management

For thousands of years, the natural world has served as a rich reservoir of medicinal compounds, with many contemporary pharmaceuticals originating from natural resources. A significant portion of these discoveries can be traced back to traditional healing practices. Plant life, in particular is abundant in secondary metabolites such as tannins, terpenoids, alkaloids, and flavonoids, which are recognized for their antimicrobial qualities. Lichens, symbiotic organisms composed of fungi and algae, show considerable promise for medical research. Research has demonstrated that metabolites derived from lichens, including depsides, depsidones, and usnic acid, are effective against mycobacteria and Gram-positive bacteria. As conventional drugs become less effective, there is a resurgence of interest in lichen-derived compounds. This investigation examines the antimicrobial and antioxidant properties of lichens found in Ooty, Tamil Nadu, India. Extracts from these lichens underwent analysis using LC-MS and various assays, such as phenol content, FRAP, ORAC, and DPPH, revealing significant antioxidant activity. In vitro experiments to inhibit pancreatic lipase, α-glucosidase, and α-amylase, coupled with in silico predictions of pharmacokinetic properties, toxicity risks, and molecular interactions, exhibited notable inhibitory potential. This study broadens our knowledge of bioactive compounds in lichens from temperate and polar regions and underscores their potential as natural alternatives for treating conditions associated with oxidative stress and metabolic syndrome.

Lichens, which represent symbiotic relationships between fungi (mycobionts) and algae or cyanobacteria (photobionts), generate a diverse array of secondary metabolites with significant biological effects. These compounds, including depsides, depsidones, dibenzofurans, and usnic acid, exhibit antimicrobial, antiviral, cytotoxic, and anti-inflammatory properties. Fundamental research involves systematic examination of lichen diversity, particularly in extreme environments, to identify and document bioactive metabolites. Historically, lichens have been utilized in various cultural practices such as dyeing, perfumery, and traditional medicine. In India, a mixture of lichen species known as 'Chharila,' comprising Parmelia, Usnea longissima, Ramalina subcomplanata, and Heterodermia tremulans, is traded for its traditional astringent, laxative, carminative, and aphrodisiac qualities. Species such as Evernia, Peltigera, Parmelia, and Cladonia are extensively used to treat fevers, skin conditions, infections, and liver ailments. For instance, Peltigera canina, which contains methionine, is used as a tonic for the treatment of liver disorders. Despite their rich pharmaceutical history, the medicinal potential of lichens remains unexplored. Challenges in species identification and large-scale production of chemical analysis hinder their broader medical applications. However, advancements in molecular biology and cultivation techniques may increase the access to these unique metabolites for novel pharmaceutical applications. Certain lichen species such as Heterodermia spp. have been investigated for their antibacterial properties. For example, H. prominens showed inhibitory effects against Mycobacterium tuberculosis, whereas A. sarmentosa showed activity against Pseudomonas aeruginosa. 

The growing demand for natural insecticides has also increased interest in lichen extracts for pest control. Lichens are used in various cultures worldwide. In Finland, Ramalina thrausta is used for skin disorders, while Usnea longissima serves as a wound dressing in Northwest countries. In India, Parmelia spp. is used in folk medicine to treat headaches, wounds, and ringworms. Cetraria islandica, which has been used as an antitussive for millennia, continues to be used in herbalist practices. It has been hypothesized that the antibiotic and preservative properties of lichen metabolites protect organisms from fungal attacks. The traditional importance of lichens and their ongoing use in medicine, perfumes, and dyes underscores their significance. Although mass collection and slow growth pose challenges, lichens have garnered increased attention in natural product research, particularly because of their antimicrobial and antioxidant properties. As molecular and experimental studies progress, lichens are likely to become increasingly valuable sources of novel bioactive compounds.

Sample Collection

TN Map

Nilgiris Map

Lichen samples were collected from Nilgiris mountain (Doddabetta) and Nanjanaad, Ooty, The Nilgiris. Foliose and Fruticose types were found commonly. The collected samples were recorded with the nature of the sample and were packed in the acid free packets and stored at - 4ºC in a deep freezer for further experiments and studies.

Spot test for the identification of lichens

Spot test is a chemical method, the chemicals were applied on the lichen fragments and the color change indicates their presence of secondary metabolites.

K test: 10% aqueous solution of potassium hydroxide (KOH) is prepared by adding 20 grams of potassium hydroxide pellets into the 100 ml of distilled water. The reagent is then sprayed with the help of a sprayer onto the lichen fragment.

C test: 5-25% of calcium hypochlorite were prepared by adding 50 grams of common bleach into 100 ml of distilled water and mixed well, this mixture is then allowed to settle down and the supernatant is used as reagent. The reagent is then sprayed with the help of a sprayer onto the lichen fragment.

I test: The iodine reagent prepared by dissolving the 0.5 grams of iodine and 1.5g of potassium iodide in 100ml of distilled water, the mixed solution reacts with the certain polysaccharides in lichen. The reagent is then sprayed with the help of a sprayer onto the lichen fragment.

Preparation of Extracts

Extracts are prepared with six different solvents: ethanol, methanol, chloroform, acetone, ethyl acetate and Water (Aqueous). The sample is made into powder and one gram of powdered sample is packed in a filter paper and immersed in 100 ml solvent and kept at room temperature for 24 hours.

Figure 1: Extract Preparation

The extracts from each solvent are collected by vaporizing each solvent using a water bath. 2 ml of extraction taken in a Petri plate and kept undisturbed at room temperature for 48 hours later the vaporized extracts form like a paste and those extracts are collected for further process.

Phytochemical Analysis of Extracts

Photochemical analysis is performed to identify the compounds that are present in the particular extracts. The tests for major compounds like alkaloids, protein and amino acids, carbohydrates, cholesterol, lignins, tannins etc., are confirmed by performing different analysis.

Antioxidant Activity

Total antioxidant capacity of extract was determined with phosphomolybdenum method using α-tocopherol as the standard expressed as µg/ml equivalent of α-tocopherol by using the standard tocopherol graph

FE3+ Reducing Power Ability

The reducing power of the lichen extract was determined by the Fe3+ to Fe2+ transformation. The reductones destroy the free radicals’ chain by donating hydrogen atoms. The Fe2+ can be identified by formation of Perl’s Prussian blue at 700 nm. Butylated hydroxytoluene (BHT) was used as the control.

(DPPH) Assay

Solution having strength of 0.3mM DPPH in ethanol and methanol was prepared and 1 ml of this solution was added to 3 ml of the extract residue dissolved in ethanol / methanol at different concentrations (10-100µg/ml). This mixture was shaken and allowed to stand at room temperature for 30 min and the absorbance was measured at 517 nm. Ascorbic acid was used as standard.

DPPH radical scavenging activity (%) = [(Abs control – Abs sample)] / (Abs control)] X 100

α-Amylase inhibition assay

For each experimental condition, including positive and negative controls, a total volume of 150 μL of the sample was prepared in an Eppendorf tube. This was supplemented with 200 μL of starch solution, 50 μL of distilled water, and 100 μL of α-amylase enzyme. The mixtures were incubated for 5 minutes at 37°C. Following this incubation, 200 μL of the resultant mixture was transferred to a new Eppendorf tube, to which 100 μL of DNS reagent was added. This solution was then incubated for 20 minutes at 100°C. After incubation, 900 μL of distilled water was introduced to the mixture and allowed to cool to room temperature. Finally, 200 μL of each prepared solution was transferred to a microplate and the absorbance was measured at 515 nm using a microplate reader.

α-Glucosidase inhibition assay

In this assay, acarbose was utilized as the standard for the calibration curve, which was constructed using concentrations ranging from 10 to 100 μg/mL in a 20 mM phosphate buffer (specifically at 10, 25, 50, 75, and 100 μg/mL). For the ethanolic extracts, working solutions were prepared at concentrations between 10 and 100 μg/mL, derived from a stock solution of 1 mg/mL (1,000 μg/mL) in phosphate buffer. A volume of 50 μL from each working solution was combined with 50 μL of 5.0 mM pNPG and incubated for 5 minutes at 37°C. Following this incubation, 100 μL of α-glucosidase enzyme (at a concentration of 0.1 U/mL) was added. The absorbance was measured at 405 nm every minute for a total duration of 20 minutes using a microplate reader.

Pancreatic lipase inhibition assay

Orlistat was employed as the standard in this study, and a calibration curve was established using concentrations ranging from 0.5 to 80 μg/mL in 70% ethanol (specifically at 0.5, 1, 5, 10, 20, 30, 50, and 80 μg/mL). For the ethanolic extracts, solutions were prepared at concentrations between 10 and 100 μg/mL, derived from a stock solution of 1 mg/mL (1,000 μg/mL) in Tris-HCl buffer. For each working solution, a volume of 25 μL was combined with 50 μL of 5.0 mM NPC and 25 μL of pancreatic lipase enzyme (10 mg/mL), followed by incubation for 5 minutes at 37°C. The absorbance was subsequently measured at 410 nm using a microplate reader.

Analysis of the pharmacological properties

The identified phytochemicals from the extracts of lichens using UHPLC-ESI-QTOF-MS were screened for their pharmacological potential and inhibitory activity against α-amylase, α-glucosidase, and human pancreatic lipase. Osiris DataWarrior software version 5.5.0 was applied to investigate their pharmacokinetic properties. In most of the cases, drug-likeness was screened as per Lipinski's Rule of Five. The rules consist of the criteria for oral bioavailability, which include molecular weight lower than 500 Da, cLogP below 5, less than 5 H bond donors, no more than 10 H bond acceptors, and not more than 10 rotatable bonds. The extra calculations involved topological polar surface area (TPSA) and

Spot Test identification

The significant colour change indicates the presence of depsides and depsones compounds in the lichen.

Figure 2: Spot Test

Sample Collection

Figure 3: sample collected, 25 Different sample were collected and transported in acid free bags and stored in - 4ºC for further identification.

Phytochemical Analysis

Among the 6 solvents used the highest activity were observed only in the ethanol and ethyl acetate extracts for the 16 phytochemical constituents (saponins, triterpenoids, anthocyanins, phlobatannins, flavonoids, carboxylic acid, reducing sugars, cholesterol, tannins, alkaloids, lignins, carbohydrates, protein, amino acids). Among 25 samples collected 10 samples were subjected for the identification of the potential activity.

Figure 4: Chemical structures of some major compounds identified in ethanolic extracts (A) 2,5DHA (2,5-dihydroxyterephthalic acid), (B) cyperine,(C) diospyrol, (D) hypoxyphenone,(E) lecanoric acid, (F) orsellinic acid, (G) prephenic acid, (H) SDA (succinyl disalicylic acid) and (I) O4BBA (o-(4- biphenylyl carbonyl benzoic acid).

DPPH Radical scavenging assay

The identified phytocomponents then subject the ethyl acetate extract to an antioxidant activity evaluation with DPPH radical scavenging assay at five different concentrations (10 μg/mL, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) using 10 replicates. The highest concentration used for samples were 75 μg/mL.

Antioxidant Activity 

The Table shows the result of the antioxidant activity of selected lichen samples.

Table 1: Antioxidant activity of the ethanolic extract 

FE3+Reducing power ability: The Table shows the result of the antioxidant activity reducing power ability of selected lichen samples and values of the standards

Table 2: FE3+ Reducing ability of the selected lichens

α-Glucosidase inhibition assay

The Table shows the result of the α-Glucosidase inhibition assay of selected lichen samples and values of the standards

Table 3: α-Glucosidase inhibition of the selected lichens

Pancreatic Lipase Inhibition Assay

The Table shows the result of the pancreatic lipase assay of selected lichen samples and values of the standards

Table 4: Pancreatic Lipase Inhibition

Pharmacological Properties analysis

The pharmacological properties of the phytochemicals identified and tabulated. 

Table 5: Pharmacology properties obtained lichens based on Lipinski’s rule.

Toxicology risk study of the extracts

Table 6: Toxicity risk of the phytochemicals obtained

Evaluation of docking α-amylase inhibition

Compounds with no toxicity risk and that passed Lipinski's rule after pharmacokinetic and toxicological evaluation were selected for further research. The said compounds were proposed as potential α-amylase inhibitors, whose binding behavior at the catalytic site of α-amylase was further evaluated by in silico molecular docking analysis. Table 3 Binding Affinities of Selected Compound Compared to Reference Inhibitor, Acarbose Evaluating whether or not the selected compounds' affinities were the same or much more potent than the reference inhibitor, acarbose. Results obtained from the molecular docking showed that the compound diospyrol, O4BBA, and lecanoric acid ranked the top as the most potent binding agents to α-amylase enzyme with values of −9.00, −8.70, and −8.10 kcal/mol, respectively. It was more than that for acarbose, with a value of −7.80 kcal/mol. This had been attributed to good ligand interaction maps as well as the stable geometries adopted by the compounds in the catalytic pocket of supporting both energetic and geometric stability at the active site.

Figure 5

Evaluation of docking α-glucosidase inhibition

In silico analysis results for the interaction of the phytochemicals with the α-glucosidase enzyme. Fig 8 In silico analysis results for the interaction of the phytochemicals with the α-glucosidase enzyme. Binding behavior of compounds in comparison to the reference inhibitor acarbose: In order to verify if the phytochemicals presented equivalent or even better performance in relation to the inhibition of the α-glucosidase enzyme, we compared the binding behavior of compounds in relation to the reference inhibitor acarbose. We calculated the binding affinities for each compound, including acarbose, as shown in Table 9. The worthy compounds were diospyrol and O4BBA, which showed the higher binding affinity at −8.80 and −8.40 kcal/mol respectively against that of −7.00 kcal/mol in acarbose. The interaction mapping showed that diospyrol presented four hydrogen bond interactions with the amino acids Asp 197, Thr 199, Asp 437, and Asp 536, besides four π-anion interactions with Asp 197 and Asp 536. These interactions made significant contributions to stabilizing the binding affinity and geometry of diospyrol at the catalytic site compared to the reference inhibitor, acarbose.

Figure 6: Docking molecular between phytochemicals and the α-glucosidase enzyme

Table 7: Binding affinities of lichen compounds with α-amylase, α-glucosidase, and pancreatic lipase. 

The lichenized fungi ethanolic extract was established to contain several bioactive compounds, including aromatic, carbohydrate, acid, lipid, and depside types, which may have potential positive effects on diverse biological activities both in vitro and in vivo. The extract exhibited relatively significant antioxidant activity that is directly proportional to the concentration of total phenols. The extracts were generally more inhibitory to α-glucosidase but less effective in the case of α-amylase and pancreatic lipase compared to the standard from the perspectives of in vitro enzyme inhibition. However, many compounds have shown very strong intermolecular interactions with α-glucosidase and α-amylase and pancreatic lipase catalytic sites, including 2,5-dihydroxyterephthalic acid, cyperine, diospyrol, hypoxyphenone, lecanoric acid, orsellinic acid, prephenic acid, succinyldisalicylic acid, and o-(4-biphenylylcarbonyl) benzoic acid in the in-silico evaluation. These extracts and compounds have shown promise in the treatment of metabolic diseases such as diabetes mellitus, and neurodegenerative disorders caused by oxidative damage, such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis.

The future will be focused on the bio-guided fractionation of the ethanolic extracts, isolation, and structural elucidation of major compounds as well as biological testing in murine models. This way, an attempt will be made to further validate the pharmacological, nutritional, and biomedical potential of the derived bioactive compounds from lichenized fungi. Compounds NN1222006 and NN1222022 represented outstanding performance in all tested parameters and, thus, can be considered promising candidates for development as natural antidiabetic agents. Its pleiotropic effects on oxidative stress, glycemic control, and lipid metabolism may place it high on the list of potential adjunctive or add-on therapies to the presently offered remedies for diabetes

Ethical Approval

This research on lichen derived compounds for diabetes management was conducted following the ethical principles established by the Hindusthan college of Arts & Science.

Funding

This work was not supported by any funding agency. The funding body had no involvement in the study design, data collection, analysis, or manuscript preparation.

Availability of Data and Material

On requirement the data’s may be set to available 

Conflict of Interest

The author declares that there are no conflicts of interest regarding the research, authorship, and publication of this study.