Phytochemical Investigation, Antioxidant and Antimicrobial Activities of Some Parmeliaceae Lichens from Western Himalaya

ISSN: 0973-7510

E-ISSN: 2581-690X

Abstract
Keywords
Introduction
Materials and Methods
Results
Discussion
Conclusion
Declarations
References

Research Article | Open Access

Phytochemical Investigation, Antioxidant and Antimicrobial Activities of Some Parmeliaceae Lichens from Western Himalaya

Kiran Pant¹, Pushpa Joshi¹ , Dalip Kumar Upreti² , Sanjeeva Nayaka²and Jyoti Darmwal¹

¹Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India.

²Lichenology Laboratory, CSIR – National Botanical Research Institute, Rana Pratap Marg, Lucknow, Uttar Pradesh, India.

Article Number: 9825 | © The Author(s). 2025

J Pure Appl Microbiol. 2025;19(1):263-274. https://doi.org/10.22207/JPAM.19.1.17

Received: 23 August 2024 | Accepted: 19 November 2024 | Published online: 07 February 2025

Issue online: March 2025

Abstract

This study aimed to examine the antioxidant and antimicrobial activities and the phytochemical composition of three Parmeliaceae lichens from the western Himalayas. Three lichen species (Everniastrum cirrhatum (Fr.) Hale, Parmotrema reticulatum (Taylor) M. Choisy, and Usnea orientalis Motyka) were analyzed for antimicrobial, antioxidant assay, and chemical analyses using Gas chromatography-mass spectrometry (GC-MS). The chemical constituents were identified, and the percentage of components obtained was calculated. Antimicrobial activity was tested with the agar well diffusion method, and the total antioxidant capacity was measured using the phosphomolybdenum method (Total Antioxidant Capacity – TAC) and the hydrogen peroxide radical scavenging test. GC-MS analysis of methanol extracts from the lichens revealed 57 compounds in all three species. Methanol extracts from these lichens demonstrated strong antimicrobial activity (inhibition zones: 8.6 ± 0.09 to 28.2 ± 0.23 mm) with the highest activity against Salmonella typhi. They also exhibited low minimum inhibitory concentrations (MICs) of 0.125 mg against many microbes. The antioxidant capacity of methanol extracts ranged from 1.256 to 1.991 mg/g ascorbic acid equivalent. Interestingly, the hexane extract of E. cirrhatum showed the highest hydrogen peroxide scavenging activity at 91.13%. This study highlights the antioxidant and antimicrobial properties of these Parmeliaceae lichens, attributed to their diverse secondary metabolites. These findings suggest promising prospects for the development of novel antimicrobial and antioxidant agents from lichen extracts, warranting further exploration in pharmacological and biomedical research.

Keywords

Biological Activity, Gas Chromatography-mass Spectroscopy, Antioxidants, Antimicrobial

Introduction

Lichens are composite organisms comprising of two microorganisms, a fungus and an algae, along with various non obligate viruses, bacteria, and fungi forming a microecosystem. Some fungi grow on the outer surface of lichen called lichenicolous fungi, while others live inside the lichen, known as endolichenic fungi. Both the main lichen and these secondary fungi produce special chemicals that have medicinal uses. Worldwide, there are about 20,000 types of lichens known, and India alone has more than 3,000 different species. This shows how diverse lichens are in India and around the world.¹ Lichens thrive in diverse and harsh environments like deserts, forests, alpine zones, tundras, and polar regions due to their unique physiology. They can grow on natural surfaces such as tree bark, soil and rocks, as well as on man-made structures like monuments and buildings. Lichens are excellent indicators of air pollution in an area.

Throughout history, lichens have been utilized for various purposes, including perfumes, dyes, food additives, fodder, and cosmetics. Many cultures around the world use lichen species for medicinal purposes. In ancient Ayurvedic medicine, species from the Parmotrema genus are commonly used to treat diarrhea, heart conditions, leprosy, skin ailments, and urinary disorders. Everniastrum cirrhatum is widely used as a crude drug² for the treatment for blood disorders, stomach ailments, leprosy, kidney stones, carminative, and aphrodisiac disorders.³ Usneas species produce usnic acid, renowned for its antibiotic properties.⁴

Lichens produce over 1050 secondary metabolites, with more than half demonstrating antibiotic properties. These substances are found as crystals or liquids in the upper cortex or among medullary hyphae.⁵ Extracts from lichens, whether crude or purified, display diverse biological activities, including antimicrobial, analgesic, antiviral, anti-inflammatory, antioxidant, hepatoprotective, antiulcer, anti-HIV, and antigen toxicity.⁶ The inefficiency of current antimicrobial drugs, along with drug resistance and emerging infectious diseases, is a major concern in medicine today.⁷ Staphylococcus aureus, for instance, has become resistant to multiple drugs, including methicillin, posing a significant global challenge. Many antibiotics developed in the mid-20^th century are no longer effective due to the pathogens’ resistance.⁸ Lichens are gaining attention for their pharmacological properties, including potent bioactivities and antioxidant compounds like phenolics.⁹ India, known for its biodiversity, hosts numerous lichen species, but only 160 have been recognized for their medicinal potential based on folklore, ancient literature, and preliminary biological screening.

Materials and Methods

Lichen collection and identification
The lichen materials were collected from Thalkedar Hills in the Pithoragarh district of Uttarakhand (India) during May 2022 and June 2023. The lichens were identified at the Lichenology Laboratory, CSIR-NBRI, in Lucknow. A voucher specimen Pamotrema reticulatum (Taylor) M. Choisy, no. 24929 (CSIR-LWG); Everniastrum cirrhatum (Fr.) Hale no. 47849 (CSIR-LWG); Usnea orientalis Motyka, no-24984/B (CSIR-LWG) was deposited at the CSIR-National Botanical Research Institute, Lucknow, India (Figure 1).

Figure 1. Lichen utilized for antimicrobial and antioxidant study. (A). Everniastrum cirrhatum, (B). Parmotrema reticulatum, and (C). Usnea orientalis

Preparation of extracts
The dried lichen materials were finely ground using an electric grinder, and pulverized material (10 g) was extracted twice by soaking with 100 ml of chloroform, hexane, and methanol for 48 h at room temperature. Further, the separated extracts were filtered through Whatman No. 1 filter paper, and the extract filtrates were condensed to dryness using a rotary evaporator at 40 °C, yielding approximately 15-20% of the extracts.

GC-MS analysis
The GC-MS analysis was performed using a Thermo GC-Trace Ultra Ver: 5.0 and Thermo MS DSQ II, equipped with a DB 35-MS Capillary Standard Non-Polar Column (30 m length, 0.25 mm ID, 0.25 µm film thickness). The instrument was initially set to 110 °C, held for 2 minutes, then gradually increased to 260 °C at a rate of 6 °C/min and maintained for 9 minutes. The injection port temperature was set to 250 °C with a helium flow rate of 1 ml/min. A 1 µl sample was injected in split mode (10:1), and ionization was done at 70 eV. The mass spectral scan range was set between 45-450 MHz. Chemical constituents were identified by comparing fragmentation patterns with the National Institute of Standards and Technology Mass Spectral database (NIST-MS), which contains over 62,000 patterns. The percentage of each component was determined from the relative peak areas in the chromatogram, and the name, molecular weight, and structure of the compounds were confirmed through GC-MS analysis. Methanol extracts of all three lichen samples were prepared separately for GC-MS analysis.

The microorganism
Pathogenic bacteria and fungi were obtained from the National Centre for Cell Science (NCCS) in Pune, including Bacillus subtilis (B. subtilis, MTCC 4755), Staphylococcus aureus (S. aureus, MTCC 4734), Klebsiella pneumonia (K. pneumonia, MTCC 7028), Salmonella typhi (S. typhi, MTCC 734) and Candida albicans (C. albicans, MTCC 10231).

Evaluation of antimicrobial activity
Agar well diffusion assay
The agar well diffusion method was used to assess the antimicrobial activity of the extracts against selected bacterial strains, including Bacillus subtilis and Staphylococcus aureus being Gram-positive, Klebsiella pneumonia and Salmonella typhi being Gram-negative strains, and Candida albicans was a fungal strain. A subculture of each strain (200 µl, equivalent to 10v CFU/ml) was uniformly spread over the surface of nutrient agar or PDA plates, and five wells were created using a sterile gel borer. Dimethyl sulfoxide (DMSO) was used as the negative control, while ampicillin (0.1 mg/ml) for bacteria and fluconazole (0.1 mg/ml) for fungi served as positive controls. The remaining wells were filled with 100 µl of hexane, chloroform, and methanol extracts, each labeled accordingly. The plates were incubated at 37 °C for 20 hours for bacteria and at 22 °C for 2 days for fungi, after which the zones of inhibition (ZOI) around the wells were measured.¹⁰

Minimum inhibitory concentration (MIC) assay
The minimum inhibitory concentration (MIC) of the extracts was determined using the microdilution method with nutrient broth and PDA broth. The MIC was tested against bacterial strains (Bacillus subtilis, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aureus, and the fungal strain Candida albicans). Five different concentrations of the extracts (1, 0.5, 0.250, 0.125, and 0.062 mg/ml) were prepared. Ampicillin and fluconazole were used as positive controls, while DMSO served as the negative control. A standardized suspension of the test bacteria and fungus was inoculated to create fresh cultures. Five milliliters of media were placed in each test tube, followed by the addition of 100 µl of the prepared extract concentrations and 20 µl of the fresh cultures. The test tubes were incubated for the appropriate time, and then turbidity was checked. The MIC value is defined as the lowest concentration of the extract at which there is complete inhibition of bacterial or fungal growth.¹⁰

Antioxidant activities
The samples were evaluated for their antioxidant potential using the following two assays:

Phosphomolybdenum method (Total antioxidant capacity-TAC)
An aliquot of 0.5 ml of the sample solution was mixed with 4.5 ml of reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. For the blank, 0.5 ml of DMSO was used instead of the sample. The extracts were added at a concentration of 1 mg/ml. The tubes were incubated in a boiling water bath at 95 °C for 90 minutes. After cooling to room temperature, the absorbance of each sample’s aqueous solution was measured at 695 nm against the blank using a UV-2450 spectrophotometer (Shimadzu, Japan). The total antioxidant capacity of the extracts was expressed as ascorbic acid equivalent (AAE), with the antioxidant capacity calculated using ascorbic acid as the standard for each extract.

Hydrogen peroxide radical scavenging test
The hydrogen peroxide (H₂O₂) scavenging activity of the extracts was determined by measuring the reduction of H₂O₂in a system containing H₂O₂and the scavenger, using the classical UV method at 330 nm. Phosphate buffer (pH 7.4) was added to all samples, each containing 0.6 ml of hydrogen peroxide. After incubating for 10 minutes, the absorbance was measured at 330 nm against a blank solution with phosphate buffer. The extracts were added at a concentration of 1 mg/mL.¹¹ The percentage of H₂O₂inhibition was calculated using the following formula:

Percentage (%) of hydrogen peroxide radical scavenging activity = [(A₀-A₁) / A₀] x 100

where A₀ is control, A₁ is test sample.

Data analysis
All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation (SD).

RESULTS

Gas chromatography-mass spectrometry (GC-MS)
The Gas chromatography-mass spectrometry (GC-MS) analysis of the methanol extracts of three lichens revealed a total of 57 pharmacological compounds (Figure 2-4). Further, comparison of obtained compounds in the NIST library and PubChem yielded their details and importance (Table 1-3). Everniastrum cirrhatum yielded 22 compounds such as Niacinamide (0.25%), Methotrexate (0.06%), Semicarbazide (0.09%), Carotene (0.15%), Lycoxanthin (0.41%), 2-Iodohiistidine (0.45%), Tetrazole (0.48%), Triprolidine (0.34%), Titanium (0.16%), Milbemycin B (0.21%), Chromone (0.22%), Carvacrol (0.12%), Androstane (0.15%), Trimethylsilyl (0.22%), Hexasiloxane (0.61%), Heptasiloxane (0.81%), Octasiloxane (0.29%), Tetradecanoic acid (0.23%), Colchicin (0.11%), Thymol (0.51%), Methadone (0.37%), W-18 (0.32%), which are of high pharmaceutical value.

Table (1):
List of compounds having pharmaceutical importance identified in Everniastrum cirrhatum

No.	RT	Compound Name	Molecular Formula	Mol. Weight	Area %	Biological activity
1	5.18	Niacinamide	C₆H₆N₂O	122	0.25	Antipruritic, antimicrobial
2	5.56	Methotrexate	C₂₀H₂₂N₈O₅	45	0.06	Anti-inflammatory actions
3	11.69	Semicarbazide	C₁₉H₂₃N₃O₂S	357	0.09	Antiviral, antitumor
4	11.81	Carotene	C₄₁H₅₈O	566	0.15	Antioxidant
5	12.79	Lycoxanthin	C₄₀H₅₆O	552	0.41	Antimicrobial
6	0.45	2-Iodohiistidine	C₆H₈IN_3O2	281	0.45	Antioxidant antiviral, antitumor
7	14.39	Tetrazole	C₁₆H₁₅N₅	277	0.48	Antiviral, antitumor
8	16.29	Triprolidine	C₁₉H₂₂N₂	278	0.34	Anticholinergic and sedative
9	16.33	Titanium	C₁₆H₁₆Br₂Ti₂	462	0.16	Antimicrobial
10	16.66	Milbemycin B	C₃₃H₄₇C₁O₇	590	0.21	Antiparasitic
11	20.41	Chromone	C₁₄H₁₆O₆	280	0.22	Antiviral, antitumor
12	21.52	Carvacrol	C₁₆H₂₈OSi	264	0.12	Antimutagenic, antigenotoxic
13	24.98	Androstane	C₂₉H₄₃NO₃Si	481	0.15	Antimicrobial
14	27.31	Trimethylsilyl	C₁₄H₂₄O₃Si₂	296	0.22	Antimicrobial
15	27.40	Hexasiloxane	C₁₂H₃₈O₅Si₆	430	0.61	Antimicrobial
16	27.61	Heptasiloxane	C₁₄H₄₄O₆Si₇	504	0.81	Antimicrobial
17	27.80	Octasiloxane	C₁₆H₅₀O₇Si₈	578	0.29	Antimicrobia
18	29.38	Tetradecanoic acid	C₃₇H₅₈O₈	630	0.23	Antielastase, antioxidant
19	29.86	Colchicine	C₃₁H₃₃NO₉	563	0.11	Anti-mitotic drug
20	30.93	Thymol	C₁₆H₂₈OSi	264	0.51	Antibacterial
21	32.43	Methadone	C₂₁H₂₇NO	309	0.37	Antagonist
22	33.24	W-18	C₁₉H₂₀ClN₃O₄S	421	0.32	Antitumor

Table (2):
List of compounds having pharmaceutical importance identified in Parmotrema reticulatum

No.	RT	Compound Name	Molecular Formula	Mol. Weight	Area %	Biological activity
1	3.76	Dialifor	C₁₄H₁₇CINO₄PS₂	393	0.32	Organophosphate
2	3.94	Tabersonine	C₂₁H₂₄N₂O₂	336	0.73	Anticancerous
3	4.02	Thiosemicarbazone	C₁₀H₁₃N₃S	207	0.07	Antiparasitic
4	4.55	Pentafluoropropionate	C₁₁H₈F₅NO₄	313	0.79	Antimicrobial
5	5.40	Hydrazinecarbothioamide	C₁₄H₁₄N₄O₃S₂	350	0.13	Antimicrobial
6	5.78	Pentanal	C₁₁H₁₄N₄O₄	266	0.14	Antifungal
7	6.65	Normethadol	C₂₀H₂₇NO	297	0.29	Antimicrobial, anticancerous
8	6.92	Propanedinitrile	C₁₈H₂₂N₂O	282	0.63	Antibacterial
9	7.01	Triprolidine	C₁₉H₂₂N₂	278	0.13	Anticholinergic and sedative
10	7.34	Lansoprazole	C₁₆H₁₄F₃N₃O₂S	369	0.13	Decreases gastricacid secretion
11	7.89	Valsartan	C₂₄H₂₉N₅O₃	435	0.25	Cardiac stimulation
12	10.34	Papaveroline	C₁₉H₂₁NO₅	343	0.33	Antiviral, cardioprotective, anti-inflammatory
13	10.34	Falcatine	C₁₆H₁₃NO₄	283	0.33	Antiviral
14	11.20	Sarreroside	C₃₀H₄₂O₁₀	562	0.12	Wound healing, antioxidant
15	11.20	Dehydrofelodipine	C₁₈H₁₇CI₂NO₄	381	0.12	Antiviral
16	11.20	Butanedial	C₂₀H₁₈O₆	354	0.12	Euphoric and sedative
17	11.50	Colchicine	C₃₁H₃₃NO₉	563	0.40	Anti-mitotic
18	14.03	Glafenin	C₁₉H₁₇CIN₂O₄	372	0.10	Anti-inflammatory
19	14.65	Buflomedil	C₁₇H₂₅NO₄	307	0.26	Antiplatelet
20	14.93	Schisandrol B	C₂₃H₂₈O₇	416	0.21	Anti-inflammatory
21	15.76	Pipemidic acid	C₁₄H₁₇N₅O₃	303	0.10	Antibacterial
22	16.01	Nitralin	C₁₃H₁₉N₃O₆S	345	0.08	Antimicrobial
23	17.07	Apraclonidine	C₉H₁₀CI₂N₄	244	0.68	Anti-inflammatory

Table (3):
List of compounds having pharmaceutical importance identified in Usnea orientalis

No.	RT	Compound Name	Molecular Formula	Mol. Weight	Area %	Biological activity
1	3.58	Tyramine	C₁₆H₁₄N₂O₄	298	0.31	Increasing blood pressure
2	3.58	Picolinamide	C₃₅H₃₄N₆O₃	586	0.31	Antifungal, antibiotic
3	3.58	Silane	C₁₄H₂₄OS	236	0.31	Antimicrobial
4	3.65	Cholic acid	C₂₄H₄₀O₅	408	0.09	Mutagenic
5	3.65	Carbonyl Oxy Camptothecin	C₂₈H₃₀N₄O₆	518	0.09	Anticancer
6	3.79	Piperidinethione	C₁₄H₁₈N₂O₂S	278	0.20	Antimicrobial, antioxidant
7	3.79	Bitropenyl	C₁₄H₁₄	182	0.20	Antioxidant, antimicrobial
8	3.91	2H-Pyran	C₁₆H₂₄O₂	248	0.12	Antibacterial, antifungal, phytotoxic
9	4.35	Hydrazine carbothioamide	C₁₄H₁₄N₄O₃S₂	350	0.20	Antibacterial and antifungal
10	4.35	Benzodioxole	C₁₁H₁₁NO₆	253	0.20	Antitumor, antibacterial
11	4.35	Hydrazone	C₁₂H₁₆N₄O₄	280	0.20	Antibacterial, anticonvulsant
12	4.53	Pyridazine	C₂₀H₁₃N₅O₂	355	0.52	Analgesic, anti-tumor, antiviral
13	4.53	Chlorendic anhydride	C₉H2CI₆O₃	368	0.52	Antibacterial
14	4.92	Mitoxantrone	C₂₂H₂₈N₄O₆	444	0.11	Antibiotics

Figure 2. Showing the GC-MS spectrum of Everniastrum cirrhatum extracted in methanol

Figure 3. Showing the GC-MS spectrum of Parmotrema reticulatum extracted in methanol

Figure 4. Showing the GC-MS spectrum of Usnea orientalis extracted from methanol

Parmotrema reticulatum yielded 23 pharmaceutically important compounds, such as Dialifor (0.32%), Tabersonine (0.73%), Thiosemicarbazone (0.07%), Pentafluoropropionate (0.79%), Hydra-zinecarbothioamide (0.13%), Pentanal (0.14%), Normethadol (0.29%), Propanedinitrile (0.63%), Triprolidine (0.13%), Lansoprazole (0.13%), Valsartan (0.25%), Papaveroline (0.33%), Falcatine (0.33%), Sarreroside (0.12%), Dehydrofelodipine (0.12%), Butanedial (0.12%), Colchicine (0.40%), Glafenin (0.10%), Buflomedil (0.26%), Schisandrol B (0.21%), Pipemidic acid (0.10%), Nitralin (0.08%), and Apraclonidine (0.68%).

Usnea orientalis yielded 14 compounds such as Tyramine (0.31%), Picolinamide (0.31%), Silane (0.31%), Cholic acid (0.09%), Carbonyloxycamptothecin (0.09%), Piperidinethione (0.20%), Bitropenyl (0.20%), 2H-Pyran (0.12%), Hydrazine carbothioamide (0.20%), Benzodioxole (0.20%), Hydrazone (0.20%), Pyridazine (0.52%), Chlorendic anhydride (0.52%) and Mitoxantrone (0.11%).

Antimicrobial activities
All the extracts demonstrated notable antibacterial activity, with zones of inhibition (ZOI) ranging from 8.3 ± 0.06 mm to 28.2 ± 0.23 mm. Among the extracts, methanol extracts from all three lichens were better than chloroform and hexane extracts. The methanol extract of P. reticulatum showed the greatest antibacterial effect against S. typhi (ZOI: 28.2 ± 0.23 mm). Methanol extracts also proved most effective for antifungal activity, with a ZOI range of 12.7 ± 0.36 mm. The methanol extract of E. cirrhatum showed the strongest activity against S. aureus (ZOI: 23.6 ± 0.08 mm). Similarly, Usnea orientalis hexane extract displayed the highest activity against S. aureus (ZOI: 24.3 ± 0.08 mm). Methanol extracts showed the highest antifungal activity, with a ZOI range of 16.3 ± 0.28 mm. Similar to antibacterial activity among the three extracts methanol, also showed better antifungal activity. It can be noted that although lichen extracts showed excellent antimicrobial activities, they are not better than the standards Ampicillin or Fluconazole. In most cases methanol extracts, of lichens showed minimum inhibitory concentrations of 0.125 mg against many microbes (Table 4).

Table (4):
Antimicrobial activity expressed as zone of inhibition for E. cirrhatum, P. reticulatum and U. orientalis extracts against S. aureus, B. subtilis, S. typhi, K. pneumonia and C. albicans (n = 3)

No.	Extract	Organism	Zone of inhibition (in mm) evaluated at 1 mg/ml	Efficiency
1	E. cirrhatum – Hexane	S. aureus	12.2 ± 0.03	++
2		B. subtilis	11.1 ± 0.05	++
3		S. typhi	14.8 ± 0.21	++
4		K. pneumoniae	12.4 ± 0.32	++
5		C. albicans	10.9 ± 0.21	+
6	E. cirrhatum – Chloroform	S. aureus	12.7 ± 0.04	++
7		B. subtilis	10.1 ± 0.16	+
8		S. typhi	15.7 ± 0.13	++
9		K. pneumoniae	10.4 ± 0.05	+
10		C. albicans	11.3 ± 0.32	++
11	E. cirrhatum – Methanol	S. aureus	23.6 ± 0.08	+++
12		B. subtilis	18.2 ± 0.14	++
13		S. typhi	23.5 ± 0.21	+++
14		K. pneumoniae	14.8 ± 0.32	++
15		C. albicans	16.7 ± 0.27	++
16	P. reticulatum – Hexane	S. aureus	10.9 ± 0.02	+
17		B. subtilis	08.3 ± 0.06	+
18		S. typhi	13.1 ± 0.04	++
19		K. pneumoniae	09.7 ± 0.24	+
20		C. albicans	09.5 ± 0.31	+
21	P. reticulatum – Chloroform	S. aureus	11.9 ± 0.13	++
22		B. subtilis	09.1 ± 0.14	+
23		S. typhi	14.6 ± 0.08	++
24		K. pneumoniae	13.9 ± 0.09	++
25		C. albicans	09.1 ± 0.33	+
26	P. reticulatum – Methanol	S. aureus	26.1 ± 0.15	+++
27		B. subtilis	20.9 ± 0.32	++
28		S. typhi	28.2 ± 0.23	+++
29		K. pneumoniae	13.8 ± 0.16	++
30		C. albicans	12.7 ± 0.36	++
31	U. orientalis – Hexane	S. aureus	24.3 ± 0.08	+++
32		B. subtilis	16.7 ± 0.04	++
33		S. typhi	20.4 ± 0.06	+++
34		K. pneumoniae	25.1 ± 0.15	+++
35		C. albicans	11.5 ± 0.27	++
36	U. orientalis – Chloroform	S. aureus	15.7 ± 0.07	++
37		B. subtilis	12.9 ± 0.05	++
38		S. typhi	24.1 ± 0.12	+++
39		K. pneumoniae	09.5 ± 0.31	+
40		C. albicans	12.7 ± 0.34	++
41	U. orientalis – Methanol	S. aureus	12.8 ± 0.05	++
42		B. subtilis	08.6 ± 0.09	+
43		S. typhi	22.2 ± 0.08	+++
44		K. pneumoniae	13.8 ± 0.21	++
45		C. albicans	16.3 ± 0.28	++
46	Amphicillin	S. aureus	32.2 ± 0.04	+++
47		B. subtilis	28.8 ± 0.23	+++
48		S. typhi	35.1 ± 0.26	+++
49		K. pneumoniae	33.9 ± 0.07	+++
50	Flucanazole	C. albicans	22.6 ± 0.30	+++

+++ is excellent, ++ is good, + is moderate

MIC activities
The minimum inhibitory concentration (MIC) values for microorganisms sensitive to the methanol extracts ranged from 62.5 to 125 µg/ml. In this study, the lowest MIC values were observed in the methanol extracts of E. cirrhatum against S. aureus and S. typhi. Similarly, the methanol extract of P. reticulatum showed the lowest MIC values against S. aureus and S. typhi. For U. orientalis, the hexane extracts exhibited the lowest MIC values against S. aureus, S. typhi, and K. pneumoniae. No MIC values were recorded for the standard antibiotics used in the study (Table 5).

Table (5):
MIC values obtained against S. aureus, B. subtilis, S. typhi, K. pneumonia and C. albicans

No.	Extract	Organism	Turbidity observed with drug concentration					MIC (mg)
No.	Extract	Organism	1 mg	0.5 mg	0.250 mg	0.125 mg	0.062 mg	MIC (mg)
1	E. cirrhatum – Hexane	S. aureus	clear	clear	clear	turbid	turbid	0.250
2		B. subtilis	clear	clear	clear	turbid	turbid	0.250
3		S. typhi	clear	clear	clear	turbid	turbid	0.250
4		K. pneumoniae	clear	clear	clear	turbid	turbid	0.250
5		C. albicans	clear	turbid	turbid	turbid	turbid	1
6	E. cirrhatum – Chloroform	S. aureus	clear	clear	clear	turbid	turbid	0.250
7		B. subtilis	clear	clear	turbid	turbid	turbid	0.5
8		S. typhi	clear	clear	clear	turbid	turbid	0.250
9		K. pneumoniae	clear	clear	turbid	turbid	turbid	0.5
10		C. albicans	clear	clear	turbid	turbid	turbid	0.5
11	E. cirrhatum – Methanol	S. aureus	clear	clear	clear	clear	turbid	0.125
12		B. subtilis	clear	clear	clear	turbid	turbid	0.250
13		S. typhi	clear	clear	clear	clear	turbid	0.125
14		K. pneumoniae	clear	clear	clear	turbid	turbid	0.250
15		C. albicans	clear	clear	turbid	turbid	turbid	0.5
16	P. reticulatum – Hexane	S. aureus	clear	clear	turbid	turbid	turbid	0.5
17		B. subtilis	clear	clear	turbid	turbid	turbid	0.5
18		S. typhi	clear	clear	clear	turbid	turbid	0.250
19		K. pneumoniae	clear	clear	turbid	turbid	turbid	0.5
20		C. albicans	clear	turbid	turbid	turbid	turbid	1
21	P. reticulatum – Chloroform	S. aureus	clear	clear	clear	turbid	turbid	0.250
22		B. subtilis	clear	clear	turbid	turbid	turbid	0.5
23		S. typhi	clear	clear	clear	turbid	turbid	0.250
24		K. pneumoniae	clear	clear	clear	turbid	turbid	0.250
25		C. albicans	clear	turbid	turbid	turbid	turbid	1
26	P. reticulatum – Methanol	S. aureus	clear	clear	clear	clear	turbid	0.125
27		B. subtilis	clear	clear	clear	turbid	turbid	0.250
28		S. typhi	clear	clear	clear	clear	turbid	0.125
29		K. pneumoniae	clear	clear	clear	turbid	turbid	0.250
30		C. albicans	clear	clear	turbid	turbid	turbid	0.5
31	U. orientalis – Hexane	S. aureus	clear	clear	clear	clear	turbid	0.125
32		B. subtilis	clear	clear	clear	turbid	turbid	0.250
33		S. typhi	clear	clear	clear	clear	turbid	0.125
34		K. pneumoniae	clear	clear	clear	clear	turbid	0.125
35		C. albicans	clear	clear	turbid	turbid	turbid	0.5
36	U. orientalis – Chloroform	S. aureus	clear	clear	clear	turbid	turbid	0.250
37		B. subtilis	clear	clear	clear	turbid	turbid	0.250
38		S. typhi	clear	clear	clear	clear	turbid	0.125
39		K. pneumoniae	clear	clear	turbid	turbid	turbid	0.5
40		C. albicans	clear	clear	turbid	turbid	turbid	0.5
41	U. orientalis – Methanol	S. aureus	clear	clear	clear	turbid	turbid	0.250
42		B. subtilis	clear	clear	turbid	turbid	turbid	0.5
43		S. typhi	clear	clear	clear	clear	turbid	0.125
44		K. pneumoniae	clear	clear	clear	turbid	turbid	0.250
45		C. albicans	clear	clear	turbid	turbid	turbid	0.5
46	Ampicillin	S. aureus	clear	clear	clear	clear	clear	NA
47		B. subtilis	clear	clear	clear	clear	clear	NA
48		S. typhi	clear	clear	clear	clear	clear	NA
49		K. pneumoniae	clear	clear	clear	clear	clear	NA
50	Fluconazole	C. albicans	clear	clear	clear	clear	clear	NA

Antioxidant activities
The lichen extracts exhibited high total antioxidant capacities, with the methanol extract of E. cirrhatum having the highest antioxidant capacity at 1.991 AAE. The methanol extract of U. orientalis had an AAE of 1.504, and the methanol extract of P. reticulatum had an AAE of 1.256. However, the percentage of hydrogen peroxide radical scavenging varied for different extracts with hexane extract, of E. cirrhatum showed maximum activity of 91.13%. The hexane extracts of P. reticulatum showed maximum activity of 90.52%. The hexane extracts of U. orientalis showing maximum activity of 65.05. It was observed that among all the lichen extracts, the hexane extracts exhibited the highest radical scavenging activity, followed by the chloroform extracts, and then the methanol extracts (Table 6).

Table (6):
Total antioxidant activity of lichen extracts through Phosphomolybdenum method and Hydrogen peroxide radical scavenging capacity (n = 3)

No.	Extracts at 1 mg/ml	Phosphomolybdenum method		Hydrogen peroxide radical scavenging capacity
No.	Extracts at 1 mg/ml	OD (695 nm)	Total Antioxidant Capacity (mg/gm AAE)	OD (330 nm)	Percentage (%)
1	E. cirrhatum – Hexane	0.176 ± 0.05	0.053	0.454 ± 0.04	91.13
2	E. cirrhatum – Chloroform	0.354 ± 0.08	0.125	1.208 ± 0.06	76.40
3	E. cirrhatum – Methanol	1.380 ± 0.14	1.991	2.989 ± 0.09	41.62
4	P. reticulatum – Hexane	0.331 ± 0.09	0.196	0.485 ± 0.12	90.52
5	P. reticulatum – Chloroform	0.402 ± 0.10	0.251	0.862 ± 0.10	83.16
6	P. reticulatum – Methanol	0.998 ± 0.16	1.256	2.983 ± 0.05	41.73
7	U. orientalis – Hexane	0.429 ± 0.19	0.258	1.789 ± 0.08	65.05
8	U. orientalis – Chloroform	0.235 ± 0.08	0.094	2.864 ± 0.16	44.06
9	U. orientalis – Methanol	1.198 ± 0.09	1.504	2.992 ± 0.12	41.56

DISCUSSION

The crude extracts or isolated compounds from lichens have shown a range of biological activities that are available in the public domain. These lichen constituents are being utilized in the development of ointments for treating fungal skin conditions, including tinea, ringworm, and athlete’s foot.¹² Usnea species are said to be used in many medicinal applications as a potent antibiotic and antifungal agent.¹³ In the study, the methanol extract of E. cirrhatum exhibited relatively low antibacterial activity against P. aeruginosa, E. coli, and S. aureus, with inhibition zones measuring 2.40 ± 0.10 mm, 2.97 ± 0.15 mm, and 2.83 ± 0.05 mm, respectively.¹⁴ In another study, the crude extract of the lichens E. cirrhatum showed significant antifungal activities against Aspergillus flavus.¹⁵ Several studies have investigated the antioxidant activities of lichen extracts using a similar set of organisms as those in the present study. The highest value of radical scavenging activity for U. orientalis and correlated it with phenolic content.¹⁶ In the recent studied, antimicrobial, antioxidant activities and phytochemical analysis for 12 lichen species which also included E. cirrhatum and U. orientalis. The antibacterial activity of E. cirrhatum ranged from zero to 12.7 ± 0.8 mm of ZOI. For U. orientalis the maximum ZOI for antibacterial activity was 13.5 ± 0.5 mm. In both cases methanol extract showed better antibacterial activity. However, in the case of antifungal both lichens showed poor activities. In case of the antioxidant study E. cirrhatum (48%) had poorer free radical scavenging activity in comparison to U. orientalis (62.87%).¹⁷

The current study demonstrates that lichen-specific substances exhibit a wide range of biological activities. This highlights the potential for these species in phytochemistry and medicine, suggesting they could be further explored through in vivo studies.¹⁸ The choice of extraction methods and solvents significantly impacts the nature and concentration of the extracts, while maintaining their physical and chemical integrity.^19,20 The methanolic extracts of lichens, in particular, show strong antioxidant activity, likely due to the high polarity of methanol, which effectively extracts these bioactive compounds.²¹ GC-MS analysis confirms the presence of antimicrobial and antioxidant compounds in the extracts, indicating their potential for pharmacological applications. Overall, these lichens serve as valuable benchmarks for their high biotechnological potential, demonstrating excellent antimicrobial and antioxidant properties.

CONCLUSION

Lichens have been utilized since ancient times for various purposes, including medicine, colorants, perfumes, food additives, and other medicinal uses, due to their diverse range of secondary metabolites. Lichen metabolites like niacinamide, methotrexate, tetrazole, chromone, carvacrol, tabersonine, pentanal, normethadol, lansoprazole, falcatine, colchicine, apraclonidine, silane, picolinamide, mitoxantrone belonging to several chemical classes and exhibit a broad spectrum of biological activities, including antibiotic, antiviral, antimycobacterial, anti-inflammatory, antipyretic, analgesic, cytotoxic, antiproliferative, and antioxidant properties. In the present study, lichen extracts have shown potent antimicrobial and antioxidant activities which may be attributed to the presence of lichen compounds. Among the three lichens E. cirrhatum has shown excellent antimicrobial and antioxidant study. Further, methanol extracts were found to contain strong antimicrobial and antioxidant effects. Therefore, more insights into in vivo studies and further research on isolation and characterization of active compounds are much needed in future research.

Declarations

ACKNOWLEDGMENTS
The authors are thankful to the Head, Department of Chemistry, D.S.B Campus, Kumaun University, Nainital, Uttarakhand and Director, CSIR – NBRI, Lucknow, for providing the laboratory facilities during the work.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
PJ and DKU designed the study. SN performed experimental analysis. KP performed the analytical calculations and numerical simulations. PJ and JD wrote the manuscript. All authors read and approved the final manuscript for publication.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.

ETHICS STATEMENT
Not applicable.

References

Nayaka S, Haridas B. Lichen forming and lichenicolous fungi of the Western Ghats, India. In Pullaiah, T. (ed.). Biodiversity Hotspots of the Western Ghats and Sri Lanka. Apple Academic Press Inc., Canada. Co-published with CRC Press (Taylor & Francis), U.K. 2024:89-107.
Crossref
Nayaka S, Upreti DK, Khare R. Medicinal lichens of India. In Drugs from plants. (Ed. PC Trivedi) Avishkar Publishers and Distributors, India. 2010:1-54
Upreti DK, Bajpai R, Nayaka S, Singh BN. Ethnolichenological studies in India: Future prosects. In: Jain AK (ed.) Emerging dimentions in Indian Ethnobotany, Scientific publishiers, Jaipur, 2015;199-237.
Cansaran D, Kahya D, Yurdakulol E, Atakol O. Identification and quantitation of usnic acid from the lichen Usnea species of Anatolia and antimicrobial activity. Zeitschriftf r Naturforschung C. 2006;61:773-776.
Crossref
Huneck S. The significance of lichens and their metabolites. Naturwissenschaften. 1999;86:559-70.
Crossref
Solarova Z, Liskova A, Samec M, Kubatka P, Busselberg D, Solar P. Anticancer potential of lichens secondary metabolites. Biomolecules. 2020;10(1):87.
Crossref
Walsh C. Where will new antibiotics come from? Nat Rev Microbiol. 2003;1:65-70.
Crossref
Kosanic M, Rankovic B. Lichen Secondary Metabolites as Potential Antibiotic Agents. In: Rankovic, B. (eds) Lichen Secondary Metabolites. Springer Cham. 2019:99-127.
Crossref
Pradhan S, Dash S, Parida S, Sahoo B, Rath B. Antioxidant and antimicrobial activities and GC/MS-based phytochemical analysis of two traditional Lichen species Trypethellium virens and Phaeographis dendritica. J Genet Eng Biotechnol. 2023;21(1):41.
Crossref
Srivastava P, Upreti DK, Dhole TN, Srivastava AK, Nayak MT. Antimicrobial property of extracts of Indian lichen against human pathogenic bacteria. Interdiscip Perspect Infect Dis. 2013;2013(1):709348.
Crossref
Hussen EM, Endalew SA. In vitro antioxidant and free-radical scavenging activities of polar leaf extracts of Vernonia amygdalina. BMC Complement Med Ther. 2023;23(1):146.
Crossref
Wilson CO, Gisvold O, Delgado JN, Remers WA. Wilson and Gisvold’s textbook of organic medicinal and pharmaceutical chemistry. Lippincott Williams & Wilkins. 2004;991
Shukla P, Upreti DK, Tewari LM. Secondary metabolite variability in lichen genus Usnea in India: A potential source for bioprospection. G-Journal of Environmental Science and Technology. 2015;2(3):44-55.
Kumar SVP, Kekuda TRP, Vinayaka KS, Swathi D, Mallikarjun N, Nishanth BC. Studies on proximate composition, antifungal and anthelmintic activity of a macro-lichen Ramalina hossei H. Magn & G. Awasthi. Int J Biotechnol Biochem. 2010;6(2):191-201.
Furmanek L, Czarnota P, Seaward MRD. The effect of lichen secondary metabolites on Aspergillus fungi. Arch Microbiol. 2022;204:100.
Crossref
Rankovic B, Rankovic D, Kosanic M, Maric D. Antioxidant and antimicrobial properties of the lichens Anaptychya ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga. Cent Eur J Biol. 2010;5(5):649-655.
Crossref
Kumar P, Nayaka S, Verma T, Niranjan A, Upreti DK. Comparative analysis of antimicrobial, antioxidant activities and phytochemicals of Himalayan lichens. Biomass Conv Bioref. 2024;1:1-21.
Crossref
Adesalu TA, Agadagba T. Isolation of symbionts and GC-MS analysis of lichens collected from Obudu mountain resort, South-South, Nigeria. Ife Journal of Science. 2016;18(2):427-434.
Dirar AI, Alsaadi DHM, Wada M, Mohamed MA, Watanabe T, Devkota HP. Effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from Sudanese medicinal plants. S Afr J Bot. 2019;120:261-267.
Crossref
Keneni YG, Bahiru LA, Marchetti JM. Effects of different extraction solvents on oil extracted from jatropha seeds and the potential of seed residues as a heat provider. Bioenerg Res. 2021;14:1207-1222.
Crossref
Azmir J, Zaidul ISM, Rahman MM, et al. Techniques for extraction of bioactive compounds from plant materials: A review. J Food Eng. 2013;117(4):426-436.
Crossref

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