BIBECHANA Vol. 22, No. 2, August 2025, 141-150 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Phytochemical analysis, total flavonoid and phenolic content, antimicrobial properties of lichens extract of Hypotrachyna cirrhata (Fr.) Divakar Nirmala Sharma1,2, Deepa Karki1, Bhanu Bhakta Neupane1,∗, Achyut Adhikari1,∗ 1Central Department of Chemistry, Tribhuvan University, Kathmandu, 44618, Nepal 2Amrit Campus,Tribhuvan University, Kathmandu, 44618, Nepal ∗Corresponding authors. Email: achyutraj05@gmail.com (AA) bhanu.neupane@cdc.tu.edu.np (BBN) Abstract Despite the significant attention lichens have received for their unique secondary metabolites, research on edible varieties remains limited. The antioxidant activity of Hypotrachyna cir- rhata was evaluated using in vitro DPPH radical scavenging assays. The total phenolic content (TPC) and total flavonoid content (TFC) of H. cirrhata were measured as 42.165 ± 0.98 mg GAE/g and 11.789 ± 0.34 mg QE/g, respectively. In this assay, ethyl acetate demonstrated strong antioxidant potential with an IC50 value of 34.14 ± 0.17 µg/mL, whereas hexane showed a higher IC50 value of 74.3 ± 1.13 µg/mL, compared to the standard quercetin, which had an IC50 value of 62.87 ± 1.02 µg/mL. The methanolic extracts of the lichen demonstrated no- table antimicrobial effects against pathogens, with minimum inhibitory concentration (MIC) values recorded at 195.312 g/mL and minimum bactericidal concentration (MBC) ranging from 195.312 to 390.625 g/mL. The study suggested that H. cirrhata may exhibit significant levels of total phenolic content (TPC) and total flavonoid content (TFC), which are closely associated with enhanced antibacterial and antioxidant activities. Keywords Anti-oxidant, anti-bacterial, total phenolic content, total flavonoid Article information Manuscript received: November 22, 2024; Revised: April 10, 2025; Accepted: May 19, 2025 DOI https://doi.org/10.3126/bibechana.v22i2.71936 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Lichens represent a form of symbiotic organism, composed of a mycobiont (fungus) in association with cyanobacteria or algae [1] . These organ- isms are capable of surviving prolonged desiccation without succumbing to starvation, thanks to their ability to sustain a slow metabolic rate. Addition- ally, lichens exhibit extraordinary adaptability to a wide range of habitats, thriving on almost any sur- face and in diverse environmental conditions, from sea level to high alpine regions. They also play a 141 http://nepjol.info/index.php/BIBECHANA achyutraj05@gmail.com bhanu.neupane@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v22i2.71936 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 142 role in forming biological soil crusts and are fre- quently found on exposed soil surfaces, walls, roofs, and rocks [2]. Lichens manifest in four fundamen- tal growth morphologies: crustose, foliose, and fru- ticose [3]. Globally, approximately 20,000 lichen species have been documented, with Nepal's cata- log identifying 1,129 species, including infraspecific taxa, distributed across 237 genera and 66 fami- lies [2]. Since prehistoric times, lichens have been uti- lized for various purposes including food, dyes, per- fumes, medicinal treatments in folk traditions, and ornamental uses [4]. Lichen species produce a di- verse array of secondary metabolites, which have been isolated and shown to exhibit a wide spectrum of biological activities [5]. Numerous lichen extracts have been employed in traditional remedies, and screening tests have frequently revealed metabolites with antibiotic [6], antimycobacterial [7], antiviral [8], antitumor [9], analgesic [10] and antipyretic [11] properties.Lichens are capable of enduring extended periods of desiccation without experiencing star- vation, thanks to their slow metabolic processes. These efforts seek to explore the possible medici- nal uses of compounds obtained from lichens. The metabolites found in any lichen species can be pro- duced through one of three major metabolic path- ways: the polymalonate pathway, the mevalonic acid pathway, or the shikimic acid pathway [12]. These pathways not only facilitate the storage of carbohydrates but also the production of secondary metabolites [13]. Lichens have garnered considerable interest from researchers due to their therapeutic potential, which is ascribed to their distinctive compounds. Despite this, comprehensive data on the edibility of lichens and detailed evaluations of their culinary and medicinal applications remain limited. To in- vestigate the biological activities of the lichen H. cirrhata (Figure 1) studies have focused on their antioxidant and antimicrobial properties, alongside the identification of phytochemical constituents and the quantification of total phenolic and flavonoid content. 2 Materials and Methods 2.1 Collection and identification of lichens Lichens Hypotrachyna cirrhata were collected from the Makwanpur District with geographical distribu- tion (27° 60 81.83 N, 85° 09 22.81 E) around 2300 meter in altitude. The collected Lichens was identi- fied from the National Herbarium and Plant Labo- ratories, Department of Plant Resources, Godawari, Lalitpur, Nepal. Figure 1: Picture of Hypotrachyna cirrhata 2.2 Preparation lichen extract After meticulously removing dust particles from the lichens, they were air-dried in the shade at room temperature for two weeks and then ground into a powder. The powdered lichens were stored in pa- per bags for future use. A 100 g portion of the dry powder was soaked in methanol at a 1:10 ratio for 24 hours. Following this, the mixture was filtered, and the filtrates were concentrated using a rotary evaporator before being dried in the air. 2.3 Phytochemicals screening The solution containing extracts from the stem was tested, revealing the presence of phytochemi- cals such as alkaloids, steroids, tannins, saponins, glycosides, terpenoids, flavonoids, reducing sugars, and coumarins. These findings were obtained us- ing the standardized procedure described by A. J. Harborne [14]. 2.4 Determination of total phenolic content The total phenolic content (TPC) in the extract was evaluated using the Folin-Ciocalteu reagent with some modifications to a previously established method [15]. In short, 20 L of the extract at a concentration of 0.5 mg/mL was added in triplicate to 96-well plates. Then, 100 L of Folin-Ciocalteu reagent and 80 L of Na2CO3 were added to each well. The absorbance was measured at 765 nm us- ing a microplate reader (Epoch2, BioTek Instru- ments, Inc., USA) and expressed in mg of gallic acid equivalents (GAE) per gram of extract, based on a calibration curve created from standard gallic acid. Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 143 2.5 Determination of total flavonoid content The total flavonoid content (TFC) in the extracts was determined using a method with slight mod- ifications from a previously reported study [15]. Briefly, 20 L of plant samples at a concentration of 0.5 mg/mL was added to 96-well plates in trip- licate. Next, 110 L of distilled water was added to each well containing the plant samples, followed by 60 L of ethanol, 5 L of AlCl3, and 5 L of CH3CO2K. The absorbance was measured at 415 nm using a microplate reader and was expressed as milligrams of quercetin equivalent per gram of extract. 2.6 DPPH radical scavenging activ- ity The antioxidant capacity of the extracts was as- sessed using a modified 96-well plate method based on the colorimetric technique [16]. Quercetin at a concentration of 20 g/mL was used as the positive control, while 50% DMSO served as the negative control. Each well in the 96-well plate received 100 L of either the positive control (quercetin), negative control (DMSO), or plant samples, all in triplicate. Following this, 100 L of DPPH reagent was added to each well, and the plate was incubated in darkness for 30 minutes. After incubation, the absorbance was measured at 517 nm using a microplate reader. The DPPH radical scavenging activity was then cal- culated using the following formula: % Inhibition = ( Acontrol −Asample Acontrol ) × 100 where, Asample = absorbance of the sample Acontrol = absorbance of the control 2.7 Antibacterial activities Lichen extracts were evaluated for antibacterial ac- tivity using the agar-well diffusion method. The test microorganisms were incubated for 12 hours at 37°C after being adjusted to a turbidity equivalent to 0.5 McFarland standards (1.5 x 10ˆ8 CFU/mL or 10ˆ8 bacteria/mL) in MHB. Using a sterile cot- ton swab, bacteria were evenly spread across MHA plates. Three wells, each approximately 4 mm deep and 6 mm in diameter, were made in the cultured MHA plates using a cork borer. Each well was then filled with 50 µL of lichen extract (50 mg/mL in 50% DMSO). Separate wells were used for the neg- ative control, containing 50 µL of 50% DMSO, and the positive control, containing 50 µL of 1 mg/mL neomycin. The plates were left at ambient temper- ature for 15 minutes to allow diffusion, then incu- bated for 18 to 24 hours at 37°C. After incubation, the zone of inhibition (ZoI) for each extract was measured in millimeters 2.7.1 Determination of minimum in- hibitory concentration (MIC) The minimum inhibitory concentration (MIC) is the smallest concentration of a substance required to prevent any visible bacterial growth, determined using a slightly modified version of a previously es- tablished method [17]. It measures the effectiveness of a chemical against a specific type of bacteria and is typically expressed in mg/mL or µg. The MIC was assessed for methanolic extracts or other sol- vent fractions of the plant extract that showed sig- nificant antibacterial activity. To prepare solutions with varying concentrations from 12.5 mg/mL to 0.097 mg/mL, 100 L of stock solutions (50 mg/mL) were serially diluted twice in 96-well plates. Then, 5 L of bacteria (106 CFU/mL) was added to all wells except the negative control. The plates were cov- ered and incubated at 37°C for 18 to 24 hours. After incubation, 0.003% resazurin solution was added to each well, followed by another incubation for three to four hours at 37°C. A blue color indicated no bacterial growth, while a pink color, resulting from the enzyme reductase, signaled bacterial presence. The well with a blue color represented the MIC, or the lowest concentration required to inhibit bacte- rial growth. 2.7.2 Determination of minimum bac- tericidal concentration (MBC) The minimum bactericidal concentration (MBC) is the lowest concentration of a substance needed to kill specific bacteria. In MBC assay, resazurin serves as a dye that changes color upon its conver- sion to resorufin, signaling the presence of viable cells (Figure 2). Using serially diluted plant ex- tracts, the microdilution method is used to deter- mine this concentration following the CLSI proto- col [18]. For the MIC, higher concentrations were applied to MHA plates and incubated for 18 to 24 hours at 37°C. Bacterial growth was then observed on the MHA plates, and the MBC was identified as the lowest concentration where no bacterial growth occurred. Figure 2: Reaction for the conversion of resazurin to resorufin during MIC determination. Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 144 3 Results and Discussions 3.1 Results 3.1.1 Phytochemicals Screening The results from the phytochemical screening, ob- tained through chemical tests, are presented in Ta- ble 1 which also can indicate the different com- pound present in H. cirrhata. Table 1: Phytochemical Screening Serial No. Group of Compounds H. cirrhata 1 Alkaloids ++ 2 Flavonoids ++ 3 Terpenoids ++ 4 Glycosides ++ 5 Polyphenols ++ 6 Quinones ++ 7 Tannins ++ 8 Steroids – 9 Saponins – 10 Fatty acids – 11 Proteins (Xanthoprotein) ++ 12 Coumarins ++ 13 Anthraquinone – 14 Reducing sugar ++ Note: (++) indicates presence; (–) indicates absence. 3.1.2 Estimation of Total Phenolic Content (TPC) Gallic acid equivalent (mg GAE/g) was used to as- sess the total phenolic content in the crude extracts of H. cirrhata. The standard used to calculate TPC is gallic acid. The calibration curve is built us- ing different final concentrations of gallic acid (10, 20, 40, 60, 80, and 100) µg/mL. Using the regres- sion equation (Y = 0.017x+ 0.0552, R2 = 0.9907) derived from the gallic acid calibration curve, the TPC of the extracts was determined. TPC value for H. cirrhata was found to be 42.165± 0.98mg GAE/g.The calibration curve of gallic acid is shown in Figure 2 and the TPC value of extracts is repre- sented in Table 2. Figure 3: Calibration curve of gallic acid Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 145 3.1.3 Estimation of Total Flavonoid Content (TFC) Using the usual technique and quercetin as a refer- ence, the total flavonoid concentration of the crude lichen extract was estimated. The quercetin equiva- lent (mg QE/g of the extract's dry weight) was used to express the TFC value. The calibration curve is constructed using varying final concentrations of quercetin (100, 80, 60, 40, 20, 10) µg/mL. Using the regression equation (Y =0.0179x+ 0.0273, R2 = 0.9997) derived from the quercetin calibration curve, the TFC of the extract was determined. H. cirrhata had a TFC value of 11.789± 0.34 mg QE/g. Figure 3 displays the quercetin calibration curve, and Table 2 displays the TFC value for the extracts of lichens H. cirrhata. Figure 4: Calibration curve of Quercetin. Table 2: Total Phenolic Content and Total Flavonoid Content with Standard Deviation of Crude Extract of H. cirrhata Name of Lichen Total Phenolic Content (mg GAE/g) Total Flavonoid Content (mg QE/g) H. cirrhata 42.165 ± 0.98 11.789 ± 0.34 3.1.4 Anti-oxidant Inhibition Activi- ties The radical scavenging activity of methanolic ex- tract of lichen and their fractions are detailed in Table 3. All extracts demonstrated concentration- dependent free radical scavenging activity in the DPPH assay. Among the different extracts, the ethyl acetate extracts of H. cirrhata exhibited the highest radical scavenging activity, with IC50 val- ues of 34.14 ± 0.17 µg/mL. Conversely, the lowest activity was observed in the methanol extract of H. cirrhata with IC50 values of 74.3 ± 1.13 µg/mL. 3.1.5 Anti-bacterial Activities Along with MIC and MBC The following zone of inhibition of lichens ex- tract were evaluated against their antibacterial properties against the given bacteria. Salmonella typhii (ATCC 14028), Shigella sonnei (ATCC 25931), Acinetobacter baumannii (ATCC19606), Escherichia coli (ATCC 3292), Staphylococcus au- reus (ATCC 25923), Klebsiellapneumoniae (ATCC 700603), and Acinetobacter baumannii (ATCC 19606) and zone of inhibition was observed which are presented in Table 4 and Figure 4. In addition, this lichen exhibited inhibition zones of 14 mm and 16 mm at identical concentrations against Shigella sonnei and Staphylococcus aureus. Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 146 Table 3: Antioxidant Activity of Different Fractions of H. cirrhata in IC50 Values Lichen (Fractions) IC50 Values (µg/mL) Crude 74.3 ± 1.13 Hexane 44.35 ± 1.23 DCM 37.56 ± 0.49 Ethyl acetate 34.14 ± 0.17 Quercetin 62.87 ± 1.02 Note: Mean values ± SD (Standard Deviation) were measured. Figure 5: Showing inhibition zone of H. cirrhata in Staphylococcus aureus b) showing inhibition zone of H. cirrhata in Shigella sonnei. Each methanolic lichen extract was evaluated against bacteria, showing the largest inhibition zones, along with corresponding MIC and MBC values (see Figure 5 and Table 5). The MIC for Shigella sonnei was determined to be 195.312 g/mL, which was also the observed MBC. For Staphylococcus aureus, the MIC was 390.625 g/mL, matching its MBC. Additional details of the MIC and MBC values are presented in Table 5, along with Figures 5 and 6.Each methanolic lichen ex- tract was evaluated against bacteria, showing the largest inhibition zones, along with corresponding MIC and MBC values (see Figure 5 and Table 5). The MIC for Shigella sonnei was determined to be 195.312 g/mL, which was also the observed MBC. For Staphylococcus aureus, the MIC was 390.625 g/mL, matching its MBC. Additional details of the MIC and MBC values are presented in Table 5, along with Figures 5 and 6. Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 147 Figure 6: MIC of lichen extracts and antibiotics against Shigella sonnei : 5–7: (A–H: 12.5–0.098 mg/mL) for H. cirrahata, 8–10: Antibiotic (Neomycin A–H: 250–1.95 g/mL), 11: Positive control (AH: Media + bacteria), 12: Negative control (A–H: Media only), and b)MIC of lichen extracts and antibiotics against Staphylococcus aureus 5–7: (A–H: 12.5–0.098 mg/mL) for H. cirrahata, 8–10: Antibiotic (Neomycin A–H: 250–1.95 g/mL), 11: Positive control (AH: Media + bacteria), 12: Negative control (A–H: Media only). Figure 7: MBC of lichen extracts and antibiotics against (a) Staphylococcus aureus and (b)Shigella sonnei Table 4: Zone of Inhibition of H. cirrhata Extract Against Bacterial Strains Serial No. Name of Bacteria Positive Control (mm) H. cirrhata Extract (mm) 1 Escherichia coli 18 – 2 Shigella sonnei 25 14 3 Salmonella typhi 20 – 4 Acinetobacter baumannii 24 – 5 Klebsiella pneumoniae 18 – 6 Staphylococcus aureus 22 16 Note: (–) indicates no zone of inhibition observed. Nirmal Sharma et al./ BIBECHANA 22 (2025) 141-150 148 Table 5: MIC and MBC Values of H. cirrhata Name of Bacteria MIC (µg/mL) MBC (µg/mL) Shigella sonnei 195.3125 195.3125 Staphylococcus aureus 390.625 390.625 4 Discussions Lichens have been traditionally employed in medicine worldwide, with much of this knowledge originating from cultural practices. The primary secondary metabolites in this species are phenolic compounds, notably depsides and depsidones [19] , [20–22]. In this study, the total phenolic con- tent (TPC) of the methanolic extract of H. cir- rhata was found to be 42.165 ± 0.98 mg GAE/g, surpassing the TPC values of acetone, water, and petroleum ether extracts of R. taitensis, which were 22.87 ± 0.12, 6.3 ± 1.06, and 12.58 ± 0.082 mg GAE/g, respectively [23]. This indicates higher phenolic content in H. cirrhata. Similarly, the to- tal flavonoid content (TFC) in the crude extract was 11.789 ± 0.34 mg QE/g. In the methano- lic extract of Ramalina lacera, the TFC was 3.97 ± 0.3 mg RE/g of dry weight (DW), while in the aqueous extract, it measured 0.01 ± 0.03 mg RE/g DW [24].The ethyl acetate extract of H. cirrhata demonstrated the highest radical scavenging activ- ity, with an IC50 value of 34.14 ± 0.17 µg/mL, likely due to its abundance of phenolic compounds with hydroxyl groups that act through various an- tioxidant mechanisms, such as free radical scaveng- ing [25]. R. hossei and R. conduplicans showed rad- ical scavenging activity at 79.05% and 72.63%, re- spectively [26], whereas C. furcata exhibited lower antioxidant activity, with percentages of 44.83% for the acetone extract, 47.39% for the methanol ex- tract, and 29.99% for the aqueous extract. In com- parison, the methanol extract of P. sulcata showed 71% activity [27,28]. The methanolic extract of H. cirrhata displayed inhibition zones of 14 mm and 16 mm against Shigella sonnei and Staphylococcus aureus, with minimum inhibitory concentration (MIC) and min- imum bactericidal concentration (MBC) values of 195.312 g/mL and 390.625 g/mL, respectively. Ac- tivity against Gram-negative bacteria (e.g., Es- cherichia coli, Salmonella typhi) was absent. The absence of activity against Gram-negative bacte- ria may be due to their unique outer membrane made of lipopolysaccharides, which acts as a bar- rier to many antimicrobial agents. Efflux pumps and selective porin channels further limit compound entry. Thus, the tested compound may either lack the ability to cross this barrier or be specific to Gram-positive bacteria.In contrast, the ethano- lic and hexane extracts of P. tinctorum exhibited smaller inhibition zones of 9.4 mm and 7.7 ± 0.2 mm, respectively, at a concentration of 300 mg/mL against methicillin-susceptible S. aureus (MSSA, ATCC 25923) [29]. Additionally, studies show that methanol, ethanol, and acetone extracts from var- ious lichens exhibit antibacterial activity against Gram-positive bacteria, such as B. subtilis and S. aureus, while showing limited inhibition against Gram-negative bacteria [29]. 5 Conclusion The study highlighted the biological potential of H. cirrhata in terms of antioxidant and antibacterial properties. Lichens have garnered significant inter- est recently due to their unique secondary metabo- lites. 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Introduction Materials and Methods Collection and identification of lichens Preparation lichen extract Phytochemicals screening Determination of total phenolic content Determination of total flavonoid content DPPH radical scavenging activity Antibacterial activities Determination of minimum inhibitory concentration (MIC) Determination of minimum bactericidal concentration (MBC) Results and Discussions Results Phytochemicals Screening Estimation of Total Phenolic Content (TPC) Estimation of Total Flavonoid Content (TFC) Anti-oxidant Inhibition Activities Anti-bacterial Activities Along with MIC and MBC Discussions Conclusion