Assessment of Antimicrobial Activity of Lichenic Compounds Isolated from Menegazzia terebrata (Hoffm.) A. Massal

Abstract

The antibiotic activity of lichenic compounds atranorin, physodic, and stictic acid isolated from Menegazzia terebrata were analyzed. The antibiotic bioactivity was assessed against eight bacterial inoculums i.e., 3g positive and 5g negative by analyzing the minimal inhibitory concentrations (MICs) using the broth tube dilution method. All lichenic compounds showed antibiotic activities. The maximum bacterial growth inhibition activity was observed by atranorin (MIC, 0.030-0.5 mg/mL) and the minimum in physodic acid (MIC, 1 mg/mL). Stictic acid (MIC, 0.25-0.5 mg/mL) exhibited moderate antibiotic activity. Among the tested bacterial inoculums, all lichenic compounds reported antibacterial activity against a streptomycin-resistant strain of Pseudomonas aeruginosa. As a result, the study referred to the antibiotic potential of lichenic compounds, which could be developed further for pharmaceutical purposes.

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RESEARCH ARTICLE
Copyright © 2022 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Biobased Materials and Bioenergy
Vol. 16, 418–423, 2022
Assessment of Antimicrobial Activity of Lichenic
Compounds Isolated from Menegazzia terebrata
(Hoffm.) A. Massal
Himanshu Rai1∗, Rajan Kumar Gupta1, Devvret Verma2, Shreesh Gupta3, Debasis Mitra4,
Pradeep Kumar Das Mohapatra4, Areej Suliman Al-Meshal5, Rokayya Sami6∗,
Amal Adnan Ashour7, and Alaa Shafie8
1Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
2Department of Biotechnology, Graphic Era Deemed to be University, Dehradun 248002, Uttarakhand, India
3WecapsulatePharma, Kandoli, Dehrdaun 248001, Uttarakhand, India
4Department of Microbiology, Raiganj University, Raiganj 733134, Uttar Dinajpur, West Bengal, India
5Department of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia
6Department of Food Science and Nutrition, College of Sciences, Taif University, P.O. 11099, Taif 21944, Saudi Arabia
7Department of Oral & Maxillofacial Surgery and Diagnostic Sciences, Faculty of Dentistry, Taif University, P.O. Box 11099,
Taif 21944, Saudi Arabia
8Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
The antibiotic activity of lichenic compounds atranorin, physodic, and stictic acid isolated from
Menegazzia terebrata were analyzed. The antibiotic bioactivity was assessed against eight bac-
terial inoculums i.e., 3 g positive and 5 g negative by analyzing the minimal inhibitory concen-
trations (MICs) using the broth tube dilution method. All lichenic compounds showed antibiotic
activities. The maximum bacterial growth inhibition activity was observed by atranorin (MIC, 0.030–
0.5 mg/mL) and the minimum in physodic acid (MIC, 1 mg/mL). Stictic acid (MIC, 0.25–0.5 mg/mL)
exhibited moderate antibiotic activity. Among the tested bacterial inoculums, all lichenic compounds
reported antibacterial activity against a streptomycin-resistant strain of Pseudomonas aeruginosa.
As a result, the study referred to the antibiotic potential of lichenic compounds, which could be
developed further for pharmaceutical purposes.
Keywords: Atranorin, Broth Tube Dilution, Lichenic Compounds, Menegazzia terebrata,
Physodic Acid, Stictic Acid.
1. INTRODUCTION
Lichens are mutualistic association of fungus and algae
[1]. They are considered as the most diverse organ-
isms found in circumpolar terrestrial habitats [2]. Lichens
have been formally mentioned in several pharmacopeias
[3, 4] and are used as traditional medicines all over the
world. About 800 lichenic compounds are produced by
lichens, with 650 (82%) being unique to them [5]. The
lichenic compounds produced by lichens are known to
have antimicrobial, anticancer, anticytotoxicity, antioxi-
dant, anti-inflammatory, and immunomodulating bioactiv-
ities [6–9]. Among the different habitats, lichens growing
∗Authors to whom correspondence should be addressed.
Emails: himanshurai08@yahoo.com, rokayya.d@tu.edu.sa
in high-altitude temperate and alpine regions harbor
higher concentrations of lichenic compounds with an
extensive range of bioactivities [10–12]. Though lichen
crude extracts and isolated lichenic acids show efficient
antibiotic activity [8, 13–17]. The bactericidal activities
of lichen secondary metabolites against antibiotic drug-
resistant bacteria are important in the current pharmaceu-
tics [18–21]. Among the various lichens, the Parmelioid
lichens exhibit effective antibacterial properties worldwide
[9, 12, 21–29]. The Himalayas are some of the lichen-
rich regions in the Indian subcontinent, and Parmelioid
lichens from these habitats have shown efficient antimicro-
bial activities [30]. The Himalayan Parmelioid lichens can
provide lichenic compounds that can be effective against
antibiotic-resistant bacteria.
418 J. Biobased Mater. Bioenergy 2022, Vol. 16, No. 3 1556-6560/2022/16/418/006 doi:10.1166/jbmb.2022.2201

RESEARCH ARTICLE
Raietal. Assessment of Antimicrobial Activity of Lichenic Compounds Isolated from Menegazzia terebrata (Hoffm.) A. Massal
The current study, therefore, analyses the antibiotic
activity of lichenic compounds isolated from a temperate-
alpine lichen Menegazzia terebrata (Hoffm.) A. Massal
against some pathogenic food spoiling and mycotoxin-
producing bacteria.
2. MATERIALS AND METHODS
2.1. Lichen Sampling, Identification, and Curation
The lichen Menegazzia terebrata (Hoffm.) A. Massal
was collected from the higher elevation of Govind
wildlife sanctuary and national park, Uttarkashi district
of Uttarakhand in western Himalaya. The lichen sample
was collected on the Osla to Harki Dun route from N
310854.20E78
2529.59 at an elevation of 3485 m,
from a rocky substratum (Fig. 1). The lichen sample was
identified up to species level using appropriate literature
and standardized diagnostic chemical analysis, i.e., spot
tests, thin layer chromatography TLC (solvent system A-
toluene: 1,4-dioxane: acetic acid, 180:45:5), and UV light
test [31, 32]. Menegazzia terebrata (Hoffm.) A. Mas-
sal (Parmeliaceae, Ascomycota) is a foliose lichen. The
chemistry of M. terebrata is constituted by three lichenic
compounds-atranorin, physodic, and stictic acid, which are
diagnostic depsides and depsidones of the species [2]. The
three lichenic compounds of M. terebrata are well diag-
nosed with TLC with specified colored bands at known Rf
values/class [32]. The identified and authenticated lichen
sample voucher specimens were deposited in the CSIR-
National Botanical Research Institute (NBRI) herbarium
in Lucknow, India.
2.2. Extraction of Lichen Crude Extracts and the
Isolation of Lichenic Compounds
The samples of M. terebrata (Hoffm.) A. Massal were
sorted, cleaned and air dried before extraction. The par-
tially powdered lichen sample was extracted in acetone.
The extraction was done in a soxhlet extractor which was
connected with a reflux-condenser below the specific boil-
ing temperature of acetone i.e., 56 C [33, 34]. The specific
lichenic compounds of M. terebrata were isolated from the
crude extracts through preparative chromatography using
Merck-Millipore preparative layer plates (2 mm). The iso-
lated lichenic compounds were further recovered by fil-
tering followed by the removal of solvents using a rotary
evaporator (BüchiRotavapor R-200™), and through freeze-
drying using a lyophilizer. The purity of isolated lichenic
compounds was checked through HPLC using diagnostic
data from Elix JA (2014).
The recovered lichen lichenic compounds were dis-
solved in 5% dimethyl sulphoxide (DMSO). The dissolved
DMSO suspension of lichenic compounds was prepared in
a dilutions series in an appropriate liquid foundation (i.e.,
2 to 0.0037 mg/mL), for the determination of the minimal
inhibitory concentration (MIC).
2.3. Microorganisms and Media
The bacterial test taken for the study consisted of
three Gram-positive bacteria i.e., Bacillus mycoides, B.
subtilis,andStaphylococcus aureus,andfivegram-
negative bacteria i.e., Enterobacter cloacae, Escherichia
coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,
and Proteus mirabilis. The bacterial inoculums were
procured from the microbial type culture collection of
the CSIR-Institute of microbial technology, Chandigarh.
The cultures were maintained on the HIMEDIA®-M173™
Müller–Hinton agar.
2.4. Antibiotic Assay
The lichenic compounds’ antibiotic activity was evaluated
using the minimal inhibitory concentration (MIC).
2.4.1. Preparation of the Inoculum
The bacterial cultures were incubated for 24 h at 37 C
on HIMEDIA®-M173™Müller–Hinton agar substrate.
The inoculum was adjusted through dilution accord-
ing to the 0.5 McFarland standard (i.e., approximately
108 CFU/mL).
2.4.2. MIC Analysis
The MIC was determined using the broth tube microdi-
lution method [35]. A dilution series ranging from 2–
0.0037 mg/mL was analyzed against the tested bacterial
inoculums. The MIC for the tested bacteria was specified
as the lichenic compound dilution without any growth. The
positive control was streptomycin, while the negative con-
trol was the solvent DMSO. All the tests were carried out
in triplicate.
2.5. Statistical Analysis
SPSS Statistics for Windows was used to conduct all sta-
tistical analyses (version 26.0, IBM Armonk, NY). A sta-
tistically significant P-value is less than 0.05.
3. RESULTS AND DISCUSSION
3.1. Lichenic Compounds
All the lichenic compounds isolated from M. terebrata
(Hoffm.) A. Massal, i.e., atranorin, physodic acid, and stic-
tic acid showed detectable antibacterial activity against the
tested bacterial inoculums. The antibacterial activity esti-
mated as MIC values was found to be different for differ-
ent lichenic compounds according to their concentrations
(Fig. 2).
3.2. Antimicrobial Activity
The MIC values of isolated lichenic compounds against
the tested bacterial inoculums ranged between 0.030–
1.0 mg/mL. The highest antibacterial activity was observed
by atranorin (0.030–0.25 mg/mL) (Fig. 3), followed by
J. Biobased Mater. Bioenergy 16, 418–423, 2022 419

RESEARCH ARTICLE
Assessment of Antimicrobial Activity of Lichenic Compounds Isolated from Menegazzia terebrata (Hoffm.) A. Massal Raietal.
Fig. 1. (A) Overview map of the sampling site (red spot); (B) Habitat of sampling site; (C) M. terebrata (Hoffm.) A. Massal colonies on rock (yellow
encirclement); (C, D) The thallus of M. terebrata (Hoffm.) A. Massal.
stictic (0.25–0.5 mg/mL) (Fig. 4) and physodic acids
(1 mg/mL) (Fig. 5), comparable to the positive control
of the streptomycin (2.10–30.81 g/mL) (Fig. 6). Besides
the broad range of antibiotic activity of isolated lichenic
compounds, the positive antimicrobial bioactivity against
streptomycin-resistant P. aeruginosa was also recorded.
3.3. Discussion
In recent years, the effective antimicrobial activities of
bioactive compounds have proven to be a boon to the ever-
increasing antibiotic-resistant strains for a variety of bac-
teria [36, 37]. In the last two decades, lichenic compounds
have been extensively studied as antibiotics [38].
420 J. Biobased Mater. Bioenergy 16, 418–423, 2022

RESEARCH ARTICLE
Raietal. Assessment of Antimicrobial Activity of Lichenic Compounds Isolated from Menegazzia terebrata (Hoffm.) A. Massal
Fig. 2. Identification of Lichen secondary metabolites from M. terebrata (Hoffm.) A. Massal through chromatography.
Fig. 3. MIC∗of lichen secondary metabolite atranorin of M. terebrata
(Hoffm.) A. Massal against the test bacteria (∗MIC values are given as
mg/mL for lichen compounds and as g/mL for antibiotics).
Fig. 4. MIC∗of lichen secondary metabolite physodic acid of M. tere-
brata (Hoffm.) A. Massal against the test bacteria.
Fig. 5. MIC∗of lichen secondary metabolite stictic acid of M. terebrata
(Hoffm.) A. Massal against different test bacteria.
Fig. 6. MIC∗of streptomycin antibiotic against the different bacterial
test.
The superior antimicrobial activity of atranorin in com-
parison to streptomycin was well established [39, 40].
Ylmaz et al. (2004) discovered antimicrobial bioactivity
in atranorin isolated from Cladonia foliacea E [41]. As
reported by Refs. [27] and [29], physodic acid has the low-
est antibacterial activity. The specific antibacterial activ-
ity of lichenic compounds against resistant P. aeruginosa
demonstrates the significance of lichenic compounds as
effective antimicrobials [18, 19, 42].
4. CONCLUSION
The results of this study effectively support lichenic com-
pounds as highly effective general antibiotics and pharma-
ceutically viable agents against resistant bacterial strains.
Ethical Compliance
There are no researches conducted on animals or humans.
Conflicts of Interest
There are no conflicts to declare.
Acknowledgments: The authors are grateful to the
Head, CAS-Botany, Institute of Science, Banaras Hindu
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RESEARCH ARTICLE
Assessment of Antimicrobial Activity of Lichenic Compounds Isolated from Menegazzia terebrata (Hoffm.) A. Massal Raietal.
University, India for providing necessary research facil-
ities. The collection of lichen samples by HimanshuRai
was supported by the Uttarakhand State Council for
Science and Technology (UCOST), India through the
MRD Project Grant (UCOST-UCS & T/R & D/LS-26/11-
12/4370 dt. 17.03.2012). Taif University Researchers
Supporting Project Number (TURSP-2020/130), Taif Uni-
versity, Taif, Saudi Arabia. In addition, the authors thank
Prince Sattam Bin Abdulaziz University, Al-Kharj for their
scientific contributions.
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Received: 3 May 2022. Accepted: 17 June 2022.
J. Biobased Mater. Bioenergy 16, 418–423, 2022 423