Bioassay-guided isolation of antimycobacterial substances from the traditionally used lichen Cladonia pyxidata (L.) Hoffm.

Abstract

The aim of the present study is to provide a scientific rationale for the folklore usage of Cladonia pyxidata (L.) Hoffm. in treating tuberculosis (Tb). Through bioassay-guided isolation, antimycobacterial metabolites were isolated from under-investigated lichen C. pyxidata and examined against M.t H37Ra and six MDR strains. Further, the cytotoxicity of all isolated metabolites was evaluated on THP-1 macrophages. Bioassay-guided isolation of acetone extract of C. pyxidata yielded four metabolites, namely usnic acid, atranorin, barbatic acid, and fumarprotocetraric acid. Among those, the MIC values of usnic acid and fumarprotocetraric acid showed more effective in inhibiting the growth of six MDR strains, compared to first-line drug rifampicin. In addition, the 50% inhibitory concentration values of these two compounds on THP-1 were found to be far higher than MIC values against tested Tb strains, indicating that THP-1 macrophages were not harmfully affected at concentrations that were effective against M.t and MDR strains. The results exposed the traditional use of C. pyxidata for treating Tb, and the key metabolites were found to be usnic acid and fumarprotocetraric acid. The current study lends the first evidence for the presence of antimycobacterial compounds in C. pyxidata.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03159-6.

Introduction

Tuberculosis (Tb) is a communicable bacterial infection affected by an obligate aerobe Mycobacterium tuberculosis (M.t) that most commonly affects the human lungs (Frothingham et al. 2005). In addition to M.t, some other Mycobacterium species, including M. africanum, M. avium, M. bovis, M. intracellularae, M. kansasii and M. microti, can also potentially cause mycobacterium ailments in humans (Brudey et al. 2006). As per the global Tb report from the World Health Organization (WHO), Tb-related illness killed about 1.5 million people in 2020 and also estimated to increase Tb death rate due to COVID pandemic situation in the period 2021–2025 (World Health Organization 2021). Besides, the drug treatment of Tb is limited due to the occurrence of many new cases of extremely drug-resistant (XDR) and multidrug-resistant (MDR) Tb strains towards first-line drugs like rifampicin (Seaworth and Griffith 2017). Approximately 484,000 novel MDR/rifampicin-resistant cases were reported by WHO in 2018 (World Health Organization 2019). The increasing cases of XDR and MDR have created a severe Tb-related health illness globally (Seaworth and Griffith 2017). Thus, a search for more effective novel anti-tubercular agents with less toxicity against MDR Tb strains is required to combat the Tb threat.

In recent years, natural sources are of scientific interest as they provide infinite possibilities for novel drug leads due to the matchless availability of chemical varieties. In addition, standardized natural extracts and their secondary metabolites have been considered promising leads for novel anti-tubercular agents (Mariita et al. 2010). In this context, a survey of lichens used in traditional medicine was carefully performed with the objective of evaluating the usage of lichen for Tb illness. As a result, twenty lichen species, namely Cetraria islandica (L.) Ach., Cladina arbuscula (Wallr.) Burgaz, Cladina rangiferina (L.) Nyl., Cladina stellaris (Opiz) Brodo, Cladonia pyxidata (L.) Hoffm., Evernia mesomorpha Nyl., Lobaria pulmonaria (L.) Hoffm., Peltigera aphthosa (L.) Willd., Peltigera spp. Willd., Rhizoplaca chrysoleuca (Sm.) Zopf., Trentepohlia jolithus, Umbilicaria esculenta (Miyoshi) Minks, Umbilicaria spp. Hoffm., Usnea ceratina Ach., Usnea diffracta Vain., Usnea florida (L.) F. H. Wigg., Usnea laevis (Eschw.) Nyl., Usnea longissima Ach., Usnea trichodeoides Vain., and Vulpicida pinastri (Scop.) J.E. Mattsson and M.J. Lai, were identified to treat Tb in various cultures across the world (Crawford 2019). Based on the availability of the lichen Cladonia pyxidata (L.) Hoffm., we have evaluated its anti-tubercular activity.

Traditionally, lichen C. pyxidata (cup moss; family: Cladoniaceae) have been consumed along with milk to treat pulmonary Tb (i.e., M.t particularly attacking the lungs) in Finland (Varita 1973). Biologically, the extracts of C. pyxidata have already been reported for antioxidant, antibacterial, antifungal and anticancer activities (Jeon et al. 2009; Kosanić et al. 2014; Sokmen et al. 2017). With this background, the present study aimed (a) to bioassay-guided isolation of C. pyxidata; (b) to screen the antimycobacterial activity of obtained metabolites against M.t H37Ra and six MDR Tb strains; and (c) to assess all compounds for cytotoxic activity against human THP-1 macrophages.

Methods

Collection of lichen

The entire lichen Cladonia pyxidata (L.) Hoffm. was collected from the rocks at Gangotri (Uttarkashi district), Uttarakhand, India, in February 2020, and a voucher specimen (20-02502) was deposited at the CSIR-National Botanical Research Institute, Lucknow, India.

Extraction and bioassay-guided isolation

Dried lichen C. pyxidata (~ 100 g) was blended into powder and extracted with acetone (200 mL × 14 days × 3) by using the maceration method (Nguyen et al. 2021) at room temperature. The combined extracts were evaporated using rotavapor (Shimadzu Rotation evaporator QR 2005-S, Japan) to obtain acetone extract of C. pyxidata (CP, 9.8 g) as a greenish-brown solid. Initially, CP was screened for antimycobacterial assay (Nguyen et al. 2021) against M.t H37Ra and found to be very active (Fig. 1).

Fig. 1.

Primary screening of anti-tubercular activity of acetone extract (CP), fractions (F1–8), and isolated compounds from Cladonia pyxidata (L.) Hoffm. against M.t H37Ra

About 5.0 g of CP was exposed to chromatography by sintered disc column (Borosil, India) over 230–400 mesh size silica gel (Merck, India) using dichloromethane-ethyl acetate gradient (0–100%) yielding eight fractions (F1–8). All these fractions were also screened against the M.t H37Ra strain (Nguyen et al. 2021). The outcomes of the screening found that four fractions (F2, F3, F5 and F7) showed above 50% inhibition against M.t strain, signifying additional purification to isolate antimycobacterial compounds. By repeated column chromatography (sintered disc column, Borosil, India), F2 (200 mg) after purification using dichloromethane-ethyl acetate gradient (0–100%) yielded usnic acid (90 mg) as yellow needles. Similarly, F3 (550 mg) yielded atranorin (250 mg) as colourless needles, F5 (300 mg) yielded barbatic acid (110 mg) as colourless needles, and F7 (800 mg) yielded fumarprotocetraric acid (350 mg) as colourless needles. For the chemical characterization, all the isolated compounds are subjected to Mass (LC/MS Triple Quad Portfolio, Agilent, China) and 2D nuclear magnetic resonance (Bruker Avance 400 Spectrometer, Germany) spectral analyses using the Robust mass spectrometry software and Bruker’s topspin software, respectively.

In vitro antimycobacterial assay

Standard cultures of non-virulent M.t H37Ra strain (ATCC 25177) and six characterized MDR clinical isolates (JAL-19049, JAL-19111, JAL-19126, JAL-19129, JAL-19187, and JAL-19188) that were resistant towards first-line anti-Tb drugs, namely rifampicin, isoniazid, and ethambutol (Gupta et al. 2018), were procured from the repository of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, India. All the strains were grown and maintained on Lowenstein Jensen medium (Hi-Media, India) at 37 ℃, while Middlebrook 7H9 broth (Merck, India) was used for the cultivation of M.t and MDR strains.

The inoculum was prepared in sterile glass vials (5 mL) containing glass beads. To this vial, 2 μL loops of mycobacterial strain suspending in 3 mL 7H9 medium was added and homogenised using a sonicator. Later, adjusted its McFarland turbidity to 1.0 and diluted it to 1:20 in Middlebrook 7H9 medium.

The stock solutions of all test samples were prepared in DMSO and filtered through a syringe filter (0.22 µM membrane) and diluted in Middlebrook 7H9 broth were used for in vitro antimycobacterial assay. Initial M.t inhibitory screening of CP, F1–8, and isolated compounds were tested at three different concentrations (100, 200 and 300 μg/mL) against M.t H37Ra strain using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction menadione assay (XRMA) at 470 nm (Singh et al. 2011) in triplicate. To 250 μL of the above prepared M.t H37Ra culture added test sample and 200 μM of XTT in 96-well plate and incubated at 37 °C for 20 min. Later, optical density was measured at 470 nm using a Spectramax plate reader. From the obtained OD values, percentage inhibition of M.t was calculated against blank.

The evaluation of the antimycobacterial activity of isolated compounds (500–1.95 μg/mL concentrations) was carried out against M.t H37Ra and six other MDR clinical isolated strains using resazurin microtitre plate assay (REMA) (Lakshmanan et al. 2011) in triplicate. In 96-well plate, the test sample and 100 μL of the above prepared MDR culture were added and incubated at 37 °C for 7 days. After, each well was treated with 30 μL of resazurin solution (0.02%) and further incubated for 2 days. The minimum inhibitory concentration (MIC) values were deliberated by a colour change from blue to pink. Rifampicin (400–0.10 μg/mL concentrations) was used as the reference drug, while DMSO (up to 2%) was used as a negative control.

Cytotoxicity assay

The isolated compounds were subjected for cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Chitturi et al. 2016) against THP-1 macrophages in triplicate. From the National Centre for Cell Science (Pune, India), the human acute monocytic leukaemia cell line (THP-1) was procured and maintained in RPMI-1640 medium (Merck, India) in a 5% CO₂ atmosphere at 37 ℃. A day before infection, differentiation of THP-1 monocytes into macrophages was induced by seeding 1 × 10⁵ cells in a 96-well plate and added with 100 nM phorbol-12-myristate-13-acetate (Sigma-Aldrich, India) and incubated at 37 ℃ overnight in cell culture medium. On the experiment day, the medium was replaced with a fresh cell culture medium and incubated with isolated compounds (50–300 μg/mL concentration dissolved in DMSO) in a 5% CO₂ atmosphere for 48 h at 37 ℃. Later, 20 μL of 5.0 mg/mL of MTT dye (Sigma-Aldrich) solubilized in DMSO was added to each well and incubated in a 5% CO₂ atmosphere at 37 ℃ for 4 h. The absorbance was recorded at 570 nm using a microplate reader (Molecular Devices, USA). From the dose–response curves, half-maximal inhibitory concentration (IC₅₀) values were deliberated against THP-1 macrophages. Doxorubicin (2.5–10 μg/mL concentration dissolved in DMSO) was used as the reference drug, while DMSO was used as a negative control.

Results and discussion

For many decades, lichens have been used in folklore medicine to treat Tb in various cultures across the world (Varita 1973; Crawford 2019). They produce characteristic metabolites such as aliphatic acids, anthraquinones, dibenzofurans, depsides, depsidones, pulvinic acids and xanthones, which are unique with respect to those of higher plants (Honda et al. 2010). Due to their unique structural integrity, various lichen metabolites have been biologically screened for antimycobacterial activity for the last two decades (Honda et al. 2010; Tatipamula and Annam 2022). Taken together, we relate here the antimycobacterial activity results of traditional lichen, C. pyxidata against M.t H37Ra and six MDR Tb strains along with cytotoxic activity against human THP-1 macrophages.

To isolate the biological active antimycobacterial substances from CP, it was initially fractionated into eight fractions namely F1–8, using column chromatography. These fractions were then screened for antimycobacterial activity against M.t H37Ra at three different concentrations using the XRMA method. The outcomes of the preliminary screening revealed that only CP, F2, F3, F5 and F7 showed above 50% inhibition against M.t (Fig. 1). Later, the bioactive fractions (F2, F3, F5 and F7) were purified using column chromatography, which yielded four metabolites. Upon characterisation using spectral and elemental analysis, these metabolites were identified as usnic acid, atranorin, barbatic acid, and fumarprotocetraric acid (Fig. 2). Based on the earlier study, it was observed that usnic acid, atranorin, and fumarprotocetraric acid were the chief chemical constituents present in the Cladonia genus and their antimicrobial activities were also well-addressed (Yılmaz et al. 2004).

Fig. 2.

Chemical representation of isolated compounds from Cladonia pyxidata (L.) Hoffm.

All four compounds obtained from bioactive fractions were further subjected to preliminary screening for inhibitory action against M.t H37Ra using the XRMA method. Among those, usnic acid and fumarprotocetraric acid showed profound mycobactericidal strength (85–90% inhibition), while atranorin and barbatic acid showed 46–68% inhibition of M.t H37Ra growth. Depending upon the outcomes of the initial screening, all four compounds at a concentration range of 1.95–500 μg/mL were further evaluated to determine MIC values against M.t H37Ra and six MDR clinical isolates of M.t, namely JAL-19049, JAL-19111, JAL-19126, JAL-19129, JAL-19187, and JAL-19188 using the REMA method. The outcomes of REMA revealed the strong antimycobacterial activity of usnic acid and fumarprotocetraric acid in the range of 3.90–31.25 μg/mL against all tested mycobacterial strains (Table 1).

Table 1.

Cytotoxicity, antimycobacterial activity, and selectivity index of isolated compounds from Cladonia pyxidata (L.) Hoffm

Sample	Cytotoxicitya (IC50 values in µg/mL)	MIC valuesb (µg/mL) [Selectivity indexc]
		M.t H37Ra	Multidrug-resistant isolates
			JAL-19049	JAL-19111	JAL-19126	JAL-19129	JAL-19187	JAL-19188
Usnic acid	134.22*	15.62 [9]	15.62 [9]	15.62 [9]	3.90 [34]	7.81 [17]	7.81 [17]	31.25 [4]
Atranorin	289.83*	250.00 [1]	250.00 [1]	62.50 [5]	62.50 [5]	250.00 [1]	250.00 [1]	250.00 [1]
Barbatic acid	247.38*	500.00 [1]	> 500 [ND]	> 500 [ND]	> 500 [ND]	> 500 [ND]	> 500 [ND]	> 500 [ND]
Fumarprotocetraric acid	94.11*	31.25 [3]	7.81 [12]	15.62 [6]	7.81 [12]	31.25 [3]	31.25 [3]	15.62 [6]
Rifampicin	91.43*	0.2 [457]	100.00 [1]	50.00 [2]	12.50 [7]	12.50 [7]	50.00 [2]	100.00 [1]
Doxorubicin	6.51	NT	NT	NT	NT	NT	NT	NT

^aValues are expressed as μg/mL (mean, n = 3), where statistical analysis determined by Student’s t-test, where *p ˂ 0.0001 was statistically significant compared to doxorubicin; IC₅₀: Half-maximal inhibitory concentration value is the lowest concentration of the sample exhibiting percentage growth inhibition of 50%, relative to the growth control. DMSO was used as a negative control

^bValues are expressed as μg/mL (n = 3); MIC: Minimum inhibitory concentration value is the lowest concentration of the sample exhibiting percentage growth inhibition of ≥ 90%, relative to the growth control; M.t: Mycobacterium tuberculosis; ND: Not determined; NT: Not tested. DMSO was used as a negative control

^cSelectivity index: Cytotoxicity (IC₅₀ value)/Antimycobacterial activity (MIC value). The higher selectivity index values indicate the more active and non-toxicity of a compound/extract

Usnic acid and fumarprotocetraric acid showed higher growth inhibitory action against M.t H37Ra with MIC values of 15.62 and 31.25 μg/mL, respectively. While, atranorin and barbatic acid were found to be active against M.t H37Ra with MIC values of 250 and 500 μg/mL, respectively. Besides, the standard drug (rifampicin) exhibited prominent inhibition of M.t with a MIC value of 0.2 μg/mL (Table 1).

Except for barbatic acid, all other compounds were found to be susceptible against six MDR strains isolated from the sputum of pulmonary Tb patients, while rifampicin was not much susceptible against tested MDR isolates may be due to the resistance of these strains against first-line anti-Tb drugs (Gupta et al. 2018) (Table 1). The MIC values of usnic acid, atranorin and fumarprotocetraric acid against MDR isolates were in the range of 3.90–250.00 μg/mL (Table 1). From the REMA method, it was noticed that usnic acid, atranorin and fumarprotocetraric acid were more effective in inhibiting the growth of MDR strains when compared to drug-sensitive M.t H37Ra strain (Table 1). Particularly, fumarprotocetraric acid exhibited noteworthy activity against most of the tested MDR strains (JAL-19049, JAL-19111, JAL-19126 and JAL-19188) with the MIC values ranged from 7.81–15.62 μg/mL. While, three MDR clinical isolates, namely JAL-19126, JAL-19129, and JAL-19187 were found to be more susceptible to usnic acid when compared to M.t H37Ra strain with MIC values of 3.90, 7.81 and 7.81 μg/mL, respectively (Table 1). Similarly, atranorin revealed better inhibitory activity against JAL-19111 and JAL-19126 strains with a MIC value of 62.50 μg/mL. Thus, the current study revealed that usnic acid, atranorin and fumarprotocetraric acid significantly inhibited the growth of M.t as well as various MDR isolated strains.

In general, the first home of M.t, an intracellular pathogen, is alveolar macrophages which initiates pathogenesis. For biological analysis, THP-1 cell line was commonly used to differentiate in vitro from macrophages. Therefore, in the present study, all isolated compounds were tested on THP-1 macrophages for their safety assessment using MTT assay. From the results, it was noticed that the IC₅₀ values of usnic acid, atranorin and fumarprotocetraric acid on THP-1 macrophages were found to be much higher than MIC values against M.t H37Ra and MDR isolated strains (Fig. 3). This indicates that THP-1 macrophages were not harmfully affected at concentrations that were effective against M.t and MDR strains. Hence, the cytotoxicity results propose the biocompatible nature of usnic acid, atranorin and fumarprotocetraric acid.

Fig. 3.

Percentage inhibition of isolated compounds from Cladonia pyxidata (L.) Hoffm. against THP-1 macrophages

Theoretically, the selectivity index (SI) value for all the four isolated compounds was calculated (IC₅₀/MIC) to estimate the efficiency and safety of any drug during in vivo/in vitro treatment for any bacterial infection (Dzoyem et al. 2016). The higher SI values indicate the more active and non-toxicity of a compound. In the present study, the SI index values of usnic acid and fumarprotocetraric acid towards MDR isolated strains were calculated to be higher than rifampicin. The SI ratio values of usnic acid (SI: 4–34) and fumarprotocetraric acid (SI: 3–12) towards all the MDR strains were far higher than rifampicin (SI: 1–7) (Table 1). This theoretical analysis indicates that usnic acid and fumarprotocetraric acid displayed strong antimycobacterial activity against MDR strains, compared to drug-sensitive strain (M.t H37Ra). These outcomes were considered very promising since these two compounds exhibited better selectivity against MDR strains than toxicity to THP-1 macrophages.

To conclude, this is the first study investigation that provides support to the traditional usage of lichen C. pyxidata in Tb and related complications via in vitro antimycobacterial activity. The bioassay-guided isolation of C. pyxidata yielded four compounds, of these, usnic acid and fumarprotocetraric acid possess significant antimycobacterial actions against non-virulent M.t H37Ra, as well as MDR strains, reveal its potential usage to combat drug resistance problem in Tb. In addition, the cytotoxicity and SI index values found to be far higher than rifampicin indicate usnic acid and fumarprotocetraric acid would be non-toxic and more effective during in vivo studies. However, a systematic investigation is further required to explore the antimycobacterial activities of usnic acid and fumarprotocetraric acid in animals and humans, after which they may be used as first-line treatment for pulmonary Tb or as an adjunctive treatment with standard antimycobacterial drugs.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

NHT proposed and monitored the biological assays and co-wrote the manuscript. HP and RA helped with isolation and biological evaluations. VBT conceived the study, analyzed the data, and wrote the manuscript. All authors have read and approved the manuscript.

Declarations

Conflict of interests

The authors declare that they have no conflict of interest in the publication.