Comprehensive Lichenometabolomic Exploration of Ramalina conduplicans Vain Using UPLC-Q-ToF-MS/MS: An Identification of Free Radical Scavenging and Anti-Hyperglycemic Constituents

In this study, we propose ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-QToF-MS/MS)-guided metabolite isolation as a choice analytical approach to the ongoing structure–activity investigations of chemical isolates from the edible lichen, Ramalina conduplicans Vain. This strategy led to the isolation and identification of a new depside (5) along with 13 known compounds (1–4, 6–14), most of which being newly described in this lichen species. The structures of the isolates were established by detailed analysis of their spectral data (IR, NMR, and Mass). The acetone extract was further analyzed by UPLC-Q-ToF-MS/MS in a negative ionization mode, which facilitated the identification and confirmation of 18 compounds based on their fragmentation patterns. The antioxidant capacities of the lichen acetone extract (AE) and isolates were measured by tracking DPPH and ABTS free radical scavenging activities. Most isolates displayed marked radical scavenging activities against ABTS while moderate activities were observed against DPPH radical scavenging. Except for atranol (14), oxidative DNA damage was limited by all the tested compounds, with a marked protection for the novel isolated compound (5), as previously noted for the acetone extract (p < 0.001). Furthermore, compound (4) and acetone extract (AE) have inhibited intestinal α-glucosidase enzyme significantly (p < 0.01). Although some phytochemical studies were already performed on this lichen, this study provided new insights into the isolation and identification of bioactive compounds, illustrating interest in future novel analytical techniques.


Introduction
Lichens are structurally complex and self-sustaining unique consortia comprised of a fungus host (mycobiont) living with algae or cyanobacteria (photobiont partner) in the framework of a unique symbiotic type of relationship. In recent years, much attention has been paid to the biological roles of lichen metabolites because of their potential applications in perfumery, cosmetics, creative crafts, the dye industry, and the pharmaceutical sector [1,2]. Moreover, many lichens and their extracts and metabolites have been utilized as ingredients in ethnic food preparations and specialties, along with ethnomedicinal applications [3]. For example, a mixture of lichens called Yangben in the Rai and Limbu communities of East Nepal is mainly composed of Ramalina species [4]. Among these fruticose epiphytic species, Ramalina conduplicans Vain is common and one of the most widely-used edible lichen of the Ramalinaceae family, this is distributed in Central and Southeastern Asian countries [5].
In Southwestern China, people used to prepare their traditional cold dishes with this lichen at their marriage banquets [6], and it also has a long history of consumption as a spice in many places in India and as a traditional food by selected communities in East Nepal [7,8]. In addition to its useful edible properties, crude extracts from this lichen are used as ethnomedicine to counteract inflammation, anthelminthic [9], and act as an anti-diabetic [10], along with antibiotic activities [3,11,12]. Many studies on this lichen have focused on their nutritional value along with the important trace elements [13] and antioxidant properties of R. conduplicans [9,14] concerning sekikaic acid and homosekikaic acid [15]. However, systematic investigations of its constituents for their bioactive potentials have not been carried out to date. Therefore, the antioxidant and alpha-glucosidase inhibiting properties of metabolites from Ramalina conduplicans were investigated here as part of our ongoing exploration of natural flora for the isolation of bioactive secondary metabolites [16,17]. Accordingly, we have designed a strategy and workflow based on the Total Ion Current Chromatography (TIC) of the acetone extract (AE) to recognize and to isolate compounds from R. conduplicans by UPLC-Q-ToF-MS/MS. AE and all the isolates were assayed for their antioxidant freeradical scavenging properties, including DNA damage protection and anti-hyperglycemic potential, through α-glucosidase inhibition.

Results and Discussion
The R. conduplicans sample was identified by morphological characteristics and thallus reactions: K+ pale yellow, KC−, P−, and also negative reaction of the medulla to calcium hypochlorite solution (C−) ( Figure S2, Supplementary Materials). These usual spot tests are based on the presence of lichen metabolites, but have to be supplemented by accurate analytical studies to reveal the metabolite content.

Chemical Profiling, Isolation, and Structure Elucidation
A HPTLC ( Figure S3, Supplementary Materials) co-migration with standards and the UPLC-PDA profile ( Figure S4, Supplementary Materials) of the acetone extract of R. conduplicans suggested the presence of a dozen of visible compounds, among which salazinic acid, usnic acid, sekikaic acid, homosekikaic acid, and divaricatic acid were identified against standards and appeared to represent the most abundant compounds. Initial LC-QToF-MS E analyses of the acetone extract of R. conduplicans indicated the presence of depsides, depsidones, and monophenolic acids based on High-Resolution Mass Spectroscopy (HRMS). Molecular formulae for C 10-35 H 10-50 O 2-15 were generated from mass ranges m/z 150-750 coupled with the fragment ions and their MS spectral data (accurate mass and fragmentation pattern) and compared to online databases (DNP, Reaxys, SciFinder).
Mass spectrometry (MS) and, particularly, quadruple time-of-flight coupled to Liquid Chromatography (UPLC-Q-ToF-MS) has been widely utilized for profiling metabolites due to its superiority in high-resolution mass, precision, and sensitivity [18], and was helpful to clearly discriminate between the depsides, depsidones, simple phenol acids, dibenzofurans, and hydroxyl fatty acids based on the fragmentation of lichen molecules [16]. Therefore, the acquired TIC of the R. conduplicans extracts, obtained within 16 min, were analyzed from spectra obtained in negative mode and, thus, are effective for characterizing trace components ( Figure 1). Metabolite assignments were made based on their polarity related to their retention time (Rt) and molecular formulae from accurate molecular weight measurement, along with adducts [M − H] − / fragment ions and Ring Double Bond Equivalence (RDBE). In the present study, a total of 18 compounds were clearly characterized from the crude extract of R. conduplicans by molecular formulae generated by ToF-MS/MS and MS/MS including their fragmentation profiles, as reported in the literature and presented in Table S1 (Supplementary Materials). within 16 min, were analyzed from spectra obtained in negative mode and, thus, are effective for characterizing trace components ( Figure 1). Metabolite assignments were made based on their polarity related to their retention time (Rt) and molecular formulae from accurate molecular weight measurement, along with adducts [M − H] − / fragment ions and Ring Double Bond Equivalence (RDBE). In the present study, a total of 18 compounds were clearly characterized from the crude extract of R. conduplicans by molecular formulae generated by ToF-MS/MS and MS/MS including their fragmentation profiles, as reported in the literature and presented in Table S1 (Supplementary Materials). Based on the fragmentation of isolates, we have identified compounds (1-5) and (7)(8)(9) along with atranorin belonging to depsides. The literature clearly indicates that sekikaic acid is an abundant molecule in Ramalina species [19]. Sekikaic acid (1) is a m-depside corresponding to the esterification of two divaricatinic acid units and is found with Rt at 11.98 min and m/z 417.1547 (C22H25O8) with fragments m/z 209 and m/z 225 corresponding to the A ring and B ring, respectively [20]. Compounds 1, 3, 5, 7, 8, and 9, having a common fragment m/z 209 ( Figures S47 and S48, Supplementary Materials), clearly indicate the difference in locating the other Bring. These depsides can be considered as ester derivatives of divaricatinic acid (11) while compound 2 is a divaric acid derivative (recognized at Rt 7.50 min, m/z 195.0657). The other identified monoaromatic compounds correspond to 2,4-di-O-methyldivaric acid (6), 4-O-methyldivaricatic acid Based on the fragmentation of isolates, we have identified compounds (1-5) and (7)(8)(9) along with atranorin belonging to depsides. The literature clearly indicates that sekikaic acid is an abundant molecule in Ramalina species [19]. Sekikaic acid (1) is a m-depside corresponding to the esterification of two divaricatinic acid units and is found with Rt at 11.98 min and m/z 417.1547 (C 22 H 25 O 8 ) with fragments m/z 209 and m/z 225 corresponding to the A ring and B ring, respectively [20]. Compounds 1, 3, 5, 7, 8, and 9, having a common fragment m/z 209 ( Figures S47 and S48, Supplementary Materials), clearly indicate the difference in locating the other Bring. These depsides can be considered as ester derivatives of divaricatinic acid (11) while compound 2 is a divaric acid derivative (recognized at Rt 7.50 min, m/z 195.0657). The other identified monoaromatic compounds correspond to 2,4-di-O-methyldivaric acid (6), 4-O-methyldivaricatic acid (10), divaricatinic acid (11), olivetolic acid (12), divarinolmonomethylether (13), and atranol (14). In this run, three additional compounds were ionized and fragmented (Rt = 8.53 min, Rt = 11.88 min, and Rt = 13.17 min) and not determined.  Table S1, including the monoaromatic divaric acid, along with the common and already-described atranorin (depside), usnic acid (related to dibenzofurans), and salazinic acid (a depsidone). The structures were concluded through MS/MS fragmentation patterns and compared with in-house standards.
Subsequently, the acetone extract was subjected to column chromatography to give eight fractions (I to VIII). An LC-MS E analysis of all fractions revealed the presence of depsides in III-VI fractions ( Figures S4-S6, Supplementary Materials). Thus, the targeted isolation and purification of III-VI fractions yielded the isolation of one new depside (5), along with other known depsides (1-4 and7-9) and monoaromatic compounds (6 and 10-14). The spectra and fragmentation patterns of these molecules were shown in the Supporting Information section ( Figures S7-S46, Supplementary Materials).
which showed the absence of a carbonyl COOH group, and its 1 H NMR spectrum indicated the presence of an additional aromatic proton at 6.53 (d, J = 1.8 Hz, 1H). A comprehensive analysis of 2D NMR (COSY, and HSQC) data, especially the 1 H-1 H COSY spectrum, revealed two discrete spin systems, including -CH-CH2-CH3-(from H-1", H-2" and H-3") and -CH-CH2-CH2-CH2-CH3 (from H-1'" to 5'"), as drawn with bold lines in Figure 3. The position of the n-pentyl group at C-6′ and n-propyl chain at C-6 was confirmed on the basis of the NOESY correlations (H-1'"/H-5', H-1'"/H-1′ and H-1"/H-5) ( Figure 3) and in comparison with the sekikaic acid data. In addition, the MS/MS spectrum of 5 showed (Figure 4) product ions m/z 209, thereby indicating the breakage of the C-O bond between two aromatic rings supported by the fragments at m/z 165 and 137 Based on these spectral characteristics, the structure of 5 was established and trivially named as decarboxyhomosekikaic acid.

Assessment of Compounds and Extract for Free Radicals Scavenging and Antioxidant Activity
The amphiphilic nature of the ABTS •+ cation was used to identify both hydrophil and hydrophobic antioxidants in dietary materials, whereas the DPPH-radical was use to measure an antioxidant's reducing power [30]. These fundamental chemical exper ments reveal the radical scavenging and reduction characteristics of the potential ant oxidant candidates.

Assessment of Compounds and Extract for Free Radicals Scavenging and Antioxidant Activity
The amphiphilic nature of the ABTS •+ cation was used to identify both hydrophilic and hydrophobic antioxidants in dietary materials, whereas the DPPH-radical was used to measure an antioxidant's reducing power [30]. These fundamental chemical experiments reveal the radical scavenging and reduction characteristics of the potential antioxidant candidates.

Protective Effect of R. conduplicans AE and Isolated Compounds on Oxidative DNA Damage
The Fenton's reaction produces the hydroxyl radical, which is a ROS that is detrimental to the human body. Hydroxyl radicals react with different nucleobases, thereby inducing the formation of mutated bases that eventually lead to DNA damage [32]. Figure 5 demonstrated that FR damaged DNA significantly (p < 0.001) compared to the control (DMSO + DNA). Though all compounds showed significant protection against hydroxyl radical-induced DNA damage (p < 0.001, cpd 10: p < 0.05), compound 14 could not prevent the oxidative damage to DNA (Figures 5 and S49). The genoprotective activity of these compounds and the AE may be attributed to the presence of free radical scavenging potential.

Assessment of In Vitro Antihyperglycemic Activity of Compounds and Extract as Intestinal α-Glucosidase Enzyme Inhibition
The α-glucosidase enzyme is a key enzyme that catalyses disaccharide digestion. The inhibition of α-glucosidase in the intestine slows digestion and the overall rate of glucose absorption into the blood. This has proven to be one of the most effective ways for lowering post-prandial blood glucose levels and, as a result, preventing the onset of late diabetes complications [33]. Sekikaic acid (1) was already recognized to inhibit α-glucosidase along with usnic acid and salazinic acid from other Ramalina species, but it is not the most effective compound [34]. As per Figure 6, it was stated that acetone extract (AE) and compound 4 have displayed better α-glucosidase inhibition (p < 0.01) than Acarbose. ure 5 demonstrated that FR damaged DNA significantly (p < 0.001) compared to the control (DMSO + DNA). Though all compounds showed significant protection against hydroxyl radical-induced DNA damage (p < 0.001, cpd 10: p < 0.05), compound 14 could not prevent the oxidative damage to DNA (Figures 5 and S49). The genoprotective activity of these compounds and the AE may be attributed to the presence of free radical scavenging potential.

Assessment of In Vitro Antihyperglycemic Activity of Compounds and Extract as Intestin α-Glucosidase Enzyme Inhibition
The α-glucosidase enzyme is a key enzyme that catalyses disaccharide digesti The inhibition of α-glucosidase in the intestine slows digestion and the overall rate glucose absorption into the blood. This has proven to be one of the most effective wa for lowering post-prandial blood glucose levels and, as a result, preventing the onset late diabetes complications [33]. Sekikaic acid (1) was already recognized to inhi α-glucosidase along with usnic acid and salazinic acid from other Ramalina species, bu is not the most effective compound [34]. As per Figure 6, it was stated that acetone extr (AE) and compound 4 have displayed better α-glucosidase inhibition (p < 0.01) th Acarbose. Figure 6. Intestinal α-glucosidase inhibitory assay. Compounds (1-8), (11)(12)(13)(14), and acetone extr (AE) were incubated with α-glucosidase enzyme and the release of p-nitrophenol was determin ***p < 0.001, **p < 0.01; vs. Acarbose. One-way ANOVA followed by Tukey's multiple comparis test was applied to compare differences. Values are represented as mean ± SD, n = 3. AE = Aceto Extract. Activity was not detected for compounds 9 and 10.
On the other hand, compounds 8, 11, 12, and 14 demonstrated inhibitory effects comparable to those of the standard Acarbose ( Figure 6). As contrasted activities can be observed between structurally-related compounds, structure-activity relationships can be considered. This is the case between depsides 3 and 4, suggesting a positive influence of the C1-pentyl chain with regard to substitution by a C1-propyl chain. When this length modification of the alkyl chain occurs on the B ring of decarboxylated derivatives (active compound 8 versus inactive compound 9) the opposite influence can be observed. The presence of a C6 -carboxylic group lowers the α-glucosidase inhibitory activity as compound 7 is less active than compound 8. Methylation of the carboxylic function of the monoaromatic divaricatinic acid 11 resulted in a complete loss of activity. Nevertheless, most of the tested compounds were found with some activity, such as compounds 1, 2, 3, 5, 6, 7, and 13, which displayed mild to moderate enzyme inhibition (p < 0.001). These results are to be pooled with the growing number of reports on the antidiabetic potential of lichen extracts or molecules [34][35][36]. The combination of activities with different mechanisms of action is of particular interest to develop potent antihyperglycemic effects. Lowering glucose absorption and limiting oxidative damages due to hyperglycemia, as expected from the lichen extract, could be promising. The challenge is to use standardized extracts that were previously checked to be safe for acute and chronic intake.

Instrumental UPLC Conditions
The instrumental conditions were set-up as per our recent report (Reddy et al., 2019) with slight modifications. Chromatographic separation was performed on an Acquity H Class UPLC system (Waters, Milford, MA, USA) with a conditioned auto sampler using an ACQUITY UPLC CSH Phenyl-Hexyl column (100 mm × 2.1 mm id., 1.7 µm particle size) (Waters, Milford, MA, USA). Column temperature was maintained at 40 • C. Highresolution masses of secondary metabolites were measured after UPLC separation. A mobile phase consisting of water with 0.1% formic acid in water (solvent A) and acetonitrile with 0.1% formic acid (solvent B) was pumped at a flow rate of 0.4 mL/min. The gradient elution program was as follows: 0 min, 5% B; 3.00 min, 20% B; 5.00min, 35% B; 7.50 min, 50% B; 10.00 min, 70% B; 12.50 min, 95% B; 17.00 min 95% B; and 21.00 min 5% B. The equilibration time was 4.0 min and the injection volume was 2 µL. The LC-QTof-MS E mode was applied to analyze the samples in both TIC as well as the MS/MS mode, where the collision energy was ramped at 15-45 eV. Eluted compounds were detected from m/z 50 to 1200 using a Xevo G2-XS Q-Tof mass spectrometer (Waters, Manchester, UK), which was connected to Electro-spray ionization (ESI) interface with a negative ion mode using the following instrument settings: capillary voltage, 2.0 KV; sample cone, 40 V; source temperature, 120 • C; desolvation temperature 350 • C; cone gas flow rate 50 L/h; desolvation gas (N 2 ) flow rate 850 L/h, argon as CID gas for MS/MS experiments. All analyses were performed using lock spray, which ensured accuracy and reproducibility. Leucine-Enkephalin (5 ng/mL) was used as a lock mass, generating a reference ion in the negative mode at m/z 554.2615, and was introduced by a lock spray at 10 µL/min for accurate mass acquisition. Data acquisition was achieved using MassLynx ver. 4.1.
Acquiring data in this manner provided information on intact precursor ions as well as fragment ions.

Lichen Sample Collection and Identification
The lichen, Ramalina conduplicans, was collected from tree bark in Bichpuri Range, Bijrani Zone of Corbett National Park, alt. N 29 • 26 40" E79 • 04 06 (1283 m) in the month of May 2019. The morphological features of lichen thallus and ascomata were observed under Magnüs MS 24/13, and spot tests for color reaction were carried out by 10% aqueous solution of potassium hydroxide (K), Steiner's stable p-phenylene diamine solution (PD), and calcium hypochlorite solution (C). For the anatomical investigation of fruiting bodies, a light microscope from ZEISS Axiostar was used. The lichen substances were identified with thin layer chromatography in solvent system 'A' following White and James's methods (1985). The standard literature [37]

Extraction and Isolation
The sorted-out lichen Ramalina conduplicans (300 g) was shade-dried, powdered, and extracted with acetone (6L) at room temperature for 48h. The result was that acetone extract was evaporated to dryness under reduced pressure, thereby affording a syrupy residue (20 g). This crude extract was subjected to gradient column chromatography (SiO 2 , 60-120 mesh) and eluted with a hexane/EtOAc mixture of increasing polarity with 10% intervals that yielded 8 fractions. These eight fractions were reconstituted in acetonitrile and subjected to UPLC Q-ToF MS E . Based on the TIC profile, we selected fractions 3-6 for purification (mass profile shown in supporting information, discussion in results section). All these fractions were subjected to semi-preparative HPLC (X Select HSST3 OBD Prep Column,5 µm, 10 mm × 250 mm), 0.1 % formic acid with water (solvent A) and acetonitrile (solvent B) as mobile phase at flow rate 4 mL/min, detected at 254 nm. Semi-preparative HPLC were conducted by gradient elution programs to obtain compounds as follows:  A DPPH radical scavenging assay was carried out as previously reported [38]. Scavenging of 2,2-diphenyl-1-picryhydrazyl (DPPH) radicals by the acetone extract (AE) (50 µg of 2 mg/mL solution dissolved in DMSO) and compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) (50 µg of 2 mg/mL solution dissolved in DMSO) was measured in 100 mM Tris-HCl buffer (pH 7.4) by recording the absorbance at 517 nm spectrophotometrically. Ascorbic acid (50 µg of 2 mg/mL solution dissolved in DMSO) served as the standard. The results were expressed as %-scavenging and calculated by using the following formula: (A c −A t )/100 × A c , where A c was the absorbance of control and A t was the absorbance of the test sample. Different concentrations of compounds were evaluated to obtain 50% scavenging activity (SC 50 ). The SC 50 was calculated based on the equation obtained from regression analysis.

ABTS Radical Scavenging Activity
Scavenging of the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS •+ ) was performed as per the earlier method [39]. Acetone extract (AE) (20 µg of 2 mg/mL solution dissolved in DMSO) and compounds (1-14) (20 µg of 2 mg/mL solution dissolved in DMSO) were incubated with ABTS •+ solution in 6.8 mM phosphate buffer (pH 8.0) as described earlier. The discoloration of the ABTS •+ solution was determined by measuring the absorbance at 734 nm spectrophotometrically. Ascorbic acid (20 µg of 2 mg/mL solution dissolved in DMSO) served as the standard. The activity was expressed as %-scavenging and calculated as follows: (A c − A t )/(100 × A c ), where A c was the absorbance of control and A t was the absorbance of the test sample. The SC 50 of compounds was calculated as per the above formula.

Free Radical Induced DNA Damage
The protective effect of acetone extract (AE) and compounds (1-14) on oxidative DNA damage was evaluated as per the previous method [40]. A total of 2 µL calf-thymus DNA mixed with 5 µL of 39 mM Tris buffer (pH 7.4) and 5 µL (10 µg) acetone extract and compounds (1-14) (10 µg of 2 mg/mL solution dissolved in DMSO) mixture was incubated at room temperature for 20 min. The reaction was initiated by adding 5 µL FeCl 3 (500 µM) and 10 µL H 2 O 2 (0.8 M) and incubated for 10 min at 37 • C. The reaction was stopped by adding 3 µL DNA loading dye. Finally, the mixture was subjected to 0.8% agarose gel electrophoresis in TAE (40 mM Tris, 20 mM acetic acid and 0.5 M EDTA) buffer (pH 7.2). A total of 3 µL of Ethidium bromide was added to agarose solution to stain DNA bands. The image was viewed under transilluminating UV light and photographed (Bio-Rad, ChemiDocTM XRS, Hercules, CA, USA with Image LabTM software (ver. 6.0.1, build34, standard edition, 2017). The band intensity of the DNA was measured by using ImageJ software (ver. 1.4.3.67, Broken Symmetry Software, Scottsdale, AZ, USA).

Intestinal α-Glucosidase Inhibition
An intestinal α-glucosidase enzyme inhibition assay was performed as per the previous method [36]. A total of 20 µL (40 µg) of acetone extract and compounds (1-14) (40 µg of 2 mg/mL solution dissolved in DMSO) were incubated with 50 µL of rat intestinal α-glucosidase enzyme (89.93 mM, prepared in 0.9% NaCl) in 100 mM phosphate buffer (pH 6.8) for 10 min. After the incubation period, 50 µL of substrate (4-nitroplenyl α-Dglucopyranoside) solution was added. The release of p-nitrophenol from substrate was measured by recording the absorbance at 405 nm spectrophotometrically. Acarbose (40 µg of 2 mg/mL solution dissolved in DMSO) was taken as the standard. The activity was expressed and calculated as follows: (A c − A t )/100 × A c , where A c was the absorbance of control and A t was the absorbance of the test sample.

Statistical Analysis
Comparisons within the groups were done by applying one-way ANOVA followed by a post-test Tukey's Multiple comparison test. Statistical significance was set at p < 0.05. Data analysis was performed by using GraphPad Prism ver. 5.01 (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
A novel UPLC-QToF-MS/MS-guided strategy was proposed here for the isolation and characterization of one new depside, decarboxyhomosekikaic acid, along with 13 known metabolites from Ramalinaconduplicans-most of them being minor metabolites that were reported on for the first time from this species. In the whole experimental design, UPLC-QToF-MS/MS was selected for multiple purposes, including targeting, finding, profiling, and isolating active constituents. Three hitherto unreferenced compounds were detected in this lichen, with their molecular formulae being deduced from HR-QToF-MS. Although in minute amounts, one isolate could be identified as an additional homosekikaic derivative. The expected major compounds atranorin, usnic acid, salazinic acid, and sekikaic acid were also obtained. However, efforts for isolating, identifying, and testing mainly targeted alkyldepsides-and monoaromatic-related compounds.
These compounds were tested for their antioxidant and α-glucosidase inhibition potential. Most of them, and the crude acetone extract (AE), have displayed antioxidant potential by scavenging ABTS and DPPH radicals and protected DNA from oxidative damage. Five compounds, and particularly hyperhomosekicaic acid, exhibited a comparable or better α-glucosidase inhibition to that of the acarbose standard. On the basis of these results, it is suggested that these lichen substances have a great potential to be used as bioresources or as structural models for novel bioactive candidate compounds. Docking experiments are necessary to document the structure-activities observed in this study along with pharmacomodulation studies to evaluate the antidiabetic properties. Acetone extract unexpectedly showed a comparable effect to that of the Acarbose standard, though it was not sufficient to consider its hypoglycemic activity in the context of the traditional use made of this edible lichen [10].
It should be kept in mind that activities obtained from the crude extract or from any of the active metabolites cannot be claimed to support a preventive or a therapeutic activity as no clinical assay has been carried out to validate an effect with a standardized dosage. Unexpected side effects can occur when preparations differ from the real traditional use, and toxicity trials have to be carried out at once.