Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of natural product chemistry and pharmaceuticals, and in specific, relates to a method for extracting lecanoric acid from Parmotrema praesorediosum to develop therapeutic agents for the treatment of antibiotic-resistant bacterial infections and fungal infections.
Background of the invention:
[0002] Natural products have historically been a cornerstone of traditional medicine and modern pharmacotherapy, providing a rich reservoir of bioactive compounds that have been utilized for the prevention and treatment of various ailments. Parmotrema perlatum, commonly known as stone flower, is a type of lichen that has been used in traditional medicine across different cultures for its supposed health-promoting properties. Despite its historical usage, there is a paucity of rigorous scientific research investigating the full spectrum of its biological activities. The current gap in the literature highlights the need for comprehensive studies to substantiate the traditional claims and explore new therapeutic potentials of this lichen.
[0003] The antioxidant properties of natural products have garnered significant attention due to their ability to neutralize free radicals, thereby mitigating oxidative stress and its associated cellular damage. Oxidative stress is implicated in the pathogenesis of numerous chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders. Therefore, identifying natural antioxidants is a priority in the development of preventive and therapeutic strategies against these conditions. Parmotrema perlatum is believed to possess antioxidant properties, yet there is a need for standardized methodologies to accurately quantify and evaluate these activities.
[0004] Antimicrobial resistance is an escalating global health concern, necessitating the discovery of novel antimicrobial agents. Natural products have historically been a valuable source of antimicrobial compounds, and there is on-going interest in identifying new agents that can effectively combat resistant strains of bacteria, fungi, and viruses. Parmotrema perlatum, with its traditional use in treating infections, presents a promising candidate for such investigations. However, comprehensive studies employing modern analytical techniques are required to validate its antimicrobial efficacy and identify the active constituents responsible for these effects.
[0005] Cancer remains one of the leading causes of morbidity and mortality worldwide, driving the continuous search for effective anticancer agents. Natural products have been pivotal in cancer drug discovery, with many current anticancer drugs being derived from or inspired by compounds found in nature. Parmotrema perlatum is purported to have anticancer properties, but robust scientific evidence is lacking. Rigorous research involving advanced bioassays and analytical methods is essential to evaluate its potential as an anticancer agent and to elucidate the mechanisms underlying its purported effects.
[0006] Antibiotic resistance has become a significant public health concern, as many bacterial strains have evolved resistance to commonly used antibiotics. The search for new antimicrobial compounds turns to natural sources, such as lichens, which are known for their diverse bioactive compounds. Lecanoric acid, a secondary metabolite found in certain lichens, has shown potential antimicrobial properties. However, its isolation and characterization from Parmotrema prasorediosum and its specific activity against antibiotic-resistant bacteria and fungal have not been extensively studied.
[0007] In existing technology, a method for quantifying and evaluating Parmotrema perlatum is known. The method involves the collection of the Parmotrema perlatum from natural habitats, followed by cleaning, drying, and grinding the samples into fine powder. The fine powder is mixed with lichen that is subjected to solvent extraction using ethanol, methanol, acetone, and water to obtain crude extracts rich in bioactive compounds. The obtained crude extract is concentrated and analysed using high-performance liquid chromatography (HPLC) and mass spectroscopy (MS) to identify and quantify the bioactive constituents. However, the method might not develop a drug based on the lecanoric acid.
[0008] Therefore, there is a need for a method that develops new drugs against antibiotic-resistant bacterial infections and fungal infections using lecanoric acid. There is also a need for a method that extracts and isolates processes described are relatively straightforward and could be scaled up, thereby leading to cost-effective production of the compound for pharmaceutical use. Further, there is also a need for a method that accelerates the development of new drugs based on lecanoric acid.
Objectives of the invention:
[0009] The primary objective of the present invention is to provide a method for extracting lecanoric acid from Parmotrema praesorediosum to develop therapeutic agents for the treatment of antibiotic-resistant bacterial infections and fungal infections.
[0010] Another objective of the present invention is to provide a method that utilizes lecanoric acid as a natural and renewable source, thereby reducing dependency on synthetic drugs and highlighting the importance of biodiversity in drug discovery.
[0011] Another objective of the present invention is to provide a method that offers the lecanoric acid to demonstrate significant antimicrobial activity, antibiotic-resistant strains of bacteria and fungal, thereby providing a potential alternative to conventional antibiotics that are losing efficacy due to resistance.
[0012] Another objective of the present invention is to provide a method that isolates bacteria such as Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) exhibiting complete inhibition at low concentrations, making it a potent and targeted antimicrobial agent.
[0013] Another objective of the present invention is to provide a method that extracts purified lecanoric acid from Parmotrema prasorediosum has demonstrated promising antimicrobial activity, modifying the need for further research using animal models and clinical trials.
[0014] Another objective of the present invention is to provide a method that accelerates the development of new drugs based on lecanoric acid.
[0015] Yet another objective of the present invention is to provide a method that enhances the scientific community's understanding of lichen-derived compounds, potentially leading to the discovery of additional bioactive substances from related natural sources.
[0016] Further objective of the present invention is to provide a method that extracts and isolates compounds, described as relatively straightforward and scalable, thereby leading to cost-effective production of pharmaceutical use.
Summary of the invention:
[0017] The present disclosure proposes a method for extracting lecanoric acid from Parmotrema praesorediosum for antimicrobial applications. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.
[0018] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for extracting lecanoric acid from Parmotrema praesorediosum to develop therapeutic agents for the treatment of antibiotic-resistant bacterial infections and fungal infections.
[0019] According to one aspect, the invention provides a method for extracting lecanoric acid from Parmotrema praesorediosum. At one step, fresh samples of Parmotrema praesorediosum are collected. Next, the collected samples are cleaned with distilled water to remove epiphytic hosts. At another step, the collected samples are dried on a filter paper at a room temperature to remove moisture and ground into a fine powder.
[0020] At another step, the fine powder is mixed with at least one organic solvent at a predetermined time to perform solvent extraction and obtain an extract. At another step, the extract is filtered to remove solid residues and obtain a crude extract, which consists of lecanoric acid. At another step, the crude extract evaporated under reduced pressure to control a purification process. The purified extract is isolated by using at least one column chromatographic technique.
[0021] Further, at another step, the isolated extract is characterized by dissolving it in a suitable solvent to obtain it in a pure crystalline form, thereby determining physical properties and chemical structure.
[0022] In one embodiment, the at least one column chromatographic technique is conducted using silica gel with a mesh size of 230–400 mm. In one embodiment, the predetermined time is at least one week to optimize the extraction process and obtain the extract.
[0023] In one embodiment, the at least one organic solvent includes acetone, ethanol, and methanol under reflux conditions. In one embodiment, the collected samples are dried at a temperature range between 40 °C and 50 °C to preserve the integrity of the lecanoric acid.
[0024] In one embodiment, the lecanoric acid is characterized by a white solid powder with a melting point of 175 °C to 176 °C, UV absorption with a maximum wavelength of 312 nm in methanol, a molecular formula, and a nuclear magnetic resonance (NMR) spectral data.
[0025] In one embodiment, the lecanoric acid is effective against bacterial strains and fungal that includes, but not limited to, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae, with complete inhibition observed at a concentration of 500 micromolar (µM) and 1 millimolar (mM) respectively. In one embodiment, the at least one chromatographic technique includes gas chromatography, high-performance liquid chromatography, thin-layer chromatography, and paper chromatography.
[0026] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0027] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.
[0028] FIG. 1 illustrates a flowchart of a method for isolating lecanoric acid from Parmotrema praesorediosum, in accordance to an exemplary embodiment of the invention.
[0029] FIG. 2A illustrates a pictorial representation of a molish test performed on a methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0030] FIG. 2B illustrates a pictorial representation of a saponins test performed on the methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0031] FIG. 2C illustrates a pictorial representation of a glycoside test performed on the methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0032] FIG. 2D illustrates a pictorial representation of an alkaloid test performed on the methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0033] FIG. 2E illustrates a pictorial representation of a terpenoid test performed on the methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0034] FIG. 2F illustrates a pictorial representation of a quinone test performed on the methanol extract of lichen powder, in accordance to an exemplary embodiment of the invention.
[0035] FIG. 3 illustrates a flowchart for isolation of lecanoric acid from crude extract Parmotrema praesorediosum using column chromatography, in accordance to an exemplary embodiment of the invention.
[0036] FIG. 4 illustrates a structure of lecanoric acid, in accordance to an exemplary embodiment of the invention.
[0037] FIG. 5A illustrates a graphical representation of a mass report of a positive mode spectrum of lecanoric acid, in accordance to an exemplary embodiment of the invention.
[0038] FIG. 5B illustrates a graphical representation of a mass report of negative mode spectrum of lecanoric acid, in accordance to an exemplary embodiment of the invention.
[0039] FIG. 5C illustrates a graphical representation of a hydrogen nuclear magnetic resonance spectrum of the lecanoric acid, in accordance to an exemplary embodiment of the invention.
[0040] FIG. 5D illustrates a graphical representation of a carbon nuclear magnetic resonance spectrum of the lecanoric acid, in accordance to an exemplary embodiment of the invention.
[0041] FIGs. 6A-6B illustrates pictorial representations of luria broth (L.B) plates with inhibition zone of tetracycline and ampicillin, in accordance to an exemplary embodiment of the invention.
[0042] FIGs. 6C-6D illustrates pictorial representations of the LB agar plates with bacterial inhibition zone of negative control and PP-05 (lecanoric acid), in accordance to an exemplary embodiment of the invention.
[0043] FIGs. 6E-6F illustrates pictorial representations of yeast plates with inhibition zone of negative control and PP-05 (lecanoric acid), in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0044] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.
[0045] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for extracting lecanoric acid from Parmotrema praesorediosum to develop therapeutic agents for the treatment of antibiotic-resistant bacterial infections and fungal infections.
[0046] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of the method for isolating lecanoric acid from Parmotrema praesorediosum. At step 102, fresh samples are collected of Parmotrema praesorediosum. Next the collected samples are cleaned with distilled water to remove epiphytic hosts. At step 104, the collected samples are dried on a filter paper at a room temperature to remove moisture and ground into a fine powder.
[0047] At step 106, the fine powder is mixed with at least one organic solvent at a predetermined time to perform solvent extraction and obtain an extract. At step 108, the obtained extract filters to remove solid residues and obtain a crude extract, which consists of lecanoric acid. At step 110, the crude extract evaporates under reduced pressure to control a purification process. The purified extract is isolated by using at least one column chromatographic technique. Further, at step 112, the isolated extract is characterized by dissolving it in a suitable solvent to obtain it in a pure crystalline form, thereby determining physical properties and chemical structure.
[0048] In one embodiment, the at least one column chromatographic technique is conducted using silica gel with a mesh size of 230–400 mm. In one embodiment, the predetermined time is at least one week to optimize the extraction process and obtain the extract. In one embodiment, the at least one organic solvent includes acetone, ethanol, and methanol under reflux conditions. In one embodiment, the collected samples are dried at a temperature range between 40 °C and 50 °C to preserve the integrity of the lecanoric acid. In one embodiment, the lecanoric acid is characterized by a white solid powder with a melting point of 175 °C to 176 °C, UV absorption with a maximum wavelength of 312 nm in methanol, a molecular formula, and a nuclear magnetic resonance (NMR) spectral data.
[0049] In one embodiment, the lecanoric acid is effective against bacterial strains and fungal that includes, but not limited to, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae), with complete inhibition observed at a concentration of 500 micromolar (µM) and 1 millimolar (mM) respectively. In one embodiment, the at least one chromatographic technique includes gas chromatography, high-performance liquid chromatography, thin-layer chromatography, and paper chromatography.
[0050] Table. 1
Chemical Test Process Colour Methanol Extract
Molisch Test Extract + water + 5% W/v a-naphthol + mixed Violet colour at junction Positive, presence of carbohydrates
Liebermann and burchard test Extract + chloroform + dissolve + add conc.sulphuric acid + acetic anhydride Blue/violet red colour Positive, Positive presence of terpenoids.
Positive, Positive presence of steroids
Ferric chloride Test Extra + methanol + add 5% ferric chloride Blue,Green and violet colour Positive, Positive presence of phenols
Shinoda test Extract + methanol + magnesium powder + conc.Hcl Pink colour Positive, Positive presence of flavanoids
Froth test Extract + water + shake for 5mins Persistent forth Positive presence of saponins
Gelatine solution test, ferric chloride Dissolve 1g of sample in purified water at about 55 °C. Dilute to 100 mL with same solvent and hold the solution at this temperature. To 2 mL of solution add 0.05 mL of a 125 g/L solution of CuSO₄.5H₂O. Mix, and add 0.5 mL of an 85 g/L solution of NaOH. Violet colour is produced Positive presence of tannins
Test for Quinones Sulphuric acid test To the 1 mL extract add few drops of conc.H2SO4 Formulation of green colour Positive presence of Quinone
Test for Alkaloids Extra + methanol + Add Brick red colour precipitate Positive presence of alkaloids
Keller killiani test To the extract add 1% ferric sulphate solution in (5%) of glacial acetic acid. Add one or two drops of concentrated sulphuric acid. Blue colour grows due to the presence of deoxy sugar Positive presence of Glycosides
Test for volatile oils Tincture alkana test Lichen extract treated with few drops of tincture alkana in the presence of volatile oils
Shows red colour Positive presence of volatile oils

[0051] In one example embodiment herein, the chemical tests are conveyed on the methanol extract of the lichen. The chemical tests include a Molisch test, libermann and burchard test, ferric chloride test, Shinoda test, froth test, gelatine solution test, ferric chloride, quinones sulphuric acid test, test for alkaloids, keller killiani test, volatile oils, and tincture alkana test. In one embodiment herein, the lecanoric acid targets the antibiotic-resistant bacteria by inhibiting their growth effectively at the specified concentration.
[0052] According to another exemplary embodiment of the invention, FIG. 2A refers to a pictorial representation 200 of a Molisch test performed on the methanol extract of lichen powder. In one embodiment herein, the Chemical tests were carried out on methanol extract of lichen Parmotrema praesorediosum. In one embodiment herein, the Molisch test is a chemical test which is used to check for the presence of carbohydrates in a given analyte. Molisch’s test involves the addition of Molisch’s reagent (a solution of α-naphthol in ethanol) to the analyte and the subsequent addition of a few drops of concentrated H2SO4 (sulphuric acid) to the mixture.
[0053] The formation of a purple or a purplish-red ring at the point of contact between the H2SO4 and the analyte + Molisch’s reagent mixture confirms the presence of carbohydrates in the analyte. In the Molisch test process include the crude extract, water, and 5 percent of 2-Naphthol are mixed efficiently and cooled down at the room temperature, and after adding the concentrated sulfuric acid, thereby obtaining the violet colour at junction.
[0054] According to another exemplary embodiment of the invention, FIG. 2B refers to a pictorial representation 202 of the saponins test performed on the methanol extract of lichen powder. Referring to Table. 1, the saponins test is secondary metabolites with high molecular weight. They present in a wide range of plant species and are distributed throughout the bark, leaves, stems, roots and even flowers. The Saponins test is composed of a lipid-soluble aglycone consisting of either a sterol or more commonly triterpenoid and water-soluble sugar residues, due to their amphiphilic nature, they are highly surface active and their biological activities are related to their chemical structures.
[0055] According to another exemplary embodiment of the invention, FIG. 2C refers to a pictorial representation 204 of the glycoside test performed on the methanol extract of lichen powder. Referring to Table 1, the glycoside test is composed by an alcoholic solution of the drug sample + few drops of NaOH + 2 percent of solution of 3,5- dinitro benzoic acid, which appearance of pink colour that indicates the presence of cardiac glycosides.
[0056] According to another exemplary embodiment of the invention, FIG. 2D refers to a pictorial representation 206 of the alkaloid test performed on the methanol extract of the lichen powder. Referring to Table 1, the alkaloid test is composed by adding 1 mL of Dragendorff”s reagent to 2 mL of extract, an orange-red precipitate, indicating the presence of alkaloids. Mayer's test is composed few drops of Mayer's reagent were added to 1 mL of extract, thereby forming a yellowish or white precipitate was formed, and indicates the presence of alkaloids.
[0057] According to another exemplary embodiment of the invention, FIG. 2E refers to a pictorial representation 208 of the terpenoid test performed on the methanol extract of lichen powder. Referring to Table 1, the terpenoid test is adapted to extract 5 mL mixed with chloroform (2 mL), and concentrated sulphuric acid (3 mL) was carefully added to form a layer. A reddish-brown coloration of the interface was formed to show positive results for the presence of terpenoids.
[0058] According to another exemplary embodiment of the invention, FIG. 2F refers to a pictorial representation 210 of the quinone test performed on the methanol extract of lichen powder. In one embodiment herein, the quinone test is performed by 1 mL of the extract is added to the few drops of concentrated H2SO4, thereby forming the green colour and the positive presence of the quinone.
[0059] According to another exemplary embodiment of the invention, FIG. 3 refers to a flowchart 300 for isolation of the lecanoric acid from crude extract Parmotrema praesorediosum using column chromatography. In one embodiment herein, the step 302 crude extract preparation from Parmotrema prasorediosum, is accomplished by extracting the lichen material with methanol, a solvent commonly used to extract a broad range of polar and semi-polar compounds. The crude extract is a complex mixture containing numerous natural compounds, which need to be isolated and purified for further analysis. The first round of the column chromatography (steps 304-318), the crude extract is subjected to column chromatography. The mixture is passed through a silica gel column (with a mesh size of 230-400), where silica gel acts as the stationary phase. Compounds in the extract are separated based on their polarity as they interact with the stationary phase and are eluted using a mobile phase consisting of a solvent mixture.
[0060] At steps 320-356, after the initial fractions are collected, selected fractions undergo further purification through additional rounds of column chromatography that allow for the isolation of pure compounds from the collected fractions. The specific fractions are chosen for further chromatography based on their composition and properties. The solvent ratios are adjusted in subsequent rounds of chromatography to allow for finer separation of individual compounds. This process ensures that compounds with similar polarities are effectively separated. The final purification and identification of pure compounds (steps 322-352) that involves isolating pure compounds, which are labeled as PP-1 302, PP-2 328, PP-3 348, PP-4 356, PP-5 326, PP-6 338, PP-7 340, PP-8 342, PP-9 346, and PP-10 354. Each label corresponds to a distinct compound isolated through the described process. The yields of the purified compounds are recorded in milligrams (mg). For example, PP-1 (400 mg) indicates that 400 milligrams of compound PP-1 were obtained after the final purification.
[0061] In one example, the solvent used a hexane that is a non-polar solvent used to evaluate the least polar compounds from the column. Ethyl acetate (EtAc) is a moderately polar solvent that elutes compounds with intermediate polarity. MeOH (Methanol) is a highly polar solvent used to elute the most polar compounds. The isolation process is to obtain pure compounds (PP-1 to PP-10), which are suitable for further analysis. These compounds can undergo chemical characterization and potentially be tested for biological activity, such as antimicrobial or antioxidant properties.
[0062] According to another exemplary embodiment of the invention, FIG. 4 refers to a structure 400 of the lecanoric acid. In one embodiment herein, the lecanoric acid is composed of two aromatic rings (benzene rings) with hydroxyl groups (-OH) and carboxyl groups (-COOH) attached to it. One of the benzene rings has a methoxy group (-OCH3) connecting it to the second ring via an ester linkage, and each ring have hydroxyl groups (-OH) and methyl groups (-CH3) as substituents, with the second ring featuring carboxyl groups (-COOH).
[0063] According to another exemplary embodiment of the invention, FIG. 5A refers to a graphical representation 500 of a mass report of the positive mode spectrum of lecanoric acid. In one embodiment herein, the mass spectrum (MS) is typically appearing in the relative abundance of ions detected at various mass-to-charge (m/z) ratios. In one embodiment herein, the x-axis represents the mass-to-charge ratio (m/z) of the ions detected during the mass spectrometry process. The y-axis represents the intensity and abundance of detected ions, measured in CPS (counts per second).
[0064] The tallest peak near m/z ~578 indicates the base peak, which is typically the most stable and abundant fragment of the molecule. In MS, the base peak is often used as a reference point for other peaks. The Peaks appearing below m/z 200 may correspond to fragmented parts of benzoic acid derivatives (for example, individual phenyl rings or carbonyl groups). The presence of smaller peaks in the higher m/z range (600-800) could suggest the presence of sodium or potassium adducts or other ion complexes that were formed during the ionization process. These peaks are common in positive ESI (electrospray ionization) mode.
[0065] According to another exemplary embodiment of the invention, FIG. 5B refers to a graphical representation 502 of the negative mode spectrum of lecanoric acid. In one embodiment herein, the positive mode spectrum is ESI-MS generally detects positively charged ions, which often include protonated molecules [M+H]+ or adducts with sodium [M+Na]+. The negative mode detects deprotonated molecules [M-H]−, or other negatively charged species, like anions.
[0066] The x-axis represents the mass-to-charge ratio (m/z) of the ions detected. This ratio gives us insight into the molecular fragments present in the sample. The y-axis represents the relative abundance of the ions detected at each m/z value, measured in CPS (counts per second). The tallest peaks represent the most abundant ion fragments. The prominent peak at around m/z 317 could suggest a significant fragment of the molecule or a specific anion species formed during the ionization process. The peak around m/z 578 may indicate a higher-order molecular fragment, or a dimeric or larger aggregate.
[0067] In one embodiment herein, the positive mode with the protonated molecular ions or sodium-adducted forms of lecanoric acid, which might result in peaks that reflect the mass of the intact or partially ionized molecule. The negative mode concentrates on deprotonated ions or fragments. The absence of smaller fragment ions could indicate a preference for larger, more stable anionic fragments.
[0068] According to another exemplary embodiment of the invention, FIG. 5C refers to a graphical representation 504 of the hydrogen nuclear magnetic resonance spectrum of the lecanoric acid. In one embodiment herein, the carboxylic acid proton is attached to the cyclic carbon. The lecanoric acid is typically appears as a singlet at around 9-12 ppm. The methyl protons are attached to carbons of the phenolic ring, which are typically appear as singlets in the region of 2-3.0 ppm. The exact chemical shifts and coupling patterns will depend on the stereochemistry of the phenolic ring and any interactions with water molecules. In one embodiment herein, the chemical shifts (6.61, 6.61, 6.23 and 6.23 ppm) are to be consistent with the expected range for protons in the phenyl ring.
[0069] According to another exemplary embodiment of the invention, FIG. 5D refers to a graphical representation 506 of the carbon nuclear magnetic resonance spectrum of the lecanoric acid. In one embodiment herein, the carbonyl (carboxylic) carbon typically appears in the range of 170-180 ppm. In one embodiment herein, the ring carbon is part of the aromatic ring, and generally appears in the range of 100-120 ppm. The exact chemical shifts will depend on the stereochemistry of the aromatic ring and any interactions with water molecules.
[0070] Table. 2
Carbon
No. PP-05 - NMR data (400 MHz, DMSO- d6)
Lecanoric Acid - NMR data (60 MHz, DMSO- d6)
1 H NMR
(δ, ppm) 13 C NMR
(δ, ppm) 1 H NMR
(δ, ppm) 13 C NMR
(δ, ppm)
1. - 117.01 - 116.3
2. Exchanged with D2O (-OH) 159.43 16.47 (1H, S)
(-OH) 159.5
3. 6.61 (1H, S) Aromatic H 108.73 6.80 (1H, S)
Aromatic H 108.7
4. - 152.76 - 153.7
5. 6.61 (1H, S)
Aromatic H 115.23 6.89 (1H, S)
Aromatic H 115.5
6. - 140.04 - 140.2
7. - 167.61 - 168.4
8. - 107.88 - 105.4
9. Exchanged with D2O (OH) 160.65 16.47 (1H, S) 162.54
10. 6.23 (1H, S)
Aromatic H 100.99 6.24 (1H, S)
Aromatic H 100.5
11. 10.01 (1H, S) (OH) 161.63 9.45 (1H, S) 163.5
12. 6.23 (1H, S)
Aromatic H 110.38 6.41(1H, S)
Aromatic H 112.3
13. - 140.83 - 144.0
14. 2.36 (3H, S)
(-CH3) 21.83 2.48 (3H, S)
(-CH3) 22.1
15. 2.37 (3H, S)
(-CH3) 21.50 2.48 (3H, S)
(-CH3) 21.8
16. 10.32 (1H, S)
(-COOH) 171..11 12.75 (1H, S)
(-COOH) 173.2

[0071] The chemical shifts in the spectrum (117.01, 159.43, 108.73, 152.76, 115.23, 140.04, 167.61, 107.88, 160.65, 100.99, 161.63, 110.38, 140.83, 21.83, 21.83, 171.11 ppm) appear to be consistent with the expected range for carbon atoms in a leconoric acid molecule. The presence of 16 peaks suggests that the molecule has 16 unique carbon environments. The presence consistent with the structure of molecule leconoric acid. In one example embodiment, the table 2 depicts the NMR data provide strongly supports the identification of PP-05 as the lecanoric acid. The high degree of similarity in both 1H and 13C NMR chemical shifts indicates that the two compounds have essentially the same structure. The minor differences observed in some chemical shifts can be attributed to factors such as solvent effects and conformational variations.
[0072] According to another exemplary embodiment of the invention, FIGs. 6A-6B refers to pictorial representations (600, 602) of luria broth (L.B) plates with inhibition zone of tetracycline and ampicillin. In one embodiment herein, the two LB plates, each containing four circular zones of inhibition, which represents area around antibiotic-containing discs where bacterial growth has been inhibited. Referring to FIG. 6A, the positive control indicates the known antibiotic (tetracycline) at a concentration of 30 mcg. A clear zone of inhibition is observed, suggesting the effectiveness of the positive control. In one embodiment herein, the tetracycline TE 30 mcg discs are used for antimicrobial susceptibility testing of bacterial cultures as per Kirby-Bauer Method, thereby indicating that phyline might not have significant antimicrobial activity against the tested bacteria.
[0073] Referring to FIG. 6B, the positive control indicates antibiotic (ampicillin) at a concentration of 30 mcg, thereby confirming the effectiveness of the positive control. The representation of ampicillin 10 mg suggest that is effective at a lower concentration than the positive control. In one example, the evaluated against E. coli and S. cerevisiae using the spread plate method. The fraction containing lecanoric acid showed complete inhibition of both organisms at a concentration of 500 µM and 1 mM respectively. This suggests that lecanoric acid is a potent antimicrobial agent. The positive control for both tetracycline and ampicillin are effective in inhibiting bacterial growth.
[0074] According to another exemplary embodiment of the invention, FIGs. 6C-6D refer to pictorial representations (604, 606) of the LB agar plates with inhibition zones of negative control and PP-05 (lecanoric acid). In one embodiment herein, the two LB agar plates. The negative control indicates a control disc without any antibiotic. A large number of bacterial colonies (greater than 100) are visible within this zone, suggesting that the bacteria are growing uninhibited. The 25 µL of PP-5 is added to 50 µL of E. coli that represents the test compound (PP-5) at a specific concentration. The clear zone of inhibition is observed, thereby indicating that PP-5 have antimicrobial activity against the tested bacteria and suggesting that the concentration or formulation of PP-5 might be different between the two plates.
[0075] According to another exemplary embodiment of the invention, FIGs. 6E-6F refers to pictorial representations (608, 610) of yeast plates, negative control and PP-05 (lecanoric acid). Referring to FIG. 6E, the negative control indicates a plate without any antibiotic. The large number of yeast colonies are visible on the negative control plate, thereby suggesting that the yeast is growing uninhibited. The 25 µL of PP-5 and 50 µL of yeast represent the test compound (PP-5) at a specific concentration. The clear zone of inhibition is observed, thereby indicating that PP-5 has antifungal activity against the tested yeast.
[0076] Referring to FIG. 6F, the large number of yeast colonies are visible within this zone, similar to FIG. 6E. The PP-5 represents the same test compound (PP-5) at the same concentration. No yeast growth is observed in this zone, thereby suggesting complete inhibition of the yeast by PP-5. In one embodiment herein, the PP-5 is more effective at inhibiting yeast growth, thereby signifying that the concentration or formulation of PP-5 might be different between the two plates.
[0077] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, the method for isolating the lecanoric acid is disclosed. The proposed method utilizes lecanoric acid as a natural and renewable source, thereby reducing dependency on synthetic drugs and highlighting the importance of biodiversity in drug discovery. The proposed method offers the lecanoric acid to demonstrate significant antimicrobial activity, in antibiotic-resistant strains of bacteria, thereby providing a potential alternative to conventional antibiotics that are losing efficacy due to resistance.
[0078] The proposed method isolates bacteria such as Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) exhibiting complete inhibition at low concentrations, making it a potent and targeted antimicrobial agent. The proposed method accelerates the development of new drugs based on lecanoric acid. The proposed method enhances the scientific community's understanding of lichen-derived compounds, potentially leading to the discovery of additional bioactive substances from similar natural sources. The proposed method extracts purified lecanoric acid from Parmotrema prasorediosum has demonstrated promising antimicrobial activity, modifying the need for further research using animal models and clinical trials.
[0079] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I/We Claim:
1. A method for extracting lecanoric acid from Parmotrema praesorediosum, comprising:
collecting fresh samples of the Parmotrema praesorediosum and cleaning the collected samples with distilled water to remove epiphytic hosts;
drying the cleaned samples on a filter paper at a room temperature to remove moisture and grinding the dried samples into a fine powder;
mixing the fine powder with at least one organic solvent at a predetermined time to perform solvent extraction and obtain an extract;
filtering the extract to remove solid residues and obtain a crude extract, which consists the lecanoric acid;
evaporating the crude extract under reduced pressure to control a purification process and isolating the purified extract by using at least one column chromatographic technique; and
characterizing the isolated extract by dissolving it in a suitable solvent to obtain in a pure crystalline form, thereby determining physical properties and chemical structure.
2. The method as claimed in claim 1, wherein the at least one column chromatographic technique is conducted using silica gel with mesh size of 230 – 400 mm.
3. The method as claimed in claim 1, wherein the predetermined time is at least one week to optimize the extraction process and obtain the extract.
4. The method as claimed in claim 1, wherein the at least one organic solvent includes acetone, ethanol and methanol under reflux conditions.
5. The method as claimed in claim 1, wherein the collected samples are dried at a temperature varies between 40 °C and 50 °C to preserve the integrity of the lecanoric acid.
6. The method as claimed in claim 1, wherein the lecanoric acid is characterized by a white solid powder with a melting point of 175 °C to 176 °C, UV absorption with a maximum wavelength of 312 nm in methanol, a molecular formula (C16H14O7), and a nuclear magnetic resonance (NMR) spectral data.
7. The method as claimed in claim 1, wherein the lecanoric acid is effective against bacterial and fungal strains that includes Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) with complete inhibition observed at a concentration of 500 micromolar (µM) and 1 millimolar (mM) respectively.
8. The method as claimed in claim 1, wherein the at least one chromatographic technique includes gas chromatography, high-performance liquid chromatography, thin-layer chromatography, and paper chromatography.