Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of natural product isolation and antimicrobial applications, and in specific relates to a method of isolating naturally occurring compound derived specifically from Parmotrema reticulatum and the application of this compound as an antimicrobial agent.
Background of the invention:
[0002] Rise of antimicrobial-resistance has become a significant global health concern, limiting the effectiveness of current treatments against bacterial, fungal, and viral infections. This issue is particularly concerning in the treatment of drug-resistant infections, such as those caused by Mycobacterium tuberculosis (TB), where existing drugs may fail to achieve the desired therapeutic outcomes. Consequently, there is an urgent need for novel antimicrobial agents that can effectively combat resistant pathogens without causing harmful side effects.
[0003] Traditional antimicrobial agents are losing efficacy as pathogens evolve and develop resistance. Drug-resistant strains, especially in diseases like tuberculosis, have led to higher treatment failure rates and increased healthcare burdens. Many synthetic antimicrobial drugs also come with the risk of toxicity, allergic reactions, and side effects, limiting their use and affecting patient compliance. Additionally, the extensive use of these drugs in both human and veterinary medicine has accelerated resistance development, making it more challenging to control infectious diseases. Thus, identifying new antimicrobial compounds with unique mechanisms of action, low toxicity, and high biocompatibility is critical to addressing these challenges.
[0004] Pharmaceuticals are drugs with modified molecular structures to evade resistance mechanisms. These include altering chemical groups to hinder pathogen enzymes that degrade or neutralize drugs. While structurally modified synthetic drugs can evade resistance mechanisms, they often require extensive R&D investment and complex manufacturing processes. Additionally, they may still have toxicity issues that can limit their use.
[0005] Combining two or more drugs to improve treatment efficacy is common. For example, drug combinations are often used in TB treatment to tackle multidrug-resistant strains. Although effective, combination therapy can lead to increased toxicity due to the additive side effects of multiple drugs. Resistance can still emerge over time, requiring new drug combinations.
[0006] Extracts from plants, fungi, and lichens are being explored as potential sources of natural antimicrobial agents. These natural sources have complex molecular structures that pathogens are less likely to resist. Although promising, natural compounds often require extensive screening, and isolating active compounds in sufficient quantities can be resource-intensive.
[0007] Novel delivery mechanisms, such as nanoparticles or liposomal formulations, aim to improve drug bioavailability and minimize side effects, ensuring that drugs reach infected areas effectively. These systems, while improving drug efficacy and safety, often require complex and costly technologies to produce. Additionally, these delivery systems may encounter regulatory challenges, delaying their entry into the market.
[0008] Therefore, there is a need for a method for isolating naturally occurring compound derived from Parmotrema reticulatum. There is also a need for utilising the naturally occurring compound as antimicrobial agents. There is also a need for a method that is effective, sustainable, and biocompatible. There is also a need for a natural antimicrobial agent that demonstrates potential in combating resistant microbial strains with reduced toxicity, offering a viable alternative to existing synthetic drugs and combination therapies.
Objectives of the invention:
[0009] The primary objective of the invention is to provide a method for isolating natural antimicrobial agent specifically from Parmotrema reticulatum and the application of this compound as an antimicrobial agent.
[0010] The other objective of the invention is to utilize a naturally occurring compound derived from Parmotrema reticulatum, as a potential antimicrobial agent, offering a sustainable and environmentally friendly alternative to synthetic drugs.
[0011] Another objective of the invention is to provide a natural antimicrobial agent that demonstrates potential in combating resistant microbial strains with reduced toxicity, offering a viable alternative to existing synthetic drugs and combination therapies.
[0012] The other objective of the invention is to reduce toxicity and improve biocompatibility in antimicrobial agents, improving the safety profile and compatibility for medical applications.
[0013] Another objective of the invention is to develop treatments effective against drug-resistant bacterial strains.
[0014] The other objective of the invention is to enable combination therapies that enhance efficacy and reduce dosages of existing drugs.
[0015] Yet another objective of the invention is to provide a method for sustainable extraction and isolation of antimicrobial compounds.
[0016] Another objective of the invention is to support drug development without the need for complex synthetic processes.
[0017] Another objective of the invention is to expand the scope of antimicrobial treatments, investigating atraric acid’s efficacy against a wide range of pathogens, including fungal and bacterial species.
Summary of the invention:
[0018] The present disclosure proposes a method for isolating atraric acid from parmotrema reticulatum for antimicrobial agents. 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.
[0019] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method of isolating atraric acid specifically from Parmotrema reticulatum and the application of this compound as an antimicrobial agent.
[0020] According to an aspect, the invention provides a method for isolating atraric acid from Parmotrema reticulatum. At first step, fresh samples of Parmotrema reticulatum are collected and cleaned with tap and distilled water. Next, the collected samples are dried at room temperature. Next, the dried samples are suspended in methanol for a time period to obtain a crude extract. Next, the crude extract is concentrated under reduced pressure to obtain a crude methanol extract of the Parmotrema reticulatum.
[0021] Next, the crude methanol extract of the Parmotrema reticulatum is subjected to column chromatography over mobile phase to obtain fractionated extracts. In one embodiment, the mobile phase comprises plurality of solvents that include hexane, ethyl acetate, and methanol. The mobile phase used in column chromatography has a mesh size of 230-400 µm. Later, the atraric acid is isolated from the fractionated extracts.
[0022] In one embodiment, an antimicrobial composition comprises the atraric acid isolated from Parmotrema reticulatum. The antimicrobial composition is configured for use in inhibiting microbial growth. The growth inhibited microbes include bacterial and fungal strains.
[0023] In one embodiment, an effective amount of an antimicrobial composition that includes atraric acid isolated from Parmotrema reticulatum is evaluated for inhibiting the growth of Mycobacterium tuberculosis. The antimicrobial composition is evaluated in combination with other antitubercular drugs to enhance efficacy and reduce dosage requirements of said drugs.
[0024] According to another aspect, the invention provides the atraric acid that is utilised for formulation of antimicrobial compositions, which are used in inhibiting microbial growth. The isolated atraric acid is characterized for antimicrobial activity against selected microbial strains. The atraric acid is formulated into a pharmaceutical composition for potential clinical use. In one embodiment, the selected microbial strains include drug-resistant strains of bacteria. The synergistic antimicrobial effect of the atraric acid is tested in combination with existing antimicrobial agents.
[0025] 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:
[0026] 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.
[0027] FIG. 1 illustrates a flowchart of a method for isolating atraric acid from Parmotrema reticulatum, in accordance to an exemplary embodiment of the invention.
[0028] FIG. 2 illustrates a FT-MS-ESI-positive mode spectrum of the atraric acid, in accordance to an exemplary embodiment of the invention.
[0029] FIG. 3 illustrates a FT-IR spectrum of the atraric acid obtained using potassium bromide (KBr) as the sample medium, in accordance to an exemplary embodiment of the invention.
[0030] FIGs. 4A to 4B illustrate a proton and carbon NMR spectra of the atraric acid, respectively, recorded in deuterated chloroform (CDCl₃) at a 400 MHz frequency, in accordance to an exemplary embodiment of the invention.
[0031] FIGs. 5A to 5D illustrate images that depict bacterial growth in various experimental conditions, in accordance to an exemplary embodiment of the invention.
[0032] FIGs. 6A to 6B illustrate images that depict the effect of the atraric acid on yeast growth, in accordance to an exemplary embodiment of the invention.
[0033] FIGs. 7A to 7B illustrate images that depict differential effects of the atraric acid on wild-type and drug-resistant bacterial strains, in accordance to an exemplary embodiment of the invention.
[0034] FIGs. 8A to 8B illustrate images that depict the effects of atraric acid in combination with antibiotics on bacterial growth, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0035] 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.
[0036] 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 of isolating atraric acid specifically from Parmotrema reticulatum and the application of this compound as an antimicrobial agent.
[0037] According to an exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for isolating atraric acid from Parmotrema reticulatum. At step 102, fresh samples of Parmotrema reticulatum are collected and cleaned with tap and distilled water. In one embodiment, the term “Parmotrema reticulatum” as used herein refers to lichen P. reticulatum that are collected from bark of plant Excoecaria agallocha. The fresh samples of Parmotrema reticulatum are cleaned to remove epiphytic hosts present on surface of the fresh samples of Parmotrema reticulatum.
[0038] At step 104, the collected samples are dried at room temperature. In one embodiment, the collected samples, which are washed, are shade dried on filter paper at room temperature for a time period of at least 7 days to obtain dried samples.
[0039] Later, at least 150 g of the dried samples are powdered to obtain a Parmotrema reticulatum powder. At step 106, the Parmotrema reticulatum powder is suspended in methanol for a time period of 7 to 10 days to obtain a crude extract. In a preferred embodiment, Parmotrema reticulatum powder is subjected to an exhaustive extraction process through maceration. This process is repeated over three consecutive cycles, with fresh methanol introduced at the beginning of each cycle to maximize the extraction efficiency to obtain the crude extract.
[0040] At step 108, the crude extract is concentrated under reduced pressure by using Rota-vapour at a temperature of 40°C to 50°C to remove methanol to obtain a crude methanol extract of the Parmotrema reticulatum. In a preferred embodiment, the crude extract is concentrated using a solvent removal technique optimized to preserve thermolabile compounds. This process involved the application of controlled conditions, such as reduced pressure at a temperature of 40°C to 50°C, to minimize thermal degradation while ensuring efficient solvent removal. The concentration process is carried out until a minimum yield of 10 g of the crude methanol extract from Parmotrema reticulatum is obtained.
[0041] At step 110, at least 10 g of the crude methanol extract of the Parmotrema reticulatum is subjected to column chromatography using mobile phase (230–400 mesh size) as the stationary phase to obtain fractionated extracts. In one embodiment, the mobile phase comprises plurality of solvents that include hexane, ethyl acetate, and methanol. The mobile phase used in column chromatography has a mesh size of 230-400 µm.
[0042] In a preferred embodiment, the column is eluted using a gradient system of hexane and ethyl acetate (Hex:EtAc). This process resulted in the generation of three distinct fractions. Fraction I is obtained with a solvent ratio of Hex:EtAc (90:10) and yield 62 mg of the fractionated extract. Fraction II is eluted with a ratio of Hex:EtAc (80:20) and produced 23 mg of the fractionated extract. Fraction III is collected with a ratio of Hex:EtAc (50:50) and yield 15 mg of the fractionated extract.
[0043] At step 112, the atraric acid is isolated from the fractionated extracts. In a preferred embodiment, each fraction is further purified by preparative column chromatography using a gradient elution system. Fraction I is purified to yield the PR-01 with a solvent ratio of Hexane:Dichloromethane (Hex:DCM, 9:1) and a final weight of 25 mg. Similarly, Fraction II is refined to obtain PR-03 using a solvent ratio of Hexane:Ethyl Acetate (Hex:EtAc, 9:1), yielding 6 mg. Fraction III is purified to produce the atraric acid (PR-06) with a solvent system of Dichloromethane:Ethyl Acetate (DCM:EtAc, 8:2) and a yield of 10 mg. This systematic multi-step purification strategy ensured the effective isolation of the atraric acid from the crude methanol extract of the Parmotrema reticulatum with high specificity and purity. In one embodiment, the atraric acid is subjected to purification using a hexane wash.
[0044] According to another exemplary embodiment of the invention, FIG. 2 refers to a FT-MS-ESI-positive mode spectrum 200 of atraric acid. The spectrum 200 is recorded using Fourier Transform Mass Spectrometry (FT-MS) coupled with Electrospray Ionization (ESI) in the positive mode, allowing for the accurate determination of the molecular mass and the identification of the ionized species of atraric acid.
[0045] Formula 1 refers to the atraric acid.

[0046] The atraric acid is obtained as a white solid powder with a melting point ranging from 143°C to 145°C. Its UV absorption spectrum showed a maximum absorption (λ max) at 285 nm in methanol. Based on elemental analysis, the molecular formula of atraric acid is determined to be C₁₀H₁₂O₂. This is further confirmed by electrospray ionization mass spectrometry (ESI-MS), which exhibited a molecular ion peak at m/z 197.05 in positive mode and m/z 195.05 in negative mode.
[0047] According to another exemplary embodiment of the invention, FIG. 3 refers to a FT-IR spectrum 300 of atraric acid obtained using potassium bromide (KBr) as the sample medium. This spectrum provides detailed information about the functional groups present in the compound, based on the absorption of infrared light at characteristic wavelengths.
[0048] According to another exemplary embodiment of the invention, FIGs. 4A to 4B refer to the Proton and Carbon NMR spectra (400 and 402) of atraric acid, respectively, recorded in deuterated chloroform (CDCl₃) at a 400 MHz frequency. These spectra (400, 402) offer valuable insights into the hydrogen atom environment within the molecule, aiding in the structural characterization of atraric acid.
[0049] Table 1:
Carbon
No. PR-06 - NMR data (400 MHz, CDCl₃) Atraric acid - NMR data (60 MHz, CDCl₃)
1 H NMR
(δ, ppm) 13 C NMR
(δ, ppm) 1 H NMR
(δ, ppm) 13 C NMR
(δ, ppm)
1 - 110.62 - 112.1
2 12.05 163.10 12.04 (OH, 1H, S) 162
3 - 105.13 - 108.6
4 Exchange with D2O 158.25 9.58, (OH, 1H, S) 161.7
5 6.22 108.67 6.32 (1H, S) 111.5
6 - 140.15 - 140.6
7 - 172.68 - 171
8 3.94 51.84 3.10 (3H, S) 51.5
9 2.46 7.69 2.48 (3H, S) 8.9
10 2.12 24.08 2.16 (3H, S) 22.1
[0050] Referring to table 1, the 1H and 13C NMR spectral data of PR-06 suggest that it is a derivative of benzoic acid. The 1H NMR spectrum (as shown in FIG. 4A) and 13C NMR (as shown in FIG. 4B) exhibited two distinct methyl proton signals: a singlet at 2.46 ppm (3H) corresponding to a carbon signal at 7.69 ppm (C-9) and another singlet at 2.12 ppm (3H) associated with a carbon signal at 24.08 ppm (C-10). Additionally, a carbonyl carbon signal is observed at 172.64 ppm (C-7).
[0051] The spectra also indicated the presence of two phenolic protons appearing as a singlet at 12.05 ppm (1H), corresponding to carbon signals at 163.16 ppm (C-2) and 158.24 ppm (C-4). A methoxy group is identified with a carbon signal at 51.81 ppm (C-8) and methoxy protons appearing as a singlet at 3.94 ppm (3H). These spectral features collectively support the structural characterization of PR-06.
[0052] The NMR spectral data revealed the presence of a single aromatic proton at 6.23 ppm (s, 1H), corresponding to a carbon signal at 105.13 ppm (C-5). A detailed comparison of the physical and spectral characteristics of atraric acid with those of PR-06 confirmed its identity as methyl 2,4-dihydroxy-3,6-dimethylbenzoate, as summarized in Table 1. Based on this analysis, the structure and absolute configuration of PR-06 were definitively assigned to atraric acid.
[0053] In one embodiment, an antimicrobial composition comprises the atraric acid isolated from Parmotrema reticulatum. The antimicrobial composition is configured for use in inhibiting microbial growth. The microbial growth inhibited includes bacterial and fungal strains.
[0054] In one embodiment, an effective amount of an antimicrobial composition that includes atraric acid isolated from Parmotrema reticulatum is evaluated for inhibiting the growth of Mycobacterium tuberculosis. The antimicrobial composition is administered in combination with other antitubercular drugs to enhance efficacy and reduce dosage requirements of said drugs.
[0055] According to another exemplary embodiment of the invention, the atraric acid that is utilised for formulation of antimicrobial compositions, which are used in inhibiting microbial growth. The isolated atraric acid is characterized for antimicrobial activity against selected microbial strains. The atraric acid is formulated into a pharmaceutical composition for potential clinical use. In one embodiment, the selected microbial strains include drug-resistant strains of bacteria. The synergistic antimicrobial effect of the atraric acid is tested in combination with existing antimicrobial agents.
[0056] According to another exemplary embodiment of the invention, FIGs. 5A to 5D refer to images (500, 502, 504, and 506) that depict bacterial growth in various experimental conditions. In another embodiment, atraric acid has been shown to be effective against both bacterial strains and fungi, including but not limited to Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae). Complete inhibition of bacterial growth was observed at a concentration of 1 micromolar (µM) for E. coli and 1 millimolar (mM) for S. cerevisiae.
[0057] FIG. 5A shows bacterial growth inhibition by tetracycline, while FIG. 5B shows inhibition by ampicillin. FIG. 5C represents the negative control, where no antibiotics are applied, resulting in bacterial growth. FIG. 5D demonstrates the effect of atraric acid (1 micromolar final concentration), which resulted in a clear plate with no bacterial growth. The bacterial strain used in this experiment was Escherichia coli.
[0058] According to another exemplary embodiment of the invention, FIGs. 6A to 6B refer to images (600, 602) that depict the effect of the atraric acid on yeast growth. FIG. 6A shows the full growth of Saccharomyces cerevisiae without the addition of atraric acid, indicating normal yeast proliferation. FIG. 6B depicts complete inhibition of yeast growth when treated with a 1 millimolar final concentration of atraric acid, resulting in no visible growth.
[0059] According to another exemplary embodiment of the invention, FIGs. 7A and 7B refer to images (700, 702) that depict differential effects of the atraric acid on wild-type and drug-resistant bacterial strains. FIG. 7A shows the growth of wild-type bacteria, where a 1 millimolar final concentration of atraric acid is applied, resulting in complete inhibition of bacterial growth. This indicates that atraric acid is highly effective in inhibiting the growth of the wild-type strain. FIG. 7B shows drug-resistant bacteria exposed to the same concentration of atraric acid. While the growth of the drug-resistant bacteria is significantly reduced, partial growth is still observed, indicating that atraric acid is less effective against these resistant strains. This comparison highlights the varying susceptibility of bacterial strains to atraric acid, with wild-type bacteria being completely inhibited and drug-resistant strains exhibiting partial resistance to its antibacterial effects.
[0060] According to another exemplary embodiment of the invention, FIGs. 8A to 8B refer to images (800, 802) that depict the effects of atraric acid in combination with antibiotics on bacterial growth. Referring to FIG. 8A, a first plate represents the negative control, containing neither bacteria nor antibiotics. A second plate shows bacteria without antibiotics, resulting in full bacterial growth. A third plate contains both bacteria and antibiotic discs, but the zones of inhibition are not clearly defined, indicating limited or no effect of the antibiotics under the conditions tested. Referring to FIG. 8B, the three plates from left to right show bacteria exposed to three antibiotic discs along with varying concentrations of atraric acid (1 nM, 10 nM, and 100 nM). As the concentration of atraric acid increases, the zones of inhibition around the antibiotic discs become more clearly visible. This suggests a potential additive and/or synergistic effect between atraric acid and the antibiotics, where atraric acid enhances the efficacy of the antibiotics in inhibiting bacterial growth. The clearer zones of inhibition with increasing atraric acid concentration indicate that the compound may boost the antibacterial activity of the antibiotics, possibly by increasing their penetration or effectiveness.
[0061] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method is disclosed herein for isolating naturally occurring compound derived from Parmotrema reticulatum.
[0062] Atraric acid is a naturally occurring compound derived from Parmotrema reticulatum that serves as a potential antimicrobial agent, offering a sustainable and environmentally friendly alternative to synthetic drugs. Using this natural compound reduces the toxicity risks commonly associated with synthetic antimicrobial agents, thus improving the safety profile and compatibility for medical applications.
[0063] The isolated atraric acid effectively targets bacterial strains, particularly drug-resistant types such as Mycobacterium tuberculosis, by leveraging its unique molecular structure and mechanism of action. Additionally, atraric acid can act as a complementary agent in antimicrobial treatments, enhancing the effects of other drugs or reducing the required dosages, potentially lowering side effects and slowing the development of resistance.
[0064] By utilizing lichens P. reticulatum as a raw material source, the proposed method emphasizes extraction techniques, providing an accessible and renewable method for producing natural antimicrobial agents. The proposed method simplifies the drug development pipeline by directly isolating atraric acid from P. reticulatum, thereby reducing the need for extensive chemical synthesis and the associated costs. The proposed method broadens the scope of antimicrobial treatments by exploring atraric acid’s efficacy against a wide range of pathogens, including both fungal and bacterial species.
[0065] 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 isolating atraric acid from Parmotrema reticulatum, comprising:
collecting and cleaning fresh samples of Parmotrema reticulatum;
drying the collected samples at room temperature;
suspending the dried samples in methanol for a time period to obtain a crude extract;
concentrating the crude extract under reduced pressure to obtain a crude methanol extract of the Parmotrema reticulatum;
subjecting the crude methanol extract of the Parmotrema reticulatum to column chromatography over mobile phase to obtain fractionated extracts; and
isolating atraric acid from the fractionated extracts.
2. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, the mobile phase comprises plurality of solvents that include hexane, ethyl acetate, and methanol.
3. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, wherein the mobile phase used in column chromatography has a mesh size of 230-400 µm.
4. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, wherein the atraric acid is utilised for formulation of antimicrobial compositions, which are used in inhibiting microbial growth.
5. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, wherein the isolated atraric acid is characterized for antimicrobial activity against microbial strains.
6. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 5, wherein the microbial strains include drug-resistant strains of bacteria.
7. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, wherein the dried samples are suspended in methanol for a time period of 7 to 10 days to obtain the crude extract.
8. The method for isolating atraric acid from Parmotrema reticulatum as claimed in claim 1, wherein the crude extract is concentrated under reduced pressure at temperature of 40°C to 50°C to obtain the crude methanol extract of the Parmotrema reticulatum.