ANTIMICROBIAL POTENTIAL OF CRUDE EXTRACTS AND FATTY ACID FRACTIONS OF FOUR PTERIDOPHYTE SPECIES FROM ASSAM, NORTHEAST INDIA, AND THEIR IDENTIFICATION BY GC-MS
HTML Full TextANTIMICROBIAL POTENTIAL OF CRUDE EXTRACTS AND FATTY ACID FRACTIONS OF FOUR PTERIDOPHYTE SPECIES FROM ASSAM, NORTHEAST INDIA, AND THEIR IDENTIFICATION BY GC-MS
S. Borkotoky * and V. V. Borah
Department of Biosciences, School of Life Sciences, Assam, Don Bosco University, Sonapur, Assam, India.
ABSTRACT: Fatty acids produced by plants protect themselves from pathogens, such as multidrug-resistant bacteria. In the present work, crude extracts, and fatty acid fractions of four pteridophyte species from Assam, Northeast India, were evaluated for their antimicrobial properties, and the bioactive compounds were identified using GC-MS analysis. The crude extract of Pteris semipinnata (Ps), Lygodium microphyllum (Lm), Lycodium flexuosum (Lf), and Lycopodiella cernua (Lc) was found to possess antibacterial and antifungal activities. The lowest MIC and MBC values were 0.25 mg/ml and 0.5 mg/ml for the fatty acid fractions against most of the microbial test strains. The GCMS analysis revealed 21 compounds in Ps, 35 in Lm, 33 in Lc, and 52 in Lf fractions. The present work supports the use of fern species in traditional medicine and therapies.
Keywords: Pteridophytes, Fatty acids, Antimicrobials, GC-MS
INTRODUCTION: About half of the deaths in tropical countries are caused by infectious diseases. Antibiotic resistance is a problem affecting the world and every nation. Antibiotic resistance occurs when bacteria become resistant and continue to proliferate in the presence of therapeutic amounts of an antibiotic, meaning the antibiotic can no longer effectively control or kill bacterial growth. Many factors contribute to antibiotic resistance. These include an inadequate grasp of how antibiotics function and how improper patient use promotes the development of resistance 1. Drug-resistant bacteria have emerged because of the overuse of antibiotics in both medicine and food production.
Utilizing plant remedies is a straightforward solution to this issue. Herbal medications are intricate biological structures that have progressively evolved and contain hundreds of active substances that interact harmoniously. Long known for their antimicrobial effects, fatty acids (FA) are produced by plants to protect themselves from pathogens, such as multidrug-resistant bacteria 2. Recently, FAs have also come to light as a possible antibiotic substitute.
Numerous FAs have been found to selectively inhibit various microbial pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Burkholderia cenocepacia, Vibrio spp., and Candida albicans suggesting their tremendous potential 3. Pteridophytes have several medicinal uses in folk medicine. In the tribal communities of Assam, pteridophytes have been used to treat various ailments, including colds and coughs, boils, cuts and wounds, respiratory problems, diarrhea, inflammation, bodily pain, hair loss and skin issues. From a single plant or a combination of plants, they create a paste, decoction, aqueous extract powder and juice 4. The majority of the tribes also manufacture their own native beer made from rice grains. Each tribe raises its unique starting culture for fermentation, made of different plant parts.
In the current work, crude extracts and fatty acids of four pteridophyte species used in starter culture for the Mishing tribe of Assam's rice beer preparation are evaluated for their antimicrobial properties. The bioactive compounds were identified using GC-MS analysis.
MATERIALS AND METHOD:
Collection of Samples: The selected plant specimens were – Pteris semipinnata, Lygodium microphyllum, Lygodium flexuosum, and Lycopodiella cernua. Fresh plants were collected in the month of March from a Mishing village in Jorhat, Assam (26º 46’ 33.012” N Latitude and 94º 15’ 29.772” E Longitude). The leaves of Pteris semipinnata (Ps), Lygodium microphyllum (Lm), Lygodium flexuosum (Lf) and leaves along with stem of Lycopodiella cernua (Lc) were collected for the study. The herbarium specimens were prepared and identified in the Weed Herbarium of Assam Agricultural University. Plant material was collected, washed, shade dried, powdered with a blender, and stored in airtight bottles.
Preparation of Extracts: Five grams of each plant powder was extracted separately using different solvents based on polarity (ethyl acetate< acetone< ethanol< methanol) in the Soxhlet apparatus for 6 hours. The crude extracts were evaporated in a rotary vacuum evaporator at 37 ºC and stored at 4 ºC.
Extraction of Fatty Acids: 2 gm of aqueous extract was treated with 10 % lead acetate solution. The supernatant was collected, diluted with water and acidified with 1 % HCl before boiling for 2-3 hours. The precipitate was then extracted with ethanol and purified by fractional crystallization to obtain the fractions Ps-F, Lm-F, Lf-F and Lc-F. These fractions were then subjected to antimicrobial screening against clinical isolates and reference strains to establish their antimicrobial potential 5.
Antimicrobial Activity:
Microorganisms: The microorganisms selected for this study were isolated from clinical samples collected from Ayursundra Superspeciality Hospital, Guwahati, Assam. These included Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Acinetoba cterbaumannii, Proteus mirabilis, Candida albicans, and C. tropicalis. The reference strains used in this study are Staphylococcus epidermidis ATCC 35984 and K. pneumoniae ATCC BAA-1705. Cultures were maintained on Luria Bertani Agar (for bacteria) and Potato Dextrose Agar (for yeast) at 4°C.
Determination of MIC and MBC: MIC and MBC were determined using resazurin microtiter assay. Two-fold serial dilutions of the crude extracts and fractionated extracts were made directly in a microtiter plate with Mueller Hinton broth (MHB) to produce varying concentrations. MHB was used to adjust microbial cultures to 0.5 McFarland turbidity standard (1.5 × 108 CFU/ml). Amoxicillin was used as a standard for positive control. The plate was sealed with a sterile sealer and kept at 37°C for 24 hours. After incubation, 0.02 % resazurin (Himedia-RM125-1G) was added to each microtiter plate well and incubated for 30 minutes at 37°C. The wells that had microbial growth became pink, whereas the wells that did not have microbial growth remained blue. The wells corresponding to the MIC and with higher concentration were streaked onto Mueller-Hinton Agar plates and incubated overnight at 37°C. The concentration corresponding to no bacterial growth was recorded as the minimum bactericidal concentration (MBC) for extract 6.
Identification of the Fatty Acid Fraction with Gas Chromatography and Mass Spectroscopy: The fatty acid fractions were re-dissolved in spectroscopy grade ethanol and filtered through 0.2 mm filter for GCMS analysis performed in a Perkin Elmer (USA) Clarus 680/600C unit fitted with Elite 5 MS column (length: 30 m, ID: 0.25 mm, film thickness: 0.25 mm). The software used in the system is TurboMassver 5.4.2. The oven program started at 60°C for 1 min and ramped at 7°C/min up to 200°C and held for 3 mins, again ramped at 10°C/min to 300°C and then held for 5 min. Next, 1.0 ml sample was injected at 280°C using He as carrier gas with a solvent delay of 5 min. The split ratio was 10: 1. The mass spectrometer (Clarus 600C; single quad) was operated in the electron ionization (EI) mode at 70 eV with a source temperature of 150°C and a continuous scan from m/z 50 to 600. The peaks were identified by matching the mass spectra with the National Institute of Standards and Technology (NIST) library, USA.
RESULTS AND DISCUSSION: The development of new drugs also depends on the study of medicinal plants. Most underdeveloped nations presently use herbal treatment, and wealthy countries are also quickly adopting it. The pharmacological effects of plants are caused by metabolites, which are organic compounds divided into primary and secondary metabolites. Plants produce secondary metabolites such as alkaloids, flavonoids, saponins, terpenoids, steroids, glycosides, tannins, volatile oils, and other substances to protect themselves from microbial infections and insect invasions7.
The experiment to ascertain the antimicrobial efficacy of the different crude extracts and fatty acid fraction utilized seven clinical and two control isolates. The antimicrobial activity of the different extracts was effective against all of the test microorganisms at dosages ranging from 0.29 to 17.75 mg/ml, as shown in Table 1-3. Fractionation sometimes leads to improved biological activity, as seen in this study. The lowest MIC and MBC values were 0.25 mg/ml and 0.5 mg/ml, respectively Table 1-3, recorded for the fatty acid fractions Ps-F, Lc-F, Lf-F, and Lm-F against most of the microbial test strains. The low MIC observed in this study further supports the potential of fatty acid as an antimicrobial resource. The results align with earlier research on the antimicrobial effectiveness of fatty acids against related species employed in this study 3, 8-9.
TABLE 1: MIC AND MBC OF THE DIFFERENT CRUDE EXTRACTS AND FRACTIONATED EXTRACTS AGAINST BACTERIAL CLINICAL ISOLATES
Sample | P. aeruginosa | S. typhi | A. baumanii | K. pneumoniae | P. mirabilis | ||||||
MIC
mg/ml |
MBC
mg/ml |
MIC
mg/ml |
MBC
mg/ml |
MIC
mg/ml |
MBC
mg/ml |
MIC
mg/ml |
MBC
mg/ml |
MIC
mg/ml |
MBC
mg/ml |
||
Pteris semipinnata | Methanol | 8.88 | 17.75 | 8.88 | 17.75 | 4.44 | 8.88 | 4.44 | 8.88 | 8.88 | 17.75 |
Ethyl acetate | 0.58 | 1.17 | 0.58 | 1.17 | 0.29 | 0.58 | 0.29 | 0.58 | 0.58 | 1.17 | |
Ethanol | 9.33 | 18.67 | 9.33 | 18.67 | 4.67 | 9.33 | 4.67 | 9.33 | 9.33 | 18.67 | |
Acetone | 2 | 4 | 2 | 4 | 1 | 2 | 1 | 2 | 2 | 4 | |
Ps-F | 0.25 | 0.5 | 0.5 | 1 | 0.25 | 0.5 | 0.25 | 0.5 | 0.5 | 1 | |
Lycopodiella cernua | Methanol | 2.48 | 9.92 | 2.48 | 9.92 | 2.48 | 4.96 | 2.48 | 4.96 | 4.96 | 9.92 |
Ethyl acetate | 0.96 | 3.84 | 0.96 | 3.84 | 0.96 | 1.92 | 0.96 | 1.92 | 1.92 | 3.84 | |
Ethanol | 2 | 8 | 2 | 8 | 2 | 4 | 2 | 4 | 4 | 8 | |
Acetone | 0.88 | 3.5 | 0.88 | 3.5 | 0.88 | 1.75 | 0.88 | 1.75 | 1.75 | 3.5 | |
Lc-F | 0.25 | 1 | 0.25 | 1 | 0.25 | 0.5 | 0.25 | 0.5 | 0.5 | 1 | |
Lygodium flexuosum | Methanol | 3.21 | 6.42 | 3.21 | 12.84 | 3.21 | 6.42 | 3.21 | 6.42 | 6.42 | 12.84 |
Ethyl acetate | 1.15 | 2.29 | 1.15 | 4.58 | 1.15 | 2.29 | 1.15 | 2.29 | 2.29 | 4.58 | |
Ethanol | 1.79 | 3.58 | 1.79 | 7.17 | 1.79 | 3.58 | 1.79 | 3.58 | 3.58 | 7.17 | |
Acetone | 1.63 | 3.25 | 1.63 | 6.5 | 1.63 | 3.25 | 1.63 | 3.25 | 3.25 | 6.5 | |
Lf-F | 0.25 | 0.5 | 0.25 | 1 | 0.25 | 0.5 | 0.25 | 0.5 | 0.5 | 1 | |
Lygodium microphyllum | Methanol | 6.15 | 12.29 | 12.29 | 24.58 | 6.15 | 12.29 | 6.15 | 12.29 | 12.29 | 24.58 |
Ethyl acetate | 1.25 | 2.5 | 1.25 | 2.5 | 0.63 | 1.25 | 0.63 | 1.25 | 1.25 | 2.5 | |
Ethanol | 9 | 18 | 9 | 18 | 4.5 | 9 | 4.5 | 9 | 9 | 18 | |
Acetone | 3.67 | 7.34 | 3.67 | 7.34 | 1.83 | 3.67 | 1.83 | 3.67 | 3.67 | 7.34 | |
Lm-F | 0.25 | 1 | 0.5 | 1 | 0.25 | 0.5 | 0.25 | 0.5 | 0.5 | 1 |
TABLE 2: MIC AND MBC OF THE DIFFERENT CRUDE EXTRACTS AND FRACTIONATED EXTRACTS AGAINST FUNGAL CLINICAL ISOLATES
Sample | C. albicans | C. tropicalis | |||
MIC mg/ml | MBC mg/ml | MIC mg/ml | MBC mg/ml | ||
Pteris semipinnata | Methanol | 8.88 | 17.75 | 4.44 | 8.88 |
Ethyl acetate | 0.29 | 0.58 | 0.29 | 0.58 | |
Ethanol | 4.67 | 18.67 | 2.33 | 4.67 | |
Acetone | 1 | 4 | 0.5 | 1 | |
Ps-F | 0.25 | 1 | 0.125 | 0.25 | |
Lycopodiela cernua | Methanol | 2.48 | 4.96 | 2.48 | 4.96 |
Ethyl acetate | 0.96 | 1.92 | 0.96 | 1.92 | |
Ethanol | 2 | 4 | 2 | 4 | |
Acetone | 0.88 | 1.75 | 0.88 | 1.75 | |
Lc-F | 0.25 | 0.5 | 0.125 | 0.25 | |
Lygodiu mflexuosum | Methanol | 3.21 | 6.42 | 3.21 | 6.42 |
Ethyl acetate | 1.15 | 2.29 | 0.57 | 1.15 | |
Ethanol | 1.79 | 3.58 | 1.79 | 3.58 | |
Acetone | 1.63 | 3,25 | 0.81 | 1.63 | |
Lf-F | 0.25 | 0.5 | 0.125 | 0.25 | |
Lygodium microphyllum | Methanol | 6.15 | 12.29 | 6.15 | 12.29 |
Ethyl acetate | 0.63 | 1.25 | 0.63 | 1.25 | |
Ethanol | 4.5 | 9 | 4.5 | 9 | |
Acetone | 1.83 | 3.67 | 3.67 | 7.34 | |
Lm-F | 0.25 | 0.5 | 0.25 | 0.5 |
TABLE 3: MIC AND MBC OF THE DIFFERENT CRUDE EXTRACTS AND FRACTIONATED EXTRACTSAGAINSTBACTERIAL CONTROL ISOLATES
Sample | S. epidermidis | K. pneumoniae | |||
MIC mg/ml | MBC mg/ml | MIC mg/ml | MBC mg/ml | ||
Pteris
semipinnata |
Methanol | 8.88 | 17.75 | 8.88 | 17.75 |
Ethyl acetate | 0.58 | 1.17 | 0.29 | 0.58 | |
Ethanol | 9.33 | 18.67 | 4.67 | 9.33 | |
Acetone | 2 | 4 | 1 | 2 | |
Ps-F | 0.25 | 0.5 | 0.25 | 0.5 | |
Lycopodiela
cernua |
Methanol | 4.96 | 9.92 | 4.96 | 9.92 |
Ethyl acetate | 1.92 | 3.84 | 1.92 | 3.84 | |
Ethanol | 4 | 8 | 2 | 4 | |
Acetone | 1.75 | 3.5 | 1.75 | 3.5 | |
Lc-F | 0.25 | 0.5 | 0.25 | 0.5 | |
Lygodium
flexuosum |
Methanol | 6.42 | 12.84 | 3.21 | 1.60 |
Ethyl acetate | 2.29 | 4.58 | 1.15 | 0.57 | |
Ethanol | 3.58 | 7.17 | 3.58 | 7.17 | |
Acetone | 3.25 | 6.5 | 3.25 | 6.5 | |
Lf-F | 0.5 | 1 | 0.5 | 1 | |
Lygodium
microphyllum |
Methanol | 12.29 | 24.58 | 12.29 | 24.58 |
Ethyl acetate | 1.25 | 2.5 | 0.63 | 1.25 | |
Ethanol | 9 | 18 | 9 | 18 | |
Acetone | 3.67 | 7.34 | 3.67 | 7.34 | |
Lm-F | 0.5 | 1 | 0.25 | 0.5 |
Based on the promising result of the antimicrobial study, the fatty acid fractions were further subjected to GCMS analysis to identify the bioactive compounds. The four pteridophytes' fatty acid fraction included 184 peaks with retention times ranging from 6.044 to 38.157 Fig. 1-4. There were 21 compounds found in the Pterissemipinnata fraction, 35 compounds in the Lygodium flexuosum fraction, 33 compounds in the Lycopodiella cernua fraction, and 52 compounds in the Lygodium microphyllum fraction after a NIST library search was conducted for the significant peaks Table 4-7.
FIG. 1: GC-MS CHROMATOGRAM OF THE PTERISSEMIPINATTA FRACTION
TABLE 4: GC-MS ANALYSIS OF FRACTIONATED EXTRACT OF PTERIS SEMIPINNATA
*Molecular structures were generated using ChemDraw Ultra 12.0
FIG. 2: GC-MS CHROMATOGRAM OF THE LYGODIUM FLEXUOSUM FRACTION
TABLE 5: GC-MS ANALYSIS OF FRACTIONATED EXTRACT OF LYGODIUM FLEXUOSUM
*Molecular structures were generated using ChemDraw Ultra 12.0
FIG. 3: GC-MS CHROMATOGRAM OF THE LYCOPODIELLA CERNUA FRACTION
TABLE 6: GC-MS ANALYSIS OF FRACTIONATED EXTRACT OF LYCOPODIELLA CERNUA
*Molecular structures were generated using ChemDraw Ultra 12.0
FIG. 4: GC-MS CHROMATOGRAM OF THE LYGODIUM MICROPHYLLUM FRACTION
TABLE 7: GC-MS ANALYSIS OF FRACTIONATED EXTRACT OF LYGODIUM MICROPHYLLUM
*Molecular structures were generated using ChemDraw Ultra 12.0
The various medicinal uses of pteridophytes 87-88 may be due to the bioactive chemicals found in the fractions, which have been documented to exhibit a variety of medicinal properties Table 4-7. With the existence of these bioactive chemicals, the MIC and MBC values of the pteridophyte extracts in the current investigation may also be confirmed. To have a better understanding of the therapeutic activities of the examined pteridophytes, it will be interesting to document the biological roles of some of the compounds present in greater quantities, such as Tridecane, 6-Cyclohexyl-, Undecane, 3-Cyclohexyl-, Ethane, 1,1-Dichloro-, 2-Chloroethyl Methyl Sulfone, (Z)-1-Chloro-2-(Methylsulfonyl) Ethylene, 2-Chloropropionyl Chloride, Propane, 1,2-Dichloro-, Disilane, 1,1,2,2-Tetrachloro-1,2-Dimethyl-, Disparlure, 4-(3,5-Di-Tert-Butyl-4-Hydroxyphenyl)Butyl Acrylate, 3-(3, 5-Di-Tert-Butyl – 4 - Hydroxyphenyl) Propyl Methacrylate, 9-Octadecen-1-Ol, (Z)-, 1,16-Hexadecanediol, 17-Octadecynoic Acid, Z,E-2-Methyl - 3, 13 – Octadecadien – 1 - Ol, 1, 21-Docosadiene, Pentadecanoic Acid, 14-Bromo-, Tetracosanoic Acid, D-Mannitol, 1-O-(22-Hydroxydocosyl)- and Decanoic Acid, Silver(1+) Salt, which has not yet been published.
CONCLUSION: The present work found that the four pteridophyte species possess antibacterial and antifungal activities. The study revealed encouraging outcomes for fatty acids as antimicrobial agents.
The GCMS analysis of the fatty acid fractions revealed bioactive compounds with reported biological activity, which supports the study's results. The present work supports the use of fern species in traditional medicine and therapies. These results will also be useful for undertaking in-silico and cell-line studies for potential pharmacological lead compounds for drug discovery in future research.
ACKNOWLEDGMENTS: The authors are grateful to Dr. Juri Bharat Kalita and Dr. Himadri Dutta from the Microbiology Department of Ayursundra superspeciality Hospital for supplying the clinical microbial isolates for antimicrobial studies. The authors are also grateful to Dr. Rajiv Chandra Goswami, Guwahati Biotech Park, for the guidance and information on GC-MS analysis.
CONFLICTS OF INTEREST: The authors (S. Borkotoky and V. V. Borah) have no conflicts of interest to declare.
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How to cite this article:
Borkotoky S and Borah VV: Antimicrobial potential of crude extracts and fatty acid fractions of four pteridophyte species from Assam, Northeast India, and their identification by GC-MS. Int J Pharm Sci & Res 2023; 14(9): 4489-08. doi: 10.13040/IJPSR.0975-8232.14(9).4489-08.
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Article Information
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4489-4508
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English
IJPSR
S. Borkotoky * and V. V. Borah
Department of Biosciences, School of Life Sciences, Assam, Don Bosco University, Sonapur, Assam, India.
shivangiborkotoky.sb94@gmail.com
12 January 2023
28 March 2023
25 April 2023
10.13040/IJPSR.0975-8232.14(9).4489-08
01 September 2023