ISOLATION, IDENTIFICATION AND ANTIMICROBIAL ACTIVITY OF ENDOPHYTES FROM ERYNGIUM FOETIDUM LEAF ALONG WITH ITS PHYTOCHEMICAL SCREENING THROUGH STANDARD PROTOCOLS

ISOLATION, IDENTIFICATION AND ANTIMICROBIAL ACTIVITY OF ENDOPHYTES FROM ERYNGIUM FOETIDUM LEAF ALONG WITH ITS PHYTOCHEMICAL SCREENING THROUGH STANDARD PROTOCOLS

Nabajyoti Sarma, Karabi Das, Manashi Garg * and Bhabana Garg

Department of Medical Laboratory Technology, GEA National College, Guwahati, Assam, India.

ABSTRACT: Eryngium foetidum commonly known as cilantro or spiny coriander is a tropical perennial herb known for its distinctive aroma and medicinal properties. While it is mainly used as a culinary herb, it has long been valued in traditional medicine for a variety of health benefits. The present study was intended to analyse the phytochemicals present in the ethanolic extract of the leaf. Qualitative analysis has revealed the presence of various bioactive compounds and the major classes of phytochemicals typically found in Eryngium foetidum were flavonoids, tri-terpenoids, steroids, tannin, saponin, carbohydrate and phenol. Also, attempt has been made to isolate endophytic bacteria from Eryngium foetidium after performing controlled surface sterilization. The isolated organisms were marked as B1 and B2 and subjected to perform antibacterial and antifungal activities against important pathogenic strains viz; Staphylococcus aureus, Pseudomonas aeroginosa, E. coli, Klebsiella pneumonia & Aspergillus sp. The B1 isolate was however found to be effective with its substantial activities with zone of inhibitions of 18mm, 17mm, 15mm and 17mm respectively along with the antifungal activity with an appreciable inhibition zone of 23mm against Aspergillus sp. The isolates were further characterized based on its morphological identification and standard biochemical tests. The morphology of both the isolated organisms showed white, irregular, wavy and mucoid texture(s) followed by positive biochemical tests for catalase, methyl red and urease. The study provides a preliminary report on the biological potential of the leaf of Eryngium foetidum and its endophytic bacteria.

Keywords: Eryngium foetidum, Endophytic bacteria, Phytochemical activities, Biochemical test, Antimicrobial activity

INTRODUCTION: Medicinal plants are a cornerstone of traditional and modern medicine, providing a natural source of remedies that contribute to human health and well-being. The study and use of medicinal plants are essential for discovering new drugs and treatments, especially as modern medicine seeks alternatives to synthetic compounds with fewer side effects.

Medicinal plants are plants with therapeutic properties or compounds that can be used to treat, prevent or manage health conditions ¹. They can provide numerous health benefits due to their bioactive compounds which include alkaloids, flavonoids, tannins, terpenoids, and glycosides.

These compounds can have antioxidant, anti-inflammatory, antibacterial, antiviral and antifungal properties making them versatile agents in healthcare. Eryngium foetidum is a versatile medicinal herb with a wide range of benefits. The essential oils present in the secretory ducts of this species present throughout the plant body are considered as the specialized cells known to contain the bioactive compounds ².

While more clinical research is required to validate some of its traditional uses its antioxidant, anti-inflammatory and antimicrobial properties makes it a valuable plant in both traditional and modern medicine ³. The active compounds such as polyphenols and flavonoids helps to reduce inflammation thereby making it beneficial for conditions like arthritis, joint pain and muscle inflammation ⁴. Studies have shown that Eryngium foetidum has natural antibacterial properties, making it useful for fighting infections. Apart from being an enormous sources of natural compounds, these plants has attracted research interest for its endophytic properties that are beneficial characteristics of endophytic microorganisms that inhabit plant tissues without causing harm ⁵. These microorganisms showing endophytic lifestyles play important roles in plant development, growth, fitness and diversification. Endophytic microorganisms can mitigate the adverse effects of insect herbivores through various mechanisms including diminished feeding, slowed development, reduced population growth as well as mortality ³. Additionally, they also are extensive producers of assorted secondary metabolites whose function ranges from outcompeting harmful organisms, anticancer, antioxidant, antidiabetic, immunosuppressive, antifungal, plant growth promotion and antibacterial activity ⁴. These properties suggest E. foetidum as a promising source for exploring endophytic microorganisms with applications in medicine, agriculture and food industries.

MATERIALS AND METHOD:

Collection and Preparation of Plant Sample: The fresh leaves of Eryngium foetidum were collected from the local market of Guwahati city of Assam through random sampling. The collected leaves were washed gently under running tap water to remove the surface dust and debris. The leaves were then cut into small pieces (1cm in length) in a sterile petriplate using scissors and forceps under laminar hood to ensure the aseptic condition. The cut pieces were further subjected to a series of surface sterilization technique using 75% Ethanol (1min), Sodium hypochlorite (2min), 75% Ethanol (30sec) and double distilled water (30 sec) ⁵.

Isolation of Endophytes: These plant segments were finally placed in Potato Dextrose Agar (PDA medium) and incubated for 24-72 hrs at 32°C for the growth of the endophytes. The endophytic bacteria growing out of the plant explants were sub cultured periodically on PDA plates at room temperature and maintained at 4°C for further use. The isolated organisms were marked as B1 and B2 ⁷.

Preliminary Characterization of the Endophytic Bacteria: The preliminary characterization of the endophytic bacteria was done by observing the phenotypic features and performing gram staining as well as biochemical tests viz; catalase, methyl red, urease for all the isolates using standard procedures ⁸.

Preparation of Plant Extract for Phytochemical Analysis: Solvent extraction was performed by weighing 50gm of the stored air tight powder of the leaves of Eryngium foetidum by using an electronic balance. The weighed leaf sample was packed in a filter paper and placed in the thimble of the Soxhlet extractor. 500ml of Ethanol was used as a solvent for the extraction. The solvent was filled in the round bottom flask of the Soxhlet extractor and extraction was carried out for 15 hours. During the process of extraction, the non-volatile substances got dissolved in the polar solvent and get concentrated at the bottom of the flask. The filtrate was finally collected and concentrated by using rotary vacuum evaporator. The concentrated leaf extract was subjected to the phytochemical screening for the identification of the phytocompounds ⁷.

Phytochemical Analysis: A qualitative phytochemical screening of the ethanolic leaf extract was performed to determine the presence of various plant metabolites using the methods of Evans et al., (1996). The extract was subjected to determination of phytochemical constituents i.e. flavonoids, tri-terpenoids, steroids, tannins, carbohydrate, saponins and phenols ⁹.

Preparation of the Endophytic Bacteria for Antibacterial and Antifungal Activity: Antibacterial as well as antifungal activity of the endophytic bacteria were performed after centrifugation of the 24 hours old culture of the growing endophytes at 5000rpm for 20 minutes. The supernatant obtained was separated by centrifugation and used as the sample for antibacterial activity and antifungal activity.

Antimicrobial Activity: The antibacterial and antifungal activity were performed by the agar well diffusion method using CLSI guidelines. The aliquots of the test organism(s) incubated with 5ml of 0.1% peptone water (w/v) were evenly swabbed onto sterilized susceptible media using sterile cotton swabs. The plates were then allowed to dry for few minutes in the laminar air flow-hood. After drying, wells of 5mm diameter were made using a cork borer at an equivalent distance of 2cm apart from the edge of the plate. For obtaining an effective inhibition zone, 50µl of endophytic supernatant were filled in all the wells. The plates were incubated at 37±1°C for 24 hours for bacterial and at 28 ± 1°C for 48 hours for fungal pathogens ^{10, 11}.

RESULTS AND DISCUSSIONS: Phytochemical investigation showed the presence of seven different constituent in the plant extract viz., flavonoids, tri-terpenoids, steroids, alkaloids, tannins, carbohydrate, saponins, phenols and fatty acids. The presence these tested phyto constituents in the ethanolic extracts of the leaf of Eryngium foetidum has been listed in the Table 1. Apparently, these compounds may be responsible for the potential activity of the leaf extract.

TABLE 1: QUALITATIVE ANALYSIS OF ETHANOLIC LEAF EXTRACT OF ERYNGIUM FOETIDUM

Characterization of the Endophytic Bacteria: The culture characteristics of isolated endophytic bacteria were recorded after 24 hours of incubation ¹² and are listed in Table 2. The morphology of both the isolated organisms showed white, irregular, wavy and mucoid texture(s) followed by positive biochemical tests for catalase, methyl red and urease. Regarding the cell shape and gram staining, both the endophytic bacteria appeared Gram-positive bacilli possibly belonging to the genus Bacillus. Also, the biochemical test result indicates that they can produce catalase and urease enzyme.

TABLE 2: COLONY MORPHOLOGY AND BIOCHEMICAL CHARACTERISTICS OF ENDOPHYTIC BACTERIA

Antimicrobial Activity: The antimicrobial activities of both the endophytes have been shown in Table 3. The antimicrobial activities were compared in terms of the inhibition zone of the isolated endophytes B1 and B2. Findings have shown that B1 exhibited maximum inhibition against the tested bacterial pathogenic strains viz; Staphylococcus aureus (ATCC-29213), Pseudomonas aeroginosa (ATCC-27853), E. coli (ATCC-25922), Klebsiella pneumonia & Aspergillus sp. with an inhibition zone of 18±0.1mm, 17±0.2mm, 15±0.1mm, 17±0.2mm in contrast to B2 with an inhibition zone of  16±0.3mm, 15±0.2mm, 15±0.1mm, 17±0.1mm respectively along with the antifungal activity with an appreciable inhibition zone of 23±0.1mm& 21.5±0.5mm against the Aspergillus sp.

TABLE 3: THE ANTIMICROBIAL ACTIVITIES OF THE ENDOPHYTES IN TERMS OF % ZONE OF INHIBITION AGAINST TESTED PATHOGENIC STRAINS

CONCLUSION: In the present study the phyto-compounds present in the ethanolic leaf extract of Eryngium foetidum were screened by standard phytochemical tests. The extract of the plant showed appreciable presence of flavonoids, tri-terpenoids, steroids, tannin, saponin, carbohydrate and phenol. These compounds were also reported to have antioxidant and anti-inflammatory activity. Further an attempt has been made to isolate endophytic bacteria from the leaf segment of the plant. Two endophytic bacteria (B1&B2) were isolated, the morphology of these isolated organisms showed white, irregular, wavy and mucoid texture(s) followed by positive biochemical tests for catalase, methyl red and urease. Both were identified as strains that belongs to Bacillus sp. Antimicrobial activity of these endophytic bacteria were reported to show substantial antibacterial as well as antifungal with an effective zone of inhibitions. However, further studies are in progress with respect to these endophytes and their biological activities.

CONFLICT OF INTEREST: None

ACKNOWLEDGEMENT: The authors would like to thank GEA National College (Affiliated under Srimanta Sankaradeva University of Health Sciences) for providing the lab facilities to carry out the research work.

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    How to cite this article:

    Sarma N, Das K, Garg M and Garg B: Isolation, identification and antimicrobial activity of endophytes from Eryngium foetidum leaf along with its phytochemical screening through standard protocols. Int J Pharm Sci & Res 2025; 16(4): 1003-06. doi: 10.13040/IJPSR.0975-8232.16(4).1003-06.

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