Introduction

The environment is home to a diverse assortment of microorganisms. They are either beneficial or pathogenic. The number of beneficial organisms is relatively limited in comparison to pathogenic microbes. Furthermore, they often undergo significant evolution and can develop resistance to drugs¹. Hence, finding new drugs is extremely necessary for mankind. Even today, one of the main sources of novel therapeutic compounds is natural ingredients such as plants, bacteria, eukaryotic microbes, and different animal species².  Various intricate and structurally varied compounds have been found in plants and other natural sources. The study of plant extracts, essential oils, isolated secondary metabolites, and newly synthesized compounds as possible antibacterial agents has attracted much attention lately^3,4,5. Especially, the coastal plants have attracted researchers recently for their novel secondary metabolites⁶.

Coastal plants survive under remarkably stressful environments such as high winds, hazardous ion concentrations, drought stress, excessive light, and inadequate nutritional availability^7,8. Hence, these plants require specific adaptations, like thick or succulent and waxy coated leaves, sharp needle-like leaves, dry seeds, as well as solute accumulation to proliferate, endure, and infiltrate dunes⁹. Plant physiological activity is also altered by environmental stress. For example, the production of secondary metabolites is correlated with the physiological activity of the plant. The Secondary Metabolites function as crucial Primary Metabolites by considerably enhancing the development and endurance of plants in a variety of environmental conditions^10,11. Since ancient times, secondary metabolites, a vital component of plants have dominated the medical industry and offered several therapeutic advantages. Many nations across the world still employ phytotherapy due to its low risk and cost-effectiveness in comparison to synthetic medications, which frequently have a wide variety of serious side effects¹². Recently multiple studies have been conducted to discover suitable antimicrobial drugs due to the multidrug- resistance of pathogens. Keita and his colleagues (2022) listed the names of the plants and their antimicrobial effectiveness against the specific organism¹³. Keeping it all in consideration the current research work focused on discovering a new antimicrobial and larvicidal agent from the coastal grass Spinifex littoreus (Burm f.) Merr.  S. littoreus is a key component of the ecology of coastal dunes. These perennial seashore plants help stabilize the sand and are prevalent on dunes across Africa, the Middle East, Asia, Australia, New Zealand, and New Caledonia¹⁴

Materials and Methods

Plant collection

Spinifex littoreus (Fig.1) was collected from Kunthukal Beach, Pamban, Tamil Nadu, India (latitude: 9.25323; longitude: 79.21829). The collected plants were authenticated and submitted to the Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph’s College, Tiruchirappalli. (Voucher nos.  J.V 001 and J.V 002).

Figure 1: Habit of Spinifex littoreus (Burm f.) Merr. (a). Male Inflorescence; (b). Female Inflorescence. Click here to view Figure

Plant extraction

The young leaves of Spinifex littoreus were separated from the plant, cleaned, and shade-dried. The dried S. littoreus leaves were mechanically pulverized. The powdered sample of S. littoreus was extracted with methanol through the Soxhlet apparatus for 16 hours. To concentrate the collected extract, it was subjected to a rotary evaporator¹⁵.

Antimicrobial activity of the methanolic extract of S. littoreus

Microorganisms

The bacterial [Escherichia coli (MTCC 443), Propionibacterium acnes (MTCC 1951), Staphylococcus aureus (MTCC 902), Aeromonas hydrophila (MTCC 12301), Streptococcus faecalis (MTCC-439), Bacteroids fragilis (ATCC 25285)] and fungal species [Cryptococcus neoformans (ATCC 32045), Aspergillus fumigatus (MTCC 343), Aspergillus niger (MTCC 281), Sporothrix schenckii (ATCC 26327)] were utilized to examine the antimicrobial effect of the methanolic extract of S. littoreus. These microorganisms were purchased from the Microbial Type Culture Collection, Chandigarh, India.

Antimicrobial Assay

The antibacterial and antifungal activity of the methanolic extract of Spinifex littoreus (SL-M) was tested using Nutrient Agar and Potato Dextrose Agar medium through agar well diffusion method. 20 ml of each medium was poured into a separate petri plate and let them solidify. After that, the surface of the agar medium was inoculated with the chosen microbial specimens over the entire surface of the agar medium. Then 6 mm diameter wells were created on the medium aseptically using a cork borer. To the well, 20 µL of different concentrations (500, 250, 100, & 50 µg/ml) of the methanolic extract of S. littoreus were introduced. Along with the test sample, the standard Gentamicin and Amphotericin B antibiotics were used as a positive control for bacterial and fungal species, respectively. Then the plates were incubated for 24 hours at 37°C for bacteria and 72 hours at 28°C for fungi. After the incubation period, the clear zone around the wells was measured and calculated the zone of inhibition^16,17.

Larvicidal bioassay methanolic extract of S. littoreus

The anti-larvicidal effect of SL-M extract was tested against the larvae of Aedes aegypti—the eggs of Ae. Aegypti were collected from the rice field stagnant water using an “O” type brush. The invitro larvicidal bioassay was conducted in compliance with WHO guidelines¹⁸. Twenty-five larvae in their third or fourth instar were placed in beakers containing 100 ml of distilled water. The larvae were treated with S. littoreus methanolic extract at different concentrations (500 µg/ml, 250 µg/ml, 100 µg/ml, 50 µg/ml, 10 µg/ml) while a control group received no treatment. We conducted experiments three times for each concentration.  The test containers had a 12:12 hour light/dark phase and were maintained at 25–28°C. Following a 24-hour exposure, larval mortality was noted. The mortality rate of the mosquito larvae was calculated by the Graph Pad Prism program (USA) and the LC50 value was calculated by the Log- probit regression model developed by Dr. O.P. Sheoran.

Results

Antimicrobial assay methanolic extract of S. littoreus

The antimicrobial analysis of the sample methanolic extract of S. littoreus extract showed dose-dependent activity for both bacteria and fungi. For the antibacterial study, the sample showed maximum inhibition for Propionibacterium acnes with 15.45 mm of the zone of inhibition (fig.2).

Figure 2: Antibacterial assay of methanolic extract of Spinifex littoreus and Gentamicin as control indicated by zone of inhibition against (A) Staphylococcus aureus, (B) Escherichia coli, Click here to view Figure

The least inhibition among the tested species was recorded in B. fragilis (7.5 mm). In the antifungal assay, A. niger (14.75 mm) and S. schenckii (14.35 mm) exhibited maximum inhibition than the other two tested organisms (fig.3).

Figure 3: Antifungal activity of methanolic extract of S. littoreus and Amphotericin-B as control showed by zone of inhibition against (A) Aspergillus niger, (B) Aspergillus fumigatus, Click here to view Figure

Table 1 enlightens the name of the organisms and their zone of inhibition values (mean ± SD) of antimicrobial activity of methanolic extract of S. littoreus.

Table 1: Antimicrobial activity of methanolic extract of Spinifex littoreus

Anti-larvicidal activity of S. littoreus methanolic extract

The preliminary examination of the larvicidal effect of the methanolic extract of S. littoreus (SL-M) suggested dose-dependent activity on Aedes aegypti larvae. 100% of mortality was recorded in the 500 µg/ml of SL-M extract. The methanolic extract of S. littoreus showed considerable larvicidal activity with an LC50 value of 67.058 ppm.

Morphological changes of Ae. Aegypti after being treated with SL-M extract

The complete body of the treated and untreated larvae was examined under a light microscope after 24 hours. The SL-CH-treated larvae suffered significant harm. Through visual inspection, it was observed that the treated larvae’s body motions and coiling were abnormal in the SL-M extract. Under a light microscope, they displayed clear morphological alterations that destroyed the thoracic and abdomen regions (oozing out of the contents of the digestive tract). In control larvae, no structural changes in their morphology were seen (Fig.4).

Figure 4: Effect of SL-M extract on the larvae of Aedes aegypti. (A) shows intestine shortening on 500 µg/ml of SL-M treatment (Arrow); (B) Thoracic rupture and gut extrusion on 250 µg/ml (Circle); Click here to view Figure

Discussion

Plants generate a range of secondary metabolites according to their environment. These substances have traditionally been employed to treat chronic diseases and infectious¹⁹. Currently, scientists are concentrating on discovering a novel and significant antibacterial substance from plant extracts due to the emerging phenomenon of multidrug resistance of pathogens²⁰. For example, alkaloids²¹, flavonoids²², tannins²³, terpenoids²⁴, phenolics²⁵, saponins²⁶, and steroids²⁷ are eventually reported as a potential antimicrobial agent against the pathogens listed as multidrug-resistant organisms. These plant secondary metabolites affect microbial cells in several ways by disturbing cytoplasmic membrane structure and functions, interacting with membrane proteins, and interrupting the transcription and translation processes^28,29,30. The plant S. littoreus also showed the presence of therapeutically important secondary metabolites such as alkaloids, flavonoids, tannins, quinones, coumarins, and steroids³¹.

In the current study, the antimicrobial assay results indicated that the bacteria Propionibacterium acnes and the fungal strain of Cryptococcus neoformans had a maximum zone of inhibition compared to the other tested species. The bacteria P. acnes are responsible for diseases like skin, joints, and lung infections³² and the fungi C. neoformans can cause Cryptococcosis – It is fatal and injures the lungs or brain³³. The chloroform extract of this S. littoreus also exhibited similar antimicrobial activities³⁴

Mosquitoes are a major public health problem, carrying illnesses such as malaria, filariasis, dengue, and Japanese encephalitis, resulting in millions of fatalities each year³⁵. This current investigation contributes to developing a novel larvicidal agent derived from S. littoreus’ methanolic extract. Results of this study showed preferable anti-larvicidal activity against the larvae of Ae. Aegypti. Similar results were recorded by Rahuman., (2008), who conducted a larvicidal bioassay for the plant Phyllanthus amarus against Aedes aegypti larvae and the LC50 value is 90.92 ppm³⁶. Plant-based extracts and their chemical components can be converted into environmentally friendly products for mosquito vector control owing to their selectivity, sustainability, and low toxic effect ^37,38. According to research by Thangam and Kathiresan (1991) marine plant extracts and synthetic insecticides work together to eradicate Aedes aegypti larvae³⁹. In the study by Manh and his colleagues., (2020), essential oils from certain terrestrial aromatic plants have larvicidal effects on Aedes aegypti larvae⁴⁰. Similar structural changes in mosquito larvae were also observed and documented by Ravi and team (2018) in the larvae of Ae. Aegypti when treated with Azolla pinnata extract⁴¹.

Conclusion

The methanolic extract of the coastal plant of Spinifex littoreus (Burm f.) Merr. exhibits potential antibacterial, antifungal, and anti-larvicidal activities. Hence the plant S. littoreus can be the alternative therapeutics for diseases like skin infections, rheumatics, and Cryptococcosis.

Acknowledgment

The authors are very much thankful to the “Trichy Research Institute of Biotechnology” for providing the necessary equipment and support rendered throughout the research. 

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Authors’ Contribution

Vedhamani John Nallaiyah and Paul Ajithkumar Issac Newton : participated in the study’s design;

Vedhamani John Nallaiyah : carried out all the experiments and drafted the manuscript.

Paul Ajithkumar Issac Newton : supervised all the work and finalized the manuscript.

All authors read and approved the final manuscript. 

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