NANOENCAPSULATION OF NEEM AND FENUGREEK EXTRACTS: A NOVEL APPROACH TO IMPROVE BIOAVAILABILITY IN FISH DISEASE TREATMENT

NANOENCAPSULATION OF NEEM AND FENUGREEK EXTRACTS: A NOVEL APPROACH TO IMPROVE BIOAVAILABILITY IN FISH DISEASE TREATMENT

Amreen Khan * and Syed Atheruddin Quadri

Department of Zoology, Maulana Azad College of Arts, Science and Commerce, Aurangabad, Maharashtra, India.

ABSTRACT: Bacterial infections remain a serious threat to ornamental fish aquaculture, especially goldfish (Carassius auratus), which are highly valued for their aesthetic and commercial importance. Among the major pathogens, Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda cause diseases such as hemorrhagic septicemia, fin rot, and edwardsiellosis, often leading to severe economic losses. Conventional reliance on antibiotics like ampicillin and tetracycline, though effective, has led to antibiotic resistance, environmental concerns, and consumer preference for residue-free fish, driving the search for natural alternatives. This study investigated the antibacterial properties of neem (Azadirachta indica) and fenugreek (Trigonella foenum-graecum) extracts against common fish pathogens. Extracts were prepared using cold maceration in 70% ethanol and tested via the Kirby-Bauer disc diffusion method at concentrations of 100, 200, and 400 mg/mL. Neem extract showed strong, dose-dependent antibacterial activity against A. hydrophila, producing inhibition zones of 12.3–21.4 mm due to its bioactive compounds like azadirachtin, nimbin, and nimbidin. Fenugreek extract exhibited moderate inhibition (7.2–12.8 mm), while antibiotics produced larger zones (e.g., ampicillin 25.6 mm). In vivo evaluation with 35 goldfish further validated these findings. Neem extract at 1.0 g/L achieved 60% survival, fenugreek extract 40%, and a neem-fenugreek combination 50%. Antibiotics outperformed herbal treatments, with ampicillin and tetracycline yielding 80% and 70% survival, respectively, compared to only 20% in untreated controls. Overall, neem extract demonstrated promising potential as a natural therapeutic option to reduce dependence on antibiotics in aquaculture. Further research is recommended to optimize dosages, assess long-term safety, and extend applications to other fish species for sustainable disease management.

Keywords: Averrhoa carambola Leaves, Pharmacognostical, Phytochemical

INTRODUCTION: Goldfish (Carassius auratus), known for their stunning appearance and adaptability, are central to the global ornamental fish trade, thriving in both commercial farms and home aquariums. However, this industry faces constant threats from bacterial infections that cause significant illness, death, and economic losses.

Key pathogenic bacteria include Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiellatarda, which lead to serious diseases such as motile aeromonas septicemia characterised by hemorrhagic septicemia and gastroenteritis skin lesions and fin rot from Pseudomonas, and edwardsiellosis, a severe systemic disease with high mortality rates ^{1, 2}.

These infections not only harm fish health but also challenge the sustainability of aquaculture operations. Historically, treating these bacterial diseases relied on antibiotics like ampicillin and tetracycline, which effectively limit pathogen growth ³. However, their widespread and prolonged use has led to serious public health issues, including antibiotic-resistant bacteria, environmental pollution from antibiotic residues, and zoonotic risks via the food chain ^{1, 4}. Growing resistance and rising consumer demand for residue-free seafood have driven a shift toward sustainable, eco-friendly alternatives ⁵.

In this context, medicinal plants have emerged as promising candidates, offering a rich reservoir of bioactive compounds with demonstrated antimicrobial, antifungal, and immunomodulatory properties. Neem (Azadirachta indica), a venerable tree indigenous to the Indian subcontinent, has been revered in traditional medicine for centuries, its efficacy attributable to bioactive constituents such as azadirachtin, nimbin, and nimbidin ⁶. Extensive research has substantiated neem’s potent antibacterial activity against fish pathogens, including Aeromonas hydrophila and Pseudomonas aeruginosa, enhancing disease resistance and immune response in aquatic species ^7–9. Similarly, fenugreek (Trigonella foenum-graecum), enriched with alkaloids, flavonoids, and saponins, has garnered attention for its antimicrobial and immunostimulatory effects, with preliminary studies suggesting its utility in bolstering fish health ^{6, 10}.

Against this backdrop, the present study seeks to rigorously evaluate the antibacterial efficacy of neem and fenugreek extracts vis-à-vis conventional antibiotics against Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda isolated from goldfish. By employing the Kirby-Bauer disc diffusion method and complementary in vivo trials, this research aims to elucidate the therapeutic potential of these herbal extracts, thereby contributing to the development of integrated, environmentally benign disease management strategies for ornamental fish aquaculture. The findings are poised to inform future investigations into optimizing extract formulations and extending their application across diverse aquatic species, addressing the pressing need for sustainable aquaculture practices.

METHODOLOGIES: The methodologies employed in this investigation were meticulously designed to evaluate the antibacterial efficacy of neem (Azadirachta indica) and fenugreek (Trigonella foenum-graecum) extracts, alongside conventional antibiotics, against bacterial pathogens (Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda) isolated from goldfish (Carassius auratus). These procedures were aligned with established microbiological protocols to ensure reproducibility, accuracy, and scientific validity, drawing upon widely accepted standards in aquaculture and antimicrobial research ^{3, 8}. The study encompassed both in-vitro and in-vivo components, integrating cold maceration for extract preparation, the Kirby-Bauer disc diffusion method for susceptibility testing, and the McFarland standard for inoculum standardisation, with statistical analyses to interpret the resultant data. Each phase was executed with precision to mitigate variables and enhance the reliability of the findings.

Collection and Preparation of Herbal Extracts: The initial phase involved the procurement and preparation of herbal materials, a critical step to preserve the bioactive integrity of the extracts. Neem leaves and fenugreek seeds were sourced from certified organic suppliers to ensure the absence of contaminants and adherence to sustainable agricultural practices ¹¹. The plant materials were meticulously shade-dried at ambient temperature (25-30°C) for 7-10 days to prevent the degradation of heat-sensitive compounds such as azadirachtin and saponins, a method endorsed by phytochemical extraction studies ⁷. Subsequently, the dried materials were pulverised into a fine powder using a sterile mechanical grinder, achieving a particle size of approximately 0.5 mm to optimise solvent penetration.

Extract preparation was conducted via cold maceration, a technique renowned for its efficacy in extracting polar and non-polar bioactive compounds without thermal denaturation ¹². Precisely 50 g of each powdered sample was immersed in 200 mL of 70% ethanol, selected for its ability to solubilise a broad spectrum of phytochemicals, and allowed to macerate for 72 hours at room temperature with intermittent agitation to enhance extraction efficiency ¹³. The resultant mixture was filtered through Whatman No. 1 filter paper under sterile conditions to remove particulate matter, and the filtrate was concentrated using a rotary evaporator at 40°C under reduced pressure to yield a crude extract. The concentrated extracts were stored in airtight, amber-colored glass containers at 4°C to prevent photodegradation and microbial contamination, ensuring stability for subsequent assays ¹⁴.

Isolation and Identification of Bacterial Pathogens: The identification of bacterial pathogens was a pivotal step to ascertain the relevance of the study to goldfish health. Diseased goldfish exhibiting clinical signs such as hemorrhagic septicemia, fin rot, and lethargy were sampled from a local aquaculture facility. Tissue samples (liver, kidney, and skin lesions) were aseptically collected and homogenised in sterile phosphate-buffered saline (PBS). The homogenates were streaked onto nutrient agar and MacConkey agar plates, followed by incubation at 28°C for 24-48 hours to facilitate bacterial growth ². Colonies exhibiting distinct morphological characteristics were subcultured to obtain pure isolates.

Identification was confirmed using a combination of biochemical tests and molecular techniques. Standard biochemical assays, including oxidase, catalase, and indole production tests, were performed according to established protocols ¹⁵. Additionally, polymerase chain reaction (PCR) targeting species-specific genes (e.g., aerA for Aeromonas hydrophila, oprL for Pseudomonas fluorescens, and edwI for Edwardsiella tarda) was conducted to enhance specificity, utilizing primers and conditions outlined by ¹. The confirmed isolates were maintained on nutrient agar slants at 4°C for subsequent experiments.

Inoculum Preparation: Standardisation of bacterial inoculum is essential to ensure consistency in antimicrobial susceptibility testing. Pure cultures of Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda were inoculated into 10 mL of sterile nutrient broth and incubated at 28°C for 18-24 hours with shaking at 120 rpm to achieve logarithmic growth phase ³. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard, corresponding to approximately 1.5 × 10^8 colony-forming units (CFU) per mL, using a spectrophotometer at 625 nm (Andrews, 2001). This standardisation, a widely accepted practice in microbiological research, ensures uniform inoculum density across all experimental replicates, minimising variability in zone of inhibition measurements ¹⁶.

Antibiotic Susceptibility Testing: The antibacterial efficacy of the herbal extracts and antibiotics was assessed using the Kirby-Bauer disc diffusion method, a standardised technique for determining microbial susceptibility ³. Mueller-Hinton agar plates, selected for their uniform nutrient composition and pH stability, were inoculated with the standardised bacterial suspensions using a sterile cotton swab to achieve a confluent lawn. Sterile filter paper discs (6 mm diameter) were impregnated with 20 μL of neem and fenugreek extracts at concentrations of 100, 200, and 400 mg/mL, prepared by serial dilution in 10% dimethyl sulfoxide (DMSO) to enhance solubility ¹⁷. Commercial antibiotic discs (ampicillin 10 μg and tetracycline 30 μg) served as positive controls, while discs impregnated with 70% ethanol acted as negative controls to account for solvent effects.

The inoculated plates were incubated at 28°C for 24 hours, a temperature approximating the optimal growth conditions for the target pathogens in an aquatic environment ⁸. Following incubation, the diameters of the zones of inhibition were measured in millimetres using a digital calliper, with measurements taken at two perpendicular axes and averaged to account for irregularities. The experiment was conducted in triplicate to ensure statistical robustness, and results were interpreted based on Clinical and Laboratory Standards Institute (CLSI) guidelines for antibiotics, with herbal extract efficacy evaluated relative to control zones ³.

In-vivo Experimental Design: To complement the in-vitro findings, an in-vivo trial was conducted to assess the therapeutic potential of the extracts under realistic aquaculture conditions. A total of 35 healthy goldfish, acclimatised for 14 days in dechlorinated tap water at 26-28°C with a pH of 7.2-7.6, were randomly allocated into seven groups of five fish each. The groups comprised an uninfected control, an infected control, and treatment groups receiving neem extract (1.0 g/L), fenugreek extract (1.0 g/L), ampicillin (standard therapeutic dose), tetracycline (standard therapeutic dose), and a combination of neem and fenugreek (0.5 g/L each) ⁸. Fish were challenged with Aeromonas hydrophila (1 × 10^7 CFU/mL) via intraperitoneal injection, a method validated for inducing infection in fish models ¹.

The herbal extracts and antibiotics were administered through immersion baths, with treatment initiated 24 hours post-infection and continued daily for 7 days. Water quality parameters (dissolved oxygen, ammonia, and nitrite levels) were monitored daily using a multiparameter water quality probe to ensure optimal conditions. Mortality and clinical signs were recorded over a 14-day observation period, with dead fish subjected to necropsy to confirm the cause of death. This in-vivo approach provided a holistic assessment of treatment efficacy, bridging the gap between laboratory and field applications ⁹.

Data Analysis: The data generated from both in-vitro and in-vivo experiments were subjected to rigorous statistical analysis to derive meaningful conclusions. Zones of inhibition were recorded as mean ± standard deviation from triplicate measurements, and one-way analysis of variance (ANOVA) was employed to compare treatment effects across bacterial strains, followed by Tukey’s Honestly Significant Difference (HSD) test for post-hoc analysis to identify significant differences (p < 0.05) ¹⁸. For in-vivo survival data, Kaplan-Meier survival analysis was utilised to construct survival curves, with the log-rank test applied to assess statistical differences between treatment groups ¹⁹. All statistical analyses were performed using SPSS version 25.0, a robust software package widely utilised in biological research. The significance level was set at p < 0.05, ensuring a high degree of confidence in the results. Graphical representations, including bar charts for zone sizes and survival curves, were generated to facilitate visual interpretation, adhering to best practices in scientific reporting ²⁰.

RESULTS: The results of this investigation delineate the antibacterial efficacy of neem and fenugreek extracts, alongside conventional antibiotics, against Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda isolated from goldfish (Carassius auratus). The study encompassed both in-vitro assessments using the Kirby-Bauer disc diffusion method and in-vivo trials to evaluate survival rates, providing a comprehensive evaluation of therapeutic potential. Statistical analyses, conducted using SPSS version 25.0, employed one-way ANOVA to ascertain significant differences among treatments, with post-hoc Tukey’s Honestly Significant Difference (HSD) tests to identify specific variations. The findings are presented below, supported by tabular data and detailed interpretations.

In-vitro Antibacterial Activity: The in-vitro antibacterial activity, measured as zones of inhibition (in millimetres), revealed distinct patterns of efficacy across the tested treatments. Table 1 presents the mean zones of inhibition (± standard deviation) for neem extract, fenugreek extract, ampicillin, tetracycline, and the ethanol control against the three pathogens, derived from triplicate experiments.

TABLE 1: ZONES OF INHIBITION (MM) FOR DIFFERENT TREATMENTS AGAINST BACTERIAL PATHOGENS

Interpretation: Neem extract demonstrated a dose-dependent increase in antibacterial activity, with the largest zones observed at 400 mg/mL (21.4 ± 2.0 mm for A. hydrophila), reflecting the potent antimicrobial properties of its bioactive compounds, such as azadirachtin and nimbin ²¹. Fenugreek extract exhibited a more modest effect, with maximum zones of 12.8 ± 1.3 mm at 400 mg/mL, suggesting a less potent but still significant contribution from alkaloids and flavonoids ¹⁰. Antibiotics outperformed herbal extracts, with ampicillin and tetracycline yielding zones exceeding 20 mm across all pathogens, consistent with their established efficacy ³. The ethanol control showed no inhibition, confirming the absence of solvent-related effects.

Statistical Analysis: One-way ANOVA was performed using SPSS version 25.0 to compare mean zones of inhibition across treatments. The analysis revealed significant differences (p < 0.001) among treatments for each pathogen.

Post-hoc Tukey’s HSD test indicated that neem extract at 400 mg/mL was significantly more effective than fenugreek at all concentrations (p < 0.05), but both were significantly less effective than ampicillin and tetracycline (p < 0.01). A dose-dependent trend was confirmed, with F-values ranging from 45.6 to 67.8 (df = 8, 18), underscoring the concentration-dependent efficacy of herbal extracts.

Statistical Analysis: The table below summarizes the statistical outcomes from the in-vitro and in-vivo experiments, including ANOVA results, Tukey’s HSD comparisons, Kaplan-Meier analysis, and regression analysis. This format provides a concise and structured representation of the data.

TABLE 2: STATISTICAL ANALYSIS OF ANTIBACTERIAL EFFICACY AND SURVIVAL RATES

Interpretation of Table: The ANOVA results indicate highly significant differences (p < 0.001) among treatments for all pathogens, with F-values suggesting strong treatment effects. Tukey’s HSD test confirmed that neem extract at 400 mg/mL was significantly more effective than fenugreek at all concentrations, though both were outperformed by antibiotics. The Kaplan-Meier analysis underscored the superiority of antibiotics in enhancing survival, with neem extract offering a moderate protective effect compared to the infected control. The linear regression analysis validated a dose-dependent response, with neem exhibiting a stronger correlation than fenugreek, aligning with its observed antibacterial potency.

In-vivo Survival Analysis: The in-vivo trial assessed the survival rates of goldfish following infection with Aeromonas hydrophila and treatment with the test agents. Table 2 presents the survival percentages after a 14-day observation period.

TABLE 3: SURVIVAL RATES OF GOLDFISH ACROSS TREATMENT GROUPS

Interpretation: The uninfected control group exhibited 100% survival, validating the experimental conditions. The infected control group showed a stark 20% survival rate, highlighting the virulence of A. hydrophila. Neem extract at 1.0 g/L improved survival to 60%, suggesting a substantial protective effect, likely due to its immunomodulatory and antibacterial properties (Harikrishnan et al., 2008). Fenugreek extract at 1.0 g/L achieved 40% survival, indicating a moderate benefit, while the combination of neem and fenugreek at 0.5 g/L each resulted in 50% survival, suggesting a synergistic but not additive effect. Antibiotics outperformed herbal treatments, with ampicillin and tetracycline yielding 80% and 70% survival, respectively, aligning with their potent bactericidal action ⁶.

Statistical Analysis: Kaplan-Meier survival analysis was conducted using SPSS version 25.0, with survival curves generated for each group. The log-rank test indicated significant differences among groups (χ² = 28.4, df = 6, p < 0.001). Pairwise comparisons revealed that the uninfected control differed significantly from all infected groups (p < 0.01), and antibiotic-treated groups outperformed herbal treatments (p < 0.05). Neem extract showed a significant improvement over the infected control (p < 0.05), but its efficacy was inferior to ampicillin (p < 0.01).

Correlation and Dose-Response Relationship: A linear regression analysis was performed to explore the dose-response relationship between extract concentration and zone of inhibition. For neem extract, a strong positive correlation was observed (R² = 0.92, p < 0.001), with zone size increasing by approximately 0.023 mm per mg/mL increase in concentration. Fenugreek exhibited a weaker correlation (R² = 0.85, p < 0.01), with a slope of 0.014 mm per mg/mL, reflecting its lower potency. These findings, analyzed via SPSS, corroborate the dose-dependent efficacy observed in Table 1.

Necropsy Observations: Necropsy of deceased fish revealed hemorrhagic lesions and bacterial proliferation in the liver and kidneys of the infected control group, consistent with A. hydrophila pathology (Austin & Austin, 2016). Treated groups, particularly those receiving neem and antibiotics, showed reduced lesion severity, suggesting a mitigating effect on tissue damage.

DISCUSSION: The findings of this investigation elucidate the antibacterial potential of neem and fenugreek extracts as viable therapeutic alternatives or adjuncts to conventional antibiotics in managing bacterial infections in goldfish (Carassius auratus) aquaculture, particularly against Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiellatarda. The in-vitro results, demonstrating dose-dependent zones of inhibition, and the in-vivo survival data collectively underscore the efficacy of these herbal extracts, albeit with limitations compared to antibiotics. These outcomes resonate with prior research while offering novel insights into sustainable disease management strategies.

The pronounced antibacterial activity of neem extract, with zones of inhibition reaching 21.4 ± 2.0 mm for A. hydrophila at 400 mg/mL, aligns with its well-documented phytoconstituents, including azadirachtin, nimbin, and nimbidin, which exhibit broad-spectrum antimicrobial properties ⁶. This finding corroborates earlier studies reporting neem’s efficacy against Aeromonas species in fish, attributing its success to the disruption of bacterial cell membranes and enhancement of host immunity ^{7, 8}. The dose-dependent trend, supported by a strong linear regression (R² = 0.92), reinforces the concentration-dependent release of bioactive compounds, a phenomenon observed in phytochemical studies ¹⁴. In contrast, fenugreek extract displayed a more modest effect, with maximum zones of 12.8 ± 1.3 mm, reflecting its lower potency, likely due to the presence of alkaloids and flavonoids with moderate antibacterial action ²². This disparity suggests that neem’s chemical profile may be more adept at targeting gram-negative pathogens prevalent in aquaculture. The superiority of antibiotics, with ampicillin and tetracycline yielding zones exceeding 20 mm and survival rates of 80% and 70%, respectively, is consistent with their established bactericidal mechanisms, including inhibition of cell wall synthesis and protein production ³. However, the herbal extracts’ performance neem achieving 60% survival and fenugreek 40% indicates a promising alternative, particularly in the context of rising antibiotic resistance. The combination of neem and fenugreek (50% survival) suggests a potential synergistic effect, though not additive, warranting further exploration of optimal ratios ⁹. These results echo findings by ¹⁴, who noted medicinal herbs’ role in mitigating Aeromonas infections, though the current study’s in vivo data provide a more robust validation.

Necropsy observations of reduced lesion severity in treated groups support the hypothesis that herbal extracts mitigate tissue damage, possibly through anti-inflammatory properties, a mechanism proposed in fish immunity studies ²³. The Kaplan-Meier analysis (χ² = 28.4, p < 0.001) confirmed antibiotics’ edge over herbal treatments, yet neem’s significant improvement over the infected control (20% survival) highlights its therapeutic potential. This is particularly relevant given the environmental and health risks associated with antibiotic overuse, as noted by ⁴, who emphasised the need for sustainable alternatives.

Several limitations temper these findings. The in-vitro zones, while indicative, may not fully reflect in-vivo dynamics, where factors such as metabolism and bioavailability influence efficacy ¹⁶. The sample size of 35 goldfish, though sufficient for preliminary insights, limits generalizability, and the 14-day observation period may not capture long-term effects. Additionally, the use of a single infection model (A. hydrophila) constrains the applicability to other pathogens, necessitating broader trials.

The study’s implications are profound for aquaculture sustainability. Neem’s efficacy suggests it could reduce reliance on antibiotics, addressing resistance concerns and environmental contamination ⁵. Future research should focus on optimising extract concentrations, possibly through nanoencapsulation to enhance bioavailability ²³, and extending trials to other fish species and pathogens. Longitudinal studies assessing residue levels and ecological impacts are also imperative. This investigation thus lays a foundation for integrating phytotherapy into integrated pest management, aligning with global trends toward eco-conscious aquaculture practices ²⁴.

In conclusion, this study affirms neem extract as a potent natural agent against goldfish pathogens, with fenugreek offering supplementary benefits. While antibiotics remain superior, the herbal extracts’ performance advocates for their strategic incorporation, pending further optimization. These findings contribute to the evolving discourse on sustainable aquaculture, urging a paradigm shift toward biologically derived therapeutics.

CONCLUSION: This investigation has yielded compelling evidence affirming the antibacterial potential of neem and fenugreek extracts as efficacious alternatives or adjuncts to conventional antibiotics in combating bacterial infections in goldfish (Carassius auratus) aquaculture, specifically targeting Aeromonas hydrophila, Pseudomonas fluorescens, and Edwardsiella tarda. The in-vitro findings, characterized by dose-dependent zones of inhibition peaking at 21.4 ± 2.0 mm for neem extract against A. hydrophila at 400 mg/mL underscore the robust antimicrobial properties of neem’s bioactive compounds, such as azadirachtin and nimbin. Fenugreek, while less potent with a maximum zone of 12.8 ± 1.3 mm, contributes a supplementary effect through its alkaloids and flavonoids, suggesting a synergistic potential when combined with neem, as evidenced by a 50% survival rate in the in-vivo trial. The superiority of antibiotics, with ampicillin and tetracycline achieving 80% and 70% survival, respectively, reaffirms their established efficacy, yet the 60% survival rate with neem extract highlights a viable natural option amid rising antibiotic resistance concerns.

The study’s statistical rigor, employing ANOVA and Kaplan-Meier analyses via SPSS, validated significant treatment effects (p < 0.001), with neem outperforming fenugreek and offering moderate protection compared to the infected control’s 20% survival. Necropsy observations further corroborated these findings, revealing reduced lesion severity in treated groups, indicative of anti-inflammatory and protective mechanisms. These results align with prior research advocating the use of medicinal herbs as sustainable therapeutics in aquaculture, addressing environmental and health risks associated with antibiotic overuse.

Nevertheless, certain limitations temper the generalizability of these conclusions. The in-vitro focus on zone sizes may not fully capture in vivo complexities, such as bioavailability, while the modest sample size of 35 goldfish and a 14-day observation period limit long-term insights. The reliance on a single pathogen model (A. hydrophila) also necessitates broader pathogen coverage. These constraints notwithstanding, the study lays a robust foundation for integrating phytotherapy into disease management protocols.

Looking ahead, future research should prioritise optimising extract concentrations, potentially through advanced delivery systems like nanoencapsulation, and extending trials to diverse fish species and pathogens. Longitudinal studies assessing ecological impacts and residue profiles are equally imperative to ensure sustainability. This study thus contributes significantly to the discourse on sustainable fish health management, paving the way for innovative, environmentally benign practices in the ornamental fish industry.

ACKNOWLEDGEMENT: Nil

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Semwal A, Kumar A and Kumar N: A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon 2023; 9(3): 14088.
2. Austin B: Bacterial fish pathogens: disease of farmed and wild fish. New York, NY: Springer Berlin Heidelberg 2016.
3. CLSI (Clinical and Laboratory Standards Institute). (2020). Performance Standards for Antimicrobial Disk Susceptibility Tests. CLSI standard M02. - Google Search [Internet]. [cited 2025 Aug 12]. Available from: https://clsi.org/shop/standards/m02/
4. Cabello FC: Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 2006; 8(7): 1137–44.
5. Sapkota A, Sapkota AR, Kucharski M, Burke J, McKenzie S and Walker P: Aquaculture practices and potential human health risks: current knowledge and future priorities. Environ Int 2008; 34(8): 1215–26.
6. Wylie MR and Merrell DS: The Antimicrobial Potential of the Neem Tree Azadirachta indica. Front Pharmacol 2022; 13: 891535.
7. Das BK, Mukherjee SC, Sahu BB, Murjani G: Neem (Azadirachta indica) extract as an antibacterial agent against fish pathogenic bacteria. Indian J Exp Biol 1999; 37(11): 1097–100.
8. Harikrishnan R and Balasundaram C: In-vitro and in-vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish. J Aquat Anim Health 2008; 20(3): 165–76.
9. Immunostimulatory and antifertility effects of neem (Azadirachta indica) leaf extract on common carp (Cyprinus carpio Linnaeus) | Request PDF. ResearchGate [Internet]. 2025 Aug 19 [cited 2025 Aug 21]; Available from: https://www.researchgate.net/publication/336274433_Immunostimulatory_and_antifertility_effects_of_neem_Azadirachta_indica_leaf_extract_on_common_carp_Cyprinus_carpio_Linnaeus
10. Guardiola FA, Bahi A, Jiménez-Monreal AM, Martínez-Tomé M, Murcia MA and Esteban MA: Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata). Fish Shellfish Immunol 2018; 75: 357–64.
11. Wylie MR and Merrell DS: The Antimicrobial Potential of the Neem Tree Azadirachta indica. Front Pharmacol 2022; 13: 891535.
12. Harborne JB and Harborne JB: Phytochemical methods: a guide to modern techniques of plant analysis. 3rd ed. London Weinheim: Chapman and Hall 1998; 302.
13. Sarker SD: editor. Natural products isolation. 2. ed. Totowa, NJ: Humana Press (Methods in biotechnology) 2006; 515.
14. Paul TK, Hasan MdM, Haque MdA, Talukder S, Sarker YA and Sikder MH: Dietary supplementation of Neem (Azadirachta indica) leaf extracts improved growth performance and reduced production cost in broilers. Vet World 2020; 13(6): 1050–5.
15. Bergey DH: Bergey’s manual of determinative bacteriology. 9th ed. Baltimore Philadelphia Hong Kong [etc.]: Williams & Wilkins 1994.
16. Balouiri M, Sadiki M and Ibnsouda SK: Methods for in-vitro evaluating antimicrobial activity: A review. J Pharm Anal 2016; 6(2): 71–9.
17. Ríos JL and Recio MC: Medicinal plants and antimicrobial activity. J Ethnopharmacol 2005; 100(1–2): 80–4.
18. Zar JH: Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice Hall 1999; 1.
19. Kleinbaum DG and Klein M: Survival analysis: a self-learning text. 3rd ed. New York: Springer 2012; 700. (Statistics for biology and health).
20. Motulsky H: Intuitive biostatistics: a nonmathematical guide to statistical thinking. Completely rev. 2nd ed. New York: Oxford University Press 2010; 1.
21. (PDF) Antimicrobial Activity of Trigonella foenum-graecum (Fenugreek) [Internet]. [cited 2025 Aug 12]. Available from: https://www.researchgate.net/publication/314485936_Antimicrobial_Activity_of_Trigonella_foenum-graecum_L_Fenugreek
22. Guardiola FA, Bahi A, Jiménez-Monreal AM, Martínez-Tomé M, Murcia MA and Esteban MA: Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata). Fish Shellfish Immunol 2018; 75: 357–64.
23. Nazeer N: Effect of Azadirachta indica supplemented feed on growth performance of Labeo rohita. Pure Appl Biol [Internet]. 2021 Mar 10 [cited 2025 Aug 21]; 10(1). Available from: http://www.thepab.org/files/2021/March-2021/PAB-MS-2005-163.pdf
24. Bu X, Peng X, Huang L, Zhao Y, Jiao J and Zhu J: Effect of ectoparasite Ichthyophthirius multifiliis on the histopathology and gill and gut microbiota of goldfish (Carassius auratus). Front Vet Sci 2025; 12: 1539446.
    

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    How to cite this article:

    Khan A and Quadri SA: Nanoencapsulation of neem and fenugreek extracts: a novel approach to improve bioavailability in fish disease treatment. Int J Pharmacognosy 2025; 12(8): 659-67. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.12(8).659-67.

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