Introduction
Snakebites are a significant public health issue in several parts of the world, especially in the regions of tropical and subtropical countries. According to the reports of WHO (World Health Organization), around 5.4 million snakebite cases occur every year, resulting in approximately 1.8 million envenomation and 138,000 deaths (WHO, 2019). However, these estimates are likely to be conservative due to underreporting and ineffective documentation. The distribution of snakebites varies geographically, with the highest numbers occurring in South Asia, Southeast Asia, and Sub-Saharan Africa, whereas, India has reported the maximum number of deaths occurring by snakebites in the world, with an estimated 58,000 deaths per year (Suraweera et al., 2020). The major snake species responsible for the maximum snakebite death cases in India is the Indian cobra (Naja naja) (Senji Laxme et al., 2021). Its venom consists of neurotoxin, and it produces systemic poisoning, thereby causing respiratory paralysis and death. It is considered a very dangerous snake among the species (Achyuthan and Ramachandran, 1981).
The genus Leucas belongs to the Lamiaceae family. The diverse pharmacological characteristics of several species in the genus Leucas, including those with antimicrobial, antioxidant, anti-inflammatory, cytotoxic, anticancer, antidiabetic, and wound-healing properties, have been the subject of in-depth research. As a result, the phytochemistry and pharmacological potential of the genus Leucas indicate that it has a promising potential as a significant source of natural compounds for drug development and discovery (Prajapati et al., 2010; Gopi et al., 2014; Hiremath et al., 2022).
The formulation of polymer-based nanoparticles has gained much attention in the field of therapeutics by increasing the efficiency of drugs via different routes such as intravenous, oral, and mucosal administration (Janes et al., 2001). Several polymer materials have been used for the formulation of nanoparticles, and among them, chitosan is reported to have more advantages for therapeutic applications. CS is a biodegradable polysaccharide known for its bioadhesive properties. Extensive research has demonstrated that CS is non-toxic and compatible with soft tissues (Bullock et al., 2000). Therefore, it has found widespread applications in pharmaceutical research and industry, serves as an efficient carrier for drug delivery, and is an effective material in biomedical applications (Mao et al., 2001). The primary amine groups contribute to the positive charge in chitosan and are important for mucoadhesive property, antimicrobial activity, and wound healing property that makes CS very useful in therapeutic applications (Berscht et al., 1994; Takeuchi et al., 1996; Mohammadpour dounighi et al., 2010).
The present study is aimed at investigating the efficiency of the methanolic extract of L. aspera (Willd.) loaded within chitosan nanoparticles against specific snake venom enzymes. To date, to the best of our knowledge, there is very little or no report on the use of the nanoparticle-loaded extract against snake venom enzymes. The present study provides an alternate option for the management of snakebites with enhanced efficiency.
Discussion
Polyphenols and flavonoids are primary antioxidants and play a crucial role in scavenging free radicals (Rahman et al., 2006). Phenol-based antioxidants effectively regulate redox homeostasis during oxidative stress, contribute to cellular function regulation, and reduce the risk of chronic diseases (Vauzour et al., 2010). Therefore, it is essential to determine the total phenolic content in plant extracts. The phenolic content estimated in this study aligns with the previously reported values (Gopi et al., 2014), indicating that the LA extract is abundant in phenolic and flavonoid compounds, which are significant contributors to oxidative defense.
The absorption spectrum for chitosan nanoparticles showed maximum absorption of approximately 200–300 nm, as reported by Serrano (2013), Vaezifar et al. (2013), and Oh et al. (2019). The peak at 226–312 nm was identified as flavonoids and their derivatives. Two peaks at 404 and 462 nm are due to the presence of tannins, which have characteristic absorption peaks in the wavelength range of 350–500 nm (Bulla et al., 2021).
Hydrodynamic dimensions and surface charge play an important role in deciding the characteristics of particles. DLS and zeta potential provide information on the nanoparticle dispersion, size, and net charge. It has been reported that by varying the concentration of chitosan solutions and other parameters such as temperature, pH of the solution, and stirring duration, the particle size and the PDI (polydispersity index) for the chitosan nanoparticles are different (Khan et al., 2016; Rasaee et al., 2016; Thandapani et al., 2017; Agarwal et al., 2018; Iswanti et al., 2019). When CS concentrations increased, there is an increased availability of protonated amine groups for ionic gelation, which indicates the increase in the particle size. For the lowest doses of STPP and CS, the minimum size of 119 nm was achieved (Koukaras et al., 2012). The size of the nanoparticles was 340 nm at a CS/TPP w/w ratio of 4:1, but the size of the nanoparticles tends to increase at other CS/TPP ratios (Zhang et al., 2004). These results align with what is typically seen in chitosan nanoparticles produced through the ionic gelation method (Bugnicourt et al., 2014; Hamdan et al., 2023).
The elevated positive zeta potential is attributed to the presence of amino groups on the chitosan particle surface, diminishing the likelihood of aggregation and thereby enhancing the nanoparticle stability. The incorporation of the surfactant Tween 80 also influenced the nanoparticle size. Despite the introduction of anionic TPP to chitosan during fabrication, the resulting nanoparticles exhibited an overall positive charge, possibly due to the substantial molecular weight of chitosan (Silva et al., 2012; Miladi et al., 2015).
The analysis of the FTIR data in the nanoparticle formulations reveals the presence of various functional group peaks, indicating the interaction between the amino groups of chitosan and the phosphate groups of STPP (sodium tripolyphosphate), confirming the fabrication of the nanoparticles. This finding is consistent with a previous study conducted by Khan et al. (2016). Similar reports of chitosan nanoparticle formation with TPP (tripolyphosphate) ratios have also been documented (Qi et al., 2004; Bhumkar and Pokharkar, 2006). The peak at 564 cm-1 corresponds to the out-of-plane bending of NH and the out-of-plane bending of C−O (Varma and Vasudevan, 2020). The peaks between 1,000 and 1,050 cm-1 indicate the stretching vibrations of C–OH and C–O–C. The peak at 1,412 cm-1 represents C–N stretching vibrations (amide III band) (Uzun and Topal, 2013), while the peak at 1,568 cm-1 corresponds to –NH2 bending vibration (Rajam et al., 2011). The peak at 1,634 cm-1 is associated with the amide I group. The absorbance peak at 1879 cm-1, related to C–O and C–C stretching, indicates the compounds containing aldehyde, ketone, organic acid, or alkene groups (Liu et al., 2016). The peak at 2,360 cm-1 is due to C=O bending vibrations (Praffulla and Bubbly, 2018), while the peak at 2,934 cm-1 is attributed to aromatic C–H bending vibration (Kunasekaran and Krishnamoorthy, 2015). The peak at 3,429 cm-1 results from the O–H stretching (Kumirska et al., 2010), and the peaks observed between 3,400 and 3,800 cm-1 are attributed to O–H and NH2 bending vibrations, as well as intramolecular hydrogen bonding (Damiri et al., 2020). The peak at 3,881 cm-1 is associated with the O–H stretching in carboxylic acids (Anandalakshmi et al., 2016).
The size observed in DLS corroborates with the FESEM results. In the present study, the size of the nanoparticles formulated in the absence and presence of the extract was measured. The encapsulated LA extract altered the surface structure and particle size of chitosan nanoparticles. The average particle size of the nanoparticle is large (260 nm) in the encapsulated form compared to the extract-free nanoparticle (119 nm). Similar observations are reported in the literature. SEM images reported in earlier studies revealed spherical morphology and uniform size distribution in both chitosan nanoparticles (CSNPs) alone and those encapsulating pomegranate peel extract (PPE). The size of the pomegranate peel extract-loaded chitosan nanoparticle is larger (127.3 nm) than that of the extract-free particle (90.6 nm) (Soltanzadeh et al., 2021). Similarly, TEM analysis revealed that chitosan nanoparticles (CNPs) exhibited spherical particles ranging in size from 9 to 25 nm, while chitosan nanoparticles (CNPs) loaded with the ethanol extract of Capparis cartilaginea (CNPs/Cc) displayed spherical shapes with a size range of 18–30.1 nm (Salman et al., 2023). It is envisaged that the loading extract could create pores or voids on the interior of chitosan nanoparticles, potentially resulting in an overall increase in particle size. The increase in the size of the nanoparticle encapsulated with the molecules, especially the crude extracts, is because the entrapment occurs by occlusion, and therefore, molecules of diverse chemical nature will be encapsulated, which results in all types of intermolecular interactions, both stabilizing and destabilizing. Similar characteristics were observed in the chitosan nanoparticle preparations, as reported previously by Luque-Alcaraz et al. (2016). According to several investigations, CNPs have a good dispersion in the size of the nanoparticles and are entangled to form a larger exposed surface area, which makes CNPs appropriate for adsorption. The morphology of the nanoparticles depends on various parameters and adopts spherical (Mohammed et al., 2017), oval (Corazzari et al., 2015), or rod-shaped (Sivakami et al., 2013) structures. In terms of morphology, the chitosan nanoparticles (CSNPs) prepared in this study exhibited a spherical shape, consistent with observations in prior research (Yang et al., 2009). Both the free chitosan nanoparticles and the chitosan nanoparticles loaded with EPE displayed a uniform distribution without any noticeable agglomeration, maintaining a spherical size of approximately 50 nm (Egil et al., 2020).
The elements present in CNPs include nitrogen, oxygen, carbon, sodium, and phosphorus. The physical interaction between TPP and chitosan nanoparticles confirms the presence of phosphorus, which is in concurrence with the previous reports (MubarakAli et al., 2018). The diffraction pattern of free CNPs displayed a broad hump, consistent with findings in similar studies that showed various diffraction peaks at 2 theta = 20, indicative of the typical identification for semi-crystalline chitosan (Author Anonymous, 2013) and CNP-LA extending over a large range of 2 theta, suggesting that CS is present in the crystalline state. Another study has reported that a weak diffraction pattern was observed at peak 2θ = 10° (Rasaee et al., 2016) and a strong diffraction characteristic peak at 2θ = 20°, for the chitosan solution, therefore revealing the high degree of crystallinity nature for the chitosan. A predominant peak (110) for CS with a large diffraction peak at 2θ = 20° was also reported (Anusha and Fleming, 2016). From the diffraction spectrum for the CS and chitosan nanoparticles, diffraction peaks of 10.18° and 20.26° were observed at 2θ, respectively. Thus, it confirms the presence of the crystalline nature of the particle present in the synthesized chitosan (Budi et al., 2020). Chitosan’s diffraction pattern has peaks at 2θ = 9.28° and 20.18°, respectively, showing its crystalline form II, and a broad band was observed at 2θ = 30° (Divya et al., 2017). The broader peaks (or the width at half-height) are due to the size of the chitosan nanoparticles, which is in congruence with the particle size observed in FESEM. The crystalline nature of the nanoparticles is different in the free and plant extract-loaded nanoparticles.
The sustained release of the encapsulated plant extract was observed after an initial burst release at both the pH (5.0 and 7.4). The difference is due to the changes in the protonation states of both CS and TPP. As the formation of nanoparticles is attributed mainly to the intermolecular electrostatic interactions, the pH dependence in the stability and, therefore, the release of the encapsulated extract is expected. Both the polymers, chitosan and TPP, used to formulate the nanoparticle are charged polymers, and therefore, pH influences the formation, stability, and porosity of the nanoparticle. The pKa of chitosan amine is 6.3, and the STPP has pH-dependent ionization behavior, owing to different pKa values (0.9, 1.9, 5.3, and 7.7) (Pati et al., 2011). Under acidic conditions (pH 5.0, which is less than the pKa), chitosan amino groups are protonated. Therefore, both highly acidic (neutralizes the charge on TPP) and near neutral pH (neutralizes charges on chitosan) will destabilize the nanoparticle and result in the fragile interaction between chitosan and TPP. This is also reflected in the higher nanoparticle sizes at these pH conditions. The same is true for the plant extract-loaded nanoparticles. This is evident from the higher release of the encapsulated plant extract at neutral pH (7.0) than that at acidic pH (5.0). The release profile follows non-Fickian diffusion, where both diffusion and polymer relaxation contribute to the release of encapsulated molecules (Yao and Weiyuan, 2011). In addition to the pH, the molecular size and other physico-chemical properties of chitosan influences the size, porosity, and stability of the nanoparticles. Previous studies have also highlighted the influence of pH on the characteristics of chitosan nanoparticles, such as particle size, surface charge, porosity, stability, and the release of the encapsulated molecules. As indicated previously, the particles are more compact at pH near 5.0 and swell while deviating from these pH conditions. The size of Rosmarinus officinalis ethanolic extract-chitosan nanoparticle (ROEE-CNPs) is 48.80 ± 6.84 nm at pH 5.0 (Kasem et al., 2024). Chitosan nanoparticle-loaded grape extract pH adjusted between 3 and 4, and the particle size was 177.5 ± 2.12 nm (Soleymanfallah et al., 2022). CNPs produced using an aqueous extract of Eucalyptus globulus Labill leaves have the smallest particle sizes (with a size range between 6.92 and 10.10 nm) at 4.8 ± 0.02 (El-Naggar et al., 2022). The average size of CNP-LA nanoparticles reported in this study is 260 nm at pH 5.0. Jatropha pelargoniifolia extract-loaded chitosan nanoparticles exhibited different release profiles at pH 2.0 and pH 7.4. Within the initial 2 h, only 12% of the JP extract was released at pH 2.0, whereas at pH 7.4, the release was 25% (Alqahtani et al., 2021). The study reported that the mitomycin C-loaded chitosan nanoparticle cumulative release at two different pH values of 6.0 and 7.4 is 47% and 53%, respectively, at the end of 1,440 min (Kavaz et al., 2017). These results indicate the potential usability of CNP-LA for sustained-release applications, which can be controlled by the difference in the local pH conditions.
Snake venom phospholipase A₂ (PLA₂) is known to elicit a diverse array of pharmacological effects, including both pro- and anti-effects on myotoxicity, neurotoxicity, and cytotoxicity (Kini, 2003; Janardhan et al., 2019). PLA₂ is a multifunctional enzyme, and thus, exploring potential compounds for developing snake venom inhibitors involves a critical examination of PLA₂ activity inhibition. Aristolochia radix has been demonstrated to inhibit PLA₂ derived from V. russelli venom in a dosage-dependent manner (Sakthivel et al., 2013). Enhancing the efficiency of neutralizing snake venom PLA₂ is achieved through soy protein nanoparticles conjugated with anti-snake venom immunoglobulins (F (ab′)2 fragments) (Renu et al., 2014). Inhibiting N. naja snake venom PLA₂ activity using biosynthesized soy protein nanoparticles (SNP) from Dryopteri cochleata (at 0.1 mg/mL) significantly neutralizes N. naja venom activity. The neutralization potential of the biosynthesized SNP surpasses that of the whole-plant extract (Singh et al., 2020).
In addition to metalloproteases and phospholipases, Elapidae snake venoms are rich in cardiotoxins, cytotoxins, hemotoxins, and myotoxins, primarily associated with hemolysis and cytolysis (Dufton and Hider, 1988). Hemolysis is a characteristic phenomenon seen in cobra venoms due to the action of multiple components. Prior studies have shown that phospholipases play a role in breaking down the intact phospholipids found on the erythrocyte membrane, leading to hemolysis (Demel et al., 1975).
Serine proteases and metalloproteases are present in snake venom. These enzymes not only degrade the extracellular matrix but also disrupt the hemostatic system (Serrano, 2013). The primary role of snake venom proteases is to disrupt the general hemostatic pathway, leading to systemic hemorrhage. Snake venom metalloproteases, however, target a diverse range of substrates, including plasma proteins, membrane proteins, endothelial cells, proteins involved in platelet aggregation, and cells associated with the inflammatory response, ultimately resulting in severe hemorrhaging at the envenomation site (Fox and Serrano, 2009). L. aspera extracts have demonstrated the ability to decrease the proteolytic activity of snake venom, with previous research highlighting significant inhibitory effects, especially in the methanolic extract of L. aspera (Gopi et al., 2014). Elevating the concentration of plant extract-loaded nanoparticles effectively suppresses the proteolytic activity of the venom.
There have been a few reports on the formulation of nanoparticles to reduce the toxic effects of snake venom proteins. Silver nanoparticles prepared in the presence of Alstonia scholaris Linn bark extract effectively neutralized Viper russelli venom (Ghosh et al., 2021). Another report states that 2-hydroxy-4-methoxybenzoic acid (HMBA) from the root extract of the Indian Sarsaparilla (Hemidesmus indicus) has viper venom neutralizing effects in animal models (Gomes et al., 2016). A previous study reported that the encapsulation of Naja naja oxiana venom within chitosan nanoparticles presents potential as an alternative to traditional adjuvant systems (Mohammadpour Dounighi et al., 2012). Cerastes cerastes venom poly lactic-co-glycolic acid nanoparticles (Cc-PLGA NPs) provide protection against high lethal doses of viper venoms (Hamzaoui and Laraba-Djebari, 2021). Therefore, there is limited information on the application of nanoparticles in neutralizing the snake venom toxicity even though it offers novel avenues for venom neutralization and targeted therapy development. Nanoparticle-based approaches show promise in enhancing antivenom efficacy and mitigating venom-induced toxicity (Gomes et al., 2018). The investigation of CNP-LA against Naja naja venom toxicity makes a substantial contribution to the field of nanoparticle-based drug delivery systems for snakebite treatment. Such targeted intervention offers a promising avenue for more effective snakebite management, addressing critical aspects of venom toxicity and providing insights into developing safer and more efficient antivenom therapies. Therefore, our study significantly advances the understanding of nanoparticle-based drug delivery systems for snakebite treatment, offering a targeted and potentially safer approach to addressing venom-induced toxicity. To the best of our knowledge, this is the first report on the use of chitosan-based nanoparticle encapsulated with the L. aspera extract to neutralize the toxicity of Indian cobra venom.
Titanium dioxide nanoparticles (TiO2-NPs) neutralize Daboia russelii venom and Naja kaouthia venom (Chakrabartty et al., 2019). Silver nanoparticles, produced through the reduction of salts using a solid dispersion of curcumin (130 nm) and concentration of 0.081 mg mL−1), were utilized to counteract the toxic effects caused by Philodryas olfersii venom (Proença-Assunção et al., 2021). Vitex negundo gold nanoparticles neutralize Naja kaouthia venom toxicity-induced reactions in animal models (Gomes, 2015). However, metal-based nanoparticles can exhibit increased toxicity as their particle size decreases, leading to adverse effects on human health through mechanisms such as immunotoxicity, inflammation, oxidative stress, DNA damage, and cytokine inductions. Prolonged exposure to metal-based nanoparticles can have toxic effects on vital organs like the brain, liver, and kidney, raising concerns about their biocompatibility and potential harm to human health (Zhang et al., 2022). Therefore, there is a need for nanoparticles based on biopolymers. In the study conducted on the Indian spectacled cobra, the authors have detailed the formulation of the chitosan nanoparticle with snake venom to test the efficiency of the nanoparticle formulation (Mohammadpour Dounighi et al., 2012). In a previous study, the anti-ophidian properties of the L. aspera methanolic extract against the Indian cobra, Naja naja, venom enzymes were evaluated (Gopi et al., 2014). In continuation of our study, we have investigated the L. aspera methanolic extract loaded within chitosan nanoparticles. Chitosan is known for its biodegradability and non-toxic properties. CNPs exhibit a non-immunogenic nature, and their capacity to deliver therapeutic agents while reducing immune reactions is a valuable attribute in the field of nanomedicine (Mohammed et al., 2017). Chitosan nanoparticles (CNPs) provide a promising platform for drug delivery, minimizing toxicity, reducing immunogenic responses linked to antivenom use, and enhancing efficacy (Veronika and Peter, 2021). Therefore, the present study highlights that the encapsulation of herbal extracts into chitosan nanoparticles can mitigate the potential toxic effects of the extracts, rendering them safer for therapeutic applications. Chitosan nanoparticles facilitate the controlled release of herbal extract, thereby enabling sustained and prolonged therapeutic effects. The CNP-LA study represents an integration of nanotechnology with traditional medicine, presenting a hopeful direction for more efficient and safer snakebite treatments compared to other nanoparticle-based therapies.