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Full Research Article

Bioactive Nanoparticles from Cassia fistula L. Seed Aqueous Extract: Assessment of Multiple Biological Activities

Rula Dhahir Al-Jayid1, Huda Jasim M. Altameme1, Ashwak Falih Kaizal1,*
1Department of Biology, College of Science for Women, University of Babylon, Babylon, Iraq.

ABSTRACT

Background: Cassia fistula L., a flowering plant of the leguminous family (Fabaceae), has been explored for its potential in synthesizing silver nanoparticles. This plant is known for its medicinal properties, making it a promising candidate for applications in nanobiotechnology.

Methods: The researchers employed a combination of hot and cold extraction procedures to synthesize silver nanoparticles using seed extracts of C.fistula. The identity and characteristics of these nanoparticles were verified through various techniques, including chromatography, imaging microscopy, X-ray diffraction and scanning electron microscopy. Biological activities such as antioxidant and antibacterial properties were tested. For antioxidant activity, a reaction mixture consisting of 50 μl of seed extract and 5 ml of a 0.04% (w/v) DPPH solution was used. Antibacterial activity was evaluated against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes at concentrations of 0.125, 0.25, 0.5 and 1 mg/ml of AgNPs. Additionally, the antibiofilm effect and hemolysis impact were studied.

Result: The synthesized silver nanoparticles demonstrated significant biological activities, including antioxidant and antibacterial effects. The diameters of the zones of inhibition for P.aeruginosa at a concentration of 1 mg/ml were 20 mm for cold extract and 18 mm for E.coli using hot extract. These findings highlight the potential of C. fistula as a sustainable source for environmentally friendly nanomaterials, contributing to advancements in medical and environmental sciences. Finally, the use of silver nanoparticles from Cassia fistula encourages sustainable and productive agricultural practices, by improving plant health and yield, potentially revolutionizing agricultural methods.

INTRODUCTION

The environmentally friendly methods have been the focus of global scientific studies since the emergence of metallic nanoparticles, which became possible through green synthesis. They have gained great attention and are known as the green revolution (Ihum et al., 2024). As per the literature, extensive research has been undertaken on the synthesis of Ag-NPs via utilizing plant extracts derived from their various (Chen et al., 2020). Herbalists have noted many pharmacological effects of Cassia fistula (Meena et al., 2022). Cassia fistula is a flowering plant of the leguminous family (Fabaceae), subfamily Caesalpiniaceae, known as Amaltas. Cassia fistula is a synonym for Cassia exceisa kunth.
       
It is a deciduous tree with opposing, pinnate leaves (Kirtikar and Basu, 2006). Flowers are arranged in scattered racemes that are 30-50 cm in length (Khan and Alam, 1996). Brown, pendulous-septate, cylindrical fruit has a diameter of 1-3 cm and a length of 25-45 cm. Reddish-brown seeds that are lenticular (Bhatnagar et al., 2010). The plant appreciates deep, well-drained, fairly rich, sandy, loamy soil (Pawar and Killedar, 2017). Cassia fistula is historically used to cure various disorders (Gupta et al., 2000; Jehangir et al., 2010). It treats asthma, leprosy, heart disease and fever. The root treats flu and colds, while the leaves relieve pain and reduce skin irritation from swelling. In addition, stem bark and fruit extracts remove blood toxins (Jung et al., 2016). In other study, Sharma et al., (2022) test Cassia fistula ethanolic extracts for antipyretic, analgesic and anti-inflammatory effects in rats. Bark extract had the greatest benefits in all experiments, whereas leaf extract was antipyretic and anti-inflammatory. Research supports its historic therapeutic usage.
       
An increasingly common strategy for attaining sustainability and protecting the environment is the “green” synthesis of NPs. (Kanwal et al., 2025). A study by Pawar and Killedar (2017) mentioned that C. fistula has several therapeutic uses, especially in traditional medicine and its flower extracts are used in the widespread manufacture of silver nanoparticles (Ag-NPs). From a medical standpoint, notable outcomes have communicated the potential for biocompatibility, non-toxicity, economic viability and environmental friendliness (AL-Musawi and AL-Abedy, 2020; Omran, 2024). When contrasted with more traditional approaches, the new area of green synthesis within nanobiotechnology offers substantial benefits to both the environment and the economy (Ganesan and Rengarajan, 2024). This study, therefore, seeks to assess the inhibitory efficacy of AgNP nanoparticles synthesized using C. fistula seed.

MATERIALS AND METHODS

Methodology for the production of cold and hot water extracts from a plant
 
Cold and hot water extracts were generated from C. fistula seeds using Harborne’s maceration technique (1998), as shown in Fig 1.

Fig 1: Cassia fistula fruits and seeds.


 
Synthesis of silver nanoparticles with C. fistula seed extract
 
To make silver nanoparticles, 5 mL of Cassia seed aqueous extract was mixed with 0.017 g of silver nitrate in 100 mL of deionized water and shaken in an incubator for 24 h. As the brownish hue deepened, it became clear that silver nanoparticles were taking shape. Alteration in colour indicates the production of Ag-NPs. After letting the solution sit at room temperature for one day, we could be sure the nanoparticles would be stable. Using a centrifuge at 15,000 rpm for 15 min, the nanoparticles were separated, washed with deionized water, frozen and subjected to further examination (Choudhary et al., 2015).
 
Synthesized Ag-NPs description
 
The morphology and size of the synthesized silver nanoparticles were determined using atomic force microscopy, which allows visualization of the surface structure by plotting the microscopic data (Bailey and Clark, 2006). The structure and size of the synthesized particles were also examined using Scanning Electron Microscopy (SEM) and the three-dimensional structure was studied by X-ray diffraction and the heterogeneous structure of the particles was evaluated by Fourier transformation spectroscopy.
 
The method described by Jothy et al., (2011) was used to quantify the radical scavenging tests. The reaction mixture included 50 ìl of seed extract (0.125-1 mg/ml) and 5 ml of a 0.04% (w/v) DPPH solution in 80% methanol. Butylated hydroxytoluene (BHT), a commercial antioxidant, was employed for comparison or as a positive control. Control and blank were DPPH without seed extract and 80% methanol, respectively. Incubation in the darkroom for 30 min produced 517 nm discolorations. The following equation determined the DPPH free radical percentage:
Where,


A0 = The absorbance of the control.
A1 = The absorbance in the presence of the seeds extract of C. fistula.

Antibacterial effect study
 
According to Prastiyanto et al. (2022), the researchers studied the inhibition zone of C. fistula seed Ag-NPs suspensions against gram-negative bacteria “Escherichia coli and Pseudomonas aeruginosa” and gram-positive bacteria “Staphylococcus aureus and Streptococcus pyogenes” using the well diffusion.
 
Antibiofilm effect study
 
To know the effect of silver nanoparticles prepared using hot and cold extracts of C. fistula seeds on the ability of bacteria to form a thin biofilm, a test of the nanomaterial was performed by making five concentrations for each sample for two types of bacterial genera, namely gram-positive bacteria such as S. aureus and gram-negative bacteria such as E. coli. According to the method of Barapatre et al., (2016), a 96-well microtiter plate technique was used to test the effectiveness of silver nanoparticles in biofilm development. The following equation was used to calculate the percentage of biofilm inhibition.
 
Study of the hemolysis impact
 
One healthy donor’s blood was subjected to hemolysis tests using the methodology of Laloy et al., (2014), Blood samples were mixed with 15 μL of nanoparticles at concentrations of (1, 0.5, 0.25 and 0.125 µg/ml ,negative control was tyrode.285 microliters of whole blood are mixed with the positive control (Triton X-100).For four hours, the suspension is incubated in an incubator shaker. Following the incubation period, the suspension is centrifuged for five minutes at 10,000 g. The Elisa Reader is used to read the supernatant in a 96-well plate at 550 nm.

The percentage haemolysis was calculated as:

RESULTS AND DISCUSSION

Characteristic of the synthesized Ag-NPs
 
As particles were deposited throughout the production process of silver nanoparticles, the colour of both the hot and cold extracts of C. fistula seeds changed from brown to a darker shade of brown and the consistency also altered. After being exposed to 1 mM of silver nitrate (AgNO3), the Cassia plant demonstrated its capacity to produce silver nanoparticles via biosynthesis. The colour change of the reaction mixture after 24 h of incubation in a shaking incubator at 37oC is clear evidence of the formation of silver nanoparticles by a group of environmentally friendly reducing agents present in cassia seeds, which reduced silver ions (Ag+) to elemental silver (Ag0) as confirmed by Mohammed et al. (2018). According to Korbekandi et al. (2013) and Benakashani et al. (2016), the colour change of the reaction mixture from light brown to dark brown indicates the successful production of silver nanoparticles.
       
Fig 2 and Fig 3 show the 2D and 3D atomic number microscopy images of silver nanoparticles synthesized using hot and cold-water extract of Cassia seeds, respectively. The average size of the silver nanoparticles is 37 and 40 nm, respectively. The atomic number microscopy technique deals with images that allow quantitative measurements of the surface of the material, such as average roughness (Ra) (Kent and Vikesland, 2012).

Fig 2: The 2D (a) and 3D (b) AFM images of silver nanoparticles synthesized by hot water extract of Cassia fistula seed.



Fig 3: The 2D (a) and 3D (b) AFM images of silver nanoparticles synthesized by cold waterextract of Cassia fistula seeds.


       
The results showed that the nanoparticle size of the hot seed extract was smaller than that of the cold seed extract. The difference in the size of the nanoparticles measured by SEM for the hot and cold extracts of silver nanoparticles from C. fistula seeds was clearly observed. This indicates that different extraction methods have different properties of bioactive silver nanoparticles. According to the nanoparticle characterization results, there was a difference in the ability of each extract to synthesize bioactive silver nanoparticles. It is clear that the hot extract was more effective than the cold extract. The smaller the size of the nanoparticles, the greater is their effectiveness (Agnihotri et al., 2014). Fig 4 shows the silver nanoparticles resulting from hot and cold extraction of C. fistula seeds, respectively.

Fig 4: Silver nanoparticles produced by (A) Hot and (B) Cold extraction of Cassia fistula seeds.


 
X-ray diffraction (XRD)
 
The materials made from silver nitrate with hot and cold seed extract were found to have a polycrystalline structure based on the results of the X-ray diffraction examination. Fig 5 shows the presence of multiple peaks that correspond to the composition of the material being worked on with a hexagonal structure. The X-axis (2θ) represents the diffraction angle, which is related to the spacing between atomic planes in the crystal lattice and the Y-axis (counts) represents the intensity of the diffracted X-rays at each angle.

Fig 5: XRD analysis of Biosynthesized nanoparticles by (A) hot and (B) cold extraction of Cassia fistula seeds.


 
Fourier transform infrared spectroscopy (FT-IR)
 
Table 1, 2 and Fig 6 and 7 indicate the peak values and functional groups for silver nanoparticles produced by hot and cold extraction of C. fistula seeds, respectively. The X-axis (wave number, cm-1) represents the frequency of the infrared light and the Y-axis (transmittance or absorbance) indicates how much light is transmitted through the sample (transmittance) or absorbed by the sample (absorbance). The IR spectrum indicates the presence of a molecule containing both O-H and C=O functional groups, suggesting a hydroxy ketone, hydroxy aldehyde, carboxylic acid or amide. Such as a strong, broad peak around 3431.17 cm-1 is characteristic of O-H stretching vibrations, typically from alcohols or carboxylic acids and the broadness suggests hydrogen bonding.

Table 1: Peak values and functional group for silver nanoparticles produced by hot extraction of Cassia fistula seeds.



Table 2: Peak values and functional group for silver nanoparticles produced by cold extraction of Cassia fistula seeds.



Fig 6: FT-IR of Biosynthesized nanoparticles by hot extraction of Cassia fistula seeds.



Fig 7: FT-IR of Biosynthesized nanoparticles by cold extraction of Cassia fistula seeds.


 
Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
 
Researchers have looked at the antioxidant properties of C. fistula seed extract to find a new natural antioxidant source. It is well-known that DPPH tests accurately measure the antioxidant capacity of the material under study. The idea behind the DPPH test is that the colour of the DPPH solution would shift from purple to yellow when the antioxidant gets rid of radicals. Fig 8 displays the screening findings showing that the C. fistula seed extract effectively scavenged the DPPH radical. Also, it shows that the scavenging percentage decreases as the concentration of the extract decreases, indicating that the extract is more effective at higher concentrations. This suggests that C. fistula seed extract possesses antioxidant properties and can effectively neutralize DPPH radicals. Therefore, the C. fistula seed extract acts as a free radical scavenger, transforming them into stable molecules.

Fig 8: Effect of the C. fistula seed extract on scavenging the DPPH radical.


 
Antibacterial activity
 
An inhibitory effect on various bacteria “Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa” was detected using two different extracts made from C. fistula seeds. The concen-trations of the extracts were 0.125, 0.25, 0.5 and 1 mg/ml.
       
According to Table 3, the study shows that the concentration of each plant extract went up, so did the size of the inhibition zones. The diameters of the inhibition zones of the hot aqueous extract towards the growth of bacteria varied from 20 mm in Pseudomonas aeruginosa at a concentration of 1 mg/ml (Fig 9A) to 11 mm in Streptococcus pyogenes and E.coli at a concentration of 0.25 mg/ml (Fig 9B and C). However, no inhibition was observed for any type of bacteria at a concentration of 0.125 mg/ml and similarly, no inhibition was observed for Staphylococcus aureus at a concentration of 0.25 mg/ml.

Table 3: The inhibition zone diameters of four bacterial species against C. fistula seeds treated with Ag-NPs.



Fig 9: Antibacterial effect of silver nanoparticles prepared from hot extract of C. fistula seeds against (A) P. aeruginosa, (B) Str. pyogenes and (C) E. coli.


       
The AgNP suspension in a cold-water extract of C. fistula seed had the largest inhibition zone, about 20 mm, against gram-positive bacteria, specifically Streptococcus pyogenes, at a concentration of 1 mg/ml (Fig 10A). On the other hand, the Pseudomonas aeruginosa bacteria had the smallest inhibition zone, about 10 mm, at a concentration of 0.125 mg/ml (Fig 10B). Also, there was no inhibition zone in E. coli or Staphylococcus aureus bacteria at a concentration of 0.125 mg/ml (Fig 10C and D) and there was no inhibition zone in Staphylococcus aureus bacteria at a concentration of 0.25 mg/ml (Fig 10D). There are a number of hypotheses regarding the potential effects of Ag-NPs on cell membrane permeability. Another theory is that nanoparticles’ large surface area, which allows them to come into contact with organisms more easily than larger particles, is responsible for their strong antibacterial activity. In addition to interacting with the surface of the membrane, Ag-NPs may also interact with the bacteria inside (Sahayaraj and Rajesh, 2011; Al-Shugeairy et al., 2021).

Fig 10: Antibacterial effect of silver nanoparticles prepared from cold extract of C. fistula seeds against (A) P. aeruginosa, (B) Str. pyogenes and (C) E.coli, (D) Staph. aureus.


       
Three lectins isolated from C. fistula seeds were shown to have antibacterial properties against several types of harmful bacteria, according to Kuo et al. (2002). Aqueous and alcoholic extracts of the stem bark have also shown promising antibacterial activity. According to Verma (2016), C. fistula contains a lot of tannins, flavonoids and glycosides, which give it a wide range of pharmacological effects, including those against diabetes, inflammation, cancer, bacteria, fungal infections and others.
       
C. fistula is the subject of phytochemical research and is widely used by traditional Indian medicinal systems (Ali, 2014). It is an important medicinal plant due to its many beneficial uses and medicinal properties; moreover, it has hepatoprotective, anti-inflammatory, antitussive, antifungal, anti-wound and antibacterial properties. Its high concentration of glycosides, flavonoids and tannins has made it famous, as noted by Danish et al. (2011).
       
Previous research has demonstrated that solvent extracts of C. fistula have antibacterial and antifungal properties. These extracts were tested against various bacteria and yeasts, including E. coli, S. aureus, P.aeruginosa and Candida albicans, Aspergillus clavatus and Aspergillus niger (Bhalodia et al., 2012). In another study by Almuhayawi et al., (2024), under in-vitro, the antibacterial activity of ZnO nanomaterials shows the maximum zone of inhibition for nanoparticles prepared from the stem against Proteus mirabilis, Staphylococcus albus and Lactobacillus.
 
Antibiofilm effect
 
The most significant characteristic of bacteria that improves their ability to adhere to instrument surfaces is biofilm. Biosynthesized Ag-NPs, their capacity to suppress biofilm formation against S. aureus and E. coli was examined using the 96-well microtiter plate technique (Barapatre et al., 2016).
       
Fig 11 demonstrates that silver nanoparticles synthesized from C. fistula seeds by hot extraction have significant antibiofilm activity against S. aureus and E.coli. These nanoparticles’ effectiveness depends on their concentration, with higher concentrations leading to greater inhibition. Staphylococcus aureus appears to be more susceptible to the anti-biofilm effects of these Ag-NPs compared to E. coli. Thus, these silver nanoparticles can be used as a very effective agent against bacterial infections, including those involving biofilm formation. The results showed a significant decrease in biofilm production and increasing the concentration of silver nanoparticles reduced the amount of biofilm development in both hot and cold seed extracts. Silver nanoparticles for hot extracts (1 mg/ml) reduced biofilm production in both S. aureus and E. coli by up to 95.53% and 78.03%, respectively (Fig 11). While the silver nanoparticles of cold extracts (1 mg/ml) reduced the biofilm production in both S. aureus and E. coli by up to 90.42 and 83.40 per cent, respectively, In Fig 12 it confirmed that the silver nanoparticles synthesized through cold extraction from C. fistula seeds possess remarkable anti-biofilm properties against both S. aureus and E.coli. The effectiveness of these nanoparticles is concentration-dependent. While both bacteria are inhibited, S. aureus generally shows a higher sensitivity to the Ag-NPs than E. coli.

Fig 11: The antibiofilm inhibition rate of silver nanoparticles synthesized by hot extraction of C. fistula seeds.



Fig 12: The antibiofilm inhibition rate of silver nanoparticles synthesized by cold extraction of C. fistula seeds.


       
The results also showed great anti-biofilm activity for both hot and cold extracts of C.fistula seeds against S.aureus as a model for gram-positive bacteria and also good activity, but to a lesser extent, against biofilm formation in gram-negative bacteria. The results showed that the effectiveness of nanoparticles made from hot fistula seed extracts was lower than that of the cold extract against Gram-negative Escherichia coli and the effectiveness of the cold extract was lower than that of the hot extract against biofilm formation of gram-positive Staphylococcus aureus.
       
The difference in inhibitory activity between silver nanoparticles of hot and cold C. fistula seed extracts can be attributed to a number of factors including antimicrobial activity, physical properties such as nanoparticle size, which affects restricted penetration and some other chemical properties such as affinity of the material to biofilms (Park et al., 2013).
       
Greeshma et al. (2024) emphasized the need to explore these antibiofilm medications in vivo following successful in vitro research. Using current antimicrobials, these antibiofilm drugs may treat biofilm-forming infections. How much sensitivity increases depends on the antibiotics and biofilm inhibitors utilized.
 
Effect of hemolysis
 
Since all substances entering the bloodstream come into contact with red blood cells, the solubility of biological materials may need to be assessed. The American Society for Testing and Materials (ASTM) states that hemolysis of less than 5% is normal (Luna-Vázquez-Gómez et al., 2021).

The results of our study showed that no hemolysis occurred even at the highest concentration used for both hot and cold extracts of C. fistula seeds, which was 1 mg/ml as shown in Table 4 and Fig 13, given that hemoglobin release can result in negative health outcomes such anemia, pulmonary hypertension and renal toxicity, these findings may have therapeutic implications (Rother et al., 2005). The non-hemolytic concentration range found here is superior to the effective concentration to develop its microbicide, anticancer and immune-stimulating effects.

Table 4: Per cent of hemolysis of the for hot and cold extracts of C. fistula seeds.



Fig 13: Per cent of hemolysis of the for both hot and cold extracts of C. fistula seeds.


               
The results indicate that the use of Ag-NPs made from Cassia fistula can result in creative farming methods that are both efficient and sustainable for the environment. This strategy encourages the shift to more environmentally friendly farming methods in addition to addressing the demand for efficient agricultural inputs. Environmentally friendly practices are promoted by using green synthesis techniques to produce silver nanoparticles from Cassia fistula. By lowering dependency on dangerous chemicals and improving agricultural resource efficiency, this supports the objective of guaranteeing sustainable patterns of use and production. Thus, achieving the twelfth goal of sustainable development: (responsible consumption and production).

CONCLUSION

The research found that Ag-NPs made from Cassia fistula seed extracts had a lot of biological activity. Although Ag-NPs were effectively generated by both the hot and cold extraction procedures, the nanoparticles were smaller and more effective when extracted using the hot approach. The structural characteristics and successful production of the nanoparticles were verified by characterization methods. The results indicate that C. fistula may be used as a sustainable and environmentally friendly source of nanomaterials, which might have medical and environmental benefits. In nanobiotechnology, this study promotes non-toxic and economically feasible alternatives to conventional nanoparticle manufacturing processes, highlighting the significance of green synthesis techniques.
       
The study’s overall findings support the feasibility of employing Cassia fistula seed extracts for the environmentally friendly synthesis of Ag-NPs, which has the potential to transform farming methods by offering a sustainable and efficient way to improve plant production and health.

CONFLICT OF INTEREST

The authors say that there is no conflict of interest.

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