Investigation of the Antimicrobial Properties of Tamarindus indica L(Red Tamarind) Fruit Pulp

Introduction

Tamarind (Tamarindus indica L.), commonly known as red tamarind, is a tropical fruit-bearing tree belonging to the Fabaceae family. It is widely cultivated in tropical and subtropical regions, including Africa, Asia, and South America, for its edible fruit, which is extensively used in culinary, medicinal, and industrial applications [1-5]. The fruit pulp of tamarind is rich in bioactive compounds, including polyphenols, flavonoids, tannins, alkaloids, and organic acids, which contribute to its diverse pharmacological properties [6]. The long utilized tamarind fruit pulp for its therapeutic benefits, such as antimicrobial, antioxidant, anti-inflammatory, and gastrointestinal health-promoting effects. The antimicrobial potential has garnered significant scientific interest due to the increasing global threat of antibiotic resistance. The presence of bioactive constituents in tamarind pulp suggests its potential efficacy against a broad spectrum of pathogenic microorganisms, including bacteria, fungi, and viruses [7-10].This study aims to evaluate the antimicrobial properties of Tamarindus indica L. fruit pulp against selected bacterial and fungal strains, elucidating its potential role as a natural antimicrobial agent [11-13]. The findings from this research may contribute to the growing body of evidence supporting the use of plant-based antimicrobials in healthcare and food industries.Recent research has highlighted the potential of medicinal plants in addressing antimicrobial resistance through their diverse bioactive compounds [14-25]. Phytochemicals such as alkaloids, flavonoids, tannins, and terpenoids have demonstrated significant antimicrobial properties against various drug-resistant pathogens [26,27]. These compounds exert their effects through multiple mechanisms, including inhibition of bacterial cell wall synthesis, disruption of membrane integrity, and interference with quorum sensing, which regulates bacterial communication and virulence [28].Several studies have focused on the synergistic potential of plant-derived compounds when used in combination with conventional antibiotics, enhancing their efficacy against resistant bacterial strains [29]. For instance, extracts from Nigella sativa, Curcuma longa, and Azadirachta indica have shown promising results in overcoming multidrug resistance by inhibiting efflux pumps and restoring antibiotic susceptibility [30,31].

Medicinal plants play a crucial role in the development of nanoparticle-based drug delivery systems. Phytosynthesized nanoparticles, such as silver and gold nanoparticles, exhibit enhanced antimicrobial activity due to their small size and high surface area, which enable better interaction with bacterial cells [32,33]. These green-synthesized nanoparticles have demonstrated potential in combating resistant pathogens, providing a sustainable and eco-friendly approach to antimicrobial therapy [34], and promoting conservation and sustainable use of medicinal plant resources is vital to ensure their availability for future drug development and traditional healthcare systems, the rich biodiversity of medicinal plants, India and other developing nations can contribute significantly to global efforts in combating AMR while preserving traditional knowledge and promoting sustainable healthcare solutions [26-29].

Materials and Methods

Plant Selection

During the rainy season of July and August 2023, fresh and healthy plant materials (fruits) were collected for research purposes from the Sri Venkateshwara Temple (Vishnu Temple), located in Panagal, Nalgonda District, Telangana, India. The temple is situated at a latitude of 17.0777892° N and longitude of 79.2859803° E. This historic structure, recognized as one of the ancient temples in Panagal, was built by the Kanduru Cholas in the 11th-12th centuries.

Authentication ofTamarindus indica L.

The collected plant specimens were meticulously prepared into herbarium samples and submitted to the Herbarium in the Department of Botany, Osmania University, Hyderabad, Telangana. Following submission, the plant species underwent a detailed taxonomic study and were formally identified and authenticated by experts of the Botanical Survey of India, Deccan Regional Centre, Hyderabad. The specimens were assigned the official herbarium voucher number: BSI/DRC/2024-25/Tech/Identification/685.

Drying

The collected plant material was washed with double-distilled water and air-dried at room temperature for 21 days in a shaded, ventilated area. The dried leaves were then finely powdered and stored in airtight containers for further analysis.

Successive extraction using Soxhlet apparatus

The Soxhlet equipment was employed for the successive extraction process. Twenty grams of Tamarindus indica (red variety) fruit pulp was placed in a Wattman No. 1 filter paper, which was then inserted into the Soxhlet thimble, and 200 ml (w/v) of solvent was added to the round bottom flask. The fruit pulp was extracted sequentially using methanol at 65°C. The extraction temperatures have been adjusted to the boiling points of the solvent to expedite the cycling of fresh solvent. The solvent was assigned a five-hour period for the hot continuous and consecutive extraction operation. Subsequent to extraction, the extracts were chilled and filtered using Whatman No. 1 filter paper[30].

Antibacterial Activity

Media Preparation for Anti-Bacterial Activity

Bacterial Strains

Bacterial strains used in the study included Gram-positive Staphylococcus aureus (ATCC 25923) and Streptococcus pneumoniae (ATCC 33400), along with Gram-negative Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli(ATCC 25922). All strains were obtained from the American Type Culture Collection (ATCC) and maintained under appropriate laboratory conditions [31-32].

Media Preparation for Antibacterial Activity

A) Nutrient Agar Media
Commercially available Nutrient Agar powder (28.0 g) was weighed and dissolved in 1000 mL of distilled water, followed by thorough mixing. The prepared solution was autoclaved at 121°C for 15 minutes to ensure sterility and then used for plate preparation to assess antibacterial activity.

B) Nutrient Broth

Commercially available Nutrient Broth powder (1.3 g) weighed, dissolved in 100 mL of distilled water, thorough mixing. The prepared solution was autoclaved at 121°C for 15 minutes and sterility and was then used for inoculum preparation.

C) Preparation of Stock Solution

The culture of each bacterial strain was prepared by subculturing the confirmed test organisms aseptically onto two separate nutrient agar slants. One slant was maintained as the stock culture, while the other served as the working set. The bacterial cultures were stored at 4°C for routine use, with an additional glycerol stock maintained at -20°C for long-term preservation.

D) Inoculum Preparation

The selected bacterial pathogens were inoculated into sterile nutrient broth and incubated at 37°C for 24 hours. The bacterial suspensions were adjusted to approximately 105105 CFU/mL to standardize inoculum density for subsequent experiments.

Antibacterial Activity of Tamarindus indica (Red Variety)

The antibacterial activity of the methanolic extract of T. indica (red variety) fruit pulp was evaluated using the agar well-diffusion method. Differenr concentrations from (25, 50, 75, and 100 µL) were tested against Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. And inoculated plates were incubated at 37°C for 18–24 hours, after which the inhibition zone diameter (mm) was measured. The activity index calculated, and measurements were taken in three fixed orientations, with the mean values recorded for accuracy.

Minimum Inhibitory Concentration

The Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial agent that inhibits visible microbial growth after overnight incubation [33].

Compound Preparation

Each test compound was individually weighed (1 mg) and dissolved in methanol to obtain a final stock concentration of 1 mg/mL. Standard amoxicillin was prepared in the same manner as a positive control.

Culture Preparation

A loopful of bacterial culture was inoculated into 3 mL of nutrient broth and incubated at 37°C overnight in a shaking incubator to ensure active bacterial growth.

Inoculum Preparation

From the overnight-grown culture, 20 µL of bacterial suspension was transferred into 1.5 mL of fresh nutrient broth. Different concentrations of the test compound were added, and the mixture was incubated at 37°C overnight. The MIC was determined by observing the lowest concentration at which no visible bacterial growth occurred.

Fungal Strains

The fungal strains Candida albicans (MTCC 183) and Aspergillus niger (MTCC) utilized in this study were sourced from the Microbial Type Culture Collection (MTCC) at the Institute of Microbial Technology (IMTECH), Chandigarh, India.

Sabouraud Dextrose Agar Preparation

Commercially obtained Sabouraud Dextrose Agar (32.5 g) was dissolved of 500 mL in distilled water and mixed thoroughly. The solution was sterilized in an autoclave at 121°C for 15 minutes and used for plate preparation to assess antifungal activity.

Antifungal Activity of Tamarindus indica (Red Variety)

The antifungal activity was evaluated using the well-diffusion method. SDA culture plates were inoculated with Candida albicans and Aspergillus niger using the spread plate technique. The plates were incubated at 37 ± 2°C for 48 hours. After incubation, the zones of inhibition around the wells were observed, and their diameters were measured in millimeters to determine antifungal efficacy.

Results and observations

Morphology of Tamarindus indicaL. (Red variety)

Tamarindus indica L., Sp. PL. 34. 1753; Baker in Hook. f., Fl. Brit. India 2: 273.1878; Gamble, Fl. Madras 1: 409. 1919.

Vem.: Chinta. English: Tamarind.

Tamarindus indica is of moderate to large in size, evergreen tree, about 15-30 m high,and 7 m in girth. bark fissured, grey, branchlets glabrous. Leaves even Trees, pinnate, 8 cm long, leaflets 12-16 pairs, oblong, 2 x 1 cm, chartaceous, glabrous or puberulous, base and apex blue, margin entire, petiole to 7 mm, petiolule reduced: stipules caducous. Flowers yellow with pink striations in terminal, few-flowered racemes; calyx tube narrowly turbinate, to 7 mm, lined by disc, lobes 4, subequal oblong, 6-8 x 2.75-3.5 mm; petals 3, oblong-oblanceolate; stamens 3, monadelphous, filaments to 6 mm, base pubescent, empty anthers 2 mm; ovary stipitate, to 5 mm, tomentose, style attenuate, 3 mm. Pod linear-oblong, falcate, thick, somewhat compressed, with brittle epicarp, pulpy mesocarp and leathary septate endocarp seeds 1-many, quadrangular, brown, shining.

Common in all plain’s districts cultivated and self-sown. It is also planted as an avenue tree.

Soxhlet extraction of T. indica(Red tamarind) fruit pulp

The Soxhlet apparatus was used for the process of successive extraction. Methanol was used to extract the fruit pulp in a step-by-step process at a temperature of 65°C. The extraction temperatures have been changed to match the boiling points of the solvent in order to speed up the cycling of new solvent. The solvent was given a five-hour duration for the hot continuous and successive extraction procedure

Antibacterial activity of Tamarindus indica fruit pulp (Red tamarind)

The antibacterial efficacy of T. indica (Red tamarind) fruit pulp methanol extract was assessed against four bacterial pathogens: Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli employing the Agar well-diffusion technique. Ampicillin functioned as the reference antibiotic for comparative evaluation. The experiment evaluated four concentrations (25 µl, 50 µl, 75 µl, and 100 µl) of the extract, with the zones of inhibition recorded following incubation at 37°C for 18–24 hours. The inhibitory zones of the methanol extract were juxtaposed with those of Ampicillin, yielding insights into the comparative efficacy of the plant-derived medication. Ampicillin exhibited zones of inhibition for Staphylococcus aureus ranging from 16 mm at 25 µg to 25 mm at 100 µg, whereas the Tamarindus extract shown moderate activity with inhibition zones increasing from 8 mm at 25 µl to 10 mm at 100 µl. For E. coli, Ampicillin exhibited inhibitory zones of 11 mm at 25 µg and 16 mm at 100 µg, while the extract displayed a consistent increase in activity, with zones ranging from 8.5 mm to 12 mm across the concentrations. The extract demonstrated inhibition zones against Streptococcus pneumonia ranging from 9.8 mm at 25 µl to 13 mm at 100 µl, with greater concentrations yielding results equivalent to Ampicillin, which displayed inhibition zones between 10 mm and 14 mm. The extract exhibited significant efficacy against Pseudomonas aeruginosa, with inhibition zones expanding from 9.5 mm at 25 µl to 14 mm at 100 µl, exceeding Ampicillin, which showed inhibition zones of 9 mm at 25 µg to 13 mm at 100 µg. This observation suggests a significant antibacterial activity of the extract, especially against this disease. The inhibitory measuredetermined by the ratio of the extract’s inhibition zone to that of Ampicillin, demonstrated superior efficacy of the T. indica (Red tamarind)fruit pulp methanolextract against Pseudomonas aeruginosa, with the inhibitory measures more than 1.0 at a concentration of 100 µl. The study underscores the concentration-dependent antibacterial efficacy of T. indica (Red tamarind)fruit pulp methanol extract, which, although generally less potent than Ampicillin, exhibited significant effectiveness against specific bacterial strains, notably Pseudomonas aeruginosa. The findings indicate the potential of T. indica (Red tamarind)as a natural antibacterial agent, warranting further research to extract and identify the bioactive components responsible for its antimicrobial capabilities. This may facilitate its use as a supplementary treatment or alternative antibacterial agent in addressing microbial infections

Minimum Inhibitory Concentration (MIC) of Tamarindus indicaL.(Red tamarind)

The Minimum Inhibitory Concentration (MIC) of T. indica (Red tamarind) fruit pulp methanol extract was assessed to ascertain its antibacterial effectiveness in comparison to the conventional antibiotic Ampicillin. The minimum inhibitory concentration (MIC), defined as the lowest concentration of a substance necessary to prevent observable microbial growth, was evaluated against four bacterial strains: Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, and Pseudomonas aeruginosa. The experimental approach entailed the preparation of a stock solution of the methanol extract and Ampicillin at a concentration of 1 mg/ml. A loopful of bacterial cultures was inoculated into 3 ml of nutrient broth and cultured overnight at 37°C in a shaking incubator. The inoculum for testing was generated by transferring 20 µl of an overnight culture into 1.5 ml of nutrient broth, to which varying quantities (5, 10, 25, 50, 100, and 200 µg/ml) of the compounds were added, followed by incubation at 37°C for 24 hours. The results were assessed by visually confirming the absence of bacterial proliferation.

Ampicillin shown showedefficacy against S. aureus and E. coli at a dosage of 200 µg/ml. The MIC values for S. aureus diminished steadily, reaching a peak activity of 0.071 µg/ml at a concentration of 200 µg/ml, whereas for E. coli, the MIC fell to 0.093 µg/ml at the same concentration of 200 µg/ml. Conversely, the methanol extract of T. indica (Red tamarind) exhibited extensive antibacterial efficacy, demonstrating effectiveness against all four bacterial strains tested at the maximum dose of 200 µg/ml. The MIC values for S. aureus varied from 0.421 µg/ml at 5 µg/ml to 0.120 µg/ml at 200 µg/ml, demonstrating a concentration-dependent decline in bacterial proliferation. For E. coli, the MIC values diminished from 0.386 µg/ml at 5 µg/ml to 0.187 µg/ml at 200 µg/ml, indicating a significant inhibitory impact. The extract exhibited notable efficacy against S. pneumoniae and P. aeruginosa, with MIC values decreasing from 0.428 µg/ml to 0.262 µg/ml and from 0.405 µg/ml to 0.351 µg/ml, respectively, at the evaluated doses. The results underscore the methanol extract’s extensive efficacy, notably its capacity to block S. pneumoniae and P. aeruginosa, which were not suppressed by Ampicillin under identical conditions.

Ampicillin had greater efficacy against S. aureus and E. coli at lower MIC values, but the methanol fruit pulp extract of T. indica (Red tamarind) showed a wider spectrum of inhibition, effective against all bacterial strains tested. This indicates that the extract possesses powerful bioactive components with considerable antibacterial efficacy, especially against isolates that exhibit reduced susceptibility to Ampicillin. The concentration-dependent decrease in MIC values further underscores its potential as a natural antibacterial agent. The results highlight the necessity for additional research to isolate and identify the active constituents responsible for the extract’s antibacterial properties, which may aid in the development of alternative or

Antifungal activity ofTamarindus indica (Red tamarind)

The antifungal efficacy of T. indica (Red tamarind)fruit pulp methanol extract was evaluated and compared to Fluconazole as the reference standard. The experiment employed the well-diffusion method, utilizing the fungal strainsCandida albicans and Aspergillus niger as test organisms. Prepared Sabouraud Dextrose Agar (SDA) culture plates were infected with fungal strains by the spread plate technique, and wells containing the extract and Fluconazole were assessed for inhibition zone formation after 48 hours of incubation at 37 ± 2°C. The inhibitory zones were quantified, and the extract’s effectiveness was evaluated at four concentrations: 25 µg/ml, 50 µg/ml, 75 µg/ml, and 100 µg/ml. Fluconazole demonstrated a gradual improvement in antifungal efficacy against Candida albicans, with inhibition zones of 18 mm at 25 µg/ml and 23 mm at 100 µg/ml. The T. indica (Red tamarind)extract exhibited significant activity, with zones expanding from 13 mm at 25 µg/ml to 20 mm at 100 µg/ml, indicating concentration-dependent antifungal effectiveness. For Aspergillus niger, Fluconazole generated inhibitory zones ranging from 12 mm at 25 µg/ml to 26 mm at 100 µg/ml, whereas the extract exhibited zones from 10 mm at 25 µg/ml to 15 mm at 100 µg/ml. Although the efficacy of the T. indica (Red tamarind)extract was consistently inferior to that of Fluconazole, it demonstrated considerable antifungal potential, especially at elevated concentrations. The results demonstrate that the fruit pulp methanol extract of T. indica (Red tamarind)inhibits the growth of fungal pathogens in a dose-dependent manner, exhibiting better efficacy against Candida albicans. The findings indicate that the methanol extract contains bioactive chemicals with antifungal characteristics, presenting potential as a natural antifungal agent. Additional research is necessary to identify and define the active components responsible for its efficacy, facilitating its prospective use in alternative antifungal treatments

Discussions

The red-fruited tamarind, scientifically classified as Tamarindus indica, is a rare variation with restricted distribution in the southern states of India. T. indica is a significant multipurpose tree species cultivated in diverse tropical and subtropical nations. Due to its pulp coloration, two phenotypes are identified as red and brown varieties. The red hue observed in unripe fruits results from the presence of anthocyanin in cell vacuoles [35,39].The T. indica(red tamarind) fruits collected for research purposes from the Sri Venkateshwara Temple (Vishnu Temple), located in Panagal, Nalgonda District, Telangana, India. The temple is situated at a latitude of 17.0777892° N and longitude of 79.2859803° E.The collected fruit was shade-dried, ground into powder, and then extracted with a Soxhlet apparatus with methanol solvent [30].The anti-bacterial activity of the ampicillin used as standard and methanolic extract of T. indica (red variety) fruit pulp was investigated using the Agar well-diffusion method, employing four concentrations (25, 50, 75, and 100 µl) against bacterial pathogens including Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. The results showed fruit pulp methanol extract of T. indica (Red tamarind) was tested for its antibacterial activity against four bacterial pathogens. With inhibitory zones ranging from 9.5 mm to 14 mm, the extract outperformed ampicillin in its moderate to substantial efficacy, particularly against P. aeruginosa. Although the extract was less effective than ampicillin, it demonstrated growing activity for other bacteria at higher concentrations. All things considered, T. indica (Red tamarind) extract demonstrated concentration-dependent antibacterial activity, showing promise as a natural antibacterial agent. It was especially effective against P. aeruginosa. Similarly [42]  reported the methanolic extract of Red Tamarindus indica fruit pulp exhibited antimicrobial activity against Rhizoctonia solani, with the highest inhibition observed at 100% concentration (14.33 mm), surpassing the positive control (fluconazole, 13 mm). The inhibition decreased with lower concentrations: 75% (11.33 mm), 50% (7.67 mm), and 25% (5 mm) [43]reported in common variety of T. indica showedthe aqueous-ethanolic extract demonstrated a wide range of antibacterial activity, suppressing both bacterial groups. Tamarind extract shown activity against all tested Gram-positive bacterial isolates, with notable efficacy against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, and Listeria monocytogenes, with inhibition zones measuring 18 mm, 19 mm, 16 mm, and 16 mm, respectively [40]. Similar studies found in T. indica (Red tamarind) according to [44] reported antimicrobial activity [41].The fruit pulp extracts exhibited a wide spectrum of activity; the cold-water extract against 95.5% of the test bacterial strains; and the hot water and ethanolic extracts against 90.9% and 86.4%, respectively [42].The study compared Ampicillin to the Minimum Inhibitory Concentration (MIC) of T. indica (Red Tamarind) fruit pulp methanol extract against S. aureus, E. coli, S. pneumoniae, and P. aeruginosa. The extract exhibited extensive antibacterial activity, with MIC values for S. aureus ranging from 0.421 to 0.120 µg/ml and E. coli from 0.386 to 0.187 µg/ml. It also significantly affected S. pneumoniae and P. aeruginosa. While Ampicillin was more effective against S. aureus and E. coli, the extract was effective against all tested strains, especially those resistant to Ampicillin, suggesting its potential as a natural antibacterial agent.As previously articulated to [43-44] the minimum inhibitory concentrations (MIC) of the extract exhibited a range from 7.81 mg/mL against B. subtilis to 31.25 mg/mL against E. coli. The Minimum Bactericidal Concentrations (MBC) varied from 125 mg/mL for P. aeruginosa to 250 mg/mL for B. subtilis ATCC 6051[42, 44].The antifungal efficacy of the methanol extract from T. indica (Red Tamarind) fruit pulp was assessed against Candida albicans and Aspergillus niger, in comparison to Fluconazole. The extract demonstrated concentration-dependent action, with inhibitory zones for C. albicans varying from 13 mm at 25 µg/ml to 20 mm at 100 µg/ml, and for A. nigerfrom 10 mm to 15 mm. While Fluconazole exhibited greater efficacy, notably against A. niger, the T. indica extract revealed significant antifungal potential, particularly at elevated dosages. The findings indicate that the methanol extract of T. indica(red tamarind) fruit pulp possesses bioactive chemicals with notable antifungal effects, necessitating additional investigation to separate the active constituents for potential application in alternative therapies.

Conclusions

The study demonstrates that the methanol extract of Tamarindus indica L. (Red tamarind) fruit pulp showed significant antibacterial and antifungal properties. The extract demonstrated concentration-dependent efficacy, especially against Pseudomonas aeruginosa and Candida albicans. Although its efficacy was generally inferior to that of the reference antibiotics Fluconazole and Ampicillin, the extract exhibited broad-spectrum activity against all examined bacterial strains and fungi, even those resistant to Ampicillin. The results indicate that the methanol extract of T. indica(Red tamarind) fruit pulp possesses bioactive compounds with significant antibacterial efficacy. The study is required to extract and identify these bioactive chemicals, which may be turned into natural medicinal agents for the treatment of multidrug-resistant illnesses.

Acknowledgments

The Auhtors express our sincere gratitude to the Head of the Department of Botany, Osmania University, Hyderabad, Telangana, for providing the necessary facilities and support throughout the course of this research. Their unwavering support and provision of resources were instrumental in the successful completion of this study. We also appreciate the department’s collaborative atmosphere, which facilitated our research efforts and contributed significantly to the outcome of this work.