Antibacterial activity of acetone extract of Boswellia serrata Resin in combination with selected phytochemicals against bovine mastitis pathogens

Introduction

The animal husbandry and dairy development sectors are integral to the national economy and the sustainability of Indian farmers. Livestock provide a vital source of employment in rural areas and contribute significantly to the nutritional needs of millions. However, the dairy industry faces numerous challenges, among which mastitis stands out as one of the most economically significant infectious diseases. Mastitis adversely affects both the quality and quantity of milk, leading to losses that include reduced milk production, premature culling of affected animals, increased veterinary expenses, and discarded or downgraded milk. Mastitis is a complex, multifactorial condition characterized by physical, chemical, and bacterial changes in milk, alongside pathological alterations in glandular tissue [1] . While it can occur in all dairy animals, it is commonly observed that nearly every cow suffers from mastitis at some stage in its life. Cows are particularly at higher risk than buffaloes, with an estimated prevalence rate of around 50% [2] . Animals that have experienced mastitis are unlikely to recover their original milk production levels, resulting in substantial economic losses [3] . Apart from its economic implications, mastitis is a significant public health concern due to medication residues in milk and the potential transmission of harmful organisms to humans https://www.biochemjournal.com [4] . The condition is caused by multiple pathogens, including Staphylococcus spp., Streptococcus spp., coliforms, Pseudomonas spp., and Mycoplasma bovis. The involvement of diverse infectious agents makes mastitis a persistent challenge for veterinarians worldwide [5] .

Effective control of mastitis is critical, as contaminated milk can transmit pathogenic organisms to humans and harbor antibiotic residues [6] . The emergence of antibiotic-resistant pathogens has intensified the need for alternative treatments.

Over the past two decades, significant attention has been directed toward developing antimicrobial agents from natural sources [7] . Medicinal plants have long been used in traditional animal healthcare for the prevention and treatment of diseases. Their secondary metabolites, including alkaloids, flavonoids, tannins, terpenes, quinones, essential oils, and resins, possess antibacterial activity [8] . These compounds exhibit a broad spectrum of activity [9] and target microbes through mechanisms distinct from conventional antibiotics, making them valuable in addressing resistant pathogens [10] .

Plant-based antimicrobials are also associated with fewer side effects and have shown significant therapeutic potential in combating infectious diseases [10,11] . Furthermore, combining plant extracts with antibiotics often enhances efficacy, producing additive or synergistic effects [13,14,15] . Boswellia serrata resin, renowned for its antibacterial properties, has been extensively used in traditional Unani and Ayurvedic medicine. It is reported to be effective in treating conditions such as arthritis, bronchitis, asthma, cough, throat infections, and intestinal disorders. Additionally, it acts as a diaphoretic, astringent, stimulant, expectorant, and diuretic, and is prescribed for syphilitic and pulmonary diseases [16,17,18,19] . In this context, the present study was undertaken to evaluate the acetone extract of Boswellia serrata resin with the objectives of phytochemical analysis, evaluation of in-vitro antioxidant activity, evaluation of in-vitro antimicrobial activity against MTCC and clinical isolates of Staphylococcus aureus and Escherichia coli, evaluation of in-vitro synergism with selected phytochemicals

Materials and Methods

This study investigated the antibacterial activity of acetone extract of Boswellia serrata resin (BSR) and its synergistic potential with enrofloxacin, amoxicillin, and phytochemicals (cashew nutshell extract, xanthone, caffeic acid, thymol, and Ficus bengalensis extract) against mastitis-causing organisms (Escherichia coli and Staphylococcus aureus). All the chemicals are of analytical grade and were procured from (Himedia and Merck) Mumbai, India. The acetone extract of Boswellia serrata resin was prepared by soaking powdered resin (20 g) in acetone (1:10 ratio) and stirring overnight at room temperature. The mixture was filtered, and the filtrate was lyophilized. The residue was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution (500 mg/mL), which was further diluted to prepare a working solution (20 mg/mL) (Fig. 1). Stock solutions of enrofloxacin, amoxicillin, cashew nutshell extract, caffeic acid, thymol, xanthone, and Ficus bengalensis extract were prepared using appropriate solvents, and working solutions were diluted to suitable concentrations for antibacterial assays.

The BSR extract was screened for phytochemicals, including alkaloids, carbohydrates, saponins, phenols, flavonoids, tannins, and proteins using standard qualitative methods as per Harbone, 1984 [20] . The presence of alkaloids was tested using Dragendorff's reagent, carbohydrates with Molisch's test, saponins with a foam test, phenols with the ferric chloride test, and flavonoids with the lead acetate test. Antioxidant activity was assessed using the phosphomolybdenum assay [21] . Reaction mixtures containing various concentrations of BSR extract, phytochemicals, and standards were incubated at 95°C for 90 minutes, and absorbance was measured at 695 nm. Results were expressed as ascorbic acid equivalents. Antibacterial activity was evaluated against E. coli and S. aureus (both clinical isolates and MTCC cultures) using the broth dilution method. Minimum inhibitory concentration (MIC) values were determined by serial dilutions in cationadjusted Mueller-Hinton broth and assessed for bacterial growth inhibition using iodonitrotetrazolium chloride (INT). MIC was defined as the lowest concentration completely inhibiting visible bacterial growth. Combined antibacterial activity was examined using the checkerboard assay [22] (Fig. 2). Interactions between BSR extract, antibiotics, and phytochemicals were evaluated, and fractional inhibitory concentration (FIC) indices were calculated to classify effects as synergistic (<0.5), additive (0.5-1.0), indifferent (1.0-4.0), or antagonistic (>4.0). Biofilm inhibition potential of the BSR extract and other phytochemicals at MIC concentrations was assessed using a crystal violet assay [23] . Treated bacterial cultures in microtitre plates were incubated, stained with crystal violet, and de-stained with ethanol. Absorbance at 550 nm was recorded to calculate percentage inhibition. Statistical analysis was performed using SPSS (version 19.0).

Results and Discussion

The acetone extract of Boswellia serrata resin revealed the presence of carbohydrates, triterpenes, flavonoids, saponins, and phenols (Table 1). These findings are consistent with previous studies, which reported the presence of carbohydrates [24] , boswellic acids [25] , flavonoids [26] , and phenols [27] . Such phytochemicals are known for their diverse pharmacological activities [28] . The antioxidant activity of Boswellia serrata resin and other phytochemicals was assessed through the phosphomolybdenum assay and are showed in Table 2. BSR extract antioxidant activity was equivalent to 44.82±0.85 µg/mL of ascorbic acid. These results align with Hartman et al. (2012) [29] , who identified limonene and esters in Boswellia resin as key contributors to antioxidant activity. Sharma et al. (2011) [30] and Gaurea et al. (2016) [27] also documented antioxidant activity in Boswellia serrata extracts using DPPH radical scavenging assays. The antioxidant activity of other tested compounds, including thymol, cashew nutshell extract, Ficus aerial roots, enrofloxacin, caffeic acid, and xanthone, ranged between 33.60±0.31 and 162±1.81 µg/mL of ascorbic acid. Similar activities have been reported by Kavoosi et al. (2013) [31] for thymol, Verma et al. (2015) [32] for Ficus aerial roots, and Sujareon et al. (2018) [33] for cashew nutshell extract. The antibacterial activity of Boswellia serrata resin extract alone and in combination with other phytochemicals and respective antibiotics such as enrofloxacin and amoxycillin was evaluated against Escherichia coli and Staphylococcus aureus (Table 3). The minimum inhibitory concentration (MIC) for Escherichia coli was 5 mg/mL, consistent with Sharma et al. (2016) [34] , who reported MIC values between 3.125 and 6.25 mg/mL using agar disc diffusion assays. Active components such as acetyl-keto boswellic acid (AKBA) and α-and β-boswellic acids were identified as key antibacterial agents [35,36] . Similarly, other phytochemicals showed varying MIC values, including cashew nutshell extract (2.5 mg/mL), Ficus aerial roots (2.5 mg/mL), xanthone (12.5 mg/mL), thymol (6.25 mg/mL), and caffeic acid (6.25 mg/mL). These results agree with studies by Mustapha et al. (2015) [37] , Miladiyah and Rachmawaty (2017) [38] , and Trombetta et al. (2005) [39] . For Staphylococcus aureus, the MIC of Boswellia serrata resin extract was 1.25 mg/mL, comparable to results from Aman et al. (2010) [40] and Sharma et al. (2016) [34] . The resin exhibited antibacterial activity due to its bioactive compounds, including boswellic acids and AKBA [36] . MIC values of other phytochemicals against Staphylococcus aureus were as follows: cashew nutshell extract (0.07 mg/mL), Ficus aerial roots (2.5 mg/mL), xanthone (25 mg/mL), thymol (6.25 mg/mL), and caffeic acid (3.125 mg/mL). These findings are supported by reports on cashew nutshell liquid [33] , xanthones [41] , and thymol [31] . The synergistic antibacterial activity of Boswellia serrata resin extract was evaluated in combination with enrofloxacin against Escherichia coli (Table 4), amoxicillin against Staphylococcus aureus (Table 5), and phytochemicals. Against Escherichia coli, the resin extract exhibited additive effects with enrofloxacin, cashew nutshell extract, thymol, and Ficus aerial roots, and synergistic effects with xanthone. The combination with caffeic acid showed indifference. The additive effects are attributed to overlapping mechanisms of action, such as cell membrane disruption by phenolic compounds [42,43] . Synergism with xanthone likely resulted from the combined disruption of bacterial membranes and inhibition of DNA synthesis [44] . Against Staphylococcus aureus, the resin extract showed additive effects with amoxicillin, xanthone, caffeic acid, thymol, and Ficus aerial roots. Similar additive antibacterial activity was reported by Rashan et al. (2021) [45] for combinations of Boswellia oleoresins with conventional antibiotics. The interaction between amoxicillin, which targets cell wall synthesis, and phenolic compounds from Boswellia serrata resin supports this additive effect [44,46] . The biofilm inhibition potential of Boswellia serrata resin extract along with other phytochemicals and respective antibiotics enrofloxacin (Table 6) and amoxicillin (Table 7) was assessed using crystal violet assays against Escherichia coli and Staphylococcus aureus. For E. coli, significant biofilm inhibition was observed with enrofloxacin, xanthone, thymol, and combinations of the resin extract with xanthone. Lower inhibition was noted with combinations involving caffeic acid, Ficus aerial roots, and cashew nutshell extract. Similar findings were reported by Raja et al. (2011) [36] , who demonstrated that AKBA could inhibit and disrupt biofilms. Ficus benghalensis aerial root extract has also shown biofilm inhibition potential against E. coli [47] . For Staphylococcus aureus, the highest biofilm inhibition was achieved with cashew nutshell extract, xanthone, caffeic acid, and combinations of the resin extract with xanthone. Moderate inhibition was observed with thymol, Ficus aerial roots, and combinations involving the resin extract with thymol or amoxicillin. Raja et al. (2011) [36] and Jorge et al. (2019) [48] have similarly reported biofilm inhibition properties for boswellic acids and caffeic acid, respectively.

Conclusion

In conclusion, Boswellia serrata resin extract demonstrated significant antioxidant and antibacterial properties, attributed to compounds such as boswellic acids, AKBA, and phenolic derivatives. The resin exhibited additive effects with enrofloxacin and amoxicillin against E. coli and S. aureus, respectively, and synergistic effects with xanthone against E. coli. It also showed promising biofilm inhibition alone and in combination with other phytochemicals, supporting its potential as a natural adjunct in combating antibiotic resistance and biofilm-associated infections.

Acknowledgement

AcknowledgmentsAuthors thank the Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh for funding the research work.

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