1. Background
Plant sterols and stanol esters, collectively known as phytosterols (PSs), are bioactive compounds with a structural similarity to cholesterol found in plant cell membranes (1). Phytosterols are naturally present in fruits, fresh vegetables, nuts, seeds, and plant oils such as canola, corn, flaxseed, olive, and sesame oil. There are no reports of adverse side effects associated with PSs, and they are generally considered safe (2). A wide range of pharmacological and biological properties has been attributed to these plant sterols, including a reduced risk of cardiovascular diseases due to their LDL cholesterol-lowering effects, as well as anti-obesity, anti-inflammatory, hypoglycemic, and immunomodulatory activities. Due to their cholesterol- and LDL-lowering benefits, plant sterols have been approved for addition to foods by organizations such as the U.S. Food and Drug Administration (FDA) (3), the European Food Safety Authority (EFSA) (4), and other official health bodies [Reviewed in (1)].
Studies on high-fat diet mice suggest that the hypolipidemic effect of PSs is linked to the regulation of cholesterol metabolism through modulation of gut microbiota (5). In vitro and in vivo studies indicate that phytosterol-enriched diets improve gastrointestinal tract function by increasing beneficial microbiota species such as Eubacterium halii and decreasing the abundance of Firmicutes bacteria (6), which are associated with enhanced calorie absorption and weight gain (7). Furthermore, PSs contribute to reduced appetite and food intake by promoting dietary fiber fermentation and increasing the production of short-chain fatty acids such as acetate and butyrate. Additionally, they reduce the production of cholesterol metabolites (8, 9). These findings highlight the therapeutic potential of PSs in addressing obesity, metabolic disorders, and gastrointestinal issues (10).
Phytosterols also support colonic epithelial cells by providing more energy and play a preventive role against colon cancer (8, 9). They improve immune responses in infants by modulating intestinal microbiota and increasing the diversity of bacterial genera such as Anaerostipes, Bacteroidetes, Firmicutesin, Staphylococcus, and Streptococcus in breast milk (9). Moreover, some studies report anticancer and antimicrobial activities of PSs (1, 2).
Global research is increasingly focused on developing novel therapies to combat antimicrobial and antiviral resistance. According to various studies, β-sitosterol extracted from Parthenium hysterophorus (11), avocado (12), the bark of Norway spruce (Picea abies) (13), and Lycorisradiata (14), as well as campesterol and its semi-synthetic derivatives (15), and stigmasterol (16), have demonstrated antibacterial activity. Pistachio nuts, in particular, are a rich source of healthy fats, fiber, protein, vitamins, and various phytochemicals, including anthocyanins, carotenoids, flavonoids, phenolic acids, and PSs (17).
2. Objectives
The antimicrobial activity of oleoresin (18), leaf phytochemicals (19), polyphenol-rich extracts from kernels (20), and lipophilic extracts from the leaves, stems, branches, hulls, woody shells, and kernels (21) of Pistacia vera L. has been previously investigated. Additionally, studies have been conducted on the antimicrobial properties of hull essential oil (22) and organic nanocomposites derived from hull extract (23) of P. vera. The objective of this study was to assess the antibacterial activities of total PSs extracted from the kernels and pistachio green hulls (PGHs) of P. vera Var. Damghan.
3. Methods
3.1. Plant Material
The kernels and PGHs of cultivars, including Abbasali, Akbari, and Khanjari, were sourced from Damghan (Iran) orchards in August 2021 and naturally sun-dried under our supervision. The cultivars were identified by Dr. Atefeh Amirahmadi (Ph.D. in Plant Biosystematics, School of Biology, Damghan University), and their specimens were preserved in the herbarium of Damghan University (Amirahmadi et al. 3086 (DU 001771), Amirahmadi et al. 3094 (DU 001780), and Amirahmadi et al. 3093 (DU 001779), respectively). High analytical grade chemicals (Merck or Sigma-Aldrich) were used in this study.
3.2. Phytosterol Extraction
Total phytosterol was extracted from the kernels and PGHs of the studied cultivars using a modified Soxhlet method as reported by Hashemi et al. [(24), under review].
3.3. Antibacterial Tests
The antibacterial activity of the extracted total PSs was assessed using the disk diffusion agar method (25) and the twofold dilution method (26). The Gram-negative and Gram-positive standard strains, namely Escherichia coli (E. coli; PTCC No: 1399; ATCC 25922) and Staphylococcus aureus (S. aureus; PTCC No: 1431; ATCC 25923), were obtained from the Iranian Research Organization for Science and Technology (IROST). Each organism was tested in duplicate on different days to ensure the reproducibility of the test. Ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), and penicillin (10 µg) were purchased from PadtanTeb Company (Iran) and used as positive controls.
3.3.1. Evaluation of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
The broth dilution method was used to determine the minimum inhibitory concentration (MIC) of the extracted total PSs against microorganisms. A 24-hour culture of each strain was examined microscopically, and 1.5 × 10⁸ bacteria, prepared from morphologically similar colonies cultured overnight, were considered equivalent to 0.5 McFarland. This equivalence was confirmed by measuring absorbance in the range of 0.08 to 0.13 at a wavelength of 625 nm using a spectrophotometer (26, 27).
Fifty microliters of bacterial suspension were added to 96-well microtiter culture plates, followed by 50 µL of PSs at different concentrations (1.6 - 10,000 mg/mL). The plates were incubated for 24 hours at 37°C. The phytosterol extracts were not sterilized; however, sterile antibiotic discs with a diameter of 6 mm were used as positive controls. Sterile distilled water served as a negative control. All tests were conducted under sterile conditions within a microbial hood. The results were evaluated by observing visible growth inhibition in the microbial tubes (absence of turbidity).
The minimum bactericidal concentration (MBC) was determined by subculture. Approximately 10 µL of each MIC tube showing no visible growth was plated onto Mueller-Hinton agar and incubated at 37°C for 24 hours. Colony growth was then examined. All tests were repeated three times to ensure consistency and reliability.
3.4. Statistical Analysis
Statistical analysis of the data was performed using SPSS software (version 27), with a significance level set at (P < 0.05).
4. Results
The evaluation of antibacterial activity of PSs extracted from Damghan pistachio varieties, assessed using the Kirby-Bauer disc diffusion method against both Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli), revealed that neither the kernel nor the PGH PSs exhibited any antibacterial activity against the tested bacteria.
5. Discussion
P. vera is the most economically significant species among the 11 species of the Pistacia genus (17). In this study, the antibacterial activity of total PSs extracted from the kernels and PGHs of three Damghan pistachio cultivars was examined for the first time. Our findings revealed that PSs extracted from neither the kernels nor the PGHs exhibited antibacterial activity against S. aureus and E. coli.
Various health benefits, including antimicrobial and antifungal properties, have been attributed to PSs (1, 2, 13). The antibacterial activity of PSs extracted from sources other than pistachio has been reported in several studies. For instance, the efficacy of β-sitosterol isolated from the chloroform extract of Parthenium hysterophorus against aquatic bacterial pathogens (11), avocado using the saponification method (12), P. abies using Soxhlet extraction and supercritical fluid extraction (13), methanol extract and solvent fractions from L. radiata (14), campesterol and its semi-synthetic derivatives (15), and stigmasterol isolated from the stem bark of Neocarya macrophylla (16) have been documented.
Ozcelik et al. investigated the antibacterial, antiviral, and antifungal activities of 15 lipophilic extracts obtained from different parts (branch, stem, leaf, kernel, hull, seeds) of P. vera and demonstrated limited antibacterial activity compared to its notable antifungal and antiviral activities (21). Smeriglio et al. found that 7.11 mg/mL of hull essential oil from P. vera Variety Bronte exhibited bactericidal effects (22). Bakhshi et al. synthesized copper nanoparticles (CuNPs) from hull essential oil of P. vera and reported their antibacterial properties (23).
To the best of our knowledge, this is the first report evaluating the antibacterial activities of total PSs extracted from the kernels and PGHs of P. vera Var. Damghan. It is also the first study to report the absence of antibacterial activity for total PSs. The choice of solvents for extraction significantly impacts the quantification and biological activity of extracted phytochemicals, including PSs (28).
In this study, total PSs extracted from the PGHs and kernels of three pistachio cultivars were analyzed, whereas previous studies have investigated either β-sitosterol (11-14), campesterol and its semi-synthetic derivatives (15), and stigmasterol (16) obtained from plants other than P. vera, or lipophilic extracts from different parts of P. vera (21). Other studies have shown bactericidal properties for hull essential oil from P. vera Variety Bronte (22) and biosynthesized CuNPs from pistachio hull essential oil (23). Further research is needed to isolate and identify extracts from P. vera with significant antimicrobial properties and to replicate existing findings on the antibacterial activity of PSs under comparable experimental conditions.
