3.2. Quantitative Measurement of Total Flavonoid and Total Phenolic Content
Polyphenolic compounds, including flavonoids, are well known for their potent antioxidant properties. Flavonoids, a diverse subclass of polyphenols, exhibit antioxidant activity by neutralizing free radicals through hydrogen or electron transfer, largely due to the presence of hydroxyl groups in their structure [24,25]. This antioxidant mechanism allows flavonoids to effectively scavenge reactive oxygen species, singlet oxygen, and various free radicals, which helps in preventing diseases associated with oxidative stress, such as cancer, inflammation, cardiovascular diseases, neurological disorders, and immune dysfunction [26,27,28]. Similarly, polyphenols play a crucial role in medicinal plants due to their hydroxyl-rich structures, enabling effective scavenging of free radicals and providing significant therapeutic potential [29,30,31].
Compared to other studies, our results revealed a higher total flavonoid and phenolic content in S. cumini seed extracts. Previous studies have reported lower flavonoid content in S. cumini aqueous seed extracts, with values such as 10.11 mg catechin equivalent (CE)/g of dried seed powder [32] and 44.1 mg quercetin equivalent (QE)/g of dried seed extract [33]. In a related study, S. cumini seeds were extracted using the three-phase partitioning (TPP) technique, which employs ammonium sulfate, water, and butanol, whereas the upper butanol and lower aqueous phase were collected to obtain extract. In this study, the total flavonoid content of obtained extract was 7.78 CE/g [34]. However, our methanol-based extraction yielded a significantly higher flavonoid content, suggesting that methanol may be more effective than aqueous extraction methods. A similar study reported 233.8 mg QE/g in a hydro-methanolic extract [25], which is higher than our findings. This variation could be attributed to differences in solvent composition and extraction methods [25]. In terms of phenolic content, in a similar study, S. cumini seeds were extracted using TPP technique, and the total phenolic content obtained was 82.66 GAE/g [34]. Similarly, another comparable study reported 100.07 mg GAE/g in an aqueous S. cumini seed extract; both are lower than our methanolic extract findings, further supporting the effectiveness of methanol in polyphenol extraction [32]. Conversely, another study recorded a much higher phenolic content of 415 mg GAE/g [33] for an aqueous extract, possibly due to differences in extraction methods, geographical factors, or the specific part of the plant used [28].
Our study highlights the efficacy of methanol as an extraction solvent for maximizing yields of bioactive flavonoids and phenolic compounds from S. cumini seeds. These compounds include quercetin, rutin, epicatechin, kaempferol, myricetin, apigenin, and catechin [25,32,33] as well as gallic acid, tannic acid, ellagic acid, chlorogenic acid, caffeic acid, p-coumaric acid, rubuphenol, tetra-decamethyl-cycloheptasiloxane, and dodecamethyl-cyclohexasiloxane, and ferulic acid [35]. These compounds are known for their antioxidant, anti-inflammatory, and disease-preventive properties [32]. By optimizing extraction processes, our findings emphasize the therapeutic potential of S. cumini seed extracts for applications in nutraceutical and pharmaceutical formulations. The significance of using both methanol and hexane lies in their complementary polarity profiles, allowing for the comprehensive extraction of both polar and non-polar compounds [11,19,21]. This approach enables a broader understanding of the phytochemical diversity in S. cumini seeds and facilitates the identification of solvent-specific extraction efficiencies, contributing to optimizing the extraction process for targeted applications.
3.3. Quantitative Measurement of Total Carbohydrate Content
Carbohydrates are categorized under the large subclass of primary metabolites, which are bountifully synthesized in the plant cells during the photosynthetic pathway, and these metabolites always serve as a key element of every plant cell. For every metabolic pathway occurring inside living human cells, carbohydrates serve several functions, such as providing an indispensable source of energy, stimulation of insulin hormone release, serving as an essential chemical entity of neurotransmitters, and regulating the optimal serotonin concentration in living cells [36,37]. In recent days, several polysaccharides isolated from medicinal plants have shown diverse decisive biological activities [38].
From the data of total carbohydrate evaluation (Table 1), it is quite evident that extraction solvent has a major role in the extraction of carbohydrates. Methanol, a polar solvent, demonstrated significantly higher efficiency than the non-polar hexane (p < 0.05), highlighting substantial differences between the two extracts. These findings align with prior studies documenting the efficacy of polar solvents in isolating carbohydrates [27,36]. Interestingly, our findings differ from a previous study in India, which reported 89.68% carbohydrates in S. cumini seeds [39]. However, the methodology and sample state used in that study were not specified. Variations in carbohydrate content may be attributed to differences in extraction methods, sample preparation, or specific part of the seed analyzed [40]. For example, whole seeds versus seed kernel extracts may yield varying carbohydrate concentrations due to the presence of structural carbohydrates in seed coats or non-extractable bound forms [41]. Additionally, regional and environmental factors such as soil composition, climate, and agricultural practices can significantly influence the biochemical profile of S. cumini seeds [40].
Determining carbohydrate content is crucial for understanding the nutritional and therapeutic potential of S. cumini [39]. The carbohydrate-rich methanolic extract of S. cumini seed warrants further investigation to isolate and characterize bioactive polysaccharides and explore their pharmaceutical applications [42].
3.4. Quantitative Measurement of Antioxidant Activity by Utilizing the DPPH Free Radical Inhibition Method
The antioxidant activity of S. cumini seed kernel extracts is attributed to polyphenolic and flavonoid compounds, which neutralize free radicals via hydrogen donation and electron transfer [24]. In this study, when hexane and methanolic extracts were added to ethanolic DPPH solution, a decrease in absorbance was observed, which indicated DPPH radical reduction. The reduction in absorbance was then quantitatively measured to assess antioxidant efficacy [25]. The methanolic extract’s superior IC50 value highlights the role of polar polyphenol and flavonoid content, likely driving its enhanced antioxidant activity. Bioactive compounds in S. cumini—such as gallic acid, ellagic acid, catechin, epicatechin, ferulic acid, gallotannins, myricetin, quercetin, and kaempferol—are potent antioxidants that stabilize free radicals by donating electrons or hydrogen atoms, preventing cellular damage [25,43].
In a previous study conducted in India, the ethanolic seed extract of S. cumini demonstrated IC50 values of 8.6 μg/mL [43] and 14.0 μg/mL [44], while the hydromethanolic extract (70% methanol) exhibited an even lower IC50 of 5.1 μg/mL [28], indicating a higher antioxidant potential compared to our methanolic extract. Conversely, aqueous extracts reported IC50 values of 35.4 μg/mL [33] and 10.59 μg/mL [32], and the TPP extract presented an IC50 value of 12.15 μg/mL [34]. All those experiments were conducted by DPPH free radical scavenging method. These higher IC50 values indicate that the aqueous and TPP extracts possess lower antioxidant activity than our methanolic extract, further emphasizing the critical role of solvent choice in determining extraction efficiency and antioxidant capacity. Variations in IC50 values across studies may stem from differences in extraction methods and sample sources [15,32]
Despite the availability of studies on ethanolic, aqueous, hydromethanolic, and TPP solvents, very few studies have utilized methanol to extract S. cumini seeds. Therefore, we selected methanol as a polar solvent for its well-established efficiency in extracting phenolic and flavonoid compounds, and the results demonstrated considerable antioxidant activity. However, when compared to previous studies [28], our findings suggest that combining methanol with varying proportions of water may enhance the extraction of antioxidant constituents. This warrants further investigation to identify the optimal solvent system for maximizing the antioxidant potential of S. cumini seed extracts.
3.5. Evaluation of Xanthine Oxidase-Inhibitory Effect of S. cumini Seed Kernel Extracts
Xanthine oxidase (XO) is a widely distributed enzyme participating in the conversion of purine bases, and its excessive production is associated with gout development [45,46]. The treatment of gout primarily focuses on increasing the elimination of uric acid or reducing its production [47,48,49]. Drugs molecules such as allopurinol, uricosuric agents, corticosteroids, and NSAIDs are commonly used for gout management. Uricase enzymes offer potential advantages but face obstacles due to antibody susceptibility [50,51]. Xanthine oxidase (XO) inhibitors are increasingly attractive due to their reduced side effect profile compared to alternative agents. Allopurinol stands out as the primary XO inhibitor in clinical use despite its related adverse effects. Seeking other antigout treatments without undesirable effects, researchers have explored various plant species known for their traditional use in treating gout and arthritis [52,53].
Our investigation about the XO-inhibitory effect of S. cumini seed revealed the significant suppressive impact of the methanol-derived extract on xanthine oxidase inhibition. Extensive studies have highlighted the remarkable in vitro and in vivo xanthine oxidase-inhibitory effect of flavonoids and polyphenols such as quercetin [54], ferulic acid [55], kampferol [56], gallic acid [57], ellagic acid [58], epicatechin [59], caffeic acid [60], p-coumaric acid [61], and chlorogenic acid [62], which are abundantly present in S. cumini seeds [35]. Polyphenols and flavonoids inhibit xanthine oxidase (XO) through structural features that allow strong interactions with the enzyme’s active site. For polyphenols, the presence of multiple hydroxyl groups enables them to form hydrogen bonds with active-site amino acids, while their ability to chelate the molybdenum cofactor in XO enhances their inhibitory activity. Polyphenols with catechol or galloyl groups are particularly potent due to their capacity to disrupt the electron transfer in XO’s catalytic cycle. Similarly, flavonoids exhibit inhibitory effects due to a planar structure, such as a flavone backbone, and hydroxyl groups at specific positions, like C3, C5, and C7, which improve binding through hydrogen bonding and π–π interactions. These structural features collectively allow polyphenols and flavonoids to reduce XO activity, lowering uric acid production and oxidative stress [47].
This study is the first to report the xanthine oxidase-inhibitory activity of S. cumini seed kernel. In contrast, previous research has documented moderate XO-inhibitory activity in the methanolic extract of S. cumini leaves [63]. Differences in extraction conditions and plant parts can significantly influence phytochemical composition and, consequently, biological activity. Previous studies have demonstrated notable variability in the distribution and concentration of phenolics, flavonoids, and other secondary metabolites among different parts of S. cumini [15,35]. In comparison to the leaf, the seed kernel is rich in ferulic acid [35], ellagic acid [35] and gallotannins [64], which are bioactive compounds with strong XO-inhibitory activity [55,65]. Moreover, bioassay-guided extensive research should be conducted in different animal models to establish a scientific connection between the XO-inhibitory effect of S. cumini seed extract and its antigout effect.
3.6. Antibacterial Potency of S. cumini Seed Kernel Extracts
In this study, hexane and methanolic extracts of S. cumini seed kernel (1.5 mg dry extract/disc) were tested against eight pathogenic bacterial strains to evaluate their broad-spectrum antibacterial potential. The selected strains included Gram-positive bacteria (S. aureus, S. pneumoniae, B. cereus, and S. epidermidis) associated with respiratory, skin, and foodborne infections and Gram-negative bacteria (E. coli, S. enteritidis, P. aeruginosa, and K. pneumoniae) commonly linked to urinary, gastrointestinal, and hospital-acquired infections [66,67,68]. Antibacterial activity was quantified by measuring the inhibition zones using a Vernier caliper. As shown in Table 2, both extracts demonstrated greater efficacy against Gram-positive bacteria compared to Gram-negative strains. Usually, plant extracts exhibit more suppressive action against Gram-positive bacteria than Gram-negative strains due to availability of drug-impenetrable lipopolysaccharide architecture in the multi-layered cell wall in the Gram-negative bacteria, which is absent in Gram-positive strains. Additionally, Gram-positive bacteria possess a peptidoglycan layer that exhibits a mesh-like structure, making it more susceptible to the penetration of extracts [68,69].
The present study demonstrated that the methanolic extract of S. cumini seed kernel exhibited significant antibacterial activity, particularly against S. epidermidis (zone of inhibition, ZOI: 19 mm), exceeding the activity of the standard antibiotic gentamicin (ZOI: 18.66 mm), whereas the same extract was moderately active against all examined Gram-negative bacteria, with the highest ZOI (18.33 mm) against K. pneumonia (ciprofloxacin; 33.66 mm). These findings align with previous reports on the antibacterial efficacy of S. cumini seed extracts. For instance, an Indian study noted the moderate activity of ethanolic extracts against S. aureus (ZOI: 8 mm) and E. coli (ZOI: 9 mm), although the extract concentration was unspecified [70]. Similarly, methanolic extracts in another study exhibited greater inhibition against Bacillus subtilis (ZOI: 20.06 mm), albeit at a higher concentration (7.5 mg per well) [71]. Additionally, a single, unidentified compound isolated from the ethyl acetate fraction of S. cumini seed methanolic extract displayed ZOIs of 20 mm, 11 mm, 17 mm, and 15 mm against E. coli, P. aeruginosa, S. aureus, and B. subtilis, respectively, using the agar well diffusion method [72]. A study from Dhaka, Bangladesh, reported moderate antibacterial activity of the methanolic extract against S. aureus (ZOI: 10 mm) and E. coli (ZOI: 10 mm) [73]. Likewise, an aqueous extract containing 22.59 mg GAE/g exhibited a ZOI of 24.5 mm against S. aureus but showed no activity against E. coli and P. aeruginosa [74]. In another study in Nepal, ethanolic extracts demonstrated similar activity against Gram-negative K. pneumoniae (ZOI: 20 mm) and Gram-positive S. aureus (ZOI: 19 mm) [75]. An additional study from Tamil Nadu, India, using 800 µg of ethanolic extract per 6 mm well, reported ZOIs of 26, 20, 19, 17, and 17 mm against P. aeruginosa, K. pneumoniae, Bacillus cereus, E. coli, and S. aureus, respectively, which were significantly higher than the inhibition zones observed in our study [76].
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values obtained in this study further underscore the broad-spectrum efficacy of methanolic extracts. The MIC value of 0.32 mg/mL against Gram-positive S. epidermidis and 0.52 mg/mL against Gram-negative K. pneumoniae highlights its potent antibacterial potential. These findings are consistent with previous studies from India and Brazil, which reported MIC values of 0.154 mg/mL to 0.648 mg/mL against S. epidermidis and S. aureus, respectively [77,78,79]. In contrast, an aqueous extract exhibited much higher MIC values of 1.41 mg/mL against S. aureus and 11.29 mg/mL against E. coli [74]. Interestingly, a study in Tamil Nadu, India, reported strikingly low MIC values for ethanolic seed extracts against B. cereus (<7.8 µg/mL), P. aeruginosa (15.6 µg/mL), K. pneumoniae (125 µg/mL), and S. aureus (125 µg/mL) [74]. These unusually low MIC values appear inconsistent with typical trends observed for plant extracts and warrant further investigation.
Overall, these discrepancies in antibacterial activity among different studies may be attributed to differences in the altitude of plant collection [80], experimental variation [81], extraction solvents [21], and extract concentrations used in prior studies, which were often unspecified. Our findings suggest that methanol might extract antibacterial compounds from S. cumini seeds more efficiently, as indicated by the comparative data. The prominent zones of inhibition exhibited by the unconfirmed isolated compound from the ethyl acetate fraction of the ethanolic extract in previous study [72] indicate that polar and semi-polar phytochemicals, which are efficiently extracted using ethanol and ethyl acetate [72], contribute to the significant inhibitory effect observed in the methanolic extract in this study. This hypothesis is consistent with the observation that methanol-based extractions consistently outperformed hexane in both ZOI and MIC/MBC analyses in our research. Moreover, numerous studies have demonstrated the potent antibacterial effects of flavonoids and polyphenols, both in vitro and in vivo. Key compounds exhibiting this activity include quercetin [82], ferulic acid [83], kaempferol [84], gallic acid [85], ellagic acid [86], epicatechin [87], caffeic acid [88], p-coumaric acid [89], and chlorogenic acid [90], all of which are abundantly found in S. cumini seeds [37]. The hexane extract, although less effective, exhibited measurable activity, particularly against E. coli (ZOI: 9 mm). This aligns with earlier findings that non-polar extracts, while generally less potent, may contain bioactive lipophilic compounds contributing to antimicrobial effects [70,72]. Importantly, the comparatively lower efficacy of hexane extract in our study highlights the importance of solvent selection in maximizing extract potency.
The novelty of our study lies in the comprehensive evaluation of MIC and MBC values across a wide range of pathogenic strains, including B. cereus, S. pneumonia, and S. enteritidis, which were not previously investigated. Notably, our study is the first to comprehensively evaluate the bactericidal activity of S. cumini hexane and methanol seed extract against both Gram-positive and Gram-negative pathogenic bacterial strains, advancing the understanding of S. cumini seed kernel’s bactericidal potential. Therefore, these findings not only expand the spectrum of antibacterial activity reported for S. cumini seed kernel extracts but also underscore their potential application as natural antibacterial agents. The methanolic extract’s consistent potency against both Gram-positive and Gram-negative bacteria suggests its potential for use in combating antibiotic-resistant pathogens. Further studies focusing on the isolation and characterization of active compounds are warranted to better understand the mechanisms underlying these effects and to develop standardized formulations for therapeutic applications.
In summary, significant differences in antibacterial, antioxidant, and xanthine oxidase-inhibitory activities between the methanolic and hexane extracts of S. cumini seeds can be attributed to the distinct phytochemical profiles of the extracts. The methanolic extract, rich in bioactive compounds such as polyphenols, flavonoids, and tannins, exhibits potent antimicrobial properties by disrupting bacterial cell walls and inhibiting enzymatic functions. In contrast, the hexane extract, with a lower yield, predominantly contains lipophilic compounds with limited bioactivity. These results underscore the effectiveness of polar solvents like methanol in extracting therapeutically relevant compounds, emphasizing the critical role of solvent selection in optimizing extraction processes for drug discovery research.
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Jitendra Pandey www.mdpi.com
