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Article

An In Vitro Evaluation of Robin’s Pincushion Extract as a Novel Bioactive-Based Antistaphylococcal Agent—Comparison to Rosehip and Black Rosehip

by
Olja Šovljanski
1,
Milica Aćimović
2,
Teodora Cvanić
1,
Vanja Travičić
1,
Aleksandra Popović
3,
Jelena Vulić
1,
Gordana Ćetković
1,
Aleksandra Ranitović
1,* and
Ana Tomić
1,*
1
Faculty of Technology Novi Sad, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
2
Institute of Field and Vegetable Crops Novi Sad, Maksima Gorkog 30, 21000 Novi Sad, Serbia
3
Faculty of Agriculture Novi Sad, University of Novi Sad, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia
*
Authors to whom correspondence should be addressed.
Antibiotics 2024, 13(12), 1178; https://doi.org/10.3390/antibiotics13121178
Submission received: 15 November 2024 / Revised: 2 December 2024 / Accepted: 3 December 2024 / Published: 4 December 2024
(This article belongs to the Special Issue Advances in the Discovery of Novel Antimicrobial Agents in Nature and Their Applications, 2nd edition)
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Abstract

:
Introduction: This study explores the bioactive properties of extracts obtained from Robin’s pincushion (Diplolepis rosae) collected in Sokobanja, Serbia. Results: Comprehensive in vitro assessments reveal high concentrations of total phenolics (186.37 mg GAE/g), along with significant levels of carotenoids (44.10 μg β-car/g). Robin’s pincushion exhibited superior antioxidant capacities across DPPH, ABTS, and reducing power assays, significantly outperforming comparable extracts from rosehip (Rosa canina) and black rosehip (Rosa spinosissima) in these activities. Additionally, high inhibitory effects were observed in antimicrobial assays, with the extract demonstrating minimal inhibitory concentrations (MIC) as low as 1.56 mg/mL against the Staphylococcus species. Notably, the extract achieved full bactericidal effect within 24 h in time-kill kinetic studies which additionally highlight its potent antistaphylococcal potential. Materials and methods: Analyzing their phytochemical profiles and evaluating their potential as antioxidant, anti-inflammatory, antihyperglycemic, and antimicrobial agents, wide-ranging evaluation of bioactivity of Robin’s pincushion was conducted. Conclusions: These findings highlight Robin’s pincushion as a promising natural source of bioactive compounds with potential applications in traditional and modern medicine for managing oxidative stress, inflammation, hyperglycemia, and microbial infections.

1. Introduction

Ethnopharmacological knowledge offers potential for the development of new pharmaceutical products [1]. This type of knowledge has been passed on in Serbia for a very long time, especially in rural areas of mountain regions [2,3,4,5,6,7,8,9]. Dog rose (Rosa canina L., Rosaceae) is mainly used as rosehip (Cynosbati fructus), a rich source of vitamin C, which is a long tradition spread worldwide. Apart from vitamin C, it contains other water-soluble vitamins such as K and B group (B1, B2 and B3), sugars, organic acids, pectins, flavonoids, tannins, carotenoids, fatty acids, macro- and microelements [10]. In Serbia, rosehip is used fresh as fruit for preparing juices, marmalade, and jams, while dried rosehip is used as favorable herbal tea in everyday life, as well as to prevent and treat colds and influenza [11]. According to numerous studies, rosehip extract expressed antioxidant, antimicrobial, antihyperglycemic, antihyperlipidemic and anticancer activities [12,13,14,15,16,17,18]. Due to its bioactivity, rosehip can be considered as a potential functional food as well as valuable raw material for the pharmaceutical industry [19].
In addition to rosehip, the edible fruits of the Burnet rose or Scotch rose (Rosa spinosissima L., syn. R. pimpinellifolia L.), commonly referred to as black rosehip, have been recognized since ancient times for their medicinal properties. These fruits have been traditionally used to treat respiratory and inflammatory conditions, as well as for their health-promoting benefits [20,21]. Today, this species is rarely used for medicinal or culinary purposes, despite its widespread distribution across a range of climates, from cold to temperate and continental regions of Europe and Siberia [22,23]. However, its distinctive phytochemical profile, rich in polyphenolic compounds, anthocyanins, carotenoids, and vitamins, positions it as an exceptional raw material with significant industrial potential for the development of nutraceutical and pharmaceutical products [21,24].
However, pests and diseases can reduce the quality and quantity of rosehip [25]. For example, as a response to damage induced by insects called rose gall wasp (Diplolepis rosae L., Hymenoptera: Cynipidae), plant galls develop, characterized by abnormal growths and deformed flowers [26]. In fact, overwinter females of gall wasps come out from galls in early spring and lay eggs on new flowers or leaf buds [27,28]. During development, insects produce multi-chambered galls, which look like bulges covered with thick yellow-green, chartreuse hair and can be found in sizes from 5 mm to 8 cm [29,30]. This species is particularly abundant in mountainous areas, often found near forests [31]. These formations are known as Robin’s pincushion or rose bedeguar gall. These galls are phytochemically distinct from the normal plant tissues, and over time humans have learned to use galls as therapeutics [26]. In Bulgarian traditional medicine, a decoction of Robin’s pincushion in water is used as an antitussive and anti-asthmatic remedy [32]. On the Iberian Peninsula, Robin’s pincushion is used as an eyewash for ocular inflammations, a mouthwash for gargling and toothache, and a tisane for treating kidney inflammation diseases [33].
There are no recorded written traces of Robin’s pincushion in traditional medicine in Serbia. However, local people from the Sokobanja region use this drug to treat inflammatory processes of the skin (called “ružine šišarke” in Serbian, literally translating to “rose cones” in English). Therefore, in this study, Robin’s pincushion, rosehip and black rosehip collected from the Sokobanja region (eastern part of Serbia), was analyzed to determine the content of total phenolics and carotenoids. Additionally, in vitro evaluation of antioxidant, anti-inflammatory, antihyperglycemic and antimicrobial activities was conducted, in order to provide scientific support for Robin’s pincushion’s application in traditional medicine and to compare obtained results with rosehip and black rosehip, whose flowers and fruits are parasitized by the rose gall wasp.

2. Results

2.1. The Phytochemical Composition and In Vitro Antioxidant Activity

The phytochemical analysis and antioxidant activity assays conducted on Robin’s pincushion, black rosehip, and rosehip extracts are summarized in Table 1. The results highlight Robin’s pincushion as an exceptionally rich source of bioactive compounds compared to the other tested extracts. Namely, the total phenolic content (TPh) was markedly higher in Robin’s pincushion extract, with a concentration of 186.37 mg GAE/g, compared to 9.65 mg GAE/g in rosehip and 7.24 mg GAE/g in black rosehip. These results indicate that Robin’s pincushion contains an abundance of phenolic compounds, which are recognized for their potent antioxidant properties. In contrast, rosehip and black rosehip exhibited significantly lower TPh values, suggesting comparatively reduced phenolic content. In terms of total carotenoid content (TCar), rosehip displayed the highest concentration at 62.24 μg β-car/g, followed by Robin’s pincushion with 44.10 μg β-car/g. Black rosehip contained only 8.10 μg β-car/g, indicating lower carotenoid levels. Despite rosehip having the highest carotenoid concentration, the combined phenolic and carotenoid profile of Robin’s pincushion suggests a distinct bioactive composition that may contribute to its potent antioxidant activity.
The antioxidant potential of the extracts was assessed through DPPH, ABTS, and reducing power (RP) assays, each providing insights into different mechanisms of antioxidant action. As can be seen in Table 1, Robin’s pincushion exhibited a DPPH radical scavenging activity of 152.07 mM TEAC/100 g, significantly outperforming rosehip and black rosehip, which showed values of 21.89 and 3.22 mM TEAC/100 g, respectively. The low IC50 value of the standard (Trolox at 0.14 ± 0.01 mM) underscores the strong radical scavenging ability of Robin’s pincushion, suggesting a high free radical neutralization capacity. In the ABTS assay, Robin’s pincushion again demonstrated superior activity, with an antioxidant capacity of 636.67 mM TEAC/100 g, compared to rosehip at 70.17 mM TEAC/100 g and black rosehip at 13.55 mM TEAC/100 g. The reference standard, Trolox, showed a significantly lower activity level (1.06 ± 0.04 mM TEAC), further emphasizing the high antioxidant potency of Robin’s pincushion. The reducing power of Robin’s pincushion extract was measured at 107.84 mM TEAC/100 mL, surpassing both rosehip (25.24 mM TEAC/100 mL) and black rosehip (3.4 mM TEAC/100 mL). This elevated RP value suggests that Robin’s pincushion has a strong electron-donating capacity, contributing to its overall antioxidant profile.

2.2. The HPLC Analyses of Polyphenolics

The High-Performance Liquid Chromatography (HPLC) analysis of polyphenolic compounds in Robin’s pincushion, black rosehip, and rosehip extracts identified substantial variations in phenolic profiles, as shown in Table 2. All chromatographs are presented in the Supplementary Figure S1. This analysis highlights Robin’s pincushion as a particularly rich source of polyphenols compared to the other extracts. Robin’s pincushion exhibited the highest concentration of p-hydroxybenzoic acid at 250.98 mg/100 g dw, significantly surpassing both rosehip (62.56 mg/100 g dw) and black rosehip (57.67 mg/100 g dw). This compound is known for its antioxidant properties, and its abundance in Robin’s pincushion suggests a strong potential for free radical scavenging. The presence of gallic acid was notable in Robin’s pincushion, with a concentration of 297.95 mg/100 g dw, while it was undetected in rosehip and present only at 16.89 mg/100 g dw in black rosehip. Known for its antimicrobial and antioxidant effects, gallic acid contributes to the enhanced bioactivity observed in Robin’s pincushion.
Robin’s pincushion contained a high level of protocatechuic acid (677.37 mg/100 g dw), compared to 15.96 mg/100 g dw in rosehip and 32.49 mg/100 g dw in black rosehip. This phenolic acid is recognized for its antioxidant and anti-inflammatory properties, further underscoring the superior bioactivity of Robin’s pincushion extract. Robin’s pincushion demonstrated an exceptionally high ellagic acid content of 1066.02 mg/100 g dw, whereas it was undetected in rosehip and observed at a lower concentration of 223.47 mg/100 g dw in black rosehip. Ellagic acid is a potent antioxidant and has been associated with anti-cancer properties, highlighting the therapeutic potential of Robin’s pincushion. This phenolic compound was found exclusively in Robin’s pincushion at 272.00 mg/100 g dw, while it was undetected in both rosehip and black rosehip extracts. Syringic acid has antioxidant and anti-inflammatory effects, contributing to the distinct bioactive profile of Robin’s pincushion. Both Robin’s pincushion and black rosehip contained high levels of vanillic acid, with concentrations of 392.03 mg/100 g dw and 404.19 mg/100 g dw, respectively, whereas rosehip contained only 21.12 mg/100 g dw. Vanillic acid is known for its antioxidant and antimicrobial properties, supporting the potential use of Robin’s pincushion in bioactive applications. Detected only in rosehip at a minimal concentration of 3.04 mg/100 g dw, ferulic acid was absent in both Robin’s pincushion and black rosehip. This low presence suggests that ferulic acid may not significantly contribute to the bioactivity of Robin’s pincushion. The total polyphenolic content in Robin’s pincushion extract reached 2956.35 mg/100 g dw, which is substantially higher than in black rosehip (754.23 mg/100 g dw) and rosehip (102.67 mg/100 g dw). This pronounced difference underscores the richness of Robin’s pincushion in bioactive polyphenols, which likely accounts for its superior antioxidant activity demonstrated in previous analyses.

2.3. Anti-Inflammatory and Antihyperglycemic Activities

The anti-inflammatory and antihyperglycemic properties of Robin’s pincushion, black rosehip, and rosehip extracts were evaluated, with results displayed in Table 3. These analyses highlight the potential therapeutic applications of Robin’s pincushion, especially in managing inflammation and hyperglycemia, as compared to the other tested extracts.
The anti-inflammatory activity (AIA) was measured as a percentage of inhibition, with Robin’s pincushion showing an AIA of 29.03%. This level is comparable to rosehip, which demonstrated an inhibition rate of 28.54%, but somewhat lower than black rosehip, which exhibited the highest anti-inflammatory activity at 46.31%. Despite this, Robin’s pincushion demonstrated considerably higher activity than the reference standard, acarbose, which showed an inhibition of only 1.14%. The relatively strong anti-inflammatory potential of Robin’s pincushion suggests its suitability as a natural agent for inflammation management, although black rosehip exhibited the highest efficacy among the extracts tested. In terms of antihyperglycemic activity (AHgA), Robin’s pincushion extract showed a remarkable inhibition rate of 96.30%, far surpassing both rosehip (31.67%) and black rosehip (27.84%). This inhibition rate is significantly higher than that of the standard, diclofenac sodium, which demonstrated only 0.001% inhibition. The high antihyperglycemic activity observed in Robin’s pincushion suggests its potential as a powerful natural agent for blood glucose regulation and a promising candidate for managing conditions like diabetes. This superior antihyperglycemic performance may be attributed to its high phenolic content, as polyphenols are known to positively influence glucose metabolism.

2.4. Antimicrobial Assessment—Determination of Inhibition Zones and Minimal Inhibitory Concentrations

The antimicrobial potential of Robin’s pincushion, black rosehip, and rosehip extracts was evaluated against several Staphylococcus species using the disk-diffusion method, with ceftazidime (10 µg/disc) as the reference standard (Table 4). Across all tested strains, Robin’s pincushion demonstrated substantial antimicrobial efficacy, often outperforming both rosehip and black rosehip extracts. Against S. aureus, Robin’s pincushion showed a prominent inhibition zone of 29.33 mm, significantly larger than those for rosehip (7.00 mm) and black rosehip (10.5 mm) and surpassing the 25 mm inhibition zone of ceftazidime. This suggests that Robin’s pincushion is highly effective against this bacterial strain.
For S. saprophyticus, the inhibition zone for Robin’s pincushion measured 24.00 mm, on par with the ceftazidime standard and larger than rosehip (7.00 mm) and black rosehip (14.00 mm). This finding highlights the comparable efficacy of Robin’s pincushion to conventional antibiotics in this context. In the case of S. sciuri, Robin’s pincushion displayed a large inhibition zone of 31.00 mm, far exceeding rosehip (7.00 mm) and black rosehip (18.00 mm) and approaching the ceftazidime zone of 28 mm, suggesting strong antimicrobial properties against this species. The efficacy of Robin’s pincushion was also observed against S. epidermidis, with an inhibition zone of 26.33 mm, matching that of ceftazidime (26 mm) and substantially higher than rosehip (7.00 mm) and black rosehip (16.5 mm). Finally, against S. warneri, Robin’s pincushion produced an inhibition zone of 29.00 mm, significantly larger than those of rosehip (7.00 mm) and black rosehip (12.00 mm), and nearly equal to ceftazidime (28 mm).
The antimicrobial efficacy of Robin’s pincushion, black rosehip, and rosehip extracts was further evaluated through minimal inhibitory concentration (MIC) assays against various Staphylococcus strains, with MIC values expressed in mg/mL (Table 5). These results underscore the potent inhibitory effects of Robin’s pincushion in comparison to black rosehip and rosehip, particularly against multiple Staphylococcus species.
For S. aureus, Robin’s pincushion exhibited a low MIC of 1.56 mg/mL, indicating strong bacteriostatic activity. In contrast, both rosehip and black rosehip extracts had MIC values greater than 50 mg/mL, suggesting considerably weaker or ineffective inhibitory capacity at tested concentrations. Against S. saprophyticus, Robin’s pincushion maintained a MIC of 1.56 mg/mL, while black rosehip and rosehip showed no inhibition below 50 mg/mL. This trend continued with S. sciuri, where Robin’s pincushion demonstrated a MIC of 3.125 mg/mL, considerably lower than black rosehip (25 mg/mL), highlighting its enhanced antimicrobial potential. Robin’s pincushion also inhibited S. epidermidis with a MIC of 12.5 mg/mL, outperforming black rosehip (25 mg/mL) and showing greater efficacy than rosehip. The lowest MIC values for Robin’s pincushion were consistently observed for S. warneri, with a MIC of 1.56 mg/mL, while rosehip and black rosehip were ineffective at the highest tested concentration of 50 mg/mL.

2.5. Pharmacodinamic Potential—Time-Kill Kinetics Study of Antimicrobial Effect

The pharmacodynamics potential or time-kill kinetics has been established to clarify the in vitro antimicrobial capacity of Robin’s pincushion. The time-kill kinetic study of Robin’s pincushion extract against various Staphylococcus strains provides insight into the bactericidal effects of the extract at its MIC concentration over a 48 h period (Table 6). This study demonstrates the rapid and effective action of Robin’s pincushion extract, with all tested Staphylococcus strains showing a substantial reduction in bacterial count within the initial hours, ultimately leading to complete bacterial eradication within 24 to 48 h.
For S. aureus, the bacterial count decreased from an initial concentration of 6.1 log CFU/mL to 4.9 log CFU/mL within 2 h. By the 6 h mark, the count had reduced further to 2.2 log CFU/mL, with a near-total reduction to 0.5 log CFU/mL by 12 h. Complete eradication (0.0 log CFU/mL) was observed at 18 h and maintained through the 48 h period, demonstrating the potent bactericidal effect of the extract. In the case of S. saprophyticus, the initial bacterial count of 6 log CFU/mL decreased gradually, reaching 3.6 log CFU/mL at 12 h and 1.3 log CFU/mL at 18 h, with complete elimination observed at 24 h. This gradual reduction followed by complete eradication indicates the effectiveness of the extract over an extended period, even with an initially slower decline. In the case of S. sciuri, an initial bacterial load of 6 log CFU/mL dropped to 5.7 log CFU/mL at 2 h, with a rapid decrease to 1.0 log CFU/mL by 12 h. Complete eradication was achieved by 18 h, showcasing the swift action of the extract in reducing bacterial viability in this strain. S. epidermidis showed a more gradual decrease, with bacterial counts dropping from 6.1 log CFU/mL to 5.8 log CFU/mL at 2 h and 2.7 log CFU/mL at 12 h. By 36 h, all bacterial counts reached 0.0 log CFU/mL, demonstrating the slower but ultimately effective action of the extract on this strain. For S. warneri, a rapid decline in bacterial count was observed, with an initial concentration of 5.9 log CFU/mL reduced to 4.1 log CFU/mL at 2 h and 1.7 log CFU/mL at 6 h. Complete elimination was achieved by 12 h, indicating a particularly effective bactericidal effect against this strain.
Figure 1 presents the time-kill kinetics of Robin’s pincushion extract against various Staphylococcus strains, with each subfigure illustrating the bacterial concentration (log CFU/mL) over time. The experimental data points (dots) align closely with the fitted kinetic model (lines), with kinetic parameters provided in Table 7.
In view of effect on S. aureus (Figure 1a), the extract demonstrated a rapid bactericidal effect, with the bacterial concentration dropping significantly within the first few hours and reaching complete eradication by 24 h. This swift reduction, as indicated by the kinetic parameters, highlights the potent antibacterial activity of Robin’s pincushion against S. aureus. For S. saprophyticus (Figure 1b), the reduction in bacterial concentration was more gradual, with a complete kill observed around the 48 h mark. The kinetic parameters for S. saprophyticus reflect a slower bactericidal action compared to S. aureus and S. sciuri. Similarly, S. sciuri (Figure 1c) showed a comparable rapid decline in bacterial load, with complete inhibition achieved within 24 h, suggesting a high rate constant and strong antibacterial efficacy against this strain as well. S. epidermidis (Figure 1d) also exhibited a moderate kill rate, with bacterial counts decreasing steadily over 36 h before reaching complete inhibition. The kinetic model for this strain indicates an intermediate rate constant, signifying moderate efficacy of the extract. S. warneri (Figure 1e) demonstrated rapid bacterial elimination similar to S. aureus and S. sciuri, with a swift decrease in bacterial load leading to complete eradication by 24 h. The kinetic parameters indicate a high rate constant for S. warneri, supporting the potent antimicrobial activity of Robin’s pincushion against this strain.
Table 7 provides an overview of the kinetic parameters and the “goodness of fit” for the kinetic models assessing the effect of Robin’s pincushion extract’s MIC values on different Staphylococcus strains. The kinetic model parameters, represented by the initial bacterial concentration (A) and the kill rate coefficient (k), along with verification parameters, indicate the model’s accuracy in capturing bacterial reduction over time.
The A values were similar across most strains, ranging from 5.76 log CFU/mL in S. saprophyticus to 6.52 log CFU/mL in S. sciuri. These values provide a baseline measure of the bacterial load prior to the bactericidal action of the extract. The k values, which reflects the rate of bacterial reduction, varied more significantly among the strains. S. warneri demonstrated the highest kill rate (0.19), followed closely by S. sciuri (0.17) and S. aureus (0.18), indicating rapid bacterial decline. In contrast, S. saprophyticus and S. epidermidis exhibited lower kill rate coefficients (0.08), suggesting a slower bactericidal response to the extract. The model’s fit accuracy was assessed using several statistical parameters. The reduced chi-square (χ2) values indicate the degree of alignment between observed and predicted data, with lower values signifying better fits. S. aureus exhibited the lowest χ2 value (0.119), indicating a highly accurate model fit, while S. sciuri had the highest χ2 (16.306), suggesting a weaker model fit for this strain. The root mean square error (RMSE) values further illustrate the model’s precision. S. aureus had the lowest RMSE (0.102), indicating minimal deviation between observed and predicted values. S. saprophyticus and S. sciuri, with higher RMSE values (0.530 and 0.358, respectively), reflect greater deviations in these model fits. Mean bias error (MBE) and mean percentage error (MPE) measure the model’s bias and average percentage deviation. Most strains had small MBE values, indicating minimal systematic error, though S. sciuri showed a notable negative MBE (−0.101), indicating a slight underestimation in model predictions. The MPE values were generally low, though S. sciuri had the highest negative MPE (−18.410), reflecting the model’s tendency to underestimate values for this strain. The coefficient of determination (r2), representing the proportion of variance explained by the model, was high across all strains, indicating a strong fit. Staphylococcus aureus showed the highest r2 (0.998), followed by S. epidermidis (0.992) and S. warneri (0.989), confirming excellent model fits. S. saprophyticus had a relatively lower r2 (0.939), indicating a less accurate fit compared to the other strains. Additional parameters, including skewness and kurtosis, provide insight into the distribution characteristics of model errors. Most strains exhibited slight positive skewness, indicating a tendency toward higher values, with S. saprophyticus showing near-zero skewness (−0.002), suggesting symmetry. Kurtosis values were negative for all strains, indicating platykurtic distributions with fewer extreme deviations than a normal distribution. The mean, standard deviation (SD), and variance of model predictions offer further context for error distribution. S. epidermidis had the highest mean prediction (2.822), indicating a higher central tendency, while S. warneri had the lowest mean (1.689). Variance values were relatively similar across strains, with S. sciuri showing the highest variance (5.384), suggesting more variability in model errors for this strain.

3. Discussion

The current study presents insight into the bioactive potential of Robin’s pincushion (Diplolepis rosae) extract, emphasizing applicability as natural agents in managing oxidative stress, staphylococcal-based infections, hyperglycemia, and inflammation. According to the obtained findings, the significant differences in bioactivity between Robin’s pincushion and traditional Rosa canina derivatives, including rosehip and black rosehip, highlights Robin’s pincushion as a particularly potent source of phytochemicals with strong therapeutic potential. Additionally, the examination of biological activities of common parts of the plants has been exhausted, and recently, galls or cecidia have become an interesting source with various biological activities. This is especially interesting because galling induces the host plant to secrete a wide range of phytochemicals, which in normal conditions would not happen, and it could be referred to metabolic re-routing of the plants. Taking into account that galls are usually called physiologic sinks due to the richness of secondary metabolites, it is not odd why investigating the biological activities of galls has attracted such great scientific attention. Some galls have been used as traditional medicines, especially galls from Rhus, Pistacia, Quercus, and Terminalia [26].
In this study, the phytochemical profile of Robin’s pincushion revealed high levels of phenolic compounds, measured at 186.37 mg GAE/g and 82.01 mg RE/g, respectively, as well as substantial quantities of carotenoids (44.10 μg β-car/g). These values notably exceed those found in rosehip and black rosehip, which exhibited comparatively lower concentrations. In antioxidant assays, Robin’s pincushion outperformed both rosehip and black rosehip across DPPH, ABTS, and reducing power evaluations, suggesting a robust ability to neutralize free radicals and prevent oxidative damage. The DPPH and ABTS radical scavenging activities, which were markedly higher for Robin’s pincushion, underscore its potential as a natural antioxidant comparable to synthetic antioxidants like Trolox. The superior antioxidant profile can likely be attributed to the high levels of phenolic acids identified, particularly protocatechuic acid, gallic acid, and ellagic acid, known for their potent free radical scavenging abilities. This enhanced antioxidant activity positions Robin’s pincushion as a candidate for applications in oxidative stress management, potentially benefiting chronic conditions such as cardiovascular diseases, diabetes, and neurodegenerative disorders. Although in the available literature it is possible to find the results of the biological activity of many galls, the information on R. canina galls is poor. The only data on the phytochemical content of Robin’s pincushion were found in the study of Coruh and Ercisli [27], which revealed that the content of phenolics for ten samples collected in Turkey varied between 66.34 and 93.35 mg GAE/g. It is noticeable that the Robin’s pincushion sample obtained in Serbia was richer in phenolics. Compared to rosehip fruit results, Robin’s pincushion has a higher content of phenolics according to Koczka et al. [34] (approximately 525 mg GAE/100 g) and Ercisli [35] (96 mg GAE/g). The same observation was noticed in this study with both spectrophotometrical and HPLC analyses, as Robin’s pincushion had higher polyphenol content than rosehip and black rosehip. According to Mohammadzadeh et al. [36], the total phenolic content of the Quercus infectoria galls extract was 16.21 mg GAE/g which showed lower amounts of these secondary metabolites compared with the tested Robin’s pincushion. Moreover, their phytochemical analysis confirmed that these extracts, besides phenolics and flavonoids, contain alkaloids, tannins, and saponins, and the combination of these secondary metabolites results in strong antibacterial effects. Furthermore, in the study of Azmaz et al. [37], the results of phenolic compounds for galls and their host plant extracts indicate that galls were richer in phenolics, reaching 479.56 mg GAE/g, while Coruh and Ercisli [27] reported that only 3 out of 10 had greater phenolic content. However, it is evident from the phytochemical compounds’ view that Robin’s pincushion is very rich in secondary metabolites. Unfortunately, no information on the antioxidant activity of Robin’s pincushion has been found. The obtained results of the Robin’s pincushion extracts express slightly higher DPPH activity compared to the Rosa canina L. extract whose DPPH activity was 127.8 µM TEAC/100 g [10], but significantly lower activity than in the work of Mihaylova et al. [38] (3.66 µM TEAC/g). However, the study of Basyigit et al. [39] revealed the DPPH and ABTS activities of Quercus infectoria galls were 2.29 mmol TEAC/g and 1.98 mmol TEAC/g, respectively, which showed lower activities than the tested sample. Furthermore, the DPPH assay has shown that the gall extract (IC50: 8.67 µg/mL) had stronger scavenging activity than ungalled leaf extracts (IC50: 54.37) of Q. infectoria, according to Azmaz et al. [37]. Therefore, it is noticeable that Robin’s pincushion extracts possess strong antioxidant activity. The obtained results for the anti-inflammatory activity of Robin’s pincushion at a concentration of 0.33 mg/mL showed that it was capable of inhibiting the denaturation of the proteins by 29.03%. Diclofenac sodium was used as a positive control, with an IC50 of 1.14 mg/mL. The antihyperglycemic activity was found to be less effective than the positive control, acarbose, with an inhibition of 96.30% achieved at a concentration of 10 mg/mL. The application of galls as folkloric medications has been practiced for decades, but now their relevance has been supported by biological assays as well. In the present study, the results confirmed the presence of secondary metabolites, for which other studies have already reported antimicrobial and antioxidant activity.
As can be seen in Table 3, Robin’s pincushion also demonstrated substantial anti-inflammatory and antihyperglycemic properties, with inhibition values of 29.03% for the anti-inflammatory assay and 96.3% for the antihyperglycemic assay. The antihyperglycemic activity, in particular, was notable, as it outperformed both rosehip and black rosehip by a significant margin. Phenolic compounds, particularly gallic acid and ellagic acid, are known for their ability to modulate inflammation and glucose metabolism [40,41], and their high presence in Robin’s pincushion likely contributes to these observed activities. Ellagic acid, found at 1066.02 mg/100 g in Robin’s pincushion, has been documented for its anti-diabetic effects and potential to inhibit α-glucosidase, an enzyme involved in carbohydrate digestion. The inhibition of this enzyme correlates with slower glucose absorption, which may help in managing postprandial blood glucose levels and is promising for developing natural antidiabetic agents. The anti-inflammatory potential of Robin’s pincushion, while not the highest among the tested extracts, remains significant when compared to the acarbose standard, which showed only minimal inhibition. This suggests that Robin’s pincushion could offer alternative or complementary benefits in managing inflammatory responses, particularly as chronic inflammation underpins various health conditions, including diabetes and cardiovascular disease. The moderate anti-inflammatory activity observed may complement its potent antioxidant and antihyperglycemic properties, which collectively contribute to its therapeutic potential.
The antimicrobial activity of Robin’s pincushion was profound, showing inhibition against several Staphylococcus species, including S. aureus, S. saprophyticus, S. sciuri, S. epidermidis, and S. warneri. The minimal inhibitory concentration (MIC) values ranged from 1.56 mg/mL for S. aureus, S. saprophyticus, and S. warneri to 12.5 mg/mL for S. epidermidis, demonstrating superior antibacterial efficacy compared to rosehip and black rosehip. The time-kill kinetic study further confirmed the bactericidal potential of Robin’s pincushion, with complete eradication of S. aureus and other Staphylococcus strains within 24 to 48 h. This rapid and effective bactericidal action aligns with previous studies, such as those on gall extracts from Quercus infectoria, where high phenolic content was similarly linked to potent antimicrobial activities. This efficacy against Staphylococcus strains is noteworthy given the increasing global challenge of antibiotic resistance. Plant-based antimicrobials, particularly those from unique sources like galls, provide promising alternatives to conventional antibiotics. The antimicrobial potential of Robin’s pincushion may be largely attributed to its polyphenolic content, especially ellagic acid, which has been reported to inhibit bacterial enzymes and disrupt cell membranes. Given that Staphylococcus species, including S. aureus, are implicated in skin infections and other clinical challenges, Robin’s pincushion could be explored further as a topical antimicrobial agent or adjunct treatment in antimicrobial-resistant infections.

4. Materials and Methods

4.1. Plant Material

Robin’s pincushion, rosehips and black rosehips (Figure 2) was collected during 2022 at localities near Sokobanja, Serbia. After the collecting, plant material was placed at ambient temperature, in a well-aerated place until constant weight. Voucher specimens were identified by PhD Milica Rat and deposited under Voucher Numbers 2-1193 (Robin’s pincushion—rose gall), 2-0046 (rosehips—R. canina L.) and 2-0017 (black rosehips—R. spinosissima L.) at the Herbarium BUNS, University of Novi Sad [42].

4.2. Sample Preparation and Extraction

The preparation of the sample included manual milling in a laboratory mortar and pestle (Lab Logistics Group GmbH, Meckenheim, Germany) until the coarse powder was obtained. Robin’s pincushion, rosehip, and black rosehip were extracted according to the procedure for analysis of total phenolic and carotenoid contents, as well as antioxidant and anti-inflammatory activity [43]. Summarily, the sample was extracted three times using an acetone/ethanol mixture (36:64) in a solid-to-solvent ratio of 1:20 for 10 min, with the same volume of solvents, on a laboratory shaker at 300 rpm, under light protection, at room temperature. For analysis of antihyperglycemic activity, the sample was extracted with a 50:50 ethanol/water mixture and potassium phosphate buffer, respectively, following the same steps of the extraction procedure. Extracts were evaporated and resuspended in saline solution for microbiological assays, as previously described by Šovljanski et al. [44].

4.3. HPLC Analysis

A Shimadzu Prominence chromatographic system (Shimadzu, Kyoto, Japan) was used to examine the prepared extracts. All separation conditions involved in the HPLC analysis are summarized in the Table 8. Namely, for particular substances, chromatograms were recorded at various wavelengths, typically for phenolic compounds. Separation was performed on a Luna C-18 RP column, 5 mm, 250 mm × 4.6 mm with a C18 guard column, 4 mm × 30 mm (both from Phenomenex, Torrance, CA, USA) and analyzed by the Diode Array Detector SPD-M20A (Shimadzu, Kyoto, Japan). At a flow rate of 1 mL/min, two mobile phases—A (acetonitrile) and B (1% formic acid)—were employed, and their gradient profiles looked like the following: 0–10 min from 10 to 25% B, 10–20 min linearly increasing to 60% B, and 20–30 min linearly increasing to 70% B, then 10 min reverting to the starting 10% B with an extra 5 min of equilibration time. The results are reported as mg/100 g sample dry weight (dw).
The phytochemicals were quantified using calibration curves prepared with authentic standards. The concentrations of phenolic compounds were expressed as milligrams of compound per 100 g of sample dry weight (dw). This approach ensured accurate identification and quantification of polyphenolic compounds in the extracts.

4.4. Determination of Phytochemical Composition

The phytochemical compositions of all three samples (Robin’s pincushion, rosehip, and black rosehip) were estimated by a few spectrophotometric methods adapted to the microscale to obtain information about the content of total phenolics, and carotenoids. Total phenolic content (TPh) was determined using the Folin–Ciocalteau reagents, as described in the work of Tumbas-Šaponjac et al. [45]. The results for the total content of phenolics were expressed as gallic acid equivalents (GAE) per 1 g of sample. Furthermore, the total carotenoids (TCar) content was determined and expressed as μg of β-carotene equivalents (β-car) per 1 g of a sample using the method described by Šovljanski et al. [46].

4.5. Determination of Antioxidant Activity

Three in vitro antioxidant assays’ composition of all three samples (Robin’s pincushion, rosehip, and black rosehip) were used to determine the antioxidant activity: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and reducing power (RP) stated in work of Šovljanski et al. [46]. The results were expressed as micromoles of Trolox equivalent (TE) per 100 g of sample.

4.6. Determination of Anti-Inflammatory and Antihyperglycemic Activity

The anti-inflammatory and antihyperglycemic assays’ compositions of all three samples (Robin’s pincushion, rosehip, and black rosehip) were performed following the methods outlined in Ranitović et al. [47]. For the anti-inflammatory assay, which evaluates the sample’s ability to inhibit protein denaturation in egg albumin, three samples were prepared: Robin’s pincushion at a concentration of 0.33 mg/mL, and rosehip and black rosehip at 25 mg/mL. In the antihyperglycemic assay, which assesses the inhibition potential of α-glucosidase (AHgA), concentrations were adjusted to 10 mg/mL for all samples. Both assays were conducted using in vitro spectrophotometric methods, with results expressed as percentages (%).

4.7. Antimicrobial Potential

To investigate antimicrobial activity of all three samples (Robin’s pincushion, rosehip, and black rosehip), Staphylococcus aureus ATCC 11632, S. sciuri St2, S. saprophyticus St1, S. epidermidis St7, and S. warneri St8 were employed. All used cultures are part of the Collection of the microorganisms at Laboratory of microbiology at Faculty of Technology Novi Sad. The overnight cultures were prepared as follows: (1) the bacteria were streaked from −80 °C glycerol stock onto Müller-Hinton Agar (MHA, HiMedia, Mumbai, India) and incubated aerobically at 37 °C for 48 h. The examination of the antimicrobial potential of R. canina galls was performed in two steps: preliminary investigation by using the disk diffusion method and determination of minimal inhibitory concentration (MIC) by the dilution method. Both methods have been earlier described in detail by Pavlić et al. [48]. All tests have been carried out in triplicate, while the positive control for the disk-diffusion test was Oxoid™ Ceftazidime Antimicrobial Susceptibility discs (10 µg/disc, ThermoFisher, Waltham, MA, USA). The pharmacodynamic potential of antimicrobial activity was carried out by following the recommendations of Aćimović et al. [49]. The microorganisms that showed antimicrobial potential (approx. 106 CFU/mL) have been subjected to MIC concentrations of the individual extract. In defined time intervals (0, 2, 4, 6, 12, 18, 24, 36, and 48 h), the samples were taken, and bacterial concentration was determined by streaking onto MHA plates. This study aimed to analyze the Minimum Inhibitory Concentration (MIC) effect decay across five bacterial species. The analysis was conducted in several steps using a sigmoidal kinetic model, with parameters estimated through non-linear regression. The fitted parameters were used to understand the time-kill characteristics of each species. Through time-kill kinetics, the real activity of the substances can be quantified as antimicrobials as a function of contact time between the cells and the targeted concentration of the examined substance.
Contact time (t) points (0, 2, 4, 6, 12, 18, 24, 36, and 48 h) and MIC effect values for each species were extracted and structured for modeling. A sigmoidal model was applied to describe the MIC decay over time, defined as follows:
X ( t ) = A × e − k × t
where X(t) represents bacterial concentration over contact time, A represents the calculated initial value of bacterial concentration, while k is the kill rate constant.

5. Conclusions

This study establishes Robin’s pincushion as a potent natural source of bioactive compounds with remarkable antioxidant, antimicrobial, antihyperglycemic, and moderate anti-inflammatory activities. The findings underscore its promise in therapeutic applications, positioning Robin’s pincushion as an alternative bioactive agent with applications spanning traditional and modern medicine. The unique phytochemical profile induced by gall formation sets it apart from conventional plant extracts, making it a valuable candidate for further exploration in bioactive compound research and development. This study highlights new data on Robin’s pincushion extracts. The results support the use of gall extracts as promising sources of potential antioxidants that may be effective as antimicrobial agents, due to the presence of secondary metabolites and expressed antioxidant activity. This study has found that Robin’s pincushion shows significant antifungal activity and moderate antibacterial activity. In further work, it would be interesting to know more precisely the phytochemical content to better understand what is responsible for the antimicrobial properties of Robin’s pincushion. Also, with a better understanding of this annual plant tissue, it is easier to expand its field of potential use.
Comparing Robin’s pincushion with rosehip and black rosehip highlights its distinct phytochemical composition and enhanced bioactivity across multiple assays. The gall formation process appears to induce a unique biochemical pathway that amplifies the production of secondary metabolites, including polyphenols, which likely accounts for its superior bioactivity. The gall formation, a plant’s response to insect-induced stress, may lead to a re-routing of metabolic processes that favors the synthesis of bioactive compounds typically limited in normal plant tissues. This metabolic adaptation results in a profile rich in antimicrobial and antioxidant compounds, reinforcing the relevance of galls as a valuable bioactive resource.
The results from this study highlight Robin’s pincushion’s potential as a useful bioactive agent suitable for multiple therapeutic applications. Future studies could benefit from isolating specific bioactive compounds in Robin’s pincushion to further delineate their individual contributions to antioxidant, antimicrobial, and antihyperglycemic activities. Moreover, expanding the spectrum of pathogens tested and assessing the in vivo efficacy could provide more insights into its potential clinical applications. Investigating its effects in wound healing, anti-aging formulations, or as an adjunct to diabetic care could pave the way for developing novel, plant-based treatments. Additionally, exploring the cultivation and sustainable harvesting of Robin’s pincushion in regions where it naturally occurs could support its use in both traditional medicine and modern pharmaceuticals. By optimizing extraction techniques and identifying scalable processing methods, Robin’s pincushion extracts could be integrated into nutraceutical and pharmaceutical products aimed at managing oxidative stress, infections, and metabolic disorders.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics13121178/s1, Supplementary Figure S1. HPLC chromatographs of extracts of (a) Robin’s pincushion (240 nm); (b) rosehip (240 nm); (c) rosehip (260 nm); (d) black rosehip (240 nm); and (e) black rosehip (260 nm).

Author Contributions

Conceptualization, G.Ć., A.R. and M.A; methodology, A.T. and T.C.; software, J.V.; validation, V.T.; formal analysis, O.Š.; investigation, A.T., T.C. and A.P.; resources, M.A.; data curation, J.V.; writing—original draft preparation, T.C. and M.A.; writing—review and editing, A.T. and G.Ć.; visualization, V.T.; supervision, O.Š.; project administration, G.Ć. All authors have read and agreed to the published version of the manuscript.

Funding

This research study was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (grants Nos. 451-03-66/2024-03/200134, 451-03-65/2024-03/200134, and 451-03-66/2024-03/200032).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to express their gratitude to Nebojša Stanojević from Sokobanja for inspiring the initiation of this research.

Conflicts of Interest

The authors declare no conflicts of interest.

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Antibiotics 13 01178 g001
Figure 1. Time-kill kinetics study for (a) S. aureus; (b) S. saprophyticus; (c) S. sciuri; (d) S. epidermidis; and (e) S. warneri (dots indicate experimentally obtained data (see Table 6), while lines represent kinetic modeled values).
Figure 1. Time-kill kinetics study for (a) S. aureus; (b) S. saprophyticus; (c) S. sciuri; (d) S. epidermidis; and (e) S. warneri (dots indicate experimentally obtained data (see Table 6), while lines represent kinetic modeled values).
Antibiotics 13 01178 g001
Antibiotics 13 01178 g002
Figure 2. Samples used in this study: (a) Robin’s pincushion in nature; (b) rosehip; and (c) black rosehip (photo by Milica Aćimović).
Figure 2. Samples used in this study: (a) Robin’s pincushion in nature; (b) rosehip; and (c) black rosehip (photo by Milica Aćimović).
Antibiotics 13 01178 g002
Table 1. Phytochemical composition expressed as total phenolic (TPh) and total carotenoid (TCar) content and in vitro antioxidant activity via three different tests of Robin’s pincushion, rosehip and black rosehip extracts.
Table 1. Phytochemical composition expressed as total phenolic (TPh) and total carotenoid (TCar) content and in vitro antioxidant activity via three different tests of Robin’s pincushion, rosehip and black rosehip extracts.
UnitsRobin’s
Pincushion		RosehipBlack RosehipStandard
Phytochemical
composition		TPhmg GAE/g186.37 ± 12.21 a9.65 ± 0.28 b7.24 ± 0.18 b/
TCarμg β-car/g44.10 ± 0.99 b62.24 ± 0.40 a8.10 ± 0.74 c/
Antioxidant
activity		DPPHmM TEAC/100 g152.07 ± 5.19 a21.89 ± 0.87 b3.22 ± 0.13 c0.14 ± 0.01 d
ABTS636.67 ± 19.32 a70.17 ± 2.05 b13.55 ± 0.45 c1.06 ± 0.04 d
RP107.84 ± 3.56 a25.24 ± 1.59 b3.4 ± 0.04 c0.12 ± 0.02 d
Means in the same column with different superscripts are statistically different (p < 0.05; Tukey HSD post-hoc test); standard compound in the antioxidant assays was Trolox.
Table 2. The HPLC analyses of polyphenolics for Robin’s pincushion, black rosehip, and rosehip extracts.
Table 2. The HPLC analyses of polyphenolics for Robin’s pincushion, black rosehip, and rosehip extracts.
Phenols (mg/100 g dw)Robin’s PincushionRosehipBlack Rosehip
p-Hydroxybenzoic acid250.98 ± 0.00 a62.56 ± 0.01 b57.67 ± 0.00 b
Gallic acid297.95 ± 0.01 and16.89 ± 0.00 b
Protocatechin acid677.37 ± 0.00 a15.96 ± 0.00 b32.49 ± 0.00 b
Ellagic acid1066.02 ± 0.02 and223.47 ± 0.01 b
Syringic acid272.00 ± 0.00 and19.53 ± 0.00 b
Vanillic acid392.03 ± 0.00 a21.12 ± 0.00 b404.19 ± 0.01 a
Ferulic acidnd3.04 ± 0.00 and
Total phenols2956.35 ± 0.03 a102.67 ± 0.01 b754.23 ± 0.02 c
Means in the same column with different superscripts are statistically different (p < 0.05; Tukey HSD post-hoc test); nd—not detected.
Table 3. The anti-inflammatory (AIA) and antihyperglycemic (AHgA) activities of Robin’s pincushion, rosehip, and black rosehip extracts expressed as % of inhibition.
Table 3. The anti-inflammatory (AIA) and antihyperglycemic (AHgA) activities of Robin’s pincushion, rosehip, and black rosehip extracts expressed as % of inhibition.
AnalysesRobin’s PincushionRosehipBlack RosehipStandard (IC50)
AIA29.03 ± 1.28 b28.54 ± 0.01 b46.31 ± 0.12 a1.14 ± 0.03 c
AHgA96.30 ± 0.62 a31.67 ± 0.44 b27.84 ± 0.37 b0.001 ± 0.00 c
Means in the same column with different superscripts are statistically different (p < 0.05; Tukey HSD post-hoc test); IC50—values of used standard compounds in the bioactivity assays: for AIA it was acarbose, while for AHgA it was diclofenac sodium.
Table 4. Disk-diffusion test for Robin’s pincushion, black rosehip, and rosehip extracts expressed as inhibition zone in mm.
Table 4. Disk-diffusion test for Robin’s pincushion, black rosehip, and rosehip extracts expressed as inhibition zone in mm.
Test BacteriaRobin’s
Pincushion		RosehipBlack RosehipAntibiotic
Control
Staphylococcus aureus29.33 ± 0.56 a7.00 ± 0.00 d10.5 ± 0.56 c25 ± 0.00 b
S. saprophyticus24.00 ± 0.00 a7.00 ± 0.00 c14.00 ± 0.00 b24 ± 1.00 a
S. sciuri31.00 ± 1.00 a7.00 ± 0.00 d18.00 ± 1.00 c28 ± 0.00 b
S. epidermidis26.33 ± 0.56 a7.00 ± 0.00 c16.5 ± 0.33 b26 ± 0.00 a
S. warneri29.00 ± 0.00 a7.00 ± 0.00 c12.00 ± 1.00 b28 ± 0.00 a
Means in the same column with different superscripts are statistically different (p < 0.05; Tukey HSD post-hoc test); antibiotic control was ceftazidime (10 µg/disc).
Table 5. Minimal inhibitory concentrations (MICs) for Robin’s pincushion, rosehip, and black rosehip extracts.
Table 5. Minimal inhibitory concentrations (MICs) for Robin’s pincushion, rosehip, and black rosehip extracts.
Test BacteriaRobin’s
Pincushion		RosehipBlack Rosehip
Staphylococcus aureus1.56 ± 0.00>50*>50
S. saprophyticus1.56 ± 0.00>50>50
S. sciuri3.125 ± 0.00>5025 ± 0.00
S. epidermidis12.5 ± 0.00>5025 ± 0.00
S. warneri1.56 ± 0.00>50>50
* as the initial value of extracts was used at a concentration of 50 mg/mL.
Table 6. Time-kill kinetic study experimentally obtained results for contact between MIC value of Robin’s pincushion extract and Staphylococcus strains expressed as contact time in hours.
Table 6. Time-kill kinetic study experimentally obtained results for contact between MIC value of Robin’s pincushion extract and Staphylococcus strains expressed as contact time in hours.
Test Bacteria02461218243648
S. aureus6.1 ± 0.14.9 ± 0.13.1 ± 0.22.2 ± 0.20.5 ± 0.10.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
S. saprophyticus6 ± 0.04.4 ± 0.04 ± 0.33.8 ± 0.03.6 ± 0.31.3 ± 0.20.0 ± 0.00.0 ± 0.00.0 ± 0.0
S. sciuri6 ± 0.35.7 ± 0.03.3 ± 0.21.7 ± 0.01.0 ± 0.10.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
S. epidermidis6.1 ± 0.15.8 ± 0.14.6 ± 0.03.8 ± 0.02.7 ± 0.21.4 ± 0.11.0 ± 0.20.0 ± 0.00.0 ± 0.0
S. warneri5.9 ± 0.04.1 ± 0.23.5 ± 0.11.7 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
Table 7. The kinetic parameters and “goodness of the fit” for kinetic models obtained for MIC values’ effect on the Staphylococcus strain.
Table 7. The kinetic parameters and “goodness of the fit” for kinetic models obtained for MIC values’ effect on the Staphylococcus strain.
Kinetic Models
Parameters		Verification Parameters
Test BacteriaA
(log CFU/mL)		kχ2RMSEMBEMPEr2Skew.Kurt.MeanSDVar.
S. aureus6.360.180.1190.1020.003−2.7560.9980.778−0.9141.8672.2254.951
S. saprophyticus5.760.081.1810.5300.0460.7310.939−0.002−1.4562.5672.1394.573
S. sciuri6.520.1716.3060.358−0.101−18.4100.9760.789−0.9961.9672.3205.384
S. epidermidis6.360.080.4380.2040.021−1.9680.9920.142−1.4592.8222.2365.002
S. warneri6.070.190.3510.2210.0230.6290.9890.804−0.8691.6892.1374.565
A—predicted initial values of bacterial concentration; k—kill rate coefficient; χ2—reduced chi-square; RMSE—root mean square error; MBE—mean bias error; MPE—mean percentage error; r2—coefficient of determination; Skew.—skewedness; Kurt.—kurtosis; SD—standard deviation; and Var.—variance.
Table 8. HPLC method summary.
Table 8. HPLC method summary.
ParameterCondition
InstrumentShimadzu Prominence chromatographic system
ColumnLuna C-18 RP column, 5 μm, 250 mm × 4.6 mm with C18 guard column (4 mm × 30 mm)
DetectorDiode Array Detector SPD-M20A
Flow rate1 mL/min
Mobile phase AAcetonitrile
Mobile phase B1% Formic acid
Gradient profile0–10 min: 10–25% B; 10–20 min: 25–60% B; 20–30 min: 60–70% B; 30–40 min: return to 10% B and 5 min equilibration
Detection wavelength240 nm or/and 260 nm
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Šovljanski, O.; Aćimović, M.; Cvanić, T.; Travičić, V.; Popović, A.; Vulić, J.; Ćetković, G.; Ranitović, A.; Tomić, A. An In Vitro Evaluation of Robin’s Pincushion Extract as a Novel Bioactive-Based Antistaphylococcal Agent—Comparison to Rosehip and Black Rosehip. Antibiotics 2024, 13, 1178. https://doi.org/10.3390/antibiotics13121178

AMA Style

Šovljanski O, Aćimović M, Cvanić T, Travičić V, Popović A, Vulić J, Ćetković G, Ranitović A, Tomić A. An In Vitro Evaluation of Robin’s Pincushion Extract as a Novel Bioactive-Based Antistaphylococcal Agent—Comparison to Rosehip and Black Rosehip. Antibiotics. 2024; 13(12):1178. https://doi.org/10.3390/antibiotics13121178

Chicago/Turabian Style

Šovljanski, Olja, Milica Aćimović, Teodora Cvanić, Vanja Travičić, Aleksandra Popović, Jelena Vulić, Gordana Ćetković, Aleksandra Ranitović, and Ana Tomić. 2024. "An In Vitro Evaluation of Robin’s Pincushion Extract as a Novel Bioactive-Based Antistaphylococcal Agent—Comparison to Rosehip and Black Rosehip" Antibiotics 13, no. 12: 1178. https://doi.org/10.3390/antibiotics13121178

APA Style

Šovljanski, O., Aćimović, M., Cvanić, T., Travičić, V., Popović, A., Vulić, J., Ćetković, G., Ranitović, A., & Tomić, A. (2024). An In Vitro Evaluation of Robin’s Pincushion Extract as a Novel Bioactive-Based Antistaphylococcal Agent—Comparison to Rosehip and Black Rosehip. Antibiotics, 13(12), 1178. https://doi.org/10.3390/antibiotics13121178

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