1. INTRODUCTION
Stroke is the second leading cause of mortality and a major cause of morbidity worldwide. The latest report released by the WHO also highlights the death and disability rates associated with stroke. The burden of stroke is greater in low-income countries than in high-income countries [1]. Among all cases of stroke, 87% are ischemic strokes, which result from insufficient blood supply due to blockage of cerebral arteries by intravascular blood clots. Rapid restoration of blood flow through techniques such as thrombolytic therapy or thrombectomy is the mainstay of treatment. However, the effectiveness of these therapies depends on their application within a narrow time window after the onset of stroke [2]. Even after recovery, many patients experience neurological problems such as motor and cognitive deficits, leading to lifelong morbidity [3]. The biochemical events during ischemia and reperfusion include oxidative stress, inflammatory reactions involving recruited leukocytes, tissue injury, and apoptosis [4]. Given the complex pathology of ischemic stroke, numerous studies have focused on developing effective neuroprotective molecules. Natural compounds from plants with antioxidant, anti-inflammatory, and antiapoptotic properties have been explored in preclinical studies as potential neuroprotective agents. However, none of these natural products have yet been translated into clinical use [5]. Therefore, there is an urgent need to identify neuroprotective compounds from plant sources to improve outcomes in stroke patients, making the identification of these compounds highly valuable for researchers [6]. Salvia officinalis L., commonly called sage, is a perennial herb in the Lamiaceae family, and this genus contains approximately 900 species. This plant is distributed not only in Asia, Europe, and North America but also in other parts of the world. Salvia officinalis is considered to have high economic value in industries such as cosmetics and perfumery because of its aromatic and medicinal properties [7]. Traditionally, it is used to treat various diseases or disorders, including memory loss, paralysis, seizures, Alzheimer’s disease, rheumatism, gout, ulcerative colitis, cancer, diarrhea, and hyperglycemia [8]. This plant has various pharmacological activities, including antioxidant, anti-inflammatory, anti-dementia, anticancer, antibacterial, antifungal, antihyperglycemic, and antihyperlipidemic effects [9]. The essential oils of the leaves of Salvia contain monoterpenes such as cineol, thujone, borneol, and camphor, and sesquiterpenes such as caryophyllene. The plant is also rich in polyphenolic tannins (salvianolic acid and rosmarinic acid), flavonoids (luteolin, quercetin, rutin, ellagic acid, chlorogenic acid, apigenin, kaempferol), phenolic acids (caffeic acid and caffeoylquinic acid), and di- and tri-terpenes (carnosic acid, carnosol, ursolic acid, and tanshinones) [10–12]. Considering its rich phytochemical and pleiotropic pharmacological properties, this study aimed to evaluate its neuroprotective properties against cerebral ischemia-reperfusion injury in rats.
2. MATERIALS AND METHODS
2.1. Materials
Trichloroacetic acid (Loba Chemie Pvt. Ltd., Mumbai, CAS Number 76-03-9), DTNB (5,5-dithiobis (2-nitrobenzoic acid) Sigma Aldrich, CAS number 69-78-3), pyrogallol (Loba Chemie Pvt. Ltd, Mumbai, CAS Number 87-66-1), thiobarbituric acid (Loba Chemie Pvt. Ltd., Mumbai, CAS Number 504-17-6), ethylene diamine tetra acetic acid (Sigma Aldrich, CAS number 60-00-4), tetrazolium salt (Loba Chemie Pvt. Ltd., Mumbai, CAS Number 298-96-4), reduced L-glutathione (GSH) (Sigma Aldrich, CAS number 70-18-8), and 2,2-diphenyl-1-picryl hydrazyl (DPPH) (Sigma Aldrich, CAS number 1898-66-4) were used. The remaining chemicals were of analytical grade.
2.2. Collection of plant material and authentication
Fresh leaves of S. officinalis were obtained from a botanical garden in Ooty, Tamil Nadu. The plant material was identified by Dr. S. Rajan, field botanist, survey of medicinal plants and collection unit, Department of AYUSH, Government of India, Ooty, Tamil Nadu. The plant material was shade-dried and then crushed into a powder via a mechanical grinder. The powdered material was stored in an airtight container.
2.3. Extraction of plant material
Three kilograms of dried powdered leaves of S. officinalis were extracted with 6 l of methanol for 7 days with intermittent shaking. The crude extract was then filtered through muslin cloth, and the filtrate was concentrated in a rotary evaporator under reduced pressure. The resulting residue was further evaporated in a glass vessel to provide a semisolid crude methanol extract, which was sealed in an air-tight container for later use. This step was repeated whenever necessary [13].
2.4. Fractionation
The methanol extract (60 g) was dissolved in 200 ml of hot water in a glass container. This water-soluble extract was transferred into a separating funnel and fractionated by using various solvents from nonpolar to polar, such as n-hexane, dichloromethane, and ethyl acetate, and the remaining one was considered the aqueous fraction. All the fractions were further concentrated via a rotary evaporator under reduced pressure. The concentrated fractions were then stored in airtight containers [14].
2.5. Evaluation of in vitro antioxidant activity
The antioxidant potential of the crude methanol extract and its fractions, such as the n-hexane, dichloromethane, ethyl acetate, and aqueous fractions, and ascorbic acid, was evaluated through in vitro methods [15].
2.6. Evaluation of in vitro anti-inflammatory activity
The anti-inflammatory activity of the crude methanol extract and its fractions, as well as diclofenac sodium, was estimated by protein denaturation inhibition assay and RBC cell membrane stabilization assay as per earlier reported protocols [16,17].
2.7. LC-MS analysis of the n-hexane fraction
The n-hexane fraction was separated on a Waters Micromass Q-ToF Micro system with a C18 reverse-phase column (150 × 2.1 mm, 3.5 μm). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), and the flow rate was 0.3 ml/min. The gradient began at 95% A and increased to 50% B over 18 minutes, then to 95% B over 5 minutes, remained at 5 minutes, returned to starting conditions in 2 minutes, and was re-equilibrated for 8 minutes. A 5.0 μl injection was made at 4°C. The source of the ESI functioned in positive ion mode (3.0 kV capillary, 120°C source temperature), using nitrogen as the cone gas (50 l/h) and desolvation gas (600 l/h, 350°C). The mass spectrometer was calibrated using sodium formate, running spectra from 50 to 2,000 m/z, while MS/MS data were obtained in DDA mode with 20–40 eV collision energies [18,19].
2.8. In silico molecular docking studies
Comprehensive docking studies were conducted for ligands identified from LC-MS data (filtered by Lipinski’s rule of 5) via Schrödinger’s Glide module in Maestro. Ligand docking was performed in standard precision mode with default settings, including van der Waals radius scaling at 0.80, a partial charge cutoff of 0.15, and Epik state penalties integrated into the docking score. The bioactive compounds identified in the NFSO via LC-MS analysis were docked for specific proteins such as Keap1–Nrf2 and MPO. The Keap1–Nrf2 signaling axis plays a crucial role in regulating oxidative stress and inflammation. Under normal conditions, Keap1 binds and promotes the degradation of Nrf2, thereby suppressing its cytoprotective activity. Targeting Keap1 to disrupt its interaction with Nrf2 is a promising therapeutic strategy to enhance antioxidant defense. Therefore, molecular docking of the selected compounds with the Kelch domain of Keap1 was performed to evaluate their potential to interfere with Keap1–Nrf2 interaction and stabilize Nrf2 activity. MPO is a major inflammatory enzyme elevated in ischemic stroke, which drives oxidative stress and inflammation. Hence, docking bioactive compounds to this protein might help predict an anti-inflammatory effect. Docking against these validated targets provides mechanistic support for NFSO’s observed antioxidant and anti-inflammatory effects [20].
2.9. Animals
Male albino Wistar rats (250–300 g) procured from Sri Raghavendra Enterprises, Bangalore, India, were used for the present study. The rats were kept in an air-conditioned room (24 ± l°C) with a 12/12-hour light/dark cycle. The animals received enough standard pellets and mineralized water for daily consumption. The study protocol was approved by the Institutional Animal Ethical Committee of Raghavendra Institute of Pharmaceutical Education and Research (RIPER)-Autonomous, Anantapur, India (Approval N.: IAEC/ XVII/01/RIPER/2021).
2.10. Acute toxicity study
The acute toxicity of the n-hexane fraction of S. officinalis (NFSO) was determined according to the guidelines of OECD 423. The NFSO was orally administered at a single dose of 2,000 mg/kg to a group of three rats. The rats were carefully observed for a period of 14 days to detect any signs of toxicity. Doses of 250 (1/8th ) and 500 mg/kg (1/4th ) were selected for the animal study, as no sign of toxicity was observed [21].
2.11. Induction of cerebral stroke
Acute ischemic stroke was induced in rats by occluding the bilateral common carotid artery (BCCA) under ketamine (60 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia. A 1-inch midline neck incision was made, and both carotid arteries were carefully separated from the vagus nerve, occluded with cotton thread for 30 minutes, followed by a 24-hour reperfusion period. The rats recovered through spontaneous breathing, with postsurgical care provided under standard conditions, including free access to food and water [22].
2.12. Study design
Thirty-six male Wistar rats were divided into six groups. Each group consists of six animals, and the animals were given therapy for 14 days.
Group 1: Normal (no surgery, no occlusion)
Group 2: Sham control (surgery without occlusion)
Group 3: Ischemic reperfusion injury (I/R injury)
Group 4: Received NFSO (250 mg/kg, p.o)
Group 5: Received NFSO (500 mg/kg, p.o)
Group 6: Received aspirin (60 mg/kg, p.o)
2.13. Assessment of parameters
After 24 hours of reperfusion, the cognitive and motor dysfunction in all the groups of animals was assessed via various methods, such as the elevated plus maze test, Y maze test, beam walking test, and wire hanging test [23].
2.13.1. Estimation of antioxidant parameters in brain tissue
The rats were euthanized after 24 hours of reperfusion. The rats’ brains were immediately removed, cleaned, homogenized, and centrifuged in ice-cold phosphate-buffered saline (pH 7.4) at 7,000 rpm for 15 minutes. The resulting supernatant was used to estimate biochemical parameters, including superoxide dismutase (SOD), catalase (CAT), reduced glutathione, malondialdehyde (MDA), and myeloperoxidase (MPO) [24].
2.13.2. Assessment of cerebral stroke area
The brains were cut into 2 mm pieces and submerged in a 1% w/v solution of 2,3,5-triphenyl tetrazolium chloride (TTC) for 30 minutes at 37°C. The slices were photographed to determine the extent of tissue infarction [25].
2.13.3. Histopathological studies
A portion of the brain was cut into 5-µm-thick pieces with a semiautomatic cryostat microtome. The brain sections were stained with hematoxylin and eosin for histological analysis. The images were captured using a camera attached to a trinocular microscope (Labomed) at a scale of 100 µm (resolution)
2.14. Statistical analysis
All the data were presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test in GraphPad Prism version 8.01. Differences were considered statistically significant at p < 0.05.
3. RESULTS
3.1. LC-MS analysis of the n-hexane fraction of S. officinalis
LC-MS analysis of the n-hexane fraction of S. officinalis (NFSO) revealed the presence of major phytoconstituents, which are represented in Table 1 and Figure 1.
 | Figure 1. LC-MS analysis of the n-hexane fraction of Salvia officinalis. [Click here to view] |
Table 1. LC-MS spectral analysis of the n-hexane fraction of Salvia officinalis.
| RT (min) | Name of the compound | Molecular formula | m/z |
|---|---|---|---|
| 12.7043 | Quercetin 3,7-dirhamnoside | C27H30O15 | 595.17 |
| 12.7043 | Luteolin-7-O-neohesperidoside | C27H30O15 | 595.17 |
| 12.7043 | isovitexin 2''-O-beta-D-glucoside | C27H30O15 | 595.17 |
| 12.7043 | Isoorientin 2''-O-rhamnoside | C27H30O15 | 595.17 |
| 15.048 | cyanidin 3-O-rutinoside betaine | C27H30O15 | 595.17 |
| 27.472 | ursolic acid | C30H48O3 | 498.39 |
| 27.472 | Betulinic acid | C30H48O3 | 498.39 |
| 26.711 | Myricetin | C15H10O8 | 341.03 |
| 29.585 | 1-O-vanilloyl-beta-D-glucose | C14H18O9 | 369.06 |
| 29.585 | Syringetin | C17H14O8 | 369.06 |
| 29.585 | Spinacetin | C17H14O8 | 369.06 |
| 39.072 | Protocatechuic acid | C7H6O4 | 192.99 |
| 39.413 | cyanin betaine | C27H30O16 | 652.19 |
| 39.413 | Rutin | C27H30O16 | 652.19 |
| 39.413 | Kaempferol 3-O-gentiobioside | C27H30O16 | 652.19 |
| 38.792 | Canthaxanthin | C40H52O2 | 529.38 |
| 32.971 | Pterostilbene | C16H16O3 | 257.12 |
| 32.411 | Limocitrin | C17H14O8 | 369.06 |
| 11.912 | Limonin | C26H30O8 | 493.18 |
| 29.585 | 3',4',5,6-tetrahydroxy-3,7-dimethoxyflavone | C17H14O8 | 369.06 |
| 20.547 | 7,8-Dimethoxyflavone | C17H14O4 | 324.14 |
LC-MS analysis revealed the presence of various bioactive compounds, including flavonoids, phenolic acids, terpenoids, and stilbenes, with potent antioxidant, anti-inflammatory, and therapeutic properties. Flavonoids such as quercetin 3,7-dirhamnoside, luteolin-7-O-neohesperidoside, isovitexin 2’’-O-beta-D-glucoside, and isoorientin 2’’-O-rhamnoside were found to have strong antioxidant and anti-inflammatory activities. Myricetin, syringetin, spinacetin, and limocitrin were found to possess anticancer and neuroprotective activities. Protocatechuic acid, a phenolic acid, exhibits antioxidant and antimicrobial activity. Anthocyanins and flavonoid glycosides (cyanidin 3-O-rutinoside betaine, rutin, kaempferol 3-O-gentiobioside) contributed to cardiovascular and metabolic well-being. Triterpenoids (ursolic acid, betulinic acid, limonin) displayed anti-inflammatory and hepatoprotective activity, and pterostilbene is reported to have neuroprotective effects. Canthaxanthin and 1-O-vanilloyl-beta-D-glucose were found to have antioxidant activity. The n-hexane fraction’s complex chemical makeup suggests that the fraction may have therapeutic value, particularly in research on anti-inflammatory, antioxidant, and anticancer properties.
3.2. In silico molecular docking studies
The LC-MS analysis identified ligands from the n-hexane fraction that were screened through ADMET software to determine whether they obey Lipinski’s Rule of Five or not based on Lipinski’s rule of five parameters (MW < 500 Da, LogP < 5, H-bond donors < 5, acceptors < 10). The ligands that obey this rule are listed in Table 2. These compounds were further screened for their binding affinity toward targeted proteins such as Keap1–Nrf2 complex and MPO. Docking analysis determined the binding affinities and interactions of different compounds with the Keap1–Nrf2 complex (Table 3 & Fig. 2). Luteolin-7-O-rhamnoside had the highest binding affinity (−5.83) and minimum glide energy (−56.74) and interacted with GLU 493, SO4 708, ASN 469, and GLU 442. Syringetin (−5.51) interacted with GLU 493, TYR 537, and GLU 542, while myricetin and spinacitin had moderate affinities (~−46.95 glide energy) with interactions at ASN 469, GLU 540, and VAL 539. Protocatechuic acid and limocitrin (−4.77) had comparable affinities but with differing interactions, binding at TYR 491, GLU 540, and GLU 542. Weak binders, 1-O-vanilloyl-beta-D-glucose and pterostilbene, displayed poor interactions, and 7, 8-dimethoxy flavanone (−3.35) and limonin (−2.23, lowest affinity) demonstrated low binding. In general, luteolin-7-O-rhamnoside was found to be the best Keap1–Nrf2 inhibitor through strong binding and several interactions.
 | Figure 2. 2D interaction of bioactive compounds of n-hexane fraction with Keap1–Nrf2 complex (1X2R). [Click here to view] |
Table 2. Lipinski’s rule for chemical compounds.
| S. No | Chemical compounds | Lipinski rule (Accepted) |
|---|---|---|
| 1. | 1-O-vanilloyl -beta-D-glucose | Yes |
| 2. | 7,8-dimethoxy flavone | Yes |
| 3. | Limocitrin | Yes |
| 4. | Limonin | Yes |
| 5. | Luteolin | Yes |
| 6. | Myricetin | Yes |
| 7. | protocatechuic acid | Yes |
| 8. | Pterostilbene | Yes |
| 9. | Spinacetin | Yes |
| 10. | Syringetin | Yes |
Table 3. Docking scores and interactions of compounds with the Keap–Nrf2 complex (1X2R) protein.
| Chemical compound | Docking score | Glide energy | Interactions |
|---|---|---|---|
| Luteolin-7-O-rhamnoside | −5.83 | −56.74 | GLU 493, SO4 708, ASN 469, GLU 442 |
| Syringetin | −5.51 | −36.52 | GLU 493, TYR 537, GLU 542 |
| Myrcetin | −4.90 | −46.95 | ASN 469, GLU 540 |
| Protocatechuic acid | −4.77 | −23.93 | TYR 491, GLU 540 |
| Limocitrin | −4.77 | −35.38 | GLU 493, TYR 491, GLU 542 |
| Spinacitin | −4.69 | −46.95 | ASN 469, VAL 539, GLU 542 |
| 1-O-vanilloyl-beta-D glucose | −4.53 | −38.59 | THR 501, MET 499, ARG 498, GLU 540 |
| Pterostilbene | −3.87 | −24.78 | SO4 708 |
| 7,8-dimethoxyflavanone | −3.35 | −25.44 | No interactions |
| Limonin | −2.23 | −28.69 | CYS 489 |
The molecular docking study with MPO (PDB ID: 1MHL) presented in Table 4 revealed that protocatechuic acid was the compound with the highest binding affinity, as evidenced by a docking score of −7.02 and significant interactions with GLU 255 and THR 369. Luteolin-7-O-rhamnoside also displayed a strong binding affinity with a docking score of −6.95 and an exceptionally low glide energy of −57.28, forming key interactions with ASP 254, GLU 255, and SER 305. Limocitrin and myricetin, with docking scores of −6.42 and −6.09, respectively, showed robust binding, interacting with residues such as VAL 187, GLN 253, and HIE 328 for limocitrin and ASN 469, GLU 493, and VAL 327 for myricetin. Interestingly, the standard reference compound, salicyl hydroxamic acid, had a lower docking score of −3.91, interacting with GLY 155, SER 156, THR 159, and ARG 161. Overall, protocatechuic acid and luteolin-7-O-rhamnoside demonstrated the most promising interactions and binding affinities, suggesting their potential as strong inhibitors of MPO. The 2D interactions of the compounds with >6 docking scores with the MPO protein are shown in Figure 3.
 | Figure 3. 2D interaction of bioactive compounds of n-hexane fraction with MPO (PDB ID: 1MHL). [Click here to view] |
Table 4. Docking scores and interactions of the compounds with the myeloperoxidase (1MHL) protein.
| Chemical compound | Docking score | Glide energy | Interactions |
|---|---|---|---|
| Protocatechuic acid | −7.02 | −35.75 | GLU 255, THR 369 |
| Luteolin-7-O-rhamnoside | −6.95 | −57.28 | ASP 254, GLU 255, SER 305 |
| Limocitrin | −6.42 | −41.82 | VAL 187, GLN 253, HIE 328 |
| Myricetin | −6.09 | −56.43 | ASN 469, GLU 493, VAL 327 |
| Spinacetin | −5.88 | −47.89 | ASP 254, ILE 306, TYR 302 |
| Syringetin | −5.57 | −44.50 | ASP 254, TYR 304, GLU 542 |
| 7,8 dimethoxy flavanone | −5.45 | −34.86 | GLN 202 |
| 1-O-vanilloyl -beta-D-glucose | −4.89 | −43.89 | GLU 255, GLN 249, LEU 262, LYS 373 |
| Pterostilbene | −4.54 | −33.45 | TYR 304 |
| Limonin | −2.73 | −33.73 | THR 307, LYS 373 |
| Salicyl hydroxamic acid (Standard) | −3.91 | −21.41 | GLY 155, SER 156, THR 159, ARG 161 |
3.3. Results of in vitro antioxidant activity of methanol extract and its fractions
The methanol extract and its fractions (n-hexane, ethyl acetate, dichloromethane, and aqueous) displayed dose-dependent inhibition, reported as percentage inhibition, according to the results of antioxidant tests such as DPPH radical and hydrogen peroxide scavenging activities (Fig. 4). The n-hexane fraction was demonstrated the highest activity among all tested samples, achieving 81.28% inhibition for the DPPH radical inhibition assay and 71.30% inhibition for the hydrogen peroxide scavenging assay at the maximum tested concentration (1,250 µg/ml). Ascorbic acid, a reference standard, achieved 95% DPPH inhibitory potential and 89.27% for hydrogen peroxide scavenging inhibitory effect. These findings suggest that the n-hexane fraction is the most potent among the tested samples and demonstrates antioxidant efficacy on par with ascorbic acid.
 | Figure 4. Results of in vitro anti-oxidant and anti-inflammatory activity of methanol and its fraction of Salvia officinalis. [Click here to view] |
3.4. Results of in vitro anti-inflammatory activity of methanol extract and its fractions
Figure 4 displays the outcomes of the anti-inflammatory properties of methanol extract and its fractions against RBC hemolysis and protein denaturation. When compared to the other fractions, the n-hexane fraction showed the highest inhibition rate in both experiments, with 77% ± 8.73% and 76% ± 0.65% inhibition, respectively, at a concentration of 1,250 μg/ml. In contrast to the methanol extract and other fractions, the n-hexane fraction demonstrated the highest potential anti-inflammatory activity. Diclofenac sodium, the reference standard, demonstrated 88.06% for RBC hemolysis inhibition and 89.95% protein denaturation inhibition. The n-hexane fraction achieved an anti-inflammatory effect close to the standard.
3.5. Results of the effects of NFSO on behavioral parameters
The neuroprotective potential of NFSO (250 and 500 mg/kg, p.o.) was evaluated through behavioral tests assessing memory retention, working memory, and motor coordination following ischemia–reperfusion (I/R) injury. No significant differences were observed between the normal and sham control groups, indicating that the surgical procedure itself did not alter behavioral performance. In the elevated plus maze test, I/R-injured rats exhibited a significant increase in transfer latency time (TLT) (55.16 ± 1.45; ***p < 0.001) compared to normal controls (11.94 ± 0.63), confirming memory impairment. Similar to aspirin, pretreatment with NFSO significantly reduced the TLT in a dose-dependent manner (39.20 ± 1.49; ***p < 0.001 at 60 mg/kg, 49.40 ± 1.32 at 250 mg/kg, *p < 0.05; 48.60 ± 0.50 at 500 mg/kg, ***p < 0.01; F (4, 20) = 118). Suggesting that higher doses of NFSO effectively preserved memory retention similar to the standard reference drug.
In the Y-maze test, spontaneous alternations reflecting working memory were markedly reduced in I/R-injured rats (24.14 ± 2.85; ***p < 0.001) compared to the normal (84.74 ± 1.04) and sham control (77.68 ± 2.03) groups. NFSO treatment significantly increased spontaneous alternations (35.30 ± 1.69, **p < 0.01at 250 mg/kg and 58.56 ± 2.86 at 500 mg/kg; ***p < 0.001) compared to the I/R group. Although aspirin (69.10 ± 0.94; ***p < 0.001) produced a slightly higher improvement, NFSO at 500 mg/kg exhibited comparable enhancement of working memory with aspirin, confirming its dose-dependent cognitive protection (F (4, 20) = 52.9). In the beam walking test, I/R injury resulted in a prolonged beam crossing time (37.66 ± 2.19; ***p < 0.001) compared to normal (12.90 ± 0.78) and sham control (14.10 ± 1.74) rats, indicating impaired motor coordination. Along with the standard drug—aspirin—NFSO treatment significantly shortened the beam crossing time (13.73 ± 0.54; **p < 0.001; 24.58 ± 1.12 at 250 mg/kg and 16.94 ± 1.05 at 500 mg/kg; ***p < 0.001, F (4, 20) = 48.4). This demonstrates that NFSO proved to improve motor coordination as that of a standard drug. In the hanging wire test, the I/R group displayed significantly reduced hanging latency (51.50 ± 1.81; ***p < 0.001) compared to the normal group (87.14 ± 2.53) and sham group (81.04 ± 0.75), indicating severe motor deficits. Pretreatment with NFSO improved the hanging latency in a dose-dependent manner (58.60 ± 0.67 at 250 mg/kg, *p < 0.05; 60.80 ± 1.02 at 500 mg/kg, **p < 0.01), while aspirin showed a slightly greater effect (70.40 ± 1.43; ***p < 0.001; F (4, 20) = 9.02). Collectively, NFSO pretreatment significantly prevented ischemia-induced cognitive and motor impairments, with effects at 500 mg/kg comparable to those of aspirin. The observed improvements in working memory, memory retention, and motor coordination confirm the neuroprotective efficacy of NFSO against cerebral injury induced by I/R conditions (Fig. 5).
 | Figure 5. Effect of the n-hexane fraction of Salvia officinalis on cognitive and motor defects associated with I/R injury in rats. A. Elevated plus maze test; B. Y maze test; C. Beam walking test; D. Hanging wire test. Data are presented as mean ± SEM (n = 6); p values were represented as follows *p < 0.05, **p < 0.01, and ***p < 0.001. [Click here to view] |
3.6. Results of biochemical estimations in the rat brain tissue
The current research proved that NFSO pretreatment successfully alleviates oxidative stress in I/R injury by increasing antioxidant enzyme levels and decreasing lipid peroxidation (LPO) in brain tissue. There was a significant decrease in SOD levels in I/R-damaged rats (0.35 ± 0.058, ***p < 0.001 ) compared to control rats (2.705 ± 0.2) and sham control rats (2.43 ± 0.12), which suggests an enhanced level of superoxide radicals that are responsible for the formation of highly toxic hydroxyl radicals. Nevertheless, NFSO (250 & 500 mg/kg) pre-treatment significantly restored SOD levels (0.82 ± 0.04; *p < 0.05 & 0.93 ± 0.10; **p < 0.01). However, at the dose of 500 mg/kg, the SOD levels restoration was equal to aspirin (1.16 ± 0.19, ***p < 0.001), indicating its antioxidant protective role (F (4, 25) = 216). CAT activity was also significantly reduced in I/R injury rats (17.04 ± 0.30; ***p < 0.001) compared to normal controls (57.42 ± 0.60) and the sham control group (54.21 ± 0.26), indicating excessive accumulation of H2O2 and oxidative stress. Similar to aspirin (35.69 ± 0.84, ***p < 0.001), NFSO treatment significantly increased the levels of CAT in a dose-dependent manner (22.05 ± 0.83, **p < 0.01 at 250 mg/kg and 32.24 ± 1.44, ***p < 0.001 at 500 mg/kg, F(4, 25) = 2,078). Furthermore, GSH content, an important indicator of antioxidant protection, was significantly decreased in I/R-damaged rats (23.55 ± 3.62; ***p < 0.001) compared to normal rats (71.6 ± 2.28) and sham rats (66.44 ± 1.75). NFSO as well as aspirin pretreatment restored GSH levels significantly (31.56 ± 1.31, **p < 0.01 at 200 mg/kg, 46.18 ± 3.2, ***p < 0.001 at 500 mg/kg and 52.43 ± 1.19, ***p < 0.001 at 60 mg/kg, F (4, 25) = 144) compared to the I/R-damaged rat, further strengthening its neuroprotective activity. Oxidative stress-mediated LPO was evaluated through the estimation of MDA levels, which were significantly increased in the I/R injury group (5.51 ± 0.66; ***p < 0.001) as compared to the normal group (1.32 ± 0.19) and sham control (1.99 ± 0.37). NFSO pretreatment significantly lowered the levels of MDA dose dependently (4.48 ± 0.16, **p < 0.01 at 250 mg/kg, 2.53 ± 1.07, ***p < 0.001 at 500 mg/kg, F (4, 25) = 48.5) and was on par with aspirin (2.33 ± 0.13, ***p < 0.001), which proved its anti-oxidative action. In summary, oxidative stress markers alter significantly in the I/R injury group without effect in the normal and sham group, as NFSO pretreatment effectively reversed oxidative stress by increasing the activity of antioxidant enzymes (SOD, CAT, and GSH) and lowering LPO, thus proving its neuroprotective potential against I/R-induced oxidative stress in brain tissue (Fig. 6).
 | Figure 6. Effect of the n-hexane fraction of Salvia officinalis on oxidative stress and inflammation markers. E. SOD; F. CAT; G. Reduced GSH; H. MDA; and I. MPO. Data are presented as mean ± SEM (n = 6); p values were represented as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. [Click here to view] |
3.6.1. Effect of NFSO on the MPO level
MPO, an inflammatory marker found in cells such as neutrophils and microglia, initiates pathological events, including free radical damage and inflammation. The brain MPO levels were significantly higher in I/R injury rats (63.62 ± 2.25; ***p < 0.001) than in normal rats (14.46 ± 1.48) and sham control (20.86 ± 4.9) without significant changes between normal and sham control. Compared with I/R-injured rats, NFSO as well as aspirin pretreated rats showed significantly lower brain MPO levels (51.11 ± 1.28, *p < 0.05 at 250 mg/kg, 48.65 ± 0.72, **p < 0.01 at 500 mg/kg; 37.79 ± 0.72, ***p < 0.001 at 60 mg/kg, F (4, 25) = 77.2) (Fig. 6).
3.7. Results of the TTC staining test
The degree of infarction after I/R injury was assessed by staining the brain slices with TTC. The brain slices that are stained red indicate the viable part, whereas the nonviable tissue appears colorless. In the control group and sham control group, the brain slices appeared red, indicating the presence of viable tissue, whereas in the I/R injury group, the brain slices appeared pale in color, as the infarct tissue failed to convert colorless to colored formazan from the TTC. Therefore, the degree of infarction is greater. In contrast, the degree of infarction was lower in NFSO (250 & 500 mg/kg) treated rats and aspirin treated rats, as indicated by a larger area that appeared pink in color (Fig. 7).
 | Figure 7. Effect of the n-hexane fraction of Salvia officinalis on infarct volume via TTC staining of rat brain slices. A. Normal; B. Sham; C. I/R injury; D. NFSO (250 mg/kg); E. NFSO (500 mg/kg); and F. Aspirin (60 mg/kg). [Click here to view] |
3.8. Results of histopathology
The above-mentioned images depict the histopathology of the brain tissue of various treatment groups, along with the control and I/R control groups. A: Rats in the normal group showing the hippocampal dentate gyrus granular cell layer with proper density and cytoarchitecture; B: Sham group showing no evidence of abnormalities in the rat brain. C: I/R group showing a disrupted arrangement and a decrease in the cellular density of the hippocampal dentate gyrus granular cell layer. Pyknotic granular cells, vacuolations, shrunken hyperchromatic nuclei, perinuclear halos, and loss of nuclear details were observed. D: At a dose of 250 mg/kg, the n-hexane fraction, despite showing much less damage, exhibited vascular congestion. E: At a dose of 500 mg/kg, the n-hexane fraction exhibited normal cytoarchitecture of neuronal tissues. F: Aspirin-treated rat’s hippocampal dentate gyrus appears normal with proper cell density, shape, and nuclear details, as to healthy tissue (Fig. 8).
 | Figure 8. Effect of the n-hexane fraction of Salvia officinalis on histopathology of rat brain tissue (H& E). A. Normal; B. Sham; C. I/R injury; D. NFSO (250 mg/kg); E. NFSO (500 mg/kg); and F. Aspirin (60 mg/kg). Yellow arrows indicate dead cells, pyknotic granular cells, and dead cells, whereas blue arrows indicate normal healthy cells with proper cellular details. [Click here to view] |
4. DISCUSSION
Ischemic stroke is a leading cause of mortality and morbidity worldwide, primarily due to oxidative stress, neuroinflammation, mitochondrial dysfunction, and blood-brain barrier disruption. These pathological events contribute to neuronal apoptosis and necrosis, exacerbating poststroke complications [26]. BCCA occlusion (BCCAO) in rats is widely recognized as a model that replicates key features of ischemic stroke, particularly cognitive impairment. Considering the central role of oxidative stress and inflammation in stroke pathology, antioxidants and anti-inflammatory agents from plants are potential therapeutic options [27]. Methanol and its fractions from Salvia officinalis leaves were investigated for their anti-inflammatory and antioxidant properties in the current study. In terms of DPPH scavenging activity, hydrogen peroxide scavenging activity, RBC hemolysis protection, and protein denaturation inhibition, the n-hexane fraction (NFSO) was shown to be the most effective. It was shown to have anti-inflammatory and antioxidant properties, and its activity was comparable to that of diclofenac sodium and ascorbic acid. These mentioned activities were due to the presence of bioactive phytochemicals such as ursolic acid, betulinic acid, luteolin, quercetin, myricetin, protocatechuic acid, and kaempferol, which were proven by LC-MS analysis. During ischemia-reperfusion, there is excessive generation of reactive oxygen species, including superoxide radicals, hydroxyl radicals, and hypochlorous acid, to the extent that it overwhelms endogenous antioxidant defenses, depleting catalase, SOD, and reduced GSH [28]. In accordance with earlier reports, the BCCAO showed a significant reduction in SOD, catalase, and GSH levels, in addition to increased MDA, a marker of LPO, compared to the normal control and sham control groups [29]. NFSO treatment markedly recovered the levels of antioxidant enzymes and lowered MDA accumulation in a dose-dependent manner, indicating its promise in reversing oxidative stress. Neuroinflammation is also a critical player in ischemic damage. Upon stroke, activated glial cells and infiltrated leukocytes release MPO, a primary pro-inflammatory enzyme that produces cytotoxic radicals at the inflammatory site. Increased MPO levels in stroke patients and preclinical models indicate its involvement in poststroke injury [30]. In the present study, BCCAO-induced increase in MPO was significantly blocked by NFSO treatment, validating its anti-inflammatory properties. NFSO treatment efficiently counters the BCCAO-induced oxidative stress and neuroinflammation as similar to aspirin, proving its effectiveness in the BCCAO acute injury. The presence of ursolic acid [31], betulinic acid [32], and limonin [33], which are known anti-inflammatory compounds, is probably responsible for this effect. Behavioral tests validated NFSO’s neuroprotective potential. The rats of BCCAO showed motor impairment in hanging wire and beam walking tests with a significant decrease in fall-off time and longer beam-crossing duration, but not in the normal control and sham control groups. Cognitive impairment was also observed in the Y-maze and plus maze tests, where ischemic rats had decreased spontaneous alternations and longer transfer latency time. Motor and cognitive dysfunctions were improved significantly after NFSO treatment in a dose-dependent manner (250 and 500 mg/kg); the improvement in cognitive dysfunctions with NFSO treatment was also similar to aspirin, a standard anti-platelet agent, which signifies its potential to counteract stroke-induced neurological impairment [34,35]. According to molecular docking studies, NFSO’s bioactive compounds may interact with MPO (1MHL) and Keap1–Nrf2 (1X2R), two important regulators of inflammation and oxidative stress, to provide its neuroprotective effects. Strong expected binding affinities on both targets were shown by luteolin-7-O-rhamnoside, suggesting that it may be a broad-spectrum modulator. Additionally, myricetin had a significant affinity for binding MPO, indicating potential anti-inflammatory properties. These results, when combined with in vivo observations, support the theory that NFSO modulates oxidative stress and inflammatory pathways to provide neuroprotective benefits. Other molecules, such as protocatechuic acid and limocitrin, showed target-specific interactions, contributing to their selective neuroprotection. Histopathological examination yielded additional confirmation of the protective effects of NFSO. TTC staining of brain sections in ischemic rats showed widespread infarction, whereas rats pretreated with NFSO had larger areas of viable (pink) tissue, denoting a smaller infarct size [36]. Ischemic rats had injury to the hippocampal dentate gyrus, a memory and cognitive function-relevant area, showing loss of granular cells and augmented pyknotic (apoptotic) neurons [37]. Treatment with NFSO kept the hippocampal architecture well maintained, reversing the granular cell loss and attenuating apoptotic changes. In summary, these results place NFSO as a potential neuroprotective agent that reduces oxidative stress, neuroinflammation, and neuronal injury after ischemic stroke. Its antioxidant and anti-inflammatory effects, as demonstrated by both in vitro and in vivo experiments, underscore its therapeutic value for poststroke recovery.
5. CONCLUSION
This study investigated the neuroprotective effects of the n-hexane fraction of Salvia officinalis on poststroke complications in rats. Pretreatment with the n-hexane fraction mitigated cognitive and motor deficits, oxidative stress, and neuroinflammation in a dose-dependent manner in a BCCAO model. LC-MS analysis revealed that luteolin, myricetin, protocatechuic acid, and limocitrin were the compounds with the best docking scores against the Keap1–Nrf2 complex and MPO. The neuroprotective effect of the n-hexane fraction may be attributed to the combined antioxidant and anti-inflammatory activities of its phytoconstituents, as suggested by LC-MS and docking analyses, which warrants further studies to delineate individual and interactive contributions.
6. ACKNOWLEDGMENTS
The authors are thankful to the AICTE-funded project under the Research Promotion Schemes (RPS) with File No.8-106/FDC/RPS (POLICY-1)/2019-20 and the DST-FIST facility of Raghavendra Institute of Pharmaceutical Education and Research (RIPER) for providing the necessary facilities for carrying out this project.
7. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
Ethical approval details are given in the ‘Materials and Methods’ section.
10. DATA AVAILABILITY
All the data is available with the authors and shall be provided upon request.
11. PUBLISHER'S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors, or the reviewers. This journal remains neutral about jurisdictional claims in published institutional affiliations.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they did not use artificial intelligence-assisted technology in writing, editing this manuscript, and no images were manipulated by AI.
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