The seeds of M. pruriens were collected from Karnataka, India. The seed powder was subjected to extraction using methanol, ethanol, petroleum ether, chloroform, and water solvents (Senguttuvan et al., 2014; Kalebar et al., 2020). The qualitative analysis of the solvent extracts revealed that the methanol extract of M. pruriens seeds possessed a superior phytochemical profile additionally supported by the in vitro antioxidant assays (Lampariello et al., 2012; Rane et al., 2019). Hence, in the present study methanol extract of M. pruriens seeds was used. Methanol extract of M. pruriens seed will be denoted as M. pruriens seed extract, henceforth.

Methanol extract of M. pruriens seeds was subjected to determination of total alkaloids, flavonoids, phenols as per standard protocol (Senguttuvan et al., 2014; Kalebar et al., 2020). The antioxidant efficiency of M. pruriens seed extract was determined by hydrogen peroxide scavenging assay and 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) radical scavenging assay as per standard protocol (Brand-Williams et al., 1995; Siddhuraju and Becker, 2003; Keser et al., 2012).

By using HPLC, the bioactive molecules, namely, L-DOPA and β-sitosterol were identified from Mucuna prurines seed extract (Kshirsagar et al., 2016). Waters HPLC system (Waters/Millipore, Milsford, MA, United States) which included a model 515 pump, a model 2,487 dual wavelength absorbance detector was employed. A C18 column was used. The mobile phase was constituted by A (water) and B (acetonitrile). The gradient range varied linearly from 50% to 90% B in 4 min with injection volume 2 mL for the RRHT column. The flow rate was 1.0 mL/min, the column temperature was maintained at 30°C, and the detection wavelength was 280 nm (Pawar et al., 2010). The extraction of bioactive molecule β-sitosterol from M. pruriens seed extract was done by Reversed-Phase Flash Chromatography. The isolation was carried out in Biotage Flash 75 system (Biotage, A Division of Dyax Corporation, Charlottsville, VA, United States) (Herrera et al., 2020).

Ethical approval for the study was obtained from Institutional Animal Ethics Committee (IAEC) (No.08/BLDE (DU)/2021). The animal care guidelines set forth by the Committee for the Purpose and Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests (Animal Welfare Division), Government of India were strictly adhered to throughout the study.

The study involved 48 male Wistar rats, aged 8–10 weeks, weighing 180–250 gm. The experimental animals were maintained at 22°C–24°C and a 12-h light/12-h dark cycle with food and water ad libitum. The animals were acclimatized to the laboratory conditions for 7 days before initiating the experimental protocol.

The experimental animals (n = 48) were allocated to one of the four groups. Group I (Untreated + LCCAO) served as control. No pre-treatment. Cerebral ischemia induced (on day 29) by LCCAO for 75 min followed by reperfusion for 12 h. Group II (β-sitosterol + Sham)—Pre-treatment with β-sitosterol, 10 mg/kg/day, (Ayaz et al., 2017), oral, 28 days + sham-operated (on day 29). Group III (β-sitosterol + LCCAO) - pre-treatment with β-sitosterol, 10 mg/kg/day, oral, 28 days + cerebral ischemia induced (on day 29) by LCCAO for 75 min followed by reperfusion for 12 h. Group IV (methanol extract + LCCAO) - pre-treatment with methanol extract of Mucuna pruriens seeds, 50 mg/kg/day (Nayak et al., 2017), oral, 28 days + cerebral ischemia induced by LCCAO for 75 min followed by reperfusion for 12 h (on day 29).

The bodyweight of rats was measured using digital weighing machine (Practum 1102-10IN, Germany). The experimental animals in each group were matched for the body weight at the study onset. Daily food consumption of each rat was monitored during the intervention period.

Cerebral ischemia was induced by left common carotid artery occlusion (LCCAO) for 75 min followed by 12 h reperfusion (Das et al., 2018). The surgical procedure for inducing cerebral ischemia was performed in the morning hours in overnight fasted rats. The animals were subjected to the procedure after the intervention period of 28 days (i.e., on day 29). Strict aseptic precautions were taken during the surgical procedure. The experimental animals were anesthetized with ketamine (60 mg/kg body weight, i. p.) and xylazine (6 mg/kg body weight). An oblique incision was taken alongside sternocleidomastoid in the neck from the base of the mandible to sternum. At the level of greater of cornu of thyroid cartilage, deep fascia and carotid sheath were incised. The common carotid artery was exposed and separated from the fascia, while protecting the vagus and jugular vein from any damage. A nylon thread (0.5 mm) was passed from behind the artery and ligated. The occlusion was maintained for 75 min following which the ligature was released and allowed for 12 h reperfusion. The deep fascia and carotid sheath were closed with proline. Superficial fascia and skin were closed with all aseptic precautions. Sham operation (group II) involved the same surgical steps except for left common carotid artery occlusion. The experimental animals were closely monitored for physiological parameters like heart rate, blood pressure and respiratory rate. Non-Invasive Blood Pressure was recorded with a tail cuff sensor (Biopac NIBP200A model). For heart rate, ECG was recorded using needle subcutaneous electrodes (Biopac Student Lab 4.1). For respiratory rate, pneumogram was recorded using respiratory pad transducer (Biopac Student Lab 4.1). Body temperature was maintained at 36°C–37.5°C using a warm pad. The surgery was uneventful and all the rats recovered following surgery.

Post-ischemic neurological deficit was assessed after 12 h of reperfusion and before sacrifice using a six point scale neurological score: 0 = no neurological deficit; 1 = failure to extend contralateral forelimb fully on lifting the animal by tail; 2 = circling to the contralateral side; 3 = falling to the contralateral side; 4 = no spontaneous walk or in a comatose state; 5 = death (Komatsu et al., 2021).

The experimental animals were sacrificed (n = 6/group) after 12 h reperfusion. The brain was carefully dissected. For gene expression analysis, the left cerebral hemisphere (occluded side) was used. Tissue was immediately stored in RNAlater at −80°C for RT-PCR analysis.

NMDA receptor, tau protein and β-actin (endogenous control) mRNA expressions were assessed by using quantitative reverse transcription-PCR (RTPCR). RNeasy kit was used to extract RNA from left cerebral hemisphere homogenates as per the manufacturer’s protocol. Reverse transcription of RNA was done using SuperScript II Reverse Transcriptase as per the prescribed protocol. cDNA was amplified with quantitative reverse transcription-PCR (Kong et al., 2021). The gene expression of NMDAR and Tau protein are presented as fold change calculated by using the formula 2^-ΔΔCq (Herur et al., 2022). PCR primers specific to each gene are as depicted in Table 1. The PCR conditions are provided as Supplementary Table S1.

The animals (n = 6/group) were sacrificed after 12 h reperfusion. Brain was carefully dissected and initially fixed in bouin’s fluid and shifted to 10% neutral buffered formalin 24 h later. For histopathological examination, 2–3 mm thick slices of cerebral hemispheres were made. Bilateral blocks of the brain were embedded in paraffin wax and 2.0 to 3.0 μ sections were cut on a rotary microtome and stained with hematoxylin and eosin (H and E). Blocks included sections from the body of the corpus callosum with coronal section of white matter, splenium of the corpus callosum, posterior limb of the internal capsule with adjacent thalamus, and rostral pons. The stained sections were examined under light microscope (Olympus CH20i) with Samsung Digital Color Camera (Model No. SDC-242, N. J 07094, United States).

HPLC chromatograms of standard solutions of L-DOPA and β-sitosterol and methanol extract of M. pruriens are presented in Figure 1. The calibration curve showed good linearity with correlation coefficients (R²- 0.9975). Based on the data, the estimates of L-DOPA and β-sitosterol in the M. pruriens seed extract was 20mg/100 gm and 12mg/100 gm respectively.

The comparison of physiological parameters before and after surgery as presented as Supplementary Table S7. Heart rate, SBP, DBP and respiratory rate were similar between groups before surgery. After surgery there was an increase in heart rate, SBP, DBP and respiratory rate across all groups. Heart rate and SBP were higher in group I (control) compared to other groups.

Table 2 presents comparison of neurological deficit scores among groups. Neurological deficit score was higher in group I (Control, Untreated + LCCAO), group III (β-sitosterol + LCCAO) and group IV (methanol extract + LCCAO) compared to group II (β-sitosterol + Sham). The scores were lower in group III (β-sitosterol + LCCAO) and IV (methanol extract + LCCAO) compared to group I (Control, Untreated + LCCAO) although not statistically significant.

Table 3 presents Real time PCR results for NMDAR and Tau protein mRNA expression in experimental animals. mRNA expression of NMDAR and Tau protein was higher in Group I (control,Untreated + LCCAO) compared to group II (β-sitosterol + sham). In the pretreated groups, group III (β-sitosterol + LCCAO) and group IV (methanol extract + LCCAO) mRNA expression of NMDAR and Tau protein was lower compared to group 1 (control, Untreated+LCCAO).

Figure 2 presents the photomicrograph of cerebral cortices stained with H&E. In Group I (control, Untreated + LCCAO), left cerebral hemisphere (occluded side) showed cerebral edema (gross alteration), altered microscopic architecture with scattered cellular layers of grey matter and white matter. Neuronal damage (red neurons) with eosinophilic cytoplasm and pyknotic nuclei with a hazy appearance were noticed. Localized cystic infarcts with features of diffuse cerebral infarction were seen (Figure 2A - i, ii). There were no microscopic alterations in the left cerebral hemisphere of group II (β-sitosterol + Sham) (Figure 2B - i, ii). In Group III (β-sitosterol + LCCAO) (Figure 2C - i, ii) and IV (methanol extract + LCCAO) (Figure 2D - i, ii), left cerebral hemisphere (occluded side) showed less ischemic changes compared to group I (control, LCCAO). Right cerebral hemisphere of the brain (non-occluded side) showed no evidence of ischemic changes in all groups (Figures 2A-D - iii, iv). However, mild increase in intracellular space was noticed with no evidence of changes in corticomedullary junction in group I (Figure 2A - iii, iv).