Free Radicals

The leaf galls in different developmental stages as well as the ungalled leaves were collected from P. pinnata trees and washed thoroughly in distilled water for removing mites and debris. The cleaned samples were cut into small pieces using a sterilized sharp blade and dried at room temperature for 10–15 days in a container covered with a cotton layer. The dried samples were then powdered in a mechanical grinding machine (Mixer grinder), to facilitate effective contact between the solvent and the samples.
Using methanol, cold extraction was carried out based on the methods of Anjali Soni [38 ] with slight modifications (48 h at 20 °C through continuous shaking at 120 rpm in a rotary shaking incubator). The crude extracts were stored in air tight vials in a refrigerator for subsequent in-vitro antioxidant DPPH free radical scavenging assay, carried out following the methods of Blois [39 (link)]. The DPPH free radical scavenging assay was the general test used to evaluate the antioxidant capacity of plant extracts. Commonly, scavenging assay was expressed as IC₅₀, (the amount of antioxidant necessary to decrease the initial concentration of DPPH by 50%). The antioxidant activity and IC₅₀ values were negatively correlated, the higher IC₅₀ value indicated the lower antioxidant capacity and vice versa [38 ]. Three independent assays were conducted and inhibition percentage (I%) of different concentrations of extracts and controls were calculated by following the equation [38 ] given below; IC₅₀ calculated by using probit-regression analysis, this indicated how much of the extracts needed to scavenge or inhibit the 50% of free-radical.
$$\mathrm{I}\%=\frac{\text{Absorbance}\text{of}\text{control}-\text{Absorbance}\text{of}\text{test}}{\text{Absorbance}\text{of}\text{control}}\times 100$$

MRS was performed at 37°C on a Bruker 11.7 T spectrometer. Hyperpolarized ¹³C in vitro: 18 mg [1-¹³C]pyruvic acid (99% isotopically enriched, Sigma-Aldrich, UK containing 15 mM trityl free radical OX63, Oxford Instruments, UK) was polarized in a HyperSense® DNP polarizer (Oxford Instruments Molecular Biotools Ltd, UK) for 1 hour. The polarized sample was dissolved in 4 ml aqueous buffer (50 mM sodium lactate, 50 mM NaOH, 1 mM EDTA) resulting in a 50 mM pyruvate solution at pH 7, 37°C. A solution of 100 µl, 50 mM hyperpolarized [1-¹³C]pyruvate was mixed with 500 µl cell suspension, and ¹³C spectra were acquired every 2 s using a single scan and a 10° flip angle.

The anthropometric characteristics were recorded as described by Stavrou et al. (16 (link)). Tanita MC-980 (Arlington Heights, IL, USA) was used for body composition assessment. We performed the 6MWT, as described in the ATS guidelines, to assess the functional status of the patients (17 (link)). The parameters O₂ saturation (SpO₂), heart rate (HR) (Nonin 9590 Onyx Vantage, USA), blood pressure (Mac, Tokyo, Japan), and self-assessed lower limb fatigue and dyspnea (via Borg Scale CR-10) (18 (link)) were recorded at predetermined time-points of the 6MWT (16 (link)). The handgrip strength test was performed using an electronic dynamometer (Camry EH 101, South El Monte, CA, USA) (19 (link)). Patients were asked to perform as many repetitions as possible at a self-regulated pace (safe and comfortable) from a sitting-to-standing position while the arms were crossed at the shoulders so as not to use them as support to assess lower limb strength (30-s Sit-to-Stand test) (20 (link)). Blood sampling of 10 mL peripheral venous blood for the determination of reactive oxygen metabolites (d-ROMs test, free radical analytical system, FRAS5, Parma, Italy) was performed 20 min before physical fitness tests (21 (link)). Pulmonary function tests were performed according to the ATS/ERS guidelines (22 (link)) in the sitting position using a MasterScreen-CPX pneumotachograph (VIASYS HealthCare, Germany). Prior to physical fitness tests, all patients answered questionnaires to measure the quality and patterns of sleep using the Pittsburgh Sleep Quality Index (PSQI) (23 (link)), cognitive impairment was assessed using the Montreal Cognitive Assessment (MoCA) (24 (link)), STOP-Bang for stratification for obstructive sleep apnea risk (25 (link)), and (iv) work ability index (WAI) to investigate the ability to return to work without restrictions (26 ).
A stationary seated bike (Toorx, Chrono Line, BRX R 300) with bluetooth capabilities was used for the measurements. It was connected to the VR application, the Meta Quest 2 (Facebook Technologies, LCC, Hacker Way, Menlo Park, CA, USA) device headset and controllers, and a computer (27 (link)). This VR training system is called VRADA (VR exercise App for Dementia and Alzheimer's patients) version 4.1 and has been developed by ORAMA-VR and Biomechanical Solutions Engineering based on interviews with older people with mild cognitive impairment. The application of the VR training system includes cognitive exercises with simple math calculations and requests users to observe and count animals that appear in their VR to enhance cognitive health and motivational techniques to address the issue of low motivation for exercise. The system gives an opportunity for each participant to choose their exercise duration, landscape in which they will cycle (forest, beach, or snowy landscape), motivating words that they want to hear during their performance (“Calmly,” “I can,” “I will do it well,” “Very nice,” or no words) and the music to enjoy while cycling. VR controllers with raycast were used as a selection mechanism that allows the user to select an answer by pointing the ray at the button and pressing the trigger button at the controller. Moreover, participants received feedback during their performance, such as indications about cycling time, distance, and speed, and could self-monitor their performance using screen-provided data. They were requested to cycle at a constant speed of between 15 and 20 km/h¹. Simultaneously, speed and distance were recorded every 45 s. At the end of the cycling procedure, participants were informed their scores in math questions, the distance they covered, and they were asked to answer four more questions assessing if they were tired, if they liked the way they exercised, how many animals they saw, and if they repeated the motivational word. In this study, all participants performed the exercise in the forest.
The HR, SpO₂, and self-assessment of lower limb fatigue and dyspnea were performed before and at the end of each exercise condition (VR, no-VR, SSE-VR, and 6MWT) for each patient.