To prepare CBD extract, the protocol outlined by Peres et al. (2018) with modifications from Flores-Sanchez and Verpoorte (2008) was followed. Approximately 10.5 kg of dried industrial hemp (C. sativa L.) biomass was procured. The sample was high in CBD and low in THC content (<0.3%). The biomass was processed at the Pilot Plant Unit at the Pakistan Council of Scientific and Industrial Research (PCSIR). The hemp was first manually shredded and air-dried in a controlled environment at 40°C to maintain cannabinoid integrity. After reaching a moisture content below 10%, the dried material was finely milled and sieved to mesh # 40, ensuring a consistent particle size for optimal extraction performance. Before extraction, the milled hemp was subjected to decarboxylation at 110°C for 60 min in a convection oven to convert cannabidiolic acid (CBDA) to its active neutral form, CBD.

The extraction was carried out using a supercritical CO₂ (SC–CO₂) extraction unit (Model SCFE-220-40-20L, Careddi Technologies, China), following parameters optimized from Kammerer et al. (2022) and Wang et al. (2021). A total of 2.5 kg of decarboxylated hemp powder was packed into the stainless steel extraction chamber. The vessel was sealed, followed by gradual heating to 50°C. Supercritical CO₂ was pumped into the vessel at a pressure of 32 MPa, well within the supercritical phase. During the extraction process, ethanol (10% w/w of CO₂) was used as a cosolvent to increase CBD solubility and extraction efficiency. CO₂ was maintained at a flow rate of 5 kg/h, and ethanol at 1 kg/h. The mixture was circulated through the hemp matrix for 3 h, after which the process was stopped upon stabilization of extract yield. The CBD-rich effluent was collected in a separation vessel where pressure was reduced to atmospheric levels, leading to the precipitation of cannabinoids and evaporation of CO₂.

The collected crude extract was transferred to a round-bottom flask. Rotary evaporation was performed at 40°C under 150 mbar vacuum pressure and 120 rpm rotation speed to remove residual ethanol. The process continued for 45–60 min until a viscous, amber-colored, CBD-rich extract was obtained. Final yield was measured by subtracting the flask’s pre- and post-weights.

Winterization was used to remove lipids and waxes from the raw cannabis extract. The crude oil was mixed with food-grade ethanol at a ratio of 10 mL of ethanol for every gram of extract. For 600 g of crude oil, approximately 6 L of ethanol was used. To ensure complete dissolution, the ethanol was preheated to about 40°C, resulting in a uniform dark solution as described by Wang et al. (2017). The solution was then stored at −80°C for 2 h to allow wax precipitation. Optimal precipitation occurred within 2 h at −80°C or 48 h at −20°C, as noted by Hazekamp (2008). Vacuum filtration was performed while the solution remained cold, initially using coarse filters (20–50 µm) followed by fine filters (5 µm), for efficient wax removal. Rotary evaporation at approximately 40°C under vacuum was then used to concentrate the winterized solution, remove ethanol to purified winterized oil, and confirm the removal of waxes, as reported by De Meijer (2009).

Analytical quantification of CBD was performed using Gas Chromatography–Mass Spectrometry (GC-MS), following derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% TMCS.

A stock solution of CBD was prepared by dissolving 10 mg of pure CBD reference standard (≥99%, Sigma-Aldrich) in 10 mL of HPLC-grade methanol, yielding a concentration of 1.0 mg/mL. Serial dilutions were prepared to obtain standard solutions in the range of 0.1–1.0 µg/mL.

Samples and standards were derivatized at 70°C for 30 min. GC-MS analysis was carried out using a Shimadzu GC-MS-QP2010 system equipped with a 5975C mass detector and an HP-5MS capillary column. The injector temperature was 280°C, and helium was used as the carrier gas at a flow rate of 1.0 mL/min. The oven was programed as initial temperature 100°C (held for 1 min), ramped to 280°C at 10°C/min, and held for 5 min. Figure 1 illustrates the calibration curve used for CBD quantification.

To assess treatment efficacy, the Unified Parkinson’s Disease Rating Scale (UPDRS) was administered by trained clinicians who were blinded to the treatment assignments. Assessments were conducted at baseline and post-intervention (8 weeks). Both motor and nonmotor symptoms were scored. Throughout the study, patient diaries, monthly check-ins, and adherence monitoring were used to record compliance and any side effects related to CBD intake.

Clinical assessments were conducted at baseline (Day 0) and at the end of the 8-week intervention period (Day 56). The primary outcome measure was the change in PD symptom severity, meticulously evaluated using the UPDRS parts I (Nonmotor experiences of daily living), II (Motor experiences of daily living), and III (Motor examination). Experienced neurologists, blinded to group allocation, administered all UPDRS assessments to minimize observer bias. Additionally, patient-reported outcomes were captured, including assessment of quality of life and perceived symptom changes, though detailed analysis of these may be beyond the scope of this focused paper. Safety and tolerability were rigorously monitored throughout the study, with all adverse events (AEs) reported by participants or observed by clinicians being recorded. Blood samples were collected at baseline and endpoint for routine biochemistry and hematology analyses to monitor systemic health and potential treatment-related side effects.

Data analysis was performed using SPSS (v20) and Statistix 8.1. For between-group comparisons of treatment effects, a two-factorial analysis of variance (ANOVA) was conducted using Statistix 8.1 software. This approach evaluated the statistical significance of differences among various treatment conditions, accounting for interactions between multiple factors. A significant threshold of P < 0.05 was used to determine meaningful improvements.

During the current study, CBD was extracted successfully from industrial hemp biomass using supercritical carbon dioxide (SC-CO₂) technology. A predecarboxylation step at 110°C was employed to convert the inactive CBDA into its active neutral form. A yield of 13.53% (w/w) was noted upon confirmation by GC-MS analysis. The cannabinoid-rich extract was further concentrated via rotary evaporation, which removed residual ethanol under controlled vacuum and temperature conditions, yielding a dense amber-colored oil. Table 1 shows all the findings related to the CBD extraction and characterization process.

The use of ethanol as a cosolvent proved critical in increasing extraction yield by disrupting cellular matrices and enhancing the solvating power of CO₂, especially for lipophilic compounds such as CBD. The sample chromatograms showed well-resolved peaks corresponding to the CBD standard, indicating high purity and accuracy of quantification (Figures 2 and 3). Based on the yield obtained, approximately 2.22 g of this extract was required to deliver a 300 mg daily dose of CBD, a quantity deemed both pharmacologically effective and clinically tolerable in PD management. This precise quantification and standardization allowed for uniform dosing across all participants, ensuring the reliability and reproducibility of the clinical outcomes.

The GC-MS results confirmed the presence of CBD in the sample. This was done by matching its retention time and characteristic fragmentation pattern with standards. The GC-MS findings are shown in Figure 4. The elution of CBD at a consistent retention time and diagnostic ions at m/z 231 and 246 reflects a cleavage at the pentyl side chain and aromatic moiety, respectively. The findings were similar to those of Alonso et al. (2020). Additionally, the absence of interfering signals confirms the CBD and demonstrates the method’s specificity and reliability in complex plant matrices.

The administration of CBD as an adjunct to levodopa (Sinemet) therapy led to a significant improvement in both motor and nonmotor symptoms among patients diagnosed with idiopathic Parkinson’s disease. This improvement was objectively measured using the UPDRS, a gold standard clinical tool. The baseline UPDRS scores were comparable between the Sinemet-only control group and the Sinemet + CBD intervention group. However, a mean improvement of 51.2 ± 4.5 points was observed when the patients were treated with the Sinemet + CBD group, compared to the control group, which showed only an average gain of 36.4 ± 3.2 points. This marked difference in mean scores underscores the potential of CBD in significantly enhancing therapeutic outcomes when used as a complementary intervention.

The therapeutic benefits of CBD extend across a wide range of symptoms. On the motor side, participants reported a substantial reduction in tremor intensity, muscular rigidity, bradykinesia, and difficulties with gait and posture. On the nonmotor front, there were clinically significant improvements in symptoms such as sleep disturbances, sensory complaints, depression, and anxiety, areas where conventional levodopa therapy often falls short. The multifaceted interaction of CBD with the endocannabinoid system (ECS), including CB1, CB2, and serotonin (5-HT1A) receptors, may account for its broad therapeutic profile. This finding reinforces the notion that CBD not only complements dopaminergic therapy but also independently addresses pathophysiological components of PD that are beyond the dopaminergic deficit. The significant improvements across both motor and nonmotor symptoms observed in the Sinemet + CBD group (Tables 2–4) are consistent with the multifaceted pharmacological profile of CBD. While levodopa primarily addresses dopaminergic deficits, CBD’s therapeutic breadth likely stems from its diverse interactions with the ECS and other non-ECS targets. For instance, the reported anxiolytic and antidepressant effects of CBD, which may contribute to the improvements in nonmotor symptoms like mood and motivation (Table 4), are often linked to its modulation of serotonin 5-HT1A receptors. Furthermore, its anti-inflammatory and antioxidant properties are crucial in mitigating neuroinflammation and oxidative stress, key pathological hallmarks of PD, thereby potentially contributing to improvements in motor symptoms by supporting neuronal integrity and function. The reduction in levodopa-induced dyskinesias, though not a primary outcome, may be related to CBD’s indirect modulation of cannabinoid receptors (CB1 and CB2) in the basal ganglia, which are implicated in motor control and dyskinesia pathophysiology. This broad symptomatic improvement suggests that CBD acts via distinct yet complementary pathways to levodopa, offering a more holistic approach to PD management.

While dietary protein intake is known to interfere with levodopa’s absorption due to competitive inhibition via large neutral amino acid transporters, the interaction between CBD and protein remains less well characterized. However, the current study revealed that a moderate interaction may exist. Patients receiving CBD alongside protein-controlled diets (i.e., evening-only high-protein meals) demonstrated a slightly greater improvement in UPDRS scores (53.6 ± 4.1) compared to those who continued with unrestricted protein intake (48.9 ± 4.8). Though the magnitude of difference was modest, the interaction reached statistical significance (P < 0.05), suggesting that structured protein timing may support optimal CBD efficacy, potentially by modulating gastrointestinal absorption or hepatic metabolism.

This finding is clinically relevant, especially given the increasing interest in nutrition-based modulation of pharmacotherapy. While CBD’s absorption is not as protein-sensitive as levodopa’s, the observed enhancement suggests a potential role of protein redistribution diets (PRD) even in non-levodopa-based therapies. These results warrant further pharmacokinetic investigations to determine the exact mechanisms underlying this interaction.

The observed subtle yet statistically significant modulation of CBD’s efficacy by dietary protein intake, as evidenced by differences in UPDRS gain between protein-controlled subgroups (Table 5), warrants deeper consideration. While the precise mechanisms underpinning this interaction require further investigation, existing literature on levodopa’s absorption provides a crucial framework. Levodopa, an amino acid, competes with dietary large neutral amino acids (LNAAs) for transport across the intestinal barrier and the blood–brain barrier via the L-type amino acid transporter 1 (LAT1). A high-protein meal can lead to increased plasma LNAA concentrations, subsequently reducing levodopa’s systemic availability and central nervous system uptake. Although CBD is not an amino acid, its absorption and metabolism could theoretically be influenced by the gut environment, which is significantly altered by macronutrient intake. For instance, changes in gastric emptying time or bile acid secretion due to protein consumption might indirectly affect CBD’s dissolution, absorption kinetics, or first-pass metabolism. This finding underscores the complex interplay between pharmacotherapy and nutritional factors in PD management, suggesting a pathway for personalized dietary strategies to potentially optimize the therapeutic window of both levodopa and adjunctive CBD.

Over the 8-week clinical intervention, CBD demonstrated a favorable safety profile, with no serious adverse events recorded across the treatment cohort. Mild side effects were reported in a small subset of patients, with the most common being drowsiness (6.7%), dry mouth (5%), and occasional gastrointestinal discomfort (3.3%). These effects were transient and did not necessitate discontinuation of treatment. Notably, there were no reported cases of psychiatric symptoms, hallucinations, or worsening of motor fluctuations—side effects occasionally seen with dopaminergic therapy or psychoactive cannabinoids like THC. This affirms CBD’s status as a nonpsychoactive, well-tolerated therapeutic compound.

Routine follow-ups, adherence checks, and patient diaries confirmed strong compliance and high acceptability of CBD among participants. This supports its feasibility for integration into long-term treatment.

The current study evidenced that CBD, when used in conjunction with the ongoing levodopa therapy, can efficiently manage the PD symptoms, particularly those that are previously inadequately addressed by dopaminergic drugs. The efficacy can be monitored in motor and nonmotor symptoms. Additionally, the potential interaction with protein intake suggests that personalized nutritional planning could further optimize outcomes.

Similarly, the chronic nature of PD and the progressive decline associated with long-term levodopa monotherapy and the integration of phytotherapeutic compounds like CBD offer a multifaceted strategy for symptom control and possibly disease modification. These findings pave the way for more robust, long-term clinical trials with larger cohorts and biomarker analyses to validate the observed effects and elucidate the molecular pathways involved. Additionally, future studies could explore CBD’s potential neuroprotective roles and its capacity to delay disease progression, beyond mere symptomatic relief.

Our findings further elucidated the role of dietary protein in modulating CBD’s efficacy. While CBD demonstrated significant improvements across various motor and nonmotor symptoms independently (e.g., as shown by average UPDRS score improvements in sensory complaints related to Parkinsonism (1.92), tremor (1.88), and walking and balance (1.83) in the CBD group, the synergistic effect was notable when CBD was administered alongside protein supplementation. For instance, in the CBD with protein group, the average improvement in sensory complaints related to Parkinsonism was 2.25, turning in bed was 2.21, and tremor was 2.08. These results suggest that while CBD is effective alone, a personalized dietary approach incorporating protein could potentially optimize therapeutic outcomes, especially for specific symptoms like turning in bed and tremors. It is critical to consider the well-established impact of dietary protein on levodopa absorption in PD management. Large neutral amino acids (LNAAs) present in protein-rich foods compete with levodopa for transport across the intestinal lumen and the blood–brain barrier. This competition can lead to fluctuations in levodopa plasma levels and reduced central nervous system availability, potentially causing motor complications. Therefore, while our study highlights CBD’s adjunctive benefits, careful dietary planning regarding protein intake is crucial to maximize levodopa’s therapeutic window and prevent motor fluctuations, alongside exploring synergistic effects with compounds like CBD.

This study demonstrated that CBD, when used alongside levodopa, offers a meaningful enhancement in both motor and nonmotor symptom control in PD. Notably, the integration of dietary protein timing further amplifies therapeutic gains, underscoring the importance of a multidimensional approach to PD management. As one of the first randomized trials to explore this triad of CBD, levodopa, and protein modulation, the findings provide a compelling foundation for rethinking adjunctive strategies in PD care. Given the favorable safety profile of CBD and its capacity to address symptoms unresponsive to conventional therapies, this research supports its consideration as part of future integrated treatment protocols. Large-scale trials are warranted to validate these results and explore long-term neuroprotective potentials.

Future research should focus on conducting larger, long-term randomized controlled trials to validate the sustained efficacy and safety of CBD as an adjunctive therapy in PD, alongside exploring optimal dosing strategies and formulations. Integrating biomarker analysis in these trials is crucial in objectively confirming symptomatic improvements and elucidating underlying neurobiological mechanisms. Furthermore, continued investigation into refined personalized nutritional strategies, particularly concerning dietary protein and its interaction with CBD and levodopa, is recommended to optimize therapeutic outcomes. Finally, delving into CBD’s potential neuroprotective effects beyond symptomatic relief should be a key area of future inquiry to assess its capacity for disease modification.

This study, though promising, has several limitations. First, the relatively small sample size of 96 participants and the 8-week intervention period limit the generalizability and long-term assessment of CBD’s efficacy and safety in PD. Second, though dietary protein was controlled, detailed pharmacokinetic analyses of CBD or levodopa to protein timing were not conducted, thus precluding a precise mechanistic understanding of their interaction. Future research should address these limitations through larger, long-term trials incorporating comprehensive pharmacokinetic studies and objective biomarker analyses to further validate and optimize CBD as an adjunctive therapy for PD.