Guar gum

Propranolol hydrochloride was purchased from Rouz Darou Pharmaceutical Co., Iran. Xanthan gum, guar gum and sodium alginate were obtained from Silverline Chemicals, India. Hydroxypropylmethyl cellulose 4000 (HPMC 4000) and sodium carboxymethyl cellulose (Na CMC) were from Shin-Estu Chemical Co., France. Carbomers including carbomer 934 (C934) and carbomer 940 (C940) were from BF Goodrich, Germany. Methanol, anhydrous citric acid, disodium hydrogen phosphate, propylene glycol and lactic acid were all purchased from Merck, Germany.
Preparation of propranolol hydrochloride gel formulationsDifferent classes of excipients usually incorporated in order to prepare vaginal gels include gelling agents, humectants, preservatives and vehicles (8 (link)). For this purpose, different concentrations of various mucoadhesive polymers including the natural polymers guar gum (in the range of 1-4% w/w), sodium alginate (in the range of 4-7% w/w) and xanthan gum (in the range of 2-5% w/w), and semi-synthetic polymers HPMC 4000 (in the range of 3-5% w/w) and Na CMC ( in the range of 4-7% w/w), as well as the synthetic polymers C934 and C940 both in the range of 0.5-2.0% w/w; were utilized to develop the gels. In order to formulate the mucoadhesive gels containing the drug, gelling agent was dispersed slowly in an aqueous-based solution containing propranolol hydrochloride (1.6% w/w, as the active ingredient), propylene glycol (5.0% w/w, as humectant) and sodium benzoate (0.25% w/w, as antimicrobial preservative), with the help of an overhead stirrer. The pH of the vagina is maintained by lactobacilli which produce sufficient lactic acid to acidify vaginal secretions to pH 3.5-4.5. The pH is important in terms of design and the efficacy of drug delivery systems (11 (link), 15 ). Hence, the pH of each formulation was adjusted to 4.0 (so as to be within the normal vaginal pH range) by the addition of lactic acid. Excipients are usually chosen from those materials which are deprived of therapeutic activity. Nonetheless, it is not always true; as it can sometimes be advantageous in the development of a pharmaceutical system (7 ). In this study, the main purpose of incorporating lactic acid into the formulations was its spermicidal activity (16 ). The composition of polymers within each of the gel formulations is given in Table 1. The prepared gel formulations were then tested on the basis of physical appearance, apparent viscosity, spreadability and strength of mucoadhesion. Then, four of these formulations were selected (named as chosen formulations) and underwent further examinations including determination of in-vitro drug release properties and drug release kinetic studies. Among these formulations, one formulation was selected as the final propranolol HCl gel formulation, which was then assessed in terms of complementary tests including propranolol HCl content within the gel as well as the duration of mucoadhesion.
Measurement of spreadability of gel formulationsThe area of spreadability of each propranolol HCl gel formuation, was determined using the following technique: five hundred milligrams (0.5 g) of the gel formulation was placed within a circle of 1 cm diameter, premarked on a glass plate, over which a second glass plate was placed. A weight of 500 g was allowed to rest on the upper glass plate for 5 min. The increase in the diameter due to spreading of the gel was noted (17 (link)) and then the spreading area was calculated using Equation 1, representing the area of a circle. This test was performed in triplicate and the data obtained expressed as mean ± standard deviation (SD).
A = π r² (Equation 1)
In the above equation, A is the area of the circle formed due to spreading of the gel (cm²), and r is the radius of the circle (cm).
Assessment of the mucoadhesive strengths of the gelsIn order to evaluate the mucoadhesive strength of the prepared propranolol HCl gel formulations, the apparatus shown in Figure 1 was used. This apparatus was principally similar to those described in previous studies (18 , 19 ). The upper stationary platform was linked to a balance, measuring the force needed to break contact between the gel and the mucosal membrane. The test cell was filled with pH 4.5 citrate-phosphate buffer, maintained at 37°C. Freshly removed sheep vaginal mucosa was used as the model mucosal membrane, and fixed in place over the two cylindrical platforms of the test apparatus and allowed to equilibrate in this solution for 2 min. Five hundred milligrams (0.5 g) of each gel formulation was then individually sandwiched between the two mucosa-covered platforms. Gels were kept in place for 5 min and then a constantly increasing force of 0.1 g/s was applied on the adhesive joint formed between the vaginal mucosa and the test gel, by gradually lowering the lower platform. This trend was continued until the contact between the test gel and the mucosa was broken and the maximum detachment force measured, was recorded. This force was taken as the strength of mucoadhesion of the test sample. Each experiment was run in triplicate, and results were expressed as mean ± SD.
Determination of in-vitro drug release profiles from the chosen propranolol HCl gel formulationsThe in-vitro release of propranolol HCl was determined from the chosen vaginal gel formulations using a dialysis tubing (MWCO of 12400 D; 99.99% retention, Sigma-Aldrich, USA) placed in the release medium under constant magnetic stirring. Five grams (5.0 g) of the gel formulations, were individually packed into sections of dialysis tubing (the length and the width of each section were 50 and 40 mm, respectively) with the ends being tightly fastened. The release medium was 200 mL of 0.1 M citrate-phosphate buffer (pH = 4.5). The medium was maintained at 37°C and stirred continuously at 100 rpm. Five mL (5.0 mL) aliquots of the release medium were withdrawn at predetermined time intervals and replaced by fresh citrate-phosphate buffer, to provide sink condition. Each withdrawn sample was further diluted with pH 4.5 citrate-phosphate buffer and it’s absorbance measured using uv-visible spectrophotometer (Shimadzu uv-visible 120A, Japan) at a λ _max of 289.2 nm. The absorbance was converted to drug concentration using the linear calibration curve constructed (Absorbance = 0.0196 Concentration (mg/L) – 0.0114; R² = 0.9995) and then cumulative percentage of propranolol HCl released was calculated with the help of a dilution factor. All measurements were performed in triplicate (n = 3).
In-vitro drug release kinetic studies of the chosen propranolol HCl gel formulationsIn order to study the release kinetics of the chosen propranolol HCl gel formulations, data obtained from in-vitro drug release studies were fitted into different kinetic mathematical models. These models were as follows: zero order (Equation 2), as cumulative percentage of drug released vs. time, first order (Equation 3), as Log cumulative percentage of drug remaining vs. time, and Higuchi’s model (Equation 4), as cumulative percentage of drug released vs. square root of time.
Q = Q₀ + K₀ t (Equation 2)
Where Q is the amount of drug released, Q₀ is the initial amount of the drug in the solution (it is usually zero), K₀ is the zero order rate constant expressed in units of concentration/time and t is the time.
LogC = LogC₀ – K₁t /2.303 (Equation 3)
Where C₀ is the initial concentration of the drug, K₁ is the first order release rate constant and t is the time.
Q_t = K_H t^1/2 (Equation 4)
Where Q_t is the amount of drug released in time t and K_H is the Higuchi’s model release rate constant reflecting the design variables of the system (20 (link)).
In order to evaluate the mechanism of drug release from the prpranolol HCl gel formulations, the first 60% drug release data were fitted in the Korsmeyer-Peppas model (Equation 5), as Log cumulative percentage of drug released vs. Log time.
M_t /M_∞ =Kt ⁿ (Equation 5)
Where M_t /M_∞ is the fraction of drug released at time t, K is the rate constant and n is the release exponent (20 (link), 21 ). The n value is used to characterize different release mechanisms, as given in Table 2 for cylindrical shaped matrices.
Determination of drug content within the final gel formulationFor determination of drug content within the final propranolol HCl gel formulation) of the gel was weighed in a 100 mL volumetric flask and then, 10.0 mL methanol was added to it (17 (link)). The content of the flask was stirred vigorously until the gel got completely dispersed to give a clear solution. The volume was adjusted to 100 mL with citrate-phosphate buffer pH=4.5. The obtained solution was diluted appropriately (dilution factor = 10) by the addition of pH 4.5 citrate-phosphate buffer and absorbance was measured in a uv-visible spectrophotometer (Shimadzu uv-visible 120A, Japan) at λ _max = 289.2 nm.
The absorbance was converted to drug concentration, using the linear calibration curve mentioned earlier. Then, the exact amount of the drug in the tested gel formulation was calculated with the help of dilution factor. This test was performed 3 times and the mean value ± SD was calculated.
Determination of duration of mucoadhesion of the final formulation The apparatus used for this study was based on that described in previous studies (19 , 22 ). The test apparatus (Figure 2) was composed of six upper and six lower cylindrical platforms within a clear jacketed perspex cell, filled with pH 4.5 citrate phosphate buffer. Freshly removed sheep vaginal mucosa (used as the model mucosal membrane) was mounted securely in place, mucosal side up-wards, on each of the platforms and allowed to equilibrate for 2 min. The test gel was then sandwiched between the two platforms and allowed to stand for 5 min. Next, through two pulley systems, a 7.0 g weight was applied on each upper platform (this weight was chosen through initial studies). As soon as the contact between the test gel and the mucosal surface broke, a small flap dropped onto a photocell detector, stopping the timer device (recording the elapsed time to 0.1 min) and measured the duration of mucoadhesion of the gel.
Statistical analysisData obtained from spreadability and strength of mucoadhesion of propranolol HCl gel formulations, were analyzed using the one way ANOVA and Tukey post-hoc test. Differences were considered to be significant at p < 0.05. The statistical package SPSS version 19.0 was used for data analysis.

The nationwide register-based Finnish Study on Parkinson’s disease register-based FINPARK study includes 22,189 people who received clinically confirmed PD diagnosis during the years 1996–2015 and were community-dwelling at the time of diagnosis.
People with PD diagnosis were identified from the Special Reimbursement Register maintained by the Social Insurance Institution of Finland (SII). Originally, 29,942 people eligible for reimbursement of anti-Parkinson drugs were identified, but as these drugs can also be used for other reasons, we excluded those who did not have ICD-10 code for PD (G20) recorded in the Special Reimbursement Register (n = 1244), those who were < 35 years old at the time of PD diagnosis (N = 53) and those who had diagnoses whose symptoms may be confused with PD (n = 6456) within 2 years of PD diagnosis, which lead to a cohort of 22,189 people. The exclusion diagnoses are listed in Supplementary Table 1. These people were excluded, as diagnosis of PD and its differential diagnostics is challenging, and false diagnoses are common in the early phase [16 (link), 17 (link)]. The proportion of excluded people (25.9%) in our study is within the range of estimated proportion of false diagnoses [16 (link), 17 (link)].
The application for special reimbursement includes anamnesis of the patient and description of the characteristic clinical features of PD including bradykinesia, rigidity and tremor. These applications are centrally reviewed in the SII. Special reimbursement for PD medications is granted if predefined criteria for PD diagnosis are fulfilled and diagnoses must be confirmed by a neurologist. Diagnosis of PD was based on United Kingdom Parkinson’s Disease Society Brain Bank’s criteria [18 (link)].
An age (+/− 1 year), sex and region-matched comparison cohort was identified from the SII database covering all residents. The index date was the date of PD diagnosis for the matched referent. The comparison people were not allowed to have purchases of PD medication (Anatomical Therapeutic Chemical classification ATC code N04) or the reimbursement code ever before the index date or 12 months after and during the diagnosis month of the referent people with PD. They also had to remain alive and community-dwelling during the month of index date. The diagnosis-based exclusion criteria of comparison people was otherwise similar to that of the PD cohort, but dementia due to PD (ICD-10 F02.3) was added to list of exclusion criteria, leaving altogether 148,009 comparison people. Dementia due to PD was added to ensure exclusion of people with PD from the comparison cohort.
Incidence of antidepressant use was investigated from 10 years before to 15 years after the PD diagnosis. Data on antidepressant purchases during 1995–2016 were gathered from the Prescription Register, which contains data on reimbursed medication purchases. Antidepressants were defined as ATC class N06A (Supplementary Table 2), and further categorized as selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), serotonin–norepinephrine reuptake inhibitors (SNRIs), mirtazapine, and other antidepressants. Incident users were identified with a one-year washout-period, starting 11 years before PD diagnosis. For those diagnosed before or during the year 2005, year 1995 was used as a one-year washout-period. People who purchased antidepressants during the washout period, those who were hospitalised for > 50% of the washout or hospitalised for the last 90 days of washout were excluded (Supplementary Figure 1). Only the first initiation of antidepressant use after washout period was included in the analysis. Hospitalisation data were obtained from the Care Register for Health Care.
Data on comorbidities since 1972 until the index date were obtained from the Special Reimbursement register: asthma or chronic obstructive pulmonary disease (code 203), cardiovascular diseases including chronic heart failure (201) hypertension (205), coronary artery disease (206, 213, 280) and rheumatoid arthritis and connective tissue diseases (code 202). Diabetes was defined as special reimbursement code 103 or purchase of antidiabetics (ATC A10, excluding guar gum A10BX01). History of depression (discharge diagnosis or diagnosis in outpatient visit in specialized health care) was identified from the Care Register for Health Care using ICD-82960, 3004, 3011; ICD-9: 2961, 2968, 3011, 3004; ICD-10 F32-F34, F38-F39.

The activity was measured using the standard 3,5-dinitrosalicylic acid (DNS)-reducing sugar assay as described previously (52 ) using 0.12 μg/ml BoMan26A or 1.6 μg/ml BoMan26B and 0.5% (w/v) LBG in 50 mm potassium phosphate buffer, pH 6.5. The incubation time was 15 min at 37 °C. Mannose was used to obtain a concentration standard curve. Temperature dependence was determined between 22 and 60 °C. pH dependence was carried out in the pH range 3–8 in 0.5-unit increments. 50 mm sodium citrate buffer was used for pH 3–5.5 and 50 mm sodium phosphate for pH 6–8. Temperature stability was tested for up to 24 h at 22, 30, 37, and 45 °C. The pH and temperature dependence and stability were conducted using the standard activity assay. Incubations for specific activities were done in the same way as the standard DNS assay but using the various substrates (LBG, guar gum, KGM, and INM) at 0.5% (w/v) concentration. Incubations with insoluble INM were centrifuged at 8000 × g for 5 min before measuring the absorbance. Specific activity units used was katal/mol, where katal was calculated as moles of produced reducing sugars/s.

In this experiment, the effect and properties of agar-agar and guar gum were evaluated with sugar and citric acid for the preparation of gummies. No artificial colouring and flavouring agents were added. The production of plant-based gummies involved a meticulously structured methodology to investigate the influence of sugar concentrations on the textural properties of the final product. The experimentation followed a systematic approach, beginning with the selection of suitable plant-based hydrocolloids, including agar-agar and guar gum, recognized for their gelling properties in food applications. To formulate the gummy base, a range of sugar concentrations (30%, 40%, and 50%) was meticulously chosen to explore the impact of varying sweetness levels on the final texture. A colloidal solution was prepared by dissolving agar-agar (4-6%) in water while continuously stirring until thorough mixing was achieved. Subsequently, citric acid (1.5–2.5%) was added to the solution. The total solution volume was standardized to 100 grams by carefully combining the recommended amounts of ingredients and adding water. The process commenced by dissolving the chosen plant-based hydrocolloids in water at specific temperatures to achieve optimal hydration and dispersion within the matrix. Subsequently, the predefined concentrations of sugar were gradually introduced into the hydrocolloid solution under controlled mixing conditions, ensuring homogeneity before gelation. The gelation process involved meticulous temperature control and timing to achieve the desired chewy texture analogous to traditional gelatin-based gummies. Each batch underwent a standardized cooling and setting phase to allow the hydrocolloid-sugar matrix to solidify into the gummy form (Figure 1). Notably, the textural attributes, including firmness, elasticity, and chewiness, were assessed using established instrumental methods (e.g., texture profile analysis) and sensory evaluations conducted by a trained panel. This methodological approach is aimed at elucidating the role of varying sugar concentrations in tandem with plant-based hydrocolloids, shedding light on their synergistic effect in mimicking the texture of gelatin-based gummies. Texture analysis was conducted using a texture analyzer to assess parameters such as chewiness, gumminess, springiness, adhesiveness, and firmness. Furthermore, each sample underwent evaluation by a semitrained sensory panel to assess various quality attributes, including texture, appearance, flavour, and overall acceptability, rated on a 9-point hedonic scale. Subsequently, formulations that received the highest scores in sensory evaluations were selected for further investigations and studies. The best formulation of plant-based hydrocolloids used in combination was selected based on chewiness and gumminess which was then used for the incorporation of turmeric and black pepper powder. Different concentrations of turmeric powder and black pepper powder were added to find the most suitable formulation based on sensory evaluation. Turmeric and black pepper-based gummies were prepared in which concentrations of turmeric powder (i.e., 1-3%) and black pepper powder (i.e., 0.2-1%) were added and prepared by the procedure mentioned in Figure 1.