Water Solubility as a Key Parameter for Rational Dose Optimization of the Nutraceutical Rutin

Abstract

Rutin (RU), a flavonoid (quercetin-3-O-rutinoside) found in dietary supplements and herbal medicines exhibits antioxidant, anti-inflammatory, antimicrobial, antidiabetic, antitumor, hepatoprotective, nephroprotective, neuroprotective activities. Despite its therapeutic potential, RU has low oral bioavailability due to poor aqueous solubility. This study evaluated RU solubility in pharmacopoeial and biorelevant media and calculated its dose number (D₀) using doses of 100, 250, and 500 mg. Thermodynamic solubility, determined by the shake-flask method at 37 °C, was assessed in purified water, 0.1 M HCl, phosphate buffer (pH 6.8), FaSSGF-v2, and FaSSIF-v2. Quantification was performed using a validated UV-Vis spectrophotometric method at 360 nm. RU solubility ranged from 27.98 to 53.07 μg/mL. D₀ values exceeded 1 in all conditions, indicating incomplete dissolution under physiological conditions. For the 500 mg dose, D₀ values ranged from 18.84 (FaSSIF-v2, 500 mL) to 71.48 (FaSSGF-v2, 250 mL), suggesting high risk of poor absorption. RU is often described in the literature as a compound exhibiting Class II-like behavior according to the Biopharmaceutics Classification System (BCS), in which dissolution may represent the rate-limiting step in oral absorption. These findings highlight the need for dose rationalization and formulation strategies to enhance solubility, as current empirical doses may exceed physiological dissolution capacity and impair therapeutic performance, guiding the development of more effective pharmaceutical or nutraceutical products.

Keywords:
Rutin; Validation Solubility; Oral bioavailability

GRAPHICAL ABSTRACT

Keywords:
Rutin; Validation Solubility; Oral bioavailability

HIGHLIGHTS

• Validated UV–Vis method quantifies rutin in supplements and biorelevant media.

• Rutin exhibits low, pH-dependent solubility and dissolution-limited absorption.

• Dose rationalization and advanced formulations may enhance rutin bioavailability.

INTRODUCTION

Rutin (RU) (quercetin-3-O-rutinoside) is recognized as one of the most important flavonoids and a valuable phytochemical in the pharmaceutical industry [¹]. It is commonly found in a wide range of plants, fruits, and vegetables, such as buckwheat, oranges, asparagus, and teas, commonly found in dietary supplements and herbal medicines. RU exhibits significant biological potential and offers numerous health benefits, including antioxidant, hepatoprotective, nephroprotective, neuroprotective, anti-inflammatory, antimicrobial, antidiabetic, antitumor, and testicular protective effects [²,³]. Also known as rutoside, sophorin, or vitamin P, RU is a naturally occurring flavonoid glycoside and one of the major secondary metabolites in plants (Figure 1).

Figure 1
General chemical structure of rutin (RU).

Chemically, it is identified as 2-(3,4-dihydroxyphenyl)-5-[α-L-rhamnopyranosyl-(1–6)-β-D-glucopyranosyloxy]-5,7-dihydroxy-3-chromen-4H, with the molecular formula C₂₇H₃₀O₁₆ and a molar mass of 610.521 g/mol. RU appears as a yellowish powder, poorly soluble in water but readily soluble in pyridine. It has a pKa ranging from 6.17 to 7.62 and a melting point of 125 °C [⁴,⁵].

Despite its pharmacological potential, RU has limited medicinal application due to poor solubility and low gastrointestinal absorption [²]. However, because of its low toxicity, it is considered safe for both human and animal use [⁶]. The oral bioavailability of a drug is influenced by several factors, including dosage form disintegration, drug dissolution, degradation within the gastrointestinal tract (GIT), interaction with enterocytes, and intestinal permeability. These processes are interdependent and modulated by the physicochemical properties of the drug, physiological variables, and formulation characteristics [⁷].

The Biopharmaceutics Classification System (BCS), developed by Amidon and coauthors in 1995, is one of the most established frameworks for predicting oral drug absorption. It classifies compounds based on solubility and intestinal permeability, which are the key determinants of drug absorption [⁸]. According to BCS, drugs are categorized into four classes: Class I – high solubility and high permeability; Class II – low solubility and high permeability; Class III – high solubility and low permeability; and Class IV – low solubility and low permeability. In addition to solubility and permeability, the dissolution rate also plays a critical role, as it may be slower than gastric emptying [⁹]. RU has been reported to exhibit low aqueous solubility and permeability characteristics associated with Biopharmaceutics Classification System (BCS) Class II-like behavior; however, its permeability and metabolic conversion in the gastrointestinal tract remain subjects of ongoing discussion [¹⁰].

In the BCS framework, solubility is defined as the highest single therapeutic dose of a compound that dissolves in 250 mL or less of aqueous media across a pH range of 1.0 to 6.8 at 37 °C [¹¹]. This definition differs from other solubility concepts such as equilibrium solubility, intrinsic solubility, kinetic solubility, and apparent solubility. Equilibrium solubility (Seq), also referred to as thermodynamic solubility, is determined from a saturated solution in equilibrium with excess solid [¹²,¹³].

To quantitatively assess the impact of solubility on absorption, the dose number (D₀) is used. This parameter relates the drug dose to its solubility and the available fluid volume in the GIT. A D₀ value greater than 1 suggests incomplete dissolution of the dose, potentially impairing absorption [¹²,¹⁴]. Recent studies have highlighted the relevance of D₀ in predicting in vivo performance, particularly for flavonoids and poorly soluble compounds [¹⁵].

Biorelevant media such as FaSSIF (fasted state simulated intestinal fluid) and FaSSGF (fasted state simulated gastric fluid) are widely used to simulate the physiological environment of the GIT in fasted and fed states. These media provide more realistic conditions compared to traditional compendial media, as they incorporate critical components like bile salts, phospholipids, and digestive enzymes [¹⁶,¹⁷]. Their use allows for more accurate prediction of solubility and dissolution behavior of compounds like RU [¹⁶].

Moreover, the intake of at least 250 mL of water during oral administration is recommended to ensure sufficient fluid volume for dissolution. This must be considered in D₀ evaluation and formulation design [¹¹]. Physiological modeling with GastroPlus™ has confirmed that the intestinal fluid volume in fasted adults can reach up to 607 mL, directly affecting the dissolution and absorption of poorly soluble drugs.

Despite its poor aqueous solubility and limited oral bioavailability, RU is widely marketed in dietary supplements, commonly in empirical doses ranging from 250 to 500 mg per serving. However, these doses are often based on market practices rather than robust clinical evidence. They may exceed the physiological dissolution capacity, potentially compromising therapeutic efficacy. Literature suggests that rational dose adjustment based on solubility data and D₀ can optimize pharmacokinetic profiles, reduce the required dose, and minimize costs and adverse effects [¹⁸].

This study aims to deepen the understanding of RU behavior in the GIT by determining its solubility in various media and calculating the dose number, with the goal of guiding future formulation strategies and clinical applications, such as nutraceuticals.

MATERIAL AND METHODS

Materials

The materials used in the study were sourced from reputable suppliers, ensuring high purity. These included NF Rutin (purity 93.45%, Shaanxi Jiahe Phytochem Co., Ltd., China), 96° GL ethanol (Jalles Machado S/A, Brazil), and methanol (purity 99%, J.T. Baker®, Mexico). Ultrapure water was produced using the ORU-2E purification system (Union, São José dos Pinhais, Brazil). Other reagents included sodium taurocholate, pepsin (Inlab, São Paulo, Brazil), sodium chloride (Biotec, Pinhais, Brazil), maleic acid (Sigma-Aldrich, Darmstadt, Germany), hydrochloric acid, and lecithin (Alfa Aesar, Massachusetts, USA).

UV-Vis spectrum analyses of rutin

Instrumentation and experimental conditions

The analyses were conducted using a UV-5100 spectrophotometer (Metash, Shanghai) equipped with quartz cuvettes with a 1 cm optical path length. Detection was performed at a wavelength of 360 nm, according to Abualhasan and coauthors, (2017) [¹⁹] with modifications. The solvent used as was methanol analytical grade. Measurements were carried out at room temperature (25 °C).

Preparation of standard and working solutions

For the validation of the analytical methodology, three stock solutions (SM1, SM2, and SM3) were prepared by dissolving the RU standard in methanol at a concentration of 1 mg/mL [¹⁹].

From these stock solutions, working solutions were prepared at appropriate concentrations for the construction of the analytical curve and for validation assays, including linearity, precision, and accuracy [²⁰].

Validation of the analytical method

The UV-Vis spectrophotometric method was validated following the International Conference on Harmonization (ICH) guidelines (Q2-R1). The validation process included assessments of selectivity, linearity, sensitivity, precision, accuracy, and robustness.

Selectivity

The selectivity of the method was evaluated by spectral scanning in the range of 200–800 nm using a RU standard solution (2.5 μg/mL) prepared in methanol to determine the maximum absorption wavelength (λmax) and establish the analytical wavelength at 360 nm [¹⁹]. Blank analyses of all media were performed to verify the absence of significant interference at the analytical wavelength.

In addition, spectral profiles obtained from samples after the 24 h equilibrium solubility experiments in the evaluated media were analyzed to assess potential matrix effects under the experimental conditions employed. Prior to analysis, samples were centrifuged at 4.000 rpm for 15 min to remove undissolved particles and minimize light scattering effects.

Linearity and homoscedasticity

Linearity was assessed by constructing a calibration curve with seven RU concentrations (2.0, 4.0, 6.0, 8.0, 10.0, 14.0, and 20.0 μg/mL), prepared in triplicate by diluting three independent standard solutions (SM1, SM2, and SM3). The concentration versus absorbance data were analyzed using linear regression, residual analysis, and a homoscedasticity test, verifying the possible presence of outliers through the Grubbs method for outlier detection [²¹].

Analysis of variance (ANOVA) was performed to determine the significance of the linear regression and the lack-of-fit analysis. The dispersion of regression residuals was examined to assess data homoscedasticity. Additionally, variance homogeneity was tested using the Cochran test at a 5% significance level. The Cochran value (C_calc) was determined and compared with the Cochran tabulated value (C_tab) [²¹,²²,²³]. C_calc was calculated by Equation 1 and the homoscedasticity was confirmed if C_cal ≤ C_tab:

(1) ${\text{C}}_{\text{calc}}\text{c}=\frac{ s²major}{{{\displaystyle \sum }}^{\text{}}s²}$

Where s² major is the largest variance and ∑s² is the sum of the variances.

RU stock solutions (1 mg/mL) were prepared in methanol (PA), and working solutions were diluted to define the calibration range. The calibration curve was assessed for coefficient of variation, correlation coefficient, lack of fit, and homoscedasticity. Method validation followed the procedures described by Abualhasan and coauthors (2017) [¹⁹] with modification.

Sensitivity

The limits of detection (LOD) and quantification (LOQ) were determined based on the standard error of the y-intercept and the slope of the calibration curve, according to the ICH guidelines and the formulas in equations 2 and 3 [²⁰].

(2) $LD= \frac{3.3 \text{x} \sigma }{s}$

(3) $LQ= \frac{10 \text{x} \sigma }{s}$

Where σ represents the standard deviation and s denotes the slope of the equation.

Precision

Precision was evaluated at two levels: repeatability and intermediate precision [²⁰]. The intra-day precision test (repeatability) was performed using three independent stock solutions at concentrations of 3.0 μg/mL, 7.0 μg/mL, 9.0 μg/mL, 15.0 μg/mL each analyzed in triplicate. Intermediate precision was assessed by analyzing the samples on two separate days and by a different analyst, and the coefficient of variation among them was calculated. The results were evaluated by ANOVA statistical analysis.

Accuracy

Accuracy of the analytical method was assessed by evaluating the closeness of the experimental results to an accepted true value. The evaluation was conducted using four concentrations of RU: 3 μg/mL, 7 μg/mL, 9 μg/mL e 15 μg/mL. Each concentration was analyzed in triplicate. This parameter is expressed as the percentage recovery of a known analyte concentration spiked into the sample or as the ratio between the experimentally determined mean concentration and the corresponding theoretical concentration [²⁰]. Accuracy was determined using the equation 4:

(4) $Recovery \left(\%\right)= \frac{Experimental concentration}{Theoretical concentration} X 100$

Robustness

To evaluate the robustness of the method, three independent stock solutions were prepared and diluted to concentrations of 3 μg/mL, 7 μg/mL, 9 μg/mL, and 15 μg/mL. These solutions were analyzed under standard conditions and with a deliberate variation in a critical parameter: the wavelength. The standard wavelength (360 nm) was adjusted by ± 5 nm, resulting in analyses at 355 nm and 365 nm. The ANOVA analysis was performed to evaluate the results [²⁰].

Solubility assessment

The solubility of RU was evaluated and quantified using the previously validated method. The analytical wavelength corresponded to the maximum absorption of rutin, a spectral region with minimal interference from common buffer and biorelevant media components. The test was performed using pharmacopoeial and biorelevant dissolution media.

Solid quantities of rutin (55 mg) were added to 250 mL of each medium, ensuring excess solid relative to its reported aqueous solubility (125 mg/L) to guarantee saturation conditions [¹¹,¹⁵]. The samples were prepared in sextuplicate, and the tested media included 250 mL of HCl (pH 1.2), phosphate buffer (pH 6.8), FaSSGF-v2 (pH 1.6), FaSSIF-v2 (pH 6.5), and ultrapure water (pH 6.2). These solutions were prepared in 250 mL Erlenmeyer flasks to achieve saturation. The solubility assays for RU were performed using the shake-flask method with an orbital shaker (Thoth 6420B, Thoth Equipamentos, Piracicaba, Brazil). During the process, samples were gently agitated to eliminate air bubbles and maintained on the orbital shaker at 37 °C with a rotation speed of 150 rpm (United states pharmacopeial convention, 2020) for 24 hours to allow the system to reach solubility equilibrium. Subsequently, the samples were aliquoted and centrifuged in a refrigerated centrifuge (Hermle Z36HK, Hermle Labortechnik GmbH, Wehingen, Germany) at 25 °C and 4,000 rpm for 15 minutes to remove undissolved RU and obtain clear supernatants, minimizing potential light scattering [²⁰]. The validated spectrophotometric method was applied consistently across all pharmacopoeial and biorelevant media evaluated in this study.

Media composition

The dissolution media evaluated included purified water (pH 6.21), pharmacopoeial media, and biorelevant media. The pharmacopoeial media consisted of 0.1 M hydrochloric acid solution (pH 1.2) and potassium phosphate buffer solution (pH 6.8), prepared according to the Brazilian Pharmacopoeia [¹⁶].

FaSSGF-v2 medium was prepared by dissolving 4.3 mg of sodium taurocholate and 396 mg of sodium chloride in 40 mL of distilled water. Subsequently, 450 μL of hydrochloric acid was added, and the mixture was stirred for 30 minutes. Then, 1.5 mg of lecithin was added, followed by stirring for 2 hours. Afterward, 10 mg of pepsin was incorporated, and the mixture was maintained under constant stirring overnight. Finally, the pH was measured and adjusted to 1.6, and distilled water was added to complete the volume to 100 mL in a volumetric flask [¹⁷].

FaSSIF-v2 medium was prepared by dissolving 161.3 mg of sodium taurocholate and 400.9 mg of sodium chloride in 40 mL of distilled water. Next, 100 μL of hydrochloric acid was added, and the mixture was stirred for 30 minutes. Subsequently, 15.16 mg of lecithin was added, and the mixture was stirred for 2 hours using a magnetic stirrer. Then, 221.76 mg of maleic acid and 50 mL of distilled water were added. The resulting mixture was maintained under constant stirring overnight. Finally, the pH was measured and adjusted to 6.5, and distilled water was added to complete the volume to 100 mL in a volumetric flask [¹⁷].

The media were prepared in 250 mL volumetric flasks and used in the equilibrium solubility experiments according to the evaluated experimental conditions.

Dose number calculation

The dose number (D₀) was calculated based on the highest single therapeutic dose, following the guidelines of the Biopharmaceutics Classification System (BCS) from the U.S. Food and Drug Administration [⁹]. For this study, doses of 100, 250, and 500 mg of RU were adopted, as they reflect the range of doses commonly found in commercially available rutin-containing nutraceutical products intended for oral administration. The solubility values (mg/mL) for each tested medium were experimentally determined, and the initial volume of gastrointestinal fluid (V₀) was estimated for the calculation, according to Equation 5 [⁹,²⁶]. An initial gastric volume of 250 and 500 mL was assumed, representing the typical volume of liquid ingested with oral medications.

(5) $D_0=\frac{Dose \left(mg\right)}{\left(V_0 \left(mL\right)x Solubility \left(\frac{mg}{mL}\right)\right)}$

Where Dose is expresses in mg, V₀ represents the gastrointestinal fluid volume (mL), and S corresponds to solubility (mg/mL).

RESULTS

Validation of the analytical method

The selectivity of the proposed UV–Vis method was assessed by comparing the spectral profiles obtained for RU and the evaluated media. As shown in Figure 2, the characteristic absorption region of RU was preserved at the analytical wavelength of 360 nm under the experimental conditions employed. Although baseline contributions from the evaluated media were observed, no critical spectral overlap compromising RU detection was identified at 360 nm.

Figure 2
UV–Vis spectral profiles used for selectivity evaluation of RU: (a) Spectral profiles of blank media (b) Spectral profiles obtained after 24 h equilibrium solubility experiments in the evaluated media containing RU.

The spectral profiles obtained after the 24 h equilibrium solubility experiments maintained consistent absorption behavior in the selected analytical region. In addition, the centrifugation step improved sample clarification and reduced potential interference associated with suspended particles and light scattering effects, supporting the applicability of the method for RU quantification in pharmacopoeial and biorelevant media.

The linearity is referred as the capacity of an analytical method to obtain results directly proportional to the analyte concentration in a sample [²⁰]. For the linearity evaluation, the interval between the upper and lower concentrations (range) has been determined as 2 μg/mL to 20 μg/mL [¹⁰]. The results of the linearity of the proposed method test were according, and proving that the method was linear, guaranteeing the reliability of the absorbance-concentration correlation. The calibration curves obtained through the Excel^® Software showed a good correlation, for RU, the linear equation obtained was y = 0.0315x + 0.0219, with determination coefficient (r²) of 0.9982. The statistical data of the regression evaluated by the ANOVA test, revealed that was possible to observe that there is a very strong correlation between the variables.

The statistical significance of the regression equation was evaluated by analyzing the model’s fit through lack-of-fit and regression significance tests. The regression was confirmed as statistically significant since the F-value (33475.2) was associated with an extremely low p-value (2.70555 × 10⁻⁸⁵), indicating a non-zero slope at the selected confidence level. The lack-of-fit test confirmed that the model fits the observed data well, with the calculated F (2.05) was lower than the corresponding critical F-value (2.244), indicating the absence of significant lack of fit. Table 1 presents the ANOVA results for the method’s linearity.

Table 1.
Result of the ANOVA Table for linearity of the method for Rutin analysis: regression significance and lack of adjustment.

Parameter	DOF	SQ	MS	Fvalue	Ftab	Pvalue
Regression	1	2.06019	2.06019	33475.2	3.998	2.70555E-85
Residue	61	0.00375	6.2E-05
Lack of fit	5	0.00058	0.00012	2.0565	2.38	0.082
Pure error	56	0.00317	5.7E-05

The homoscedasticity was evaluated using the Grubbs test and the results were satisfactory because the calculated G values were lower than the tabulated G value for n=9 (2.21), and none of the values obtained was discrepant to be. Homoscedasticity was also confirmed by the Cochran test where the value of C_cal (0.1970) ≤ C_tab (0.3384), which did not detect any outliers. In this way it was considered homoscedastic.

The sensitivity of the method was evaluated and the limits of detection (LOD) and quantification (LOQ) were determined to be 0.1947 and 0.5901, respectively. These values were calculated using Equations 1 and 2, confirming the method’s sensitivity.

The precision of the method was assessed at two levels: repeatability and intermediate precision. In this regard, the methodology proved to be reproducible and accurate, with results from both evaluations (Table 2) demonstrated that the method exhibited relative standard deviation (RSD) values within acceptable limits (5%), analyzed in six replications on the same day (reproducibility), and with the comparison of the sample analyzed on different days by different analysts (intermediate precision). Thus, indicating excellent precision and reproducibility. The small differences observed between repeated results fell within the thresholds established in the literature for routine analytical methods, further validating the method’s precision.

Table 2.
Precision data from absorbance against concentration (μg/mL) of RU reference standard analyzed on two separate days to determine intra-day, inter-day precision and precision with distinct analysts using the relative standard deviation (RSD, %)

RU (μg/mL)	Repeatability		Intermediare precision
	Intra-day	RSD (%)	Inter-day	RSD (%)
3	3.27 ± 0.14	4.45	2.99 ± 0.03	1.22
7	7.53 ± 0.28	3.76	7.51 ± 0.09	1.29
9	9.64 ± 0.34	3.60	9.66 ± 0.09	0.18
15	15.53 ± 0.58	3.79	15.83 ± 0.11	0.70

Accuracy, defined as the ability of an analytical procedure to produce results consistent with and close to the expected value across the reportable range, was also assessed [²⁰]. The accuracy of the method was evaluated and the method was proven to be accurate (Table 3) for the quantification of RU, with a recovery rate of less than 105%.

Table 3.
Accuracy data from spectrophotometry method for determination of RU (360 nm)

Concentration (μg/mL)	Theoretical (μg/mL)	Measured (μg/mL)
3	3.09 ± 0.02	103.10 ± 0.61
7	7.30 ± 0.03	104.35 ±0.45
9	9.41 ± 0.02	104.56 ± 0.20
15	15.05 ± 0.00	100.36 ± 0.00

The robustness of an analytical method is the ability of the method to withstand small variations in some parameters [²⁰]. Robustness was assessed by varying an experimental parameter, specifically the wavelength. The coefficient of variation (CV%) was calculated for each condition, and the recovery of the rutin was analyzed. The wavelength variation was performed with a change of 5 nm, and the wavelengths of 355 and 365 nm were evaluated, in addition to the 360 nm described in the method. The results were satisfactory for the variation of 355 nm, proving the robustness of the method in the tested wavelength range. However, for the variation of the wavelength of 365 nm, the method was not considered robust. Therefore, the method proved to be robust for variations in the wavelength of 355 nm, with a coefficient of variation of less than 5% in the face of small variations in the direction of decreasing the wavelength [²⁰].

Solubility assessment

The solubility of RU was evaluated in different dissolution media, representing both pharmacopoeial, biorelevant solutions and purified water. The results revealed significant variations depending on the pH of the medium used (Table 4). In the acidic medium (pH 1.2), represented by hydrochloric acid solution, RU exhibited a solubility of 29.12 ± 0.66 μg/mL, indicating limited dissolution under conditions simulating the gastric environment. A similar result was observed in FaSSGF-v2 (pH 1.6), a medium simulating fasting gastric conditions, in which RU solubility was 27.98 ± 3.39 μg/mL, statistically comparable to that observed in hydrochloric acid solution. When solubility was analyzed in purified water (pH 6.0), the obtained value was 32.88 ± 0.64 μg/mL, showing an intermediate behavior between the acidic and alkaline media. In the phosphate buffer (pH 6.8), simulating intestinal conditions, a significant increase in solubility was observed, reaching 41.96 ± 2.15 μg/mL. In FaSSIF-v2 (pH 6.5), a biorelevant medium simulating fasting intestinal conditions and representing a more physiologically relevant gastrointestinal environment, RU solubility increased significantly, reaching 53.07 ± 2.80 μg/mL.

Table 4.
Dose Number (D₀) of Rutin in Adults at 100 mg, 250 mg and 500 mg Using 250 mL and 500 mL Across Different Biorelevant and Pharmacopoeial Media

Medium	Solubility (μg/mL)	D0 (100mg/250 mL)	D0 (250mg/500 mL)	D0 (500mg/250mL)	D0 (500mg/500mL)
HCl buffer (pH 1.2)	29.12 ± 0.66	13.74	17.17	68.68	34.34
Phosphate buffer (pH 6.8)	41.96 ± 2.15	9.53	11.92	47.66	23.83
Purified water (pH 6.0)	32.88 ± 0.64	12.17	15.21	60.83	30.41
FaSSGF-v2 (pH 1.6)	27.98 ± 3.39	14.30	17.87	71.48	35.74
FaSSIF-v2 (pH 6.5)	53.07 ± 2.80	7.54	9.42	37.69	18.84

Dose Number Calculation

Table 4 also presents the Dose Number (D₀) values calculated for rutin at doses of 100 mg, 250 mg, and 500 mg, considering simulated physiological fluid volumes of 250 mL and 500 mL across the different tested media. In all conditions evaluated, D₀ values remained above 1. At the 100 mg dose and 250 mL volume, D₀ values ranged from 7.52 (FaSSIF-v2) to 14.30 (FaSSGF-v2). For the 250 mg dose and 500 mL volume, ranged from 9.42 (FaSSIF-v2) to 17.87 (FaSSGF-v2). At the 500 mg dose and 250 mL volume, D₀ ranged from 37.69 (FaSSIF-v2) to 71.48 (FaSSGF-v2).

Overall, the lowest D₀ values were observed in FaSSIF-v2, while the highest were found in acidic media, particularly FaSSGF-v2 and HCl buffer. Increasing the fluid volume from 250 mL to 500 mL led to a reduction in D₀ values across all media; however, D₀ consistently remained above 1 regardless of dose or medium.

DISCUSSION

The analytical method used in this study proved to be selective, linear, homoscedastic, precise, accurate, and robust under the evaluated conditions. Linearity showed an excellent correlation (r² = 0.9982) with no lack of fit, and precision results were within acceptable limits for both repeatability and intermediate precision. Accuracy values were satisfactory, with recoveries below 105%, and the method demonstrated adequate sensitivity and homoscedasticity. The method demonstrated acceptable robustness for small wavelength variations around the analytical wavelength; however, statistically significant differences were observed at 365 nm.

The results demonstrate that RU exhibits limited solubility in both pharmacopoeial and biorelevant media, directly reflecting its known limitations in oral bioavailability. Given that oral drug absorption fundamentally depends on dissolution within the gastrointestinal tract, the low solubility of RU under physiological conditions reinforces the interpretation that RU exhibits biopharmaceutical behavior consistent with compounds exhibiting Class II-like characteristics, in which dissolution may represent a limiting step for oral absorption [⁸]. In this scenario, quantifying solubility in media that mimic physiological environments provides not only a descriptive parameter, but a practical framework to anticipate whether commonly administered doses can realistically dissolve in vivo [¹⁷,²⁷].

Table 4 presents consolidated D₀ values for different doses (100 mg, 250 mg, and 500 mg) in various media and under two commonly used physiological volumes (250 mL and 500 mL). D₀ values greater than 1 indicate that complete dissolution of the dose may not occur, implying limited absorption [²⁷]. Considering the most common marketed dose of rutin (500 mg), D₀ values were high across all media, even at 500 mL. For example, in FaSSIF-v2 (pH 6.5), simulating the fasted intestinal environment, D₀ reached 37.69 with 250 mL and 18.84 with 500 mL, indicating incomplete dissolution even under optimal conditions. In more acidic media such as HCl (pH 1.2), the D₀ exceeded 68.68 at 250 mL, highlighting dissolution limitations in the gastric phase. These findings are consistent with literature describing compounds exhibiting Class II-like characteristics as frequently facing dissolution-limited absorption [²⁸]. Taken together, these values illustrate that the limitation is not merely theoretical: the available physiological volumes are insufficient to dissolve typical rutin doses [²⁹].

Even at the reduced 100 mg dose, D₀ remained well above the ideal limit of 1 [⁹,¹²] in all tested media. In FaSSIF, D₀ was 7.54, indicating that even a fivefold dose reduction does not fully overcome dissolution barriers. This reinforces that dose adjustments should be based on biopharmaceutical parameters rather than empirical selection [¹⁸]. Therefore, D₀ provides a quantitative criterion that can support rational dose definition in nutraceutical development, replacing empirical dose escalation approaches [¹⁸,²⁹].

Table 4 also highlights the impact of fluid intake on dissolution. Doubling the fluid volume from 250 mL to 500 mL nearly halved the D₀ values for the 500 mg dose, yet values remained above the desirable limit, particularly in media with lower solubility such as FaSSGF and HCl. These observations align with EMA guidelines recommending at least 250 mL of water for oral solid dosage forms [¹¹]. However, even under this recommended intake, dissolution capacity remains insufficient, indicating that increasing water volume alone is unlikely to overcome the limitation [¹⁷,²⁸].

D₀ has proven to be an effective predictive tool for in vivo performance of poorly soluble drugs, assisting not only in forecasting dissolution but also in guiding formulation strategies such as dose reduction, solubility-enhancing excipients, or modified-release systems [²⁷]. Recent literature further emphasizes that elevated D₀ values can be used in early development as decision-making triggers, indicating when advanced formulation technologies or re-evaluation of target doses are required. In this context, the present findings position D₀ as a practical screening parameter to prioritize development pathways for rutin-based products [²⁹].

In this context, the appropriateness of marketed rutin doses, commonly defined empirically (100–500 mg), should be questioned. The D₀ values observed far exceed 1 in all scenarios, suggesting that these doses surpass physiological dissolution capacity and may compromise therapeutic performance [¹⁸,³⁰]. A 100 mg dose, while still associated with a D₀ above 1, represents a meaningful improvement and can be further optimized using solubilizing excipients or advanced delivery systems such as self-emulsifying drug delivery systems (SNEDDS), nanocarriers, or cyclodextrin complexation [³⁰]. Thus, instead of increasing content per unit, development strategies should prioritize technologies capable of increasing the dissolved fraction of rutin within the gastrointestinal tract [³⁰].

Importantly, despite its low solubility, rutin is considered safe and well tolerated at high doses, supporting its widespread use. However, safety should not be conflated with efficacy; D₀ values indicate that oral absorption may be limited by insufficient dissolution. This distinction is particularly relevant for nutraceuticals, where high-dose products are often marketed under the assumption that greater content results in greater benefit, which may not hold true when dissolution is the limiting step [¹⁸].

Notably, in the FaSSIF medium, which presented the highest solubility among tested biorelevant media (53.07 μg/mL), and considering the physiological intestinal volume estimated in GastroPlus (607 mL), the maximum amount of rutin that can dissolve is approximately 32 mg. This value was obtained by multiplying solubility by estimated intestinal volume (53.07 μg/mL × 607 mL = 32,213.5 μg = 32.2 mg). These calculations highlight that even in the most favorable intestinal environment, only a fraction of the commonly marketed doses can dissolve. Therefore, simply increasing rutin content in commercial formulations will not improve absorption, as in vivo dissolution is the limiting factor. This reinforces the need for biopharmaceutically guided dose selection and formulation strategies that enhance solubility rather than relying on empirical dose escalation. Additionally, these findings provide a quantitative explanation for the consistently low systemic exposure reported for rutin, despite the use of high oral doses [⁵].

Overall, the solubility data and D₀ calculations consolidate the role of evidence-based approaches in determining appropriate RU doses. The validated analytical method, combined with biorelevant media such as FaSSIF, allows a more accurate prediction of in vivo behavior, guiding the development of more effective formulations. Nevertheless, equilibrium solubility experiments do not capture dynamic processes such as supersaturation, precipitation, or intestinal permeability [¹⁷,¹⁸]. Future studies should therefore integrate dissolution-permeation testing, physiologically based biopharmaceutic modeling, and formulation prototypes to further evaluate the translational impact of the proposed dosing rationale [¹¹,¹⁶,¹⁸].

The findings demonstrated that RU exhibits low aqueous solubility under physiologically relevant conditions, resulting in high D₀ values across the evaluated media. These results reinforce the importance of considering gastrointestinal fluid volume and solubility during the development and optimization of RU containing nutraceutical products.

CONCLUSION

The RU determination method was validated as linear, homoscedastic, selective, precise, robust, and accurate, and was successfully applied to evaluate solubility and dose number (D₀). The results demonstrated that RU exhibits limited aqueous solubility in both pharmacopoeial and biorelevant media, with D₀ values exceeding 1 under all tested conditions, supporting the interpretation that rutin presents dissolution-limited behavior under physiological conditions, consistent with compounds exhibiting Class II-like behavior. Notably, in FaSSIF, the medium with the highest solubility (53.07 μg/mL), and considering a physiological intestinal volume of 607 mL, only approximately 32 mg of RU can dissolve, indicating that even the most favorable intestinal environment can accommodate only a fraction of the commercially relevant doses evaluated in this study (100/250/500 mg).

The solubility results and D₀ determinations reinforce the role of biopharmaceutical criteria in defining appropriate rutin dosing. The integration of a validated analytical method with biorelevant media enables more precise predictions of in vivo outcomes, thereby supporting the development of improved formulations. By translating solubility measurements into dose-related constraints through D₀, this study provides a practical framework to question empirical dosing practices and prioritize formulation strategies that enhance dissolution.

These finding highlights that increasing the rutin content in commercial formulations does not enhance absorption, emphasizing the need for dose rationalization and solubility-enhancing strategies, such as modified-release systems or complexation, to optimize oral bioavailability. Altogether, the data support the use of D₀ as an initial decision-making tool for nutraceutical development and offer guidance for future translational studies focused on linking formulation design, dose selection, and clinical performance.

Funding:

The authors are thankful to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Coordination for the Improvement of Higher Education of Brazil; Finance code001). CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico/ National Counsel of Technological and Scientific Development of Brazil.

Institutional Review Board Statement:

Not applicable

Informed Consent Statement:

Not applicable

Data Availability Statement:

Research data are available in the body of the manuscript.

Acknowledgments:

The work was financially supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Coordination for the Improvement of Higher Education of Brazil; Finance code 001).

Use of Generative Artificial Intelligence

The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The authors declare that generative artificial intelligence (AI) or AI-assisted tools were used under full human supervision. The tool(s) and version(s) used, and their purpose, are described here: ChatGPT (OpenAI, GPT-5 series) was used to assist with English language editing, grammar refinement, improvement of scientific writing, and text organization during manuscript preparation. No confidential or sensitive data were uploaded to such tool(s), and all AI-assisted content was checked, corrected and approved by the authors, who take full responsibility for the integrity and originality of the manuscript.

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Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Paulo Vitor Farago

*

Correspondence: [e-mail]; Tel.: [phone] (F.B.B.P.)

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Publication Dates

• Publication in this collection
  28 Sept 2026
• Date of issue
  2026

History

• Received
  17 Jan 2026
• Accepted
  01 June 2026