Therapeutic equivalence of a formulation of purified micronized flavonoid fraction of diosmin/hesperidin 450 mg/50 mg in healthy adults: an open-label, randomized, single-dose, crossover study
Published on 07 October 2025
Generics and Biosimilars Initiative Journal (GaBI Journal). 2025;14(2).
Introduction/Study objectives: Given that the purified micronized flavonoid fraction (diosmin/hesperidin 450 mg/50 mg) is a formulation with low water solubility, granulated form, and low intestinal membrane permeability, the present bioequivalence study was conducted to compare Dipemina (T: tested) with Daflon (R: reference product). |
Introduction/Study objectives
Diosmin (diosmetin 7-O-rutinoside) is a natural flavonoid, specifically a flavone glycoside, commonly found in citrus plants belonging to the Rutaceae family, whose most well-known fruits include lemon, orange, grapefruit, pomelo, and mandarin [1]. It is biosynthesized through the oxidation of the flavonoid hesperidin.
The organic synthesis of diosmin begins with hesperidin, and an oxidation step also occurs during the process. The result of its purification is a flavonoid fraction enriched to approximately 90% diosmin and 10% of various flavonoids expressed as hesperidin [2]. Finally, the micronization process ensures that the particle size of the flavonoid fraction is less than 2 μm, thereby enhancing its intestinal absorption [3].
Since the 1990s, a series of double-blind, randomized, controlled clinical trials have been published aiming to demonstrate the therapeutic effects of daily administration of two 500 mg tablets of a purified micronized flavonoid fraction (PMFF), commercially known as Daflon®, in patients with chronic venous insufficiency. A reduction was observed in ankle circumference, leg pain, heaviness, cramps, and swelling sensation in the lower limbs [4]. Concurrently, the effect of administering two PMFF tablets daily was evaluated in patients with hemorrhoidal symptoms, resulting in significant relief of itching, heaviness, bleeding, pain, and anal discharge [5]. These findings established the phlebotropic effect of PMFF, with Daflon® serving as the reference brand.
Diosmin has very low water solubility, which significantly limits its absorption. Before crossing the intestinal epithelial cell membranes, it is hydrolyzed by the intestinal microbiota into diosmetin. Diosmetin is thought to passively diffuse across these membranes more easily. However, given the low plasma concentrations observed and the considerable inter-individual variability in bioavailability, the possibility of active transmembrane transport cannot be excluded [6].
Regarding the biotransformation of diosmin and diosmetin, an animal study identified 64 metabolites for diosmin and 46 for diosmetin, involving enzymatic reactions such as methylation, glycosylation, glucuronidation, demethylation, hydroxylation, and decarboxylation [7]. This metabolic diversity presents a challenge in delineating the pathways responsible for the anti-inflammatory and antioxidant activities that contribute to their cardiovascular clinical benefits [8], as well as in understanding the patient-specific factors that may influence bioavailability. The low solubility and membrane permeability of flavonoids may result in variable absorption, making comparative pharmacokinetic testing essential for the development of generic products.
Considering the manufacturing and purification process of diosmin—together with smaller amounts of other biologically active flavonoids such as hesperidin [9]—its specialized pharmaceutical formulation (micronization), and its extensive metabolism, this study aimed to assess the therapeutic bioequivalence of a new PMFF formulation Dipemina® (test) in comparison to Daflon (reference), under fasting conditions in healthy adult volunteers.
Given the high cost of branded flavonoid products, developing cost-effective generics like Dipemina® is important for improving access to vascular disease treatment in Chile and the rest of Latin America.
Methodology
Drugs
The active ingredient of the test drug (T) is a micronized and purified mixture of diosmin and hesperidin, with diosmin as the principal flavonoid (450 mg) and a flavonoid fraction expressed as hesperidin (50 mg), formulated as film-coated tablets for oral administration. It is manufactured by Laboratorio Dr Lazar y Cía, Química e Industria (Argentina). The flavonoid composition results from a purification process carried out by Laboratoire Bioresine Internationale (Morocco). The product is marketed in Chile under the brand name Dipemina® (ISP Registration No. F-14971/20) by Laboratorio Raffo SA.
The bioequivalence study was conducted in comparison with the reference drug, commercially known as Daflon® (ISP Registration No. F-8621/16), which consists of a 500 mg flavonoid fraction enriched with 450 mg of diosmin and 50 mg expressed as hesperidin. This product is manufactured by Les Laboratoires Servier (France) and served as the reference product (R) in the present study.
Study design
This was an open-label, randomized, single-dose, two-period, two-sequence crossover study under fasting conditions with a 14-day washout Participants who received the test product (T) during the first period were administered the reference product (R) in the second period, or vice versa. The study was conducted in accordance with the current G-BIOF 01 technical guideline issued by the Public Health Institute of Chile (ISP) on therapeutic bioequivalence [10].
The study was conducted by Centro Innolab Spa (Contract Research Organization) in full compliance with International Council Harmonization GCP E6 R2 guidelines [11, 12], Technical Standard No. 57 [13], and the Chilean Public Health Institute’s bioequivalence guidance (G-BIOF 01) [10].
Sample size determination
Taking into account the study design, with logarithmic transformation of the data, a statistical power of 80%, a significance level of 5%, and coefficients of variation for Cmax of diosmin (34.45%) and hesperidin (20.38%) obtained from a previous study with Daflon®, and in accordance with the recommendations issued by the Department of Pharmaceutical Regulatory Affairs for Bioequivalent Products, ISP (Exempt Resolution RW No. 27620/20), the calculated sample size using RStudio software was 56 participants [14].
Participants
This study was not prospectively registered due to administrative reasons. Ethics approval: Metropolitan North Health Service REC, Chile (No. 2022/180). All participants provided written informed consent.
A series of laboratory tests were performed, including complete blood count and urinalysis, blood glucose, urea, total protein, alkaline phosphatase, bilirubin, aspartate and alanine transaminases, lipid profile, creatinine, HIV, hepatitis B and C serologies, and human chorionic gonadotropin (hCG). Additionally, participants underwent an electrocardiogram and a chest X-ray. Individuals whose results were classified as healthy (or fit for study inclusion) by a medical examination, and who met all inclusion criteria without falling under any exclusion criteria, were randomized into the study.
Key inclusion criteria were males or females (not pregnant or breastfeeding), aged between 18 and 55 years, with a body mass index between 18.51 and 29.99 kg/m², non-smokers, and without a history of drug or alcohol abuse.
Key exclusion criteria included: smoking, intake of vitamin supplements within 7 days before drug administration, history of drug or alcohol abuse, recent changes in dietary or exercise habits, consumption of foods or beverages containing grapefruit and/or citrus or flavonoids within 7 days prior to each drug administration, and a history of any significant cardiovascular, hepatic, renal, pulmonary, haematologic, gastrointestinal, endocrine, dermatologic, immunologic, muscular, or neurologic disease, as well as a history of gastrointestinal surgery that could affect drug absorption.
Oral administration of pharmaceutical products
In each study period, participants underwent a fasting period of at least 10 hours prior to drug administration. Each subject received a single dose consisting of one tablet of diosmin/hesperidin 450 mg/50 mg, administered with 250 mL of potable water while seated at a 45° angle. Participants remained in relative rest for the first 4 hours post-dosing.
Diet composition
Participants were provided with breakfast, lunch, an afternoon snack, and dinner. The diet was standardized and nutritionally balanced according to the dietary guidelines for the Chilean population, with specific restrictions on beverages containing xanthines, grapefruit juice, and other citrus-based juices such as orange or pomelo juice. Given that diosmetin exposure can be modulated by intestinal/hepatic CYP3A and transport processes, avoiding grapefruit and other citrus reduced exogenous flavonoid inhibitor confounding [15].
Participant care
Throughout the study, participants were under medical supervision, which included monitoring of vital signs (heart rate, respiratory rate, oxygen saturation, blood pressure, and body temperature) at 1.0, 3.0, 4.0, 11.0-, and 23.0-hours post-dose, as well as at each ambulatory sampling point. The most reported adverse reactions associated with diosmin/hesperidin treatment include diarrhoea, dyspepsia, nausea, or vomiting [16].
Sample collection
Serial blood sampling was performed via the insertion of a forearm cannula equipped with a bidirectional silicone cap in each participant. Each participant received a single dose of test or reference with 14-days washout period. Serial sampling occurred at pre-dose and 1.00, 1.33, 1.67, 2.00, 2.33, 2.67, 3.00, 3.33, 3.67, 4.00, 4.33, 4.67, 5.00, 5.33, 5.67, 6.00, 8.00, 10.00, 12.00, 24.00, 48.00, and 72.00 h post-dose.
Bioanalytical analysis of diosmetin
Plasma concentrations of the aglycones derived from diosmin and hesperidin (expressed as diosmetin) were determined using a liquid–liquid extraction method followed by high-performance liquid chromatography (HPLC) (Shimadzu Nexera model) with tandem mass spectrometry detection (LC-MS/MS) (Applied Biosystem, API 6500 model). Diosmetin-d3 (deuterium-labelled) was used as the internal standard. The lower limit of quantification (LLOQ) was 50 pg/mL of diosmentin according to LLOQ requirements outlined in the G-VMBA guideline (17). The complete method was validated for linearity, selectivity, sensitivity, accuracy and precision, stability, dilution integrity, and recovery in accordance with the G-VMBA guideline published by the Department of the National Agency of Medicines (ANAMED) from the Institute of Public Health of Chile (ISP) [17].
Pharmacokinetic analysis
Based on the relative bioavailability study—i.e., the plasma concentration–time profile of diosmetin for the test product (T) compared to the reference (R)—pharmacokinetic parameters were calculated. Given that diosmetin has an average half-life greater than 24 hours, the pharmacokinetic parameters evaluated included: maximum plasma concentration (Cmax), time to reach maximum concentration (Tmax), area under the plasma concentration–time curve up to 72 hours (AUC0–72), and elimination half-life (T½). These parameters were calculated using PHOENIX® WINNONLIN® version 8.1 [18, 19]. All concentrations and exposures are reported in pg/mL and pg·h/mL
Statistical analysis
Pharmacokinetic and statistical analyses will be performed on data from eligible and evaluable participants who completed the study.
Safety Set included all dosed participants. Pharmacokinetic (PK) Set included participants with sufficient samples to derive Cmax and AUC0–72 in both periods. Bioequivalent (Per-Protocol) Set excluded major protocol deviations and non-completers; one participant withdrew and was excluded from PK and Bioequivalent sets. Concentrations below LLOQ were treated as zero prior to Tmax and missing thereafter.
Primary endpoints were ln(Cmax) and ln(AUC0–72). Geometric mean Test/Reference ratios and 90% confidence intervals were estimated using ANOVA on log-transformed data. Bioequivalence was concluded if both 90% CIs lay within 80.00%–125.00%. Tmax was summarized as median (range) and compared descriptively; it was not used for BE decision [20].
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Funding sources
The sponsor funded the study. The sponsor (Laboratorios Raffo Chile) had no direct participation in design, data collection, data analysis, data interpretation and manuscript writing. Backer Health Care Agency provided support in manuscript writing process. The authors had full access to the data and final responsibility for the decision to submit.
Competing interests: During the execution of the study, Katherine Bouyer Sáez worked as a clinical research and coordinator of the bioequivalence office of InnoLab Laboratory, Santiago, Chile and Francisco Fuentes worked as Technical Director of Laboratorios Raffo Chile.
Provenance and peer review: Not commissioned; externally peer reviewed.
Authors
Katherine Bouyer Sáez
Biochemist, Operations Manager, Centro Innolab SpA, Diagonal, Paraguay 486, Región Metropolitana, 8330049 Santiago, Chile
Francisco Fuentes Poblete
Pharmaceutical Chemist, Technical Director, Laboratorio Raffo Chile, 253 José Luis Araneda, oficina 202, Región Metropolitana, 7770609 Ñuñoa, Santiago, Chile
Author for correspondence: Francisco Fuentes Poblete, Pharmaceutical Chemist, Technical Director, Laboratorio Raffo Chile, 253 José Luis Araneda, oficina 202, Región Metropolitana, 7770609 Ñuñoa, Santiago, Chile |
Disclosure of Conflict of Interest Statement is available upon request.
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