Daflon and the protection of venous valves

by L . Pascarel la, USA

Division of Vascular Surgery
Department of Surgery,
The University of Iowa Hospitals
and Clinics
Iowa City, Iowa

Increased venous pressure underlies all the clinical manifestations of chronic venous disorders. Venous hypertension is the result of incompetent venous valves in the superficial veins for most patients. A strong link between venous hypertension, valve failure, and venous inflammation has been evoked in pharmacological studies and confirmed in a variety of animal models. A cascade of inflammatory reactions, resulting in adverse changes in venous valves and the venous wall, eventually produces venous hypertension. Symptoms, telangiectasias, varicose veins, and ultimately venous leg ulcers appear to be a consequence of the changes induced by venous hypertension. Treatment to inhibit inflammation and hamper the development of venous hypertension may offer the greatest opportunity to prevent progression of chronic venous disease and related complications. Inflammation-dependent valve failure is considered a target for drugs. Daflon, a venoactive drug containing purified micronized diosmin, hesperidin, linarin, isorhoifolin, and diosmetin at optimized dosages, is the only drug with evidence for the preservation of valve structures in animal models and for the suppression of commissural transitory reflux that occurs in symptomatic patients after prolonged standing. Daflon has also been shown to protect the microcirculation in animal models. This protection translates into clinical benefits, such as reduction in edema, hematoma resorption after surgery, acceleration of ulcer healing, and improvement in lymphatic drainage. Although the role of Daflon in the attenuation of the various elements of venous inflammation is now better known, it is still worthy of a more in-depth exploration in future.

Most veins of the superficial and deep system in the lower extremities are equipped with a series of one-way bicuspid valves that open to allow flow toward the heart and close to prevent reverse blood flow toward the feet. Venous valves, which were first described by Dutch physician Jacques Dubois and whose true function was later described by William Harvey,1 ensure that blood flows in the correct direction, particularly when the body is upright, traveling against gravity and other pressures.2 Venous pathology for most patients develops when venous pressure increases and blood return is impaired by incompetent valves in the deep or superficial axial veins, perforator veins, or venous tributaries. Chronic venous disorders may also result from venous obstruction or a combination of both valve incompetence and obstruction. These mechanisms lead to global or regional venous hypertension, particularly with standing or walking.3 The subsequent macro- circulatory hemodynamic disturbances contribute to the large variety of clinical presentations seen in chronic venous disorders. In turn, prolonged periods of venous hypertension in the legs alter the microcirculation, resulting in dermal changes with hyperpigmentation, lipodermatosclerosis, and eventual ulceration.

The presentation of chronic venous disorders includes symptoms and signs. A recent large-scale epidemiological study has shown that the most commonly expressed chronic venous disorder–related symptoms include (in order of frequency): heaviness; leg pain; swelling sensation; nighttime cramps; sensation of “pins and needles” in the legs; and sensation of burning and itching.4 The CEAP (Clinical-Etiological-Anatomical-Pathophysiological) classification system is widely used in venous medicine to describe the signs of chronic venous disorders, including telangiectasia, varicose veins, edema, skin changes, and healed or active venous leg ulcers.5

Venous hypertension and valve failure
Venous hypertension is, in most cases, caused by reflux through incompetent venous valves (Figure 1).3,6 Close inspection of surgical specimens removed from limbs with chronic venous insufficiency and, more recently, direct observation with angioscopy have revealed that lesions involve the venous wall, valvular annulus, and valve cusps.7,8 By maintaining venous hypertension or further increasing it, valve failure and failure of the valvular annulus are responsible for disease progression. Monocyte/ macrophage infiltration into the valve leaflets and venous wall of C2 patients with varicose veins has been demonstrated in immunohistochemical studies using a monoclonal antibody specific for monocytes and macrophages.9 Leukocyte infiltration appears to be greater at both the base of the valve leaflets and in the proximal venous wall, according to monoclonal antibody studies.

Figure 1. Visualization of competent and incompetent venous valves.
Competent (panel A) and incompetent (panel B) venous valves as schematic (left) and B-flow ultrasound
images (right). In panel B, the valve sinus is distorted. The cusp above the dilatation is frozen
and the adjacent cusp is prolapsed. The high-velocity retrograde streaming deviates laterally above a
prolapsing cusp.
From reference 6: Lane et al. Phlebolymphology. 2007;14:105-115. Image courtesy of the author.

Regions of low shear stress with venous eddies and recirculation contain substantial numbers of venous valves (Figure 2),10,11 and these phenomena may explain why leukocytes are preferentially deposited in these regions. In the long run, macrophage-induced tissue damage softens the venous wall rendering the valve liable to damage and/or destruction.12 Venous valve failure, and the subsequent reflux that results in distal venous hypertension, is likely to contribute to chronic hypertension, which activates leukocytes in the endothelium of veins and promotes leukocyte-mediated destruction of skin and subcutaneous tissues in the lower limb.

In addition to leukocyte activation, mast cells infiltrate into the venous wall and this infiltration may play a role in the development of varicosity. Increased expression of intercellular adhesion molecule 1 (ICAM-1) and CD68 on the endothelial surface of venous walls in patients with venous insufficiency has been demonstrated, and this increased expression may be related to the development of varicose veins.12

Figure 2. Selected mechanisms that may control inflammation of the vein wall and
valve leaflet.
A normal vein valve and wall are shown in panel A. Valve leaflets may be subject to inflammatory damage
by alteration in the magnitude and direction of fluid shear stress on the endothelium (panel B). Venous
valve leaflets may become unable to close due to vein wall distension caused by elevated venous
pressure (panel C) or by weakening of the vein wall due to proteolytic degradation of its extracellular
matrix (panel D).
Abbreviation: MMP, matrix metalloproteinases.
From reference 11: Schmid-Schönbein. Medicographia. 2008;30:121-126. Image courtesy of the author.

This finding suggests a continuous inflammatory reaction related to venous wall remodeling.13,14
In order for leukocytes to migrate through the endothelial cell layer into tissue, endothelial cells must be activated.12 Endothelial stretching of the vein, due to changes in blood flow and fluid shear stress, may induce activation of the endothelium. Fluid shear stress is a key regulator for endothelial cells, and a decrease in shear stress makes the adhesion of leukocytes to the endothelium easier.9

Clinical observations confirmed in animal models
Since the mechanisms responsible for venous valve failure in primary chronic venous disorders cannot be investigated in vivo in human beings, animal models have been developed for experimental research. Lalka et al described a simple, reproducible model of hind-limb valve disruption in the greyhound. 15 After acute valve degeneration, animals developed an immediate increase in poststimulation segmental venous pressure that lasted up to 14 weeks. Despite establishing that reflux occurred in segments with disrupted valves, reflux did not extend into the tributaries and no evidence of varicose vein development was found. It was hypothesized that in these quadrupeds the hydrostatic column of the hind limb was relatively short, which could explain these findings.16

To elucidate possible mechanisms for valve remodeling in chronic venous disorders, an arteriovenous fistula (AVF) model has also been tested. Unfortunately, an arterialized pressure profile was observed in the distal veins, making this model unsuitable for studying this chronic disease. The combination of outflow obstruction and AVF to produce a model of sustained venous hypertension was developed by van Bemmelen 17 and applied to the study of reflux development by Bergan’s team.18 In a series by Takase et al, rat saphenous vein valves were examined after prolonged exposure to venous hypertension; femoral venous hypertension was elevated for a period of 3 weeks using the van Bemmelen model. In this model, venous reflux developed in response to venous hypertension around 100 mm Hg.

Examination of vein morphology revealed that valve failure occurred as a result of venous wall dilation and valve leaflet shortening. As the leaflets shortened, complete valve closure became more and more difficult until this was no longer possible and reflux subsequently ensued. Evaluation of the valves for molecular markers of inflammation revealed that leukocyte infiltration with granulocytes, monocytes, and T lymphocytes had been enhanced. In addition, endothelial cells of the saphenous vein wall expressed more P-selectin and ICAM-1, endothelial cell membrane adhesion molecules.18 The leaflets were still able to close properly in the early stages of this trial after AVF placement, which indicates that pressure per se may not be responsible for compromising the leaflets. It was later observed, however, that a reduction in leaflet dimensions had taken place by the time the leaflets failed and reflux occurred.

It has been suggested that with dilation of the venous wall, there comes a moment when reflux develops across the leaflets. Long-term exposure to irregular fluid shear stress at the leaflet surface during venous reflux would be inflammation- inducing for the endothelial cells of the valve leaflets. Eventually, leaflets would be irreparably harmed and then destroyed, leading to a deleterious spiral of venous hypertension and venous inflammation.

A new low-flow/high-pressure animal model in veins is being developed by Bouskela’s team to avoid the pitfalls of previous models. The objectives of such a model are to: achieve long periods of observation; study alterations in venous pressure over time; assess changes in microcirculatory parameters; and determine the inflammatory profile of the model. It will allow for the assessment of venous pressure and how it evolves with time and for the exploration of microcirculatory parameters, with a Cytoscan® device and intravital microscopy.

Inflammation-dependent valve failure as a new drug target: the example of Daflon
Intervention in the inflammatory reaction that occurs as part of the progress of chronic venous disorders may be a new pharmacological target and, for this reason, the models of Bergan and Bouskela have been used to assess the effect of Daflon,19 a venoactive drug (VAD) consisting of micronized purified flavonoid fraction.

♦ Chemical family of Daflon
Daflon is produced from a plant extract from the epicarp of Citrus aurantium var amara. It belongs to the chemical family of flavonoids that are included in the six main categories of venoactive drugs (Table I, page 204). Daflon contains purified micronized diosmin, hesperidin, linarin, isorhoifolin, and diosmetin at optimized dosages.20 Each of the active ingredients in Daflon contributes to its action and explains its superior beneficial effect in reducing capillary permeability versus other VADs.20

♦ Daflon’s mode of action
The pharmacodynamic effects of Daflon and their clinical consequences are summarized in Table II (page 205).21-24

Table I. The prinicipal categories of venoactive drugs.

♦ Preservation of venous valve structure
Daflon is the only VAD that has evidence for preserving valve structure in animal models and for suppressing commissural transitory reflux that occurs in symptomatic patients after prolonged standing.25 In two trials of pharmacological postoperative recovery in patients with varicose veins who underwent phlebectomy, Daflon helped attenuate postoperative pain and improve quality of life.26-29

♦ Protection of the microcirculation
Experimental in vivo models have been used to study the effect of drugs on the microcirculation. Microcirculatory preparations include: hamster cheek pouch; hamster or mouse skinfold; rat or hamster mesentery; rat, hamster, or mouse cremaster; etc.30 VADs enhance capillary resistance and decrease capillary filtration, both of which help check capillary leakage. Daflon improves microvascular reactivity and increases functional capillary density after ischemia-reperfusion injury31; following this type of injury, it also induces a significant dose-related reduction in macromolecular permeability.32 These protective microcirculatory properties of Daflon result in clinical benefits. In a recent meta-analysis, ten studies published between 1975 and 2009 including 1010 patients were analyzed to determine whether Daflon, hydroxyethylrutoside, Ruscus extracts, and diosmin reduced edema. Mean reduction in ankle circumference was –0.80±0.53 cm with Daflon, –0.58±0.47 cm with Ruscus extract, –0.58±0.31 cm with hydroxyethylrutoside, –0.20±0.5 cm with single diosmin, and –0.11±0.42 cm with placebo. Daflon reduced ankle edema more than other VADs (P<0.0001).33When used after varicose vein stripping, Daflon helped decrease postoperative hematomas and accelerate their resorption.26-29 Complications of chronic venous disorders are related to chronic venous hypertension and are visualized in the skin, the final target of chronic venous hypertension. This hypertension is a cause of chronic inflammation, which manifests as the result of persistent and sustained injury. Ultimately, in limbs with chronic venous insufficiency the most severely impaired circulation is the dermal capillary circulation. In a metaanalysis of 5 randomized controlled trials containing 723 C6 patients, micronized purified flavonoid fraction was efficacious for healing venous ulcers when used as an adjunct treatment to compression therapy and appropriate local therapy, particularly for large (>5 cm2 in area) and/or persistent (>6-month duration) ulcers.34

♦ Improvement in lymphatic drainage
The drainage function of lymphatic vessels is very important; lymphatic vessels transport 4 liters of efferent lymph into the bloodstream daily. Fluid turnover every 24 hours (including the volume of fluid reabsorbed in the lymph nodes) is up to two thirds of the total volume of interstitial fluid.35 The skin of the lower extremities contains a more dense and extensive network of lymphatic capillaries than the skin of the upper extremities.36 Natural human posture, which is upright, means that the lower extremities have higher filtration pressure and fluid influx. Greater lymph transport in the lower extremities counterbalances the higher volume of interstitial fluid produced by orthostatism and gravity.

Table II. Overview of the pharmacodynamic effects and clinical properties of Daflon. Based on references 21-24.

Lymphatic vessels spontaneously contract twice to four times a minute to transport lymph. In human legs, the contraction of prenodal lymphatic vessels propels the flow of lymph.37 In-ternal extensions of lymphatic endothelial cells act as valves to prevent the return of lymph.35 Varicose veins are associated with lymphatic dysfunction and structural damage to the lymphatic network. When the transportation of lymph is disrupted, the lymph stasis that follows can promote inflammation. 38 Lipid accumulation in the media of diseased veins may further damage adventitial lymphatic vessels.

Protein and extracellular fluid accumulation in lymphedema may be reduced by treatment with Daflon,39 which can also stimulate lymph contractility and flow and reduce excess protein in tissues with high protein edema.40 In a study investigating Daflon (n=46) or placebo (n=48) in the treatment of lymphedema over 6 months,41 patients in the Daflon group experienced a 7% reduction in lymph volume versus a 10% increase for patients in the placebo group. Discomfort was reduced in both groups, but the Daflon group also reported a significant reduction in the sensation of heaviness. In addition, Daflon was found to be efficacious in reducing edema in Bancroftian filarial lymphedema.42

♦ Potent anti-inflammatory effect
Disturbed venous flow patterns and chronic venous inflammation are two interlinked phenomena. It is thought that mediators resulting from disturbed blood flow, and subsequent inflammation, have an important role in the occurrence of venous pain. Locally released proinflammatory mediators, resulting from hemodynamic changes and hypoxia, can activate nociceptors located in close proximity to the microcirculation, including those in the venous wall, space between endothelial and smooth muscle cells of the media, and perivenous space.43

The primary site of activation of venous and/or perivenous nociceptors may not be in large venous vessels, which is suggested by the fact that the occurrence of pain does not correlate closely with objective parameters of varicose vein remodeling, incompetent venous valves, and inflammation. The efficacy of Daflon in the treatment of patients with symptoms of chronic venous disease has been widely evaluated in comparative and noncomparative clinical trials.21,24

There is substantial evidence from meta-analyses 44 and the RELIEF study (Reflux assEssment and quaLity of lIfe improvEment with micronized Flavonoids), a large observational study,45 that Daflon effectively relieves venous symptoms and lower limb edema. In the latest recommendations for the management of active venous ulcers, the evidence for using Daflon as adjuvant therapy was assigned the grade 1B. It should not be forgotten that Daflon is also capable of reducing associated pain.24

Figure 3. Reflux flow rates across the valve of the saphenous vein.
Reflux flow rates across the saphenous venous valve measured after 3 weeks
of venous hypertension in a control group (Vehicle) and two Daflon treatment
groups at doses of 50 and 100 mg/kg/day (MPFF). n is the number of rats in
each treatment group. *PAbbreviation: MPFF, micronized purified flavonoid fraction.
From reference 48: Takase et al. Eur J Vasc Endovasc Surg. 2004;28:484-493.
© 2004, Elsevier Ltd.

♦ Protection against inflammation-related valve damage in chronic venous disease with Daflon
♦ Pharmacological studies
Animal models have revealed Daflon’s ability to attenuate or block chronic inflammatory effects in the circulation, whether at the micro- or macrocirculatory level. In a venous occlusion/ reperfusion model, in which increased venous blood pressure augmented the inflammatory cascade and tissue injury, 46 markers of inflammation decreased in a dose-dependent manner in Daflon-treated animals. Parenchymal cell death and leukocyte rolling, adhesion to postcapillary venules, and migration were also significantly reduced by Daflon.47 Important data supporting the macrocirculatory protective effect of Daflon have been provided by Takase et al.48 Reflux rate was significantly reduced in a dose-dependent manner in animals treated with Daflon (Figure 3). Many inflammatory indicators were lowered dose-dependently by Daflon, including leukocyte infiltration, expression of P-selectin and ICAM-1, as was the degree of apoptosis. By delaying or blocking the inflammatory reaction in venous hypertension, it appears that in this rat model Daflon might slow the development of reflux and thus slow the damage to valve structures. These findings were confirmed by a new study in rats that showed that edema and fistula blood flow produced by an acute AVF were reduced by Daflon. Daflon also reduced granulocyte and macrophage infiltration into the valves, which is consistent with observations from the previous study.49

Figure 4. Venous valves with and without reflux.
Illustrations of a normal venous valve without reflux (panel A), a valve with nonpathological commissural
reflux usually seen in the evening after a prolonged time in an upright position (panel B), and a valve
with pathological intervalvular reflux (panel C).
Modified from reference 25: Tsoukanov et al. Phlebolymphology. 2015;22:18-24. Image courtesy of
the author.

♦ Clinical trials
Treatment with Daflon 1000 mg/day over 2 months resulted in the elimination of transitory commissural reflux (Figure 4) observed in patients presenting with subjective leg symptoms without visible signs of chronic venous disorders, so-called C0S subjects.25 Transitory elimination of reflux occurred in parallel with pain relief and improvement in quality of life. In this trial, consecutive C0S subjects were enrolled and assessed for the following: (i) symptom intensity using a visual analog scale; (ii) quality of life using the Chronic Venous Insufficiency quality of life Questionnaire (CIVIQ-20); and (iii) saphenous reflux duration and saphenous vein diameter, with a Duplex scan examination performed twice a day (morning and evening). A total of 41 C0S patients were enrolled in the study and, of these patients, 15 had no reflux in either the morning or evening and 26 had transitory evening reflux with 22 being commissural and 4 intervalvular. Saphenous vein diameter was greater in the subgroup of patients with transitory reflux compared with patients without reflux (P<0.05).

After Daflon treatment, there was a trend toward a reduction in intervalvular reflux length, while transitory commissural refluxes (n=22) no longer appeared. Additionally, vein diameters returned to normal values. These results mirror the protective effect of Daflon on venous valve structure.

♦ Venous valve protection and targeted pharmacological intervention
A practical purpose of elucidating the molecular steps involved in the development of valve lesions is to identify ways of intervening with a targeted treatment. Studies have focused on available molecules known to modify the sequence of events involving leukocyte adhesion, interaction with endothelium, activation, and migration, and the subsequent valvular damage in large veins, mainly the great saphenous vein, with which these processes are associated. However, studies on the pathophysiology of chronic venous disorders have not yet shown that this sequence of events extends down to venules, where valves and microvalves play an important role in venous hemodynamics, and that this sequence is not just limited to large veins, including the saphenous veins.

We know from recent findings that the majority of microvalves in lower limbs are present in channels with a luminal diameter <100 μm.50 The role that microvalves play is still unclear, and their location and arrangement in normal lower limbs suggest that they prevent blood flow back into the capillary bed (Figure 5). Although there appears to be no difference between lower limbs with venous ulcers and normal limbs with respect to the number and density of microvalves,50 microvalves in diseased limbs are stretched and incompetent, allowing retrograde flow from large veins into the dermal capillary bed. Vincent and coworkers have proposed two hypotheses linking the failure of microvalves with skin changes in venous insufficiency: (i) degenerative changes in very small veins in leg skin may be related to the appearance of telangiectasias, reticular veins, and corona phlebectatica; and (ii) valve incompetence in both larger proximal vessels and small superficial veins, at the level of microvalves, would account for the appearance of severe skin changes in the event of venous insufficiency.51

This review shows that Daflon currently possesses the most appropriate profile to protect venous valves, and perhaps microvalves, even if its role in vivo still remains to be explored in more depth.

Daflon is also registered as Ardium®, Alvenor®, Arvenum® 500, Capiven®, Detralex®, Elatec®, Flebotropin®, Variton®, and Venitol®.

1. Caggiati A. The venous valves of the lower limbs. Phlebolymphology. 2013; 20:87-95. 
2. Eberhardt RT, Raffetto JD. Chronic venous insufficiency. Circulation. 2014;130: 333-346. 
3. Bergan JJ, Schmid-Schönbein GW, Coleridge-Smith PD, Nicolaides AN, Boisseau MR, Eklof B. Chronic venous disease. N Engl J Med. 2006;355: 488-498. 
4. Rabe E, Guex JJ, Puskas A, Scuderi A, Fernandez Quesada F; VCP Coordinators. Epidemiology of chronic venous disorders in geographically diverse populations: results from the Vein Consult Program. Int Angiol. 2012;31:105-115. 
5. Rabe E, Pannier F. Clinical, aetiological, anatomical and pathological classification (CEAP): gold standard and limits. Phlebology. 2012;27(suppl 1):114-118. 
6. Lane RJ, Graiche JA, Cuzzilla ML, Coroneos JC. Incompetent venous valves: ultrasound imaging and exo-stent repair. Phlebolymphology. 2007;14:105-111. 
7. Corcos L, De Anna D, Dini M, Macchi C, Ferrari PA, Dini S. Proximal long saphenous vein valves in primary venous insufficiency. J Mal Vasc. 2000;25:27-36. 
8. Van Cleef JF, Hugentobler JP, Desvaux P, Griton P, Cloarec M. Endoscopic study of reflux of the saphenous valve. J Mal Vasc.1992;17:113-116. 
9. Ono T, Bergan JJ, Schmid-Schönbein GW, Takase S. Monocyte infiltration into venous valves. J Vasc Surg.1998;27:158-166. 
10. Lurie F, Kistner RL, Eklof B, Kessler D. Mechanism of venous valve closure and role of the valve in circulation: a new concept. J Vasc Surg. 2003;38:955-961. 
11. Schmid-Schönbein GW. Triggering mechanisms of venous valve incompetence. Medicographia. 2008;30:121-126. 
12. Takase S, Bergan JJ, Schmid-Schönbein GW. Expression of adhesion molecules and cytokines on saphenous veins in chronic venous insufficiency. Ann Vasc Surg. 2000;14:427-435. 
13. Jacobs MP, Badier-Commander C, Fontaine V, Benazzoug Y, Feldman L, Michel JB. Extracellular matrix remodeling in the vascular wall. Pathol Biol (Paris). 2001; 49:326-332. 
14. Badier-Commander C, Jacobs MP, Michel JB. Varicose remodeling [in French]. Med Ther. 2000;6:718-723. 
15. Lalka SG, Unthank JL, Dalsing MC, Cikrit DF, Sawchuk AP. Venous hemodynamics in a chronic venous valvular insufficiency model. Arch Surg. 1990;125: 1579-1583. 
16. Jones GT. Animal models in chronic venous disease. Medicographia. 2008;30: 154-156. 
17. van Bemmelen S, Hoynck van Papendrecht AA, Hodde K, Klopper PJ. A study of valve incompetence that developed in an experimental model of venous hypertension. Arch Surg.1998;121:1048-1052. 
18. Takase S, Pascarella L, Bergan J, Schmid-Schönbein GW. Hypertension-induced venous valve remodeling. J Vasc Surg. 2004;39:1329-1334. 
19. Pascarella L. Daflon and the preservation of venous valves. Phlebolymphology. 2016;23:20-30. 
20. Paysant J, Sansilvestri-Morel P, Bouskela E, Verbeuren TJ. Different flavonoids present in the micronized purified flavonoid fraction (Daflon® 500 mg) contribute to its anti-hyperpermeability effect in the hamster cheek pouch microcirculation. Int Angiol. 2008;27:81-85. 
21. Lyseng-Williamson KA, Perry CM. Micronised purified flavonoid fraction: a review of its use in chronic venous insufficiency, venous ulcers and haemorrhoids. Drugs. 2003;63:71-100. 
22. Perrin M, Ramelet AA. Efficacy of venoactive drugs in primary chronic venous disease. Survey of evidence, synthesis and recommendations. In: Bergan JJ, Bunke N, eds. The Vein Book. 2nd ed. New York, NY: Oxford University Press; 2014:514-527. 
23. Perrin M, Ramelet AA. Pharmacological treatment of primary chronic venous disease: rationale, results and unanswered questions. Eur J Vasc Endovasc Surg. 2011;41:117-125. 
24. Nicolaides A, Kakkos S, Eklof B, et al. Management of chronic venous disorders of the lower limbs - guidelines according to scientific evidence. Int Angiol. 2014;33:87-208. 
25. Tsoukanov YT, Tsoukanov AY, Nikolaychuk A. Great saphenous vein transitory reflux in patients with symptoms related to chronic venous disorders, but without visible signs (C0s), and its correction with MPFF treatment. Phlebolymphology. 2015;22:18-24. 
26. Pokrovsky AV, Saveljev VS, Kirienko AI, et al. Surgical correction of varicose vein disease under micronized diosmin protection (results of the Russian multicenter controlled trial DEFANS). Angiol Sosud Khir. 2007;13:47-55. 
27. Pokrovsky AV, Saveljev VS, Kirienko AI, et al. Stripping of the great saphenous vein under micronized purified flavonoid fraction (MPFF) protection (results of the Russian multicenter controlled trial DEFANCE). Phlebolymphology. 2008; 15:45-51. 
28. Veverkova L, Kalac J, Jedlicka V, et al. Analysis of surgical procedures on the vena saphena magna in the Czech Republic and an effect of Detralex during its stripping [article in Czech]. Rozhl Chir. 2005;84:410-412. 
29. Veverkova L, Kalac J, Jedlicka V, et al. Analysis of the various procedures used in great saphenous vein surgery in the Czech Republic and benefit of Daflon 500 mg to postoperative symptoms. Phlebolymphology. 2006;13:195- 201. 
30. Virgini-Magalhaes CE, Bottino DA, Bouskela E. Microcirculation and chronic venous insufficiency: from production of pharmacological models to discovery of new therapies. Phlebolymphology. 2001;35:16-19. 
31. Bouskela E, Cyrino FZ, Lerond L. Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischemia/ reperfusion in the hamster cheek pouch. Br J Pharmacol. 1997;122: 1611-1616. 
32. Cyrino FZ, Bottino DA, Lerond L, Bouskela E. Micronization enhances the protective effect of purified flavonoid fraction against postischaemic microvascular injury in the hamster cheek pouch. Clin Exp Pharmacol Physiol. 2004;31:159- 162. 
33. Allaert FA. Meta-analysis of the impact of the principal venoactive drugs agents on malleolar venous edema. Int Angiol. 2012;31:310-315. 
34. Coleridge-Smith P, Lok C, Ramelet AA. Venous leg ulcer: a meta-analysis of adjunctive therapy with micronized purified flavonoid fraction. Eur J Vasc Endovasc Surg. 2005;30:198-208. 
35. Rovenská E, Rovenský J. Lymphatic vessels: structure and function. Isr Med Assoc J. 2011;13:762-768. 
36. Stanton AW, Patel HS, Levick JR, Mortimer PS. Increased dermal lymphatic density in human leg compared with forearm. Microvasc Res.1999;57:320-328. 
37. Olszewski WL, Engeset A. Intrinsic contractility of prenodal lymph vessels and lymph flow in human leg. Am J Physiol. 1980;239:H775-H783. 
38. Tanaka H, Zaima N, Sasaki T, et al. Loss of lymphatic vessels and regional lipid accumulation is associated with great saphenous vein incompetence. J Vasc Surg. 2012;55:1440-1448. 
39. Casley-Smith JR, Casley-Smith JR. The pathophysiology of lymphedema and the action of benzo-pyrones in reducing it. Lymphology.1988;21:190-194. 
40. Clement DL. Management of venous edema: insights from an international task force. Angiology. 2000;51:13-17. 
41. Pecking AP, Fevrier B, Wargon C, Pillion G. Efficacy of Daflon 500 mg in the treatment of lymphedema (secondary to conventional therapy of breast cancer). Angiology.1997;48:93-98. 
42. Das L, Subramanyam Reddy G, Pani S. Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema. Filaria J. 2003;2:5. 
43. Vital A, Carles D, Serise JM, Boisseau MR. Evidence for unmyelinated C fibers and inflammatory cells in human varicose saphenous veins. Int J Angiol. 2010; 19:e73-e77. 
44. Martinez MJ, Bonfill X, Moreno RM, Vargas E, Capella D. Phlebotonics for venous insufficiency. Cochrane Database Syst Rev. 2005;(3):CD003229. 
45. Jantet G. Chronic venous insufficiency: worldwide results of the RELIEF study. Reflux assEssment and quaLity of lIfe improvEment with micronized Flavonoids. Angiology. 2002;53:245-256. 
46. Takase S, Lerond L, Bergan JJ, Schmid-Schönbein GW. Enhancement of reperfusion injury by elevation of microvascular pressures. Am J Physiol Heart Circ. 2002;282:H1387-H1394. 
47. Takase S, Delano FA, Lerond L, et al. Inflammation in chronic venous insufficiency: is the problem insurmountable? J Vasc Res. 1999;36(suppl 1):3-10. 
48. Takase S, Pascarella L, Lerond L, Bergan JJ, Schmid-Schönbein GW. Venous hypertension, inflammation and valve remodeling. Eur J Vasc Endovasc Surg. 2004;28:484-493. 
49. Pascarella L, Lulic D, Penn AH, et al. Mechanisms in experimental venous valve failure and their modification by Daflon 500 mg. Eur J Vasc Endovasc Surg. 2008;35;102-110. 
50. Phillips MN, Jones GT, van Rij AM, Zhang M. Micro-venous valves in the superficial veins of the human lower limb. Clin Anat. 2004;17:55-60. 
51. Vincent JR, Jones GT, Hill GB, van Rij AM. Failure of microvenous valves in small superficial veins is a key to the skin changes of venous insufficiency. J Vasc Surg. 2011;54:62S-69S.