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 2013
NPC Natural Product Communications Vol. 8
No. 4
Diosmin – Isolation Techniques, Determination in Plant Material 545 - 550
and Pharmaceutical Formulations, and Clinical Use
Anna Bogucka – Kockaa, Michał Woźniaka, Marcin Feldob, Janusz Kockic and Katarzyna Szewczyka,*
a
Chair and Department of Pharmaceutical Botany, Medical University, Lublin, 20-093 Chodźki 1, Poland
b
Chair and Department of Vascular Surgery and Angiology, Medical University, Lublin, 20-081 Staszica 11,
Poland
c
Department of Clinical Genetics, Medical University, Lublin, 20-080 Radziwiłłowska 11, Poland

k.szewczyk@umlub.pl

Received: December 23rd, 2012; Accepted: February 5th, 2013

Diosmin is a naturally occurring flavone glycoside used in the treatment of venous diseases. In this review, we present the clinical aspects of the use of diosmin
preparations in venous stasis, hemorrheologic disorders and vein wall remodeling. Because of its multiple applications in biology and its many therapeutic
activities, research on isolation and identification of diosmin is of high relevance. The aim of this review is to present an overview of techniques of isolation
and separation of diosmin in plant material, pharmaceutical formulations such as Daflon®, Diosed® and Dioven® tablets, and biological fluids.

Keywords: Diosmin, Venous stasis, Hemorrhagic disorders, Vein wall remodeling, Plant material, Pharmaceutical formulations.

Diosmin, the 7-rhamnoglucoside of 5,7,3-trihydroxy-4-

HO OH

methoxyflavone; IUPAC name: {5-hydroxy-2-(3-hydroxy-4- HO

O
O
OH

methoxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R, O
O O
O
4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2- HO OH HO CH3

yl]oxychromen-4-one} and its flavanone analog hesperidin, are

O
HO OH

common flavonoids in the Rutaceae family [1a]. Diosmin differs
from hesperidin by the presence of a double bond between two
carbon atoms in the C-ring of the diosmin molecule. Diosmin can
be manufactured by extracting hesperidin from citrus rinds,
followed by conversion of hesperidin to diosmin [2a].
Diosmin was first isolated in 1925 from Scrophularia nodosa L.,
and first introduced as a therapeutic agent in 1969 [3]. Nowadays it
is widely used for its phlebotonic and vascular-protecting activity in
treatment of chronic venous insufficiency, lymphedema and
varicose veins [1a,3]. The therapeutic action of diosmin is attributed
to the inhibitory effect on capture and metabolism of noradrenalin
by the venous walls. This results in an increase in the vascular tonus
with favorable consequences, reduction of elasticity and
permeability [4]. As a flavonoid, diosmin also exhibits anti-
inflammatory, free-radical scavenging, and antimutagenic
properties [1a,3]. Diosmin possesses an antihypertensive effect,
which is evidenced by lowered blood pressure, lipid peroxides,
improved nitric oxide availability and antioxidant status.
Scavenging of superoxide anions by diosmin is attributed to its
antioxidant effect and leads to the increase in nitric oxide
availability which infers relaxation of the vessel wall and controls
blood pressure. The antihypertensive effect of diosmin was
evaluated in deoxycorticosterone acetate (DOCA)-salt induced
hypertension in male Wistar rats and the maximum efficacy was
reached with a medium dose of 50 mg/kg body weight [5].
Diosmin is very popular in the treatment of chronic venous
insufficiency (CVI), which affects an ever increasing number of
people. CVI is characterized by pain, leg heaviness, a sensation of
swelling, and cramps, and is correlated with varicose veins. The
complications in CVI are usually associated with inflammatory
and thromboembolic processes. The progress of the disease can be
Figure 1: Structure of diosmin.
slowed down, especially if adequate treatment is used. After the
administration of 600 mg of diosmin for 60 days, parameters of pain
and edema in four groups of patients with different severity of
clinical and pathomorphological findings were significantly
improved [2a,3]. It is worthy of notice that diosmin in a dose of 600
mg is usually prescribed in CVI, but a dose 1000 mg per day would
be more adequate [2b].
Several large clinical trials have demonstrated diosmin to be
effective in the treatment of acute and chronic symptoms of
hemorrhoids [3]. Diosmin significantly reduced cell death caused
by lipopolysaccharide in a concentration-dependent manner.
Diosmin also dose-dependently suppressed LPS-induced tumor
necrosis factor-alpha TNF-α expression, and the apoptotic
mechanisms in rat pheochromocytoma PC12 cells via the inhibition
of a caspase-dependent apoptotic pathway [6]. Oral treatment with
diosmin (100 mg/kg/day) for a period of 45 days showed significant
ameliorative effects on all the biochemical parameters in oxidative
stress in streptozotocin-nicotinamide (STZ-NA)-induced diabetic
rats. Oxidative stress has been suggested as a contributory factor in
development and complication of diabetes [7]. Glinski et al.
proposed diosmin as being effective in treatment of venous
ulceration and delayed healing [8].
After oral administration, diosmin is rapidly hydrolyzed by
enzymes of the intestinal microflora into its flavone aglycone,
diosmetin, which is subsequently absorbed into the systemic
circulation. After 26-43 h diosmetin is degraded to phenolic acids or
their glycine-conjugated derivatives and eliminated through the
urine. Diosmin and diosmetin that are not absorbed are eliminated
in the feces [2c,3,9].
546 Natural Product Communications Vol. 8 (4) 2013
Determination in plant extracts and pharmaceutical
formulations: Diosmin has a wide range of biological activities,
including anti-inflammatory, blood lipid lowering,
antihyperglycemic, antioxidant and free radical scavenging effects
[1e,6,7]. Recent studies have shown the use of diosmin for the
treatment of venous leg ulcer and, as a chemopreventive agent, in
urinary – bladder and colon carcinogenesis [1a,e,6]. It is also a
potential agent for the treatment of neurodegenerative diseases [6].
Because diosmin has many applications in biology and therapeutics,
methods for isolation and identification of this compound are of
high practical relevance. In this part of our work, an overview of the
possible methods of isolation of diosmin is given.
Diosmin typically appears as a greyish–yellow or light yellow
hygroscopic powder [10]. The choice of an optimal extraction
procedure for obtaining pure diosmin from biological material is
very important and depends on the goals of the conducted research.
Different methods of isolation may be applied, and the utilization of
various strategies is dependent on the origin of the biological
material from which diosmin is to be extracted.
Diosmin, unlike most flavonoid compounds, is very poorly soluble
both in polar and nonpolar protic solvents, but freely soluble in
some aprotic solvents, for example, dimethyl sulfoxide. This
procedure makes it possible to separate the target compound rather
easily from accompanying flavonoids. According to the method
proposed by Ivashev et al. [4] plant material (Hyssopus officinalis
L., Lamiaceae), after defatting with chloroform, was treated with
aqueous ethanol or water (and subsequently with 96% ethanol), and
then diosmin was extracted from the plant material with dimethyl
sulfoxide DMSO. The extracts obtained were combined and poured
into a tenfold amount of water and a crystalline precipitate of
diosmin was separated within 48 h. Purification by recrystallization
from DMSO-water and DMSO-methanol mixtures and washing
with ethanol was suggested to further purify diosmin from
accompanying compounds [4].
Diosmin, hesperidin and other flavonoids from citrus peels were
extracted and the effect of water content in the citrus peel (0% and
75%), the extraction time (30 and 90 min) and the citrus peel/water
ratio were evaluated. The citrus peels were obtained from three
species: tahiti lime {Citrus latifolia (Tanaka ex Yu.Tanaka) Tanaka,
sweet orange [C. sinensis (L.) Osbeck], and oneco tangerine (C.
reticulata Blanco)}. The highest yield was obtained from dry
material with 30 min of extraction. In contrast, with 30 min and wet
material the lowest yield was obtained. With wet material it was
demonstrated that the yield increased with extraction time. The
yields from dry material were higher than those from wet material
and the extraction time had no influence on the phenolic content.
According to these results the optimal ultrasound-assisted extraction
conditions were set as follow: frequency 60 kHz, extraction time 30
min, temperature 40˚C, citrus peel/water ratio (g/mL) 1/10, Ca(OH)2
as basifying agent, and water as solvent [1b].
For the isolation of diosmin from Buchu {(Barosma betulina
(Thunb.) Bartl. and H.L.Wendl., Rutaceae} leaves, El-Shafae et al.
[1a] proposed refluxing with methanol containing 10%
dimethylsulfoxide (4 x 15 min). These authors determined diosmin
from tablets, too. In this case, weighed and powdered tablets were
refluxed with the same solvent (3 x 5 min). Incorporation of 10%
dimethylsulfoxide in methanol provided the most efficient solvent
for complete extraction of diosmin. A different method of extraction
from pharmaceutical formulations was suggested by Moldovan et
al. [11a]. Tablets containing diosmin were ground into powder and
extracted with 0.5 M NaOH. Then samples were filtered and
Bogucka – Kocka et al.
quantitatively transferred into calibrated flasks with doubly –
distilled water. Diosmin, together with other flavonoids, was
extracted from citrus peels using optimal ultrasound-assisted
extraction with water. After acidification to pH 7, precipitates were
obtained by centrifugation and were dissolved in dimethyl
sulfoxide: methanol (2:1, v/v) and filtrated through 0.45 µm nylon
membrane and used for HPLC analysis [1c].
Commercial citrus fruit juices and freshly prepared hand-squeezed
citrus fruit juices were sonicated at room temperature for 15 min
and filtered before use. Five Daflon tablets (a mixture of micronized
flavonoid glycosides consisting of 450 mg of diosmin and flavonoid
extract equivalent to 50 mg of hesperidin) were powdered, and
sonicated at 30˚C with 90 mL of methanol/DMSO (1:1) for 30 min
[1e].
A sensitive, accurate and precise bioanalytical method involving a
simple liquid-liquid extraction of diosmin from plasma samples was
developed. Plasma samples were incubated with β-
glucuronidase/sulfatase in order to release diosmetin from its
glucuronic acids conjugate. The analytes were isolated by liquid–
liquid extraction with tert-butyl methyl ether at pH 2. Tert-butyl
methyl ether in acidic media proved to be the best in terms of high
extraction recovery, negligible matrix effects, and absence of
endogenous interference at the retention times in the chromatogram
[1f].
Polyamide as a sorbent has been used for cleaning samples before
analysis of diosmin in natural samples. Lime (Citrus latifolia) juice
containing diosmin and hesperidin was centrifuged for 10 min,
mixed with 25 mL of double distilled deionized water, adjusted to
pH 3 with concentrated HCl and filtered to remove solid particles.
The SPE polyamide cartridge was sequentially conditioned with 5
mL of n-hexane, 5 mL of methanol and 10 mL of double distilled
deionized water without allowing the cartridge to dry. The authors
had also studied the effects on extraction efficiency of some
parameters including percentage of methanol in washing solution,
pH of washing solution, type and volume of elution solvent and
flow rate of sample solution through the cartridge. For optimizing
the washing solution, different percentages of methanol in water at
different pHs ranging from 2.5 to 7.5 were examined. To obtain a
suitable solvent for elution of analytes from the cartridge, different
solvents such as methanol, acetonitrile, ethylacetate, diethylether, n-
hexane and acetone were examined [1c].
Separation and detection: There are a number of methods for
diosmin determination in pharmaceutical formulations, plant
material and biological fluids. These methods include voltammetry,
thin–layer chromatography, high performance liquid
chromatography (HPLC), Fourier transform infrared spectroscopy
and spectrodensitometry [1a,e,g,11a,b,12a]. Comparison of many
techniques used for determination of diosmin in different samples is
shown in Table 1.
Spectroscopic and spectrophotometric methods: A Fourier
transform infrared (FT-IR) spectroscopic method was developed for
the rapid, direct measurement of diosmin in different
pharmaceutical drugs. Beer’s law and/or chemometric methods
(partial least squares PCR+, principal component Regression PLS1
or PLS2) were used in order to minimize the effects of sample pre-
treatment steps and provide direct FT-IR measurement. The method
proposed is simple, precise and not time-consuming compared with
the chromatographic methods that exist in the literature.
Quantification was achieved in about 10–15 min, including sample
preparation and spectral acquisition [11b].
Diosmin Natural Product Communications Vol. 8 (4) 2013 547

A rapid and sensitive spectrophotometric method for the Adam et al. [12c] suggested and optimized an electrochemical
determination of diosmin has been developed. The method is based technique for the determination of diosmin and other selected
on the reaction of the target compound with 4-aminoantipyrine flavonoids in body liquids. The most suitable conditions for determi-
(AAP) in the presence of hexacyanoferrate (III) (HCF) in an nation of the flavonoids were the following: frequency 180 Hz, step
alkaline medium and subsequent formation of a purple colored potential 1.95 mV/s and phosphate buffer of pH 7 as supporting
product having a maximum absorption (λmax) at 524.5 nm. The electrolyte. The limit of detection and limit of quantification of
reaction with AAP in the presence of an oxidant such as diosmin were 2.66 and 8.88 nM, respectively. The suggested
hexacyanoferrate (III) in alkaline solution is one of the most widely method could be used for analysis of body liquids such as urine.
used reactions for the determination of phenols. The conditions
affecting the reaction (reagents concentration, pH, order of addition Table 1: The methods used for determination of diosmin.
Method Matrix Reference
of reagents, stability in time) were optimized. Under the optimum Adsorptive stripping differential pulse
conditions, Beer’s law was proven to be valid in the range of 0.3 – Dioven® 500 [12a]
voltammetry
35 μg mL-1 diosmin with a correlation coefficient of 0.9993, LOD FT-IR ®
Dioven 500 [11b]
(calculated as three times the standard deviation of the blank) = 0.1 GC/MS Human plasma, urine [1j]
Daflon®, Citrus fruit
µg mL-1 and LOQ = 0.3 µg mL-1 . The proposed method was HPLC-DAD [1e]
Citrus latifolia, C. sinensis, C.
successfully applied to the analysis of drug tablet formulations. This HPLC-DAD-ESI-MS
reticulata
[1b]
method is inexpensive, fairly rapid and sensitive [11c]. A simple, HPLC-ESI/MS Citrus juices [1i]
rapid, economical and sensitive method for the spectrophotometric HPLC-MS-MS Human plasma [1f]
determination of diosmin has been developed using sodium HPLC-UV
Dioven®, Diosed®, Daflon®,
[1a]
Barosma betulina
nitroprusside as a chromogenic reagent. Diosmin, as a phenolic
HPLC-UV Human plasma [1d]
compound, reacts with sodium nitroprusside and hydroxylamine HPTLC - densitometry Diosminin® Citrus fruit [1g,h]
hydrochloride in a basic medium to give a green-colored product SPE/HPLC-UV Lime juice [1c]
measured at an analytical wavelength of 763 nm. The proposed Spectrophotometry UV-VIS Dioven® 500 [11a,c]
method was applied to determine diosmin in a commercial dosage Square wave voltammetry Human urine [12b,c]
form. The limits of detection (LOD) and quantification (LOQ) were
0.05 and 0.2 µg mL-1, respectively. The method can be successfully Chromatographic methods: For the fast determination of diosmin
applied as an alternative to the AAP method for the routine analysis in complex pharmaceutical preparations a thin-layer
of diosmin in pharmaceutical formulations [11a]. chromatographic method with densitometric UV detection was
developed. Chromatographic separation on silica gel was performed
Electroanalytical methods: An investigation of the oxidative with chloroform–methanol–water (23 + 12 + 2, v/v), as mobile
behavior of diosmin at a glassy carbon electrode (GCE) using cyclic phase and the developed spots were analyzed by densitometry at λ =
voltammetry (CV) and differential pulse voltammetric (DPV) 344 nm. Under the experimental conditions established, repeatable
techniques, and also an optimization of the experimental conditions and accurate results were obtained. The method is characterized by
for the determination of this compound in pharmaceutical dosage high sensitivity; the limit of detection was 20 ng [1g]. A HPTLC-
forms was performed. Tablets containing 150 mg diosmin were densitometric method was used for the simultaneous quantitative
dissolved in NaOH (0.02 M). A series of Britton–Robinson (BR) determination of diosmin in citrus fruit extracts and in
universal buffer solutions of various pH values ranging from pH 2.0 pharmaceutical preparations. The determination was carried out at
– 10.0 were prepared and used as supporting electrolytes. The BR 330 nm in the reflection mode. A mobile phase of ethyl acetate –
buffer solutions were prepared by mixing an acid mixture methanol – water – acetic acid (25:2:2:1, v/v/v/v) was used [1h].
containing acetic (0.04 M), orthophosphoric (0.04 M), and boric
acids (0.04 M), and adjusting the pH with an appropriate volume of Reverse-phase high-performance liquid chromatography combined
sodium hydroxide (0.2 M) solution to the required pH. The with different detectors is the commonly used analytical method for
oxidation process of the drug was found to be quasi-reversible with the separation and identification of flavonoids. A method has been
an adsorption controlled step. The adsorption stripping response developed for the simultaneous determination of diosmin,
was evaluated with respect to various experimental conditions, such hesperidin and naringin in different citrus fruit juices and
as the pH of the supporting electrolyte, the accumulation potential pharmaceutical preparations. Compounds were separated using
and the accumulation time. The proposed voltametric methods at tetrahydrofuran/water/acetic acid (21:77:2, v/v/v) as the mobile
the GCE provided the advantages of simplicity, precision and phase at 34˚C, using a C8 reversed-phase column and with a UV-
accuracy for the analysis of diosmin compared with other reported VIS detector. The method has been successfully applied to the
methods (spectrophotometric and HPLC). The methods are also free determination of all three flavonoid glycosides in several samples of
from any interference of commonly used excipients and additives in different citrus fruit juices and for the determination of diosmin and
the formulations of the drug [12a]. hesperidin in pharmaceutical preparations. The technique is simple,
specific, precise and accurate [1e]. An HPLC method was
Hanuštiak et al. [12b] suggested an electrochemical technique for established for the simultaneous determination of diosmin,
the determination of diosmin and other flavonoids by square wave hesperidin and eriocitrin in Iranian lime juice. The filtrate was
voltammetry using a carbon paste electrode as a working electrode. passed through a polyamide cartridge, washed with 8 mL
The measurements were performed using the following parameters: water/methanol (90:10 v/v, adjusted at pH = 3 with concentrated
initial potential 0.1 V, end potential 1.2 V, pulse amplitude 49.85 HCl) to remove interferences and eluted with 4 mL HPLC grade
mV, step potential 1.95 mV, frequency 180 Hz. As a supporting methanol. The eluate was dried and dissolved in 1 mL of
electrolyte phosphate buffer (0.1 M NaH2PO4 + 0.1 M Na2HPO4, acetonitrile, filtered and injected into the HPLC system. HPLC
pH 7) was used. The carbon paste was made of graphite powder and separation of hesperidin and diosmin was performed using a mobile
mineral oil (70:30, w/w). The limit of detection of diosmin was 2.66 phase consisting of water/acetonitrile/acetic acid (78:19:3, v/v) with
µM. The proposed method is a simple, low cost and sensitive a flow rate of 0.8 mL/min at room temperature. The analytes were
technique for the determination of diosmin in biological material monitored at 280 nm. Limit of detection (LOD; 0.0283 µg/mL),
[12b]. limit of quantification (LOQ; 0.0857 µg/mL), recovery (93.3±4.1-
548 Natural Product Communications Vol. 8 (4) 2013
95.4±3.9), repeatability of retention times (4.7%) and peak areas
(3.6%) were determined after eight injections. The results showed
good sensitivity, linearity and repeatability, and the method was
found to be suitable for quality control and routine analysis of
diosmin in natural samples [1c].
High pressure liquid chromatography was used for determination of
diosmin and other flavonoids in extracts from citrus peels. The
HPLC analysis was performed using a C8 column (250 x 4,6; 5
µm). A photodiode array detector was set to 290 and 360 nm
monitoring wavelength. The mobile phase consisted of water with
0.1% formic acid and acetonitrile and was used in an isocratic
program with 25% acetonitrile. The flow-rate was 0.75 mL/min.
The ESI-MS were acquired in positive mode. In order to obtain a
Collision Induced Dissociation (CID), a fragmentor voltage of 70–
190 V was used [1b]. The structural information about carbohydrate
sequence, aglycone moiety and the diglycosidic linkage was
achieved. A simple, selective and sensitive reversed – phase HPLC
method was developed for the determination of diosmin and
hesperidin in Buchu leaves and tablets containing these flavonoids.
The mobile phase, consisting of methanol – water (60:40, v/v) in a
simple isocratic mode, gave optimum chromatographic separation
of diosmin and hesperidin [1a].
A sensitive, accurate and precise bioanalytical method involving a
simple liquid–liquid extraction of plasma samples and a LC–
MS/MS determination of diosmin and diosmetin was developed and
validated to meet the requirements for pharmacokinetic
investigations. The analytes were separated on a C18 reversed-
phase column using a mixture of methanol/1% aqueous formic acid
(58:42, v/v) at a flow rate of 0.5 mL/min. APCI in the positive ion
mode and multiple reactions monitoring (MRM) was employed.
Although diosmin and diosmetin have similar structures their
physicochemical properties represented a problem for their
extraction from biological samples. Previous HPLC methods
involved the use of sodium hydroxide solution for diosmin
extraction, but strong alkalis may convert flavanones to the
corresponding chalcone derivatives, causing misleading results. The
limit of detection of diosmin in plasma was 0.13 ng/mL (S/N=3)
and the estimated LOQ was calculated as low as 0.34 ng/mL
(S/N=10) [1f]. The proposed method was quicker and gave better
LOD and LOQ values than the method described by Kanaze et al.
[1d]. In this method, optimum chromatographic separation of
diosmin, hesperidin, naringenin and the internal standard (rhoifolin)
were achieved using a C8 reversed–phase column and tetrahydro-
furan/water/acetic acid (21:77:2, v/v/v) as the mobile phase.
Also a micro HPLC-ESI/MS method was established as a rapid and
easy technique. The micro HPLC was coupled on-line with an MS
detector equipped with an ESI source operating in the negative
mode. The use of microcolumn HPLC greatly enhanced detection
performance. The method can be very useful in the analysis of the
natural complex of citrus flavonoids, for the identification of
components for which corresponding standards are not available,
and for the quantitation of components present in low amounts due
to the lower limit of detection of this method [1i].
Additionally, a GC/MS method for the simultaneous determination
of diosmetin and hesperetin in human plasma and urine has been
developed and validated. Samples were separated on an HP-5MS
fused-silica capillary column (30 m × 0.25 mm, film thickness 0.25
μm). The injector and interface temperatures were set at 280°C. The
column temperature was initially held at 80˚C for 1 min, and then
raised to 310˚C at a rate of 20˚C per min and held for 10 min. The
injection volume was 1 μL and the total run time was 24.5 min. The
dried extracts were derivatized by the addition of pyridine and the
Bogucka – Kocka et al.
derivatization reagent BSTFA + TMCS 1% and heated at 80˚C for 3
h. Finally, the trimethylsilyl (TMS) derivative mixture was injected
into the gas chromatograph-mass spectrometer (GC-MS) system for
analysis [1j].
Venous stasis, hemorrheologic disorders and vein wall
remodeling: The complex activity of diosmin was shown to impact
on all the above elements of venous pathology and to exert a
synergic effect on vein walls and lymphatic vessels. Managing
venous stasis therefore requires that venous return be stimulated
first [2a,8,13a]. Venous stasis increases the risk of thromboembolic
complications - some hemodynamic factors play a role in the
development of venous thrombi. It was demonstrated that ADP
released by pathological, deformation-resistant erythrocytes, leads
to platelet aggregation. In the face of the risk of thrombus
formation, combating venous stasis and the factors that lead to it
becomes crucial [13a-c].
Diosmin and other flavonoids, such as chrysin, apigenin, luteolin
and myricetin, extend the effect of noradrenaline on the vein wall.
Noradrenaline, when released from neurovascular synapses, causes
a contraction of vascular smooth muscle in the wall of the vein,
which promotes venous return. Noradrenalin-releasing cells are also
located in the walls of lymphatic vessels. These vessels are
characterized by spontaneous contractile activity and respond to
noradrenaline by increasing the frequency of contractions [13d].
The vasodilatory effects of flavonoids are related to their structure.
The presence and position of hydroxyl groups on the B ring seems
to be particularly important. For the vasodilatory effect, two ortho –
positioned hydroxyl groups on the B ring are essential. Moreover,
the presence of a double bond between C2 and C3 of the
benzopyran ring system increases the vasodilatory potency [15a,b].
Extending the duration of the effect of noradrenaline on vascular
walls therefore stimulates not only venous return, but also the
contractility of lymphatic vessels [13d,e]. All these are synergistic
effects that counteract venous stasis: stimulating lymphatic vessel
contractility enables previously developed edemas to be absorbed,
while stimulating venous return reduces the excessive venous blood
pressure that is responsible for the development of edemas [13e-g].
In addition, activated inflammatory mediators and endothelial cell
activation may degrade extracellular matrix constituents by release
of oxygen free radicals and matrix metalloproteinases (MMPs)
activity [14a]. Venous hypertension induces morphological changes
in valves, including fibrosis, with strong MMP-2 and MMP-9
activity in valves and during remodeling [14a,16a].
The mechanical properties of the vessel wall depend to a great
extent on extracellular matrix composition. Extensive changes of
these mechanical properties have been demonstrated in the wall of
varicose veins [14b]. Tissue remodeling is a complex process,
which is controlled by numerous factors, including peptide growth
factors. Increased content of transforming growth factor-β (TGF-β)
and fibroblast growth factor (FGF) have been demonstrated in the
wall of varicose veins. Furthermore, increased blood concentration
of vascular endothelial growth factor (VEGF) was found in patients
with varicose veins [14c,d]. VEGF levels decreased in patients with
C4 venous insufficiency treated with a micronized purified
flavonoid fraction (MPFF) (98 pg/mL to 58 pg/mL) [2d]. In light of
these mechanisms for venous remodeling, future therapy schemes
may have to be directed against the fundamental trigger mechanism,
including leucocyte and endothelial cell activation [2e,16b].
Diosmin (Daflon®) administration reduced the edema, the valve
annulus diameter, and fistula blood flow produced by the acute
AVF in line with its ability to block elevation of microvascular
permeability in inflammation [13h,17]. The MPFF also reduces
granulocyte and macrophage infiltration in line with observations in
Diosmin Natural Product Communications Vol. 8 (4) 2013 549

experimental acute ischemia and reperfusion or ischemia produced the ratio of completely healed ulcers [13j]. Diosmin was shown to
by venous hypertension [13i,16b]. Fluid shear stress on the reduce significantly lower limb edema as measured by several
endothelium, if accompanied by unsteady retrograde or even different methods (limb circumference, volumetry) [2f,13k,l]. The
turbulent (with random fluctuation) stress, may be an inflammatory efficacy of diosmin compared with placebo in reducing the severity
stimulus for the endothelial cells on a valve leaflet and may trigger of lower limb edema was demonstrated in a clinical trial of 200
cell apoptosis [18,19]. In such conditions the degradation of the patients with functional or organic chronic venous insufficiency
collagen matrix of the valve, and consequently mechanical [13j,n].
softening and restructuring of the leaflets and the venous wall may
occur [13d,e,18,19]. Administration of diosmin and other flavonoids led to an
improvement in skin lesions associated with chronic venous
Pathophysiology of venous insufficiency and the clinical aspect insufficiency in 88% of patients after 2 months of treatment,
of the use of diosmin preparations: As mentioned above, compared with only 21% of patients in the placebo group A multi-
phlebological pharmacotherapy plays an important role in treating center, placebo-controlled trial involving 107 patients with venous
venous stasis, edema and pain. Venous stasis not only initiates ulceration showed that after 2 months of treatment with a product
chronic ischemia of the lower limbs, but also exacerbates pre- containing diosmin at a dose of 2 tablets (one tablet = 450 mg
existing pathological conditions. This favors the development of micronized purified diosmin) per day, combined with simultaneous
edemas, initially by means of increased venous blood pressure, and compression therapy and topical treatment, the ratio of healed ulcers
later by the outflow of fluid from the vascular bed [13j]. The edema was 32% as compared with 13% in the placebo group (p = 0.028)
fluid is initially absorbed by the lymphatic system. However, the [13o]. Moreover, healing proceeded twice as fast in the diosmin
lymphatic drainage route is quickly overloaded, and the retention of group.
fluid leads to the development of edema, which in turn leads to the
development of further adverse effects [13j,k]. The "tension band" Conclusions: Diosmin has a wide range of biological activity,
effect leads to reduced oxygenation of peripheral tissues and fosters including an anti-inflammatory effect and an inhibition of
the accumulation of toxic metabolites. These cause trophic skin prostaglandin synthesis [11c,12a]. Previous research on the
disorders. Venous stasis is also a cause of hemodynamic disorders: phlebotropic effect of diosmin still does not give a clear answer
increased hematocrit caused by a loss of protein and fluid from the whether the clinical effect is indeed conditioned by this action. Our
vascular bed, increased blood viscosity, and increased resistance of review is an introduction to the choice of analytical methods for the
morphotic blood elements to deformation. These hemodynamic determination of diosmin and its metabolites in peripheral blood of
disorders exacerbate venous stasis. This, combined with venous patients with chronic venous insufficiency. From the data reported
valve incompetence, creates a vicious circle. This is mainly driven in this short review, it can be seen that there are numerous
by erythrocyte aggregation, as both blood viscosity and resistance to techniques for the isolation and separation of diosmin from plant
deformation are largely dependent on this phenomenon. material, pharmaceutical formulations and biological fluids. Various
analytical approaches exist for the detection of diosmin and the
Diosmin products are characterized by their effects on venous and analytical methods should meet the following prerequisites: short
lymphatic vessel systems and their protective effect on time, be relatively inexpensive, highly accurate, and precise for a
microcirculation vessels [13l,m]. This explains the efficacy of variety of applications. This review may be helpful in choosing a
diosmin and other flavonoids in improving the function and suitable method for diosmin isolation and analysis.
protecting the structure of veins and lymphatic vessels in the
treatment of chronic venous insufficiency [13j,k,m]. Volumetric Acknowledgements - The authors wish to thank Prof. Dr Christian
measurements demonstrated significant efficacy of diosmin in Zidorn (Innsbruck and Brussels) for first proof reading. This work
reducing the severity of lower limb edema in another clinical trial. was developed using the equipment purchased within the Project
After 2 months of treatment, significant reduction in lower limb “The equipment of innovative laboratories doing research on new
circumference was observed in diosmin-treated patients compared medicines used in the therapy of civilization and neoplastic
with the placebo group. Combination treatment with diosmin diseases” within the Operational Program Development of Eastern
preparations and standard compression therapy was shown to Poland 2007-2013, Priority Axis I Modern Economy I.3 Innovation
accelerate the healing of venous ulceration of shanks and increase Promotion.
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 Natural Product Communications Vol. 8 (4) 2013
Published online (www.naturalproduct.us)

Volatile Composition of Six Horsetails: Prospects and Perspectives

Françoise Fons, Didier Froissard, Jean-Marie Bessière, Alain Fruchier, Bruno Buatois and Sylvie Rapior 509
Chemical Compositions of the Rhizome, Leaf and Stem Oils from Malaysian Hornstedtia leonurus
Nor Akmalazura Jani, Hasnah Mohd. Sirat, NorAzah Mohamad Ali and Azrina Aziz 513
Effect on Emotional Behavior and Stress by Inhalation of the Essential oil from Chamaecyparis obtusa
Hikaru Kasuya, Erika Hata, Tadaaki Satou, Masaki Yoshikawa, Shinichiro Hayashi, Yoshinori Masuo and Kazuo Koike 515
Chemical Composition and Antibacterial Activity of Rhizome Oils from Five Hedychium Species
Ratchuporn Suksathan, Siriwoot Sookkhee, Somboon Anuntalabhochai and Sunee Chansakaow 519
Chemical Composition and Antimicrobial Activity of Three Essential Oils from Curcuma wenyujin
Jingjing Zhu, Agnieszka D. Lower-Nedza, Meng Hong, Song Jiec, Zhimin Wang, Dong Yingmao, Christine Tschiggerl,
Franz Bucar and Adelheid H. Brantner 523
Essential Oil Composition and Antimicrobial Activity of Aerial Parts and Ripe Fruits of Echinophora spinosa (Apiaceae)
from Italy
Daniele Fraternale, Salvatore Genovese and Donata Ricci 527
Composition and in vitro Anticancer Activities of the Leaf Essential Oil of Neolitsea variabillima from Taiwan
Yu-Chang Su, Kuan-Ping Hsu, Eugene I-Chen Wang and Chen-Lung Ho 531

Review/Account
Natural Products from Marine Algae of the Genus Osmundaria (Rhodophyceae, Ceramiales)
Kelvin Osako and Valéria Laneuville Teixeira 533
Phenols, Alkaloids and Terpenes from Medicinal Plants with Antihypertensive and Vasorelaxant Activities. A Review
of Natural Products as Leads to Potential Therapeutic Agents
Francesco Maione, Carla Cicala, Giulia Musciacco, Vincenzo De Feo, Anibal G. Amat, Armando Ialenti and Nicola Mascolo 539
Diosmin – Isolation Techniques, Determination in Plant Material and Pharmaceutical Formulations, and Clinical Use
Anna Bogucka – Kocka, Michał Woźniak, Marcin Feldo, Janusz Kocki and Katarzyna Szewczyk 545
 Natural Product Communications
2013
Volume 8, Number 4
Contents
Original Paper Page
Anti-melanogenesis Constituents from the Seaweed Dictyota coriacea
Ryeo Kyeong Ko, Min-Chul Kang, Sang Suk Kim, Tae Heon Oh, Gi-Ok Kim, Chang-Gu Hyun, Jin Won Hyun and Nam Ho Lee 427
Methyl Carnosate, an Antibacterial Diterpene Isolated from Salvia officinalis Leaves
Elisa Climati, Fabio Mastrogiovanni, Maria Valeri, Laura Salvini, Claudia Bonechi, Nilufar Zokirzhonovna Mamadalieva,
Dilfuza Egamberdieva, Anna Rita Taddei and Antonio Tiezzi 429
Cytotoxicity of Meroterpenoids from Sargassum siliquastrum against Human Cancer Cells
Jung Im Lee, Myoung K. Kwak, Hee Y. Park and Youngwan Seo 431
Isolation of Methyl 27-caffeoyloxyoleanolate – A New Oleanane Triterpenoid from the Roots of Hibiscus vitifolius
Duraisamy Ramasamy and Ariamuthu Saraswathy 433
Synthesis and Cytotoxic Activity of New Betulin and Betulinic Acid Esters with Conjugated Linoleic Acid (CLA)
Barbara Tubek, Paweł Mituła, Natalia Niezgoda, Katarzyna Kempińska, Joanna Wietrzyk and Czesław Wawrzeńczyk 435
Analysis of Pyrrolizidine Alkaloids and Evaluation of Some Biological Activities of Algerian Senecio delphinifolius (Asteraceae)
Soukaina Tidjani, Philippe N. Okusa, Amar Zellagui, Laetitia Moreno Y Banuls, Caroline Stévigny, Pierre Duez and Salah Rhouati 439
Berbanine: a New Isoquinoline-isoquinolone Alkaloid from Berberis vulgaris (Berberidaceae)
Anna Hošťálková, Zdeněk Novák, Milan Pour, Anna Jirošová, Lubomír Opletal, Jiří Kuneš and Lucie Cahlíková 441
Dicentrine Production in Callus and Cell Suspension Cultures of Stephania venosa
Tharita Kitisripanya, Jukrapun Komaikul, Nirachara Tawinkan, Chuennapha Atsawinkowit and Waraporn Putalun 443
New Flavan and Alkyl α,β-Lactones from the Stem Bark of Horsfieldia superba
Nabil Ali Al-Mekhlafi, Khozirah Shaari, Faridah Abas, Ethyl Jeyaseela Jeyaraj, Johnson Stanslas, Shaik Ibrahim Khalivulla and
Nordin H. Lajis 447
New Flavonol Triglycosides from the Leaves of Soybean Cultivars
Yoshinori Murai, Ryoji Takahashi, Felipe Rojas Rodas, Junichi Kitajima and Tsukasa Iwashina 453
Melitidin: A Flavanone Glycoside from Citrus grandis ‘Tomentosa’
Wei Zou, Yonggang Wang, Haibin Liu, Yulong Luo, Si Chen and Weiwei Su 457
Two New Chalcones from the Flowers of Clerodendrum inerme
Shaik Khadar Shahabuddin, Rachakunta Munikishore, Golakoti Trimurtulu, Duvvuru Gunasekar, Alexandre Deville and Bernard Bodo 459
A Novel Phenolic Compound from Phyllanthus emblica
Gaimei She, Ruiyang Cheng, Lei Sha, Yixia Xu, Renbin Shi, Lanzhen Zhang and Yajian Guo 461
Anti-austeric Activity of Phenolic Constituents of Seeds of Arctium lappa
Yasuhiro Tezuka, Keiichi Yamamoto, Suresh Awale, Feng Li, Satoshi Yomoda and Shigetoshi Kadota 463
Bioactive Lignans from the Leaves and Stems of Schisandra wilsoniana
Guang-Yu Yang, Rui-Rui Wang, Zhong-Hua Gao, Yin-Ke Li, Liu-Meng Yang, Xiao-Nian Li, Shan-Zhai Shang, Yong-Tang Zheng,
Wei-Lie Xiao and Han-Dong Sun 467
Antioxidative / Acetylcholinesterase Inhibitory Activity of Some Asteraceae Plants
Ivana Generalić Mekinić, Franko Burčul, Ivica Blažević, Danijela Skroza, Daniela Kerum and Višnja Katalinić 471
Antioxidant and Antimicrobial Activities, and Phenolic Compounds of Selected Inula species from Turkey
Alper Gökbulut, Onural Özhan, Basri Satılmış, Kadir Batçıoğlu, Selami Günal and Engin Şarer 475
Two New Dihydrostilbenoid Glycosides Isolated from the Leaves of Litsea coreana and their Anti-inflammatory Activity
Wenjian Tang, Weili Lu, Xiaoqing Cao, Yilong Zhang, Hong Zhang, Xiongwen Lv and Jun Li 479
Inhibitory Activity of Benzophenones from Anemarrhena asphodeloides on Pancreatic Lipase
Yang Hee Jo, Seon Beom Kim, Jong Hoon Ahn, Qing Liu, Bang Yeon Hwang and Mi Kyeong Lee 481
Identification and Quantification of Furanocoumarins in Stem Bark and Wood of Eight Algerian Varieties of
Ficus carica by RP-HPLC-DAD and RP-HPLC-DAD-MS
Samia Rouaiguia-Bouakkaz, Habiba Amira-Guebailia, Céline Rivière, Jean-Claude Delaunay, Pierre Waffo-Téguo and
Jean-Michel Mérillon 485
UPLC-Q-TOF/MS Coupled with Multivariate Statistical Analysis as a Powerful Technique for Rapidly Exploring
Potential Chemical Markers to Differentiate Between Radix Paeoniae Alba and Radix Paeoniae Rubra
Nian-cui Luo, Wen Ding, Jing Wu, Da-wei Qian, Zhen-hao Li, Ye-fei Qian, Jian-ming Guo and Jin-ao Duan 487
Antimicrobial Activity of Crude Methanolic Extract from Phyllanthus niruri
Darah Ibrahim, Lim Sheh Hong and Ninthianantham Kuppan 493
Cellulose Contents of Some Abundant Indian Seaweed Species
Arup K. Siddhanta, Sanjay Kumar, Gaurav K. Mehta, Mahesh U. Chhatbar, Mihir D. Oza, Naresh D. Sanandiya,
Dharmesh R. Chejara, Chirag B. Godiya and Stalin Kondaveeti 497
Anti-inflammatory Potential of Silk Sericin
Pornanong Aramwit, Pasarapa Towiwat and Teerapol Srichana 501
Composition of Essential Oil from Aerial and Underground Parts of Geum rivale and G. urbanum Growing in Poland
Aleksandra Owczarek, Jan Gudej and Agnieszka Kice 505
Continued Inside backcover

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