Ruscus (Rusci rhizoma)

TABLE OF CONTENTS

I. REGULATORY STATUS OVERVIEW ...........................................................................................5

II. ASSESSMENT REPORT FOR HERBAL SUBSTANCE(S), HERBAL PREPARATION(S) OR

COMBINATIONS THEREOF WITH WELL-ESTABLISHED USE AND/OR TRADITIONAL USE ....6

II.1 INTRODUCTION ............................................................................................................................... 7

II.1.1 Description ............................................................................................................................ 7

II.1.2 Information on period of medicinal use in the Community regarding the specified

indication ............................................................................................................................... 8

II.2 N ON - CLINICAL DATA ................................................................................................................... 10

II.2.1 Pharmacology ...................................................................................................................... 10

II.2.1.1 Primary pharmacodynamics ............................................................................................ 10

II.2.1.2 Safety pharmacology studies ........................................................................................... 22

II.2.1.3 Pharmacodynamic interactions ........................................................................................ 22

II.2.1.4 Assessor’s overall conclusions on pharmacology ........................................................... 23

II.2.2 Pharmacokinetics ................................................................................................................. 24

II.2.2.1 Preparation of the extracts ............................................................................................... 24

II.2.2.2 Absorption ....................................................................................................................... 24

II.2.2.3 Distribution ...................................................................................................................... 26

II.2.2.4 Metabolism ...................................................................................................................... 26

II.2.2.5 Elimination ...................................................................................................................... 26

II.2.2.6 Assessor’s overall conclusions on pharmacokinetics ...................................................... 27

II.2.3 Toxicology ........................................................................................................................... 28

II.2.3.1 Acute toxicity .................................................................................................................. 28

II.2.3.2 Repeat-dose toxicity ........................................................................................................ 29

II.2.3.3 Genotoxic and carcinogenic potentials ............................................................................ 29

II.2.3.4 Reproductive toxicity ...................................................................................................... 29

II.2.3.5 Assessor’s overall conclusions on toxicology ................................................................. 29

II.3 C LINICAL DATA ............................................................................................................................ 31

II.3.1 Clinical pharmacology ........................................................................................................ 31

II.3.1.1 Pharmacodynamics .......................................................................................................... 31

II.3.1.2 Pharmacokinetics ............................................................................................................. 32

II.3.2 Clinical efficacy .................................................................................................................. 32

II.3.2.1 Dose response studies ...................................................................................................... 32

II.3.2.2 Clinical studies (case studies and clinical trials) ............................................................. 33

II.3.2.3 Clinical studies in special population (e.g. elderly and children) .................................... 38

II.3.2.4 Assessor’s overall conclusions on clinical efficacy ........................................................ 38

II.3.3 Clinical Safety/Pharmacovigilance ..................................................................................... 38

II.3.3.1 Patient exposure .............................................................................................................. 38

II.3.3.2 Adverse events ................................................................................................................ 38

II.3.3.3 Serious adverse events and deaths ................................................................................... 40

II.3.3.4 Laboratory findings ......................................................................................................... 40

II.3.3.5 Safety in special populations and situations .................................................................... 40

II.3.3.6 Assessor’s overall conclusions on clinical safety ............................................................ 41

II.4 ASSESSOR’S OVERALL CONCLUSIONS .............................................................................. 42

A NNEXES ................................................................................................................................................. 44

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TABLE OF ILLUSTRATIONS

Table 1 : estrogen and progesterone measured in serum levels of ovariectomised dogs, according to their

hormonal supplementation .......................................................................................................................... 12

Figure 1: proposed mechanism of action for Ruscus and effect of temperature ........................................ 12

Figure 2: effect of Ruscus extract on the permeability of pig vascular ear vein ......................................... 20

Figure 3: blood kinetics of tritiated compounds of Ruscus extract in 2 rats after oral administration ........ 25

Figure 4: radioactivity levels measured in blood of Wistar rats after oral administration of a 3 H-labelled

Ruscus extract ............................................................................................................................................. 25

Figure 5: time course of plasma concentration of degluconeoruscin after oral ingestion of 1 g Ruscus

extract by 3 human volunteers ..................................................................................................................... 32

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ABBREVIATIONS

Afssaps

Agence Française de Sécurité Sanitaire des Produits de Santé (French Health Products

Safety Agency)

ATP

Adenosine Tri-Phosphate

AUB

Area Under Baseline

CVD

Chronic Venous Disease

CVI

Chronic Venous Insufficiency

DOMA

3,4-dihydroxymandelic acid

DOPEG

3,4-dihydroxyphenylglycol

EMEA

European Medicines Agency

ESCOP

European Scientific Cooperative On Phytotherapy

ETDRS

Early Treatment Diabetic Retinopathy Study

FLQA

Freiburger Life Quality Assessment

HPLC

High Pressure Liquid Chromatography

HUVEC

Human Umbilical Vein Endothelial Cell

IV

Intravenous

LD50

Lethal Dose 50% (dose that kills 50% of experimental animals)

LOCF

Last Observation Carried Forward

LSC

Liquid Scintillation Counting

MOPEG

3-methoxy-4-hydroxyphenylglycol

NMN

Normetanephrine

PGF2α

Prostaglandin F2α

PLA2

Phospholipase A2

PSUR

Periodic Safety Update Report

Pt

Total protein concentration

PTS

Post-Thrombotic Syndrome

THMC

Trimethylhesperidin chalcone

TLC

Thin Layer Chromatography

VAD

Vaso-Active Drug

VAS

Visual Analog Scale

VMA

3-methoxy-4- hydroxymandelic acid

WBA

Whole Body Autoradiography

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I. REGULATORY STATUS OVERVIEW 1

MA: Marketing Authorisation;

TRAD: Traditional Use Registration;

Other TRAD: Other national Traditional systems of registration;

Other: If known, it should be specified or otherwise add ’Not Known’

Member State

Regulatory Status

Comments 2

Austria

MA

TRAD

Other TRAD

Other Specify:

Belgium

MA

TRAD

Other TRAD

Other Specify:

Bulgaria

MA

TRAD

Other TRAD

Other Specify:

Cyprus

MA

TRAD

Other TRAD

Other Specify:

Czech Republic

MA

TRAD

Other TRAD

Other Specify:

Denmark

MA

TRAD

Other TRAD

Other Specify:

Estonia

MA

TRAD

Other TRAD

Other Specify:

Finland

MA

TRAD

Other TRAD

Other Specify:

France

MA

TRAD

Other TRAD

Other Specify:

Germany

MA

TRAD

Other TRAD

Other Specify:

Greece

MA

TRAD

Other TRAD

Other Specify:

Hungary

MA

TRAD

Other TRAD

Other Specify:

Iceland

MA

TRAD

Other TRAD

Other Specify:

Ireland

MA

TRAD

Other TRAD

Other Specify:

Italy

MA

TRAD

Other TRAD

Other Specify:

Latvia

MA

TRAD

Other TRAD

Other Specify:

Liechtenstein

MA

TRAD

Other TRAD

Other Specify:

Lithuania

MA

TRAD

Other TRAD

Other Specify:

Luxemburg

MA

TRAD

Other TRAD

Other Specify:

Malta

MA

TRAD

Other TRAD

Other Specify:

The Netherlands

MA

TRAD

Other TRAD

Other Specify:

Norway

MA

TRAD

Other TRAD

Other Specify:

Poland

MA

TRAD

Other TRAD

Other Specify:

Portugal

MA

TRAD

Other TRAD

Other Specify:

Romania

MA

TRAD

Other TRAD

Other Specify:

Slovak Republic

MA

TRAD

Other TRAD

Other Specify:

Slovenia

MA

TRAD

Other TRAD

Other Specify:

Spain

MA

TRAD

Other TRAD

Other Specify:

Sweden

MA

TRAD

Other TRAD

Other Specify:

United Kingdom

MA

TRAD

Other TRAD

Other Specify:

1 This regulatory overview is not legally binding and does not necessarily reflect the legal status of the products in

the MSs concerned.

2 Not mandatory field

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II.

ASSESSMENT REPORT FOR HERBAL SUBSTANCE(S), HERBAL

PREPARATION(S) OR COMBINATIONS THEREOF WITH WELL-ESTABLISHED

USE AND/OR TRADITIONAL USE

Herbal substance(s) (binomial scientific name of

the plant, including plant part)

Ruscus aculeatus L., rhizoma

Herbal preparation(s)

Traditional use

Dried powdered root

Dry extract (2.5-6.5 : 1 ; water)

Dry extract (5-8.5 : 1 ; 80% V/V ethanol)

Dry extract (6-9 : 1 ; primary solvent 96 % V/V

ethanol followed by water.)

Dry extract (15-20 : 1 ; 60% V/V methanol)

Pharmaceutical forms

Solid forms for oral use

Rapporteurs

Antoine Sawaya

Jacqueline Viguet Poupelloz

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II.1 I NTRODUCTION

The aim of this report is to assess the preclinical and clinical available data on Ruscus aculeatus for

preparing a Community herbal monograph. This report is based on the documentation provided by the

European Medicines Agency (EMEA) completed by additional researches and information taken from

recently revised monographs on Ruscus aculeatus (Commission E Monographs, 2001; ESCOP, 2003).

Recent investigations on the efficacy and safety of Ruscus aculeatus have also been published in reviews

specializing in phytotherapy and alternative medicines (Commission E Monographs, 2001; Bone, 2003).

This report takes up parts of these works. However, as far as Ruscus aculeatus alone is concerned with the

monograph,

for

relevant

safety

data,

the

studies

performed

with

Ruscus aculeatus combinations such as Cyclo 3® or Phlebodril® are not discussed in this report.

This report focuses on findings with preparations based on aqueous-alcoholic extracts of the rhizome since

clinical experience has been collected with these types of extracts, and they were used in most preclinical

and clinical trials. The extracts used in the trials are specified as far as possible. Unfortunately, correct

specifications of solvent and drug-extract ratio are missing in most of the publications. No detail can be

given if the extract could not be identified.

II.1.1

D ESCRIPTION

• Herbal substance

Ruscus aculeatus L . (Liliaceae) is a widely distributed European plant native from Western Europe. This

plant has numerous appellations. The English name - Butcher’s Broom - derives from the use by the

European butchers of the stems to clean their cutting board not only because of their stiffness and solidity,

but also because of the essential oil which was credited with antibacterial properties. Ruscus aculeatus is a

small, clump-forming shrub with erect shoots bearing stiff, ovate, leaf-like phylloclade. Tiny green

flowers appear in late winter and spring on the phylloclade. Both root and stem are used in preparations.

The European Pharmacopoeia prescribes not less than 1.0 per cent of total sapogenins expressed as

ruscogenins (mixture of neoruscogenin and ruscogenin).

• Herbal preparations

It is around the middle of the twentieth century that steroidal saponins (ruscogenin and neoruscogenin)

were isolated from the rhizome. Besides primary steroidal saponins the extract of the rhizome contains

also several other minor compounds such as other steroidal sapogenins and saponins, sterols, triterpenes,

flavonoïds, coumarins, sparteïne, tyramine and glycolic acid (Dunouau et al. , 1996; Mimaki et al. , 1998).

Both the above ground and below ground parts of the plant contain ruscogenins, although concentrations

are higher in the below ground parts of the plant (Nikolov et al. , 1976). In the present time, preparations

are mainly based on aqueous and aqueous-alcoholic extracts of the rhizome. The dried powdered root is

also on the market. Ruscus aculeatus extracts are also used in commercial products combined with other

products such as trimethylhesperidin chalcone (TMHC), ascorbic acid or Melilotus officinalis extract.

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except

Ruscogenin

Neoruscogenin

II.1.2 I NFORMATION ON PERIOD OF MEDICINAL USE IN THE C OMMUNITY REGARDING THE

SPECIFIED INDICATION

This plant was used during the ancient times as a diuretic or to relieve menstrual pains and water retention

discomfort. Ruscus aculeatus was also used throughout Europe for the treatment of constipation, urinary

disorders and abdominal pains. Ruscus aculeatus sinks into oblivion in the early 1900s. The reported

vasoconstrictive effect of the ruscogenin and neoruscogenin (see paragraph II.2 Non-clinical data)

accounted for its current use in chronic venous insufficiency, haemorrhoids and varicose veins.

Ruscus aculeatus has been used over a decade as herbal substance or herbal preparations notably in France

or Germany, since 1986 and 1978, respectively. At present, with regard to the different available

monographs, Ruscus aculeatus is listed as follows:

¾ French Monograph : cahiers de l’Agence n° 3 (Afssaps, 1998)

Adults recommendations given for dried powered root and dry aqueous extracts in capsules for oral

administration corresponding to a daily amount of total ruscogenins around 10 mg.

¾ ESCOP Monograph (ESCOP, 2003)

Adults recommendations given for solid or liquid extracts in amounts corresponding to 7-11 mg of

total ruscogenins for oral administration taken once a day.

“Supportive therapy for symptoms of chronic venous insufficiency, such as painful, tired and heavy

legs, tingling and swelling.

• It has to be noted that the ESCOP Monograph takes only into account the available data on Ruscus

aculeatus alone.

¾ Commission E Monograph (Commission E Monographs, 2001)

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“Traditionally used in subjective symptoms of chronic insufficiency such as sensation of heavy legs

and in haemorrhoids symptoms”.

Supportive therapy for symptoms of haemorrhoids, such as itching and burning.”

Adults’ recommendations given for alcoholic extracts of the whole plant or standardized for

ruscogenins as determined by the total amount of ruscogenin and neoruscogenin (ranges 7-11 mg)

in capsules for oral administration.

“Supportive therapy for discomforts of chronic venous insufficiency, such as pain and heaviness,

Supportive therapy for complaints of haemorrhoids, such as itching and burning. “

• It has to be noted that the Commission E Monograph takes into account the whole data available on

Ruscus aculeatus including non-clinical and clinical data referring to combinations.

¾ Bfarm (BfArM information, 2007)

Adults recommendations given for dry extracts for oral administration:

Ethanolic 80 % (V/V); (5-8.5 : 1) in coated tablets and Ethanolic 96 % (V/V); (6-9 : 1) in soft

capsules, respectively .

“Traditional herbal medicinal product to ease/soothe the feeling /sensation of heavy legs. The

product is a traditional herbal product for use in specified indications exclusively based on long-

standing use.”

• Daily dosage and corresponding daily amount of total ruscogenins are not given.

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as well as cramps in the legs, itching and swelling.

II.2

N ON - CLINICAL DATA

Non clinical data presented in this assessment report should be taken into account according to the kind of

preparations registered in the European Union. Request of information concerning Ruscus aculeatus

showed that in France and Germany, preparations consist of hydro-alcoholic extracts, aqueous extracts,

and plant powder.

Assessor’s comment

The non clinical assessment report was mainly based on bibliographic data submitted by the European

Medicines Agency (EMEA). Some references were occasionally added by the non clinical assessor on

specific points.

In general, the characteristics of the extract used (mode of extraction, content in active substances, etc.)

were not provided. Such a lack of data does not allow an adequate assessment of non clinical studies, and

clear conclusion on efficacy and safety of the extract

In most of the studies, the extract was provided by Pierre Fabre Médicament (Castres, France) and/or the

authors were employed by this company. Therefore, we will consider that the extract produced by Pierre

Fabre Médicament was used, unless otherwise indicated. It should be noted that no precision on this

extract is given by any author (mode of extraction, content in active substances, etc.)

II.2.1

P HARMACOLOGY

II.2.1.1 P RIMARY PHARMACODYNAMICS

Ruscus aculeatus being reported to be used in against venous insufficiency-related feeling/sensation of

heavy leg, several in vitro / in vivo studies aimed at demonstrating its contractile properties on veins or

lymphatic vessels as well as its capacity to improve vascular permeability.

Assessor’s comment

It should be noted that studies conducted with the combination used in Cyclo 3 ® ( Ruscus extract and

hesperidine methylchalcone) were not taken into consideration. Indeed, the potential beneficial effect of

the flavonoids or of the combination should not be attributed to Ruscus extract taken separately.

II.2.1.1.1 C ONTRACTILE EFFECT ON VEINS

A. In vitro studies

• Studies performed on rings of canine saphenous veins

This model was first described by Marcelon et al (Marcelon et al. , 1983b; Marcelon and Vanhoutte,

1984). Rings of canine saphenous veins were mounted in organ chambers filled with a physiological salt

solution and connected to a force transducer for continuous recording of isometric tension. Prior to

experimentation with Ruscus extract, the segments were placed at the optimal point of their length-tension

relationship using standard electrical stimulation. Preparations were then allowed to equilibrate at their

optimal length for 90 minutes. Thereafter, Ruscus extract was applied and the tension measured. It showed

that at concentrations ≥ 3.10 -5 g/ml, Ruscus extract caused a dose-dependent increase in tension.

The effects of the following various pharmacological agents on the contractile effect induced by Ruscus

were studied in an attempt to determine the mechanism of action underlying this contractile effect:

–

Phentolamine (3.10 -6 M), an α-adrenolytic agent, nearly abolished the contractile response to

Ruscus ;

–

Cocaine (3.10 -5 M), a blocker or the recapture of norepinephrine in the inter-synaptic gap, reduced

the contractile response to Ruscus ;

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– 6-hydroxydopamine (10 -6 M), inducing chemical denervation, reduced the contractile response to

Ruscus similarly to cocaine;

– Tetrodotoxin (10 -7 M), atropine (10 -8 M), methysergide (10 -7 M), indomethacin (3.10 -5 M) did not

impact on the contractile response to Ruscus ;

– Adenosine (2.10 -5 M) and verapamil (2.10 -6 M) produced relaxation of the rings contracted by

Ruscus (-37% and -50%, respectively);

– Acetylcholine (10 -7 M, 10 -6 M) caused further increase in tension (+23% and +37%, respectively).

In parallel, the same authors examined the effect of Ruscus extract on helical strips of canine Saphenous

veins connected to a force transducer and incubated in a moist tunnel-shaped chamber superfused with a

Krebs-Ringer solution containing 7-1-[ 3 H]-norepinephrine. Ruscus extract (5.10 -4 g/ml) caused an increase

in tension. In addition, the release of intact [ 3 H]-norepinephrine and the overflow of all metabolites

(DOPEG, DOMA, MOPEG, NMN) except VMA were increased.

From these findings, it was concluded that the venoconstrictor response to Ruscus extract is not due to the

activation of cholinergic, serotoninergic, or prostaglandinergic receptors. The effect being inhibited by

phentolamine but persisting after chemical denervation with 6-hydroxodopamine, a direct effect on

postjunctional α-adrenergic receptors of the smooth muscle cells is suggested. However, it seems that the

release of norepinephrine stored in adrenergic nerve endings is also involved in the contractile effect of

Ruscus extract in view of the responses obtained with 6-hydroxodopamine, and cocaine, and in view of

the increased overflow of [ 3 H]-norepinephrine in veins previously incubated with this neurotransmitter.

The release of norepinephrine from adrenergic nerve endings does not involve the initiation of spike

electrogenesis, since tetrodotoxin had no influence on the effect provoked by Ruscus extract. The authors

considered that the effects of adenosine, verapamil and acetylcholine reflected interference with the

postjunctional effect rather than with its action on the adrenergic nerve endings. However, the effect by

acetylcholine may be artificially biased by damaging of the endothelium in the experimental model; in a

non de-endothelialised vessel a relaxing effect by NO release would be expected.

In another study, Rubanyi et al confirmed that Ruscus extract could provoke the contraction of rings from

canine saphenous veins. The tension increased as Ruscus extract concentration increased from 10 -4 to

10 -3 g/ml; the maximal contraction averaged 80% of the response to 10 -4 M norepinephrine. It should be

mentioned that a factor 10 within a dose relationship from basic tension to maximum has to be considered

as very narrow in pharmacological terms. However, the contractile effect of Ruscus was depressed by

prazosin or rauwolscine (α1- and α2-antagonist, respectively) from 5.10 -8 M and in a dose-dependent

fashion, while it was abolished when both substances were used concomitantly. The same response was

observed when phentolamine, a non selective α-antagonist was used in the experiment described above

(Marcelon et al. , 1983b; Marcelon and Vanhoutte, 1984). This suggests that the contractile response of

Ruscus in this model is due to α-adrenergic activation only (Rubanyi et al. , 1984).

At 5.10 -6 M, the inhibitory effect of rauwolscine was higher than that of prazosin. This difference was not

observed when prazosin or rauwolscine was used with cocaine, both combinations depressing the

contractile activity of Ruscus in the same extent. According to the authors, this suggests that

norepinephrine released from adrenergic nerve endings activates preferentially postjunctional

α2-adrenoceptors (Rubanyi et al. , 1984).

The same authors reiterated the study using Ruscus extract (2.10 -4 g/ml), prazosin and rauwolscine

(5.10 -6 M) at 24°C, 37°C, and 41°C. Compared to the results obtained at 37°C, cooling decreased while

warming increased the contractile response to Ruscus extract in rings of canine saphenous veins. These

results were explicated in details by other authors. They acknowledged that these differences are due to

the preferential modulation by temperature of the α1-adrenergic component of the response. At 37°C, the

α1- and α2-adrenergic components of the response to Ruscus are equivalent (Rubanyi et al. , 1984;

Marcelon and Vanhoutte, 1988).

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Abbreviations: U1=neuronal uptake; NE=norepinephrine; +=activation

Figure 1: proposed mechanism of action for Ruscus and effect of temperature (Marcelon and

Vanhoutte, 1988)

To study the influence of the hormonal status of the animals on the contractile effect obtained with Ruscus

extract, Miller et al treated ovariectomized female dogs with subcutaneous pellets containing a carrier

substance (untreated), 17β-estradiol, progesterone, or 17β-estradiol and progesterone for 16-25 days. After

that treatment period, the animals underwent surgery. The serum levels of hormones were measured and

the carotid arteries and lateral saphenous veins removed and cut into rings after having been cleaned of

connective tissue. The endothelium was removed in all rings. Thereafter, isometric tension was

determined in absence or presence of a combination of prazosin or rauwolscine (10 -7 M) following in vitro

exposure to norepinephrine (10 -8 to 10 -4 M), tyramine (10 -8 to 10 -4 M), or Ruscus extract (10 -6 to 10 -3 g/ml)

(Miller et al. , 1991a). Serum hormone levels are reported in the table below.

Progesterone level

(ng/100 mL)

Untreated 1 80

Estrogen 30 400

Progesterone – 750

Estrogen+Progesterone 30 2300

Table 1 : estrogen and progesterone measured in serum levels of ovariectomised dogs, according to

their hormonal supplementation (Miller et al. , 1991a)

Estrogen level

(ng/100 mL)

The contractile effect induced by electrical stimulation, norepinephrine and tyramine (indirect

sympathomimetic substance, releases norepinephrine from adrenergic nerve endings) were not influenced

by hormonal treatment. On the contrary, the contractile effect observed with Ruscus tended to be

augmented in the progesterone group compared to the untreated group, the difference being significant

compared to the group treated with estrogen and progesterone. Subsequent assays showed no effect of

adrenergic blockade (prazosin plus rauwolscine) on the contractile effect of Ruscus in rings from untreated

animals, whereas this effect was decreased in progesterone group and increased in estrogen and estrogen

plus progesterone groups (Miller et al. , 1991a).

The same authors published further work performed on the same animals, which consisted in studying the

influence of hormonal status and endothelium on the contractile effect of Ruscus (Miller et al. , 1991b).

In coronary arteries, Ruscus extract was able to initiate the release of endothelium-derived factors whose

action is inhibitory to contractions initiated by the extract itself, PGF2α and norepinephrine. The release

of this factor could involve the stimulation of muscarinic receptors because the relaxing effect was

12/44

Group

inhibited by atropine. Considering the nature of this relaxing factor, it is not probably prostacyclin because

the experiment was performed in presence of indomethacin in the medium. Similarities with nitric oxide

are evoked because the relaxing effect attributed to this relaxing factor is inhibited by haemoglobin (which

inactivates NO) and by methylene blue (which inactivates guanylate cyclase). The hormonal status of the

animals had no effect on the results obtained.

In femoral veins, the constrictor effect of Ruscus was influenced by the integrity of the endothelial cells

and by the hormonal status of the animals. Indeed, the contractile effect obtained with Ruscus was greater

in veins without endothelium, and greater in rings from animals receiving oestrogen plus progesterone.

This result is contradictory to previous results obtained by these authors and described above [see (Miller

et al. , 1991a)]. In varicose veins, the endothelium may be dysfunctional. Therefore, excitatory effect of the

extract would prevail in this situation. Elevated oestrogen and progesterone serum levels would potentate

this effect (Miller et al. , 1991b).

• Study performed on rings of saphenous veins taken from rabbits

The influence of the hormonal status of female rabbits on the contractile effect obtained with Ruscus

extract, Harker et al. treated ovariectomized rabbits with either placebo or 17β-oestradiol (subcutaneous

implants) for 14 to 21 days. After that treatment period, the animals underwent surgery. The serum level

of oestradiol was measured and the lateral saphenous veins removed and cut into rings after having been

cleaned of connective tissue. The endothelium was removed in all rings which were then suspended in

organ chambers to measure isometric tension after application of various agents. The effect of

α-adrenergic antagonists and of temperature (24°C, 37°C, 41°C) on the contractile effect of Ruscus extract

was also studied (Harker et al. , 1988).

The serum oestradiol level reported by the authors amounted to 1.0 ng/ml or less in control animals, while

it reached 54 ng/dl in estradiol treated animals. It should be noted that these figures may be wrong as

1.0 ng/ml is equivalent to 100 ng/dl.

In both groups, a dose-dependent contractile effect of Ruscus was observed. The involvement of

postjunctional adrenoceptors was dependent upon the hormonal status of the animals. Indeed, contractions

were not affected by adrenergic blockade in control animals, and partially inhibited by prazosin and

rauwolscine in estradiol-treated animals suggesting that hormone permits the expression of postjunctional

α-adrenergic effect of Ruscus .

Cooling increased tension mediated by α2-adrenoceptors (rauwolscine) after application of Ruscus extract

on rings of control rabbits, but not on rings of estradiol-treated animals. Moreover, it was concluded that

warming did not affect the contractile response to Ruscus extract (Harker et al. , 1988).

• Studies performed on rings of human saphenous and varicose veins

The contractile effect of Ruscus extract was investigated on rings of human varicose veins collected from

females undergoing varicectomy (Marcelon et al. , 1988b). To study the influence of the hormonal status,

three groups were considered:

– Rings from women at the end of menstrual cycle (14th to 25th day) : n=3;

– Rings from women at the beginning of cycle (1st to 6th day): n=4;

– Rings from post-menopausal women: n=6.

As previously described, isometric tension was measured in organ chambers (at 37°C).The contractile

effect of Ruscus extract (10 -5 to 10 -3 g/ml) was compared to that obtained with noradrenaline (10 -4 M).

In these conditions, the variation of hormone levels occurring during the menstrual cycle or in post-

menopausal women did not influence the contractile effect of Ruscus extract which reached 43 to 52% of

the contractile effect obtained with norepinephrine (Marcelon et al. , 1988b).

In another study, the role of endothelium in the contractile effect of Ruscus extract (10 -6 to 10 -3 g/ml) was

evaluated on rings of varicose and saphenous veins taken from patients undergoing surgery for primary

varicose veins (Miller et al. , 1994). In this study, Ruscus caused a concentration-dependent contractile

13/44

effect to which varicose veins were more sensitive than saphenous veins. Additionally, contractions to

Ruscus were not affected by removal of the endothelium. It was also confirmed in human veins that a

major component of the contraction results from activation of adrenergic receptors because the blockade

of adrenergic receptors significantly reduced that contractile effect. It is unlikely that Ruscus extract

stimulates contraction by endothelin-A receptors in veins of these patients as the selective antagonist of

these receptors (BQ-123) did not reduce the contractions either in absence or presence of adrenergic

blockade. It is also unlikely that endothelin-B receptors are stimulated by Ruscus extract as varicose

tributaries do not contract to sarafotoxin S6c (selective endothelin-B agonist). Therefore, the authors

concluded that contractions to Ruscus in human varicose veins are independent of the endothelium and

mediated by activation of adrenergic (but not endothelin-A) receptors on the smooth muscle (Miller et al. ,

1994).

• Studies performed on segments of canine and human saphenous/varicose veins

Branco and Osswald (1988) studied the influence of Ruscus extract on the uptake and metabolism of

norepinephrine in segments of canine lateral saphenous veins, and of human varicose/saphenous veins. In

these tissues, the endogenous norepinephrine and dihydroxymandelic acid (DOMA) contents were

determined. Thereafter, removal, accumulation and metabolism of [ 3 H]-norepinephrine were studied

without and with application of Ruscus extract (10 -5 , 10 -4 , 10 -3 g/ml).

Compared to the normal canine vein, the normal human veins appeared sparsely innervated by the

sympathetic system as shown by 10-fold lower norepinephrine content. However, it should not be

concluded that it is not endowed with efficient adrenergic mechanism. Indeed, its capacity to metabolize

norepinephrine is rather high and its high reactivity to adrenergic agonists is well known; in this study, the

capacity of human vein to remove, accumulate and metabolize norepinephrine was one-half of that

exhibited by the canine vein. Normal canine and human veins also differed in the pattern in which

noradrenaline was metabolized. In human veins, O-methylation by Catechol-O-methyltransferase

(COMT) was of lesser importance than in canine veins. From this finding, the authors concluded that in

what concerns disposition of noradrenaline, extrapolation from vessels of experimental animals to those of

humans is not permissible.

The comparison of human normal and varicose veins showed that varicose veins contain and accumulate

less norepinephrine. They have a high endogenous content and a raised rate of formation of DOMA.

Concerning the effect of Ruscus extract i n human normal veins, it produced a concentration-dependent

reduction of [ 3 H]-norepinephrine accumulation (-50% at 10 -3 g/ml, compared to control). Changes were

less marked in the varicose veins (-50% at 10 -3 g/ml, compared to control). The authors concluded that the

highest concentration of Ruscus extract only may have released norepinephrine. However, all three

concentrations affected the metabolism of norepinephrine: a concentration-dependent reduction of the

formation of all norepinephrine metabolites was observed, and was similar in normal and varicose veins

(DOMA formation was more affected in varicose veins).

Overall, quantitative but not qualitative differences were noted between human normal and varicose veins

concerning the effect of Ruscus . It provoked a depression of the metabolism of norepinephrine and

reduced its accumulation. This led to an increase in the concentration of norepinephrine in the biophase.

This could explain the potentiation of the action of norepinephrine (Branco and Osswald, 1988).

Assessor’s comments

The contractile effect of Ruscus extract was investigated on rings of saphenous veins taken from dogs

and rabbits, and on human saphenous and varicose veins by recording of the isometric tension. Studies on

the canine model showed that a concentration-dependent contractile effect was obtained within the

concentration range of 10 -5 -10 -3 g/ml for Ruscus extract. This was confirmed in the human model.

To determine the mechanism underlying this effect, further experimentations with various

pharmacological agents and a study of tritiated norepinephrine metabolism were performed. They showed

that in the canine venous rings, this effect was mediated by direct activation of postjunctional α1- and α2-

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adrenergic receptors, and by stimulation of the release of norepinephrine form adrenergic nerve endings. It

should be noted that α-adrenergic activation only was involved. The involvement of adrenergic receptors

in the contractile effect of Ruscus extract was confirmed in human veins (saphenous, varicose).

These results are in accordance with those obtained on segments of human normal and varicose veins,

where Ruscus extract was shown to reduce norepinephrine accumulation and metabolism. Although less

marked in varicose veins, the reduction of norepinephrine accumulation was significant from 10 -4 g/ml, a

concentration compatible with those inducing a contractile effect on venous rings.

The influence of temperature was evaluated. In canine venous rings, cooling decreased while warming

increased the contractile response to Ruscus extract obtained at 37°C. It was hypothesized that this

differences is due to the preferential modulation by temperature of the α1-adrenergic component of the

response. However, in venous rings taken from rabbits, cooling increased the contraction induced by

Ruscus depending on the hormonal treatment, while warming had no effect.

Regarding the influence of the hormonal status on the contractile effect induced by Ruscus extract,

contradictory results were obtained in animals. In one study performed with rings of varicose veins

collected from women, the hormonal status did not impact on the contractile effect obtained. However, the

number of samples tested was too low and the grade of varicosis of the veins was not studied, so that no

definitive conclusion can be drawn. Similarly, conflicting results were obtained about a possible role of

endothelium in the Ruscus effect in canine venous rings. In a human model, it was shown that

“contractions to Ruscus in human varicose veins are independent of the endothelium and mediated by

activation of adrenergic […] receptors on the smooth muscle” (Miller et al. , 1994).

Rings of human varicose veins were more sensitive than saphenous veins to the contractile effect obtained

with Ruscus extract.

B. In vivo studies

• Studies performed in dogs

To confirm in vivo the adrenergic mechanism of action of Ruscus extract, this drug was administered

intravenously to anaesthetised dogs at doses ranging from 1 to 10 mg/kg (Marcelon et al. , 1983a). The

lateral saphenous vein was transilluminated and its diameter measured by a photoelectric cell. Ruscus

caused a dose-dependent constriction of the saphenous vein and potentiated the dose-response curve to

local norepinephrine. These responses were antagonized by phentolamine. In the conditions of the study,

the venoconstriction caused by Ruscus was the result of α-adrenergic activation.

• Studies performed on the hamster cheek pouch

A team of the University of Lund, Sweden, aimed at examining the effects of Ruscus extract on the

microcirculation of the hamster cheek pouch and at gaining knowledge about the mechanism(s)

underlying these effects. They recognized that “the usefulness of the healthy hamster cheek pouch

preparation to study chronic venous insufficiency is debatable, but in vivo animal models for such study

are clearly lacking” (Bouskela et al. , 1994).

In the first experiment, six male hamsters were treated with Ruscus extract solution for 28 days by oral

route at 0 and 150 mg/kg/day. At the end of the treatment period, animals were anaesthetised and an area

of the cheek pouch isolated for measurement of vascular diameter (arterioles and venules) 3 by means of a

3 Anesthesia was induced by an intraperitoneal injection of 0.1-0.2 ml of sodium pentobarbital and maintained with a-chloralose (100 mgjkg)

administered intravenously. The femoral artery and vein were cannulated for pressure measurements, anesthetic and Ruscus extract injections.

Throughout the surgery and subsequent experiment, the animal rested on a heating pad controlled by a rectal thermistor and body temperature was

maintained at 36.5°C. A tracheal tube was inserted to facilitate spontaneous breathing. The hamster was placed on a stage containing a chamber

with a silicon rubber ring surrounding a transillumination window. This chamber was preceded by another one which pre-heated the superfusion

solution. Both chambers were mounted with Peltier elements for temperature control, allowing easy change and regulation of the superfusate's

temperature. The cheek pouch was carefully everted with the aid of a moist cotton stick and the distal, non-muscular, part of it identified and

pinned to a silicon ring. Dissection was performed under a stereomicroscope: a crescent-shaped incision was made in the top layer, the flap was

pinned to the side and the areolar connective tissue removed to expose the bottom layer vasculature for microscopic observations. During the

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videotape recording device. The measurements were made on the same region in every animal. In these

conditions, no significant difference was noted between control and treated animals regarding the body

weight and mean arterial blood pressure. In the group receiving Ruscus extract, constriction of venules

(diameter decreased by 30% compared to controls) and dilation of arterioles (diameter increased by 37%

compared to controls) were observed (Bouskela, 1991).

IV injection (5 mg/kg) of Ruscus extract caused a venular constriction but did not significantly affect

either the arteriolar diameter or the mean arterial blood pressure. It should be noted that the measurement

of vascular diameters were made before and after the injection of Ruscus extract. When the extract was

added to the superfusate (topical application), venular constriction and arteriolar dilation were reported.

Cooling to 25°C induced dilation of both types of vessels, whereas warming to 40°C produced the

opposite effect (constriction) at 50.10 -3 mg/ml and higher (Bouskela, 1991).

In a similar study, arteriolar and venular diameters were measured before (3 times, separated by 10 min

intervals) and after (every 10 min for 60 min) intravenous injection of Ruscus extract at the dose of

5 mg/kg to 7 male hamsters. No effect on mean arterial blood pressure was detected. In these animals,

venular but not arteriolar injection was observed at the cheek pouch level. When applied topically to

18 male hamsters, the Ruscus extract produced venular constriction and arteriolar dilation at the cheek

pouch level. The effect of temperature was similar to what was reported above by Bouskela (1991)

(Bouskela et al. , 1993b).

In an attempt to further characterize the mechanism of action of Ruscus extract on the hamster cheek

pouch, another study was performed with male animals. Ruscus extract was used at concentrations ranging

from 5 to 1000 µg/ml/min applied topically (in the superfusate). At 50 µg/ml/min and above, Ruscus

extract application induced venular but not arteriolar constriction. The venular constriction amounted to

approximately 10%. At the concentration of 0.2 mg/ml/min, the venular constriction remained for 120 min

(determined every 10 min) whereas the internal diameter of arterioles was not modified. Therefore, the

concentration of 0.2 mg/ml/min was used in further explorations. The venular constriction evoked by

Ruscus was blocked by low concentrations (10 -9 M) of prazosin and diltiazem (these drugs did not induce

significant venular dilation at this concentration), and only by high concentrations (10 -6 M) of rauwolscine.

The authors postulated that at high concentration, rauwolscine may not be selective only for

α2-adrenoceptors. It was concluded that venular constriction in the hamster cheek pouch was mediated by

calcium and preferentially by α1-adrenoreceptors. Additionally, the authors hypothesized that the lack of

effect on arterioles could be explained by augmented liberation of endothelium-derived relaxing factors on

the arteriolar side which probably overrides its constrictor properties (Bouskela and Cyrino, 1994;

Bouskela et al. , 1994).

• Complementary mechanistic studies

A team of the Faculdade de Medicina, Porto tested the effect of Ruscus extract on segments of veins.

They compared at the microscopic level human varicose and normal veins, saphenous vein taken from

dogs aged 4 months to 7 years and above, and canine saphenous vein obtained after surgical denervation.

Human varicose vein and canine denervated saphenous veins shared common characteristics: thickening

of the vessel wall due to increase in extracellular material and smooth muscle cells hypertrophy,

abnormalities of smooth muscle cells (signs of increased protein synthesis). In dogs, impact of surgical

sympathetic denervation on the structure of the vein was independent of the age of the animals.

Additionally, the [ 3 H]-norepinephrine content was determined in vessels of dogs (4 months to 7 years and

above), and in human varicose/normal veins. The authors showed that sympathetic innervation was

lowered in veins taken from aged dogs, and in human varicose veins (compared to normal veins).

Therefore, it was concluded that human varicose vein behaves like a partially denervated vessel and many

of its structural and biochemical peculiarities appear to be linked to the reduced sympathetic supply of the

preparation and throughout the experiment, the cheek pouch was constantly superfused with a bicarbonate buffered saline solution at a rate of 4.6

ml/min.

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vein. The authors considered that the denervated canine vein represents an interesting model for the study

of the human varicose vein (Azevedo et al. , 1991).

Texeira and Osswald (1988) produced denervation of lateral saphenous vein in anaesthetized dogs by

using artery clamps applied down- and upstream of the segment for 5 min. During the surgical

intervention, osmotic minipumps were implanted in the subcutaneous tissue of dogs’ leg and connected by

a catheter to the plantar branch of the denervated vein. These minipumps allowed IV injection of saline

(containing Na2-EDTA + heparin) ± Ruscus extract (50 µg/kg/h). Animals recovered for 5 days and were

then re-operated to remove the segment of the denervated vein and of the contralateral vein (to serve as

control). This allowed to study the effect of denervation (injection of saline), of Ruscus on denervated vein

(injection of Ruscus and saline). For the morphological study of the vein, the dimension of smooth muscle

cells was taken as the most important criterion of morphological alterations caused by denervation (at the

extraneuronal level). In collected segments, the endogenous norepinephrine content was measured by

HPLC; denervation was considered successful and complete when the norepinephrine content was

decreased by 95%. Additionally, the O-methylating capacity of these tissues was reflected by the total

amount of O-methylated metabolite (OMI), determined after incubation of vein strips with

3 H-7-(±)-isoprenaline for 30 minutes (Texeira and Osswald, 1988).

This study showed that intravenous infusion of Ruscus extract protected the extraneuronal component of

the venous tissue against the deleterious consequences of denervation. Indeed, it induced prevention of

increase in smooth muscle cell diameter, and partial prevention of the impairment of O-methylating

capacity of tissues. However, Ruscus extract administration had no effect on the denervation process itself

(Nad depletion of at least 95% in denervated veins) (Texeira and Osswald, 1988).

Assessor’s comments

The venoconstricting property of Ruscus extract shown in vitro was confirmed in in vivo models. In

anaesthetised dogs, a dose-dependent contractile effect was shown for Ruscus extract doses ranging from

1 to 10 mg/kg given intravenously, and attributed to result of α-adrenergic activation only.

The IV administration of Ruscus extract (5 mg/kg) to hamsters induced venular constriction at the level of

cheek pouch microcirculation, without any impact on the diameter of arterioles or mean arterial blood

pressure. However, topical application of the extract resulted in venular constriction and arteriolar

dilation. Cooling was shown to induce dilation of both types of vessels, whereas warming produced their

constriction. Studies conducted with pharmacological agents showed that venular constriction in the

hamster cheek pouch was mediated by calcium and preferentially by α1-adrenoreceptors.The dilation of

arterioles was considered to result from augmented liberation of endothelium-derived relaxing factors on

the arteriolar side.

The oral route was also investigated in this experimental model; the dose of 150 mg/kg/day for 28 days

induced venular constriction (internal diameter decreased by 30%) and arteriolar dilation (internal

diameter increased by 37%) which was also attributed to liberation of endothelium-derived relaxing

factors on the arteriolar side.

Briefly, it can be concluded that the contractile effect of Ruscus extract on veins was shown in two in vivo

experimental models by intravenous route (anaesthetised models, hamster cheek pouch), topical

application (hamster cheek pouch), and oral route (hamster cheek pouch, 150 mg/kg/day). The

involvement of the α-adrenergic system was confirmed. Arteriolar dilation was noted in some of these

studies, and particularly when Ruscus extract was administered orally, but no effect on the mean arterial

pressure was detected. It could result from induction of liberation of endothelium-derived relaxing factors

on the arteriolar side. Additionally, in a canine model of human varicose vein, IV injection of Ruscus

extract for 5 days prevented against the occurrence morphological alteration of smooth muscle cells, and

against the impairment of O-methylating capacity of tissues.

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The animal models used to demonstrate the venoconstricting activity of Ruscus extract are acceptable,

taking into consideration the lack of experimental models reproducing the physiopathological complexity

of the chronic venous insufficiency. These models are those commonly used for the evaluation of drugs

belonging to this therapeutic class, even if the use of functional exploration methods (e.g. Doppler) could

have been used to complete the weight of evidence.

II.2.1.1.2 C ONTRACTILE EFFECT ON LYMPHATIC VESSELS

A. In vitro studies

• Study performed on rings of canine lymphatic vessels

Thoracic ducts were removed from dogs, cleaned of connective tissue, and cut into rings. Those rings

were put in organ chambers filled with a physiological salt solution and connected to a force transducer

for continuous recording of isometric tension. The substances tested, directly added to the bath solution,

were norepinephrine (10 -8 to 10 -4 M) and Ruscus extract (10 -5 to 2.10 -3 g/ml). To clarify the mechanism of

action of Ruscus extract, some α-adrenergic antagonists were used: phentolamine (3.10 -6 M), prazosin

(3.10 -7 M) and rauwolscine (3.10 -7 M) (Marcelon et al. , 1988a).

In these experimental conditions, it was shown that both norepinephrine and Ruscus extract caused a

concentration-dependent contraction of the lymphatic thoracic duct rings. The contractile activity of

Ruscus was partially inhibited by prazosin or rauwolscine, and completely eliminated by phentolamine.

Taking into account that venous smooth muscle cells have been isolated in mesenteric and thoracic

lymphatics, the importance of the presence of adrenergic nerve endings at this level for the control of

lymphatic pumping, and the results obtained in this study, the authors concluded that Ruscus causes a

similar adrenergic activation of both lymphatic collectors and cutaneous veins. The contractile effect

obtained on lymphatic rings was obtained at similar concentrations than those inducing contraction of

venous rings (Marcelon et al. , 1988a).

• Study performed on bovine lymphatic vessels

In order to determine the mechanism of noradrenaline action on lymphatic vessels, various studies were

performed using electric stimulation on bovine mesenteric lymphatic vessels. Among those studies, one

was performed to gain knowledge about the desensitization of adrenoceptors after application of the

endogenous transmitter (norepinephrine) or Ruscus extract (McHale, 1991).

While application of norepinephrine (10 -6 M) induced an increase of contraction frequency which returned

rapidly to control values as the result of receptors desensitization, application of Ruscus extract (30 µg/ml,

i.e. 3.10 -5 g/ml) had similar excitatory effect which was sustained for the duration of drug perfusion.

According to the author, this might suggest that Ruscus is acting on different receptors in these vessels

from those through which norepinephrine is acting (McHale, 1991).

Assessor’s comment

Ruscus extract was shown to exert contractile effect in vitro on dog thoracic and bovine mesenteric

lymphatic vessels. The results concerning the mechanism involved are contradictory between both

experiments. Indeed, results obtained with canine tissues suggest that the mechanism of action is similar to

the one described at the venous level, i.e. α-adrenergic activation. According to its authors, the study

performed on bovine tissue suggests that pathways involved differ from those of norepinephrine.

However, they did not further discuss on the role of chemical stability to explain their results

(norepinephrine is probably less chemically stable than Ruscus ).

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B. In vivo studies

A study was conducted in anaesthetised dogs, after isolation of a lymph vessel parallel to the saphenous

vein at the ankle level and ligature of other vessels (Pouget et al. , 1991). After Ruscus extract intravenous

injection, the total protein concentration (Pt) was measured by UV-spectrometry in lymph collected at

10 min intervals. This allowed the calculation of oncontic pressure (Ponc) using the following formula:

Ponc = 0.45 Pt + 0.078 Pt. The doses of Ruscus extract administered were 1, 2 and 5 mg/kg because they

were in the range of the doses reported as veinotonic [see (Marcelon et al. , 1983a)].

In some animals, the activity of Ruscus was studied in presence of calcium antagonism by nifedipine.

Ruscus extract (5 mg/kg) was administered and its activity followed for 1 hour. Then, nifedipine was

administered intravenously (0.1 mg/kg, the activity of this dose was maintained for more than 1 hour).

After 30 minutes, a second injection of Ruscus extract was performed, and its activity again followed for 1

hour.

At 2 and 5 mg/kg, Ruscus extract caused a rise in lymph flow without affecting lymph pressure. This

reflected the augmentation of the contractility of lymph vessels. This is in agreement with the results of an

in vitro study performed on peripheral lymphatic vessels [see (McHale, 1991)]. It was also shown that

Ruscus caused contraction of the isolated thoracic duct of the dog in vitro [see (Marcelon et al. , 1988a)].

This adds weight to the hypothesis of an enhancement of the “pumping system” suggested by the results

obtained in vivo in this study, with enhanced propulsion of peripheral lymph toward the heart.

Ruscus also caused a rise in oncotic pressure which suggests that it induces a favourable effect on edema.

The activity of Ruscus extract remained unchanged after nifedipine injection. This suggests that the

mechanism underlying the effect of Ruscus on peripheral lymphatics does not involve the opening of

voltage operated calcium channels.

Assessor’s comment

In anaesthetised dogs, administration of Ruscus extract improved the contractility of peripheral lymph

vessels. Taken together with in vitro results, this suggests that Ruscus extract enhances the lymphatic

pumping system thus favouring a better return of peripheral lymph to the heart. Additionally, a rise in

oncotic pressure was detected, which could suggest a favourable effect on edema.

However, it should be noted that no study was conducted by the intended therapeutic route of

administration, i.e. the oral route. It is questionable whether active substance(s) involved in this effect

would 1) be absorbed by oral route and 2) undergo significant hepatic metabolism impairing its(their)

activity at the lymphatic level.

II.2.1.1.3 E FFECT ON VASCULAR PERMEABILITY

A. In vitro studies

An experiment was conducted using segment of the lateral ear vein of the pig canulated on both sides and

put into an organ bath of Krebs solution. Ruscus extract (0.05%) was applied intraluminally for

15 minutes. Then, ethacrynic acid (0.1%) was added to damage the endothelium (2.5 minutes). Finally, the

permeability to low- and high molecular substances was measured. To quantify the water edema, an

aqueous solution of the dye Evansblue was applied, and the protein edema was measured with bovine

serum marked by the dye, each being applied under a pressure of 30 cm H 2 O for 30 minutes. The content

of Evansblue was then determined by photometry (Hönig and Felix, 1989).

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Figure 2: schematic representation of the experiment (Hönig and Felix, 1989)

The results obtained showed that Ruscus extract reduced the water- and protein permeability induced by

ethacrynic acid. The drug had to be present when the damaging agent was applied; if it was washed 1 min.

before, no protective effect was observed. The authors also showed that no chemical binding occurred

between the drug and the damaging agent.

To elucidate this edema-protective mechanism, saponins from Ruscus were applied for 15 minutes and

then washed. The permeability of these vessels was increased compared to controls. According to the

authors, the surface-active saponins so strongly bind to the endothelium that they leave holes when being

removed.

Assessor’s comment

In vitro on ear vein of pig, Ruscus extract had protective effects against ethacrynic-induced edema.

Saponins may play an important role by anchoring in the endothelium and taking part to the membrane

structure.

B. In vivo studies

• Study performed on the hamster cheek pouch

The number of leakage sites on hamster cheek pouch 4 was measured by fluorescence microscopy after IV

injection of fluorescein-labelled dextran. The number of leakage sites (diameter of the fluorescent spot >

100 µm) was determined 2, 5, 7, 10, 15, 20, and 20 minutes after the beginning of each topical application

of histamine. Each histamine application lasted 5 minutes with a minimum interval of 40 minutes between

each application. The first application of histamine, after injection of FITC-dextran, was made prior to any

drug treatment and thus served as a positive control. Ruscus extract was then applied topically in the

4 Experiments were performed on the cheek pouch of male hamsters 7 to 10 weeks old. Anesthesia of the animals was induced by intraperitoneal

injection of 0.1-0.2 ml of sodium pentobarbital and maintained with α-chloralose (100 mg/kg) administered through the femoral vein. The femoral

artery was cannulated for pressure measurements, anesthetic and Ruscus extract injections. Throughout the surgery and subsequent experiment,

the animal rested on a heating pad controlled by a rectal thermistor and body temperature was maintained at 37.5°C. A tracheal tube was inserted

to facilitate spontaneous breathing. The hamster was placed on a microscope stage. The cheek pouch was gently everted and pinned with 4-5

needles into a circular well filled with silicone rubber to give a plane bottom layer, thus avoiding stretching of the tissue but preventing shrinkage.

In this position, the pouch was submerged in a superfusion solution that continuously flushed the pool of the microscope stage. Before the pouch

was pinned, large arterioles and venules were located with the aid of a Zeiss binocular microscope. Fashioning of a single layer preparation started

with incision of the upper layer to swing a triangular flap to one side. The exposed area was dissected and the fibrous, almost avascular,

connective tissue covering the vessels was removed. The dissected part of the pouch was 120-150 µm thick.

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following concentrations: 0.002, 0.02, 0.2 and 2 mg/ml/min before the subsequent application of

histamine. In the group dosed at 0.2 mg/ml/min, the influence of prazosin, rauwolscine and diltiazem

(10 -9 M to 10 -5 M) on Ruscus effect was studied (Bouskela and Cyrino, 1994).

In this experimental model, histamine increased the number of fluorescent vascular leakage sites from

postcapillary venules, which evidence an increase in macromolecular permeability (quantified as the

number of leaky sites in the prepared area). The topical application of Ruscus extract induced a dose-

dependent inhibition of the macromolecular permeability-increasing effect of histamine (-25% at 0.002

mg/ml/min, almost -50% at 0.2 mg/ml/min).This effect was blocked by prazosin and diltiazem, but not by

rauwolscine. Overall, it was concluded that Ruscus extract inhibited the microvascular permeability

induced by histamine. This effect would be mediated by calcium and preferentially by α1-adrenoreceptors

(Bouskela and Cyrino, 1994).

These findings confirm the results previously obtained in a similar study which showed that topical

application of Ruscus extract has a protective effect against leakage of FITC-dextran in the cheek pouch of

hamsters after administration of various permeability-increasing substances, i.e. bradykinin, histamine,

and leukotriene B4 (Bouskela et al. , 1993a).

• Study performed on a feline model of edema

Edema was induced in anaesthetised cats by IV injection of ethacrynic acid. Ruscus extract was

administered one hour before edema induction. Its antiedema effect was evaluated in hindlegs by

measuring the water and protein content of the edema (Felix et al. , 1984).

Compared to control animals, the protein content of the edema was decreased in Ruscus pre-treated cats.

Water content was not affected, which would suggest that Ruscus extract only influences the passage of

plasma protein in the interstitium. However, in Ruscus pre-treated animals, it was demonstrated that

waters flows into the tissues more slowly than in control animals. Indeed, the capillary filtration

coefficient increased to a smaller degree in Ruscus pretreated animals, compared to control ones.

Therefore, it was concluded that Ruscus extract inhibited the destruction of the endothelium by ethacrynic

acid without totally suppressing it.

The effective dosage was 20 mg/kg by IV route, 10-20 times higher by oral route (administration 4 hours

before induction of edema). By oral route, the effective dose decreased after subchronic administration

and reached 20-40 mg/kg (4-6 days).

It is also reported that a protective effect was observed with ruscogenin at 4 mg/kg by IV route. This effect

was weaker than the effect reported with the whole extract at 20 mg/kg. This last contained 2.5%

ruscogenin, leading to a ruscogenin dose of 0.5 mg/kg (Felix et al. , 1984).

Assessor’s comment

The antiedema effect reported for Ruscus extract in vitro was confirmed in two in vivo studies. After IV

administration, Ruscus extract inhibited the microvascular permeability induced by histamine on hamster

cheek pouch, an effect suggested to be mediated by calcium and preferentially by α1-adrenoreceptors. In a

feline model of ethacrynic acid-induced edema, IV administration of 20 mg Ruscus extract/kg decreased

the protein content of the edema and slowed the water flow into the tissues. This is in agreement with the

increase in oncotic pressure reported by Pouget et al (1991) in lymphatic vessels of anaesthetised dogs

administered 2 and 5 mg/kg Ruscus extract intravenously (see II.1.2.2. above).

However, none of these studies was performed by the oral route. In the study performed by Felix et al.

(1984), the anti-edema effect of Ruscus extract was investigated by oral route. It can be concluded that the

oral effective dose approximates 200-400 mg/kg, or 20-40 mg/kg/day after repeated administrations for 4-

6 days (the criteria used by the authors to decrease the dose were not detailed). Additionally, it should also

be noted that this effect is not only due to ruscogenin, but also to other components of the extract.

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II.2.1.1.4 E FFECT ON HYPOXIA - INDUCED ACTIVATION OF ENDOTHELIAL CELLS

Endothelium plays a role in the development of varicose veins. Hypoxic conditions which develop during

blood stasis are able to activate endothelial cells to release inflammatory mediators and growth factors.

Inflammatory mediators induce neutrophil adherence and activation. Those activated neutrophils may then

infiltrate and induce damage in the subendothelial layers. On the other hand, the growth factos trigger

smooth muscle cell proliferation, leading to a thickening and dizorganisation of the media as observed in

the wall of varicose veins. Therefore, a study was performed on human umbilical vein endothelial cells

(HUVEC) to determine whether Ruscus extract could prevent endothelial cell activation by hypoxia

(Bouaziz et al. , 1999).

Ruscus extract was shown to prevent the activation of endothelial cells (HUVEC) incubated in hypoxia

conditions for 2 hours. This was demonstrated by measuring the ATP content of control and treated cells.

The hypoxia-induced decrease in ATP was concentration-dependently inhibited by Ruscus extract, the

effect being maximal at 50 µg/ml.

Hypoxia strongly increased Phospholipase A2 (PLA2) activity, more than 2-fold the value obtained in

normoxic conditions (↓ ATP → cytosolic calcium → PLA2 activation). When HUVEC cells were

incubated with Ruscus extract under hypoxia, PLA2 activation was inhibited (50% inhibition at 0.05

µg/ml). Additionally, Ruscus extract inhibited neutrophil adherence to HUVEC (PLA2 activity →

prostaglandin, platelet activating factor → ↑ in HUVEC adhesiveness for neutrophils).

II.2.1.2 S AFETY PHARMACOLOGY STUDIES

No study available.

Assessor’s comment

Considering the pharmacological profile of Ruscus extract, i.e. stimulation of α-adrenergic system, the

lack of a safety pharmacology study evaluating its potential effects on the cardiovascular function gives

cause for concern. No toxicology study evaluating this endpoint is available.

In the studies performed on the hamster cheek pouch, the mean arterial blood pressure was not modified

after IV administration of 5 mg/kg Ruscus extract, and oral administration at the dose of 150 mg/kg. The

need of such a safety pharmacology study addressing cardiovascular aspects should be discussed in light

of clinical safety data.

II.2.1.3 P HARMACODYNAMIC INTERACTIONS

No study available.

Assessor’s comment

Ruscus extract contains substances having α-adrenergic stimulating properties. Moreover, it was shown

to displace norepinephrine from adrenergic nerve endings. Therefore, pharmacodynamic drug-drug

interactions could occur with:

– any drug potentiating the α-adrenergic system;

– any drug antagonizing the α-adrenergic system.

As a precaution measure, it may be relevant to include a warning for patients treated with any of those

drugs.

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II.2.1.4 A SSESSOR ’ S OVERALL CONCLUSIONS ON PHARMACOLOGY

Primary pharmacodynamics studies performed in vitro and in vivo using various experimental models

showed that Ruscus extract possess a contractile activity on veins . This activity is mediated by

stimulation of the α-adrenergic system. In vitro mechanistic studies showed that direct activation of

postjunctional α1-and α2-adrenergic receptors, and stimulation of the release of norepinephrine from

adrenergic nerve endings were involved. Although this effect does not appear to be clearly influenced by

the hormonal status (estrogens, progesterone), it seems potentiated by temperature.

In in vivo studies, this venoconstricting activity was shown after intravenous and oral routes; in the

hamster cheek pouch model, local application of the extract ( i.e . in the superfusate) was also effective. It

should be noted that only one study was conducted by the oral route: at the level of hamster cheek pouch

microcirculation, the dose of 150 mg/kg/day administered for 28 days induced venular constriction

(internal diameter decreased by 30%) and arteriolar dilation (internal diameter increased by 37%) without

any impact on the mean arterial blood pressure, the latter effect being attributed to liberation of

endothelium-derived relaxing factors on the arteriolar side.

Similarly, other primary pharmacodynamics studies showed that Ruscus extract exerts a contractile effect

on lymphatic vessels in anaesthetised dogs at 2 and 5 mg/kg administered intravenously. A rise in

oncontic pressure suggested a favourable effect on edema . This was confirmed in a feline model of

ethacrynic acid-induced edema. The effective dosage amounted to 20 mg/kg by intravenous route, and 10-

20 times higher by oral route. However, after subchronic administration (4-6 days), the oral effective

dosage decreased to reach 20-40 mg/kg/day. The same study showed that ruscogenin was also effective,

but that other components of the extract were involved to obtain maximal activity.

Due to the mechanisms underlying the effect of Ruscus extract, pharmacodynamic drug-drug

interactions could occur with any drug potentiating or antagonizing the α-adrenergic system. As a

precaution measure, it may be relevant to include a warning for patients treated with any of those drugs.

Considering the pharmacological profile of Ruscus extract, i.e. stimulation of α-adrenergic system, the

lack of a safety pharmacology study evaluating its potential effects on the cardiovascular function gives

cause for concern. No toxicology study evaluating this endpoint is available. In the studies performed in

the hamster cheek pouch model, the mean arterial blood pressure was not modified after IV administration

of 5 mg/kg Ruscus extract, and oral administration at the dose of 150 mg/kg. Therefore, the need of a

safety pharmacology study addressing cardiovascular aspects should be discussed in light of clinical safety

data.

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II.2.2

P HARMACOKINETICS

II.2.2.1 P REPARATION OF THE EXTRACTS

Three studies were performed in rats and dogs to gain knowledge in the pharmacokinetics of Ruscus

extract (Chanal et al. , 1978; Chanal et al. , 1981; Bernard et al. , 1985).

In each study, the extract was tritiated according to the Wilzbach technique. Briefly, this labelling method

involved an incubation period with tritium gas (5-7 days) followed by the elimination of labile tritium.

Analysis by TLC was then performed and showed each time that most of the radioactivity was associated

with the sapogenins (80 to 94%, depending on the study).

According to the authors, other labelling methods could not be applied:

– Carbon 14 labelling was not appropriate because the extract contains a multiplicity of sapogenins;

– Labelling by biosynthesis would have required years of growth (the extract is made from the

rhizome);

– These sapogenins cannot be chemically synthesized because of the lack of any precursor.

Assessor’s comment

As indicated by other authors, the Wilzbach method used to label the extract is controversial (Bernard et

al. , 1985). Tritium labelling of a complex mixture such as the Ruscus extract used in this study presents

some disadvantages which preclude a full confidence in the results obtained. In particular, the stability of

the labelling is not justified (possible 3 H- 1 H exchanges after the labelling, etc.).

Additionally, no quantification of 3 H 2 O was performed in any sample of the studies. After 3 H- 1 H exchange

or metabolism, 3 H 2 O mix with the body water pool. The elimination of tritiated water from the organism

being particularly slow, the monitoring of 3 H radioactivity to study the pharmacokinetics of Ruscus

extract’s compounds is likely to produce biased results. Overall, these studies should not be taken into

account for regulatory purposes (however main results will be described below) .

II.2.2.2 A BSORPTION

Blood kinetics of radioactivity were determined in a study involving 2 male Wistar rats administered the

labelled-extract orally (Chanal et al. , 1978). Blood samples were collected at 0.25, 0.5, 1, 2, 3.5, 5, 6, 8

and 24 hours after administration. Radioactivity level, expressed as per cent radioactivity per 10 ml of

blood in relation to the dose administered, was measured by Liquid Scintillation Counting (LSC).

In both rats, blood radioactivity reached a level close to the maximum 2 hours after the administration of

the extract. Thereafter, radioactivity level remained practically constant over the remaining 22 hours (see

figure 3). The authors concluded that the half-lives of the tritiated compounds were relatively long, on the

order of several days.

In another study involving oral administration of the labelled extract to 6 male Wistar rats, blood samples

were collected at 0.25, 0.75, 1.75, 2.25, 3.5, 5, 6.5, 8, 24 and 48 hours after administration (Chanal et al. ,

1981). Radioactivity level was expressed as per cent radioactivity per 10 ml of blood in relation to the

dose administered, and the figures given are the mean for the 6 rats (see figure 4). Relatively high

radioactivity levels were reached from 3.5 hours and the Tmax amounted to 8 hours. Blood levels declined

slowly and remained high after 24 hours.

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Figure 3: blood kinetics of tritiated compounds of Ruscus extract in 2 rats after oral administration

(from Chanal et al ., 1978)

1

0,1

Rat A

Rat B

0,01

0

5

10

15

20

25

30

Time (hr)

Figure 4: radioactivity levels measured in blood of Wistar rats after oral administration of a 3 H-

labelled Ruscus extract (graph comprising a logarithmic trend curve elaborated from data of

Chanal et al , 1981)

Assessor’s comment

Two studies performed on a limited number of rats after oral administration of a labelled Ruscus extract

showed that radioactivity was detected in the blood for more than 24 hours. The authors suggest that the

tritiated compounds of the extract would have relatively long half-lives. However, such a conclusion is

speculative because the tritiated water, which is slowly eliminated, was not quantified in the samples

collected. Therefore, no conclusion can be drawn from these studies.

It should be noted that the results obtained in the studies performed by Chanal et al in 1978 and in 1981

are contradictory; in the first study, blood radioactivity remained stable over 25 hours, whereas in the

second one, a decrease of blood radioactivity starts after 8 hours.

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II.2.2.3 D ISTRIBUTION

Tissue distribution was investigated by whole body autoradiography (WBA) in macaca monkeys

administered a labelled extract of Ruscus by either intravenous (n=1, extract dissolved in sodium chloride

0.9%), or oral (n=4, extract dissolved in the content of one Cyclo 3 oral ampoule) routes (Bernard et al. ,

1985).

WBA performed 24 hours after IV administration showed intense labelling in the bile, the contents of the

distal digestive tract and to a lesser extent the urine. Moderate label was found in the circulating blood,

and relatively substantial label in the renal and hepatic parenchyma, spleen, bone marrow, and adrenals

(cortex). The intense binding of radioactivity at the hepatic and renal levels was confirmed by LSC.

Two hours after oral administration, highest radioactivity levels were detected in the bile, digestive and

urinary contents; liver and kidney were labelled uniformly. Blood activity was slightly lower than after IV

administration. The radioactivity levels decreased the following 5 hours except for digestive organs (urine,

bile, feces). After 24 hours, non negligible radioactivity levels persisted in circulating blood, renal and

hepatic parenchyma, bone marrow, spleen, adrenal cortex, contents of the distal digestive tract, bile and

urine.

Assessor’s comment

Radioactivity was mainly found at the hepatic and renal levels 24 hours after both intravenous and oral

administration of the labelled Ruscus extract. It should be noted that intense radioactivity was found in the

bile. Again, it remains unknown if the radioactivity arises from any of the compounds of the extract, or

from tritiated water. Therefore, no conclusion can be drawn.

However, deep distribution of radioactivity in the bone marrow after IV and oral administrations

was shown, thus underlying the need of genotoxicity studies.

II.2.2.4 M ETABOLISM

To determine the nature of plasma radioactivity, blood samples collected 2 hours after oral administration

of a radio-labelled extract to 2 rats were extracted with methanol, and analysed by TLC (Thin Layer

Chromatography). Results indicate that 39% of the radioactivity deposited corresponded to the sapogenin

spot. The authors conclude that there was a “considerable fraction of the unchanged product” in the

plasma after 2 hours (Chanal et al. , 1978).

Assessor’s comment

This experiment does not allow drawing any conclusion on the metabolism of Ruscus extract due to

concerns already expressed on labelling method and experimental procedures, and to the poor separating

capacity of the method employed (TLC).

II.2.2.5 E LIMINATION

Ninety six hours after oral administration of the labelled extract to 2 rats, approximately 18% and 29% of

radioactivity was recovered in the urine and feces, respectively. It should be noted that excretion occurred

mainly during the first 24 hours. A non negligible part of the fraction recovered in the faeces arised from

notable biliary excretion (approx. 10% of the administered radioactivity). (Chanal et al. , 1978).

Urinary excretion amounted to 32-35% in Wistar and Atrichis rats administered a labelled extract orally,

while the corresponding figure for faecal excretion was slightly higher, i.e. 39-45%. Most of the

radioactivity was excreted within the first 24 hours. The authors report that the existence of an

enterohepatic cycle was confirmed in a study conducted by intravenous administration (one third of total

radioactivity eliminated in the faeces)(Chanal et al. , 1981).

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In monkeys, radioactivity was excreted in the urine (26%) and in the feces (6.5%) 24 hours after IV

administration (n=1). Those figures amounted to 20% and 23% for urine and feces, respectively, 24 hours

after oral administration. The authors suggested the existence of an enterohepatic cycle considering the

extent of faecal elimination and bile activity (Bernard et al. , 1985).

Assessor’s comment

First, it should be noted that these figures were obtained from a limited number of animals. Additionally,

it seems that the amount of radioactivity recovered from urine and faeces appears to be insufficient in all

studies (less than 50% to 80% of the administered dose). Taking into consideration the relatively long

half-lives of the labelled compounds which was hypothesised from blood kinetics, an incomplete

radioactivity collection after either 24 hours or 96 hours seems coherent.

However, as most of the studies showed that excretion occurred mainly during the first 24 hours, and in

view of the stable blood radioactivity levels after 24 hours, accumulation of radioactivity could occur after

repeated administrations.

Radioactivity was excreted in urine and faeces, with faecal elimination slightly above. The authors suggest

that Ruscus extract labelled components undergo an enterohepatic cycle. However, the excretion of a

compound in the bile and a high amount of faecal elimination are not sufficient to prove the existence of

an enterohepatic cycle. This suggestion should be rather regarded as a hypothesis to be further

investigated.

This experiment does not allow to draw any conclusion on the metabolism of Ruscus extract due to

concerns already expressed on labelling method and experimental procedures.

II.2.2.6 A SSESSOR ’ S OVERALL CONCLUSIONS ON PHARMACOKINETICS

The pharmacokinetics of Ruscus extract was investigated in rat (2 studies) and in monkey (1 study). In

each study, the extract was tritiated according to the Wilzbach technique. Briefly, this labelling method

involved an incubation period with tritium gas (5-7 days) followed by the elimination of labile tritium.

Afterwards, the extract could be administered to animals. However, this labelling method presents some

disadvantages. For example, the stability of the labelling remains unknown (possible 3 H- 1 H exchanges

after the labelling, etc.).

Additionally, the authors did not perform a quantification of tritiated water in the samples collected. This

represents a major bias because the radioactivity measured in samples cannot be attributed without any

doubt to the compounds of the Ruscus extract. Indeed, 3 H 2 O can mix with the body water pool after 3 H- 1 H

exchange or metabolism, and the elimination of tritiated water from the organism is particularly slow.

Overall, it is concluded that these studies should not be taken into account for regulatory purposes

because they are endowed with major bias precluding a full confidence in the results obtained.

One concern arises from the monkey distribution study, where in-depth distribution of radioactivity was

reported at the bone marrow level. While it remains unknown if radioactivity is due to any component of

the extract or to tritiated water, the worst-case scenario should be considered. Therefore, this finding

strongly underlies the need of genotoxicity studies with Ruscus extract.

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II.2.3

T OXICOLOGY

II.2.3.1 A CUTE TOXICITY

The acute toxicity of an ethanolic extract of Ruscus was investigated in dogs and guinea pigs (Caujolle et

al. , 1953; ESCOP, 2003):

– In 6 male and female dogs, death occurred within 1 hour following intravenous infusion of the extract

at doses ranging from 0.83 g/kg to 1.8 g/kg. The frequency of cardiac contractions was progressively

decreased but was not attributed by the authors to a toxic effect on the myocardium because the hearts

of treated dogs could react normally to epinephrine. Additionally, the blood pressure was decreased.

The monitoring of respiratory function showed that at toxic doses, tachypnea occurred and was

sometimes associated with rhythm perturbation. At lethal doses, hyperventilation was followed by fatal

apnea. The authors considered that at high doses, the Ruscus ethanolic extract that they administered to

dogs produced cardiovascular and respiratory reactions. The respiratory centres were deeply affected

and apnea always preceded the cardiac arrest so that death was attributed to respiratory alteration.

Moreover, at high doses, hyperglycemia was reported (1.82 to 2.84 g/l).

– In 8 male guinea pigs, the intraperitoneal injection of the ethanolic extract induced no toxic symptoms

at doses lower than 1.5 g/kg. Animals receiving 2g/kg and above died.

Estimation of the oral and intraperitoneal LD50 values of an ethanolic fluid extract of Ruscus in rats and

mice revealed differences depending on the harvest time of the plant, the route of administration, and the

use of roots or rhizomes. The oral LD50 of the rhizome extract could not be determined in rats because

administration of doses inducing 100% mortality could not be reached. In mice, it amounted to 24.69-

33.73 ml/kg in mice. After intraperitoneal administration, the DL50 of the rhizome extract reached 1.15-

1.70 ml/kg in mice, and 2.07-2.39 ml/kg in rats. Root extract was found to be more toxic than rhizome

extract in both species. The observed symptoms of intoxication were convulsion, paralysis and gastro-

intestinal inflammation with dysentery. Animals died following respiratory failure. Autopsies revealed

pronounced irritation of the mucosa and strong visceral congestion (Boucard et al. , 1967; ESCOP, 2003).

Assessor’s comment

Extracts used in both studies differed from the extract produced by the company Pierre Fabre Médicament

used in pharmacology and pharmacokinetic studies. They were obtained by ethanolic extraction but

contents in ruscogenin and neoruscogenin remain unknown.

The first study in dogs reported a mean LD0 of 1.20 g/kg (0.83 – 1.8 g/kg) by intravenous route. The

authors attribute the cardiovascular findings observed at high dose (decreased frequency of cardiac

contractions, decreased blood pressure) to be secondary to alteration of respiratory centres. However, this

is not sufficiently established, particularly if the α-adrenergic activity of Ruscus components is taken into

consideration. In the second study, the oral LD50 of mice reached 25-34 ml/kg. Death occurred by

respiratory failure in rats and mice treated by oral and intraperitoneal routes.

As described in section II.2.1.2. no safety pharmacology study is available on cardiovascular and

respiratory systems. The need of such studies should be discussed in light of clinical safety data.

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II.2.3.2 R EPEAT - DOSE TOXICITY

The ESCOP monograph reports the findings of a 26-week toxicity studies performed in male rabbits by

administration in the diet (Roux, 1969; ESCOP, 2003). A Ruscus extract was administered to 17 animals

at 2 g/kg, and to 19 animals at 5 g/kg. Five animals served as controls. It is stated that body weight and

blood counts did not reveal any difference between treated animals and controls.

Assessor’s comment

The lack of information available on this (unpublished) study precludes its use in safety evaluation.

Indeed, no precision on the extract administered to the animals is given (mode of extraction, content in

active substances, etc.), and the choice of the rabbit as a toxicology species is amazing. Usually, rat or

rabbit are used as rodent, and dog or monkey as non-rodent. The parameters monitored in treated animals

are not indicated, except body weight and blood count which is insufficient.

II.2.3.3 G ENOTOXIC AND CARCINOGENIC POTENTIALS

No information available.

Assessor’s comment

The lack of genotoxicity study precludes the listing of Ruscus aculeatus . Additionally, as no long-term

study is available, the carcinogenic risk cannot be appreciated.

II.2.3.4 R EPRODUCTIVE TOXICITY

The ESCOP monograph reports the findings of an unpublished study conducted in female pregnant rat by

administration of a preparation containing ethanolic Ruscus extract, trimethylhesperidin methylchalcone,

methyl-4-esculetol and acorbic acid (Labie, ; ESCOP, 2003). Twenty female rats received a daily dose of

2.4 ml of the preparation corresponding to 0.24 ml of Ruscus extract and equivalent to 25 times the

recommended dose for humans. Twenty animals served as control. Treatment started one week before

conception and continued until delivery. No sign of intoxication was noted in treated animals. The fertility

of females in the treatment group was comparable to that in the control group and this offspring did not

show any teratogenic sign.

Assessor’s comment

The lack of information available on this (unpublished) study precludes its use in safety evaluation (route

of administration, number of resorptions, viable foetuses, number of foetuses examined for evaluation of

visceral/skeletal abnormalities, etc.). Only one dose level was tested, which is not acceptable. The study

was performed on an association of a mixture ( Ruscus extract) with other components; a conclusion on the

teratogenic risk of Ruscus extract only cannot be drawn.

It is considered that the embryo-fetotoxic risk of Ruscus extract is unknown; adequately conducted

reproduction toxicity studies are lacking.

II.2.3.5 A SSESSOR ’ S OVERALL CONCLUSIONS ON TOXICOLOGY

Extracts used in two acute toxicity studies were obtained by ethanolic extraction. Their characteristics

(content in various compounds, e.g. ruscogenin and neoruscogenin) remain unknown. Potential variability

compared to the extract(s) intended for therapeutic cannot be evaluated. In dogs, a mean intravenous

LD0 of 1.20 g/kg was measured, while the oral LD50 of mice reached 25-34 ml/kg with another extract.

The authors attribute the cardiovascular findings observed in dogs at high doses (decreased frequency of

cardiac contractions, decreased blood pressure) to be secondary to alteration of respiratory centres. In rats

and mice, death occurred by respiratory failure too. No safety pharmacology study is available on

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cardiovascular and respiratory systems. Considering the α -adrenergic activity of Ruscus

components, the need of such studies should be discussed in light of clinical safety data.

No other toxicity studies are available. The ESCOP monograph reports repeat-dose toxicity and

reprotoxicity studies performed in rabbits and rats, respectively. However, these studies remain

unpublished so that the information available is very sparse. Therefore, they cannot be taken into

consideration for safety evaluation.

The lack of adequately conducted genotoxicity and embryo-fetal toxicity studies precludes the

listing of Ruscus aculeatus. Additionally, as no long-term study is available, the carcinogenic risk cannot

be appreciated.

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II.3 C LINICAL DATA

Clinical data on efficacy and safety of Ruscus aculeatus alone are very limited.

II.3.1 C LINICAL PHARMACOLOGY

Clinical pharmacology on Ruscus aculeatus is not well documented. Two publications have been

identified.

II.3.1.1

P HARMACODYNAMICS

II.3.1.1.1 O VERVIEW OF AVAILABLE DATA

In a randomized, placebo-controlled, double-blind, crossover, 4-arm study, 20 healthy volunteers

(11 men and 9 women aged between 20 and 43 years) took a single dose of four different treatments

immediately before the first measurements: 450 mg Ruscus extract, 450 mg trimethylhesperidin chalcone

(TMHC), 900 mg of a combination of the two substances or a placebo. A 1-week wash-out phase was

provided between the treatments. The venous function was determined by plethysmography. Volumetric

measurements were performed in orthostatic conditions with normal blood flow (foot volume) and after a

pronounced ischemia (tissue volume). The difference between foot volume and tissue volume corresponds

to the blood volume. Heamodynamic and volumetric reactions were monitored before intake and after

70, 90, 120 and 150 minutes. Ruscus extract caused a significant decrease in venous capacity and venous

outflow. Ruscus extract also significantly reduced tissue volume compared to placebo whereas the

decrease in blood volume was not significant (Rudofsky, 1991).

Assessor’s comments:

The characteristics of the Ruscus extract are unknown.

A long term study was also performed with 141 patients, with chronic venous insufficiency (CVI), who

were recruited to a randomized, double-blind, multicentre study. The cause of late CVI was either primary

varicosis or post-thrombotic syndrome (PTS). After a two weeks washout, they were given 4 weeks of

treatment with 3 x 2 and then 8 weeks with 2 x 2 capsules of Ruscus extract or placebo.

The patients venous pump function during toe-stand exercises were also investigated by plethysmography.

In CVI patients there was a continuous decrease in the foot and ankle volume after a 12-week treatment

with active substance whereas the volume increased under placebo. The tissue volume was reduced by the

same degree as the foot volume in PTS patients. In varicosis patients, the reduction in leg swelling was

due to a decrease in tissue volume and volume of blood stored in the veins during orthostasis (Rudofsky,

1991).

Assessor’s comments:

The characteristics of the Ruscus extract are unknown.

II.3.1.1.2 A SSESSOR ’ S OVERALL CONCLUSIONS ON PHARMACODYNAMICS

On the basis of publications, the quality of the two available pharmacodynamics studies cannot be

evaluated. For example, the characteristics of the patients are incomplete as well as the design of the

studies. The statistical analysis is not given. Moreover, the characteristics of the Ruscus extract are not

specified. However, the findings corroborate the preclinical pharmacological properties described in

section II.2.1 that acknowledge to Ruscus extract (manufactured by Pierre Fabre) an alpha-adrenergic

effect and thus a venous vasoconstrictive effect that account for a reduction in volume of blood stored in

the veins and for a stimulating effect on the lymphatic drainage. These two pharmacological properties

assume a positive effect in patients suffering from chronic venous insufficiency.

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Dose-effect studies are missing which preclude from justifying the dosage regimen used in the clinical

studies.

Other methods of functional exploration could have been used to evaluate the effect on veins (e.g.

Doppler).

II.3.1.2

P HARMACOKINETICS

II.3.1.2.1 O VERVIEW OF AVAILABLE DATA

During a pilot study involving three volunteers, the major spirostanol glycosides of Ruscus aculeatus

(degluconeoruscin and deglucoruscin) were detected in human plasma after an oral administration of 1g of

Ruscus extract (Rauwald and Grunwidl, 1991) (see figure 5 below).

Figure 5: time course of plasma concentration of degluconeoruscin after oral ingestion of 1 g Ruscus

extract by 3 human volunteers (Rauwald and Grunwidl, 1991)

Assessor’s comments:

The only conclusion that can be drawn from this study is that degluconeoruscin and deglucoruscin seems

to be absorbed after Ruscus extract oral administration. Of note, the characteristics of the extract are not

given. The publication wasn’t detailed enough to determine the grade of CVI, the dose administered, the

statistical analysis and the Doppler procedure.

II.3.1.2.2 A SSESSOR ’ S OVERALL CONCLUSIONS ON PHARMACOKINETICS

Available pharmacokinetics data are too scarce. The pharmacokinetics of Ruscus extract should further

studied.

II.3.2

C LINICAL EFFICACY

II.3.2.1 D OSE RESPONSE STUDIES

According to the provided literature no dose-finding studies have been conducted with Ruscus extract

alone neither in patients with chronic venous insufficiency nor in patients suffering from haemorrhoids.

Monographs dosage recommendations are empirical.

With respect to the ESCOP and the Commission E monographs an adult daily dose should be equivalent

to an amount of 7 to 11 mg of total ruscogenins. The French herbal preparations containing Ruscus

aculeatus alone recommend a daily dose equivalent to an amount of around 10 mg of total ruscogenins

with regard to a traditional use in subjective symptoms of chronic insufficiency such as sensation of heavy

legs.

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II.3.2.2 C LINICAL STUDIES ( CASE STUDIES AND CLINICAL TRIALS )

The efficacy of Ruscus aculeatus has been proposed in the different following indications. A review of the

literature for each indication has been performed.

1. Chronic venous insufficiency

Chronic Venous Insufficiency (CVI): symptoms and therapeutic measures Chronic venous insufficiency

is a common disease which is characterised by symptoms due to disorders of the venous return in the

lower leg and in foot. The situation leads to an increased pressure in the veins and lack of blood flow to

the legs and feet. The main symptoms of chronic venous insufficiency are: leg heaviness, aching, dilated

or unsightly veins and oedema (swelling). In the more severe cases, patients can have skin colour changes,

recurrent skin infections and chronic ulcers.

Two main options to treat symptoms due to chronic venous insufficiency are firstly compression therapy

which has demonstrated an efficacy to reduce leg volume, to hinder progression and to reduce symptoms.

This therapy can be completed or replaced by the use of systemic veno-active drugs. Physical activity such

as for example walking or swimming can also be suitable.

Clinical efficacy and safety data:

Study from Vanscheidt and al. (2002) (submitted during the first step of the procedure)

Only one clinical study, with Ruscus extract alone, performed in patients with chronic venous

insufficiency (CVI) and involving Ruscus aculeatus alone has been found in the literature.

Methodology:

This multicenter double-blind, randomized, placebo-controlled clinical study has been recently performed

with a Ruscus extract alone and published (Vanscheidt et al. , 2002) . The aim of this trial was to confirm

the efficacy and safety of a Ruscus extract (Fagorutin® Ruscus Kapseln) according to the latest scientific

standards.

Design: The study enrolled women suffering from chronic venous insufficiency (Widmer classification

grades I and II, CEAP 3-5) and was conducted at 10 different centres in Germany. Randomisation was

carried out after a 2-week placebo run-in phase. The treatment phase lasted 12 weeks. Checkpoints were

scheduled after 4, 8, and 12 weeks of treatment.

Treatments : Active treatment and placebo were taken twice daily (morning and evening) orally with some

fluid. One capsule active treatment contained 36.0-37.5 mg dry extract from Ruscus aculeatus rhizome

with a drug extract ratio of 15-20 : 1 (excipient methanol 60%) corresponding to an amount of around

4.5 mg of total ruscogenins.

Outcomes :

- The primary variable of the study was the change in foot and lower leg volume, measured as the area

under the curve of volume changes over the 12-weeks treatment-time of (AUB 0-12 , area under baseline).

- Among the secondary variables “changes in leg volume” and “changes in subjective symptoms” were

evaluated after 12 weeks of treatments.

All measurements were carried out at the same time of the respective days in the late afternoon or early

evening. Before the measurements the patients underwent a 45-min temperature and cardiovascular

equilibrium period in a sitting position. The leg volume was determined by water displacement. The ankle

and lower leg circumference were measured using a measuring tape. The measurements were carried out

at the lateral and medial ankle and the middle of the middle of the lower leg.

The subjective symptoms : tiredness and heaviness, sensation of tension, tingling sensation, and pain were

assessed at each visit before the volume measurements. The subjective symptoms were evaluated by a

10 cm Visual Analog Scale (VAS) where 0 was equivalent to “no complaints” and 10 to “strongest

complaints”.

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The quality of life was investigated at visits 1 and 5 by a disease specific and validated questionnaire on

the quality of life which included subjective symptoms and life style judgement (Launois et al. , 1996).

It ca be compared with FLQA (Freiburger Life Quality Assessment). Global efficacy was assessed by the

investigator with a four point scale (very good, good, moderate and bad).

Results : Overall, 167 patients were screened, of whom 166 were randomised and included in the study.

Eighteen had insufficient data and were excluded from the efficacy analysis.

Efficacy on leg volume: For the AUB 0-12 , the median values (min/max) were – 656 [ml x day]

(- 13972/5908) for the Ruscus extract and 175 [ml x day] (-22795/4970) for placebo which reflected a

decrease of leg volume in the Ruscus group but an increase in the placebo group. Statistical analysis

revealed a significant treatment contrast (p < 0.001). Moreover, the median values of all parameters

showed a decrease over time reflecting an increasing reduction of leg volume, ankle circumference, lower

leg circumference, and subjective symptoms in the Ruscus extract group. With the placebo treatment

generally, the baseline values were maintained for all parameters. Significant differences between the

treatment groups were seen for the volume changes of the lower leg after 8 and 12 weeks, ankle

circumference after 4, 8 and 12 weeks, lower leg circumference for 8 and 12 weeks, and finally after

12 weeks for the subjective symptom 1 (heaviness and tiredness) and subjective symptom 2 (sensation of

tension).

Evaluation of subjective symptoms : The global efficacy of the Ruscus extract was evaluated by the

investigator as very good and good more frequently, whereas placebo was more frequently assessed as

moderate and bad (p = 0.0498). A significantly positive correlation coefficient was calculated for the

changes of each of the subjective symptoms 1 (heaviness and tiredness), 2 (sensation of tension) and

3 (tingling tension) and leg volume changes.

Quality of life: The Quality of life did not reveal any changes after 12 weeks of treatment for both groups.

Assessor’s comments:

The study of Vanscheidt and al is deserving of being the first and the only one clinical study performed

with a Ruscus extract alone. . Nevertheless, it is always rather difficult to appreciate the real quality of a

study through a publication. Several methodological insufficiencies remain unsolved and information is

missing such as: lack of complete protocol with modalities of randomisation, sample calculation and

power of the study.

Moreover, the respect of the double-bind procedure can not be evaluated.

18 patients have been excluded from the analysis for insufficient data of which no information is given. At

12 weeks, all missing data have been replaced by a LOCF value (last observation carried forward).

However, we do not know how many data are missing.

Lastly, the study has been conducted in 10 centres in Germany; however, the homogeneity between

centres has not been evaluated in terms of recruitment or clinical practices. Taking into account these

overall comments, efficacy results should be interpreted with caution.

According to the author, the study was designed in accordance with the guidelines for testing drugs for

chronic venous insufficiency (CVI), i.e. study design with oedema reduction as the primary variable. It has

to be noted that these guidelines were written by the author himself and published in “Phlebologie” after

the beginning of this study i.e. April 1999 (Vanscheidt et al., 2000) and that oedema is not considered as a

cardiovascular risk factor in the recommendations made by the European Society of Hypertension and the

European Society of Cardiology in 2007.

Although we can agree with most of the proposed design of this study (e.g. inclusion and exclusion

criteria, duration of the study), the clinical relevance of the primary criterion is debatable . Even if

oedema reduction as primary variable can be considered a reliable quantitative primary end-point to

evaluate one of the pharmacological effects of Ruscus aculeatus, the clinical relevance of this primary

variable is questionable. According to the assessor’s opinion,, improvement in subjective symptoms such

as sensation of heaviness or tiredness, tingling or pain should be of more clinical relevance . As stated by

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the author himself, any reduction of oedema is only regarded as clinically relevant if it is accompanied by

an improvement in patient’s quality of life.

Thus, despite significant results regarding the primary variable and the positive correlation shown

between leg oedema and all the subjective symptoms except pain, the clinical relevance of these results

remains questionable. Indeed, the positive effect relative to the subjective symptoms is very limited. The

difference between both groups for the subjective symptoms “tingling sensation” and “pain” are not

statistically significant and the significativity of the difference for the two other subjective symptoms

“heaviness and tiredness” and “sensation of tension” is debatable taking into account the multiplicity of

the analyses. Moreover, the treatment response measured by the disease specific questionnaire on the

quality of life appeared negative at the end of this study as results on this questionnaire did not reveal any

changes in both arms).

In conclusion, the credit that we can attribute to this study is to have tested a Ruscus extract alone. The

posology of the extract used in the study is in adequacy with the one recommended by the available

monographs i.e. a daily dose equivalent to an amount of 7 to 11 mg of total ruscogenins. However, the

level of evidence of Ruscus extract efficacy in relieving symptoms of chronic venous insufficiency given by

this study is low. It has to be noted that the efficacy was not evaluated in men. Another study to confirm

these results is deemed necessary. Evaluation of a sustained efficacy over a longer period (up to 1 year)

has not been studied.

Other Clinical Efficacy Data (submitted in response to the list of Questions by the AESGP)

- Human pharmacological studies . Rudofsky G and al.Wirkung eines Kombinationspräparates auf die

Venenkapazität [Effect of a drug combination on venous capacity]. Fortschr Med 1982; 100: 1217-20.

Rudofsky G. and al Die Wirkung der Kombination aus Ruscus -Extrakt und Trimethylhesperidinchalkon

bei gesunden Probanden unter Wärmebelastung [Improving venous tone and capillary sealing. Effect of a

combination of Ruscus extract and hesperidine methyl chalcone in healthy probands in heat stress].

Fortschr Med 1989; 107: 52, 55-2, 58.

Additional studies were provided in addition to the only clinical study to sustain efficacy results. We are

of the opinion that these results, despite the fact that they demonstrate an increase of the tonus of the

venous wall, are of limited interest as Ruscus aculeatus extract was not tested alone but in combination

with other substances. It is therefore difficult to differentiate a specific effect of Ruscus aculeatus extract

from the effect of the combinations tested.

- Meta-analysis . Boyle P and al. Meta-analysis of clinical trials of Cyclo 3 Fort in the treatment of

chronic venous insufficiency. Int Angiol 2003; 22: 250-62.

Clinical data for Ruscus aculeatus have also been published in a meta-analysis that included 20 placebo-

controlled randomised double-blind studies. However, the conclusions of the meta-analysis by Boyle and

al cannot be taken into account. Indeed, this publication is related to Cyclo 3 which contains Ruscus

aculeatus extract (150 mg per capsule), hesperidine chalcone (150 mg) and ascorbic acid (100 mg) and not

to Ruscus extract alone; this demonstration of the clinical efficacy cannot be attributed to Ruscus .

- International Consensus Symposium. Ramelet AA, and al Veno-active drugs in the management of

chronic venous disease. An international consensus statement: current medical position, prospective views

and final resolution. Clin Hemorheol Microcirc 2005; 33: 309-19.

The International Consensus symposium, held during the 13 th Congress of European Society for Clinical

Hemorheology (ESCH) in Siena (Italy), 2005, concluded that vaso-active drugs (VAD) are effective and

may be applied in chronic venous disease (CVD), when symptomatic, at any class of CEAP. The

Consensus statement of the international experts also declares, that “in some cases VAD may replace

compression and/or complement its effects”. The experts classified, based on the available data, both the

horse chestnut extract and Ruscus extract to “Grade B” of their recommendation explained in the

statement published by Ramelet and al . Despite this conclusion, we still consider that the efficacy of

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Ruscus aculeatus to relieve symptoms of chronic venous insufficiency is not demonstrated due to the lack

of relevance, for this procedure, of the study and the meta-analysis.

- Comparative study with another herbal: Horse Chestnut Extract Diehm C and al Comparison of leg

compression stocking and oral horse-chestnut seed extract therapy in patients with chronic venous

insufficiency. Lancet 1996; 347: 292-4.

Comparison with horse chestnut extract Diehm and al study “ Comparison of leg compression stocking

and oral horse-chestnut seed extract therapy in patients with chronic venous insufficiency ”.

Results of a randomised placebo-controlled three-armed study performed with another herbal substance:

oral horse chestnut extract, were provided. This study demonstrated that on the criterion oedema

protection (leg volume reduction), results are comparable between horse chestnut extract, and leg

compression stockings.

However, this extrapolation of results to Ruscus aculeatus extract is not acceptable. This indirect

comparison is hazardous and cannot be accepted as no direct comparison has been performed between

Ruscus Aculeati Extract and leg compression stockings.

2. Haemorrhoids

No clinical studies are available in the treatment of haemorrhoids with Ruscus aculeatus alone. Most of

the studies are performed with a Ruscus combination with various flavonoïds. One can not rule out a

positive synergistic effect of the combinations. Thus the proper efficacy of Ruscus aculeatus is difficult to

assess (Abascal and Yarnall, 2005).

Experiments on single cases: patients treated with a 10% hydro-alcoholic (alcohol 30°) Ruscu s extract

given per os were reported (Caujolle et al. , 1953). Over 11 cases followed during a few months, 10 cases

showed an improvement of the symptoms whereas in one case Ruscus extract was ineffective. However,

this later patient was suffering from haemorrhoids associated with haemorrhage. Another successful case

corresponding to a pregnant women treated locally with suppository was reported.

In addition, the author reported 13 out of 15 observations from rural physicians corresponding to patients

treated either per os or locally using suppository.

Assessor’s comments:

Despite the identified Ruscus aculeatus pharmacological effects i.e. enhancement effect on venotonicity,

no well-conducted studies are available with Ruscus aculeatus alone.

Even if there is evidence to suggest that Ruscus extract is effective in relieving symptoms of haemorrhoids,

this evidence is of very low level. Further researches in that field are needed . However, due to the long-

term use of ruscus extract-containing products in this indication, Ruscus aculeatus can be considered as

traditional herbal medicinal product for symptomatic relief of itching and burning associated with

haemorrhoids.

3. Orthostatic hypotension

One case of profound refractory orthostatic hypotension treated for longer than 2 years has been reported

(Redman, 2000). The author recommends one “standard” 470 mg capsule of Ruscus aculeatus every hour

from waking till evening until blood pressure is high enough that it is no longer needed. This corresponds

to doubling the normal “recommended” dose of two capsules three times daily. It has to be noted that the

author recommends even so combining the intake of Ruscus with non pharmacological measures and other

natural products medicines.

Assessor’s comments:

According to the author, with regard to its identified pharmacological properties Ruscus application and

extension to the treatment of orthostatic hypotension is obvious. In the assessor’s opinion this application

should be clearly better documented. To date, with regard to the available data, no recommendation can

be given. Further researches in that field are needed.

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4. Diabetic retinopathy

One study involving Ruscus aculeatus alone has been found in the literature

(Archimowicz-Cyrylowska et al. , 1996).

The study was carried out with 60 patients (32 women, 28 men), aged from 20-75 years, mostly suffering

from non-insulin dependent diabetes mellitus (type II) for 1-27 years, characterized by non-proliferative

diabetic retinopathy. They were randomly assigned to three equal groups: Group I - treated with troxerutin

(Venoruton Zygma, GmbH) 1 tablet containing 0.5g of 0-(beta-hydroxyethyl)-rutoside 2 times a day;

Group II - treated with 1 capsule containing 0.0375g of Ruscus extract (Fagorutin- Ruscus , Fink GmbH)

2 times a day, and Group III – treated with 2 tablets of pressed buckwheat herb (each tablet containing

0.5g Fagopyrum esculentum herb and 0.03 troxerutin (Fagorutin Buchweisen tabl., Fink GmbH)) 3 times

a day. During the study period of 3 months, all subjects remained on stable diabetic diet, unchanged

hypoglycaemic medication for the period of treatment. If administered earlier, any hypolipaemic

medication was withdrawn at least 4 weeks before the onset of the study.

At the beginning, as well as on the last day of the investigation each patient was subjected to an

ophtalmological examination and clinical biochemistry. The oscillating potentials of the electroretinogram

were also recorded.

→ Results:

Group I, medicated with troxerutin, was characterized by a decrease in amplitude of oscillating potentials

by 21 % considering both eyes. In contrast, in group II and group III, treated with Ruscus and buckwheat

herb, an increase in amplitude of oscillating potentials (by 15 % and by 18 % respectively) was observed.

The changes in the amplitude detected were statistically insignificant when compared with the initial

values.

In all patients treated for 3 months with troxerutin, Ruscus and buckwheat herb preparations, a slight

statistically insignificant increase in visual acuity was observed.

Examination of the anterior segment of the eyeball after 3 months of pharmacotherapy did not show any

differences when compared with the initial picture in all the groups evaluated.

A regression of changes located in fundus of eye was demonstrated in 27.8% while a progression in 5.6 %

of patients treated with troxerutin was observed. Evaluation of the fundus of the eye in group II ( Ruscus

extract) revealed a quite distinct improvement in 23.1 % of patients and no cases with progression, while

in patients receiving buckwheat herb (group III) an improvement was demonstrated in 26.7 % and a

deterioration in 3.3 % of the examined diabetics.

Mean blood serum concentrations of glucose significantly decreased by 12.7% in the troxerutin treated

group, by 10.6 % in the Ruscus group and by 15.1 % in subjects medicated with buckwheat herb.

Similarly, concentrations of glycosylated haemoglobin were lower after the 3-month period of treatment

in all groups studied.

Assessor’s comments:

The design of the study is not acceptable for many reasons, such as:

– The patients are not adequately defined: the stage of the non-proliferative diabetic retinopathy is

not given; no reference is made to an approved European diabetic retinopathy classification

such as the ETDRS classification; neither baseline blood pressures nor baseline glycosylated

haemoglobin concentrations are given.

– With regard to the pathology, the duration of the study is too short.

– Only 20 patients were enrolled in each group of treatment.

– With regard to the pathology, the choice of the comparators is not justified and cannot be

considered as relevant. Moreover, there is no comparison with a placebo.

Thus, the design of this study is not relevant and cannot be taken into account.

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II.3.2.3

C LINICAL STUDIES IN SPECIAL POPULATION ( E . G . ELDERLY AND CHILDREN )

Elderly

According to the provided literature, no clinical studies have been conducted with Ruscus extract alone in

elderly.

Children

According to the provided literature, no clinical studies have been conducted with Ruscus extract alone in

children.

Pregnancy

The use of Ruscus extract alone has not been evaluated in pregnant women.

II.3.2.4

A SSESSOR ’ S OVERALL CONCLUSIONS ON CLINICAL EFFICACY

To date, the clinical data on Ruscus aculeatus extract alone that can be taken into consideration are limited

to only one publication of a randomized placebo-controlled study performed in patients with chronic

venous insufficiency (Vanscheidt et al. , 2002).

The results obtained from this clinical study suggest an efficacy in the relief of symptoms such as

“heaviness and tiredness” and “sensation of tension” in patients suffering from chronic venous

insufficiency. However, in the opinion of the assessor, the provided evidence is insufficient to implement

the Ruscus aculeatus monograph for a well-established use in relieving symptoms of chronic venous

insufficiency.

In the treatment of haemorrhoids, no clinical data are available with Ruscus aculeatus alone; only

pharmacological effects, data provided by studies with Ruscus in combination and the long-term use

suggest that Ruscus extract is effective to relieve symptoms of haemorrhoids.

There is no sufficient data to sustain the indication of Ruscus aculeatus extract in orthostatic hypotension

and in diabetic retinopathy.

Based on the available data, the monograph information should remain limited to the traditional use :

- for relief of symptoms of heavy legs.

- for symptomatic relief of itching and burning associated with haemorrhoids.

The use Ruscus extract should be limited to adults. Of note, no data are available in men.

The use of Ruscus extract alone has not been evaluated in pregnant women.

II.3.3

C LINICAL S AFETY /P HARMACOVIGILANCE

II.3.3.1

P ATIENT EXPOSURE

According to the provided literature no data are available.

II.3.3.2

A DVERSE EVENTS

Results from the study published by Vanscheidt and al (2002)

This study is a multi-centre, double-blind, randomized, placebo-controlled trial with women suffering

from chronic venous insufficiency to investigate the efficacy and safety of an extract of Ruscus aculeatus

rhizome. Randomization was carried out after a 2-week run-in phase at visit 2. During the run-in period,

all patients received placebo. The following treatment phase with either Ruscus extract or placebo in the

two parallel groups lasted 12 weeks. Checkpoints were scheduled after 4, 8 and 12 weeks of treatment.

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The daily dosage of the Ruscus extract (72-75 mg dry extract from butcher’s broom rhizome) was chosen

according to the German monograph.

166 patients from 30 to 89 years-old were included. 37 patients experienced one or more treatment

emergent adverse events:

– 17 patients experienced 22 adverse events in the Ruscus extract group, including 2 cases of calf

cramps;

– 20 patients experienced 26 adverse events in the placebo group, including 4 cases of calf cramps.

The tolerability was assessed as very good in 76.8% in the Ruscus extract group versus 78.8% in the

placebo group, good in 23.2% versus 20.0% and moderate in 0% versus 1.3%.

Assessor’s comments:

37 patients experienced adverse effects, of whom 17 were in the Ruscus extract group. No information is

available on these cases regarding the nature and the seriousness, excepted for six of them (2 in the

Ruscus extract group and 4 in the placebo group), which were calf cramps. No conclusion with regards to

the safety of this extract can be drawn from these data.

Data from the literature

Three publications and one abstract presented during a French conference regarding the safety of Ruscus

aculeatus have been identified.

(Valnet-Rabier et al. , 2004)

The abstract reports one case of collagenous colitis which occurred in one 52 years-old female patient.

This patient initiated Ruscus aculeatus therapy about 10 months before the diagnosis, which has been

histologically confirmed. At the onset of the colitis, other fluid extracts were taken by the patient, but none

had a known colorectal toxicity.

Assessor’s comments:

It is of note that Cyclo 3 ® , a Ruscus aculeatus containing medicinal product has been associated with

cases of lymphocytic colitis and diarrhea. From 1991 to 2003, several publications reported such cases in

the literature.

(Landa et al. , 1990)

This first publication reports one case of papulo-erythematous eruption of both legs, that spread within a

few days to the entire skin, with oedema of the eyelids in a 30 years-old pregnant female patient, after the

application of a vasoconstrictor cream for the treatment of varices. Patch tests revealed positive results to

Ruscus aculeatus and thimoresal, 2 ingredients of the cream.

(Elbadir et al. , 1998)

In this second publication, 8 cases of contact allergy were collected from 1986 to 1995, in patients

receiving ruscogenins containing medications. Ruscogenins are components of Ruscus aculeatus. These 8

cases involve 6 women and 2 men aged from 28 to 55 years-old who experienced eczema at the

application site, after the use of a topic medication (7 cases) or a cosmetic cream (1 case). Prick tests or

patch tests were performed in 7 patients and all were positive for ruscogenins or Ruscus.

Assessor’s comments :

One of these cases is described in the publication by N. Landa.

(Ramirez-Hernandez, 2006)

The last publication, and the most recent one, reports one case of perianal eczema in one 34 years-old

patient following the local application of an antihaemorrhoidal cream. The outcome was favourable after

therapy withdrawal. Several months later, the patient developed a generalized eczematous cutaneous

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eruption one day after the application of an anticellulitis product on the lower limbs. Patch tests for both

creams revealed a positive reaction to ruscogenins.

Assessor’s comments :

According to these data, the local application of Ruscus aculeatus or ruscogenins has been associated

with allergic reactions, mainly represented by contact eczema. In one case, the event spread to the entire

skin. In all cases, the outcome was favourable after therapy withdrawal and administration of corticoids.

Other data

The review of the PSURs regarding Ruscus aculeatus medicinal products (topics and capsules) did not

allow to identify other safety issues, but confirms that Ruscus containing creams may potentially be

associated with allergic reactions, mainly eczemas, and that the capsules may cause diarrhoea and

lymphocytic colitis.

II.3.3.3 S ERIOUS ADVERSE EVENTS AND DEATHS

According to the provided literature no data are available.

II.3.3.4 L ABORATORY FINDINGS

According to the provided literature no data are available.

II.3.3.5

S AFETY IN SPECIAL POPULATIONS AND SITUATIONS

II.3.3.5.1 I NTRINSIC ( INCLUDING ELDERLY AND CHILDREN ) / EXTRINSIC FACTORS

II.3.3.5.2 D RUG INTERACTIONS

According to the provided literature no data are available.

II.3.3.5.3 U SE IN PREGNANCY AND LACTATION

In an open study involving 9 pregnant women, 3 of the pregnant women applied 2 to 3 grams of a Ruscus

containing cream (100 mg of cream contains 1.6 grams Ruscus extract and 1.6 g Melilot extract) twice

daily during the 3 rd trimester of pregnancy. No embryotoxic effects were noted by the author (Berg, 1991).

Assessor’s comments:

The study was performed with a combination of Ruscus extract and another product, thus a conclusion on

the safety of the Ruscus extract alone cannot be drawn. Furthermore, as the combination has been

administered during the third trimester of pregnancy, the embryotoxicity cannot be ruled out, only the

foetotoxicity is addressed.

In an open study, 20 pregnant women have been enrolled (Baudet et al. , 1991). The women have taken

two capsules per day of Cyclo 3 Fort (a combination of Ruscus extract 150 mg, trimethylhesperidin

chalcone 150 mg and ascorbic acid 100 mg) after 21 to 24 weeks of amenorrhea. Fetal development was

measured through the pulse Doppler method of the cord. The authors conclude that this test shows “an

absolute harmlessness for the infant, assessed with the usual clinical and ultrasonographic criteria of

pregnancy surveillance, with Doppler’s velocimetry at the level of the umbilical artery and with the state

of the infant and the anatomopathologic aspect of the placenta after birth.”

Assessor’s comments:

As for the prior study, this study was performed with a combination of Ruscus extract and other products.

The combination administered from the second trimester of pregnancy has shown neither foetotoxic effect

nor harmful effect for the new born.

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In a review of the botanical treatments for haemorrhoids, some authors concluded that the available

studies in pregnant women treated with a Ruscus combination do not establish the safety of Ruscus in

pregnancy conclusively (Abascal and Yarnall, 2005).

Assessor’s comments:

No data are available with Ruscus extract alone. Even considering Ruscus combinations, data are too

limited to allow any recommendations.

Lactation

As there are no clinical or animal data available on the use of Ruscus extract during lactation and due to

the potential harmful effect on the breast fed new born, particularly with regard to gastrointestinal

disorders, the use of Ruscus extract should be avoided during the lactation.

II.3.3.5.4 O VERDOSE

According to the provided literature no data are available.

II.3.3.5.5 D RUG ABUSE

According to the provided literature no data are available.

II.3.3.5.6 W ITHDRAWAL AND REBOUND

According to the provided literature no data are available.

II.3.3.5.7 E FFECTS ON ABILITY TO DRIVE OR OPERATE MACHINERY OR IMPAIRMENT OF MENTAL ABILITY

According to the provided literature no data are available.

II.3.3.6 A SSESSOR ’ S OVERALL CONCLUSIONS ON CLINICAL SAFETY

No conclusion with regards to the safety of this extract can be drawn from the study published by W

Vanscheidt et al . The data are too scarce and insufficient. No information is available on the adverse

effects regarding the nature and the seriousness, excepted for six of them (2 in the Ruscus extract group

and 4 in the placebo group), which were calf cramps.

Data from the literature highlight two kinds of adverse effects which have been associated with the intake

of Ruscus aculeatus or ruscogenins containing products. The topical forms have been associated with

contact dermatitis . Although the patient received medicinal products containing multiple substances, prick

tests and patch tests allowed confirming, for the topical use, the responsibility of Ruscus /ruscogenins in

the occurrence of the allergic reaction. The second well identified risk concerns the oral route and the

administration of capsules. Therapy with Ruscus aculeatus containing capsules has been associated with

diarrhea/lymphocytic colitis . The literature data seem to be supported by spontaneous reports regarding

different medicinal products and collected data in the PSURs.

From the available studies, a conclusion on the safety of the use of Ruscus extract alone during pregnancy

cannot be drawn. The women were exposed to a mixture containing R uscus extract from the second

trimester of pregnancy. Thus, the only conclusions which can be drawn relates to the foetotoxicity or the

new born effects. For a very limited number of pregnant women (23) no foetotoxic effect appeared to date

but complementary data are necessary to conclusively establish the safety of the Ruscus extract alone

during the latter pregnancy . No data on exposure during the first trimester of pregnancy are available. So,

no conclusion can be drawn about the teratogenic potential of the Ruscus extract alone.

As there are no clinical or animal data available on the use of Ruscus extract during lactation and due to

the potential harmful effect on the breast fed new born, particularly with regard to gastrointestinal

disorders, the use of Ruscus extract during breast feeding should be avoided.

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II.4

ASSESSOR’S OVERALL CONCLUSIONS

Non-clinical aspects

Pharmacology

Primary pharmacodynamics studies performed in vitro and in vivo using various experimental models

showed that Ruscus extract possess a contractile activity on veins . This activity is mediated by

stimulation of the α-adrenergic system. In vitro mechanistic studies showed that direct activation of

postjunctional α1-and α2-adrenergic receptors, and stimulation of the release of norepinephrine form