PHYTOTHERAPY RESEARCH

Phytother. Res.

17

, 537–541 (2003)Published online in Wiley InterScience (www.interscience.wiley.com).

DOI

: 10.1002/ptr.1203

Copyright © 2003 John Wiley & Sons, Ltd.

Received 12 July 2001Accepted 10 December 2001

John Wiley & Sons, Ltd.

Reducing Effect of

Salvia miltiorrhiza

Extracts

on Alcohol Intake: Influence of Vehicle

VEHICLES FOR SALVIA MILTIORRHIZA EXTRACT

Giovanni Vacca,

1

Giancarlo Colombo,

2

* Giuliana Brunetti,

1

Samuele Melis,

1

Daniela Molinari,

1

Salvatore Serra,

1

Roberto Seghizzi,

3

Paolo Morazzoni,

3

Ezio Bombardelli,

3

Gian Luigi Gessa,

1,2,4

and Mauro A. M. Carai

1

1

Neuroscienze Scarl, Cagliari, Italy;

2

CNR Institute of Neurogenetics and Neuropharmacology, Cagliari, Italy;

3

Scientific Department and Formulation Development Laboratory, Indena SpA, Milan, Italy;

4

‘Bernard B. Brodie’ Department of Neuroscience, University of Cagliari, Cagliari, Italy.

A previous study demonstrated that an extract of

Salvia miltiorrhiza

, a medicinal herb highly valued inChinese folk medicine for the treatment of different pathologies, including insomnia, was capable of redu-cing voluntary alcohol intake in selectively bred Sardinian alcohol-preferring (sP) rats. The present study wasdesigned to evaluate the suitability of different emulsifying, suspending agents and solvents as vehiclesthrough which

Salvia miltiorrhiza

extracts can exert their reducing effect on alcohol intake. A single dose(100 mg/kg) of a standardised extract of

Salvia miltiorrhiza

was dissolved in either pure Polysorbate 80,arachis oil, PEG 400, or Polyoxyl 35 castor oil, or suspended in 0.5% CMC in water, and administeredacutely by gavage to sP rats. A significant and specific reduction in alcohol intake was recorded only in ratstreated with the combination of Polysorbate 80 plus the

Salvia miltiorrhiza

extract. A further experimentdemonstrated that the ability of the combination of Polysorbate 80 in water plus the

Salvia miltiorrhiza

extract to decrease alcohol intake was dependent upon the concentration of Polysorbate 80. The results ofthe present study demonstrate that Polysorbate 80 is a proper vehicle for unravelling the reducing effect of

Salvia miltiorrhiza

extracts on alcohol intake. The ability of Polysorbate 80 to form micelles with the activeingredient(s) of the

Salvia miltiorrhiza

may explain these results. They may also offer relevant informationfor pharmaceutical preparation of

Salvia miltiorrhiza

extract to be used in future clinical trials. Copyright© 2003 John Wiley & Sons, Ltd.

Keywords: Salvia miltiorrhiza

extract; Sardinian alcohol-preferring (sP) rats; Voluntary alcohol intake; Vehicles; Polysorbate 80.

INTRODUCTION

Salvia miltiorrhiza

Bge. (Labiatae) is a native plant inChina. Its dried roots (known as Danshen) are officiallylisted in the Chinese Pharmacopoeia and used inChinese folk medicine for the treatment of differentpathologies, including haematological abnormalities,heart disease, hepatitis, haemorrhage, menstrual disorders,miscarriage, oedema and insomnia (see Tang andEisenbrand, 1992; Huang, 1993; State PharmacopoeiaCommission of the People’s Republic of China, 2000).The major chemical constituents of the roots of

Salviamiltiorrhiza

are diterpene pigments, of orange-redcolour, with a phenanthrenequinone structure, such astanshinone I and II and cryptotanshinone.

A recent study from this lab demonstrated that astandardised extract of

Salvia miltiorrhiza

was effectivein reducing voluntary alcohol intake in Sardinian alcohol-preferring (sP) rats (Colombo

et al.

, 1999), one of the ratlines selectively bred world-wide for excessive alcoholpreference and consumption. Interestingly enough, sP

rats appear to be a useful model in the search of novel,potentially effective drugs for the treatment of alcoholism;indeed, drugs that reduce the consumption of alcoholicbeverages in human alcoholics also reduce voluntaryalcohol intake in sP rats. In the study testing

Salviamiltiorrhiza

(Colombo

et al.

, 1999), the extract was dis-solved in an acqueous solution with the emulsifyingagent, polyoxyethylene sorbitanmonoleate (Polysorbate80), and administered intragastrically at the dose of200 mg/kg to rats trained to consume alcohol in daily4 h drinking sessions. Together with the reduction involuntary alcohol intake, the

Salvia miltiorrhiza

extractinduced a marked inhibition of alcohol absorption fromthe gastrointestinal tract, as evidenced by significantlylower blood alcohol levels after the intragastric, but notintraperitoneal, administration of alcohol (Colombo

et al.

, 1999).The present study was designed to further investigate

the reducing effect of

Salvia miltiorrhiza

extracts onalcohol intake, mainly focusing on the suitability of thevehicle in which the

Salvia miltiorrhiza

extract was dis-solved or suspended. To this aim, the present studycompared the anti-alcohol effect of a specific dose ofthe

Salvia miltiorrhiza

extract when it was dissolved orsuspended in five different non-toxic vehicles [namely:Polysorbate 80, arachis oil, polyethylene glycol 400(PEG 400), polyether of castor oil and ethylene oxide(Polyoxyl 35 castor oil) and carboxymethylcellulose

* Correspondence to: Dr G. Colombo, CNR Institute of Neurogenetics andNeuropharmacology, c /o ‘Bernard B. Brodie’ Department of Neuroscience,University of Cagliari, S. S. 554, Km 4.5, I-09042 Monserrato (CA), Italy.Tel: +39 070 675-4325. Fax: +39 070 675-4320. E-mail: colomb@unica.it

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

17

, 537–541 (2003)

538

G. VACCA

ET AL

.

sodium (CMC)], which are widely used in the commonpractice of pharmaceutical preparations (see Zografi

et al.

, 1990; Martindale, 1999). These vehicles werechosen on the basis of their capability to dissolve or sus-pend lipophylic molecules, as expected to be containedin the

Salvia miltiorrhiza

extract. Organic solvents suchas alcohol and dimethylsulphoxide, which would easilydissolve the

Salvia miltiorrhiza

extract, were excludedfrom the study because of their interference with alcoholdrinking behaviour and high toxicity (Kocsis

et al.

, 1975;Fossom

et al.

, 1985; Castro

et al.

, 1995), respectively.The results of the present study may offer potentiallyuseful information for a suitable formulation of the

Salvia miltiorrhiza

extract to be possibly tested in clinicaltrials on human alcoholics.

MATERIALS AND METHODS

Plant material

Roots of

Salvia miltiorrhiza

were collected in 1998 inthe eastern part of China and identified using the genustreatment found in

Flora of China

(Xi-wen and Hedge,1994) and Chinese Pharmacopoeia (State Pharmaco-poeia Commission of the People’s Republic of China,2000). A voucher specimen is kept at Indena SpA, Milan,Italy under the registry no. BA 59986.

Extraction procedure

Roots of

Salvia miltiorrhiza

were dried and then extractedwith acetone. After concentration under vacuum, the res-idue was re-dissolved in alcohol and the solution defattedwith

n

-hexane. The acqueous layer was concentrated andextracted with ethyl acetate. The final extract (namedIDN 5082 – batch no. 38838/1) was obtained after removalof ethyl acetate by alcohol and drying under vacuum at50

°

C for approximately 48 h. The

Salvia miltiorrhiza

extract used in the present study contained 28.9% totaltanshinones and 14.7% tanshinone IIA.

Alcohol intake studies

Animals.

Male sP rats, from the 47th generation andweighing 500–600 g, were used. Rats derived from apopulation of sP rats which had undergone caesarianderivation at Charles River (Lyon, France) for produc-tion of Specific Pathogen Free (SPF) individuals. Ratswere individually housed in standard plastic cages [sizes425

×

266

×

150 (h) mm] with wood chip bedding. Theanimal facility was under an inverted 12:12 h light-darkcycle (lights on at 21:00), at a constant temperature of22

±

2

°

C and relative humidity of approximately 60%.All rats used in the present study fulfilled the selectioncriteria adopted in this lab for qualification as sP rats(see Colombo, 1997).

Experimental procedure.

The rats were continuouslyoffered two bottles containing alcohol (10% v/v, in tapwater) and tap water, respectively. Food pellets (MILMorini, San Polo d’Enza, RE, Italy) were alwaysavailable. Daily alcohol, water and food intakes were

monitored by weighing the bottles and food pellets(0.1 g accuracy). Rats were habituated to handling andintragastric gavage infusion.

After approximately one week of habituation to theexperimental regimen, all rats showed stable dailyintakes of alcohol, water and food. On the test days, ratswere divided into two groups (

n

= 6–8) matched fordaily alcohol, water and food intakes during the threepreceding days. In the first series of tests, the

Salviamiltiorrhiza

extract was dissolved in 2 ml/kg of eitherPolysorbate 80 [Galeno, Comeana (FI), Italy], arachisoil [Galeno, Comeana (FI), Italy], PEG 400 [Galeno,Comeana (FI), Italy], or Polyoxyl 35 castor oil (BASFdistributed by Fluka, Bucks, Switzerland), or suspendedin 0.5% CMC [Galeno, Comeana (FI), Italy] in water.In these experiments, the

Salvia miltiorrhiza

extract wasadministered acutely at the single dose of 100 mg/kg;rats from the control group received an equal volumeof vehicle. Drug injection occurred intragastrically bymeans of a stainless steel, 95-mm long gavage 60 minbefore lights off.

In the further experiment, 100 mg/kg

Salvia miltiorrhiza

extract were suspended in water solutions containingeither 5, 10, 25, 75 or 100% Polysorbate 80 and admin-istered acutely by gavage (injection volume: 2 ml/kg) togroups of

n

=

7 rats 60 min before lights off. Concen-trations of Polysorbate 80 comprised between 25 and75% were not tested because they yielded to suspensionswhich were not fluid enough to pass through the gavage.Control rats received 2 ml/kg of either tap water (

n

=

7)or pure Polysorbate 80 (

n

=

7).All solutions or suspensions were prepared immedi-

ately prior to administration, with the only exception ofthe solution with 100% Polysorbate 80, which was keptunder continuous agitation, at room temperature, for 12consecutive hours. In all experiments, precipitation ofthe extract was limited by a continuous agitation, at roomtemperature and in a dark container, of the solution orsuspension until administration. Analysis at the micro-scope of the distribution of the coloured (orange-red)component of the extract was performed to evaluatepreparation solubility.

Data Analysis.

It should be pointed out that the effectof the single dose of 100 mg/kg

Salvia miltiorrhiza

extractdissolved in each vehicle was evaluated in one of fiveseparate experiments, each including two independentgroups of rats (vehicle- and

Salvia miltiorrhiza

extract-dosed rats). Data on alcohol (expressed in g/kg), water(ml/kg) and food (g/kg) intakes in vehicle- and

Salviamiltiorrhiza

extract-treated rats from each experimentwere compared using the unpaired two-tailed Mann-Whitney U test.

Data on alcohol, water and food intakes from theexperiment comparing the effect of the

Salvia miltiorrhiza

extract suspended in different concentrations of Poly-sorbate 80 were evaluated by a one-way ANOVA.

Blood alcohol level study

Animals

Male SPF sP rats, from the 47th generationand weighing 500–600 g, were used. Rats were housedas specified above. Prior to the experiment, rats had freeaccess to alcohol (10% v/v) for a period of time comparableto that of rats employed in the alcohol intake study.

VEHICLES FOR

SALVIA MILTIORRHIZA

EXTRACT

539

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

17

, 537–541 (2003)

Alcohol and food pellets were removed 24 and 12 hbefore the start of the experiment, respectively.

Experimental procedure

The

Salvia miltiorrhiza

extractwas dissolved in 2 ml/kg pure Polysorbate 80 as describedabove and administered intragastrically at the doses of0 (

n

=

10) and 100 (

n

=

10) mg/kg 60 min prior to theintragastric administration of 2 g/kg alcohol (15% w/v,in distilled water). Blood samples (50

µ

l) were collectedfrom the tip of the tail of each rat at 0, 30, 60, 120, 180,240 and 360 min post-alcohol infusion. Blood sampleswere analysed by means of an enzymatic system [AM1Analyser (Analox Instruments Ltd, London, UK)] basedon measurement of oxygen consumption in the alcohol-acetaldehyde reaction.

Data analysis

Data on blood alcohol levels were expressedin mg/100 ml and analysed by a two-way (drug treat-ment; time interval) ANOVA with repeated measureson time intervals, followed by the Newman-Keuls testfor multiple comparisons.

RESULTS

In all tests, the average daily intake of alcohol in thevehicle-treated groups varied between 4.8 and 6.4 g/kg,well within the range of alcohol amounts usually consumedby sP rats under the two-bottle free choice regimenwith unlimited access (see Colombo, 1997), and sug-gestive of a minimal influence of each vehicle tested onvoluntary alcohol intake in sP rats.

When dissolved in pure Polysorbate 80, 100 mg/kg

Salvia miltiorrhiza

extract exerted a marked reducingeffect on alcohol intake; as shown in Fig. 1A, alcoholintake in

Salvia miltiorrhiza

extract-treated rats wasapproximately 40% lower than that recorded invehicle-treated rats (

p

= 0.025, Mann-Whitney U test).A compensatory, significant increase in water intake(

p

=

0.002, Mann-Whitney U test; Fig. 1A) left totalfluid intake virtually unchanged (

p

> 0.05, Mann-Whitney U test; data not shown). Food intake wasnot affected by the administration of the

Salvia

Figure 1. Effect of the acute administration of the Salvia miltiorrhiza extract, dissolved in pure Polysorbate 80 (panel A), arachis oil(panel B), polyethylene glycol 400 (PEG 400) (panel C), or Polyoxyl 35 castor oil (panel D), or suspended in carboxymethylcellulose(CMC) (panel E) on daily alcohol (g/kg), water (ml/kg) and food (g/kg) intakes in Sardinian alcohol-preferring (sP) rats. Alcohol(10%, v/v) and water were offered under two-bottle, free choice regimen with unlimited access for 24 h per day. Food pellets were alwaysavailable. The Salvia miltiorrhiza extract was administered intragastrically, at the dose of 100 mg/kg (injection volume: 2 ml/kg),60 min before lights off. Bars are the mean ± SEM of n = 6–8 subjects. In panel A, *: p = 0.025 and +: p = 0.002 with respect tovehicle-treated rats (Mann-Whitney U test).

Copyright © 2003 John Wiley & Sons, Ltd.

Phytother. Res.

17

, 537–541 (2003)

540

G. VACCA

ET AL

.

miltiorrhiza

extract (

p

> 0.05, Mann-Whitney U test;Fig. 1A).

In contrast, when dissolved in arachis oil (Fig. 1B),PEG 400 (Fig. 1C), or Polyoxyl 35 castor oil (Fig. 1D),or suspended in CMC (Fig. 1E), 100 mg/kg

Salviamiltiorrhiza

extract failed to induce any significantalteration of voluntary intake of alcohol (

p

> 0.05, Mann-Whitney U test); similarly, neither water nor food intakesresulted to be significantly modified by administrationof the

Salvia miltiorrhiza

extract (

p

> 0.05, Mann-Whitney U test; Fig. 1, panels B–E).

The reducing effect on alcohol intake of

Salvia milti-orrhiza

extract in Polysorbate 80 resulted to be depend-ent upon the concentration of Polysorbate 80. Indeed,as shown in Fig. 2, alcohol intake in

Salvia miltiorrhiza

extract-treated rats gradually decreased as the concen-

tration of Polysorbate 80 was increased [

F

(6;42) = 4.053,

p

= 0.0027]. Conversely, water intake increased signi-ficantly as the concentration of Polysorbate 80 wasincreased [

F

(6;42) = 3.189,

p

= 0.0114] (Fig. 2). In contrast,treatment with the

Salvia miltiorrhiza extract did notsignificantly alter food intake [F(6;42) = 1.044, p = 0.4113](Fig. 2).

Combination of 100 mg/kg Salvia miltiorrhiza extractand 2 g/kg alcohol resulted in a significant reduction,by approximately 40% with respect to vehicle-treatedrats, in blood alcohol levels [Ftreatment(1;85) = 11.949, p =0.0030] (Fig. 3). Post hoc comparisons revealed significantdifferences at 60 to 360 min time intervals betweenSalvia miltiorrhiza extract- and vehicle-treated rats.

DISCUSSION

These results confirm those of a previous investigationfrom this lab (Colombo et al., 1999) and demonstratethat extracts of Salvia miltiorrhiza are capable of redu-cing voluntary alcohol intake in sP rats, an animal modelof excessive alcohol drinking behaviour. In the presentstudy, the acute administration of 100 mg/kg Salviamiltiorrhiza extract dissolved in Polysorbate 80 induceda reduction of approximately 40% of daily alcoholintake. Remarkably enough, the decreasing effect ofSalvia miltiorrhiza extract in Polysorbate 80 on alcoholintake was accompanied by a compensatory increase inwater intake; in contrast, food intake (a variable usuallyrecorded in animal studies where alcohol intake ispharmacologically manipulated as a measure of possibleanimal malaise and non-selectivity of the drug action)

Figure 2. Effect of the acute administration of the Salvia milti-orrhiza extract, dissolved in an ascending series of concen-trations of Polysorbate 80, on daily alcohol (g/kg), water (ml/kg)and food (g/kg) intakes in Sardinian alcohol-preferring (sP) rats.Alcohol (10%, v/v) and water were offered under two-bottle,free choice regimen with unlimited access for 24 hours per day.Food pellets were always available. The Salvia miltiorrhizaextract was dissolved in a water solution with 5, 10, 25, 75or 100% Polysorbate 80 and administered intragastrically atthe constant dose of 100 mg/kg (injection volume: 2 ml/kg)60 min before lights off. Bars are the mean ± SEM of n = 7subjects.

Figure 3. Effect of the acute administration of the Salvia milti-orrhiza extract, dissolved in pure Polysorbate 80, on bloodalcohol levels (mg/100 ml) in Sardinian alcohol-preferring(sP) rats. Alcohol was administered by gavage at the doseof 2 g/kg 60 min after the intragastric administration of theSalvia miltiorrhiza extract. Rats were fasted for 24 h beforealcohol administration. Blood samples were collected fromthe tip of the tail at different time intervals from alcoholtreatment. Each point is the mean ± SEM of n = 10 subjects.*: p < 0.05 with respect to vehicle-treated rats (Newman-Keulstest).

VEHICLES FOR SALVIA MILTIORRHIZA EXTRACT 541

Copyright © 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 537–541 (2003)

was completely unaffected by the administration of theSalvia miltiorrhiza extract.

As in the initial study (Colombo et al., 1999), combina-tion of the Salvia miltiorrhiza extract (in Polysorbate80) and alcohol resulted in a significant reduction ofblood alcohol levels, suggestive of a likely inhibition ofalcohol absorption from the gastrointestinal tract. Theextent to which the capability of Salvia miltiorrhizaextracts to decrease alcohol absorption may contributeto its reducing effect on alcohol intake is a matter ofpresent investigation.

The present study was primarily designed to comparethe suitability of different vehicles in unravelling thereducing effect of the Salvia miltiorrhiza extract onalcohol intake. In order to facilitate a comparison betweenthe different tests, they were conducted under identicalexperimental conditions, with the vehicle being the onlydifference. Quite unexpectedly, and in sharp contrastwith the results of the test where pure Polysorbate80 was used as vehicle, 100 mg/kg Salvia miltiorrhizaextract failed to induce any reducing effect on alcoholintake when it was dissolved in arachis oil, PEG 400,or Polyoxyl 35 castor oil, or suspended in CMC. Fur-thermore, the efficacy of the combination of the Salviamiltiorrhiza extract and Polysorbate 80 in reducingalcohol intake was dependent on the concentration ofPolysorbate 80, with low concentrations exerting aminimal effect and those in a higher range (75–100%)inducing a 35–40% reduction.

These results, taken together, suggest that although itis necessary to dissolve the Salvia miltiorrhiza extractwhen attempting to unravel its anti-alcohol effect, aloneit is not a sufficient condition. Indeed, the reducingeffect of Salvia miltiorrhiza on alcohol intake was evident

when the extract was combined with the solvent, Poly-sorbate 80, but not with the suspending agent, CMC;however, not all the tested solvents were effective, andeven the emulsifying agent, Polyoxyl 35 castor oil,which should result in a degree of dissolution of theextract – when the formulate is exposed to gastric fluids– comparable to that of Polysorbate 80, was ineffective.A possible explanation for these apparently odd, diver-gent results may reside in the capability of the differentsolvents to form micelles with the active principle(s)contained in the extract, influencing therefore theirstability and bioavailability. Accordingly, it may beproposed that Polysorbate 80 is the most effective vehicle,among those tested in the present study, in formingmicelles with the Salvia miltiorrhiza extract. The con-centration-dependency nature of the reducing effect ofcombination of Polysorbate 80 plus Salvia miltiorrhizaextract on alcohol intake apparently supports thishypothesis.

Salvia miltiorrhiza extracts may constitute a novelpharmacotherapy for alcoholism. Future clinical studieswill assess whether the reducing effect of Salvia milti-orrhiza extracts on alcohol intake reported in alcohol-consuming rats generalises to human alcoholics. Inview of this, the results of the present study may offerpotentially useful information on the pharmaceuticalpreparation to be tested.

Acknowledgements

The authors are grateful to Mrs M. Elena Vincis, Mrs GabriellaManca and Mr Antonio Pilleri for animal breeding and care, andMrs Anne Farmer for language editing of the manuscript.

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