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Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats
Accepted Manuscript Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol selfadministration in Sardinian alcohol-preferring rats Paola Maccioni , Daniela Vargiolu , Maura Falchi , Paolo Morazzoni , Antonella Riva , Walter Cabri , Mauro A.M. Carai , Gian Luigi Gessa , Giancarlo Colombo PII:
S0741-8329(14)00102-5
DOI:
10.1016/j.alcohol.2014.06.002
Reference:
ALC 6408
To appear in:
Alcohol
Please cite this article as: MaccioniP., VargioluD., FalchiM., MorazzoniP., RivaA., CabriW., CaraiM.A.M., GessaG.L. & ColomboG., Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats, Alcohol (2014), doi: 10.1016/ j.alcohol.2014.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats
Giancarlo Colomboa
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Paola Maccionia, Daniela Vargiolua, Maura Falchia, Paolo Morazzonib, Antonella Rivab, Walter Cabrib, Mauro A.M. Caraia, Gian Luigi Gessaa,
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Neuroscience Institute, National Research Council of Italy, Section of Cagliari, I-09042 Monserrato (CA), Italy
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Indena S.p.A., I-20139 Milan (MI), Italy
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Corresponding author: Giancarlo Colombo Neuroscience Institute National Research Council of Italy Section of Cagliari S.S. 554, km. 4,500 I-09042 Monserrato (CA), Italy Telephone: +39 070 675 4342 Fax: +39 070 675 4320 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract The dried roots of Salvia miltiorrhiza are highly valued in Chinese folk medicine for use in the prevention and treatment of a series of ailments. Previous studies have demonstrated that administration of standardized extracts of Salvia miltiorrhiza selectively reduced excessive
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alcohol drinking and relapse-like drinking in selectively bred Sardinian alcohol-preferring (sP) rats. The present study was designed to extend these findings on the “anti-alcohol” properties of Salvia miltiorrhiza extracts to operant procedures of oral alcohol self-administration. Two
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independent groups of sP rats were trained to lever-respond on an FR4 schedule of
reinforcement for alcohol (15%, v/v) or sucrose (1–3%, w/v) in daily 30-min sessions. Once
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responding had stabilized, rats were tested under the fixed ratio 4 (FR4) schedule of reinforcement (index of alcohol reinforcing properties) and the progressive ratio (PR) schedule of reinforcement (index of alcohol motivational properties). Treatment with Salvia miltiorrhiza extract (0, 50, 100, and 200 mg/kg, intragastrically [i.g.]) markedly reduced lever responding
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for alcohol, amount of self-administered alcohol, and breakpoint for alcohol (defined as the lowest response requirement not achieved in the PR experiment). No dose of Salvia miltiorrhiza extract altered any parameter of sucrose self-administration. These results a)
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demonstrate that treatment with Salvia miltiorrhiza extract selectively reduced the reinforcing and motivational properties of alcohol in sP rats and b) extend to operant procedures of
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alcohol self-administration previous data on the “anti-alcohol” effects of Salvia miltiorrhiza extracts. These data strengthen the notion that novel pharmacological approaches for treatment of alcohol use disorders may stem from natural substances. Keywords: Salvia miltiorrhiza extract; alcohol self-administration; fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement; reinforcing and motivational properties of alcohol; Sardinian alcohol-preferring (sP) rats
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ACCEPTED MANUSCRIPT Introduction In the search for new pharmacological remedies for alcohol use disorders (AUDs), particular attention has recently been focused on extracts or preparations derived from several medicinal plants (for review, see Carai et al., 2000; Li, 2010; Liu, Lawrence, & Liang,
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2011; Rezvani, Overstreet, Perfumi, & Massi, 2003; Tomczyk, Zovko-Končić, & Chrostek, 2012). The most striking example in this research field is likely represented by the discovery and development of extracts of and ingredients from the roots of Pueraria lobata (the kudzu
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plant). Initial preclinical studies unanimously demonstrated that administration of extracts from Pueraria lobata, or its active constituents (the isoflavones daidzin, daidzein, and
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puerarin), markedly reduced several alcohol-related behaviors – including alcohol drinking and operant, oral alcohol self-administration – in rats and hamsters (e.g., Heyman, Keung, & Vallee, 1996; Keung, Lazo, Kunze, & Vallee, 1995; Keung & Vallee, 1993; Lin et al., 1996; Overstreet et al., 1996). Subsequent clinical studies generalized to humans the “anti-alcohol”
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properties of Pueraria lobata extracts observed in rodents: treatment with a Pueraria lobata extract a) decreased the number of alcohol drinks in heavy drinkers exposed to a naturalistic laboratory setting (Lukas et al., 2005), and b) decreased the number of weekly alcohol
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drinks, decreased the number of heavy drinking days, increased the percentage of days of abstinence, and increased the number of consecutive days of abstinence in heavy drinkers
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recruited in a recent double-blind, placebo-controlled survey (Lukas et al., 2013). These effects occurred in the absence of any potentiation of the subjective or psychomotor effects of alcohol (Penetar, Maclean, McNeil, & Lukas, 2011). Finally, a recent double-blind, placebo-controlled survey demonstrated that treatment with the active constituent, puerarin, was likewise capable of reducing alcohol drinking in heavy drinkers, reproducing the effects of the whole extract (Penetar et al., 2012). In recent years, this laboratory has contributed to the characterization of the “antialcohol” profile of root extracts from Salvia miltiorrhiza Bunge (Labiatae), a medicinal plant the dried roots of which (known as Danshen) are officially listed in the Chinese 3
ACCEPTED MANUSCRIPT Pharmacopoeia and have been used for hundreds of years in Chinese folk medicine for the treatment of different pathologies, including cardiovascular disease, hematological abnormalities, hyperlipidemia, hepatitis, hemorrhage, menstrual disorders, miscarriage, edema, and insomnia (State Pharmacopoeia Commission PRC, 2000; Tang & Eisenbrand,
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1992; Zhou, Zuo, & Chow, 2005). Specifically, acute or repeated administration of several standardized extracts of Salvia miltiorrhiza have been found to reduce a) acquisition (Brunetti et al., 2003) and maintenance (Colombo et al., 1999; Colombo, Serra, et al., 2006;
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Vacca et al., 2003) of high alcohol drinking and b) relapse-like alcohol drinking (Serra et al., 2003) in selectively bred, Sardinian alcohol-preferring (sP) rats exposed to the standard,
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homecage 2-bottle “alcohol vs. water” choice regimen. Notably, an additional study likely identified the active constituent underlying the reducing effect of Salvia miltiorrhiza extracts on alcohol intake. The reducing effect of four different extracts of Salvia miltiorrhiza on alcohol intake in sP rats was positively and highly significantly correlated with their content of
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the diterpene, miltirone. In addition, pure miltirone – given at doses comparable to its content in the effective doses of the active extracts – reduced alcohol intake in sP rats (Colombo, Serra, et al., 2006).
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The present study was designed to extend the investigation of the “anti-alcohol” properties of Salvia miltiorrhiza extracts to operant procedures of oral alcohol self-
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administration. Under these procedures, alcohol is made available to rats via completion of a behavioral response (usually, responding on a lever a given number of times), to allow the reinforcing and motivational properties of alcohol to be assessed in addition to its mere consumption. The present study employed two different procedures of alcohol selfadministration: a) fixed ratio (FR) schedule of reinforcement, in which the response requirement (RR; i.e., the “cost” of each alcohol presentation in terms of number of responses on the lever) is predetermined and kept fixed throughout the session (providing a measure of alcohol intake and of the reinforcing properties of alcohol), and b) within-session 4
ACCEPTED MANUSCRIPT progressive ratio (PR) schedule of reinforcement, in which – over the same single session – RR is progressively increased after the delivery of each reinforcer, and the lowest ratio not completed (named breakpoint) is taken as the measure of motivational properties of alcohol (see Markou et al., 1993).
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The present study was conducted using male sP rats as animal model of AUDs. These rats have been selectively bred for high alcohol preference and consumption under the 2-bottle “alcohol vs. water” choice regimen (Bell et al., 2012; Colombo, Lobina, Carai, &
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Gessa, 2006). When exposed to sessions of operant, oral alcohol self-administration, sP rats display robust lever responding for alcohol, self-administer large amounts of alcohol, and
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achieve high levels of breakpoint for alcohol (see Colombo, Lobina, et al., 2006). Materials and methods
All experimental procedures employed in the present study were in accordance with the Italian law on the “Protection of animals used for experimental and other scientific
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reasons”.
Plant material and preparation of plant extract
Roots of Salvia miltiorrhiza were collected in 2005 in the eastern part of China and
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identified using the genus treatment described in Flora of China (Xi-wen & Hedge, 1994) and
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Chinese Pharmacopoeia (State Pharmacopoeia Commission PRC, 2000). A voucher specimen is kept at Indena S.p.A., Milan, Italy, under the registry no. 137127. Roots of Salvia miltiorrhiza were dried and subsequently extracted with acetone. After concentration under vacuum, the residue was re-dissolved in alcohol and the solution defatted with n-hexane. The aqueous layer was concentrated and extracted with ethyl acetate. The final extract (named IDN5655) was obtained after removal of ethyl acetate by alcohol and drying under vacuum at 50 °C for approx imately 48 h. The Salvia miltiorrhiza extract used in the present study contained 21% total tanshinones and 4.3% miltirone. Animals 5
ACCEPTED MANUSCRIPT Male sP rats from the 80th generation and 60 days old at the start of the study were utilized. Rats were alcohol-naïve before the start of the study. Rats were housed 4 per cage in standard plastic cages with wood chip bedding. The animal facility was under an inverted 12:12-hour light-dark cycle (lights on at 9:30 PM), at a constant temperature of 22 ± 2 °C and
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relative humidity of approximately 60%. Over the 2-week period preceding the start of the study, rats were extensively habituated to handling and intragastric infusions (by a metal gavage). Food pellets (Harlan, San Pietro al Natisone, Italy) and water were always available
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in the homecage, except as noted.
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Rats were divided into two groups: “alcohol” (n = 32) and “sucrose” (n = 32) groups. Apparatus
Self-administration sessions were conducted in modular chambers (Med Associates, St. Albans, VT, USA) located in sound-attenuated cubicles, with fans for ventilation and background white noise. The front panel of each chamber was equipped with a) two
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retractable response levers, b) one dual-cup liquid receptacle positioned between the two levers, and c) two stimulus lights (one green and one white) mounted above each lever. The liquid receptacle was connected by polyethylene tubes to two syringe pumps located outside
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the chamber. A white house light was centered at the top of the back wall of each chamber.
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For half of the rats, the right lever was associated with alcohol (or sucrose), and achievement of RR a) activated the alcohol (or sucrose) pump, resulting in the delivery of 0.1 mL alcohol (or sucrose) solution, and b) switched on the green light for the 2-s period of alcohol (or sucrose) delivery. For these rats, the left lever was associated with water, and achievement of RR a) activated the water pump, resulting in the delivery of 0.1 mL water, and b) switched on the white light for the 2-s period of water delivery. The study design was counterbalanced so that the opposite condition was applied to the other half of the rats (left lever: alcohol [or sucrose]; right lever: water). Experimental procedure 6
ACCEPTED MANUSCRIPT 2-bottle choice phase Rats of the “alcohol” group were initially exposed to the homecage 2-bottle “alcohol vs. water” choice regimen with unlimited access for 24 hours/day over 10 consecutive days. The alcohol solution was presented at a concentration of 10% (v/v). This initial phase was a) part
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of the conventional procedure of alcohol self-administration employed in this laboratory with sP rats (e.g., Maccioni et al., 2012) and b) conducted to allow the rats to become accustomed to the taste of alcohol and start to experience its pharmacological effects in order to possibly
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shorten the subsequent auto-shaping phase once rats are introduced into the operant chambers. During this phase, daily alcohol intake averaged approximately 6.5 g/kg.
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Shaping, training, and maintenance phases
Immediately after the 2-bottle choice regimen, rats of the “alcohol” group were introduced into the operant chambers and trained to lever-respond for alcohol. Selfadministration sessions lasted 30 min (with the sole exception of the very first session that
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lasted 120 min) and were conducted 5 days per week (Monday to Friday) during the first 4 to 6 h of the dark phase of the daily light/dark cycle. Rats were water-deprived during the 12 h prior to the first session in the operant chamber. Rats were initially exposed to an FR1
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schedule of reinforcement for 10% alcohol (v/v) for 4 consecutive daily sessions. FR was then increased to FR2 and FR4 over 4 consecutive sessions. In sessions 9 and 10, the alcohol
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solution was presented at the final concentration of 15% (v/v). Rats were then exposed to 4 consecutive sessions during which the water lever alone or the alcohol lever alone was available every other day; water and alcohol were available on FR1 and FR4, respectively. From then on, both levers were concomitantly available (maintenance phase) for a total of 20 sessions conducted with FR4 and FR1 on the alcohol and water lever, respectively. During these 20 sessions, “alcohol” rats displayed an average number of lever responses for alcohol of approximately 180 per session; the resulting mean amount of self-administered alcohol was approximately 0.90 g/kg per session. Conversely, a negligible number of lever responses 7
ACCEPTED MANUSCRIPT for water (averaging ≤ 5 per session) were recorded in each session. After the 20 selfadministration sessions of the maintenance phase, rats were selected for inclusion in the two experiments (“FR” and “PR”, see below). Specifically, the 24 rats displaying the most stable responding behavior over the last 5 daily sessions (less than 20% daily difference) were
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selected and divided into two groups (n = 12 in the “FR” experiment; n = 12 in the “PR” experiment), matched for total number of lever responses on the alcohol lever and amount of self-administered alcohol.
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Rats of the “sucrose” group were trained to lever-respond for a sucrose solution. Selfadministration sessions lasted 30 min and were conducted 5 days per week (Monday to
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Friday) during the first 4 to 6 h of the dark phase of the daily light/dark cycle. Lever responses were initially shaped under FR1 and 3% (w/v) sucrose for 2 consecutive sessions. On the subsequent 4 sessions, FR was increased to FR2 and FR4. Rats were then randomly divided into two subgroups (n = 16 in the “FR” experiment; n = 16 in the “PR” experiment) for
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subsequent inclusion in the two experiments. Specifically, in one rat group (“FR” experiment), the sucrose concentration was reduced to 1% (w/v) over two consecutive sessions; in the other rat group (“PR” experiment), the sucrose concentration was maintained at 3% (w/v).
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These two sucrose concentrations were selected on the basis of previous results (e.g., Maccioni et al., 2009) in order to obtain, in the test sessions with both FR and PR schedules
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of reinforcement, lever-responding behaviors comparable to those usually performed by sP rats to obtain 15% alcohol. Rats were then exposed to 4 consecutive sessions during which either the water lever or the sucrose lever alone was available every other day; water and sucrose were available on FR1 and FR4, respectively. From then onward, both levers were concomitantly available (maintenance phase); a total of 20 sessions was conducted with FR4 and FR1 on the sucrose and water lever, respectively. During these 20 sessions, “sucrose” rats exposed to 1% and 3% sucrose displayed an average number of lever responses for sucrose of approximately 210 and 850, respectively, per session; the resulting mean amount 8
ACCEPTED MANUSCRIPT of self-administered sucrose solution was approximately 7.0 and 35.0 mL/kg, respectively, per session. Conversely, a negligible number of lever responses for water (averaging < 5 per session) were recorded in each session. After the 20 self-administration sessions of the maintenance phase, rats of both subgroups (“FR” and “PR”) were selected for inclusion in the
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two experiments; specifically, the 12 rats of the “FR” experiment and the 12 rats of the “PR” experiment displaying the most stable responding behavior over the last 5 daily sessions (less than 25% daily difference) were selected.
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Test sessions
During the test sessions of the “FR” experiments, RR on the alcohol (or sucrose) and
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water lever was maintained at FR4 and FR1, respectively. Test sessions of the “FR” experiments lasted 30 min. During the test sessions of the “PR” experiments, RR on each lever was increased progressively over the session according to the procedure described by Richardson & Roberts (1996); namely, RR was increased as follows: 4, 9, 12, 15, 20, 25, 32,
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40, 50, 62, 77, 95, 118, 145, 178, 219, etc. During the test sessions of the “PR” experiments, the water lever was inactive (lever responses were recorded, but had no programmed consequences). Test sessions of the “PR” experiments lasted 60 min; longer times were not
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required as previous data (e.g., Maccioni et al., 2009, 2012) indicated that lever responding
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for alcohol in sP rats exposed to the PR schedule of reinforcement used in the present study reached its plateau within 10–15 min. In each experiment (“FR” and “PR”), test sessions were conducted on Fridays and all doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Four consecutive (Monday to Thursday) self-administration sessions (with FR4 on the alcohol or sucrose lever and FR1 on the water lever and no drug treatment) elapsed between test sessions. After each test session, alcohol (or sucrose) selfadministration rapidly recovered to baseline levels. Salvia miltiorrhiza extract was dissolved in a 1:1 mixture of polysorbate 80 (Tween 80) plus polyethylene glycol 600 (PEG 600). The solution was kept under continuous agitation, at 9
ACCEPTED MANUSCRIPT room temperature and in a dark container, for at least 12 consecutive hours before administration. Salvia miltiorrhiza extract was administered intragastrically (infusion volume: 2 mL/kg), at the doses of 0, 50, 100, and 200 mg/kg, 60 min before the start of the selfadministration session. Dose range of Salvia miltiorrhiza extract, treatment time, and route of
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administration were chosen based on the results of previous studies testing the effects of Salvia miltiorrhiza extracts on different alcohol-related behaviors in sP rats (Brunetti et al., 2003; Colombo et al., 1999; Colombo, Serra, et al., 2006; Serra et al., 2003; Vacca et al.,
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2003). Measured variables and data analysis
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In “FR” experiments, measured variables were as follows: a) number of responses on each lever (alcohol [or sucrose] and water levers); b) amount of self-administered alcohol (or sucrose) and water (expressed in g/kg pure alcohol, mL/kg sucrose solution, and mL/kg water, respectively, and determined from the number of earned reinforcers).
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In “PR” experiments, measured variables were as follows: a) number of responses on each lever (alcohol [or sucrose] and inactive levers); b) breakpoint for alcohol (or sucrose), defined as the lowest RR not achieved by the rat on the alcohol (or sucrose) lever.
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Data on the effect of Salvia miltiorrhiza extract on each variable were statistically
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analyzed by 1-way ANOVA for repeated measures, followed by the Newman-Keuls test for post hoc comparisons. Comparisons between number of lever responses and breakpoint values for alcohol and sucrose in vehicle-treated rat groups were performed using the Student t test. Results “FR” experiment Treatment with Salvia miltiorrhiza extract significantly reduced the number of lever responses for alcohol [F(3,33) = 9.66, p < 0.0001] (Fig. 1, panel A). A post hoc test revealed that all three doses of Salvia miltiorrhiza extract decreased – relative to values of the vehicle10
ACCEPTED MANUSCRIPT treated rat group – the number of lever responses for alcohol (p < 0.001, Newman-Keuls test). The magnitude of the reducing effect on the number of lever responses for alcohol was similar among the three doses of Salvia miltiorrhiza extract, averaging 50–60% in comparison to vehicle-treated rats. Reduction in number of lever responses for alcohol
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resulted in a proportional decrease in the amount of self-administered alcohol [F(3,33) = 9.98, p < 0.0001] (Fig. 1, panel C). Lever responding for water was negligible (averaging ≤ 4 per session in all rat subgroups) and not altered by treatment with Salvia
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miltiorrhiza extract [F(3,33) = 0.97, p > 0.05] (data not shown).
In the vehicle-treated rats of the “sucrose” group, number of lever responses for
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sucrose was similar to those observed in the vehicle-treated, “alcohol” rat group (p > 0.05, Student t test). Treatment with Salvia miltiorrhiza extract failed to alter a) number of lever responses for sucrose [F(3,33) = 0.32, p > 0.05] (Fig. 1, panel B), and b) amount of selfadministered sucrose [F(3,33) = 0.27, p > 0.05] (Fig. 1, panel D). Lever responding for water
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was negligible (averaging ≤ 3 per session in all rat subgroups) and not altered by treatment with Salvia miltiorrhiza extract [F(3,33) = 0.13, p > 0.05] (data not shown). “PR” experiment
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Treatment with Salvia miltiorrhiza extract resulted in a significant reduction of the
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number of lever responses for alcohol [F(3,33) = 4.01, p < 0.05] (Fig. 2, panel A) and breakpoint for alcohol [F(3,33) = 4.48, p < 0.01] (Fig. 2, panel C). A post hoc analysis revealed that a) the number of lever responses for alcohol was significantly reduced by treatment with only 200 mg/kg Salvia miltiorrhiza extract (p < 0.01), and b) breakpoint for alcohol was significantly reduced by treatment with both 100 (p < 0.05) and 200 mg/kg (p < 0.01) Salvia miltiorrhiza extract. The reducing effect of Salvia miltiorrhiza on both variables tended to be dose-related and averaged approximately 50%, in comparison to vehicle-treated rats, in the rat group treated with 200 mg/kg Salvia miltiorrhiza extract. Responding on the inactive lever was significantly increased by treatment with Salvia 11
ACCEPTED MANUSCRIPT miltiorrhiza extract [F(3,33) = 4.33, p < 0.05]; it averaged 5.3 ± 1.4, 6.8 ± 2.4, 25.7 ± 7.8, and 24.7 ± 6.2 in the rat groups treated with 0, 50, 100, and 200 mg/kg Salvia miltiorrhiza extract, respectively, with significant differences at the post hoc test between the vehicle-treated rat group and the two rat groups treated with 100 (p < 0.05) and 200 mg/kg (p < 0.05) Salvia
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miltiorrhiza extract. In the vehicle-treated rats of the “sucrose” group, the number of lever responses for sucrose and value of breakpoint for sucrose were similar to those observed in the vehicle-
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treated, “alcohol” rat group (p > 0.05, Student t test). Treatment with Salvia miltiorrhiza extract altered neither the number of lever responses for sucrose [F(3,33) = 0.35, p > 0.05]
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(Fig. 2, panel B) nor breakpoint for sucrose [F(3,33) = 0.26, p > 0.05] (Fig. 2, panel D). Responding on the inactive lever was negligible (averaging ≤ 7 per session in all rat subgroups) and not altered by treatment with Salvia miltiorrhiza extract [F(3,33) = 0.25, p > 0.05] (data not shown).
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Discussion
As expected, sP rats displayed robust lever-responding behaviors for alcohol under both experimental procedures: when exposed to the FR4 schedule of reinforcement, sP rats
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responded on the alcohol lever an average of 165 times and consumed an average of
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0.80 g/kg alcohol in 30-min sessions (for example, see the values collected in the vehicletreated rat group in the “FR” experiment). When exposed to the PR schedule of reinforcement, sP rats achieved a breakpoint value of approximately 50 (see values collected in the vehicle-treated rat group in the “PR” experiment). These data a) are highly similar to those collected in several previous alcohol self-administration studies using sP rats (e.g., Maccioni et al., 2009, 2012), b) suggest that alcohol has strong reinforcing and motivational properties in sP rats, and c) confirm the validity of sP rats as an animal model for investigations aimed at identifying new pharmacotherapies for AUDs (see Bell et al., 2012). 12
ACCEPTED MANUSCRIPT The results of the present study demonstrate that treatment with a standardized extract of the Chinese medicinal plant, Salvia miltiorrhiza, effectively reduced oral alcohol self-administration in alcohol-preferring sP rats. The effect of Salvia miltiorrhiza extract was evident under two different operant procedures, known to provide a measure of the strength
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of the reinforcing (FR schedule of reinforcement) and motivational (PR schedule of reinforcement) properties of alcohol (see Markou et al., 1993). These data a) extend to operant alcohol self-administration the capacity of different Salvia miltiorrhiza extracts to
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decrease several alcohol-related behaviors, including acquisition and maintenance of high alcohol drinking and relapse-like drinking, in sP rats (Brunetti et al., 2003; Colombo et al.,
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1999; Colombo, Serra, et al., 2006; Serra et al., 2003; Vacca et al., 2003), and b) provide additional elements to the preclinical “anti-alcohol” profile of Salvia miltiorrhiza extracts. Additional information on the reducing effect of Salvia miltiorrhiza extract on alcohol reinforcing and motivational properties may be provided by the analysis of cumulative
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response patterns. Data from both “FR” (Fig. 3) and “PR” (Fig. 4) experiments suggest that, in comparison to treatment with vehicle, treatment with all three doses of Salvia miltiorrhiza extract resulted in a) less steep curves (suggestive of a reduced frequency in lever-pressing),
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particularly at the beginning of the session, and b) lower plateau values (suggesting that fewer ratios were completed before responding ended). Together, these data suggest that
alcohol.
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treatment with Salvia miltiorrhiza extract weakened the drive of rats to access and consume
The reducing effect of Salvia miltiorrhiza extract on lever responding was selective for alcohol, as treatment with the same dose range of Salvia miltiorrhiza extract failed to affect – in both “FR” and “PR” experiments – lever responding for an alternative, non-drug reinforcer such as a sucrose solution (the concentrations of which were selected in order to produce reinforcing and motivational properties highly comparable to those produced by the alcohol solution). Moreover, the results of the two “sucrose” experiments tend to exclude that 13
ACCEPTED MANUSCRIPT reduction of alcohol self-administration induced by treatment with Salvia miltiorrhiza extract was secondary to possible malaise or motor-incapacitating and sedative effects that would disrupt the normal rates of lever responding. Additionally, in the alcohol “PR” experiment, treatment with Salvia miltiorrhiza extract resulted in a significant increase in the number of
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responses on the inactive lever (i.e., the water lever in the training sessions that was inactivated in the test sessions under the PR schedule of responding). Although this increase in lever responding on the inactive lever was unexpected and presently somewhat
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inexplicable, it strengthens the conclusion that the reducing effect of Salvia miltiorrhiza extract on lever responding for alcohol was not associated with any nonspecific, disrupting effect on
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the rat motor performance. Together, these data complement those collected in the previous alcohol-drinking studies conducted using the 2-bottle “alcohol vs. water” choice regimen, in which the reducing effect of alcohol intake produced by treatment with Salvia miltiorrhiza extracts was associated with a) a compensatory increase in water intake and b) no effect on
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food intake (Brunetti et al., 2003; Colombo et al., 1999; Colombo, Serra, et al., 2006; Vacca et al., 2003).
Previous studies found that treatment with different extracts of Salvia miltiorrhiza
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reduced blood alcohol levels (BALs) (Colombo et al., 1999; Vacca et al., 2003). Notably, this effect was evident when alcohol was administered intragastrically but not intraperitoneally,
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suggesting that Salvia miltiorrhiza extracts may be capable of altering alcohol absorption from the gastrointestinal system. However, this action had likely minimal, if any, impact on alcohol self-administration: as indicated by cumulative responding patterns, most of the reducing effect of Salvia miltiorrhiza extract occurred during the initial part of the sessions, suggesting that Salvia miltiorrhiza extract affected primarily the drive to lever-respond for alcohol. Salvia miltiorrhiza extracts contain several diterpenes, including tanshinone IIA, cryptotanshinone, and miltirone, that may constitute the active ingredients underlying the “anti-alcohol” effects elicited. Accordingly, a previous investigation found that a) the reducing 14
ACCEPTED MANUSCRIPT effect on alcohol intake of four different extracts of Salvia miltiorrhiza in sP rats was positively correlated to their content of miltirone and b) miltirone itself, when administered alone, reduced alcohol intake in sP rats (notably, the magnitude of this reduction was comparable to that produced by the active extracts of Salvia miltiorrhiza with a proportional content of
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miltirone) (Colombo, Serra, et al., 2006). Although not investigated in the present study, it is highly likely that miltirone was also responsible for the observed reducing effect of Salvia miltiorrhiza extract on operant alcohol self-administration. This premise may help to formulate
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a hypothesis on the mechanism of action underlying the observed reducing effect of Salvia miltiorrhiza extract on alcohol self-administration (as well as on other alcohol-related
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behaviors). Miltirone has indeed been found to exert anxiolytic effects in rodents, likely due to its capacity to activate the GABAA receptor (Lee et al., 1991). Several studies have demonstrated that sP rats display a unique “emotional” profile comprising high levels of anxiety-related behaviors (e.g., Colombo et al., 1995; Richter, Zorrilla, Basso, Koob, & Weiss,
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2000; Roman et al., 2012) and voluntary alcohol drinking markedly reduced these anxietyrelated behaviors (Colombo et al., 1995; Lobina, Gessa, & Colombo, 2013), suggesting that anxiolysis is likely one of the alcohol effects that drive sP rats to seek and consume alcohol.
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Accordingly, it may be proposed that the anxiolytic effect of miltirone might substitute for the anxiolytic effect of alcohol usually sought by the sP rats, contributing toward the reducing
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effects of miltirone and Salvia miltiorrhiza extracts on alcohol intake and, as in the present study, the reinforcing and motivational properties of alcohol. Additional studies are now needed to assess the possible contribution of other diterpenes to the “anti-alcohol” effects of Salvia miltiorrhiza extracts. In summary, the results of the present study demonstrate that treatment with a standardized extract of the Chinese medicinal plant, Salvia miltiorrhiza, selectively reduced operant, oral alcohol self-administration in selectively bred, alcohol-preferring sP rats, extending to the reinforcing and motivational properties of alcohol the capacity of Salvia 15
ACCEPTED MANUSCRIPT miltiorrhiza extracts to prevent different alcohol attributes. Clinical studies should now be designed to assess whether the “anti-alcohol” profile of Salvia miltiorrhiza extracts, as depicted by the several preclinical studies conducted to date, may generalize to humans affected by AUDs.
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Acknowledgments
The authors are grateful to Mrs. Carla Acciaro for animal breeding and care, and Ms.
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Anne Farmer for language editing of the manuscript.
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ACCEPTED MANUSCRIPT References Bell, R. L., Sable, H. J., Colombo, G., Hyytiä, P., Rodd, Z. A., & Lumeng, L. (2012). Animal models for medications development targeting alcohol abuse using selectively bred rat lines: neurobiological and pharmacological validity. Pharmacology, Biochemistry, and Behavior, 103, 119–155.
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Brunetti, G., Serra, S., Vacca, G., Lobina, C., Morazzoni, P., Bombardelli, E., et al. (2003). IDN 5082, a standardized extract of Salvia miltiorrhiza, delays acquisition of alcohol drinking behavior in rats. Journal of Ethnopharmacology, 85, 93–97. Carai, M. A., Agabio, R., Bombardelli, E., Bourov, I., Gessa, G. L., Lobina, C., et al. (2000). Potential use of medicinal plants in the treatment of alcoholism. Fitoterapia, 71 Supplement 1, S38–42.
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Colombo, G., Agabio, R., Lobina, C., Reali, R., Zocchi, A., Fadda, F., et al. (1995). Sardinian alcohol-preferring rats: A genetic animal model of anxiety. Physiology and Behavior, 57, 1181–1185.
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Colombo, G., Agabio, R., Lobina, C., Reali, R., Morazzoni, P., Bombardelli, E., et al. (1999). Salvia miltiorrhiza extract inhibits alcohol absorption, preference, and discrimination in sP rats. Alcohol, 18, 65–70. Colombo, G., Lobina, C., Carai, M. A., & Gessa, G. L. (2006a). Phenotypic characterization of genetically selected Sardinian alcohol-preferring (sP) and -non-preferring (sNP) rats. Addiction Biology, 11, 324–338.
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Colombo, G., Serra, S., Vacca, G., Orrù, A., Maccioni, P., Morazzoni, P., et al. (2006b). Identification of miltirone as active ingredient of Salvia miltiorrhiza responsible for the reducing effect of root extracts on alcohol intake in rats. Alcoholism: Clinical and Experimental Research, 30, 754–762. Heyman, G. M., Keung, W. M., & Vallee, B. L. (1996). Daidzin decreases ethanol consumption in rats. Alcoholism: Clinical and Experimental Research, 20, 1083–1087.
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Keung, W. M., Lazo, O., Kunze, L., & Vallee, B. L. (1995). Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism. Proceedings of the National Academy of Sciences of the United States of America, 92, 8990–8993.
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Keung, W. M., & Vallee, B. L. (1993). Daidzin and daidzein suppress free-choice ethanol intake by Syrian golden hamsters. Proceedings of the National Academy of Sciences of the United States in America, 90, 10008–10012. Lee, C. M., Wong, H. N., Chui, K. Y., Choang, T. F., Hon, P. M., & Chang, H. M. (1991). Miltirone, a central benzodiazepine receptor partial agonist from a Chinese medicinal herb Salvia miltiorrhiza. Neuroscience Letters, 127, 237–241. Li, T. K. (2010). From herbal roots to synthetic medicines: a historical perspective. Alcoholism: Clinical and Experimental Research, 34, 1–3. Lin, R. C., Guthrie, S., Xie, C. Y., Mai, K., Lee, D. Y., Lumeng, L., et al. (1996). Isoflavonoid compounds extracted from Pueraria lobata suppress alcohol preference in a pharmacogenetic rat model of alcoholism. Alcoholism: Clinical and Experimental Research, 20, 659–663. Liu, Q., Lawrence, A. J., & Liang, J. H. (2011). Traditional Chinese medicine for treatment of alcoholism: from ancient to modern. The American Journal of Chinese Medicine, 39, 1–13. 17
ACCEPTED MANUSCRIPT Lobina, C., Gessa, G. L., & Colombo, G. (2013). Anxiolytic effect of voluntarily consumed alcohol in Sardinian alcohol-preferring rats exposed to the social interaction test. Journal of Alcoholism and Drug Dependence, 1, 132. Lukas, S. E., Penetar, D., Su, Z., Geaghan, T., Maywalt, M., Tracy, M., et al. (2013). A standardized kudzu extract (NPI-031) reduces alcohol consumption in nontreatmentseeking male heavy drinkers. Psychopharmacology, 226, 65–73.
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Lukas, S. E., Penetar, D., Berko, J., Vicens, L., Palmer, C., Mallya, G., et al. (2005). An extract of the Chinese herbal root kudzu reduces alcohol drinking by heavy drinkers in a naturalistic setting. Alcoholism: Clinical and Experimental Research, 29, 756–762. Maccioni, P., Carai, M. A., Kaupmann, K., Guery, S., Froestl, W., Leite-Morris, K. A., et al. (2009). Reduction of alcohol’s reinforcing and motivational properties by the positive allosteric modulator of the GABA(B) receptor, BHF177, in alcohol-preferring rats. Alcoholism: Clinical and Experimental Research, 33, 1749–1756.
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Maccioni, P., Zaru, A., Loi, B., Lobina, C., Carai, M. A., Gessa, G. L., et al. (2012). Comparison of the effect of the GABAB receptor agonist, baclofen, and the positive allosteric modulator of the GABAB receptor, GS39783, on alcohol self-administration in 3 different lines of alcohol-preferring rats. Alcoholism: Clinical and Experimental Research, 36, 1748–1766. Markou, A., Weiss, F., Gold, L. H., Caine, S. B., Schulteis, G., & Koob, G. F. (1993). Animal models of drug craving. Psychopharmacology, 112, 163–182. Overstreet, D. H., Lee, Y. W., Rezvani, A. H., Pei, Y. H., Criswell, H. E., & Janowsky, D. S. (1996). Suppression of alcohol intake after administration of the Chinese herbal medicine, NPI-028, and its derivatives. Alcoholism: Clinical and Experimental Research, 20, 221–227.
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Penetar, D. M., Maclean, R. R., McNeil, J. F., & Lukas, S. E. (2011). Kudzu extract treatment does not increase the intoxicating effects of acute alcohol in human volunteers. Alcoholism: Clinical and Experimental Research, 35, 726–734.
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Penetar, D. M., Toto, L. H., Farmer, S. L., Lee, D. Y., Ma, Z., Liu, Y., et al. (2012). The isoflavone puerarin reduces alcohol intake in heavy drinkers: a pilot study. Drug and Alcohol Dependence, 126, 251–256.
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Rezvani, A. H., Overstreet, D. H., Perfumi, M., & Massi, M. (2003). Plant derivatives in the treatment of alcohol dependency. Pharmacology, Biochemistry, and Behavior, 75, 593–606. Richardson, N. R., & Roberts, D. C. (1996). Progressive ratio schedules in drug selfadministration studies in rats: a method to evaluate reinforcing efficacy. Journal of Neuroscience Methods, 66, 1–11. Richter, R. M., Zorrilla, E. P., Basso, A. M., Koob, G. F., & Weiss, F. (2000). Altered amygdalar CRF release and increased anxiety-like behavior in Sardinian alcoholpreferring rats: a microdialysis and behavioral study. Alcoholism: Clinical and Experimental Research, 24, 1765–1772. Roman, E., Stewart, R. B., Bertholomey, M. L., Jensen, M. L., Colombo, G., Hyytiä, P., et al. (2012). Behavioral profiling of multiple pairs of rats selectively bred for high and low alcohol intake using the MCSF test. Addiction Biology, 17, 33–46. Serra, S., Vacca, G., Tumatis, S., Carrucciu, A., Morazzoni, P., Bombardelli, E., et al. (2003). Anti-relapse properties of IDN 5082, a standardized extract of Salvia miltiorrhiza, in alcohol-preferring rats. Journal of Ethnopharmacology, 88, 249–252. 18
ACCEPTED MANUSCRIPT State Pharmacopoeia Commission of the People’s Republic of China (2000). Pharmacopoeia of the People’s Republic of China, English edition. (Beijing, China: Chemical Industry Press). Tang, W., & Eisenbrand, G. (1992). Chinese Drugs of Plant Origin – Chemistry, Pharmacology and Use in Traditional and Modern Medicine. (Berlin, Germany: Springer-Verlag).
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Tomczyk, M., Zovko-Končić, M., & Chrostek, L. (2012). Phytotherapy of alcoholism. Natural Product Communications, 7, 273–280. Vacca, G., Colombo, G., Brunetti, G., Melis, S., Molinari, D., Serra, S., et al. (2003). Reducing effect of Salvia miltiorrhiza extracts on alcohol intake: influence of vehicle. Phytotherapy Research, 17, 537– 541.
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Xi-wen, L., & Hedge, I. C. (1994). Lamiaceae (Labiatae). In Flora of China, W. Zheng-yi and P.H. Raven, eds. (St. Louis, MO: Science Press and Missouri Botanical Garden), pp. 195–222.
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Zhou, L., Zuo, Z., & Chow, M. S. (2005). Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. Journal of Clinical Pharmacology, 45, 1345–1359.
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ACCEPTED MANUSCRIPT Figure legends
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Figure 1. “FR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on number of responses for alcohol (panel A) or sucrose (panel B) and amount of self-administered alcohol (panel C) or sucrose (panel D) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (1% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions. When self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under the same FR schedule of reinforcement. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Each bar is the mean ± SEM of n = 12 rats. *p < 0.001 in comparison to vehicle-treated rats (Newman-Keuls test).
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Figure 2. “PR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on number of responses for alcohol (panel A) or sucrose (panel B) and breakpoint for alcohol (panel C) or sucrose (panel D) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (3% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; when self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under a progressive ratio (PR) schedule of reinforcement, in which the response requirement (RR) was increased progressively over a 60-min session. Breakpoint was defined as the lowest RR not achieved by the rat. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Each bar is the mean ± SEM of n = 12 rats. *p < 0.05, and **p < 0.01 in comparison to vehicle-treated rats (Newman-Keuls test).
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Figure 3. “FR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on cumulative response patterns of self-administration for alcohol (top panel) or sucrose (bottom panel) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (1% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; when self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under the same FR schedule of reinforcement. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Sessions were divided into 60 intervals of 30 s each. Each point is the mean ± SEM of n = 12 rats.
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Figure 4. “PR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on cumulative response patterns of self-administration for alcohol (top panel) or sucrose (bottom panel) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (3% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; once self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under a progressive ratio (PR) schedule of reinforcement, in which the response requirement was increased progressively over a 60-min session. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Sessions were divided into 120 intervals of 30 s each. Each point is the mean ± SEM of n = 12 rats.
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S0741-8329(14)00102-5
DOI:
10.1016/j.alcohol.2014.06.002
Reference:
ALC 6408
To appear in:
Alcohol
Please cite this article as: MaccioniP., VargioluD., FalchiM., MorazzoniP., RivaA., CabriW., CaraiM.A.M., GessaG.L. & ColomboG., Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats, Alcohol (2014), doi: 10.1016/ j.alcohol.2014.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats
Giancarlo Colomboa
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Paola Maccionia, Daniela Vargiolua, Maura Falchia, Paolo Morazzonib, Antonella Rivab, Walter Cabrib, Mauro A.M. Caraia, Gian Luigi Gessaa,
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Neuroscience Institute, National Research Council of Italy, Section of Cagliari, I-09042 Monserrato (CA), Italy
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Indena S.p.A., I-20139 Milan (MI), Italy
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Corresponding author: Giancarlo Colombo Neuroscience Institute National Research Council of Italy Section of Cagliari S.S. 554, km. 4,500 I-09042 Monserrato (CA), Italy Telephone: +39 070 675 4342 Fax: +39 070 675 4320 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract The dried roots of Salvia miltiorrhiza are highly valued in Chinese folk medicine for use in the prevention and treatment of a series of ailments. Previous studies have demonstrated that administration of standardized extracts of Salvia miltiorrhiza selectively reduced excessive
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alcohol drinking and relapse-like drinking in selectively bred Sardinian alcohol-preferring (sP) rats. The present study was designed to extend these findings on the “anti-alcohol” properties of Salvia miltiorrhiza extracts to operant procedures of oral alcohol self-administration. Two
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independent groups of sP rats were trained to lever-respond on an FR4 schedule of
reinforcement for alcohol (15%, v/v) or sucrose (1–3%, w/v) in daily 30-min sessions. Once
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responding had stabilized, rats were tested under the fixed ratio 4 (FR4) schedule of reinforcement (index of alcohol reinforcing properties) and the progressive ratio (PR) schedule of reinforcement (index of alcohol motivational properties). Treatment with Salvia miltiorrhiza extract (0, 50, 100, and 200 mg/kg, intragastrically [i.g.]) markedly reduced lever responding
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for alcohol, amount of self-administered alcohol, and breakpoint for alcohol (defined as the lowest response requirement not achieved in the PR experiment). No dose of Salvia miltiorrhiza extract altered any parameter of sucrose self-administration. These results a)
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demonstrate that treatment with Salvia miltiorrhiza extract selectively reduced the reinforcing and motivational properties of alcohol in sP rats and b) extend to operant procedures of
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alcohol self-administration previous data on the “anti-alcohol” effects of Salvia miltiorrhiza extracts. These data strengthen the notion that novel pharmacological approaches for treatment of alcohol use disorders may stem from natural substances. Keywords: Salvia miltiorrhiza extract; alcohol self-administration; fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement; reinforcing and motivational properties of alcohol; Sardinian alcohol-preferring (sP) rats
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ACCEPTED MANUSCRIPT Introduction In the search for new pharmacological remedies for alcohol use disorders (AUDs), particular attention has recently been focused on extracts or preparations derived from several medicinal plants (for review, see Carai et al., 2000; Li, 2010; Liu, Lawrence, & Liang,
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2011; Rezvani, Overstreet, Perfumi, & Massi, 2003; Tomczyk, Zovko-Končić, & Chrostek, 2012). The most striking example in this research field is likely represented by the discovery and development of extracts of and ingredients from the roots of Pueraria lobata (the kudzu
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plant). Initial preclinical studies unanimously demonstrated that administration of extracts from Pueraria lobata, or its active constituents (the isoflavones daidzin, daidzein, and
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puerarin), markedly reduced several alcohol-related behaviors – including alcohol drinking and operant, oral alcohol self-administration – in rats and hamsters (e.g., Heyman, Keung, & Vallee, 1996; Keung, Lazo, Kunze, & Vallee, 1995; Keung & Vallee, 1993; Lin et al., 1996; Overstreet et al., 1996). Subsequent clinical studies generalized to humans the “anti-alcohol”
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properties of Pueraria lobata extracts observed in rodents: treatment with a Pueraria lobata extract a) decreased the number of alcohol drinks in heavy drinkers exposed to a naturalistic laboratory setting (Lukas et al., 2005), and b) decreased the number of weekly alcohol
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drinks, decreased the number of heavy drinking days, increased the percentage of days of abstinence, and increased the number of consecutive days of abstinence in heavy drinkers
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recruited in a recent double-blind, placebo-controlled survey (Lukas et al., 2013). These effects occurred in the absence of any potentiation of the subjective or psychomotor effects of alcohol (Penetar, Maclean, McNeil, & Lukas, 2011). Finally, a recent double-blind, placebo-controlled survey demonstrated that treatment with the active constituent, puerarin, was likewise capable of reducing alcohol drinking in heavy drinkers, reproducing the effects of the whole extract (Penetar et al., 2012). In recent years, this laboratory has contributed to the characterization of the “antialcohol” profile of root extracts from Salvia miltiorrhiza Bunge (Labiatae), a medicinal plant the dried roots of which (known as Danshen) are officially listed in the Chinese 3
ACCEPTED MANUSCRIPT Pharmacopoeia and have been used for hundreds of years in Chinese folk medicine for the treatment of different pathologies, including cardiovascular disease, hematological abnormalities, hyperlipidemia, hepatitis, hemorrhage, menstrual disorders, miscarriage, edema, and insomnia (State Pharmacopoeia Commission PRC, 2000; Tang & Eisenbrand,
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1992; Zhou, Zuo, & Chow, 2005). Specifically, acute or repeated administration of several standardized extracts of Salvia miltiorrhiza have been found to reduce a) acquisition (Brunetti et al., 2003) and maintenance (Colombo et al., 1999; Colombo, Serra, et al., 2006;
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Vacca et al., 2003) of high alcohol drinking and b) relapse-like alcohol drinking (Serra et al., 2003) in selectively bred, Sardinian alcohol-preferring (sP) rats exposed to the standard,
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homecage 2-bottle “alcohol vs. water” choice regimen. Notably, an additional study likely identified the active constituent underlying the reducing effect of Salvia miltiorrhiza extracts on alcohol intake. The reducing effect of four different extracts of Salvia miltiorrhiza on alcohol intake in sP rats was positively and highly significantly correlated with their content of
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the diterpene, miltirone. In addition, pure miltirone – given at doses comparable to its content in the effective doses of the active extracts – reduced alcohol intake in sP rats (Colombo, Serra, et al., 2006).
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The present study was designed to extend the investigation of the “anti-alcohol” properties of Salvia miltiorrhiza extracts to operant procedures of oral alcohol self-
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administration. Under these procedures, alcohol is made available to rats via completion of a behavioral response (usually, responding on a lever a given number of times), to allow the reinforcing and motivational properties of alcohol to be assessed in addition to its mere consumption. The present study employed two different procedures of alcohol selfadministration: a) fixed ratio (FR) schedule of reinforcement, in which the response requirement (RR; i.e., the “cost” of each alcohol presentation in terms of number of responses on the lever) is predetermined and kept fixed throughout the session (providing a measure of alcohol intake and of the reinforcing properties of alcohol), and b) within-session 4
ACCEPTED MANUSCRIPT progressive ratio (PR) schedule of reinforcement, in which – over the same single session – RR is progressively increased after the delivery of each reinforcer, and the lowest ratio not completed (named breakpoint) is taken as the measure of motivational properties of alcohol (see Markou et al., 1993).
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The present study was conducted using male sP rats as animal model of AUDs. These rats have been selectively bred for high alcohol preference and consumption under the 2-bottle “alcohol vs. water” choice regimen (Bell et al., 2012; Colombo, Lobina, Carai, &
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Gessa, 2006). When exposed to sessions of operant, oral alcohol self-administration, sP rats display robust lever responding for alcohol, self-administer large amounts of alcohol, and
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achieve high levels of breakpoint for alcohol (see Colombo, Lobina, et al., 2006). Materials and methods
All experimental procedures employed in the present study were in accordance with the Italian law on the “Protection of animals used for experimental and other scientific
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reasons”.
Plant material and preparation of plant extract
Roots of Salvia miltiorrhiza were collected in 2005 in the eastern part of China and
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identified using the genus treatment described in Flora of China (Xi-wen & Hedge, 1994) and
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Chinese Pharmacopoeia (State Pharmacopoeia Commission PRC, 2000). A voucher specimen is kept at Indena S.p.A., Milan, Italy, under the registry no. 137127. Roots of Salvia miltiorrhiza were dried and subsequently extracted with acetone. After concentration under vacuum, the residue was re-dissolved in alcohol and the solution defatted with n-hexane. The aqueous layer was concentrated and extracted with ethyl acetate. The final extract (named IDN5655) was obtained after removal of ethyl acetate by alcohol and drying under vacuum at 50 °C for approx imately 48 h. The Salvia miltiorrhiza extract used in the present study contained 21% total tanshinones and 4.3% miltirone. Animals 5
ACCEPTED MANUSCRIPT Male sP rats from the 80th generation and 60 days old at the start of the study were utilized. Rats were alcohol-naïve before the start of the study. Rats were housed 4 per cage in standard plastic cages with wood chip bedding. The animal facility was under an inverted 12:12-hour light-dark cycle (lights on at 9:30 PM), at a constant temperature of 22 ± 2 °C and
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relative humidity of approximately 60%. Over the 2-week period preceding the start of the study, rats were extensively habituated to handling and intragastric infusions (by a metal gavage). Food pellets (Harlan, San Pietro al Natisone, Italy) and water were always available
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in the homecage, except as noted.
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Rats were divided into two groups: “alcohol” (n = 32) and “sucrose” (n = 32) groups. Apparatus
Self-administration sessions were conducted in modular chambers (Med Associates, St. Albans, VT, USA) located in sound-attenuated cubicles, with fans for ventilation and background white noise. The front panel of each chamber was equipped with a) two
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retractable response levers, b) one dual-cup liquid receptacle positioned between the two levers, and c) two stimulus lights (one green and one white) mounted above each lever. The liquid receptacle was connected by polyethylene tubes to two syringe pumps located outside
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the chamber. A white house light was centered at the top of the back wall of each chamber.
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For half of the rats, the right lever was associated with alcohol (or sucrose), and achievement of RR a) activated the alcohol (or sucrose) pump, resulting in the delivery of 0.1 mL alcohol (or sucrose) solution, and b) switched on the green light for the 2-s period of alcohol (or sucrose) delivery. For these rats, the left lever was associated with water, and achievement of RR a) activated the water pump, resulting in the delivery of 0.1 mL water, and b) switched on the white light for the 2-s period of water delivery. The study design was counterbalanced so that the opposite condition was applied to the other half of the rats (left lever: alcohol [or sucrose]; right lever: water). Experimental procedure 6
ACCEPTED MANUSCRIPT 2-bottle choice phase Rats of the “alcohol” group were initially exposed to the homecage 2-bottle “alcohol vs. water” choice regimen with unlimited access for 24 hours/day over 10 consecutive days. The alcohol solution was presented at a concentration of 10% (v/v). This initial phase was a) part
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of the conventional procedure of alcohol self-administration employed in this laboratory with sP rats (e.g., Maccioni et al., 2012) and b) conducted to allow the rats to become accustomed to the taste of alcohol and start to experience its pharmacological effects in order to possibly
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shorten the subsequent auto-shaping phase once rats are introduced into the operant chambers. During this phase, daily alcohol intake averaged approximately 6.5 g/kg.
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Shaping, training, and maintenance phases
Immediately after the 2-bottle choice regimen, rats of the “alcohol” group were introduced into the operant chambers and trained to lever-respond for alcohol. Selfadministration sessions lasted 30 min (with the sole exception of the very first session that
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lasted 120 min) and were conducted 5 days per week (Monday to Friday) during the first 4 to 6 h of the dark phase of the daily light/dark cycle. Rats were water-deprived during the 12 h prior to the first session in the operant chamber. Rats were initially exposed to an FR1
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schedule of reinforcement for 10% alcohol (v/v) for 4 consecutive daily sessions. FR was then increased to FR2 and FR4 over 4 consecutive sessions. In sessions 9 and 10, the alcohol
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solution was presented at the final concentration of 15% (v/v). Rats were then exposed to 4 consecutive sessions during which the water lever alone or the alcohol lever alone was available every other day; water and alcohol were available on FR1 and FR4, respectively. From then on, both levers were concomitantly available (maintenance phase) for a total of 20 sessions conducted with FR4 and FR1 on the alcohol and water lever, respectively. During these 20 sessions, “alcohol” rats displayed an average number of lever responses for alcohol of approximately 180 per session; the resulting mean amount of self-administered alcohol was approximately 0.90 g/kg per session. Conversely, a negligible number of lever responses 7
ACCEPTED MANUSCRIPT for water (averaging ≤ 5 per session) were recorded in each session. After the 20 selfadministration sessions of the maintenance phase, rats were selected for inclusion in the two experiments (“FR” and “PR”, see below). Specifically, the 24 rats displaying the most stable responding behavior over the last 5 daily sessions (less than 20% daily difference) were
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selected and divided into two groups (n = 12 in the “FR” experiment; n = 12 in the “PR” experiment), matched for total number of lever responses on the alcohol lever and amount of self-administered alcohol.
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Rats of the “sucrose” group were trained to lever-respond for a sucrose solution. Selfadministration sessions lasted 30 min and were conducted 5 days per week (Monday to
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Friday) during the first 4 to 6 h of the dark phase of the daily light/dark cycle. Lever responses were initially shaped under FR1 and 3% (w/v) sucrose for 2 consecutive sessions. On the subsequent 4 sessions, FR was increased to FR2 and FR4. Rats were then randomly divided into two subgroups (n = 16 in the “FR” experiment; n = 16 in the “PR” experiment) for
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subsequent inclusion in the two experiments. Specifically, in one rat group (“FR” experiment), the sucrose concentration was reduced to 1% (w/v) over two consecutive sessions; in the other rat group (“PR” experiment), the sucrose concentration was maintained at 3% (w/v).
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These two sucrose concentrations were selected on the basis of previous results (e.g., Maccioni et al., 2009) in order to obtain, in the test sessions with both FR and PR schedules
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of reinforcement, lever-responding behaviors comparable to those usually performed by sP rats to obtain 15% alcohol. Rats were then exposed to 4 consecutive sessions during which either the water lever or the sucrose lever alone was available every other day; water and sucrose were available on FR1 and FR4, respectively. From then onward, both levers were concomitantly available (maintenance phase); a total of 20 sessions was conducted with FR4 and FR1 on the sucrose and water lever, respectively. During these 20 sessions, “sucrose” rats exposed to 1% and 3% sucrose displayed an average number of lever responses for sucrose of approximately 210 and 850, respectively, per session; the resulting mean amount 8
ACCEPTED MANUSCRIPT of self-administered sucrose solution was approximately 7.0 and 35.0 mL/kg, respectively, per session. Conversely, a negligible number of lever responses for water (averaging < 5 per session) were recorded in each session. After the 20 self-administration sessions of the maintenance phase, rats of both subgroups (“FR” and “PR”) were selected for inclusion in the
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two experiments; specifically, the 12 rats of the “FR” experiment and the 12 rats of the “PR” experiment displaying the most stable responding behavior over the last 5 daily sessions (less than 25% daily difference) were selected.
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Test sessions
During the test sessions of the “FR” experiments, RR on the alcohol (or sucrose) and
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water lever was maintained at FR4 and FR1, respectively. Test sessions of the “FR” experiments lasted 30 min. During the test sessions of the “PR” experiments, RR on each lever was increased progressively over the session according to the procedure described by Richardson & Roberts (1996); namely, RR was increased as follows: 4, 9, 12, 15, 20, 25, 32,
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40, 50, 62, 77, 95, 118, 145, 178, 219, etc. During the test sessions of the “PR” experiments, the water lever was inactive (lever responses were recorded, but had no programmed consequences). Test sessions of the “PR” experiments lasted 60 min; longer times were not
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required as previous data (e.g., Maccioni et al., 2009, 2012) indicated that lever responding
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for alcohol in sP rats exposed to the PR schedule of reinforcement used in the present study reached its plateau within 10–15 min. In each experiment (“FR” and “PR”), test sessions were conducted on Fridays and all doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Four consecutive (Monday to Thursday) self-administration sessions (with FR4 on the alcohol or sucrose lever and FR1 on the water lever and no drug treatment) elapsed between test sessions. After each test session, alcohol (or sucrose) selfadministration rapidly recovered to baseline levels. Salvia miltiorrhiza extract was dissolved in a 1:1 mixture of polysorbate 80 (Tween 80) plus polyethylene glycol 600 (PEG 600). The solution was kept under continuous agitation, at 9
ACCEPTED MANUSCRIPT room temperature and in a dark container, for at least 12 consecutive hours before administration. Salvia miltiorrhiza extract was administered intragastrically (infusion volume: 2 mL/kg), at the doses of 0, 50, 100, and 200 mg/kg, 60 min before the start of the selfadministration session. Dose range of Salvia miltiorrhiza extract, treatment time, and route of
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administration were chosen based on the results of previous studies testing the effects of Salvia miltiorrhiza extracts on different alcohol-related behaviors in sP rats (Brunetti et al., 2003; Colombo et al., 1999; Colombo, Serra, et al., 2006; Serra et al., 2003; Vacca et al.,
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2003). Measured variables and data analysis
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In “FR” experiments, measured variables were as follows: a) number of responses on each lever (alcohol [or sucrose] and water levers); b) amount of self-administered alcohol (or sucrose) and water (expressed in g/kg pure alcohol, mL/kg sucrose solution, and mL/kg water, respectively, and determined from the number of earned reinforcers).
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In “PR” experiments, measured variables were as follows: a) number of responses on each lever (alcohol [or sucrose] and inactive levers); b) breakpoint for alcohol (or sucrose), defined as the lowest RR not achieved by the rat on the alcohol (or sucrose) lever.
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Data on the effect of Salvia miltiorrhiza extract on each variable were statistically
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analyzed by 1-way ANOVA for repeated measures, followed by the Newman-Keuls test for post hoc comparisons. Comparisons between number of lever responses and breakpoint values for alcohol and sucrose in vehicle-treated rat groups were performed using the Student t test. Results “FR” experiment Treatment with Salvia miltiorrhiza extract significantly reduced the number of lever responses for alcohol [F(3,33) = 9.66, p < 0.0001] (Fig. 1, panel A). A post hoc test revealed that all three doses of Salvia miltiorrhiza extract decreased – relative to values of the vehicle10
ACCEPTED MANUSCRIPT treated rat group – the number of lever responses for alcohol (p < 0.001, Newman-Keuls test). The magnitude of the reducing effect on the number of lever responses for alcohol was similar among the three doses of Salvia miltiorrhiza extract, averaging 50–60% in comparison to vehicle-treated rats. Reduction in number of lever responses for alcohol
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resulted in a proportional decrease in the amount of self-administered alcohol [F(3,33) = 9.98, p < 0.0001] (Fig. 1, panel C). Lever responding for water was negligible (averaging ≤ 4 per session in all rat subgroups) and not altered by treatment with Salvia
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miltiorrhiza extract [F(3,33) = 0.97, p > 0.05] (data not shown).
In the vehicle-treated rats of the “sucrose” group, number of lever responses for
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sucrose was similar to those observed in the vehicle-treated, “alcohol” rat group (p > 0.05, Student t test). Treatment with Salvia miltiorrhiza extract failed to alter a) number of lever responses for sucrose [F(3,33) = 0.32, p > 0.05] (Fig. 1, panel B), and b) amount of selfadministered sucrose [F(3,33) = 0.27, p > 0.05] (Fig. 1, panel D). Lever responding for water
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was negligible (averaging ≤ 3 per session in all rat subgroups) and not altered by treatment with Salvia miltiorrhiza extract [F(3,33) = 0.13, p > 0.05] (data not shown). “PR” experiment
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Treatment with Salvia miltiorrhiza extract resulted in a significant reduction of the
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number of lever responses for alcohol [F(3,33) = 4.01, p < 0.05] (Fig. 2, panel A) and breakpoint for alcohol [F(3,33) = 4.48, p < 0.01] (Fig. 2, panel C). A post hoc analysis revealed that a) the number of lever responses for alcohol was significantly reduced by treatment with only 200 mg/kg Salvia miltiorrhiza extract (p < 0.01), and b) breakpoint for alcohol was significantly reduced by treatment with both 100 (p < 0.05) and 200 mg/kg (p < 0.01) Salvia miltiorrhiza extract. The reducing effect of Salvia miltiorrhiza on both variables tended to be dose-related and averaged approximately 50%, in comparison to vehicle-treated rats, in the rat group treated with 200 mg/kg Salvia miltiorrhiza extract. Responding on the inactive lever was significantly increased by treatment with Salvia 11
ACCEPTED MANUSCRIPT miltiorrhiza extract [F(3,33) = 4.33, p < 0.05]; it averaged 5.3 ± 1.4, 6.8 ± 2.4, 25.7 ± 7.8, and 24.7 ± 6.2 in the rat groups treated with 0, 50, 100, and 200 mg/kg Salvia miltiorrhiza extract, respectively, with significant differences at the post hoc test between the vehicle-treated rat group and the two rat groups treated with 100 (p < 0.05) and 200 mg/kg (p < 0.05) Salvia
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miltiorrhiza extract. In the vehicle-treated rats of the “sucrose” group, the number of lever responses for sucrose and value of breakpoint for sucrose were similar to those observed in the vehicle-
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treated, “alcohol” rat group (p > 0.05, Student t test). Treatment with Salvia miltiorrhiza extract altered neither the number of lever responses for sucrose [F(3,33) = 0.35, p > 0.05]
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(Fig. 2, panel B) nor breakpoint for sucrose [F(3,33) = 0.26, p > 0.05] (Fig. 2, panel D). Responding on the inactive lever was negligible (averaging ≤ 7 per session in all rat subgroups) and not altered by treatment with Salvia miltiorrhiza extract [F(3,33) = 0.25, p > 0.05] (data not shown).
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Discussion
As expected, sP rats displayed robust lever-responding behaviors for alcohol under both experimental procedures: when exposed to the FR4 schedule of reinforcement, sP rats
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responded on the alcohol lever an average of 165 times and consumed an average of
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0.80 g/kg alcohol in 30-min sessions (for example, see the values collected in the vehicletreated rat group in the “FR” experiment). When exposed to the PR schedule of reinforcement, sP rats achieved a breakpoint value of approximately 50 (see values collected in the vehicle-treated rat group in the “PR” experiment). These data a) are highly similar to those collected in several previous alcohol self-administration studies using sP rats (e.g., Maccioni et al., 2009, 2012), b) suggest that alcohol has strong reinforcing and motivational properties in sP rats, and c) confirm the validity of sP rats as an animal model for investigations aimed at identifying new pharmacotherapies for AUDs (see Bell et al., 2012). 12
ACCEPTED MANUSCRIPT The results of the present study demonstrate that treatment with a standardized extract of the Chinese medicinal plant, Salvia miltiorrhiza, effectively reduced oral alcohol self-administration in alcohol-preferring sP rats. The effect of Salvia miltiorrhiza extract was evident under two different operant procedures, known to provide a measure of the strength
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of the reinforcing (FR schedule of reinforcement) and motivational (PR schedule of reinforcement) properties of alcohol (see Markou et al., 1993). These data a) extend to operant alcohol self-administration the capacity of different Salvia miltiorrhiza extracts to
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decrease several alcohol-related behaviors, including acquisition and maintenance of high alcohol drinking and relapse-like drinking, in sP rats (Brunetti et al., 2003; Colombo et al.,
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1999; Colombo, Serra, et al., 2006; Serra et al., 2003; Vacca et al., 2003), and b) provide additional elements to the preclinical “anti-alcohol” profile of Salvia miltiorrhiza extracts. Additional information on the reducing effect of Salvia miltiorrhiza extract on alcohol reinforcing and motivational properties may be provided by the analysis of cumulative
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response patterns. Data from both “FR” (Fig. 3) and “PR” (Fig. 4) experiments suggest that, in comparison to treatment with vehicle, treatment with all three doses of Salvia miltiorrhiza extract resulted in a) less steep curves (suggestive of a reduced frequency in lever-pressing),
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particularly at the beginning of the session, and b) lower plateau values (suggesting that fewer ratios were completed before responding ended). Together, these data suggest that
alcohol.
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treatment with Salvia miltiorrhiza extract weakened the drive of rats to access and consume
The reducing effect of Salvia miltiorrhiza extract on lever responding was selective for alcohol, as treatment with the same dose range of Salvia miltiorrhiza extract failed to affect – in both “FR” and “PR” experiments – lever responding for an alternative, non-drug reinforcer such as a sucrose solution (the concentrations of which were selected in order to produce reinforcing and motivational properties highly comparable to those produced by the alcohol solution). Moreover, the results of the two “sucrose” experiments tend to exclude that 13
ACCEPTED MANUSCRIPT reduction of alcohol self-administration induced by treatment with Salvia miltiorrhiza extract was secondary to possible malaise or motor-incapacitating and sedative effects that would disrupt the normal rates of lever responding. Additionally, in the alcohol “PR” experiment, treatment with Salvia miltiorrhiza extract resulted in a significant increase in the number of
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responses on the inactive lever (i.e., the water lever in the training sessions that was inactivated in the test sessions under the PR schedule of responding). Although this increase in lever responding on the inactive lever was unexpected and presently somewhat
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inexplicable, it strengthens the conclusion that the reducing effect of Salvia miltiorrhiza extract on lever responding for alcohol was not associated with any nonspecific, disrupting effect on
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the rat motor performance. Together, these data complement those collected in the previous alcohol-drinking studies conducted using the 2-bottle “alcohol vs. water” choice regimen, in which the reducing effect of alcohol intake produced by treatment with Salvia miltiorrhiza extracts was associated with a) a compensatory increase in water intake and b) no effect on
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food intake (Brunetti et al., 2003; Colombo et al., 1999; Colombo, Serra, et al., 2006; Vacca et al., 2003).
Previous studies found that treatment with different extracts of Salvia miltiorrhiza
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reduced blood alcohol levels (BALs) (Colombo et al., 1999; Vacca et al., 2003). Notably, this effect was evident when alcohol was administered intragastrically but not intraperitoneally,
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suggesting that Salvia miltiorrhiza extracts may be capable of altering alcohol absorption from the gastrointestinal system. However, this action had likely minimal, if any, impact on alcohol self-administration: as indicated by cumulative responding patterns, most of the reducing effect of Salvia miltiorrhiza extract occurred during the initial part of the sessions, suggesting that Salvia miltiorrhiza extract affected primarily the drive to lever-respond for alcohol. Salvia miltiorrhiza extracts contain several diterpenes, including tanshinone IIA, cryptotanshinone, and miltirone, that may constitute the active ingredients underlying the “anti-alcohol” effects elicited. Accordingly, a previous investigation found that a) the reducing 14
ACCEPTED MANUSCRIPT effect on alcohol intake of four different extracts of Salvia miltiorrhiza in sP rats was positively correlated to their content of miltirone and b) miltirone itself, when administered alone, reduced alcohol intake in sP rats (notably, the magnitude of this reduction was comparable to that produced by the active extracts of Salvia miltiorrhiza with a proportional content of
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miltirone) (Colombo, Serra, et al., 2006). Although not investigated in the present study, it is highly likely that miltirone was also responsible for the observed reducing effect of Salvia miltiorrhiza extract on operant alcohol self-administration. This premise may help to formulate
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a hypothesis on the mechanism of action underlying the observed reducing effect of Salvia miltiorrhiza extract on alcohol self-administration (as well as on other alcohol-related
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behaviors). Miltirone has indeed been found to exert anxiolytic effects in rodents, likely due to its capacity to activate the GABAA receptor (Lee et al., 1991). Several studies have demonstrated that sP rats display a unique “emotional” profile comprising high levels of anxiety-related behaviors (e.g., Colombo et al., 1995; Richter, Zorrilla, Basso, Koob, & Weiss,
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2000; Roman et al., 2012) and voluntary alcohol drinking markedly reduced these anxietyrelated behaviors (Colombo et al., 1995; Lobina, Gessa, & Colombo, 2013), suggesting that anxiolysis is likely one of the alcohol effects that drive sP rats to seek and consume alcohol.
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Accordingly, it may be proposed that the anxiolytic effect of miltirone might substitute for the anxiolytic effect of alcohol usually sought by the sP rats, contributing toward the reducing
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effects of miltirone and Salvia miltiorrhiza extracts on alcohol intake and, as in the present study, the reinforcing and motivational properties of alcohol. Additional studies are now needed to assess the possible contribution of other diterpenes to the “anti-alcohol” effects of Salvia miltiorrhiza extracts. In summary, the results of the present study demonstrate that treatment with a standardized extract of the Chinese medicinal plant, Salvia miltiorrhiza, selectively reduced operant, oral alcohol self-administration in selectively bred, alcohol-preferring sP rats, extending to the reinforcing and motivational properties of alcohol the capacity of Salvia 15
ACCEPTED MANUSCRIPT miltiorrhiza extracts to prevent different alcohol attributes. Clinical studies should now be designed to assess whether the “anti-alcohol” profile of Salvia miltiorrhiza extracts, as depicted by the several preclinical studies conducted to date, may generalize to humans affected by AUDs.
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Acknowledgments
The authors are grateful to Mrs. Carla Acciaro for animal breeding and care, and Ms.
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Anne Farmer for language editing of the manuscript.
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ACCEPTED MANUSCRIPT References Bell, R. L., Sable, H. J., Colombo, G., Hyytiä, P., Rodd, Z. A., & Lumeng, L. (2012). Animal models for medications development targeting alcohol abuse using selectively bred rat lines: neurobiological and pharmacological validity. Pharmacology, Biochemistry, and Behavior, 103, 119–155.
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ACCEPTED MANUSCRIPT Lobina, C., Gessa, G. L., & Colombo, G. (2013). Anxiolytic effect of voluntarily consumed alcohol in Sardinian alcohol-preferring rats exposed to the social interaction test. Journal of Alcoholism and Drug Dependence, 1, 132. Lukas, S. E., Penetar, D., Su, Z., Geaghan, T., Maywalt, M., Tracy, M., et al. (2013). A standardized kudzu extract (NPI-031) reduces alcohol consumption in nontreatmentseeking male heavy drinkers. Psychopharmacology, 226, 65–73.
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Maccioni, P., Zaru, A., Loi, B., Lobina, C., Carai, M. A., Gessa, G. L., et al. (2012). Comparison of the effect of the GABAB receptor agonist, baclofen, and the positive allosteric modulator of the GABAB receptor, GS39783, on alcohol self-administration in 3 different lines of alcohol-preferring rats. Alcoholism: Clinical and Experimental Research, 36, 1748–1766. Markou, A., Weiss, F., Gold, L. H., Caine, S. B., Schulteis, G., & Koob, G. F. (1993). Animal models of drug craving. Psychopharmacology, 112, 163–182. Overstreet, D. H., Lee, Y. W., Rezvani, A. H., Pei, Y. H., Criswell, H. E., & Janowsky, D. S. (1996). Suppression of alcohol intake after administration of the Chinese herbal medicine, NPI-028, and its derivatives. Alcoholism: Clinical and Experimental Research, 20, 221–227.
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Penetar, D. M., Maclean, R. R., McNeil, J. F., & Lukas, S. E. (2011). Kudzu extract treatment does not increase the intoxicating effects of acute alcohol in human volunteers. Alcoholism: Clinical and Experimental Research, 35, 726–734.
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ACCEPTED MANUSCRIPT Figure legends
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Figure 1. “FR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on number of responses for alcohol (panel A) or sucrose (panel B) and amount of self-administered alcohol (panel C) or sucrose (panel D) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (1% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions. When self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under the same FR schedule of reinforcement. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Each bar is the mean ± SEM of n = 12 rats. *p < 0.001 in comparison to vehicle-treated rats (Newman-Keuls test).
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Figure 2. “PR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on number of responses for alcohol (panel A) or sucrose (panel B) and breakpoint for alcohol (panel C) or sucrose (panel D) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (3% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; when self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under a progressive ratio (PR) schedule of reinforcement, in which the response requirement (RR) was increased progressively over a 60-min session. Breakpoint was defined as the lowest RR not achieved by the rat. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Each bar is the mean ± SEM of n = 12 rats. *p < 0.05, and **p < 0.01 in comparison to vehicle-treated rats (Newman-Keuls test).
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Figure 3. “FR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on cumulative response patterns of self-administration for alcohol (top panel) or sucrose (bottom panel) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (1% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; when self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under the same FR schedule of reinforcement. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Sessions were divided into 60 intervals of 30 s each. Each point is the mean ± SEM of n = 12 rats.
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Figure 4. “PR” experiment – Effect of intragastric administration of a standardized extract of roots from Salvia miltiorrhiza on cumulative response patterns of self-administration for alcohol (top panel) or sucrose (bottom panel) in selectively bred, Sardinian alcohol-preferring rats. Rats were initially trained to lever-respond for oral alcohol (15% v/v, in water) or sucrose (3% w/v, in water) [fixed ratio (FR) 4 (FR4)] and water (FR1) in daily 30-min sessions; once self-administration behavior had stabilized, rats were tested with Salvia miltiorrhiza extract under a progressive ratio (PR) schedule of reinforcement, in which the response requirement was increased progressively over a 60-min session. All doses of Salvia miltiorrhiza extract were tested in each rat under a Latin-square design. Sessions were divided into 120 intervals of 30 s each. Each point is the mean ± SEM of n = 12 rats.
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Salvia miltiorrhiza (mg/kg)
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Amount of self-administered sucrose (ml/kg)
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B
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150
Number of responses for sucrose
200
300
M AN U
250
0
Amount of self-administered alcohol (g/kg)
A
AC C
Number of responses for alcohol
300
0
50
100
200
12
D
10 8 6 4 2 0
0
50
100
200
Salvia miltiorrhiza (mg/kg)
ACCEPTED MANUSCRIPT
150
50 0
0
50
100
50
40
EP
30 20 10 0
TE D
C
AC C
Breakpoint for alcohol
60
0
50
100
200 150 100 50
0
200
200
Salvia miltiorrhiza (mg/kg)
0
50
100
200
60
Breakpoint for sucrose
100
250
RI PT
200
B
SC
250
Number of responses for sucrose
A
300
M AN U
Number of responses for alcohol
300
D
50 40 30 20 10 0
0
50
100
200
Salvia miltiorrhiza (mg/kg)
300 250
RI PT
200 150
SC
100 50 0 3
6
9
12
15
18
21
24
27
30
TE D
300 250
EP
200 150
AC C
Cumulative response pattern of self-administration for sucrose
0
M AN U
Cumulative response pattern of self-administration for alcohol
ACCEPTED MANUSCRIPT
100 50
0 mg/kg Salvia miltiorrhiza 50 mg/kg Salvia miltiorrhiza 100 mg/kg Salvia miltiorrhiza 200 mg/kg Salvia miltiorrhiza
0 0
3
6
9
12
15
18
21
24
Min of self administration session
27
30
300 250
RI PT
200 150
SC
100 50 0 3
6
9
12
15
18
21
24
27
30 60
TE D
300 250
EP
200 150
AC C
Cumulative response pattern of self-administration for sucrose
0
M AN U
Cumulative response pattern of self-administration for alcohol
ACCEPTED MANUSCRIPT
100 50
0 mg/kg Salvia miltiorrhiza 50 mg/kg Salvia miltiorrhiza 100 mg/kg Salvia miltiorrhiza 200 mg/kg Salvia miltiorrhiza
0 0
3
6
9
12
15
18
21
24
Min of self-administration session
27
30 60