Composition comprising Raphanus, Theobroma and Passiflora for treating opioid and alcohol abuse

Abstract

The present invention relates to a composition comprising a) an extract of a plant belonging to the genus Raphanus, wherein said extract is obtainable or obtained by extracting at least the air roots, seeds and/or bulbs with a hydrophilic, medium-polar and/or lipophilic solvent; b) an extract of a plant belonging to the genus Theobroma, wherein said extract is obtainable or obtained by extracting at least the fruit with a hydrophilic and/or medium-polar solvent, and c) an extract of a plant belonging to the genus Passiflora, wherein said extract is obtainable or obtained by extracting at least the flower with a hydrophilic, medium-polar and/or lipophilic solvent, its use in treating opioid abuse, opioid dependency, alcohol dependency and/or alcohol abuse and/or for use in treating the symptoms of opioid and/or alcohol withdrawal, and a method for its production.

Claims

1. A pharmaceutical composition for treating opioid and alcohol abuse, and/or symptoms of opioid and alcohol withdrawal comprising: (a) 2.5 parts of an extract of Raphanus sativus, (b) 3 parts of an extract of Theobroma cacao, (c) 2.5 parts of an extract of Passiflora incarnata, and (d) 1.5 parts of an extract of Crocus sativus.

2. The pharmaceutical composition of claim 1 further comprising a carrier.

3. The pharmaceutical composition of claim 2, wherein the carrier is olive oil.

4. The pharmaceutical composition of claim 3, wherein olive oil is present in the composition at a ratio of about 1 part of olive and about 4 parts of said extracts in mass.

5. The pharmaceutical composition of claim 1, wherein said extract of (a) is obtainable or obtained by extracting at least the air roots, seeds and/or bulbs with a hydrophilic, medium-polar and/or lipophilic solvent; and said extract of (b) is obtainable or obtained by extracting at least the fruit with a hydrophilic and/or medium-polar solvent; said extract of (c) is obtainable or obtained by extracting at least the flower with a hydrophilic, medium-polar and/or lipophilic solvent; and said extract of (d) is obtainable or obtained by extracting at least the flower with a hydrophilic, medium-polar and/or lipophilic solvent.

6. The pharmaceutical composition of claim 5, wherein said solvent of (a) is; and/or wherein said solvent of (b) is a water/alcohol mixture.

7. The pharmaceutical composition of claim 1, further comprising: (e) black cumin seed oil.

8. A method for treating opioid abuse or dependency, and/or alcohol abuse or dependency, and/or symptoms of opioid and alcohol withdrawal in a subject in need, comprising administering to the subject a pharmaceutical composition of claim 1.

9. The method of claim 8, wherein the pharmaceutical composition is administered to the subject at a dose of about 18.5 to about 77.5 mg/kg per day.

10. The method of claim 9, wherein said dose is administered for about 7 days to about 5 weeks.

11. The method of claim 10, wherein said dose is administered for at least two weeks.

12. The method of claim 8, wherein the symptoms of alcohol withdrawal include agitation, alcoholic hallucinosis, anorexia, anxiety, panic attacks, catatonia, confusion, delirium tremens, depersonalization, depression, derealization, diaphoresis, diarrhea, euphoria, fear, gastrointestinal upset, headache, hypertension, hyperthermia, insomnia, irritability, migraines, nausea and vomiting, palpitations, psychosis, rebound REM sleep, restlessness, seizures, sweating, tachycardia, tremors, and/or weakness.

13. The method of claim 8, wherein the symptoms of opioid withdrawal include tremors, cramps, muscle and bone pain, chills, perspiration, priapism, tachycardia, itch, restless legs syndrome, flu-like symptoms, rhinitis, yawning, sneezing, vomiting, diarrhea, weakness and/or akathisia; whereas psychological symptoms can include dysphoria, malaise, cravings, anxiety, panic attacks, paranoia, insomnia, dizziness, nausea, and/or depression.

14. The method of claim 8, wherein the pharmaceutical composition is administered orally.

Description

(1) The figures show:

(2) FIG. 1:

(3) Dose—response effect of orally administered extract composition on the intraperitoneal administration of morphine or heroine to re-establish motor behavior in rats. Each data point represents the mean (±SD) of five rats. * Significant differences between baseline and each treatment p<0.001; † significant difference between that dose to its lesser dose (p<0.001).

(4) FIG. 2:

(5) % Dependence reduction to morphine or heroin following oral administration of extract composition. Each data point represents the mean (±SD) of five rats. * Significant differences between baseline and each treatment p<0.001; † significant difference between that dose to its lesser dose (p<0.001).

(6) FIG. 3:

(7) HIC score for mice exposed for 72 h of ethanol. Mice were treated at 1 and 4 h with different doses of the extract or vehicle and the HIC scores were recorded overtime for 64 h. HIC scores were significantly less (p<0.001) in 40 or 60 mg/kg extract treated groups for the first 12 h.

(8) FIG. 4:

(9) Brief spindle episodes (BSE) activities were recorded in mice following chronic exposure to ethanol. Mice treated with 40 and 60 mg/kg of the extract composition have significantly less BSE from 2 to 72 h following ethanol withdrawal than vehicle treated mice. The BSE activities for mice treated with 40 or 60 mg/kg of the extract composition were significantly less (p<0.001) than vehicle treated mice.

(10) The examples illustrate the invention:

EXAMPLE 1: SELF-ADMINISTRATION MODEL

(11) 1.1 Material and Methods

(12) 1.1.1 Extract Composition

(13) The extract composition used in example 1 and all other examples was made up as follows: 2.5 parts of a Raphanus sativus extract, said extract being obtained by extraction as follows: Adding 500 ml of 30% methanol/water mixture to 90 g dried powdered radish in a glass beaker Heating solution to 40 to 50° C. for 30 min Filtering solution and evaporating filtrate under vacuum to dryness Freeze-drying the dried residue for 24 hrs (the resulting native extract quantity was 36.3 g dried extract) Adding 40% excipients (37 parts maltodextrin and 3 parts silica) for standardization purposes so that the extract is standardized to contain at least 1.5% total flavonoids 3 parts of a Theobroma cocoa extract, said extract being obtained by extraction as follows: Adding 1 Liter purified water to 200 g of dried powdered cacao fruits in a glass beaker Heating solution between 70-80° C. for 45 min. Filtering solution and evaporating the filtrate to a volume of 100 mL Freezing and freeze drying for 24 hours. Adding excipients to standardize the extract to contain a min. concentration of procyanidins. 2.5 parts of a Passiflora incarnata extract, said extract being obtained by extraction as follows: Adding 750 mL methanol to 175 g of dried powdered passion flowers in a glass beaker Heating solution between 40-50° C. for 30 min. Filtering solution and evaporating to dryness. Freeze drying the residue for 24 hours. Adding excipients to standardize the extract to contain a min. concentration of flavonoids. 1.5 parts of a Crocus sativus extract, said extract being obtained by extraction as follows: Adding 300 mL ethanol to 10 g of dried powdered saffron flowers in a glass beaker Heating solution between 30-45° C. for 30 min. Filtering solution and evaporating to dryness. Freeze drying the residue for 24 hours. Adding excipients to standardize the extract to contain a min. concentration of safranal. The dried extracts were mixed in a mixture of water and olive oil.
1.1.2 The Intravenous Self-Administration Apparatus

(14) Responses on either of two levers (mounted 15 cm apart on the front wall of each operant test cage) were recorded on an IBM compatible computer with a Med Associates interface. The intravenous self-administration system consisted of polyethylene silicone cannulas con-structed according to the design of Weeks (1972) (Weeks J R. Long-term intravenous infusion. In: Myers R D, editor. Methods in Psychobiology. Vol. 2. Academic Press; New York: 1972. pp. 155-168), Instech harnesses and swivels, and Harvard Apparatus infusion pumps. Shaping of the bar-press response was initially accomplished by training rats to bar-press for water. Cannulas were then implanted in the external jugular vein according to procedures described by Weeks (1972) (see above). Self-administration testing began with a 16-h nocturnal session followed by daily 1-h sessions, 6 days a week. A lever-press response produced a 10-ml infusion of drug solution (0.01 mg of morphine sulfate) in about 0.2 s or a 50-ml infusion of drug solution.

(15) 1.1.3 The Plus Maze Procedure

(16) The apparatus was made of black Plexiglas and consisted of two runways that intersected at the center at right angles. Each arm of the maze measured 40×10 cm (length by width). Two of the arms that were opposed to each other had walls that measured 40 cm in height (closed arms), whereas the other two arms had no walls (closed arms). The maze was elevated 52 cm above the floor. It was located in a darkened room so that only the open arms were illuminated, each with its own 40-W incandescent light. Animals were placed in the center of the maze and the number of entries into each type of arm was counted (all four paws in the arm defining an entry) as was the time spent on each type of arm. The test was terminated 5 min after the animal was placed in the center. The following measures were calculated: total number of arm entries, entries into open and closed arms, time in open and closed arms, and percent of total time spent in open arms. Changes in the total number of arm entries reflect a general index of activity, whereas changes in the percent measure constitute an index of anxiety. Increased percent open-arm time reflects an anxiolytic state, while decreased percent open-arm time reflects an anxiogenic state. Animals' movements were recorded by using an overhead video camera and VCR. They were subsequently scored by a “blind” observer.

(17) 1.1.4 Microdialysis Study

(18) Under pentobarbital anesthesia (50 mg/kg ip), the rats were implanted stereotaxically with a microdialysis guide cannula (CMA: 8309010; Acton, Mass.) over the nucleus accumbens and with bilateral injector guides 0.5 mm above the interpeduncular (Paxinos and Watson, 1986, The rat brain in stereotaxic coordinates, Ed 2. (Academic Press, London)). Animals were monitored for proper recovery but otherwise left undisturbed for 4 days after surgery. The afternoon prior to the in vivo microdialysis experiment, the rats were placed in a cubical microdialysis chamber with free access to food and water. With the rats briefly anesthetized with Brevital (45 mg/kg ip), dialysis probes were inserted through the guide cannulas. Artificial cerebrospinal fluid containing 146 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl.sub.2, and 1.0 mM MgCl.sub.2 was delivered continuously by a Harvard syringe pump at a flow rate of 1 ml/min. Collection of perfusates began the next day. Twenty-minute fractions were collected in vials containing 2.0 ml of 1.1 M perchloric acid solution (containing 50 mg/l EDTA and 50 mg/l sodium metabisulfite). After 2 h of baseline collections, 18-MC (10 mg) or vehicle was locally administered into the interpeduncular nucleus and the rats received a dose of morphine (5 mg/kg ip) or saline. The collection of dialysate samples was then continued for 3 h. Upon completion of an experiment, rats were killed by an overdose of pentobarbital. Each brain was removed, frozen, and sliced in a cryostat. The tracks left by the probes were identified and their exact positions determined by reference to the atlas of Paxinos and Watson (1986) (The rat brain in stereotaxic coordinates, Ed 2. (Academic Press, London)).

(19) Dialysate samples were assayed for dopamine, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) by high-pressure liquid chromatography (HPLC) with elec-trochemical detection. The HPLC system consisted of an ESA autosampler, an ESA solvent delivery system, an C18 column. The mobile phase consisted of 0.075 mM sodium dihy-drogen phosphate, monohydrate, 0.0017 mM octane sulfonic acid, and 25 mM EDTA in 10% HPLC grade acetonitrile, adjusted to pH 3.0 with phosphoric acid. The flow rate was set at 0.53 ml/min.

(20) 1.1.5 Microinjection Study

(21) Rats were stereotaxically implanted under sodium pen-tobarbital anesthesia (50 mg/kg) with bilateral gauge injector guides (Plastics One, Roanoke, Va., USA) 0.5 mm above the interpeduncular nuclei (Paxinos and Watson, 1986, see above). Obturators were screwed into the injector guides. The injector guides were fastened to the skull using stainless steel screws and cranioplastic cement. Rats were returned to individual cages and were provided with food and water ad libitum. The cages were kept on a heating pad overnight, and the following day the rats were returned to the colony room. Rats were allowed at least 4-5 days of recovery from surgery before being utilized in microinjection studies. Morphine and extract composition (or vehicle) were locally administered into the interpeduncular nucleus using an infusion pump (Harvard Apparatus); all such treatments were administered in a 1-ml volume during a 1-min infusion to prevent reflux through the guide cannula; the injection cannula was kept in place for an additional minute after a treatment was administered.

(22) 1.2 Results

(23) The extract composition reduced the self-administration of morphine abuse; at 40 mg/kg, these effects generally lasted for 18-64 h. The extract mixture (40 mg/kg) appeared to produce downward shifts, without any displacement to the left or right, in the entire unit infusion dose—response curve of a self-administered drug, indicating that reinforcing efficacy was reduced (i.e., morphine and other drugs of abuse like nicotine were less reinforcing in the presence of the extract composition). It should also be noted that the extract mixture was potent at reducing self-administration. The extract mixture showed very good activity on the opium receptors.

EXAMPLE 2: MOTOR BEHAVIOUR TEST

(24) 2.1 Material and Methods

(25) Rats weighing 250 g±10% were utilized in the experiment. Each group consisted of 5 animals.

(26) The rotarod performance test is the test used herein to assess the motor coordination of rats following the induction of dependence on morphine or heroin. Rat's response is delivered following administration of 0.04 mg/kg of morphine, 0.04 mg/kg of heroin following a 10-ml infusion of drug solution (0.01 mg of morphine sulfate) in about 0.2 s or a 50-ml infusion of drug solution (0.015 mg of heroin sulfate) in the same concentration.

(27) To assess the effects of experimental treatments, experiments were performed when baseline of administration rates stabilized within 10% variation from one day to the next 5 days. This took 20 days of testing to show dependence and consistent motor performance using the rotarod performance test. Morphine or heroin i.p. doses were administered daily into rats to equilibrate behavior. Following the oral administration of extract, the doses of morphine or heroin were adjusted to equilibrate the motor coordination similar to the “normal” condition.

(28) Performance measures of motor behavior were conducted using a commercially designed and constructed accelerating rotarod (Rotamex). Motor coordination was quantified as the animal's ability to remain in place on an accelerating rotating rod. Before drug dosing, rats were gradually trained to stay on the rod by providing them six to eight 3-min training periods per day. The rats were trained at increasing rod speeds of 3, 6, 9, 12, and 15 rpm for periods of 3 min. Electric shock (2 mA, AC) was present on the apparatus floor to encourage the animal to stay on the rod. The animal's time of falling was recorded automatically by photocell detection. An individual animal was considered trained when it reached a criterion of two successive trials at 15 rpm wherein it stayed on the rod for the full 3-min session. Only those rats that met this criterion after 2 days of training were used in these experiments. All testing was conducted at least 2 h after the final training trial. Drugs were given by intraperitoneal injection 30 min prior to the testing session. Animals were placed on the rod while it was rotating at 10 rpm. Upon placement, the rod's rotational speed was increased at a rate of 8.3 rpm/min. Total possible test session duration was 3 min. Time of falling and revolutions per minute at time of fall were recorded for each animal.

(29) All data were presented as a mean±standard deviation (SS) as either mg/Kg or % and assessed by using one way ANOVA analysis followed by a post hoc test (Tukey's test) (95% confidence) for multiple comparisons (SPSS version 17). P value less than 0.05 (<0.05) is considered statistically significant.

(30) 2.2 Results and Conclusions

(31) Increasing doses of extract composition administration reduced significantly (p<0.001) the doses of morphine and heroin in order to stabilize rats motor coordination according to the rotarod performance test (FIG. 1). This latter effect was dose-dependent and the percentage of reduction of morphine or heroin was reduced significantly with higher doses (FIG. 2). The 50% dependency reduction values were 9.8 and 12.5 mg/kg for heroin and morphine respectively (FIG. 2). The results indicate that the extract reduced morphine and heroin dependence in dose-dependent manner but without showing superiority of either of the opioid narcotics used.

EXAMPLE 3: CNS HYPER EXCITABILITY ASSOCIATED WITH ALCOHOL WITHDRAWAL IN A MOUSE MODEL OF Ethanol Dependence

(32) 3.1 Materials and Methods

(33) Animals

(34) Adult male mice (80-90 days of age) were used in these experiments. The mice weighed 25-30 g at the start of the experiments.

(35) Extract Preparation and Administration

(36) Extract composition was dissolved in saline, and saline alone was used as the vehicle. Extract composition was administered orally.

(37) Ethanol Exposure and Measurement

(38) Mice were chronically exposed to ethanol vapor in Plexiglas inhalation chambers (60×36×60 cm.sup.3). Briefly, ethanol (95%) was volatilized by passing air through an air stone submerged in ethanol. The ethanol vapor was mixed with fresh air and delivered to the chambers at a rate of 10 l/min, which maintained the ethanol concentration in the chamber in the range of 10-13 mg/L air. Prior to entry into the ethanol chambers, intoxication was initiated by administration of ethanol (1.6 g/kg; 8% w/v; ip.) and blood ethanol concentration (BEC) was stabilized by administration of the alcohol dehydrogenase inhibitor, pyrazole (1 mmol/kg). Mice maintained in the control (air) chamber received injections of saline and pyrazole. The housing conditions in the inhalation chambers were identical to that in the colony room. Immediately after removing mice from the inhalation chambers, blood samples were collected for determination of BEC. Chamber ethanol concentration was determined daily by collecting air samples (2 ml) with a gas-tight syringe through a port in the chamber wall. The samples were then transferred to Venoject™ tubes for later analysis using an enzymatic spectrophotometric assay procedure. Ethanol concentration in the chambers is expressed as mg/L air. Blood samples were collected from the retro-orbital sinus with heparinized capillary tubes. The samples were centrifuged for phase separation and 5 μl of plasma were injected into an Analox Instrument analyzer (Lunenburg, Mass.). BEC (in mg/dl) was recorded by measuring oxygen uptake generated by the oxidation of ethanol to acetaldehyde and hydrogen peroxide by ethanol oxidase.

(39) Handling-Induced Convulsion (HIC)

(40) Mice were randomly assigned to ethanol treatment conditions and then separated into several groups (control and concentration-dependent groups). Mice were continuously exposed for 72 h to ethanol vapor in inhalation chambers. Upon removal from the inhalation chambers, blood samples were collected for determining blood ethanol concentration (BEC). At 1 and 4 h following ethanol withdrawal, mice received oral administration of the extract composition (0, 20, 40 or 60 mg/kg). The convulsions induced following withdrawal of alcohol were recorded as handling-induced convulsion (HIC) response. HIC response has proven to be a sensitive and reliable index of CNS hyper-excitability associated with ethanol withdrawal.

(41) Electroencephalographic Activity

(42) Separate groups of mice were used to assess electrographic measures of ethanol withdrawal. Mice were stereotaxically implanted with chronic indwelling electrodes, as previously described. Briefly, monopolar stainless steel, semi-micro electrodes (120 mm) were implanted into hippocampus (AP: −1.65 mm; L: 1.5 mm; V: −2.25 mm), amygdala (AP: −0.7 mm; L: −2.25 mm; V: −5.25 mm), and visual cortex (AP: −3.0 mm; L: −2.0 mm), along with a stainless steel screw in the nasal area (AP: +4.0 mm; L: +0.5 mm) for use as a reference electrode. The coordinates are given relative to bregma. Electrodes were connected to a four-pin MicroTech plug and fixed to the skull using dental acrylic and a light-cured resin composite. Three to five days following the surgery, baseline electroencephalographic (EEG) activity was recorded from freely moving mice every 2 h over an 8 h period. The next day, mice were placed in the inhalation chambers and received 72 h continuous exposure to ethanol vapor. At 1 and 4 h following ethanol withdrawal, mice received orally the extract composition (0, 40 or 60 mg/kg). EEG data were collected during withdrawal with additional samples recorded at 24, 32, 48, and 72 h post-withdrawal. Recording sessions were conducted in electrically-shielded chambers, with electrode cables connected to Grass amplifiers. Spontaneous EEG data were digitized by a CED analog-to-digital converter and trains of high-voltage electrographic activity, known as brief spindling episodes (BSE), were identified by a computer program (Spike2). Briefly, automated analysis entailed identifying and classifying bursts of EEG activity with a frequency between 7 and 9 Hz and duration of at least 1 s. Data are presented as percent BSE activity (i.e. cumulative duration of all BSE events relative to entire duration of each recording session).

(43) Data Analysis

(44) Data was presented as HIC scores or BSE activity and the data between groups were analyzed by analysis of variance (ANOVA) followed by a post-hoc test. P<0.05 was considered significant.

(45) 3.2 Results

(46) Extract composition was administered at 1 and 4 h following ethanol withdrawal. The extract at 40 and 60 mg/kg doses reduced significantly (P<0.001) HIC scores in comparison to vehicle treated mice (FIG. 3). This reduction was observed during the first 12 h post withdrawal. The 20 mg/kg extract dose exhibited a pattern of reduction in HIC scores; however, they were not significant. Furthermore, there were no differences in HIC scores between 40 and 60 mg/kg treated groups (FIG. 3). Similarly, the extract composition at 40 or 60 mg/kg reduced significantly the BSE activity in a time dependent fashion (FIG. 4). The maximum reduction in BSE activities were observed during the first 36 h post alcohol withdrawal. After 36 h however, the BSE activity in the vehicle-treated group started to decline. Finally, the extract composition treatment of repeated withdrawals was effective in blocking the development of withdrawal sensitization observed in vehicle-treated mice.

EXAMPLE 4: TREATMENT OF ALCOHOL DEPENDENCE IN PATIENTS WITH DEPRESSIVE DISORDER

(47) The extract composition has serotonin re-uptake inhibitors factors. Thus, alcohol-dependent patients with co-morbid major depressive disorder were compared.

(48) 4.1 Methods

(49) Four alcohol-dependent patients comorbid with major depressive disorder in municipal alcohol clinics were given the extract composition (40 mg/kg) in a small pilot study. During the 6-week study period patients continued their routine treatment at the clinics. Abstinence was not required but encouraged. The patients attended visits weekly during the first two weeks, and then at 4 and at 6 weeks. Outcome measures were Alcohol Use Disorders Identification Test (AUDIT), Obsessive Compulsive Drinking Scale (OCDS) and Drinking Diary.

(50) 4.2 Study Participants

(51) Three men and one woman aged 31 to 47 years who were voluntarily seeking outpatient treatment for alcohol problems at two Jordanian municipal alcohol-clinics. Patients with a history of heavy drinking (averaging four or more daily drinks for men and three or more daily drinks for the women) for at least five years, significant depression defined by the Beck Depression Inventory II (BDI-II>16), and who were interested in voluntarily taking part in the study were recommended by their clinic doctor to be screened by the study physician. The patients were interviewed by the study doctor (psychiatrist LM) applying the Structured. The time since the last prior inpatient detoxification had to be at least four weeks. In addition, the eligible patients had to be currently in a depressive episode lasting for more than two weeks. The exclusion criteria included other substance use dependence screened by urine test (amphetamine, benzodiazepines, cocaine, tetrahydrocannabinol and opiates) schizophrenia or other psychotic disorder, and bipolar I and II disorder, acute risk of suicide, pregnancy or breastfeeding, a severe untreated somatic problem, or a serious dysfunction of the liver and mental disability. Other medications prescribed by participants' physicians were allowed, with the exception of other antidepressants. All patients were Jordanian. The mean length of the present depressive period was 22 months. Informed consent was obtained from all patients participating in the study.

(52) 4.3 Study Design

(53) Four patients were initially screened. A screening interview (SCID) was conducted to confirm the diagnoses of MDD and alcohol dependence. Patients completed questionnaires including the Obsessive-Compulsive Drinking Scale (OCDS; Anton R F: Obsessive compulsive aspects of craving: development of the Obsessive Compulsive Drinking Scale, Addiction 2000, 95: (211-217)) and the Alcohol Use Disorders Identification Test (AUDIT; Saunders J B et al., 1993, Development of the alcohol Use disorders identification test (AUDIT): WHO collaborative project on early detection of persons with harmful alcohol consumtion II, Addition 1993, 88(6):791-804), AUDIT-QF (Aalto M et al., 2006, Alcohol Clin Exp. Res., effectiveness of structured questionnaires for screening heavy drinking in middle aged women, 30(11): 1884-1888), and AUDIT-3 (Gual A et al., 2002, Alcohol (37(6):591-596, Audit-3 and Audit-4: effectiveness of two short forms of the alcohol) were used for a detailed drinking analysis. The recording of alcohol use disorders identification test, consumption during the 6-week treatment period was done with a personal drinking diary for all days.

(54) Eligible patients received orally 40 mg/day extract composition. Patients were instructed to take the study medication in the morning. Patients were permitted to telephone the study physician at any time. If the patient did not appear at a scheduled visit, a new appointment was offered.

(55) During the 6-week treatment period, the patients returned to the study site at weeks 2, 4±2, and 6±2 for data collection and for medication checking and dispensing. At each visit, the drinking diary and the study medication intake since the previous visit were recorded from the medication diary. The study medication was ensured by pill count from the returned used bottle. Outcomes were recorded on specific weeks: OCDS (weeks 0, 2, 4 and 6); AUDIT (week 0, 2 and 6). Clinical laboratory tests (MCV, AST, ALT, CDT, and GGT) were taken at the beginning of the study and were repeated at weeks 2, 4, and 6, to ensure the safety of the medication. No breath or blood test for alcohol was performed, but if the patient was obviously intoxicated, a new appointment was offered.

(56) Statistical Analysis

(57) All primary and secondary outcome statistical analysis was performed by an independent source.

(58) 4.4 Results

(59) The baseline AUDIT and alcohol use histories are similar in AUDIT scores decreased from baseline, from 27.6±6.3 to 12.47±7.9 in the extract composition group (40 mg/kg). The overall reduction was highly significant (p<0.0001).

(60) Alcohol consumption measured by the AUDIT QF (quantity-frequency) score was significantly reduced: extract composition (40 mg/kg) from 6.1±1.4 to 3.7±2.3 and from 6.0±1.5 to 4.1±2.1 (p<0.0001). The treatment by time interaction was not significant. The number of heavy drinking days measured by the AUDIT-3 score was also diminished significantly: for the 40 mg extract mixture/kg from 2.7±0.9 to 1.6±1.1 and from 3.0±0.8 to 2.2±1.1 (p>0.0001). The treatment by time interaction was not significant.

(61) 4.5. Results Summary

(62) Alcohol consumption measured by the AUDIT QF (quantity-frequency) score was significantly reduced with these people to whom the extract composition was given.

EXAMPLE 5: EXTRACT OF RAPHANUS INCREASES BRAIN DOPAMINE CONCENTRATIONS IN RATS

(63) Materials and Methods

(64) 2.1. Experimental Animals

(65) Male Wistar rats (250-300 g, Amman-Jordan) were used throughout the study (8 rats for each experiment). Animals were housed in groups of 4/cage in a 12/12 h light-cycle (lights on at 07.00 a.m.), with ad-lib food and water available. The animals were randomly allocated to different groups of the experiment. All experiments were conducted in accordance with standard ethical guidelines and approved by the local ethical Committee University of Medical Committee on the Use and Care of Animals, 81/021, Jul. 10, 2011 (Petra University, Amman, Jordan)).

(66) 2.2. Drugs

(67) Fluoxetine hydrochloride [N-methyl-3-[(4-trifluoromethyl) phenoxy]-3-phenylpropylamine hydrochloride] and desipramine hydrochloride [10-11-dihydro-N-methyl-5H-dibenz (Z) [b, f] azepine-5-propanamine hydrochloride] (TOCRIS Bioscience, UK) were dissolved in sterile saline and administered intraperitoneally at a concentration of 1 ml/kg; the extract was prepared immediately before use. The control groups were administered saline.

(68) 2.3. Plant Material

(69) The Raphanus sativus extract used was prepared in Jordan. To prepare the extract, 100 g of dried and milled stigma was extracted with 1000 ml distilled water by maceration. The extract was dried at 35° C.-40° C., and the yield of extraction was 23 mg of freeze-dried powder per 100 mg dry stigma. The extract was dissolved in normal saline and immediately administered to the animals.

(70) 2.4. Brain Preparation

(71) Thirty minutes after drug and/or extract injection, animals were killed in a CO.sub.2 box, beheaded by a guillotine, and their brains were removed by a specialist in less than a minute. Brains were homogenized in a Falcon tube containing 10 ml of cool (0° C.) sterile saline and centrifuged at 3000 rpm/min for 5 min at 4° C. The supernatant was used for subsequent neurotransmitter detection by ELISA. On the basis of previous studies, an interval time of 30 min was selected; this time was considered to be sufficient for extract action.

(72) 2.5. Statistical Analysis

(73) Data are represented as means±standard error of mean (SEM) of the neurotransmitters concentration. One way analysis of variance (One-Way ANOVA) 3.

(74) Results

(75) 3.1 Effects of Raphanus Extract on Brain Serotonin Concentration

(76) The effect of different doses of Raphanus extract (2, 8, 32, 64, 128 and 256 mg/kg, i.p.) on brain serotonin was studied. The animals received either saline (1 ml/kg, i.p.), or fluoxetine (10 mg/kg, i.p.), desipramine (50 mg/kg, i.p.), Raphanus extract (different concentrations) and were sacrificed 30 min later. One way ANOVA indicated that fluoxetine can increase brain serotonin levels significantly but neither desipramine nor Raphanus extract can increase brain serotonin levels.

(77) 3.2 Effects of Raphanus Extract on Brain Dopamine Concentration

(78) Our results indicated that both fluoxetine (10 mg/kg, i.p.) and desipramine (50 mg/kg, i.p.) can increase dopamine concentration in the brain.

(79) Interestingly, the Raphanus extract can increase dopamine concentration in the brain in a dose-dependent manner with the extract dose of 256 mg/kg, i.p. being the most potent in this study.

(80) 3.3 Effects of Raphanus Extract on Brain Glutamate Concentration

(81) In the last part of the experiments, the effect of the Raphanus extract on brain glutamate concentration was investigated. The results indicated that there were fluctuations in brain glutamate level in dependence from the doses of the extract. The extract increased the glutamate concentration in the brain significantly, e.g., using the dose of 264 mg/kg, i.p.