Microsoft Word - م.م.صعاب عماد Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 9   STUDY THE ANALGESIC AND SEDATIVE EFFECT OF Ocimum basilicum ALCOHOLIC EXTRACT IN MALE RATS. Seab Emad Sahib Al-Ghurabi* * Dept. of Health Community-Al-Kut Technical Institute-Foundation of Technical Education seabemad2@gmail.com ABSTRACT This study was carried out on male rats to evaluate the analgesic and sedative effect of different doses of Alcoholic Extract of Ocimum basilicum leaves. The analgesic effect studied by using formalin test, 20 male rats divided to a four groups (5 each), first group exposed water orally only before injection of formalin, second group exposed orally with Diclofenac at a dose 0.71mg/kg B.W before injection of formalin, Third and fourth groups orally incubated with Alcoholic Extract of Ocimum basilicum leaves at a dose (50 and 100mg/kg BW) respectively. While the sedative effect studied by using pentobarbitone sleeping time test and open field test, in each test 15 male rats used and divided to a three groups one of them treated with distillated water and the other two groups treated with Alcoholic Extract of Ocimum basilicum leaves at a dose (50 and 100mg/kg BW) respectively. Results of formalin test showed a significant reduction in the mean value of nociceptive response in Diclofenac group specially at late phase while, groups treated with Alcoholic Extract of Ocimum basilicum leaves revealed a significant reduction in the nociceptive response value at the both phases (early and late phase) moreover the group treated with 100mg/kg showed the highest attenuation in the mean value of nociceptive response compared to 50mg/ kg treated group. Furthermore, the dose 100mg/kg produced the potent sedative effect in pentobarbitone sleeping time test and open field test. These results pointed to analgesic and sedative effect of Ocimum basilicum may duo to its consistence of the active analgesic and sedative compounds and its effect are in a dose dependent manner. Key words: analgesic, sedative, Ocimum basilicum, alcoholic extract, male rats. ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Dec. 24 , 2012 . Accepted for publication May 9 , 2013 . Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 10   INTRODUCTION Traditional medicinal plants are a therapeutic resource used by the population of the African, Asian continent specifically for health care, which may also serve as starting materials for drugs (Iwn et al.,1999). A medicinal plant” is any plant which in one or more of its part contains substances that can be used for the therapeutic purposes or which are precursors for the synthesis of useful drugs (WHO ,2001). Basil (Ocimum basilicum) of the family Lamiaceae The plant tastes somewhat like anise, with a strong, pungent, sweet smell. Typically called sweet basil or Holy basil. The plant grows in several regions around the world. The genus Ocimum is ranked high among some of the astonishing herbs for having enormous medicinal potentialities (Klima´nkova et al.,2008). Ocimum basilicum has also been used in the treatment of a number of ailments like bronchitis, rheumatism and pyrexia (Keita et al., 2000). The fixed oil of O. basilicum was found to possess significant anti-inflammatory (Chaurasia, and Vyas, 1977) along with anti-microbial (Rana et al., 1997), analgesic and spasomlytic properties without any noticeable toxicity or hypoglycemic effects (Sethi, 1979) and is an effective anti-diarrheal, antioxidant, anti-depressant and anti-helmentic and enhances wound healing (Vohora and Rizwan, 1973). In traditional medicine, Ocimum basilicum has been used as an antioxidant, antiseptic, preservative, sedative, digestive regulator and diuretic. It also has been recommended for the treatment of headaches, coughs, infections of upper respiratory tract, kidney malfunction and to eliminate toxins (Evans et al.,2007). It is also known the leaves of basil are suitable for the treatment of cough and pain (Basilico and Basilico, 1999). The latter use was based on observational impression rather than on clinical or experimental studies. Therefore, the aim of the present study was to investigate the analgesic and the sedative effect of different doses the alcoholic extract of Ocimum basilicum in male rats. So the aim of this study is to evaluate the analgesic and sedative effect of alcoholic extract of Ocimum basilicum leaves and to investigate which dose of the extract is more potent. Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 11   MATERIAL AND METHODS PLANT MATERIALS AND EXTRACT PREPARATION: Fresh Ocimum basilicum leaves were purchased from a local market in al-kut city. Later these plant leaves were washed under tap water, and then dried in room temperature at shade. The dried leaves were grinding to a fine powder by an electrical grinder. The plant classification was done in the Ministry of Agriculture/ State Board for Seeds Testing and Certification S.B.S.T.C in Abu Graib /Baghdad. Organic solvent extraction of the Ocimum basilicum leaves was carried out by using ethanol (95% ethyl alcohol) which is considered as very effective in extracting the active ingredients of the plant according to method described by (Effraim et al., 2000) this was done by using Soxhlet apparatus. 50 gm of plant leaves powder was put inside the thumble and 500 ml of 95% ethanol was put inside the flask. The extraction was carried out for 24 hours by heating temperature that kept the solvent at 50-60 Co until a clear and colorless solvent appeared in the extracting unit. After that, the extract was dried by using an electric oven at temperature 40-45 Co until dry extract was obtained. The dry extract was placed in an incubator under 38-40 Co for complete dryness of the sample .The final extract was kept frozen at –20 Co until use. ANIMALS: Fifty mature male Albino Wister rats with average weight of 280-300g and age of 10-12 weeks were used in this study; all animals were obtain from the central animal house of collage of veterinary medicine university of Baghdad, animals in all stages of the experiments were housed in plastic cages in a conditioned room (22-25 °C) also had free access to standard pellet diet throughout the experimental period. EXPERIMENTAL DESIGN: a-STUDY THE ANALGESIC EFFECT OF OCIMUM BASILICUM ALCOHOLIC EXTRACT (FORMALIN TEST). Formalin test was carried out in this experiment to investigate the analgesic effect of Ocimum basilicum alcoholic extract. Formalin test is important in studying the effect of analgesic after acute long-lasting pain (Dubuisson and Dennis, 1977; Francesca, 2004). 20 male rats divided to four Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 12   groups (each of 5 rats) first group (control group) exposed orally with distill water, second group exposed orally with Diclofenac 0.71mg/kg before injection of formalin, third group exposed Oral with O.B.E at a dose 50mg/kg B.W (Sirhan, 2011) before injection of formalin, fourth group exposed Oral with O.B.E at a dose 50mg/kg B.W before injection of formalin. 5% formaldehyde was used as described (Dubuisson and Dennis ,1977). Each rat received 10µl of formalin subcutaneously into the right hind paw by using insulin syringe with a 30-gauge needle. Immediately after formalin injection, animals were placed individually in open roof cage (50×50×30 cm) of flat floor to allow clear observation. Prior to this procedure, each rat was allowed for 5 minutes to adapt the testing box and left freely moving and exploring. The formalin test which is sensitive for various classes of analgesic drugs has two distinct phases, reflecting different types of pain. The early phase (initial pain) that occurs about 5 minutes after formalin injection reflects a direct effect of formalin on nociceptors (neurogenic pain) whereas the late phase that occurs between 15 and 45 minutes following injection reflects tissue injury or inflammatory pain (Elisabetsky et al.,1995). An interphase period depression to pain response was noticed where a decrease in pain behavior occurred between (5) and (15) minutes after injection (auto analgesia). In the formalin test, several mediators such as histamine, kinin, serotonin and prostaglandins are released from damaged cells which take part in the inflammatory response and are able to stimulate nociceptors and induction of pain (Le Bars et al.,2001). b- STUDY THE SEDATIVE EFFECT OF OCIMUM BASILICUM ALCOHOLIC EXTRACT. i. PENTOBARBITAL SLEEPING TIME TEST: This study was conducted according to that done by (Gilani and Janbaz , 1995) on Webster albino rats. 15 male rats were divided to three groups (5each), the rats of first group exposed orally with distill water and after 1 hour injected intra peritonealy with pentobarbitone 35mg/kg B.W. While, second group exposed orally with 50gm/kg B.W of Ocimum basilicum alcoholic extract and after 1 hour injected intra peritonealy with pentobarbitone 35mg/kg B.W. Third group, exposed orally with 100gm/kg B.W of Ocimum basilicum alcoholic extract and after 1 hour injected intra peritonealy with pentobarbitone 35mg/kg B.W. and the time of sleeping were recorded for each group. Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 13   ii. OPEN FIELD TEST: The Open Field Test (OFT) is an experiment used to assay general locomotor activity levels and anxiety in rodents in scientific research (Hall and Ballachey, 1932) this test developed by Calvin S. Hall to test emotionality of rodents (Victor, 1969). The open field test (OFT) is a commonly used qualitative and quantitative measure of general locomotor activity and willingness to explore in rodents (Stanford, 2007). Changes in these measures are often used to assess the sedative or stimulant effects of pharmacological agents. This basic behavioral assessment is used in almost every study involving rodent behavior. 15 male rats used in this parameter divided to three groups (5each); first group exposed to 1 ml of distillated water only and considered as control group, Second group exposed 50mg/kg B.W of Ocimum basilicum alcoholic extract, third group exposed 100mg/kg B.W of Ocimum basilicum alcoholic extract. the measurement taken after 1 hour from exposure of extract for each rat individually in a wooden box which setup manually and it have surrounding walls to prevent escape, the floor of the wooden box marked in a grid for a 16 square, each square dimensions are (15x15cm). The observation recorded for five minutes as the following: a) Number of squares or lines cross that each animal moved over it. b) Number of jumping or trying to escape. RESULTS AND DISSCUSION At the early phase of formalin test the oral exposure of O.B.E at a dose 100mg/kg B.W (T3 group) cause the higher significant attenuatetion to the nociceptive response value (33.82 + 1.27) compared to C and T1group values (41.43 + 1.12 and 38.20+ 1.61). On the other hand, at the late phase of the formalin test, diclofenac group (T1) recorded the highest significant reduction in the nociceptive response value (20.63 + 1.51) compared to the other groups. While (T3and T2 group) showed slight attenuatetion in nociceptive response values (37.12 + 2.13 and 42.34 + 2.24) respectively compared to T1 group value (20.63 + 1.51). Within group, result of the C group and T2 group showed that the late phase recorded a significant elevation in the nociceptive response when compared to the each group corresponding early phase. Inversely, T1 group showed a significant reduction in the nociceptive response value (20.63 + 1.51) Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 14   at late phase when compared to early phase value (38.20+ 1.61) of the same group. While, T3 group showed no significant difference between the nociceptive response value of early phase and late phase (33.82 + 1.27 and 37.12 + 2.13) respectively (table 1). In addition to the sedative effect of Ocimum basilicum leaves extract (table 2) pointed out to an elevation in time of sleeping in both treated group. But the G3 group which treated with 100 mg /kg BW of Ocimum basilicum leaves extract recorded highest significant elevation in the mean value (81.40+ 3.69) when compared to (G2 group) with mean value (57.60+ 2.69) which treated with 50 mg /kg BW of Ocimum basilicum leaves extract and control group with mean value (48.40 + 4.63) (table 2). Table 1: Nociceptive response in male rats treated orally with the alcoholic extract of Ocimum basilicum leaves, Diclofenac. (Formalin test). L.S.D. = 4.32, n =5. Figures represent mean ± standard error. Different capital letters represent significant difference between groups vertically at p<0.05. Different small letters represent significant difference within phase horizontally at p<0.05. Phases Groups Nociceptive response (Number of licking & flinching ) early phase (0-5)minutes late phase (15-45)minutes C (control group) Oral administration of distill water before injection of formalin 41.43 + 1.12 Ab 56.58 + 1.62 Aa T1(treated group) Oral administration of Diclofenac at a dose 0.71mg/kg B.W before injection of formalin 38.20+ 1.61 Ba 20.63 + 1.51 Db T2 (treated group) Oral administration of O.B.E at a dose 50mg/kg B.W before injection of formalin 36.23 + 0.94 BCb 42.34 + 2.24 Ba T 3 (treated group) Oral administration of O.B.E at a dose 100mg/kg B.W before injection of formalin 33.82 + 1.27 Ca 37.12 + 2.13 Ca Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 15   Table 2: Effect of alcoholic extract of Ocimum basilicum leaves (50, 100 mg/kg BW) on time of sleeping induced by pentobarbitone in male rats. L.S.D. =17.8, n =5. Figures represent mean ± standard error. Different letters represent significant difference between groups at p<0.05. Furthermore, within open field test, treatment with 100gm/kg B.W of O.B.E (G3 group) recorded significant reduction in the mean values (5.80±0.58 and 1.40±0.24) of movement activity at the both behaviors measurement (no. of square which crossed and no. of escape jumping) when compared with G2 mean values (11.80±0.86 and 3.00±0.32) and G 1 mean value (17.40±1.03 and 7.20±0.58) respectively. While the G2 group which treated with 50gm/kg B.W of O.B.E recorded a slight significant reduction in the mean values of the both behaviors measurement (no. of square which crossed and no. of escape jumping) when compared with G 1 mean values (17.40±1.03 and 7.20±0.58) respectively. Groups Time of sleeping G1 group Exposed orally with distill water and after 1 hour injected intra peritonealy with pentobarbitone 35mg/kg B.W. 48.40 + 4.63 b G2 group exposed orally with 50gm/kg B.W of O.B.E and after 1 hour injected intra peritonealy with pentobarbitone 35mg/kg B.W. 57.60+ 2.69 b G3 group exposed orally with 100gm/kg B.W of O.B.E and after 1 hour injected intra peritonealy with pentobarbeton 35mg/kg B.W. 81.40+ 3.69 a Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 16   Table 3 : Effect of alcoholic extract of Ocimum basilicum leaves (50, 100 mg/kg BW) on the movement activity in open field test. Behavioral measurements Groups No. of square which crossed No. of escape jumping G1 group (Control group) Exposed orally with distill water before 1h from test 17.40±1.03 a 7.20±0.58 a G2 group Exposed orally with 50gm/kg B.W of O.B.E before 1h from test 35mg/kg B.W 11.80±0.86 b 3.00±0.32 b G3 group Exposed orally with 100gm/kg B.W of O.B.E before 1h from test 35mg/kg B.W 5.80±0.58 c 1.40±0.24 c L.S.D of (no. of square which crossed) bar = 3.84 L.S.D of (no. of escape jumping) bar = 1.23 n =5. Figures represent mean ± standard error. Different letters represent significant difference between groups at p<0.05. The results of the present study demonstrate that alcoholic extract of Ocimum basilicum, have a significant effect on pain in the current antinociceptive model in rat (formalin test), these results also showed that the analgesic effect of alcoholic extract was in the range of 50 to 100mg/kg. But the dose of (100mg/kg) was the most effective and more potent and seems to qualified the effect of analgesic agent (Diclofenac). In formalin test, the centrally acting drugs such as narcotics inhibited both phases equally, while peripherally acting drugs only inhibited the second phase (Shibatta et al.,1989). It is also well known that the formalin model may involve sensorial C-fibers (Heapy et al., 1987) in early phase and a combined process generated by peripheral inflammatory tissue and functional changes in the dorsal horn in late phase (Dalal et al.,1999). In fact, the effect of the extract on both phases of formalin test showed that they contain active analgesic principles acting both centrally relates to antagonistic action of the nociceptors (neurogenic pain) and peripherally (inhibitory actions or released prostaglandins) (inflammatory pain). On the other hand, our result showed that diclofenac inhibit the nociceptive stimuli in both Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 17   phase in the formalin test especially in late phase due to its well known analgesic and anti-inflammatory effect (Rang et al., 2003). While, the sedative effect of Ocimum basilicum alcoholic extract was confirmed by the significant increase in pentobarbitone sleeping time in a sleeping time test and suppression in the movement activity in open field test specially after pretreatment with the alcoholic extract at the dose 100gm/kg B.W. There is always a question that which of the active components of whole extract of a medicinal plant is more important. There are many reports about the beneficial effects of Ocimum basilicum and their compounds. In Ocimum basilicum it has been reported that three main terpenes including linalool, 1,8- cineol, and eugenol comprise the most important components of the extract (Ismail, 2006). However, the centrally acting of Ocimum basilicum may contribute to the modulation of glutamergic and GABAergic transmission which is one mechanisms of action of monoterpenes (Szabadiss and Erdelyi, 2000). The second major terpene is cineole present in the essential oil of Ocimum basilicum. It has been reported that 1,8-cineole exerts anticonvulsant activity, potentiates pentobarbitone sleeping time, and has an inhibitory effect on locomotor activity (Santos and Rao, 2000). Also it’s well documented that the most abundant constituent of the Ocimum basilicum alcoholic extract was linalool. Linalool may have analgesic effect through suppression of voltage-gated currents (Narusuye et al., 2005). Additionally, it has been reported that linalool has a locomotor inhibitory action as well as hypnotic action (Robbers et al., 1996). Eugenol also reported is a one of the Ocimum basilicum essential oil (Aoshima and Hamamoto ,1999).Reported that eugenol action through potentiation of binding of GABA to its receptor and by increasing the affinity of these receptors to bind GABA. , it has been reported that eugenol has anesthetic, sedative, and muscle relaxant effects (Boissier et al.,1967).Thus, when GABA is a one of the inhibitory neurotransmitters (Goodman and Gilmans, 2001), the current study suggested that the activation of GABA neurotransmitter by eugenol may attributed to inhibition of the nociceptive response at the both phases in formalin test with a degree of sedation which clarified in pentobarbitone sleeping time test and open field test. Flavonoids were also documented (Sirhan, 2011), as a main compound in Ocimum basilicum alcoholic extract. The flavonoids are known for their antinociceptive and /or anti-inflammatory activity (Meyre-Silva et al., 1999). Flavonoids which belongs to Qurectine group has an inhibitory effect on 5- Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 18   LOX (LIPOOXYGENASE) pathway (the main pathway for production of chemical mediators important in pain and inflammatory process) (Ficarra et al.,1995).In our study the extract has inhibitory effects on peripheral antinociceptive (late phase) this effect may be due to components such as flavonoids in Ocimum basilicum extract. Another compound in Ocimum basilicum extract are Tannins and Caffeic acid (Sirhan, 2011) , Tannins and Caffeic acid were reported had anti- inflammatory effect by inhibiting of prostaglandins release (Neradil et al., 2003; Seeram et al., 2005), so depending on the above fact these compounds may attributed the peripheral antinociceptive effect in the current study. 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Al-Ghurabi Diyala Agricultural Sciences Journal, 6 ( 1 ) 9 – 22 ,2014 22   . دراسة التأثير المسكن والمهدئ للمستخلص الكحولي لنبات الريحان في ذآور الجرذان *صعاب عماد صاحب الغرابي seabemad2@gmail.com . هيئة المعاهد الفنية- المعهد لتقني الكوت - قسم صحة المجتمع -مدرس مساعد * المستخلص التأثير المسكن والمهدئ لجرع مختلفة من هذه الدراسة على ذآور الجرذان لتقيم أنجزت ، اختبار الفورمالين باستخدامتمت دراسة التأثير المسكن . نبات الريحان ألوراقالمستخلص الكحولي األولىالمجموعة جرعة ، )جرذ لكل مجموعة 5( موعاتمج أربع إلىقسمت ًاذآر ًاجرذ 20استخدم عن طريق الفم ةجرعفقد المجموعة الثانية أما ، مالينقبل الحقن بالفورالماء فقط عن طريق الفم المجموعة الثالثة وجرعة ، وزن الجسم قبل الحقن بالفورمالينآلغم من \ملغم 0.71بالدايكلوفيناك بجرعة آلغم من وزن \ملغم 100و 50(الريحان بجرعة ألوراقبالمستخلص الكحولي عن طريق الفم والرابعة اربيتون اختبار وقت التنويم للبنتوب باستخدامبينما التأثير المسكن فقد تمت دراسته . على التوالي) الجسم بالماء تعرضتمجاميع احدهما 3 إلىجرذ قسمت 15في آل اختبار استخدم ،واختبار الميدان المفتوح 50(بجرعة والريحان ألوراقبالمستخلص الكحولي تعرضت األخرىالمقطر فقط بينما المجموعتين . على التوالي) آلغم من وزن الجسم\ملغم 100و معدل االستجابة لأللم لمجموعة ةأظهرت نتائج اختبار الفورمالين وجود انخفاض معنوي في قيم خالل الطور المتأخر من االختبار بينما المجاميع التي عولجت بالمستخلص الكحولي ةالدايكلوفيناك خاص في قيم االستجابة لأللم خالل آال الطورين من االختبار ًامعنوي ًاانخفاض تأظهرالريحان فقد ألوراق آلغم من المستخلص \ملغم 100ذلك فأن المجموعة التي عولجت بـ فضال عن) الطور المبكر والمتأخر( .آلغم\ملغم 50بالمجموعة المعالجة بـ ةمعدل االستجابة لأللم مقارن ةانخفاض بقيم أدنىأظهرت تأثير مهدئ في اختبار أعلى إلىدت آلغم أ\ملغم 100ذلك فأن المجموعة المعالجة بـ فضال عنو التأثير المسكن والمهدئ نأ إلىهذه النتائج أشارت. وقت التنويم للبنتوباربيتون واختبار الميدان المفتوح كن ومهدئ مس تأثيرفعالة ذات احتوائه على مرآبات إلىيعود الريحان ألوراقللمستخلص الكحولي .ةتأثير معتمد على الجرعوهذا ال .ذآور الجرذان تأثير مسكن ، تأثير مهدئ ، مستخلص آحولي ، نبات الريحان ، :الكلمات المفتاحية