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Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L.

Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Trees and shrubs of the genus Cordia are widely distributed in the warmer regions, including Indonesia. The aim of this study was to evaluate the analgesic and anti-inflammatory properties of the ethanolic extract of plant leaves in Wistar albino rats. The analgesic activity was evaluated using the hot plate method and acetic acid-induced writhing, and the anti-inflammatory activity was determined using carrageenan-induced paw oedema. The results showed that the Cordia sebestena ethanol extract (100, 200 and 400 mg/kg) exhibited significant analgesic effects in a dose-dependent manner in the two pain models tested. The extract also exhibited significant anti-inflammatory effects in the carrageenan-induced inflammation test. The data obtained support the traditional folklore therapeutic claim about its analgesic and anti-inflammatory properties. Nonetheless, further scientific investigation is required to establish its analgesic and anti-inflammatory properties in other experimental models and clinical settings. Keywords analgesic – anti-inflammatory – carrageenan – Cordia sebestena – hot plate – writhing INTRODUCTION Cordia sebestena L. belongs to the Boraginaceae family, a Preliminary phytochemical screening of the flowers, stem native plant from the Bahamas to the tip of northern South bark and fruit was done by some researchers (Adeosun et al., America, which has been erroneously listed as a Florida 2012, 2013; Dai et al., 2013). Osho et al. (2015) mention that native (Osho et al., 2015). C. sebestena L. is an evergreen tree, the ethyl acetate extract of the leaf indicates the presence of also known as the Geiger tree, Kou Haole (means a ‘foreign a mixture of non-polar aliphatic and aromatic compounds plant’) in Hawaiian and as ‘Geiger’ in Indonesia. The plant can and polar hydroxyl aliphatic and aromatic compounds. grow up to 10 m tall and cultivated largely in tropical and However, the chemical composition of the analgesic and anti- subtropical areas, where it is widely distributed due to its inflammatory activities of the leave extract of C. sebestena extensive use in landscaping. The flowers are dark orange in have not been studied to date. The isolation of the pure colour, appear as clusters at branch tips, throughout the year, compounds of the bioactivity of individual compounds of the especially in June–July and have a pleasant fragrance. The leaf extract will give much information about the medicinal fruits are oval shaped and green and white in colour (Atolani values of this plant. et al., 2014). This plant originated in Hawaii and has been The C. sebestena ethyl acetate leaf extract has been shown used as traditional medicine. In Nigeria, C. sebestena is used to have antibacterial activity against Bacillus cereus and in traditional medicine for the treatment of gastrointestinal Staphylococcus aureus, as well as a relatively low toxicity disorders (Osho et al., 2015). profile in the liver of rats (Osho et al., 2015) . The ethanolic * E-mail: siska@uhamka.ac.id © European Pharmaceutical Journal OR 26 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. extract of the whole plant of C. sebestena possesses significant Veterinary, Bogor Agriculture Institute, Bogor. They were hepatoprotective activity (Chandana et al., 2014), while acclimatised to the laboratory conditions for 10 days before C. sebestena root has anti-inflammatory and analgesic the studies, maintained in normal conditions and fed with activities (Trivedi et al., 2017). The leaves of the plant possess standard pellet and water ad libitum. The room temperature anti-hyperglycemic properties in streptozotocin-induced was maintained at 25±1°C, and the animals were kept under diabetes, and it is hypolipidemic and a potent antioxidant a 12 h light and dark cycle. The experimental protocol was (Sarathchandiran and Gnanavel, 2013). The essential oil of C. approved by the Institutional Animal Ethical Committee of sebestena stem bark obtained through hydrodistillation was Universitas Prof. Dr HAMKA, Reg. no. 08/18.09/003. For all analysed using GC–MS, identifying a total of 19 compounds, pharmacology experiments (analgesic, anti-inflammatory), including aliphatic hydrocarbons (72.73%) and cyclic male Wistar albino rats were used and divided into five hydrocarbons (13.89%). The essential oil is a potent antioxidant groups of five animals each: Group I was the control group, in vitro (Adeosun et al., 2013). Sebestenoids A–D (1–4) were Group II was the standard group treated with a standard drug isolated from bioassay-guided fractionation prepared from (tramadol, Kimia Farma, Indonesia), Groups III, IV and V were the C. sebestena fruit extract (Dai et al., 2013). The chloroform, treated with CSE at a dose of 100, 200 and 400 mg/kg orally, ethyl acetate and methanol extract of C. sebestena root showed respectively, for 5 days. The extract and standard drug were significant anti-inflammatory and analgesic activities at both given once a day between 08:00 and 09:00. dose levels of 100 and 200 mg/kg (Trivedi et al., 2017). This study investigated the analgesic and anti-inflammatory Analgesic screening activities of an ethanolic extract of C. sebestena L. cultivated in Indonesia. Analgesic activity was assessed using a hot plate Hot plate method method, acetic acid-induced writhing and formalin-induced The hot plate method was modified from those described by paw licking. Anti-inflammatory activity was determined using Ojewole (2006). Group I served as the control group and was carrageenan-induced rat paw oedema. orally administered 2 mL of 0.5% carboxymethyl cellulose (CMC) sodium (Na) suspension; Group II was treated with MATERIALS AND METHODS the standard drug tramadol (5.14 mg/kg) and Groups III, IV and V were treated with CSE at a dose of 100, 200 and Plant materials 400 mg/kg, respectively. All test samples were prepared by suspending in 0.5% CMC Na solution immediately before Fresh leaves of C. sebestena in the flowering stage were collected the start of the experiments. The animals were in a fasting in September 2018 from the Universitas Muhammadiyah Prof. condition for 12 h before starting the experiments. Rats Dr HAMKA regional, East Jakarta, Indonesia. The leaves were were placed on a hot plate maintained at 55±1°C. The identified and authenticated taxonomically as Herbarium responses were recorded in the form of jumping or licking of Bogorience, Bogor, Indonesia. A voucher specimen was the paws, with the reaction time recorded at 15, 30, 45 and deposited at the Pharmacognosy laboratory, Universitas 60 min intervals after the administration of the treatments. Muhammadiyah Prof. Dr HAMKA as a record. The plant material A cut-off time of 30 s was selected to avoid tissue damage. was dried in the shade and ground to a coarse powder. Inhibition of hot plate paw-licking responses was expressed as the percentage of the maximal possible effect (% MPE), Preparation of ethanol extract calculated as: The dried powder (2 kg) was subjected to repeated extraction %MPE= T−× TT / 100, a bb by maceration at room temperature with petroleum ether (60– 80°C) as a solvent for 2 days. The plant material was separated by filtration. The marc was dried and extracted continuously where T and T represent the hot plate paw-licking latencies a b (two times, 2 days) with 1.5 L of dichloromethane (DCM; after and before the administration of test drug or vehicle, Merck, Germany) and filtered. The final marc was extracted respectively. with ethanol 70% (Brataco, Indonesia) and concentrated by a vacuum evaporator (under reduced pressure). The Writhing method percentage yield of the ethanol extract was 8.88% w/w. The The method described in Koster et al. (1959) was used. In final 70% ethanol C. sebestena leaves extract (CSE) was used this method, pain-causing acetic acid was injected in the to evaluate the analgesic and anti-inflammatory activities. peritoneal cavity, which induced writhing (abdominal pain and constrictions). Group I (the control group) was treated Animals with 2 mL of 0.5% CMC Na suspension; Group II was treated with the standard drug acetylsalicylic acid intraperitoneal Healthy male Wistar albino rats aged 8–9 weeks and weighing injection (i.p) given 15 min before i.p. injection of 0.75% acetic 180–200 g were obtained from the animal house, Faculty of acid solution (Merck, Germany) and Groups III, IV and V were 27 28 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. Table 1: Analgesic effects of CSE as determined by the hot plate method in rats Time after drug administration Treatment Dose (mg/kg) 0 min 15 min 30 min 45 min 60 min Control --– 2.32±1.36 2.56±0.97 2.26±1.11 2.40±1.00 1.66±0.61 3.94±1.79 Tramadol 5.14 2.54±0.69 3.58±2.28 3.44±1.74 3.94±1.98 (57.87) 3.34±2.13 CSE 100 5.00±1.17 4.96±1.75 3.80±2.81 2.70±0.89 (50.30) 3.98±1.60 CSE 200 3.20±1.09 5.16±2.02 4.24±0.66 4.40±0.74 (58.29) 3.94±1.00 CSE 400 3.64±1.21 5.52±1.98 4.20±2.44 5.46±2.95 (57.87) The latency for licking of the hind paw or jumping off from the surface was expressed as mean±SEM, (n=5). Values in brackets denote percentage of maximum possible effect (MPE) of the latency for licking of the hind paw or jumping off. CSE, C. sebestena ethanol extract. p<0.05 compared with the control. p<0.05 compared with the control group. treated orally with CSE at doses of 100, 200 and 400 mg/kg, standard treated group. The anti-inflammatory effect was respectively. Afterwards, the abdominal writhing was counted expressed as percentage inhibition of oedema: for 15, 30, 45 and 60 min, and the rate of inhibition of writhing was determined. The number of writhing and stretching was Percentage inhibition= VV−× /V 100, ( ) tt observed, recorded and expressed as percentage inhibition. The effectiveness of the treatment was evaluated by the decrease in the number of writhes compared with the control where V is the volume of control and V is the volume of the group. The percentage of inhibition of the number of writhing test. episodes was calculated as: Statistical analysis %inhibiton rate= (VV−× ) /V 100%, The experimental data were expressed as mean±SEM. Data were analysed by one-way analysis of variance followed by where ‘V’ is the average number of stretching of control per Tukey, and the significance of the difference between means group and ‘V ’ is the average number of stretching of test per was determined when the values of p<0.05 were considered group. significant. RESULTS AND DISCUSSION Anti-inflammatory screening Carrageenan test Two different analgesic testing methods were employed to Acute anti-inflammatory activity was evaluated by identify possible peripheral and central effects of the test carrageenan-induced rat paw oedema as previously substance. In this study, the analgesic reactivity to thermal described by Turner et al. (1965). Male Wistar albino rats stimuli in rats was assessed using the hot plate test, which is (150–200 g) maintained under environmental conditions had a sensitive acute pain test for detecting opiate analgesia. The free access to standard diet and were fasted for 10 h before hot plate test is a simple and sensitive method for studying starting the experiments. Group I (the negative control analgesic and hyperanalgesic reactions in rats. Using the group) was treated with 2 mL of 0.5% CMC Na suspension hot plate test, it was shown that oral administration of the and diclofenac Na (Kimia Farma, Indonesia) 5 mg/kg was used ethanolic extract (100, 200 and 400 mg/kg) significantly as a standard control. The CSE (100, 200 and 400 mg/kb) was prolonged the reaction time 15 min after treatment compared administered to Groups III, IV and V. The treatment of Groups I, with the corresponding control groups (Table 1), and these II, III, IV and V were given 30 min before carrageenan injection. effects were dose independent. At 30 min after sample administration, oedema was induced The analgesic effects of 100, 200 and 400 mg/ kg of CSE by injecting 0.1 mL of 1% λ-carrageenan in sterile saline into were investigated. Table 1 presents the analgesic activity of the subplantar surface of the right hind paw. The paw volume the ethanol extract assessed using the hot plate. Tramadol was measured using a micrometre screw gauge at 0, 1, 2, 3 showed no statistically significant increase in the reaction and 4 h after carrageenan injection and compared with the time, but there were significant differences in the thermal 27 28 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. Table 2. Analgesic activity of CSE determined by the writhing test method Reaction time (min) Treatment Dose (mg/kg) 15 30 45 (%inhibition) 60 (% inhibition) Control --– 68.6±10.64 59.0±7.91 56.2±6.87 39.0±5.95 Acetosal 5.14 29.4±1.34 23.8±3.70 20.6±2.51 (63.35) 14.4±2.96(63.07) CSE 100 38.6±7.02 36.6±12.17 36.0±14.31(35.94) 31.2±10.89(20.00) CSE 200 32.6±7.02 31.0±4.41 29.0±3.74 (48.40) 25.4±2.61 (34.87) CSE 400 33.2±4.86 29.0±4.94 27.8±1.48 (50.53) 22.2±5.93(43.08) The data were expressed as mean±SEM, (n=5). Values in brackets denote the percentage of inhibition rate of the number of writhing. CSE, C. sebestena ethanol extract. p<0.05 compared with the positive group. Table 3: Anti-inflammatory effects of CSE assessed by the carrageenan-induced rat paw oedema methodCSE, C. sebestena ethanol extract Volume of hind paw (mm) Treatment Dose (mg/kg) 0 h 1 h 2 h 3 h 4 h Control --– 7.73±6.02 13.18±0.72 23.68±7.52 25.68±1.91 20.56 ±0.65 Diclofenac 19.37±9.39 14.74±7.90 5 5.25±0.56 10.01±6.56 16.57±7.90 sodium (72.90) (64.38) 24.78±9.39 16.86±7.90 CSE 100 6.26±0.58 11.89±0.68 23.51±0.92 (74.74) (61.32) 21.45±8.80 16.18±1.18 CSE 200 5.31±1.00 10.02±6.56 14.88±6.23 (75.24) (67.18) 23.69±0.93 16.46±6.30 CSE 400 5.53±0.85 12.36±5.89 18.51±0.68 (76.66) (66.40) The values of paw oedema were expressed as mean±SEM, n=5. Values in brackets denote inhibition percentage of the oedema paw volume. p<0.05 compared with the positive group. stimulus observed in rats treated with the different doses the pain threshold but also alter the physiological response to of CSE compared with normal saline (negative control) pain, suppressing animal anxiety and apprehension (Kumar throughout the 60 min. et al., 2014). From Table 1, it is clear that oral administration of 100, 200 The results presented in Table 2 show that 100, 200 and 400 and 400 mg/kg of CSE significantly prolonged the reaction mg/kg of CSE exhibited significant (p<0.05) inhibition of the time 15 min after treatment in comparison with the control writhing compared with the standard drug (acetosal, 5.14 groups. Although the extracts appeared to induce higher mg/kg i.p). Indeed, CSE exerted a dose-dependent decrease analgesic activity in most cases, there was no consistent in abdominal constriction in rats stimulated with 1% acetic pattern of activity among the three extracts, that is, the effects acid solution. At 60 min, the number of writhing of all extracts were not dose dependent. The oral administration of ethanol decreased significantly compared with the control. The oral extract of C. sebestena (100, 200 and 400 mg/kg) significantly administration of ethanol extract (100, 200 and 400 mg/ attenuated hot plate thermal stimulation. kg) significantly attenuated the number of writhing in the In this study, the analgesic activity was evaluated through writhing test method. the hot plate and writhing assay in rats, whereas anti- The acetic acid-induced writhing method is not only simple inflammatory activity was assessed via carrageenan-induced and reliable but also affords rapid evaluation of peripheral paw oedema in rats. Tissue damage or injury is associated with analgesic action. In this experiment, the animals react with pain and inflammation. Analgesics can act on the peripheral characteristic stretching behaviour, which is called writhing. or central nervous system. Peripherally acting analgesics act The abdominal constriction is related to the sensitisation of by blocking the generation of impulses of chemoreceptors at nociceptive receptors to prostaglandins (Victoria et al., 2012). the site of pain, while centrally acting analgesics not only raise In this model, pain is generated indirectly via endogenous 29 30 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. mediators, such as bradykinin, serotonin, histamine, function (Trivedi et al., 2015, 2017). The carrageenan-induced substance P and PGs which all act by stimulation of peripheral oedema method has been commonly used as an experimental nociceptive neurons (Garcia, 2004). The ethanolic extract animal model for acute inflammation. This model is mainly (100, 200 and 400 mg/kg) administered orally significantly mediated by histamine, serotonin and increased synthesis inhibited the acetic acid-induced writhing in rats. of prostaglandins in the damaged tissue, followed by In the carrageenan-induced oedema test, a maximum prostaglandin release mediated by bradykinin, leukotrienes oedema paw volume of 25.68±1.91 and 20.56±0.65 mm and polymorphonuclear cells (Kaushik, 2012). In this study, (Table 3) was observed in the control rats, 3 and 4 h after the oral treatment with all the CSE doses significantly inhibited carrageenan injection. Administration of CSE (100, 200 and the paw oedema, suggesting that the anti-inflammatory 400 mg/kg) significantly (p<0.05) inhibited the development actions of the ethanol extracts are related to inhibition of one of pad swelling at 4 h after carrageenan injection. All doses or more intracellular signalling pathways involved with these of CSE were potent and produced anti-inflammatory effects mediator effects. similar to diclofenac Na. CONCLUSION The oral administration of CSE (100, 200 and 400 mg/kg) significantly attenuated the volume of carrageenan-induced oedema similar to diclofenac Na, indicating that the ethanolic The present study demonstrated that the ethanol extract extract of C. sebestena leaves possesses anti-inflammatory from C. sebestena leaves (100, 200 and 400 mg/kg) possess activity. significant potential to inhibit pain and suppress inflammation. This result has a similar effect to the previous research ( Trivedi The increase in dose is proportional to an increase in effect. et al., 2017). It could be the phytochemical composition of However, further studies should be conducted to ensure the root that attributed the analgesic and anti-inflammatory efficacy and safety. The experiments could explore the properties the same as in the leaves.  In this experiment, we fractions of the extract for further pharmacological and did not explore the phytochemical components and could toxicological characterisation. not find the reference of active ingredients from C. sebestena CONFLICTS OF INTEREST leaves that attributed the analgesic and anti-inflammatory properties. Inflammation is a pathophysiological response of living tissue The authors declare that they have no competing interests. to injury that leads to the local accumulation of plasmatic ACKNOWLEDGEMENT fluid and blood cells. There are various components to an inflammatory reaction, such as oedema formation, leukocyte infiltration and granuloma formation that can contribute to All authors are grateful to the Dean of Faculty of Pharmacy the associated symptoms and tissue injury. Inflammation and Sciences, Universitas Muhammadiyah Prof. Dr HAMKA types are acute and chronic. The initial cardinal signs of Jakarta Indonesia, for providing necessary facilities and inflammation include redness, heat, swelling, pain and loss of support to carry out the research work. References [1] Adeosun CB, Olaseinde S, Opeifa AO, Atolani O., Essential oil from Inflammatory activity of Pinus roxburghii Sarg.Advances the stem bark of Cordia sebestena scavenges free radicals. J. of inPharmacological Sciences. 2012; 1-6. Acute Medicine. 2013;(3): 138-141. [7] Koster R, Andersons M, DeBeer E., Acetic acid analgesic screening. [2] Atolani O, Kayode OO, Adeniyi O and Adeosun CB. In vitro Federation Proceeding. 1959;(18): 418-420. antioxidant potential of fatty acids obtained by direct [8] Kumar CNS, Das A, Ray AGR., Anti-inflammatory activity of transmethylation from fresh Cordia sebestena flowers. Annals of Sapindus laurifolius leaf extract in wistar rats. J. of Medicinal Plants Tropical Research. 2014; 36(2): 104-114. Studies. 2014; 2 (1): 1-5. [3] Chandana R, Basini J, Reddy VJS., Hepatoprotective and [9] Ojewole, JAQ., Analgesic , anti-inflammatory and hypoglycaemic antioxidant effect of Cordia sebestena in animal model. effects of ethanol extract of Zingiber officinale (Roscoe.) Rhizomes International J. of Pharmacology & Toxicology. 2014;4(3): 181-189. (Zingiberaceae) in mice and rats. Phytother. Res.2006; (20): 764- [4] Dai J, Sorribs A, Yoshida WY, Williams PG., Sebestenoids A-D, 772. BACE1 inhibitors from Cordia sebestena. J. of Phytochemistry. [10] Osho A, Otuechere CA, Adeosun CB, Oluwagbemi T and Atolani 2013; 71 (17-18): 2168-2173. O. Phytochemical, sub-acute toxicity, and antibacterial evaluation [5] Garcia MD,Fernandez MA, Alvarez A, Saenz MT., Antinociceptive of Cordia sebestena leaf extracts. J Basic Clin Physiol Pharmacol. and anti-inflammatory effect of the aqueous extract from leaves 2015: 1-8. of Pimenta racemosa var.ozua (Mirtaceae). J. Ethnopharmacol. [11] Sarathchandiran I and Gnanavel M., Investigation on 2004; (91): 69-73. hypoglycemic, antioxidant and hypolipidemic activity of [6] Kaushik D,Kumar A, Kaushik P, Rana AC., Analgesic and anti- ethanolic leaf extract of Cordia sebestena in Streptozotocin – 29 30 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. induced diabetic rat. International J. of Research in Pharmaceutical Sciences. 2013; 4 (3): 336-343. [12] Trivedi MH, Ramana KV, and Rao CV., Anti-inflammatory and analgesic activities of Cordia monoicaRoxb.Stem. World J. of Pharmacy and Pharmaceutical Sciences. 2017; 6 (9): 1530 -1536. [13] Trivedi MH, Ramana KV, Rao CV., Evaluation of antiinflammatory and analgesic activities of Cordia sebestena L. Roots. Indo American J. of Pharm. Research. 2015;5(08): 2765-2768. [14] Turner RA., Screening Methods in Pharmacology. Academic Press Inc, London. 1965; 152. [15] Victoria SH, Das S, Lalhlenmawia H, Phucho L, and Shantabi L., Study of analgesic, antipyretic and anti-inflammatory activities of the leaves of Thunbergia coccinea Wall. International Multidisciplinary Research J. 2012; 2 (2): 83-88. 31 OR http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L.

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Abstract

Trees and shrubs of the genus Cordia are widely distributed in the warmer regions, including Indonesia. The aim of this study was to evaluate the analgesic and anti-inflammatory properties of the ethanolic extract of plant leaves in Wistar albino rats. The analgesic activity was evaluated using the hot plate method and acetic acid-induced writhing, and the anti-inflammatory activity was determined using carrageenan-induced paw oedema. The results showed that the Cordia sebestena ethanol extract (100, 200 and 400 mg/kg) exhibited significant analgesic effects in a dose-dependent manner in the two pain models tested. The extract also exhibited significant anti-inflammatory effects in the carrageenan-induced inflammation test. The data obtained support the traditional folklore therapeutic claim about its analgesic and anti-inflammatory properties. Nonetheless, further scientific investigation is required to establish its analgesic and anti-inflammatory properties in other experimental models and clinical settings. Keywords analgesic – anti-inflammatory – carrageenan – Cordia sebestena – hot plate – writhing INTRODUCTION Cordia sebestena L. belongs to the Boraginaceae family, a Preliminary phytochemical screening of the flowers, stem native plant from the Bahamas to the tip of northern South bark and fruit was done by some researchers (Adeosun et al., America, which has been erroneously listed as a Florida 2012, 2013; Dai et al., 2013). Osho et al. (2015) mention that native (Osho et al., 2015). C. sebestena L. is an evergreen tree, the ethyl acetate extract of the leaf indicates the presence of also known as the Geiger tree, Kou Haole (means a ‘foreign a mixture of non-polar aliphatic and aromatic compounds plant’) in Hawaiian and as ‘Geiger’ in Indonesia. The plant can and polar hydroxyl aliphatic and aromatic compounds. grow up to 10 m tall and cultivated largely in tropical and However, the chemical composition of the analgesic and anti- subtropical areas, where it is widely distributed due to its inflammatory activities of the leave extract of C. sebestena extensive use in landscaping. The flowers are dark orange in have not been studied to date. The isolation of the pure colour, appear as clusters at branch tips, throughout the year, compounds of the bioactivity of individual compounds of the especially in June–July and have a pleasant fragrance. The leaf extract will give much information about the medicinal fruits are oval shaped and green and white in colour (Atolani values of this plant. et al., 2014). This plant originated in Hawaii and has been The C. sebestena ethyl acetate leaf extract has been shown used as traditional medicine. In Nigeria, C. sebestena is used to have antibacterial activity against Bacillus cereus and in traditional medicine for the treatment of gastrointestinal Staphylococcus aureus, as well as a relatively low toxicity disorders (Osho et al., 2015). profile in the liver of rats (Osho et al., 2015) . The ethanolic * E-mail: siska@uhamka.ac.id © European Pharmaceutical Journal OR 26 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. extract of the whole plant of C. sebestena possesses significant Veterinary, Bogor Agriculture Institute, Bogor. They were hepatoprotective activity (Chandana et al., 2014), while acclimatised to the laboratory conditions for 10 days before C. sebestena root has anti-inflammatory and analgesic the studies, maintained in normal conditions and fed with activities (Trivedi et al., 2017). The leaves of the plant possess standard pellet and water ad libitum. The room temperature anti-hyperglycemic properties in streptozotocin-induced was maintained at 25±1°C, and the animals were kept under diabetes, and it is hypolipidemic and a potent antioxidant a 12 h light and dark cycle. The experimental protocol was (Sarathchandiran and Gnanavel, 2013). The essential oil of C. approved by the Institutional Animal Ethical Committee of sebestena stem bark obtained through hydrodistillation was Universitas Prof. Dr HAMKA, Reg. no. 08/18.09/003. For all analysed using GC–MS, identifying a total of 19 compounds, pharmacology experiments (analgesic, anti-inflammatory), including aliphatic hydrocarbons (72.73%) and cyclic male Wistar albino rats were used and divided into five hydrocarbons (13.89%). The essential oil is a potent antioxidant groups of five animals each: Group I was the control group, in vitro (Adeosun et al., 2013). Sebestenoids A–D (1–4) were Group II was the standard group treated with a standard drug isolated from bioassay-guided fractionation prepared from (tramadol, Kimia Farma, Indonesia), Groups III, IV and V were the C. sebestena fruit extract (Dai et al., 2013). The chloroform, treated with CSE at a dose of 100, 200 and 400 mg/kg orally, ethyl acetate and methanol extract of C. sebestena root showed respectively, for 5 days. The extract and standard drug were significant anti-inflammatory and analgesic activities at both given once a day between 08:00 and 09:00. dose levels of 100 and 200 mg/kg (Trivedi et al., 2017). This study investigated the analgesic and anti-inflammatory Analgesic screening activities of an ethanolic extract of C. sebestena L. cultivated in Indonesia. Analgesic activity was assessed using a hot plate Hot plate method method, acetic acid-induced writhing and formalin-induced The hot plate method was modified from those described by paw licking. Anti-inflammatory activity was determined using Ojewole (2006). Group I served as the control group and was carrageenan-induced rat paw oedema. orally administered 2 mL of 0.5% carboxymethyl cellulose (CMC) sodium (Na) suspension; Group II was treated with MATERIALS AND METHODS the standard drug tramadol (5.14 mg/kg) and Groups III, IV and V were treated with CSE at a dose of 100, 200 and Plant materials 400 mg/kg, respectively. All test samples were prepared by suspending in 0.5% CMC Na solution immediately before Fresh leaves of C. sebestena in the flowering stage were collected the start of the experiments. The animals were in a fasting in September 2018 from the Universitas Muhammadiyah Prof. condition for 12 h before starting the experiments. Rats Dr HAMKA regional, East Jakarta, Indonesia. The leaves were were placed on a hot plate maintained at 55±1°C. The identified and authenticated taxonomically as Herbarium responses were recorded in the form of jumping or licking of Bogorience, Bogor, Indonesia. A voucher specimen was the paws, with the reaction time recorded at 15, 30, 45 and deposited at the Pharmacognosy laboratory, Universitas 60 min intervals after the administration of the treatments. Muhammadiyah Prof. Dr HAMKA as a record. The plant material A cut-off time of 30 s was selected to avoid tissue damage. was dried in the shade and ground to a coarse powder. Inhibition of hot plate paw-licking responses was expressed as the percentage of the maximal possible effect (% MPE), Preparation of ethanol extract calculated as: The dried powder (2 kg) was subjected to repeated extraction %MPE= T−× TT / 100, a bb by maceration at room temperature with petroleum ether (60– 80°C) as a solvent for 2 days. The plant material was separated by filtration. The marc was dried and extracted continuously where T and T represent the hot plate paw-licking latencies a b (two times, 2 days) with 1.5 L of dichloromethane (DCM; after and before the administration of test drug or vehicle, Merck, Germany) and filtered. The final marc was extracted respectively. with ethanol 70% (Brataco, Indonesia) and concentrated by a vacuum evaporator (under reduced pressure). The Writhing method percentage yield of the ethanol extract was 8.88% w/w. The The method described in Koster et al. (1959) was used. In final 70% ethanol C. sebestena leaves extract (CSE) was used this method, pain-causing acetic acid was injected in the to evaluate the analgesic and anti-inflammatory activities. peritoneal cavity, which induced writhing (abdominal pain and constrictions). Group I (the control group) was treated Animals with 2 mL of 0.5% CMC Na suspension; Group II was treated with the standard drug acetylsalicylic acid intraperitoneal Healthy male Wistar albino rats aged 8–9 weeks and weighing injection (i.p) given 15 min before i.p. injection of 0.75% acetic 180–200 g were obtained from the animal house, Faculty of acid solution (Merck, Germany) and Groups III, IV and V were 27 28 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. Table 1: Analgesic effects of CSE as determined by the hot plate method in rats Time after drug administration Treatment Dose (mg/kg) 0 min 15 min 30 min 45 min 60 min Control --– 2.32±1.36 2.56±0.97 2.26±1.11 2.40±1.00 1.66±0.61 3.94±1.79 Tramadol 5.14 2.54±0.69 3.58±2.28 3.44±1.74 3.94±1.98 (57.87) 3.34±2.13 CSE 100 5.00±1.17 4.96±1.75 3.80±2.81 2.70±0.89 (50.30) 3.98±1.60 CSE 200 3.20±1.09 5.16±2.02 4.24±0.66 4.40±0.74 (58.29) 3.94±1.00 CSE 400 3.64±1.21 5.52±1.98 4.20±2.44 5.46±2.95 (57.87) The latency for licking of the hind paw or jumping off from the surface was expressed as mean±SEM, (n=5). Values in brackets denote percentage of maximum possible effect (MPE) of the latency for licking of the hind paw or jumping off. CSE, C. sebestena ethanol extract. p<0.05 compared with the control. p<0.05 compared with the control group. treated orally with CSE at doses of 100, 200 and 400 mg/kg, standard treated group. The anti-inflammatory effect was respectively. Afterwards, the abdominal writhing was counted expressed as percentage inhibition of oedema: for 15, 30, 45 and 60 min, and the rate of inhibition of writhing was determined. The number of writhing and stretching was Percentage inhibition= VV−× /V 100, ( ) tt observed, recorded and expressed as percentage inhibition. The effectiveness of the treatment was evaluated by the decrease in the number of writhes compared with the control where V is the volume of control and V is the volume of the group. The percentage of inhibition of the number of writhing test. episodes was calculated as: Statistical analysis %inhibiton rate= (VV−× ) /V 100%, The experimental data were expressed as mean±SEM. Data were analysed by one-way analysis of variance followed by where ‘V’ is the average number of stretching of control per Tukey, and the significance of the difference between means group and ‘V ’ is the average number of stretching of test per was determined when the values of p<0.05 were considered group. significant. RESULTS AND DISCUSSION Anti-inflammatory screening Carrageenan test Two different analgesic testing methods were employed to Acute anti-inflammatory activity was evaluated by identify possible peripheral and central effects of the test carrageenan-induced rat paw oedema as previously substance. In this study, the analgesic reactivity to thermal described by Turner et al. (1965). Male Wistar albino rats stimuli in rats was assessed using the hot plate test, which is (150–200 g) maintained under environmental conditions had a sensitive acute pain test for detecting opiate analgesia. The free access to standard diet and were fasted for 10 h before hot plate test is a simple and sensitive method for studying starting the experiments. Group I (the negative control analgesic and hyperanalgesic reactions in rats. Using the group) was treated with 2 mL of 0.5% CMC Na suspension hot plate test, it was shown that oral administration of the and diclofenac Na (Kimia Farma, Indonesia) 5 mg/kg was used ethanolic extract (100, 200 and 400 mg/kg) significantly as a standard control. The CSE (100, 200 and 400 mg/kb) was prolonged the reaction time 15 min after treatment compared administered to Groups III, IV and V. The treatment of Groups I, with the corresponding control groups (Table 1), and these II, III, IV and V were given 30 min before carrageenan injection. effects were dose independent. At 30 min after sample administration, oedema was induced The analgesic effects of 100, 200 and 400 mg/ kg of CSE by injecting 0.1 mL of 1% λ-carrageenan in sterile saline into were investigated. Table 1 presents the analgesic activity of the subplantar surface of the right hind paw. The paw volume the ethanol extract assessed using the hot plate. Tramadol was measured using a micrometre screw gauge at 0, 1, 2, 3 showed no statistically significant increase in the reaction and 4 h after carrageenan injection and compared with the time, but there were significant differences in the thermal 27 28 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. Table 2. Analgesic activity of CSE determined by the writhing test method Reaction time (min) Treatment Dose (mg/kg) 15 30 45 (%inhibition) 60 (% inhibition) Control --– 68.6±10.64 59.0±7.91 56.2±6.87 39.0±5.95 Acetosal 5.14 29.4±1.34 23.8±3.70 20.6±2.51 (63.35) 14.4±2.96(63.07) CSE 100 38.6±7.02 36.6±12.17 36.0±14.31(35.94) 31.2±10.89(20.00) CSE 200 32.6±7.02 31.0±4.41 29.0±3.74 (48.40) 25.4±2.61 (34.87) CSE 400 33.2±4.86 29.0±4.94 27.8±1.48 (50.53) 22.2±5.93(43.08) The data were expressed as mean±SEM, (n=5). Values in brackets denote the percentage of inhibition rate of the number of writhing. CSE, C. sebestena ethanol extract. p<0.05 compared with the positive group. Table 3: Anti-inflammatory effects of CSE assessed by the carrageenan-induced rat paw oedema methodCSE, C. sebestena ethanol extract Volume of hind paw (mm) Treatment Dose (mg/kg) 0 h 1 h 2 h 3 h 4 h Control --– 7.73±6.02 13.18±0.72 23.68±7.52 25.68±1.91 20.56 ±0.65 Diclofenac 19.37±9.39 14.74±7.90 5 5.25±0.56 10.01±6.56 16.57±7.90 sodium (72.90) (64.38) 24.78±9.39 16.86±7.90 CSE 100 6.26±0.58 11.89±0.68 23.51±0.92 (74.74) (61.32) 21.45±8.80 16.18±1.18 CSE 200 5.31±1.00 10.02±6.56 14.88±6.23 (75.24) (67.18) 23.69±0.93 16.46±6.30 CSE 400 5.53±0.85 12.36±5.89 18.51±0.68 (76.66) (66.40) The values of paw oedema were expressed as mean±SEM, n=5. Values in brackets denote inhibition percentage of the oedema paw volume. p<0.05 compared with the positive group. stimulus observed in rats treated with the different doses the pain threshold but also alter the physiological response to of CSE compared with normal saline (negative control) pain, suppressing animal anxiety and apprehension (Kumar throughout the 60 min. et al., 2014). From Table 1, it is clear that oral administration of 100, 200 The results presented in Table 2 show that 100, 200 and 400 and 400 mg/kg of CSE significantly prolonged the reaction mg/kg of CSE exhibited significant (p<0.05) inhibition of the time 15 min after treatment in comparison with the control writhing compared with the standard drug (acetosal, 5.14 groups. Although the extracts appeared to induce higher mg/kg i.p). Indeed, CSE exerted a dose-dependent decrease analgesic activity in most cases, there was no consistent in abdominal constriction in rats stimulated with 1% acetic pattern of activity among the three extracts, that is, the effects acid solution. At 60 min, the number of writhing of all extracts were not dose dependent. The oral administration of ethanol decreased significantly compared with the control. The oral extract of C. sebestena (100, 200 and 400 mg/kg) significantly administration of ethanol extract (100, 200 and 400 mg/ attenuated hot plate thermal stimulation. kg) significantly attenuated the number of writhing in the In this study, the analgesic activity was evaluated through writhing test method. the hot plate and writhing assay in rats, whereas anti- The acetic acid-induced writhing method is not only simple inflammatory activity was assessed via carrageenan-induced and reliable but also affords rapid evaluation of peripheral paw oedema in rats. Tissue damage or injury is associated with analgesic action. In this experiment, the animals react with pain and inflammation. Analgesics can act on the peripheral characteristic stretching behaviour, which is called writhing. or central nervous system. Peripherally acting analgesics act The abdominal constriction is related to the sensitisation of by blocking the generation of impulses of chemoreceptors at nociceptive receptors to prostaglandins (Victoria et al., 2012). the site of pain, while centrally acting analgesics not only raise In this model, pain is generated indirectly via endogenous 29 30 Eur. Pharm. J. 2019, 66(2), 26-31. Evaluation of analgesic and anti-inflammatory activities of ethanolic extract of Cordia sebestena L. Sunaryo H. et al. mediators, such as bradykinin, serotonin, histamine, function (Trivedi et al., 2015, 2017). The carrageenan-induced substance P and PGs which all act by stimulation of peripheral oedema method has been commonly used as an experimental nociceptive neurons (Garcia, 2004). The ethanolic extract animal model for acute inflammation. This model is mainly (100, 200 and 400 mg/kg) administered orally significantly mediated by histamine, serotonin and increased synthesis inhibited the acetic acid-induced writhing in rats. of prostaglandins in the damaged tissue, followed by In the carrageenan-induced oedema test, a maximum prostaglandin release mediated by bradykinin, leukotrienes oedema paw volume of 25.68±1.91 and 20.56±0.65 mm and polymorphonuclear cells (Kaushik, 2012). In this study, (Table 3) was observed in the control rats, 3 and 4 h after the oral treatment with all the CSE doses significantly inhibited carrageenan injection. Administration of CSE (100, 200 and the paw oedema, suggesting that the anti-inflammatory 400 mg/kg) significantly (p<0.05) inhibited the development actions of the ethanol extracts are related to inhibition of one of pad swelling at 4 h after carrageenan injection. All doses or more intracellular signalling pathways involved with these of CSE were potent and produced anti-inflammatory effects mediator effects. similar to diclofenac Na. CONCLUSION The oral administration of CSE (100, 200 and 400 mg/kg) significantly attenuated the volume of carrageenan-induced oedema similar to diclofenac Na, indicating that the ethanolic The present study demonstrated that the ethanol extract extract of C. sebestena leaves possesses anti-inflammatory from C. sebestena leaves (100, 200 and 400 mg/kg) possess activity. significant potential to inhibit pain and suppress inflammation. This result has a similar effect to the previous research ( Trivedi The increase in dose is proportional to an increase in effect. et al., 2017). It could be the phytochemical composition of However, further studies should be conducted to ensure the root that attributed the analgesic and anti-inflammatory efficacy and safety. The experiments could explore the properties the same as in the leaves.  In this experiment, we fractions of the extract for further pharmacological and did not explore the phytochemical components and could toxicological characterisation. not find the reference of active ingredients from C. sebestena CONFLICTS OF INTEREST leaves that attributed the analgesic and anti-inflammatory properties. Inflammation is a pathophysiological response of living tissue The authors declare that they have no competing interests. to injury that leads to the local accumulation of plasmatic ACKNOWLEDGEMENT fluid and blood cells. There are various components to an inflammatory reaction, such as oedema formation, leukocyte infiltration and granuloma formation that can contribute to All authors are grateful to the Dean of Faculty of Pharmacy the associated symptoms and tissue injury. Inflammation and Sciences, Universitas Muhammadiyah Prof. Dr HAMKA types are acute and chronic. The initial cardinal signs of Jakarta Indonesia, for providing necessary facilities and inflammation include redness, heat, swelling, pain and loss of support to carry out the research work. References [1] Adeosun CB, Olaseinde S, Opeifa AO, Atolani O., Essential oil from Inflammatory activity of Pinus roxburghii Sarg.Advances the stem bark of Cordia sebestena scavenges free radicals. J. of inPharmacological Sciences. 2012; 1-6. Acute Medicine. 2013;(3): 138-141. [7] Koster R, Andersons M, DeBeer E., Acetic acid analgesic screening. [2] Atolani O, Kayode OO, Adeniyi O and Adeosun CB. 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Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Nov 1, 2019

Keywords: analgesic; anti-inflammatory; carrageenan; Cordia sebestena; hot plate; writhing

References