Anti-Pyretic and Analgesic Potentials of Aqueous Extract of Phragmanthera capitata S. Balle in Albino Rats Lapah Pierre Takem*, Noa Pierre Abe and O. John Ogbonna Department of Pharmacology, Faculty of Basic Medical Sciences, University of Calabar, Calabar-Nigeria *Corresponding author (Email: lapietak@yahoo.com) Abstract - Phragmanthera capitata is parasitic plant (mistletoe) that colonizes many plants including avocado trees. The whole plant infusion/decoction is used in Cameroon folk medicine to relief fever and abdominal pain. This study was aimed at assessing anti-pyretic and analgesic potentials of aqueous extract of Phragmanthera capitata (AEPC) in Wistar rats at doses of 100, 200 and 300 mg/kg body weight. To assess anti-pyretic potential; 2, 4-dinitrophenol (DNP) and turpentine were used to induce pyrexia with standard drug being diclofenac sodium (50 mg/kg). To assess peripheral analgesic effect, acetic acid-induced writhing test was used. To assess central analgesic potential, formalin-induced licking test was used with standard drug being pethidine (5 mg/kg). Data obtained were analyzed using analysis of variance (ANOVA) with Tukey test used as post hoc. In DNP-induced pyrexia, results revealed that AEPC at 200 and 300 mg/kg significantly (P <0.05 and P <0.01) reduced rat body temperature by 0.80±0.02 and 1.20±0.10 OC respectively as compared to 0.90±0.05 OC for standard drug. In turpentine induced pyrexia, same inhibition trend was shown as in DNP induced pyrexia. In peripheral analgesic activity, AEPC (300 mg/kg) maximally inhibited (P <0.05) number of writhes to 4.25±0.10 as compared to 9.27±0.51 for standard drug and 31.50±2.32 for control. In central analgesic activity, the number of licks was reduced to 4.99±0.13 as compared to 4.21±0.09 for standard drug and 39.25±3.13 for control. Therefore, AEPC possesses enormous anti-pyretic and analgesic properties in a dose-dependent manner. These properties corroborate the extract being used in Cameroon folk medicine to relief fever and abdominal pain. Keywords - Phragmanthera Capitata, Anti-Pyretic, Analgesic, Dose-Dependent, Loranthaceae
1. Introduction
Phytopharmaceuticals have been utilized over the centuries by different communities of the world [1]. Local communities use medicinal plants as a primary source of their health care system [2] and this account for about 80% of black African culture [3]. Consequently, medicinal plants are assuming greater importance in their well-being [4]. In Cameroon, there is a rich tradition in the use of herbal medicine for the treatment of several ailments [5] though it is poorly funded and needs organization [6]. This is so because poverty cannot be successfully combated without proper health care services [7]. Yet, curative health spending is not always well targeted to the poorest in Africa including Cameroon which has about 50.5% population under the poverty line [8]. This makes orthodox medicine unaffordable, so much so that in Cameroon only 3 out of 20 patients are able to buy prescribed drugs and 1 out of every 1000 patients is able to see a specialist [9]. This trend is similar in other developing countries, however. On this backdrop, it is essential and crucial that research into indigenous plant medicine should be encouraged and fortified to sustain primary health care of the communities.
Phragmanthera capitata is mistletoe (parasitic plant) of loranthaceae family [10] with woody shrub and pendent branches to 2 m, often associated with ants’ nests [11]. It is found in West Tropical Africa including Nigeria; West-Central Africa including Cameroon and Angola in South Tropical Africa [12, 13]. Aqueous extract of P. capitata possesses anti-diarrheal properties [14], anti-secretory, gastroprotective and anti-ulcer activities [15]. Infusion of leaves has anti-diabetic activities [16] and is also employed in the treatment of a variety of other ailments including fever and abdominal pains in traditional practices in Cameroon [17]. Our goal was, therefore, to assess anti-pyretic
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and analgesic activities of AEPC in experimental animal model and to relate these activities to their traditional claims.
2. Materials and Methods
2.1. Drugs and Chemicals
Diclofenac sodium (Tianjin Bofa Pharmaceutical Co, Lit., China), Acetic acid and pethidine (BDH limited, England).
2.2. Plant Material and Preparation of Extract
Phragmanthera capitata plant or “ntsalar” as it is called in Babadjou vernacular, was harvested from avocado trees in Konka, Baligham village in North Western Region of Cameroon in October, 2013. Authentication of the plant was by Cameroon National Herbarium (CNH) with Voucher No. 24673/SRF/CAM. The fresh plant sample was washed in tap water, rinsed in distilled water and shade dried at room temperature (25±2oC) for fifteen days. The dried plant sample was ground and water extracted (1kg in 7.5 litres) by maceration for 3 days, filtered and sun-dried for another 3 days to obtain the extract with a yield of 15.5%.
2.3. Experimental Animals
Sprague Dawley rats of either sex weighing between 200-250 g were selected from Animal House Unit of Department of Pharmacology, University of Calabar. The rats were kept in polypropylene cages of four animals per cage and maintained under standard laboratory conditions of temperature (28 ± 20C) and relative humidity (50±5%) and a 12 hour dark/light cycle and received standard pellet diet (Agro Feeds, Calabar) and water ad libitum. Animals were handled during the experimentation as recommended by OECD's guidelines on animal care [18].
2.4. Phytochemical Screening
Preliminary qualitative screening of AEPC for the presence of phytoconstituents such as flavonoids, alkaloids, tannins, saponins, glycosides and polyphenols were done following standard tests procedures [19, 20].
2.5. Acute Toxicity Evaluation
Acute toxicity evaluation of AEPC was carried out in female albino mice in accordance with the Organization for Economic Co-operation and Development guidelines no. 425 Up and Down Procedure [21, 22].
2.6. Pyretic Activity
2.6.1. DNP-Induced Pyrexia
Pyrexia was induced by administration of 10 ml/kg of DNP intraperitoneally [23] to overnight fasted rats but with access to water ad libitum until the start of the experiment. Basal rectal temperatures were taken with clinical thermometer (Wuxil Medical, China) pre DNP injection and 30 minutes (min) post injection. Only rats that showed hyperthermia of at least 0.7°C were selected and randomized into 5 groups of 6 rats. Group I (control) received saline (10 ml/kg), Group II (standard) received diclofenac sodium (50 mg/kg), and Groups III-V (tests) received AEPC (100, 200, 300 mg/kg) respectively per oral. Rectal temperatures of the animals were obtained at 30, 60, 90, 120 and 150 min post treatment [24].
2.6.2. Turpentine-Induced Pyrexia
Basal temperatures of rats were taken before administration by subcutaneous injection of 2.3 ml/kg turpentine [25] into the dorsum region of rats. At 11 h post administration, rectal temperatures were taken and only rats that showed febrile were selected and grouping, treatment and rectal temperature recording were same as in DNP-induced pyrexia above except that control group received 2.3 ml/kg of normal saline.
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2.7. Analgesic Activity
2.7.1. Writhing Test
Animals were randomized into 5 groups of 6 animals and treated thus: Group I (control) received saline (10 ml/kg), Group II (standard) received diclofenac sodium (50 mg/kg), Groups III-V (tests) received AEPC (100, 200 and 300 mg/kg) respectively per oral. After 30 min, 10 ml/kg acetic acid (0.6%, v/v in saline) were injected intraperitoneally. After 5 min, writhes were counted [26, 27] for the next 20 min. A writhe is defined as abdominal muscles contraction followed by body and hind limb(s) elongation.
2.7.2. Formalin Test
Rats were randomly divided into 5 groups of 6 rats with Group I (control) receiving saline (10 ml/kg), Group II (standard) receiving pethidine (5 mg/kg) and Groups III-V (tests) receiving AEPC (100, 200, and 300 mg/kg) respectively per oral. Forty five minutes post treatment, each rat received 20 μl formalin (5% v/v in saline) injected into sub-plantar region of right hind paw. Licking score counts/unit time in response to pain were recorded [28].
2.8. Statistical Analysis
Data were expressed as means ± SD (n = 6) and were statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey test as post hoc. Values of P <0.05 were considered statistically significant [29].
3. Results
Preliminary phytochemical screening of AEPC revealed high presence of tannins and terpenoids; moderate presence of glycosides, saponins, flavonoids and anthraquinones; and low presence of phenols and alkaloids. In the acute toxicity assessment, AEPC was observed safe up to a concentration of 3000 mg/kg in mice during the limit test.
Fig. 1. Temperature change with time after induction of pyrexia with 2, 4-dinitrophenol (10 ml/kg). Each point represents the mean ± SEM (n=6). *P < 0.05, ⃰ ⃰P < 0.01
Fig. 1 shows temperature change with time from induction of pyrexia with DNP (10 ml/kg) 30 min before treatment with 50 mg/kg diclofenac sodium as standard drug, 10 ml/kg normal saline as control and AEPC (100, 200, 300 mg/kg) as tests. Thirty minutes post treatment or 60 min post induction of pyrexia the temperature of the rats decreased variably except Group III. One hour post treatment or 90 min post induction, only Groups IV (200 mg/kg) and V (300 mg/kg) further had decrease in temperature. One and a half hours post treatment, Groups IV
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and V had a slight increase in temperature while Groups I-III had elevated temperature. Two hours post treatment, AEPC at 200 and 300 mg/kg significantly (P <0.05 and P <0.01) reduced rat body temperature by 0.80±0.02 and 1.20±0.03OC respectively as compared to 0.90±0.02 OC for standard drug. Fig. 2 shows temperature change with time associated with induction of pyrexia by subcutaneous injection of 2.3 ml/kg of turpentine into the dorsum region of rats 11 h before treatment with 10 ml/kg normal saline as control, 50 mg/kg diclofenac sodium as standard drug, and AEPC (100, 200, 300 mg/kg) as tests.
Fig. 2. Temperature change with time after induction of pyrexia with turpentine (2.3 ml/kg ). Each point represents the mean ± SEM (n=6). *P < 0.05, ⃰ ⃰P < 0.01
Fig. 3. Mean number of writhes per experimental group after injecting (i.p) 10 ml/kg acetic acid (0.6%, v/v in saline). Data were presented as mean ± SEM (n=6). *P < 0.05
The Fig. 3 shows mean number of writhes in experimental groups. Visceral pain was induced by intraperitoneal injection of 10 ml/kg acetic acid (0.6%, v/v in saline) 30 min post treatment with 10 ml/kg saline as control, 50 mg/kg diclofenac sodium as standard drug, AEPC (100, 200 and 300 mg/kg) as tests. At a concentration of 300 mg/kg, the number of writhes was significantly (P <0.05) reduced to 4.25 from 31.50 for control and 9.25 for
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diclofenac sodium.
Fig. 4. Mean number of licks per experimental groups after injecting (s.p) 20 μl formalin (5% v/v in saline). Data were presented as mean ± SEM (n=6). *P < 0.05. Fig. 4 shows mean number of licks in various experimental groups. AEPC (300 mg/kg) significantly (P <0.05) inhibited mean number of licks to 4.99 while standard had 4.21 as compared to 29.25 for control group.
4. Discussion
Pyrexia was induced by administration of 10 ml/kg of DNP intraperitoneally. It is known that DNP induces febrile through uncoupling oxidative phosphorylation [30] resulting in fast consumption of energy without generating adenosine triphosphate. The energy of the proton gradient is then lost as heat (pyrexia) [31]. DNP does elevate resting metabolic rates to 50% [32].
Thirty minutes post treatment or 60 min post induction of pyrexia the temperature of the rats decreased variably except Group III. This could be due to physical cooling effect from the volume of water contained in the solutions. One hour post treatment, only Groups IV (200 mg/kg) and V (300 mg/kg) further had decrease in temperature. This suggests that phytoactive compounds in the extract were blocking heat production. Two hours post treatment, Groups III-V had temperature decreased significantly (P <0.05) compared to control. This might be attributed to production of free radical scavenging activity by flavonoid in the plant extract since DNP acts as a protonophore by allowing protons to bypass ATP synthase through leakage across the inner mitochondrial membrane. In addition, the extract might have anti-prostaglandin activity since prostaglandin signals to the hypothalamus to increase body's thermal set point [33]. Diclofenact sodium, however, acts via inhibition of prostaglandin synthesis through inhibition of cyclooxygenase-2 [34] and it might be why the extract performed better. Pyrexia was also induced by subcutaneous injection of 2.3 ml/kg turpentine into the dorsum region of rats. Turpentine induces febrile slowly reaching peak 11 h post injection. Macrophages release protein signals interleukin-1 and interleukin-6 to counteract the turpentine (pyrogen) [35]. This is achieved through interleukin acting on the temperature-regulating hypothalamus to increase body temperature (pyrexia) [36], stimulating hepatocystes to secrete acute-phase proteins [37] and increasing the quantity of circulating eosinophils and neutrophils to neutralize turpentine. AEPC significantly decreased body temperature of rats in turpentine-induced pyrexia at peak (11 h) suggesting that the extract might have blocking potential one temperature-regulating receptors of the hypothalamus.
Writhing test was used to assess peripheral analgesic activity of plant extract by intraperitoneal inject of 10
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ml/kg acetic acid (0.6%, v/v in saline). Mediators involved in visceral pain transmission include acetylcholine, kinin, calcitonin-gene-related peptide, substance P and prostaglandin [38]. At a concentration of 300 mg/kg, the number of writhes was significantly reduced. This suggests that bioactive chemical found in the extract antagonized peripheral mediators of visceral pain transmission by blocking mediators’ transmission of pain. Formalin test is a selective test for accessing centrally acting analgesia because its effect is mediated through opoid receptors widely distributed in the CNS. AEPC (300 mg/kg) significantly inhibited mean number of licks which suggests that the extract acts centrally and probably has increased activation threshold or has desensitized nociceptors. In evaluating peripheral analgesia, acetic acid-induced writhing test is well known model which is mediated by peritoneal mast cells, prostaglandin pathways and acid sensing ion channel [39, 40]. AEPC (300 mg/kg) maximally inhibited mean number of writhes suggesting that the extract interfered with writhes excitation since it contains flavonoid which might have produced free radical scavenging activity because free radicals have been implicated in pain excitation. From the present study, aqueous extract of Phragmanthera capitata has enormous anti-pyretic and analgesic properties which corroborate the extract being used to relief fever and abdominal pain in Cameroon folk medicine.
Acknowledgement
Authors wish to thank Mr. Takem Bertrand Mokom for assistance in sourcing and preparing whole plant sample following standard procedures.
