Journal of Ethnopharmacology 158 (2014) 271–275
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Research Paper
Evaluation of antinociceptive and anti-inflammatory activities of standardised rootbark extract of Xeromphis nilotica Bulus Adzu a,n,1, Mohammed Barau Amizan b, Samuel Ehiabhi Okhale c a
Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria Directorate of Pharmaceutical Services, Ministry of Health, Damaturu, Yobe State, Nigeria c Department of Medicinal Plant Research and Traditional Medicine, NIPRD, Abuja, Nigeria b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 August 2014 Received in revised form 11 October 2014 Accepted 17 October 2014 Available online 29 October 2014
Ethnopharmacological relevance: Xeromphis nilotica (Stapf) Keay (Rubiaceae), popularly known as ‘barbaji’ (in Nigeria), is a lowland shrub that grows wild in tropical areas of Africa and Asia. The plant's extract is used for the treatment of various diseases in folk medicine including pain related ailments. Important bioactive constituents have been isolated from the plant among them are coumarin, alkaloids, flavonoids, saponins, and terpenes. This study is aimed to evaluate the analgesic and anti-inflammatory efficacy of standardised aqueous extract of the plant using in vivo models of pain and inflammation in mice and rats. Materials and methods: Aqueous extract of Xeromphis nilotica root bark was prepared and standardised using HPLC technique. Three dose levels (25, 100 and 400 mg/kg) of the extract were used, administered orally to laboratory mice and rats. Acetylsalicylic acid (100 mg/kg, p.o.) was used as the positive control. Nociception was induced in laboratory rodents: chemically using acetic acid and formalin, and mechanically using analgesy meter; while inflammation was induced using fresh raw egg albumin. Results: The extract showed 11 constituents peak profiles in the HPLC analysis. The extract alleviates mice response to acetic acid-induced writhing, analgesy-meter and formalin tests. It significantly decreased the oedema induced by egg albumin induced inflammation, but failed to show significant effect beyond 80 min of the test. Conclusion: The extract has antinociceptive effect and short acting anti-inflammatory activities. The results justify its usage in the treatment of pain and inflammatory conditions, and also provided evidence of its potential as source of new pain relief drug prototype. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Xeromphis nilotica Aqueous extract Antinociceptive Anti-inflammatory
1. Introduction Medicinal plants used in folkloric treatment of pain related ailments have a long and popular usage (Almeida et al., 2001) especially in developing countries (Jager, 2005). Interestingly, the World Health Organization (WHO) has recommended the integration of traditional medicines proved to be useful into national health care programmes, and developed a strategy to address issues of policy, safety, efficacy, quality, access and rational use (WHO Traditional Medicine Strategy 2014–2023). Xeromphis nilotica (Stapf) Keay – Bull. Jard. Bot. État Bruxelles 28: 39. 1958 (IK) family: Rubiaceae (IPNI, 2012), is a lowland shrub that grows wild in the savannah and other tropical regions of Africa (Dalziel, 1937; Hedberg et al., 1983; El Ghazali, 1993; El-Kamali, 2009) n
Corresponding author. Tel.: þ 55 65 8146 1081. E-mail address: [email protected] (B. Adzu). 1 Current address: Laboratório de Farmacologia, Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil. http://dx.doi.org/10.1016/j.jep.2014.10.030 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
and Asia (Farooqui et al., 2013). The plant is locally known as either ‘barbaji’, ‘gial-goti’, ‘kwanarya’, or ‘tsibra’ in northern Nigeria (Dalziel, 1937; Danjuma et al., 2009), and is reputed to be of medicinal value in the folkloric treatment of various ailments that include; pain, fever, epilepsy and mental disorder (Hedberg et al., 1983; Chhabra et al., 1987). Biological evaluations of the plant material showed that it exhibited active antischistosomal (Sulaiman et al., 1988), and molluscidal (El Kheir and Salih, 1980) activities, and preliminary pharmacological studies in our laboratory and elsewhere show that the plant rootbark extracts' exhibited CNS activities that were sedative in nature (Adzu et al., 2008; Danjuma et al., 2009). The plant was also reported to contain useful phytochemicals that include carbohydrate, coumarin, alkaloids, flavonoids, saponins, and terpenes (El Kheir and Salih, 1980; Lemmich et al., 1995; Farid et al., 2002). The scientific basis for its folkloric use as pain remedy has not been evaluated to the best of our knowledge. In this report, we investigated the plant's use in the treatment of pain related ailments (Hedberg et al., 1983; Adzu et al., 2008) by evaluating the antinociceptive and anti-inflammatory activities of
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its aqueous extract. The extracts' constituent profiles were also standardised using HPLC.
2. Materials and methods 2.1. Plant extract The plant material was collected around Funtua (111320 0″ N; 71190 0″ E), Katsina State, Nigeria through herbal medicine practitioner by co-author (M.B. Amizan). It was authenticated at the Department of Biological Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria, where voucher specimen (no. 2867) is being kept at the Herbarium of the department. Xeromphis nilotica is not endangered plant species and therefore its collection for purposes of use and scientific study does not require prior authorisation. The collected roots of the plant were dried under shade, powdered, and 200 g of the powdered material was extracted in 1 l of distilled water. The mixture was filtered, filtrate freeze dried (Lyovac GT2, Germany) and standardised as described below (Section 2.2.) to yield a dark brown standardised aqueous extract of Xeromphis. nilotica (SAEXn). 2.2. Standardisation of SAEXn using high performance liquid chromatography analysis This analysis was performed to establish the extract constituents' marker peaks for subsequent quality and stability checks. The test was performed using the procedure reported by Chindo et al. (2014). The system used (Shimadzu Corporation, Kyoto Japan) consist of Ultra-Fast LC-20AB prominence with the following accessories: SIL-20AC auto-sampler; DGU-20A3 degasser; SPDM20A UV-diode array detector; VP-ODS 5 mm column; CTO-20AC column oven, CBM-20Alite system controller and Windows LCsolution software. The chromatographic conditions were made up of a mobile phase: solvent A: 0.2% v/v formic acid and solvent B: acetonitrile. The mode was linear gradient, flow rate was 0.6 ml/ min, injection volume; 10 ml of 300 mg/ml solution of SAEXn in HPLC grade water; the oven temperature was 40 1C, and detection wavelength: UV 254 nm. Quercetin-3-O-rutinoside (rutin) (Fluka, Germany) (50 mg/ml in methanol) was used as the internal standard. The HPLC operating conditions were programmed to give the following: at 0.01 min, 10% solvent B; at 7.5 min, 20% solvent B; at 15 min, 60% solvent B; at 20 min, 80% solvent B; at 25 min, 100% solvent B. The total run time was 25 min. Analysis was carried out for rutin only, SAEXn only, and SAEXn with rutin. 2.3. Animals Swiss albino mice and Wistar rats of both sexes obtained from the Animal Facility Centre, NIPRD, Abuja, Nigeria, were used for the study. The animals were maintained in propylene cages with sawdust as bedding and kept on standard laboratory feeds. They were however fasted prior to each experiment, but had water ad libitum that was withdrawn 1 h to commencement of experiments. We certify that the animals were used in accordance with Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals (Zimmermann, 1983), as contained in NIPRD's standard procedures for laboratory animal usage (SOP no. 05:3:06). 2.4. Acetic acid-induced writhing The model described by Koster et al. (1959) as modified for our laboratory settings (Adzu et al., 2002) was adopted. Mice were grouped into five groups (n¼ 5) and treated p.o. with water (10 ml/ kg), SAEXn (25, 100 and 400 mg/kg), or acetylsalicylic acid (ASA) (100 mg/kg). Nociception was induced in each mouse by injecting
0.75% of an aqueous solution of acetic acid (10 ml/kg, i.p.) 1 h after treatment, and placed in transparent observational cage. The number of writhes (stretching of the hind limbs accompanied by abdominal muscle constriction) exhibited by each mouse was cumulatively counted for 15 min, 5 min (lag time) after the aqueous acid injection. SAEXn percentage protection was calculated using the ratio: [(negative mean treated mean)/negative mean] 100% (Chen et al., 2012). 2.5. Analgesy meter test This experiment is a mechanical model of nociception; a modification of the Randall and Selitto (1957) test in which a meter exerts force at a constantly increased rate on rat paw monitored by a pointer moving along a linear scale. The test was carried out using Ugo Basile Analgesy-Meter (no. 7200, Italy) on normal rats as described by Vongtau et al. (2004). Twenty five rats were grouped into five groups (n¼5) and treated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), or ASA (100 mg/kg). The rat paw was gently placed between the plinth and plunger of the instrument 1 h after treatment and increased pressure (exerted by 20 g) applied to the middle dorsum of the rat's left hind paw. Stimulus was terminated and force threshold readings taken as soon as nociceptive response were elicited by the rats. Readings were taken pre- and at 15, 30 and 60 min after treatment (Adzu and Haruna, 2007). 2.6. Formalin test The method described by Tjolsen et al. (1992) was adopted with little modification (Adzu et al., 2003). The formalin test model is biphasic, and measures pain of both neurogenic (first phase), and of inflammatory origin (second phase). Five groups of rats (n ¼5) were pretreated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), or ASA (100 mg/kg), and were injected s.c. with 50 ml solution of 2.5% formalin into the sub-plantar surface of rat left hind paw 1 h after the treatment. Severity of pain was rated in two distinct phases for 60 min: the first phase (0–10 min) taking every 2 min and late phase (15–60 min) every 5 min using 3 prominent pain-induced behaviour in the following scoring manner: 0 – normal weight baring on the injected paw; 1 – light resting on the paw on the floor; 2 – elevation of the injected paw and 3 – for licking, biting or grooming of the injected paw. The mean (7SEM) of the readings was recorded as the pain score. This model is accompanied by the development of oedema in the injected left paw due to release of inflammatory mediators (Akah et al., 1993). And to evaluate the anti-inflammatory activity of SAEXn on this process, the left paw oedema volume was measured and compared with that of the right hind paw using a LETICA (Spain) digital plethysmometer (LE 7500) calibrated with 0.1% Triton X-100 (BDH, UK) at the end of the duration of the assay, 1 h after formalin injection. 2.7. Egg albumin-induced oedema This test is based on the principle that oedema induced by phlogistic agents can be used to evaluate the anti-inflammatory effects of drugs (Winter et al., 1963). The test was performed using fresh raw egg albumin to induce oedema as described by Akah and Nwambie (1994). Rats were divided into five groups (n¼5) and treated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), and ASA (100 mg/ kg). 1 h after this treatment, inflammation was induced by injecting 0.1 ml of fresh egg albumin into sub-plantar of the rat's left hind paw. Oedema volume was measured with the LETICA digital plethysmometer calibrated with 0.1% Triton X-100. Readings were taken 5 min after oedema induction and at an interval of 20 min for a total of 120 min after treatment (Adzu and Haruna, 2007).
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2.8. Data analysis Results were expressed as mean7SEM. The data were analysed using GraphPad Prism Version 5.01 for Windows, GraphPad Prism Software (San Diego California). Student t-test was used to analyse level of statistical significance between groups and one way-ANOVA among groups followed by Dunnett's test for multiple comparisons.
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Preliminary phytochemical test earlier performed on aqueous extract of Xeromphis nilotica showed the presence of saponins, tannins, glycosides, alkaloids and flavonoids (Adzu et al., 2008) and these phytochemicals might be responsible for the peaks, and the biological activities of the extract. The in vivo acute toxicity (LD50) of aqueous extract of Xeromphis nilotica was established to Table 1 Effect of SAEXn on acetic acid-induced writhing test in mice.
3. Results 3.1. HPLC analysis The HPLC analysis detected 11 peaks due to SAEXn beside that of the reference standard (rutin) as shown in Fig. 1.
Treatment
Dose (mg/kg, p.o.)
% Inhibition of nociceptiona
SAEXn
25 100 400 100
28.3 45.7 69.6 80.8
ASA a
Mean 7 SEM, n¼5.
3.2. Acetic acid-induced writhing SAEXn significantly inhibited the noxious stimulus induced by acetic acid, by 28–70% when compared with the control. ASA however gave superior (81% inhibition) activity (Table 1).
Table 2 Effect of SAEXn on analgesy-meter test in rats. Treatment
3.3. Analgesy The result expressed as threshold time showed that SAEXn exhibited significant activity against the mechanically induced pain at 100 and 400 mg/kg, p.o., as from 30 min after treatment (Table 2).
VehicleSAEXn
ASA
3.4. Formalin test
a
SAEXn inhibited both phases of the formalin test in the groups that received 100 and 400 mg/kg, p.o. It however failed to show significant inhibition of the subsequent oedema induced by formalin (Table 3).
n
The result showed that SAEXn exhibited anti-inflammatory activities in this model, but failed to elicit significant effect beyond 80 min in the group that received 25 and 100 mg/kg, p.o. (Table 4).
Vehicle SAEXn
a
The HPLC analysis shows 11 peaks due to metabolic classes in SAEXn or their synergistic interactions identified by the technique.
4.767 1.5 4.077 1.2 3.96 7 0.4 4.60 7 0.5 4.247 0.8
Threshold (mean 7SEM)a 15 min
30 min
60 min
3.8 7 1.1 4.5 7 0.9 5.6 7 1.3 6.6 7 1.8n 7.6 7 0.9n
4.80 7 0.9 5.767 0.9 6.17 1.6n 6.7 7 1.0n 8.60 7 1.7n
3.8 7 0.9 5.2 7 2.1n 9.3 7 1.0n 6.0 7 2.2n 9.7 7 1.7n
Table 3 Result of the formalin induced noxious stimulus test in rats.
ASA
4. Discussion
25 100 400 100
Pre-treatment
x 20 g, n ¼5. Significantly different from vehicle po 0.05.
Treatment
3.5. Egg albumin-induced inflammation
Dose (mg/kg, p.o.)
b n
Dose (mg/kg, p.o.)
– 25 100 400 100
Pain scorea,b
Oedema volume (cm3)
First phase
Second phase
2.56 7 0.2 2.2 7 0.2 1.75 7 0.2n 0.88 7 0.2n 1.9 7 0.3
2.487 0.1 1.98 7 0.1 1.727 0.2n 1.917 0.1n 1.157 0.1n
Maximum score¼ 3. Mean 7 SEM, n¼5. Significantly different from vehicle po 0.05.
Fig. 1. HPLC profile of SAEXn.
0.277 0.07 0.187 0.02 0.2 7 0.02 0.2 7 0.02 0.32 7 0.04
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Table 4 Result of the egg albumin-induced paw oedema test in rats. Treatment
Vehicle SAEXn
ASA a n
Dosea
– 50 100 400 100
Experimental time (min) 20
40
60
80
100
120
0.52 70.04 0.47 70.02 0.36 70.02 0.32 70.07 0.31 70.03
0.46 7 0.02 0.447 0.03 0.32 7 0.06n 0.337 0.07n 0.317 0.07n
0.417 0.04 0.34 7 0.04n 0.28 7 0.03n 0.25 7 0.06n 0.167 0.05n
0.32 7 0.08 0.26 7 0.04 0.247 0.07n 0.23 7 0.07n 0.157 0.01n
0.277 0.04 0.26 7 0.05 0.217 0.03 0.187 0.02n 0.12 7 0.04n
0.22 7 0.03 0.2 7 0.06 0.197 0.05 0.167 0.04n 0.117 0.02n
mg/kg, p.o.; values in parenthesis are paw oedema volume (cm3). po 0.05.
be 1549 mg/kg in mice (Adzu et al., 2008), meaning that the experimental doses used in this study (25, 100 and 400 mg/kg) were within safe margin. ASA was chosen as the standard drug in this study because of its ability to irreversibly inhibit COX-1 and modifies the enzymatic activity of COX-2. COX-2 normally produces prostanoids, most of which are pro-inflammatory (Day et al., 1988). When administered, ASA rapidly deacetylates producing salicylate that blocked prostaglandin synthesis thereby preventing sensitization of pain receptors to both mechanical and chemical stimulus (Clark et al., 2012); the pain models used in this study. In vivo experiments using laboratory animal systems have been used in pharmacological evaluations to generate reliable data and gives high predictive value of efficacy in humans (Normandin, 2007). The in vivo experimental models were chosen in such a way that the existence of both central and peripherally mediated pain relief could be measured (Adzu and Haruna, 2007). SAEXn exhibited significant activity on the acetic acid-induced writhing test, indicating antinociception potency. The test is associated with increased levels of PGE and PGF2α in peritoneal fluids (Deraedt et al., 1980), and sympathetic nervous system mediators (Duarte et al., 1988; Borsato et al., 2000) leading to production of algogenic substances at the peritoneal receptors (Bentley et al., 1981). It is a sensitive test that can detect activity that may appear inactive in other methods (Collier et al., 1968). However, the test cannot indicate the exact mechanism of analgesic effect of test substances (Stai et al., 1995) with false positive occurring with agents like sedatives (Elisabetsky et al., 1995). Thus, other models were explored in this study. SAEXn showed potency on the mechanical model using analgesy-meter. The test measures analgesic activity based on the principle that inflammation increases the sensitivity to nociception and this sensitivity is susceptible to modification by analgesics (Vogel, 2002). The fact that SAEXn increased the threshold of the intact paw suggests involvement of both peripheral and centrally mediated activity (Vongtau et al., 2004). SAEXn exhibited potent analgesic activity on both phases of the formalin test. This test which is biphasic is recommended as a basic pain research tool for studying the mechanisms of analgesic agents (Hunskaar and Hole, 1987) because of its connection to tissue injury (Tjolsen et al., 1992). The first phase involves stimulation of nociception in the paw mediated centrally, while the second is the activation of local inflammatory processes that stimulates pain sensation and to some degree, sensitization of nociceptive neurons similar to clinical pain (Tjolsen et al., 1992). Suppression of both phases of pain by SAEXn as observed in this study provide further evidence of dual activity involving both centrally mediated and peripherally localised pain relief mechanisms; plausibly by prostaglandin synthesis inhibition, and/or the central action of the plant extract earlier reported (Adzu et al., 2008; Danjuma et al., 2009). However, SAEXn failed to significantly inhibit the oedema induced by formalin in the test. Inflammation, a common occurrence in infective condition is a
complex array of responses of tissue to injury (Vane and Bolting, 1995) usually associated with pain as a secondary process. The result indicates that SAEXn might not be useful in long lasting neurogenic pain; unless perhaps multiple dosing is employed. This observation corroborates well with its inhibition of oedema induced by egg albumin in rat paw, which was observed in the first 80 min but was not maintained for the 120 min duration of the experiment. Egg albumin-induced oedema model is used to screen agents for an anti-inflammatory effect (Akah et al., 1993). The effect of SAEXn on this test suggests the presence of a short acting anti-inflammatory agent. Conclusively, SAEXn most likely acts through an intertwine action of blocking the stimulus propagation in the pain nervous fibres and/or decrease in the production of prostaglandins responsible for pain mediation (Matheus et al., 2005). Whereas the result is in agreement with the folkloric experience of its crude usage, it is also a pointer to its worth as potential source of new analgesic; especially for the fact that important analgesic prototypes like salicylic acid and morphine were originally derived from plant kingdom (Elisabetsky et al., 1995) and cases have been built against many of them, demanding that they should be changed (Argoff, 2013) for better alternative.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Evaluation of antinociceptive and anti-inflammatory activities of standardised rootbark extract of Xeromphis nilotica Bulus Adzu a,n,1, Mohammed Barau Amizan b, Samuel Ehiabhi Okhale c a
Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria Directorate of Pharmaceutical Services, Ministry of Health, Damaturu, Yobe State, Nigeria c Department of Medicinal Plant Research and Traditional Medicine, NIPRD, Abuja, Nigeria b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 August 2014 Received in revised form 11 October 2014 Accepted 17 October 2014 Available online 29 October 2014
Ethnopharmacological relevance: Xeromphis nilotica (Stapf) Keay (Rubiaceae), popularly known as ‘barbaji’ (in Nigeria), is a lowland shrub that grows wild in tropical areas of Africa and Asia. The plant's extract is used for the treatment of various diseases in folk medicine including pain related ailments. Important bioactive constituents have been isolated from the plant among them are coumarin, alkaloids, flavonoids, saponins, and terpenes. This study is aimed to evaluate the analgesic and anti-inflammatory efficacy of standardised aqueous extract of the plant using in vivo models of pain and inflammation in mice and rats. Materials and methods: Aqueous extract of Xeromphis nilotica root bark was prepared and standardised using HPLC technique. Three dose levels (25, 100 and 400 mg/kg) of the extract were used, administered orally to laboratory mice and rats. Acetylsalicylic acid (100 mg/kg, p.o.) was used as the positive control. Nociception was induced in laboratory rodents: chemically using acetic acid and formalin, and mechanically using analgesy meter; while inflammation was induced using fresh raw egg albumin. Results: The extract showed 11 constituents peak profiles in the HPLC analysis. The extract alleviates mice response to acetic acid-induced writhing, analgesy-meter and formalin tests. It significantly decreased the oedema induced by egg albumin induced inflammation, but failed to show significant effect beyond 80 min of the test. Conclusion: The extract has antinociceptive effect and short acting anti-inflammatory activities. The results justify its usage in the treatment of pain and inflammatory conditions, and also provided evidence of its potential as source of new pain relief drug prototype. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Xeromphis nilotica Aqueous extract Antinociceptive Anti-inflammatory
1. Introduction Medicinal plants used in folkloric treatment of pain related ailments have a long and popular usage (Almeida et al., 2001) especially in developing countries (Jager, 2005). Interestingly, the World Health Organization (WHO) has recommended the integration of traditional medicines proved to be useful into national health care programmes, and developed a strategy to address issues of policy, safety, efficacy, quality, access and rational use (WHO Traditional Medicine Strategy 2014–2023). Xeromphis nilotica (Stapf) Keay – Bull. Jard. Bot. État Bruxelles 28: 39. 1958 (IK) family: Rubiaceae (IPNI, 2012), is a lowland shrub that grows wild in the savannah and other tropical regions of Africa (Dalziel, 1937; Hedberg et al., 1983; El Ghazali, 1993; El-Kamali, 2009) n
Corresponding author. Tel.: þ 55 65 8146 1081. E-mail address: [email protected] (B. Adzu). 1 Current address: Laboratório de Farmacologia, Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil. http://dx.doi.org/10.1016/j.jep.2014.10.030 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
and Asia (Farooqui et al., 2013). The plant is locally known as either ‘barbaji’, ‘gial-goti’, ‘kwanarya’, or ‘tsibra’ in northern Nigeria (Dalziel, 1937; Danjuma et al., 2009), and is reputed to be of medicinal value in the folkloric treatment of various ailments that include; pain, fever, epilepsy and mental disorder (Hedberg et al., 1983; Chhabra et al., 1987). Biological evaluations of the plant material showed that it exhibited active antischistosomal (Sulaiman et al., 1988), and molluscidal (El Kheir and Salih, 1980) activities, and preliminary pharmacological studies in our laboratory and elsewhere show that the plant rootbark extracts' exhibited CNS activities that were sedative in nature (Adzu et al., 2008; Danjuma et al., 2009). The plant was also reported to contain useful phytochemicals that include carbohydrate, coumarin, alkaloids, flavonoids, saponins, and terpenes (El Kheir and Salih, 1980; Lemmich et al., 1995; Farid et al., 2002). The scientific basis for its folkloric use as pain remedy has not been evaluated to the best of our knowledge. In this report, we investigated the plant's use in the treatment of pain related ailments (Hedberg et al., 1983; Adzu et al., 2008) by evaluating the antinociceptive and anti-inflammatory activities of
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its aqueous extract. The extracts' constituent profiles were also standardised using HPLC.
2. Materials and methods 2.1. Plant extract The plant material was collected around Funtua (111320 0″ N; 71190 0″ E), Katsina State, Nigeria through herbal medicine practitioner by co-author (M.B. Amizan). It was authenticated at the Department of Biological Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria, where voucher specimen (no. 2867) is being kept at the Herbarium of the department. Xeromphis nilotica is not endangered plant species and therefore its collection for purposes of use and scientific study does not require prior authorisation. The collected roots of the plant were dried under shade, powdered, and 200 g of the powdered material was extracted in 1 l of distilled water. The mixture was filtered, filtrate freeze dried (Lyovac GT2, Germany) and standardised as described below (Section 2.2.) to yield a dark brown standardised aqueous extract of Xeromphis. nilotica (SAEXn). 2.2. Standardisation of SAEXn using high performance liquid chromatography analysis This analysis was performed to establish the extract constituents' marker peaks for subsequent quality and stability checks. The test was performed using the procedure reported by Chindo et al. (2014). The system used (Shimadzu Corporation, Kyoto Japan) consist of Ultra-Fast LC-20AB prominence with the following accessories: SIL-20AC auto-sampler; DGU-20A3 degasser; SPDM20A UV-diode array detector; VP-ODS 5 mm column; CTO-20AC column oven, CBM-20Alite system controller and Windows LCsolution software. The chromatographic conditions were made up of a mobile phase: solvent A: 0.2% v/v formic acid and solvent B: acetonitrile. The mode was linear gradient, flow rate was 0.6 ml/ min, injection volume; 10 ml of 300 mg/ml solution of SAEXn in HPLC grade water; the oven temperature was 40 1C, and detection wavelength: UV 254 nm. Quercetin-3-O-rutinoside (rutin) (Fluka, Germany) (50 mg/ml in methanol) was used as the internal standard. The HPLC operating conditions were programmed to give the following: at 0.01 min, 10% solvent B; at 7.5 min, 20% solvent B; at 15 min, 60% solvent B; at 20 min, 80% solvent B; at 25 min, 100% solvent B. The total run time was 25 min. Analysis was carried out for rutin only, SAEXn only, and SAEXn with rutin. 2.3. Animals Swiss albino mice and Wistar rats of both sexes obtained from the Animal Facility Centre, NIPRD, Abuja, Nigeria, were used for the study. The animals were maintained in propylene cages with sawdust as bedding and kept on standard laboratory feeds. They were however fasted prior to each experiment, but had water ad libitum that was withdrawn 1 h to commencement of experiments. We certify that the animals were used in accordance with Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals (Zimmermann, 1983), as contained in NIPRD's standard procedures for laboratory animal usage (SOP no. 05:3:06). 2.4. Acetic acid-induced writhing The model described by Koster et al. (1959) as modified for our laboratory settings (Adzu et al., 2002) was adopted. Mice were grouped into five groups (n¼ 5) and treated p.o. with water (10 ml/ kg), SAEXn (25, 100 and 400 mg/kg), or acetylsalicylic acid (ASA) (100 mg/kg). Nociception was induced in each mouse by injecting
0.75% of an aqueous solution of acetic acid (10 ml/kg, i.p.) 1 h after treatment, and placed in transparent observational cage. The number of writhes (stretching of the hind limbs accompanied by abdominal muscle constriction) exhibited by each mouse was cumulatively counted for 15 min, 5 min (lag time) after the aqueous acid injection. SAEXn percentage protection was calculated using the ratio: [(negative mean treated mean)/negative mean] 100% (Chen et al., 2012). 2.5. Analgesy meter test This experiment is a mechanical model of nociception; a modification of the Randall and Selitto (1957) test in which a meter exerts force at a constantly increased rate on rat paw monitored by a pointer moving along a linear scale. The test was carried out using Ugo Basile Analgesy-Meter (no. 7200, Italy) on normal rats as described by Vongtau et al. (2004). Twenty five rats were grouped into five groups (n¼5) and treated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), or ASA (100 mg/kg). The rat paw was gently placed between the plinth and plunger of the instrument 1 h after treatment and increased pressure (exerted by 20 g) applied to the middle dorsum of the rat's left hind paw. Stimulus was terminated and force threshold readings taken as soon as nociceptive response were elicited by the rats. Readings were taken pre- and at 15, 30 and 60 min after treatment (Adzu and Haruna, 2007). 2.6. Formalin test The method described by Tjolsen et al. (1992) was adopted with little modification (Adzu et al., 2003). The formalin test model is biphasic, and measures pain of both neurogenic (first phase), and of inflammatory origin (second phase). Five groups of rats (n ¼5) were pretreated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), or ASA (100 mg/kg), and were injected s.c. with 50 ml solution of 2.5% formalin into the sub-plantar surface of rat left hind paw 1 h after the treatment. Severity of pain was rated in two distinct phases for 60 min: the first phase (0–10 min) taking every 2 min and late phase (15–60 min) every 5 min using 3 prominent pain-induced behaviour in the following scoring manner: 0 – normal weight baring on the injected paw; 1 – light resting on the paw on the floor; 2 – elevation of the injected paw and 3 – for licking, biting or grooming of the injected paw. The mean (7SEM) of the readings was recorded as the pain score. This model is accompanied by the development of oedema in the injected left paw due to release of inflammatory mediators (Akah et al., 1993). And to evaluate the anti-inflammatory activity of SAEXn on this process, the left paw oedema volume was measured and compared with that of the right hind paw using a LETICA (Spain) digital plethysmometer (LE 7500) calibrated with 0.1% Triton X-100 (BDH, UK) at the end of the duration of the assay, 1 h after formalin injection. 2.7. Egg albumin-induced oedema This test is based on the principle that oedema induced by phlogistic agents can be used to evaluate the anti-inflammatory effects of drugs (Winter et al., 1963). The test was performed using fresh raw egg albumin to induce oedema as described by Akah and Nwambie (1994). Rats were divided into five groups (n¼5) and treated p.o. with water (10 ml/kg), SAEXn (25, 100 and 400 mg/kg), and ASA (100 mg/ kg). 1 h after this treatment, inflammation was induced by injecting 0.1 ml of fresh egg albumin into sub-plantar of the rat's left hind paw. Oedema volume was measured with the LETICA digital plethysmometer calibrated with 0.1% Triton X-100. Readings were taken 5 min after oedema induction and at an interval of 20 min for a total of 120 min after treatment (Adzu and Haruna, 2007).
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2.8. Data analysis Results were expressed as mean7SEM. The data were analysed using GraphPad Prism Version 5.01 for Windows, GraphPad Prism Software (San Diego California). Student t-test was used to analyse level of statistical significance between groups and one way-ANOVA among groups followed by Dunnett's test for multiple comparisons.
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Preliminary phytochemical test earlier performed on aqueous extract of Xeromphis nilotica showed the presence of saponins, tannins, glycosides, alkaloids and flavonoids (Adzu et al., 2008) and these phytochemicals might be responsible for the peaks, and the biological activities of the extract. The in vivo acute toxicity (LD50) of aqueous extract of Xeromphis nilotica was established to Table 1 Effect of SAEXn on acetic acid-induced writhing test in mice.
3. Results 3.1. HPLC analysis The HPLC analysis detected 11 peaks due to SAEXn beside that of the reference standard (rutin) as shown in Fig. 1.
Treatment
Dose (mg/kg, p.o.)
% Inhibition of nociceptiona
SAEXn
25 100 400 100
28.3 45.7 69.6 80.8
ASA a
Mean 7 SEM, n¼5.
3.2. Acetic acid-induced writhing SAEXn significantly inhibited the noxious stimulus induced by acetic acid, by 28–70% when compared with the control. ASA however gave superior (81% inhibition) activity (Table 1).
Table 2 Effect of SAEXn on analgesy-meter test in rats. Treatment
3.3. Analgesy The result expressed as threshold time showed that SAEXn exhibited significant activity against the mechanically induced pain at 100 and 400 mg/kg, p.o., as from 30 min after treatment (Table 2).
VehicleSAEXn
ASA
3.4. Formalin test
a
SAEXn inhibited both phases of the formalin test in the groups that received 100 and 400 mg/kg, p.o. It however failed to show significant inhibition of the subsequent oedema induced by formalin (Table 3).
n
The result showed that SAEXn exhibited anti-inflammatory activities in this model, but failed to elicit significant effect beyond 80 min in the group that received 25 and 100 mg/kg, p.o. (Table 4).
Vehicle SAEXn
a
The HPLC analysis shows 11 peaks due to metabolic classes in SAEXn or their synergistic interactions identified by the technique.
4.767 1.5 4.077 1.2 3.96 7 0.4 4.60 7 0.5 4.247 0.8
Threshold (mean 7SEM)a 15 min
30 min
60 min
3.8 7 1.1 4.5 7 0.9 5.6 7 1.3 6.6 7 1.8n 7.6 7 0.9n
4.80 7 0.9 5.767 0.9 6.17 1.6n 6.7 7 1.0n 8.60 7 1.7n
3.8 7 0.9 5.2 7 2.1n 9.3 7 1.0n 6.0 7 2.2n 9.7 7 1.7n
Table 3 Result of the formalin induced noxious stimulus test in rats.
ASA
4. Discussion
25 100 400 100
Pre-treatment
x 20 g, n ¼5. Significantly different from vehicle po 0.05.
Treatment
3.5. Egg albumin-induced inflammation
Dose (mg/kg, p.o.)
b n
Dose (mg/kg, p.o.)
– 25 100 400 100
Pain scorea,b
Oedema volume (cm3)
First phase
Second phase
2.56 7 0.2 2.2 7 0.2 1.75 7 0.2n 0.88 7 0.2n 1.9 7 0.3
2.487 0.1 1.98 7 0.1 1.727 0.2n 1.917 0.1n 1.157 0.1n
Maximum score¼ 3. Mean 7 SEM, n¼5. Significantly different from vehicle po 0.05.
Fig. 1. HPLC profile of SAEXn.
0.277 0.07 0.187 0.02 0.2 7 0.02 0.2 7 0.02 0.32 7 0.04
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Table 4 Result of the egg albumin-induced paw oedema test in rats. Treatment
Vehicle SAEXn
ASA a n
Dosea
– 50 100 400 100
Experimental time (min) 20
40
60
80
100
120
0.52 70.04 0.47 70.02 0.36 70.02 0.32 70.07 0.31 70.03
0.46 7 0.02 0.447 0.03 0.32 7 0.06n 0.337 0.07n 0.317 0.07n
0.417 0.04 0.34 7 0.04n 0.28 7 0.03n 0.25 7 0.06n 0.167 0.05n
0.32 7 0.08 0.26 7 0.04 0.247 0.07n 0.23 7 0.07n 0.157 0.01n
0.277 0.04 0.26 7 0.05 0.217 0.03 0.187 0.02n 0.12 7 0.04n
0.22 7 0.03 0.2 7 0.06 0.197 0.05 0.167 0.04n 0.117 0.02n
mg/kg, p.o.; values in parenthesis are paw oedema volume (cm3). po 0.05.
be 1549 mg/kg in mice (Adzu et al., 2008), meaning that the experimental doses used in this study (25, 100 and 400 mg/kg) were within safe margin. ASA was chosen as the standard drug in this study because of its ability to irreversibly inhibit COX-1 and modifies the enzymatic activity of COX-2. COX-2 normally produces prostanoids, most of which are pro-inflammatory (Day et al., 1988). When administered, ASA rapidly deacetylates producing salicylate that blocked prostaglandin synthesis thereby preventing sensitization of pain receptors to both mechanical and chemical stimulus (Clark et al., 2012); the pain models used in this study. In vivo experiments using laboratory animal systems have been used in pharmacological evaluations to generate reliable data and gives high predictive value of efficacy in humans (Normandin, 2007). The in vivo experimental models were chosen in such a way that the existence of both central and peripherally mediated pain relief could be measured (Adzu and Haruna, 2007). SAEXn exhibited significant activity on the acetic acid-induced writhing test, indicating antinociception potency. The test is associated with increased levels of PGE and PGF2α in peritoneal fluids (Deraedt et al., 1980), and sympathetic nervous system mediators (Duarte et al., 1988; Borsato et al., 2000) leading to production of algogenic substances at the peritoneal receptors (Bentley et al., 1981). It is a sensitive test that can detect activity that may appear inactive in other methods (Collier et al., 1968). However, the test cannot indicate the exact mechanism of analgesic effect of test substances (Stai et al., 1995) with false positive occurring with agents like sedatives (Elisabetsky et al., 1995). Thus, other models were explored in this study. SAEXn showed potency on the mechanical model using analgesy-meter. The test measures analgesic activity based on the principle that inflammation increases the sensitivity to nociception and this sensitivity is susceptible to modification by analgesics (Vogel, 2002). The fact that SAEXn increased the threshold of the intact paw suggests involvement of both peripheral and centrally mediated activity (Vongtau et al., 2004). SAEXn exhibited potent analgesic activity on both phases of the formalin test. This test which is biphasic is recommended as a basic pain research tool for studying the mechanisms of analgesic agents (Hunskaar and Hole, 1987) because of its connection to tissue injury (Tjolsen et al., 1992). The first phase involves stimulation of nociception in the paw mediated centrally, while the second is the activation of local inflammatory processes that stimulates pain sensation and to some degree, sensitization of nociceptive neurons similar to clinical pain (Tjolsen et al., 1992). Suppression of both phases of pain by SAEXn as observed in this study provide further evidence of dual activity involving both centrally mediated and peripherally localised pain relief mechanisms; plausibly by prostaglandin synthesis inhibition, and/or the central action of the plant extract earlier reported (Adzu et al., 2008; Danjuma et al., 2009). However, SAEXn failed to significantly inhibit the oedema induced by formalin in the test. Inflammation, a common occurrence in infective condition is a
complex array of responses of tissue to injury (Vane and Bolting, 1995) usually associated with pain as a secondary process. The result indicates that SAEXn might not be useful in long lasting neurogenic pain; unless perhaps multiple dosing is employed. This observation corroborates well with its inhibition of oedema induced by egg albumin in rat paw, which was observed in the first 80 min but was not maintained for the 120 min duration of the experiment. Egg albumin-induced oedema model is used to screen agents for an anti-inflammatory effect (Akah et al., 1993). The effect of SAEXn on this test suggests the presence of a short acting anti-inflammatory agent. Conclusively, SAEXn most likely acts through an intertwine action of blocking the stimulus propagation in the pain nervous fibres and/or decrease in the production of prostaglandins responsible for pain mediation (Matheus et al., 2005). Whereas the result is in agreement with the folkloric experience of its crude usage, it is also a pointer to its worth as potential source of new analgesic; especially for the fact that important analgesic prototypes like salicylic acid and morphine were originally derived from plant kingdom (Elisabetsky et al., 1995) and cases have been built against many of them, demanding that they should be changed (Argoff, 2013) for better alternative.
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