Analgesic, Anti-Inflammatory and Anti-Oxidant Activities of Clausena anisata leaf and Stem Extracts and Quantitative and Qualitative Phytochemical Analysis of their ContentsKumatia EK1*, Ayertey F1, Appiah AA1 and Ocloo A2
2Pharmacology Department, Centre for Plant Medicine Research, Mampong-Akwapim, Ghana
Kumatia EK, Department of Phytochemistry, Centre for Plant Medicine Research, P.O. Box 73, Mampong-Akwapim, Ghana, Tel: +233 342195766, Email: firstname.lastname@example.org, email@example.com
Clausena anisata is a medicinal plant use to treat diseases including inflammatory, pain and oxidative stress conditions. Nonetheless, these activities have not been evaluated for its stem. This study aimed to evaluate and compared analgesic, anti-inflammatory and anti-oxidant activities of ethanol extracts of leaf (CLE) and stem (CSE) of C. anisata. Anti-inflammatory and anti-oxidant activities were assessed by carrageenan induced paw edema and DPPH free radical scavenging assays respectively. Hot plate and writhing assays were used to test the analgesic activity. Total phenolics and flavonoids were quantified using UV-Vis spectrophotometry methods. CLE and CSE produced (p<0.05) dose-depended anti-inflammatory activity (76.35-85.83%) comparable to that of indomethacin (78.26%) and (p< 0.05) analgesic activity in the writhing assay. The extracts also produce (p<0.0001) overall analgesic activity in the hot plate assay comparable to tramadol, although, their activities were short lived. EC50 values obtained in the anti-oxidant activity showed that ascorbic acid (standard) was 86 and 74 times potent than CLE and CSE respectively. Total flavonoids and phenolics obtained per 100 mg of CLE or CSE were 2.46 ± 0.08 and 3.93 ± 0.122 mg QE (Quercetin Equivalent) respectively and 6.69 ± 0.54 and 7.45 ± 0.64 mg GAE (Gallic Acid Equivalent) respectively. Flavonoids and phenolic contents of CSE were higher than CLE. This may partly explain where CSE was more active than CLE in all the assays. These results show that CLE and CSE possessed significant anti-inflammatory and analgesic activities and weak anti-oxidant activity.
Pain, Inflammation, Alkaloid, Coumarin, Triterpene.
Pain is a protective mechanism that helps a living organism to get rid of obnoxious stimuli in other to avoid harm. However, alterations in the pain pathway produce hypersensitivity, so that pain metamorphosize from short-term warning signal to chronic devastating condition . Likewise, inflammation is a key element of the immune system that is activated by any stimulus that poses actual or apparent hazard to tissue homeostasis . Acute inflammatory reaction is self-limiting and leads to tissue repair and return of tissue homeostasis. Conversely, chronic inflammation is implicated in diseases such as diabetes, fatty liver, obesity, rheumatoid arthritis and atherosclerosis [3-4].
Moreover, free radicals are valuable substances in living systems that aid in gene expression, receptor activation, and signal transduction . Nonetheless, excess free radicals produce oxidative stress which is implicated in cardiovascular disease, cancers, diabetes and inflammation [6,7]. Anti-oxidants molecules derived from plants are known to obstruct the generation of free radicals and relieve diseases caused by oxidative stress [6,8]. Moreover, some investigators have shown that plant-based products with high quantity of flavonoids and phenolics also have high antioxidant and anti-inflammatory activities [6,9,10].
Clausena anisata (Wild.) Hook f. ex. Benth (Rutaceae) is a small perennial tree that grows up to 10 m high. Various parts of the plant are use as remedy for several diseases such as epilepsy, malaria, gut disturbances, convulsion, toothache, oral candidiasis, fungal infections, and high blood pressure in traditional medicine [11-13]. Anti-bacterial, anti-hypertensive, anti-pyretic, anti-inflammatory, larvicidal, anti-oxidant, wound healing and in vitro anti-diabetic activities were evaluated for leaf extracts of the plant [14-19]. Furthermore, extracts of the root of C. anisata were reported to demonstrate analgesic, anti-inflammatory, anti-pyretic and hypoglycemic activities [20-22]. Finally, antimicrobial and cytotoxic activities of the fruit, leaf, stem bark, twigs and root of C. anisata were also evaluated . However, anti-inflammatory, analgesic and anti-oxidant activities have not been performed on C. anisata stem. Moreover, total flavonoid and phenolic content of the leaf and stem of the plant have also not been determined. We therefore sought to evaluate and compare these activities of C. anisata stem and leaf extracts in other to provide scientific justification for some of their uses in traditional medicine.
Materials and Methods
Collection and extraction of the plant materials
C. anisata was collected in March 2012, from the arboretum of Centre for Plant Medicine Research (Centre) at Ayikumah in the Greater Accra Region of Ghana and authenticated by Mr. Ofori Lartey, a botanist at the Centre. Samples (CPM 0112 and CPM 0212) of the leaf and stem respectively were deposited in the Centre’s herbarium. The leaf and the stem were separated from the plant respectively and chopped into pieces.The plant materials were separately air dried for 34 days and pulverized with hammer mill. The leaf (0.5 kg) was macerated with ethanol (16 l × 2) for 4 days and filtered and combined. The extract was dried at 45oC with rotary evaporator to obtain a dark green semi solid labeled CLE (49.58 g; yield-9.92 %w/w). The stem (0.5 kg) was also taken through a similar process to obtain brown solid labeled CSE (3.817 g; yield-0.76 %w/w).
The three species of animals (Swiss albino rats, ICR mice and C3HP mice) used in this study were obtained from the Animal House Unit of the Pharmacology Department of the Centre. The animals were housed in aluminum cages under standard environmental conditions of 25 ± 2°C, 40-60% humidity with 12 h light/12 h dark cycle with free access to sterile water and feed. Mice used in the acetic acid-induced assays were fasted overnight just before the experiment. The animals were handled and cared for according to the guidelines of the Committee for the Update of the Guide for the Care and use of Laboratory Animals, 2011.
Drugs and chemicals
Carrageenan and Acetic acid were procured from Sigma Chemical Co. (St. Louis, USA). Indomethacin and diclofenac sodium salt powders were purchased from Cayman Chemical Company (Ann Arbor, USA) and Sigma-Aldrich (St. Louis, USA) respectively. Tramadol hydrochloride was purchase from Bristol Laboratories Limited. DPPH was purchased from Tokyo Chemical Industries Limited (Tokyo, Japan). AlCl3 and NaNO3 were supplied by Park Scientific Limited (Northampton, U.K.). Gallic acid and Folin - Ciocalteu reagent were also supplied by Nice Chemicals Limited, (Kerala, India) and Research – Lab Fine Chem Industries, (Mumbai India) respectively. NaOH was also obtained from Daejung Chemicals and Metals Co. Ltd (Gyeonggi-do Korea). Finally, NaCl was purchased from Timstar Laboratory Supply Ltd (Cheshire, U.K.). Propylene glycol was used as an emulsifier and analytical grade ethanol was used for extraction.
Phytochemical screening of the extracts
About 100 ml each of the extract were added to 50 ml of distilled water in ceramic evaporating dishes and evaporated on water bath to remove the ethanol. The aqueous portions of the extracts obtained were added to 50 ml of distilled water and screened for the presence or absence of thirteen (13) classes of phytochemical constituents .
Determination of total phenolic content
Phenolic content of CLE and CSE were determined as previously reported  with slight modifications. Briefly, absorbance of triplicate concentrations of 100-0.195 μg/ml of Gallic acid (standard compound) at 765 nm λmax was measured and used to obtain a standard calibration curve. The extracts, 800 μl of 5-0.3125 mg/ml of CLE or CSE was added to 4 ml of Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water). The mixture was neutralized with 3.2 ml of sodium carbonate solution (7.5% w/v) after 8 min and incubated in the dark at room temperature for 2 h. Absorbance was measured at the stated wavelength using double beam UV/VIS spectrophotometer (Biobase BK – D590). The total phenolic contents of CLE or CSE was determined from the linear equation of a standard curve. The content of total phenolic compounds was expressed as 100 mg/mg gallic acid equivalent (GAE) ± SEM.
Determination of total flavonoid content
Flavonoid content in CLE or CSE was measured using the aluminum chloride UV spectrophotometry assay as described previously  with slight modifications. Quercetin solution (1 ml) of concentrations ranging from 3.9 ×10-3-1 mg/ml was added to 4 ml of distilled water in 10 different test tubes. NaNO3 (5% w/v; 0.3 ml) was added and incubated for 5 min. AlCl3 (10% w/v; 0.3 ml) was added to the mixture, followed by addition of 2 ml of 1 M NaOH at the 6th min. The volume was made up to 10 ml with distilled water and mixed thoroughly. CLE or CSE (0.3125-5 mg/ml) was dissolved in methanol and treated separately just like the standard. The absorbance was determined at 510 nm using UV/VIS spectrophotometer (Biobase BK – D590) and in triplicates. Calibration curves were plotted and their linearity determined in order to estimate the flavonoid contents. The flavonoid content was expressed as mg of quercetin equivalence (QE) per 100 mg ± SEM of CLE or CSE respectively.
DPPH free radical scavenging assay – anti-oxidant activity
The DPPH free radical scavenging assay was used to evaluate the antioxidant activity of CSE and CLE as described by Oyaizu  with slight modifications. Briefly, 2-fold serial dilutions of CSE or CLE was prepared to obtain 7 different concentrations (2.5-0.039 mg/ml). The standard, ascorbic acid was also prepared in 3-fold dilutions (1-1.5 × 10-4 mg/ml). DPPH solution (20 μg/ml) 3 ml was added to 1 ml of each sample and incubated in the dark for 30 min at room temperature. The absorbance was read at 517 nm against a blank using UV/VIS spectrophotometer (Biobase BK – D590) in triplicates. The negative was prepared by adding 3 ml of DPPH solution to 1 ml of methanol and treated under the same conditions as the samples. Percentage inhibition was calculated as:
Antioxidant activity = (A-B)/A × 100%
Where A is optical density of blank and B is the optical density of sample.
The effective concentration (EC50) of each sample was estimated from a regression curve.
Anti-inflammatory activity assay
Anti-inflammatory activity of the extracts was evaluated using carrageenan induced paw oedema model in rats . Male Sprague-Dawley rats (254-294 g) were divided into 8 Groups (n=5). Group 1-3 were given CLE 50-200 mg/kg p.o. Group 4-6 were also given CSE 50-200 mg/kg p.o. Group 7-8 were given 10 mg/kg p.o. of indomethacin as standard drug or 1 ml each of 1% propylene glycol aqueous solution as vehicle control respectively. Inflammation was induced by the sub-plantar injection of 0.1 ml carrageenan in normal saline (1% w/v) in the right hind paw of each animal. Baseline paw volume (Vo) of the right hind limbs were measured prior to induction of inflammation and at an hourly interval (Vt) until the 5th h by volume displacement using plethysmometer after extract or drug was given. (Vt-Vo) was taken as oedema (Olivera de Lima et al.,). Total oedema induced during the 5-hour period was measured as net Area Under the time course Curves (AUC). Anti-inflammatory activity was calculated using the formula:
Antiinflammatory activity = ((AuC Control-AUC Treated)/ AUC Control) × 100%
Analgesic activity assays
Acetic acid induced writhing assay
Analgesic activity of the extracts was also evaluated in the acetic acid induced-writhing model as previously described . In short, 40 male Swiss albino mice (30-36 g) were divided into 8 Groups (n=5). Group 1-3 were given reconstituted CLE 50-200 mg/kg p.o. in 1% propylene glycol aqueous solution. Moreover, Group 4-6 were also given CSE 50-200 mg/kg p.o. Group 7 and 8 received diclofenac sodium, 10 mg/kg p.o. as standard drug and 0.2 ml of 1% propylene glycol aqueous solution as vehicle control respectively. Each mouse was then injected with 1 ml/100 g (i.p.) of 1 % v/v aqueous acetic acid 45 min after treatment and quarantined in transparent Perspex cages. Writhing movements and stomach contortions made by each mouse was counted for 20 min after induction. Analgesic activity (AE) was calculated using the formula:
AE = ((MRc-MRt)/MRc) × 100%
Where: MRc=average writhing count of the vehicle treated control group; MRt=average writhing count of treated group.
Hot plate assay
The mouse hot plate test used to evaluate the analgesic activity was performed as previously described . Briefly, 40 male C3HP mice (23-33 g) were divided into 8 Groups (n=4). Each mouse was placed on an electric hot plate (UGO Basile hot/cold plate 35 100) maintained at 55 ± 0.5°C. Latency time (To), recorded as analgesic response was measured as the time taken for a mouse to lick, lift, shake or stamp any of the hind limbs or jump. Baseline latencies (To) were calculated as means of two pre-treatment determination of each mouse. Mean baseline latency of mice used for this test ranged from 5.4-10.6 s. Plant extracts were reconstituted with 1% propylene glycol aqueous solution and administered as follows: Group 1-3 were given CLE 5-200 mg/kg p.o. Group 4-6 also received CSE 5-200 mg/kg p.o., whereas Group 7 was given tramadol 5 mg/kg p.o. (as standard drug) and Group 8 served as vehicle control respectively. Latency of each mouse was then measured at 0.25, 0.5 and 1 h. Thereafter, the latency was measured at hourly (Tt) intervals for 4 h after each treatment per mouse. Analgesic activity was calculated as the sum of percentage maximal probable effect (% MPE) over the experimental period. % MPE was calculated as :
%MPE=((To-Tt)/To) × 100
Graphpad Prism 5 was used for all statistical analysis. Data obtained for the time course curves were analyzed using Two Way ANOVA followed by Bonferroni’s multiple comparison test to determine statistically significant. Whereas, One-way ordinary ANOVA was employed to analyse data in the writhing assay followed by Dunnett’s multiple comparison test for statistically significant. Results were considered to be statistically significant when p < 0.05 by comparing the test group with the control group.
CLE and CSE possessed significant anti-inflammatory and analgesic activities and weak anti-oxidant activity. However, CSE demonstrated higher analgesic, anti-inflammatory and anti-oxidant activities than CLE. Flavonoids and phenolic content of CSE were higher than CLE. This may partly explain where CSE was more active than CLE in all the assays. These results lend credence to the ethnopharmacological utilization of leaf and stem of C. anisata.
The authors wish to acknowledge the technicians of the Animal House Unit of the Centre for Plant Medicine Research for their technical support.
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