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Comparative analysis of analgesic and anti- GossMa*
inflammatory activity of bark and leaves of Acacia ferruginea DC.
Samriti FaujdaraSwapnil Sharma a, Bhawna Satia, A.K. Pathak b, Sarvesh Kumar Paliwala
a Department of Pharmacy, Banasthali University, Rajasthan 304022, India b Department of Pharmacy, Barkatullah University, Bhopal, Madhya Pradesh 462001, India
ARTICLE INFO
Article history:
Received 10 November 2015 Received in revised form 5 February 2016
Accepted 10 February 2016 Available online 19 February 2016
Keywords: Acacia ferruginea Analgesic activity Carrageenan
Acetic acid-induced writhings Flavonoids
ABSTRACT
The aim of the present study was to investigate and compare the analgesic and antiinflammatory activities of hydroalcoholic extracts of bark and leaves of Acacia ferruginea DC. Hydroalcoholic extracts of bark and leaves were evaluated for analgesic activity using hot plate method and acetic acid-induced writhing test, whereas the anti-inflammatory activity was evaluated by carrageenan-induced paw oedema method. Hydroalcoholic extract of the bark at the dose of 50 mg/kg (6.10 ± 0.30) and leaves at a dose of 100 mg/kg (5.72 ± 0.39) after 45 min exhibited significant (P < 0.001) analgesic activity in hot plate test, which was comparable to Tramadol (6.11 ± 0.31) at a dose of 10 mg/kg. However, in acetic acid-induced writhing test, hydroalcoholic extract of both bark (90%) and leaves (90.91%) showed maximum protection from acetic acid at the dose of 100 mg/kg as compared to standard drug (50.91%) at a dose of 5 mg/kg. In the evaluation of anti-inflammatory activity, hydroalcoholic extract of leaves at a dose of 400 mg/kg had significantly (74.68%) inhibited the inflammation as comparable to indomethacin (82.8%) after 3 h of induction of carra-geenan. It is concluded that hydroalcoholic extracts of bark and leaves have central analgesic and peripheral analgesic effects, respectively. Both hydroalcoholic extracts of the bark and leaves significantly reduced the paw oedema at a dose of 400 mg/kg and exhibited anti-inflammatory activity.
© 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
* Corresponding author. Department of Pharmacy, Banasthali University, Tonk, Rajasthan 304022, India. Tel.: +91 9887636909; fax: 01438 228365.
E-mail address:faujdar.samriti@rediffmail.com (S. Faujdar). http://dx.doi.Org/10.1016/j.bjbas.2016.02.002
2314-8535/© 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
In spite of the progress made in medical research during the past decade, the treatment of some serious diseases remains problematic due to the side effect and high cost associated with it. Inflammation is a local response of animals towards injury. Basically, it is a body defence system to inhibit the spread of infection. It is characterized by formation of oedema, leucocytes infiltration and granuloma formation, tissue injury and repair (Bairagi et al., 2012). Inflammation causes triggers in inflammatory mediators such as TNF-a, interleukins and prostaglandins. Anti-inflammatory agents are capable of inhibiting the cyclooxygenase COX-1 and COX-2 pathway of arachidonic acid metabolism, which produces prostaglan-dins (Tasleem et al., 2014). Although a large number of remedies are available in the market, like immunosuppressants, NSAIDs, corticosteroids and histamines, side effects associated with them limit their use. Osteoporosis, gastric lesions, high blood pressure and allergy are the common side effects associated with them. Therefore, attention is being focused on the efficacy of plant based medicines with lesser side effects. According to WHO, 80% of the world population rely on plant based medicine for primary healthcare. It is estimated that in 1997, the world market for over the counter phytopharmaceuticals was US$ 10 billion with an annual growth of 6.5%. WHO includes phytotherapy in its health programmes and gives special emphasis on the validation of drugs from plant origins in developing countries (Ullah et al., 2014). Pain is frequently associated with inflammation (Bihani et al., 2014).
Pain is an unpleasant and emotional experience associated with tissue damage. Analgesics are the drugs used to relieve pain. Classical analgesics of natural origin include opiates and non-steroidal anti-inflammatory drugs but they are associated with side effects such as gastric lesions and tolerance and dependence (Ezeja et al., 2011). So, there is a need to explore natural available alternative sources to NSAIDs and opiates.
Secondary metabolites of plants such as steroids, flavo-noids, alkaloids, terpenoids and glycosides have gained importance due to their diverse pharmacological activities such as anti-inflammatory, analgesic and antipyretic, etc. Acacia is a large genus containing 1300 species belonging to the family Mimosoideae. Different species of this genus have been reported to have anti-malarial, antifungal, antibacterial, anti-diarrhoeal, anti-oxidant, antiviral, hepatoprotective and antispasmodic activities. From ancient times, different species of Acacia have been used by the tribal community in Rajast-han to treat different ailments such as syphilis, dental pain, urinary tract bleeding and hypertension. A species of Acacia, i.e. Acacia ferruginea, is one of the important drugs of Ayurveda. A.ferruginea DC., a drought resistant, deciduous tree belonging to the family Mimosoideae, is native to Peninsular India from Gujrat to Gunjam in the east (Orwa et al., 2009). The bark of the plant is bitter and traditionally used as astringent and cure for itching, leucoderma, ulcers, stomatitis and diseases of the blood. Traditionally, the leaf extract has been in use as astringent and as a treatment for dysentery, gonorrhoea, urinary tract disorders, and useful in the diseases of the eye and liver. Bark decoction of A. ferruginea is one of the active ingredients of a gargle preparation. Ethanolic extract of A. ferruginea leaves is
reported to have hepatoprotective (Akare et al., 2009), larvici-dal (Vahitha et al., 2002), antiulcer (Sowndhararajan and Kang, 2013), and anti-tumour (Sakthivel and Guruvayoorappan, 2013) activities. Chemical constituents of A. ferruginea include fla-vonoids, phenols, alkaloids, terpenoids, anthraquinones and tannins. Glycosides and saponins were also present in trace amounts (Sakthivel and Guruvayoorappan, 2013). Earlier phy-tochemical studies indicated that this species acts as a rich source of tannins (catechin, epigallocatechin), terpenoids, polyphenolics (gallic acid) and saponins. It is noticeable that the aforementioned chemical constituents have been reported to possess analgesic and anti-inflammatory properties. Since no scientific data are available just yet to justify the anti-inflammatory and analgesic activities of this plant, the present study was designed to compare the anti-inflammatory and analgesic activities of hydroalcoholic extracts of the bark and leaves of A. ferruginea.
2. Materials and methods
2.1. Plant material
Bark and leaves of A. ferruginea plant were collected from Raholi village of the district of Tonk, Rajasthan, India in the month of August 2012 and were authenticated at the Department of Botany (Ref. RUBL21147), University of Rajasthan, Rajasthan, India. A specimen was submitted to the Department of Botany, Rajasthan University, for further reference. The bark and leaves were shade dried, coarsely powdered and stored in an air tight container for further use.
2.2. Preparation of extract
The coarsely powdered leaves and bark were extracted with 70% methanol using soxhlation. The solutions were filtered and concentrated under vacuum using rotary evaporator (Heidolph, Schwabach, Germany). Yields of extracts were calculated on the basis of percentage w/w. Hydroalcoholic extracts of leaves and bark were used in comparing the analgesic and anti-inflammatory activities of A. ferruginea.
2.3. Phytochemical screening
Hydroalcoholic extracts of bark and leaves were observed for the presence of alkaloids, carbohydrates, flavonoids, gum and mucilages, tannins, terpenoids, steroids and saponins (Harborne, 1998).
2.4. Experimental animals
Wistar albino rats (180-220 g, Male) and Swiss albino mice (2540 g) were kept in polypropylene cages (3 in each cage) at an ambient temperature of 25 ± 2 °C and 55-65% relative humidity. A 12 h light/dark cycle was maintained in the animal house. The rats and mice had free access to water and feeds ad libitum. The approved protocol (BU/BT/627/14-15) of animal study was carried out as per the guidelines of IAEC and CPCSEA.
2.5. Chemicals and drugs
Carrageenan was procured from Sigma-Aldrich Chemical Company, Steinheim, Germany. Indomethacin and tramadol were procured from Chem-Impex International, Kolkata, and Abbott Healthcare Pvt. Ltd., Mumbai, India, respectively. Aspirin and acetic acid were procured from Merck Specialities Pvt. Ltd., Mumbai. All the chemicals used for the study were of analytical grade. The rotary evaporator used to concentrate the extract was procured from Heidolph, Schwabach, Germany. UV-visible spectrophotometer (Shimadzu, UV-1800) used to calculate the concentration was purchased from Shimadzu, Japan. Eddy's hot plate analgesiometer used to evaluate analgesic activity was purchased from The Quality Apparatus, Ambala Cantt.The plethysmometer used to calculate the paw volume was procured from IITC Life Sciences, USA. HPTLC used for quantification was procured from CAMAG.
2.6. Estimation of phytoconstituents
2.6.1. Estimation of total phenolic content
Total phenolic content present in the hydroalcoholic extract of bark and leaves of A. ferruginea was estimated using the method reported by Singleton et al. (1999). 0.2 mL of the hydroalcoholic extract solution and 0.2 mL of the Folin-Ciocalteu reagent were mixed thoroughly. After 4 min, 1 mL of 15% Na2C03 was added, and the mixture was allowed to stand for 2 h at normal temperature. The absorbance was taken at 760 nm using a UV-visible spectrophotometer (Shimadzu, UV-1800). The concentration of total phenolics was calculated as milligram of gallic acid equivalent by using an equation obtained from the gallic acid calibration curve. The determination of total phenolic content in the extracts was carried out in triplicate and the results were averaged.
2.6.2. Estimation of total flavonoids
Total flavonoids present in the hydroalcoholic extract of bark and leaves of A. ferruginea were determined as per the reported protocol of Woisky and Salatino (1998). Hydroalcoholic extract (0.5 mL), 10% aluminium chloride (0.1 mL), 1M potassium acetate (0.1 mL) and distilled water (2.8 mL) were mixed and incubated at room temperature for 30 min. The absor-bance was measured at 415 nm using a UV-visible spectrophotometer (Shimadzu, UV-1800). Rutin was used to make the calibration curve. The calculation of total flavo-noids in the extract was carried out in triplicate and the results were averaged and expressed as milligram rutin equivalent per gram dry weight of extract (mg RE/g d. w.).
2.6.3. Estimation of total saponins
The total saponins present in the bark and leaves were estimated by the method described by Rajpal (2002). Five grams of bark powder and leaves were refluxed with 70% methanol for 30 min. The hydroalcoholic extract obtained was filtered. The dried marc was successively extracted three times with methanol (25 mL).The extracts were combined and solvent was distilled off. Residue remained after distillation was again re-fluxed with petroleum ether. Petroleum ether extract was cooled and the solvent was distilled off. The soft extract (semisolid
mass) was treated successively with chloroform (25 mL) and ethyl acetate (25 mL) and solvent was decanted off after cooling. This soft extract was then dissolved in 90% methanol. The solution was filtered and concentrated to 5 mL. This solution was added dropwise with constant stirring in acetone to precipitate saponins. The precipitates were filtered, collected and dried to a constant weight at 105 °C. The determination of total saponin content in the bark and leaves was carried out in triplicate and the results were averaged and expressed in percentage.
2.7. Acute toxicity
Acute toxicity of bark and leaf extracts was performed as per the 0ECD-420 guidelines. Wistar albino rats and Swiss albino mice were divided into different groups comprising six animals each. The control groups received normal saline (2 mL/kg, p.o.). The other groups of Wistar rats and mice received 100-2000 mg/ kg of the test extract respectively. Immediately after dosing, the animals were observed continuously for the first 4 hours for any behavioural changes. Thereafter, they were then kept under observation up to 14 days after drug administration to find out the mortality, if any (Ganapaty et al., 2002).
2.8. Analgesic activity
The analgesic activity of hydroalcoholic extracts of bark and leaves was evaluated by using two models: (1) Hot plate method based on pain sensation at 55 °C and (2) Acetic acid-induced writhing based on chemical induced algesia.
2.8.1. Hot plate method
The analgesic activity of hydroalcoholic extracts of the bark and leaves were evaluated in mice by the method given by Eddy and Leimback (1953). This method was specifically used to evaluate central action of both the hydroalcoholic extracts of bark and leaves. The mice were divided into eight groups comprising six animals each. Group I received only vehicle and served as control. Group II received tramadol (10 mg/kg b.wt, i.p.) and served as standard. Groups III, IV and V were administered with hydroalcoholic extract of bark of A. ferruginea in different doses of 30, 50 and 100 mg/kg b.wt, i.p. respectively. Groups VI, VII and VIII were administered with hydroalcoholic extract of leaves of A. ferruginea in different doses of 30, 50 and 100 mg/kg b.wt, i.p. respectively. Each mice was placed on the hot plate maintained at 55 ± 2 °C, and the response time was recorded as the time at which animals reacted to pain stimulus by either paw licking response or jumping response, whichever appeared first at 15, 30,45,60 and 90 min after treatment. The cut off time for the reaction was 15 seconds.
2.8.2. Acetic acid-induced writhing method
The analgesic activity of hydroalcoholic extracts of bark and leaves against acetic acid-induced writhings was evaluated according to the method given by Dey et al. (2010). This method was used preferentially to evaluate the peripheral action of hydroalcoholic extracts of the bark and leaves. The animals were divided into eight groups comprising six mice in each group. Group I received distilled water and served as control, Group II received aspirin, peripheral acting analgesic (5 mg/kg b.wt,
i.p.) and served as standard. Groups III, IV and V were intra-peritoneally administered with hydroalcoholic extracts of the bark in different doses of 30, 50 and 100 mg/kg b.wt., respectively. Groups VI, VII and VIII were intraperitoneally administered with hydroalcoholic extracts of leaves in different doses of 30, 50 and 100 mg/kgb.wt., respectively. Thirty minutes after the extract administration, 0.1 mL of 0.6% acetic acid was injected for the induction of writhings in mice. The writhing effect was indicated by stretching of at least one hind limb. This response was observed for 30 min after the acetic acid administration, and reduction in number of writhings in the treated groups and standard were compared with animals in the control group.
The percentage protection of abdominal constrictions was calculated by the formula:
Protection (%) = Wc - Wt/Wc x 100
Wc = Mean no. of writhes (Control), Wt/Ws = Mean no. of writhes (Test/Standard).
2.9. Anti-inflammatory activity
Anti-inflammatory activity of hydroalcoholic extracts of both bark and leaves against carrageenan was studied according to the method given by Winter et al. (1962). Animals were divided into eight groups comprising six rats in each group. Group I received 0.9% NaCl solution (0.1 mL) and served as control, Group II received indomethacin (10 mg/kg b.wt, i.p.) and served as standard. Groups III, IV and V were administered with hydroalcoholic extracts of the bark (100,200 and 400 mg/kg b.wt., i.p.) and Groups VI, VII and VIII were administered with hydroalcoholic extracts of leaves (100, 200 and 400 mg/kg b.wt., i.p.) respectively.
All the animals were fasted overnight prior to the start of experiment and only water was allowed ad libitum. A mark was made on the tibio-tarsal junction in the right hind paw of the rat, so that each time the paw was dipped to a fixed volume. Acute inflammation was produced in all the groups by administering 1% w/v, 0.1 mL carrageenan in normal saline into the sub-plantar tissue of the right hind paw, and the linear paw circumference was observed at 1 h, 2 h and 3 h by volume displaced by paw using Plethysmometer (IITC, Life Science, USA). Extracts and standard were administered intraperitoneally, 1 h before the administration of carrageenan. The average increase in paw volume of each group was calculated and compared with the control group and the standard group.
2.10. HPTLC profiling of bark extract
2.10.1. Chromatographic method
HPTLC profiling of the bark extract and crude saponins was carried out according to the method given by Harborne (1998). The fingerprinting of bark extract and crude saponin was executed by spotting 6 |L solution of bark extract and crude saponin on an HPTLC plate. The chromatography was performed using 4.0 x 10.0 cm HPTLC plate silica gel 60 F254 (E. Merck, KGaA, Darmstadt, Germany). The bark extract and crude saponins were applied as band length 6 mm wide and 8 mm
apart by means of Camag (Muttenz, Switzerland) Linomat 5 sample applicator with a 100 |L syringe, which was programmed through winCATS software. The plates were dried at 100 °C for 3 min. The plates were kept in a photo documentation chamber. The Rf values and fingerprint data were recorded by winCATS software.
2.10.2. Solvent system selection
A large number of solvent systems were tried but the best resolution was observed in chloroform:glacial acetic acid:methanol:water in the ratio of 6:2:1:1.
2.10.3. Detection of spots
The developed plates were dried at 100 °C in hot air oven for 3 min. The plates were kept in a photo-documentation chamber. The Rf values and finger print data were recorded by winCATS software.
2.11. Statistical analysis
Results were expressed as average of triplicate readings with ± S.E.M. Bonferroni t-test was used to evaluate the significance of results.
3. Results
3.1. Phytochemical screening
The results of phytochemical screening of the hydroalcoholic extracts of bark and leaves of A. ferruginea revealed the presence of alkaloids, flavonoids, triterpenoids, saponins, tannins and phenolic compounds.
3.2. Estimation of phytoconstituents
Total phenolic content and total flavonoid content were estimated by the method described by Singleton and Woisky respectively. Total flavonoid content present in hydroalcoholic extract of bark and leaves, respectively, were found to be 18.66 mg/g RE and 64.6 mg/g RE, whereas total phenolic content estimated in bark and leaves, respectively, were 17.55 mg/g GAE and 9.32 mg/g GAE. Total saponin content estimated in bark and leaves extract, respectively, were 34% w/w and 23% w/w.
3.3. Acute toxicity
Hydroalcoholic extracts of both bark and leaves were evaluated for acute toxicity in rats and mice by oral administration of extracts. No behavioural changes were observed after 4 h. It was found that both the extracts were safe at the highest dose of 2000 mg/kg and no mortality was shown even after 14 days of extract administration. Moreover, no mortality was observed during the toxicity study.
3.4. Analgesic activity 3.4.1. Hot plate method
The analgesic effects of hydroalcoholic extracts of bark and leaves of A. ferruginea using hot plate method are presented
Fig. 1 - Analgesic activity of hydroalcoholic extracts of Acacia ferruginea bark and leaves using hot plate method (Mean ± SD).
in Fig. 1. It revealed that intraperitoneal administration of both the extracts at the dose of 100 mg/kg significantly reduced the thermal stimulation in the hot plate. Analgesic activity of both the extracts after 45 min was comparable to standard drug Tramadol, central analgesic (10 mg/kg). Among all the doses tested above, bark extract at the same dose of 100 mg/kg showed higher inhibition [(8.85 ± 0.45)min] of thermal stimulation as compared to leaf extract [(6.79 ± 0.29)min] at a response time of 90 min, whereas tramadol (10 mg/kg) showed maximum inhibition [(6.11 ± 0.31)min] after 45 min.
3.4.2. Acetic acid induced writhing test in mice The results of the analgesic effects of hydroalcoholic extracts of A. ferruginea bark and leaves were evaluated by acetic acid-induced writhing method, depicted in Fig. 2. Both the extracts exhibited excellent analgesic activity at all doses of 30, 50 and
100 mg/kg as compared to control and standard. It was observed that the number of writhings statistically attenuated with increase in concentration of extracts. The maximum protection was observed at a dose of 100 mg/kg in both leaf (90.91%) and bark extracts (90%) against acetic acid, which was comparable to standard aspirin (50.91%) at the dose of 5 mg/kg.
Anti-inflammatory activity
3.5.1. Carrageenan induced paw oedema The anti-inflammatory activity of the hydroalcoholic extracts of bark and leaves were evaluated by using carrageenan induced paw oedema method, presented in Fig. 3. Carrageenan induced peritonitis was used to access fluid extravasation and leucocyte migration due to inflammation. A significant dose-dependent inhibition in paw oedema was
Fig. 2 - Analgesic activity of hydroalcoholic extracts of Acacia ferruginea bark and leaves using acetic acid induced writhing test in mice (Mean ± SD).
Inhibition of Paw Oedema
Degrees of freedom 7,40; P < 0.05 of ANOVA followed by Bonferroni t-test compared with control
Fig. 3 - Anti-inflammatory activity of hydroalcoholic extracts of Acacia ferruginea bark and leaves using carrageenan induced paw oedema in rats (Mean ± SD).
shown by both the hydroalcoholic extracts in different doses of 100, 200 and 400 mg/kg and comparable to indomethacin (10 mg/kg). The hydroalcoholic leaf extract (400 mg/kg) of A. ferruginea and indomethacin (10 mg/kg) exhibited statistically significant (P < 0.05) inhibition in paw oedema of 74.68% and 82.28% at 3 h of induction of carrageenan. At a dose of 400 mg/kg, hydroalcoholic extract of leaves (74.68%) produced highly significant (P < 0.05) acute anti-inflammatory activity as compared to hydroalcoholic extract of bark (67.09%) of A. ferruginea at 3 h.
3.6. HPTLC profiling
Figs. 4 and 5 showed HPTLC profiling of bark extract and crude saponin, which showed several peaks. While comparing bark extract with crude saponins, results revealed that same Rf values were observed at 0.07, 0.13, 0.18, 0.31, 0.35 and 0.73, which confirmed the presence of similar types of saponins in the bark extract. The Rf values and area of different phytoconstituents and saponins present in bark extract and crude saponins were presented in Table 1a and Table 1b respectively.
Results of HPTLC analysis showed that crude saponins and bark extract exhibited maximum area at Rf 0.68 and 0.37 respectively, when executed in the same solvent system.
The findings of the comparative analysis of bark and leaf extracts of A. ferruginea are presented in Table 2.
4. Discussion
Plants are the potential source of new drugs for human benefit. Plants used in traditional medicine contain a wide variety of chemical constituents that can be used to treat chronic and
acute diseases (Sermanni, 2011). Phytochemical screening of hydroalcoholic extracts of bark and leaves of A. ferruginea revealed the presence of alkaloids, carbohydrates, flavonoids, tannins, terpenoids, steroids and saponins. Significant analgesic activity was shown by plants that contain organic acids and flavonoids (Sasikala et al., 2011). Flavonoids may attribute to a number of pharmacological activities. Some flavonoids are reported to possess significant analgesic and anti-inflammatory activity. Some flavonoids can significantly interfere with inflammatory mediators.Terpenoids have been reported to show significant analgesic and anti-inflammatory activity (Ullah et al., 2014).
Acute toxicity study showed that both hydroalcoholic extracts of plant caused no mortality up to a dose of 2000 mg/ kg, and no behavioural changes were observed in any group. In the present study, analgesic activity of hydroalcoholic extracts of bark and leaves of A. ferruginea was evaluated by two models, i.e. Hot plate method and acetic acid-induced writhings in mice, while anti-inflammatory activity was evaluated by using carrageenan induced paw oedema.
The peripheral analgesic effect may be mediated through inhibition of cyclooxygenase and/or lipoxygenases, whereas central analgesic action may be mediated through inhibition of central pain receptors (Shulan et al., 2011). Therefore, central (Hot plate method) and peripheral (acetic acid-induced writhing) models were selected for the evaluation of the analgesic action. Eddy's hot plate test involved marked central analgesic effect as evidenced by significant increase in reaction time. Results depicted that both extracts have shown increase in latency period in a dose dependent manner. Both hydroalcoholic extracts at the dose of 100 mg/kg after 45 min significantly reduced the pain compared to tramadol; this central analgesic effect may be due to inhibition of prostaglandins synthesis. Acetic acid-induced writhing test is the second most com-
Fig. 4 - HPTLC chromatogram of bark extract.
monly used test to assess peripherally acting analgesics. Algesia is produced in mice by the release of free arachidonic acid from the tissue phospholipid via. cyclooxygenase and prostaglandins production which results in localized oedema. The increase in prostaglandin production further enhances the vascular permeability. The decrease in the number of writhings infers attenuation of prostaglandins synthesis which results in sig-
nificant analgesic activity. Hydroalcoholic extracts of bark and leaves of A. ferruginea reduced the writhing at all doses as compared to standard, aspirin (5 mg/kg) and control (Tasleem et al., 2014).
Carrageenan induced paw oedema is the most widely used method for the assessment of anti-inflammatory activity of plant extracts. Mechanism of action of carrageenan is biphasic,
Fig. 5 - HPTLC chromatogram of crude saponins.
Table 1 - HPTLC profiling of (a) bark extract and (b) crude
saponins.
Peak Maximum Rf Height area
1 0.01 186.2
2 0.07 3,451.9
3 0.11 5,083.0
4 0.13 4,242.2
5 0.14 4,373.6
6 0.16 5,062.8
7 0.18 3,857.0
8 0.26 14,687.3
9 0.29 8,441.2
10 0.31 5,972.6
11 0.34 8,183.2
12 0.35 5,970.0
13 0.37 12,256.7
14 0.40 8,027.9
15 0.43 7,559.7
16 0.46 8,019.0
17 0.51 4,450.6
18 0.53 8,418.3
19 0.56 5,855.9
20 0.59 7,383.8
21 0.63 3,271.3
22 0.65 6,175.4
23 0.69 2,136.7
24 0.71 1,194.0
25 0.73 4,197.6
26 0.78 480.9
27 0.79 447.4
28 0.82 106.8
1 0.05 4,177.2
2 0.07 4,107.3
3 0.10 1,027.0
4 0.13 148.6
5 0.15 426.0
6 0.17 381.1
7 0.18 612.2
8 0.22 1,987.9
9 0.25 1,398.8
10 0.27 2,612.3
11 0.30 2,225.4
12 0.31 3,146.2
13 0.35 5,999.0
14 0.36 6,067.5
15 0.68 34,126.8
16 0.73 8,198.5
17 0.77 3,461.9
Table 2 - Comparative analysis of bark and leaf extracts
of Acacia ferruginea.
S. No. Parameters Bark Leaves
1. Total phenolic content 17.55 mg/g GAE 9.32 mg/g GAE
2. Total flavonoid content 18.66 mg/g RE 64.60 mg/g RE
3. Total saponin content 34% 23%
4. Reaction time 6.10 min 5.72 min
5. Maximum protection 90% 90.91%
from acetic acid
6. Inhibition of paw 67.09% 74.68%
oedema
which involves the metabolism of cyclooxygenase products of arachidonic acid and the production of reactive free oxygen species. The first phase is marked by release of serotonin, histamine and kinins in the first hour. The second phase is characterized by release of prostaglandins and lysosome enzymes in the second to fourth hour. Most drugs show their anti-inflammatory response at second phase. The results showed that hydroalcoholic extracts of bark and leaves significantly inhibited the inflammation induced by carrageenan in the third hour of the study. In comparison to the bark extract, leaf extract at the same dose of 400 mg/kg significantly attenuated the inflammation comparable to the standard, indomethacin, at the same time (Ullah et al., 2014). As both
the extracts contain a higher concentration of flavonoids and phenols, which are proven to have good anti-inflammatory potential (Zhang et al., 2006), it maybe postulated that the extracts reduce inflammation by inhibiting COX.
5. Conclusion
It was concluded from the present study that both hydroalcoholic extracts of bark and leaves of A.ferruginea have significant analgesic and anti-inflammatory activities. On comparison, bark extract revealed central analgesic activity and leaf extract showed peripheral analgesic activity, which may attribute to more amount of flavonoids and terpenoids present in the plant. Leaf extract showed good anti-inflammatory activity as compared to the bark. Secondary metabolites like phenolic compounds, tannins, saponins, steroids, and terpe-noids found in the phytochemical screening might be responsible for these activities. These findings support the traditional use of this plant in relieving algesia and inflammation. However, further work has to be done to isolate the compound responsible for these activities and to evaluate the possible mechanism associated with these activities.
Acknowledgement
The authors are highly thankful to the vice chancellor of the university for providing all the facilities for the present study. The authors are also thankful to the Department of Pharmacy, Banasthali University for their continuous support.
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