Abstract
The anti-inflammatory effect of the ethanol extract of Stereospermum suaveolens (Roxb.) DC (Bignoniaceae) bark given orally at the dose of 200 and 400 mg/kg body weight was studied in rats using the carrageenan-, dextran-, and histamine-induced hind paw edema, and cotton pellet-induced granuloma formation models. Indomethcin at the dose of 10 mg/kg body weight was used as a standard drug. The extract (400 mg/kg body weight per os) showed maximum inhibition of edema 64.6, 53.48, and 50.06% at the end of 3 h with carrageenan-, dextran-, and histamine-induced rat paw edema, respectively. The extract (400 mg/kg) exhibited significant reduction (34.77%) in granuloma weight in the cotton pellet-induced granuloma model. From these results it could be concluded that, the ethanol extract of Stereospermum suaveolens possesses maximum anti-inflammatory activity in a dose-dependent manner, in various experimental models.
Introduction
Stereospermum suaveolens (Roxb.) DC (Bignoniaceae), popularly known as “padiri”, is a large deciduous tree found throughout the moist parts of India. Various parts of the plant are used in native medicine. Traditionally, a decoction of the root is used for the treatment of inflammation, pain, fever, asthma, and vomiting (CitationCSIR, 1976; CitationKirtikar & Basu, 1988). The flowers mixed with honey are given orally for the control of hiccups (CitationChopra et al., 1999). In southern India the bark is used in folk medicine for the treatment of diabetes (CitationCSIR, 1976). The fruit is useful for the treatment of leprosy (CitationNadkarni, 1976). The root extract is known to possess anticancer activity due to the presence of lapachol (CitationCSIR, 1976; CitationRamachandran & Mohandoss, 1988). Previous phytochemical studies showed presence of higher carboxylic acids, β-sitosterol and saponin (CitationCSIR, 1976) in the roots, lapachol, dehydro-α-lapachone (CitationJoshi et al., 1977), sterekunthal B, sterequinone C, stereochenols A and B (CitationHaque et al., 2005, Citation2006) in the bark, and stereolensin (CitationRamachandran & Kotiyal, 1979), scutellarein, 6-hydroxy luteolin (CitationSankara Subramanian et al., 1972), dinatin (4,5,7-trihydroxyl-6-methoxyflavon), and dinatin-7-glucuroniside (CitationGhani, 1998) in the leaves. However, based on the literature survey, there is no scientific report proving the anti-inflammatory efficacy of this particular species. Keeping this view, the present investigation was to study the anti-inflammatory activity of Stereospermum suaveolens in various experimental animal models.
Materials and methods
Plant material
The bark of Stereospermum suaveolens was collected during October 2006 from Palode forest, Thiruvananthapuram District, Kerala, India. The plant was identified and authenticated by N. Mohanan, scientist at the Tropical Botanical Garden and Research Institute, Palode, Tiruvananthapuram District, Kerala, and a voucher specimen (TBS-1) has been deposited in our laboratory for further reference. The bark of the plant was shade dried and powdered with a mechanical grinder. The powdered plant material was then passed through a 40-mesh sieve and stored in an air-tight container for future use.
Preparation of plant extract
The shade-dried coarse powdered bark of Stereospermum suaveolens (500 g) was packed in the Soxhlet apparatus and extracted with 1.5 L of 95% ethanol at a temperature of 40°–50°C for 72 h. The extract was filtered and the filtered extract was then concentrated to dryness in a rotary evaporator under reduced pressure at a temperature of 40°C. The resultant black residue was stored in a desiccator for use in subsequent experiments and considered as the crude ethanol extract. The yield of the extract was 11% w/w. The preliminary phytochemical screening was performed for the extract (CitationKokate et al., 1998; CitationTrease & Evans, 2002; CitationHorbone, 1998).
Test compounds
The ethanol extract of Stereospermum suaveolens (EESS 200 and 400 mg/kg body weight p.o.) and indomethacin (10 mg/kg body weight p.o.) were used in this study.
Chemicals and reagents
Indomethacin was purchased from Fourrts (India), Chennai. Carrageenan was purchased from SD Fine Chemicals, Mumbai, India. Dextran and histamine were purchased from Anilax Enterprises, Florham Park, New Jerrsy, USA. The solvents and chemicals used were analytical grade.
Animals
Male Wistar albino rats (130–180 g) and female Swiss albino mice (20–25 g) were purchased from M/S BN Ghose, Kolkata. The animals were grouped and housed in poly acrylic cages (38 × 23 × 10 cm) with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25° ± 2°C) with 14 h dark:10 h light cycle. They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata) and water ad libitum. The animals were acclimatized to laboratory conditions for one week before commencement of the experiment. Ethical clearance was obtained from Jadavpur University Animals Ethical Committee for using animals in the present study.
Acute oral toxicity study
An acute oral toxicity study was performed as per OECD-423 guidelines (CitationEcobichon, 1997). Female Swiss albino mice (20–25 g) were randomly distributed to six groups (n = 6). The animals were fasted overnight and the drug was administered orally at doses of 100, 200, 400, 800, 1600, and 3200 mg/kg body weight. The animals were closely observed for the first 24 h for any toxic symptoms and for 72 h for any mortality.
Anti-inflammatory study
Carrageenan-induced rat paw edema model
The rats were divided into four groups containing six rats in each group. Acute inflammation was induced according to the method of CitationWinter and Porter (1957). An aliquot of 0.1 mL of 1% carrageenan in normal saline (0.9% w/v NaCl) was injected in to the sub-plantar region of the right hind paw. The extract was dissolved in distilled water. The extract (200 and 400 mg/kg body weight p.o.), indomethacin (10 mg/kg body weight p.o.) and control vehicle (normal saline 0.5 mL/100 g body weight) were administered orally 1 h before carrageenan injection.
Different groups were treated as follows:
Group I: Carrageenan + normal saline (0.5 mL/100 g body weight p.o.)
Group II: Carrageenan + EESS (200 mg/kg body weight p.o.)
Group III: Carrageenan + EESS (400 mg/kg body weight p.o.)
Group IV: Carrageenan + indomethacin (10 mg/kg body weight p.o.)
The paw volume was measured by plethysmometer just before and 1, 2, 3 and 4 h after administration of carrageenan (CitationWinter et al., 1962). Edema inhibitory values were calculated using the formula:
Inhibition (%) = Inhibition control (Vt–V0) – (Vt–V0) treated/ (Vt–V0) control × 100
where Vt is the paw volume at t-hour after carrageenan injection and V0 is the paw volume before carrageenan injection.
Dextran-induced rat paw edema model
The animals were treated exactly the same way as in the carrageenan-induced paw edema model but instead of carrageenan, here 0.1 mL of dextran (1% w/v in normal saline) was used as the edemogen (CitationWinter et al., 1962). The paw edema was measured as mentioned in the carrageenan-induced paw edema model.
Histamine- induced rat paw edema model
The anti-inflammatory activity of the extract was measured with a phlogistic agent (histamine) which acts as a mediator of inflammation. The animals were treated exactly the same as in the carrageenan-induced model but instead of carrageenan, here 0.1 mL of histamine base (10−3g/mL normal saline) was used as the edemogen. The paw edema was measured as mentioned in the carrageenan-induced paw edema model. In the edema models, animals who did not receive any drug were used as control. The data obtained for the various groups were reported as means ± SEM. Percentage edema inhibition was calculated.
Cotton pellet-induced granuloma formation in rats
This study was carried out as described by CitationAmpai and Duangta (2004). The absorbent cotton wool was cut in to small pieces, weighing 10 mg and made up to a pellet. The pellets were then sterilized in a hot air oven at 120°C for 2 h. The rats were divided into four groups of six animals, anesthetized and 10 mg of sterile cotton pellets were implanted subcutaneously in each axilla of the rats under sterile conditions.
The animals were treated for seven consecutive days as follows:
Group I: normal saline (5 mL/kg body weight p.o.)
Group II: EESS (200 mg/kg body weight p.o.)
Group III: EESS (400 mg/kg body weight p.o.)
Group IV: indomethacin (10 mg/kg body weight p.o.)
The animals were anesthetized again on day 8 and cotton pellets were removed surgically free from extraneous tissue and weighed immediately for the wet weight. The cotton pellets were incubated at 37°C for 24 h and dried at 60°C for 18 h and weighed after cooling. The transudative weight, granuloma formation and the percentage granuloma inhibition of the test compound were calculated. The change in body weight of the animals from the first and the last day of the experimental was also recorded.
Results
Acute oral toxicity study
The ethanol extract of Stereospermum suaveolens does not produced any mortality up to 3200 mg/kg body weight. Further dosing was not performed to estimate the LD50 (lethal dose) value. According to the OECD guidelines for the acute toxicity, an LD50 dose of 2000 mg/kg and above is categorized as unclassified and hence the drug is found to be safe.
Anti-inflammatory activity
The results of the mediator-induced acute rat paw edema indicate that the ethanol extract of Stereospermum suaveolens has anti-inflammatory activity (, , and ). The EESS (200 and 400 mg/kg body weight p.o.) exhibited significant anti-inflammatory activity and the effects were comparable to that of indomethacin, a potent non-steroidal anti-inflammatory drug (NSAID).
Table 1. Anti-inflammatory effect of ethanol extract of Stereospermum suaveolens on carrageenan-induced rat paw edema.
Table 2. Anti-inflammatory effect of ethanol extract of Stereospermum suaveolens on dextran-induced rat paw edema in rats.
Table 3. Anti-inflammatory activity of ethanol extract of Stereospermum suaveolens on histamine-induced rat paw edema in rats.
The EESS (200 mg/kg body weight p.o.) showed a maximum activity of 51.94% inhibition in carrageenan, 46.44% inhibition in dextran and 50.61% inhibition in histamine-induced rat paw edema volume. The EESS (400 mg/kg body weight p.o.) demonstrated the maximum 64.60% inhibition in carrageenan-induced rat paw edema, 53.48% inhibition in dextran and 59.59% inhibition in histamine-induced rat paw edema volume. The standard drug (indomethacin 10 mg/kg body weight p.o.) exhibited 68.33% inhibition in carrageenan, 60.87% inhibition in dextran and 62.88% inhibition in histamine-induced rat paw edema volume.
As shown in , in the chronic inflammation model (cotton pellet-induced granuloma), the EESS (200 mg/kg body weight p.o.) significantly (P < 0.05) reduced transudative weight and granuloma formation with a percentage inhibition of 29.24%. The EESS (400 mg/kg body weight p.o) significantly (P < 0.01) reduced transudative weight and granuloma formation with a percentage inhibition of 34.77%, while the standard drug (indomethacin 10 mg/kg body weight. p.o.) significantly (P < 0.001) reduced transudative weight and granuloma formation with a percentage inhibition of 45.44%. The EESS (400 mg/kg body weight p.o.) significantly (P < 0.05) reduced the body weight gain of animals while the EESS (200 mg/kg body weight p.o.) not significantly (P >0.05) reduced the body weight gain compared to control group. The standard drug (indomethacin 10 mg/kg body weight. p.o.) significantly (P < 0.01) reduced the body weight gain of animals.
Table 4. Effect of ethanol extract of Stereospermum suaveolens on cotton pellet-induced granuloma in rats.
Discussion
The anti-inflammatory effects of the EESS were studied using several standard pharmacological methods such as carrageenan-, dextran-, and histamine-induced acute rat hind paw edema models and cotton pellet-induced granuloma formation test in rats (chronic test).
Carrageenan-induced paw edema is commonly used as an experimental model of acute inflammation (CitationDella Loggia et al., 1986). The edema formation is a biphasic event, the first phase (during the first hour) is due to the release of histamine and 5-HT and the second phase is caused by the release of bradykinin, protease, prostaglandin, and lysosome (CitationCastro et al., 1968). EESS (200 and 400 mg/kg body weight p.o.) showed significant and dose-dependent anti-inflammatory activity in this model. In this model EESS (200 and 400 mg/kg body weight p.o.) showed anti-inflammatory activity of 27.01% and 30.34% edema inhibition, respectively, at 1 h after treatment and of 51.94% and 64.60% edema inhibition, respectively, at 3 h after treatment. The EESS reduced the paw edema produced by dextran which is known to be mediated histamine and 5-HT. Dextran also induces fluid accumulation which contains little protein and few neutrophils, whereas carrageenan induces protein-rich exudation containing large numbers of neutrophils (CitationGupta et al., 2003). The EESS also effectively suppressed the inflammation produced by histamine. So it may be suggested that its anti-inflammatory activity is due to its inhibition on histamine and 5-HT release at the site of inflammation or by blocking their action responsible for prostaglandin synthesis or by inhibiting prostaglandin synthesis through COX-2 inhibition mechanism.
In the cotton pellet-induced granuloma formation test, EESS was moderately effective in inhibiting the transudative formation and the granuloma formation. This suggests an inhibitory effect of the extract on vascular permeability. The EESS and the standard drug indomethacin slightly reduced the body weight gain and the anti-inflammatory effect is comparable to that of indomethacin. It therefore may be suggested that the anti-inflammatory effect of EESS occurs either in a similar fashion as indomethacin or other NSAIDs, by inhibition of prostaglandin synthesis through COX pathway or by inhibiting various mediators of inflammation.
The preliminary phytochemical screening of the EESS has revealed presence of flavonoids, saponin and terpenoids which were suggested to be responsible for the anti-inflammatory property of the plant.
Whatever the type of exudation may be, the EESS effectively suppressed inflammation in all the studied models. This indicates that EESS probably has activity against the inflammatory mediators such as histamine and 5-HT and against prostaglandin synthesis. However, different and specific models, using various mediators such as edemogen should be employed to confirm this notion.
Conclusion
From the present study it can be concluded that the ethanol extract of Stereospermum suaveolens bark exerted dose-dependent anti-inflammatory effects. The exact mechanism of the action is not fully understood. More detailed phytochemical studies are, however, necessary to identify the active principle(s) and exact mechanism(s) of action.
Acknowledgements
Balasubramanian. T, is greateful to Bharathi College of Pharmacy, Bharathinagara, Karnataka, India for providing necessary facilities.
Declaration of interest
We declare that the performed research work “Anti-inflammatory effect of ethanol extract of medicinal plant Stereospermum suaveolens (Roxb.) DC in rats” has not been published, and that it has not been submitted simultaneously for publication, elsewhere. We assure that the work has been conducted as per the university animal ethical committee guidelines.
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