Evaluation of antinociceptive activity of two Bangladeshi medicinal plants, Kalanchoe pinnata (Lam.) Pers. and Lagerstroemia speciosa (L.) Pers.
Kalanchoe pinnata (Lam.) Pers. (Family: Crassulaceae, local name: Pathorkuchi) is an herb found ubiquitously in Bangladesh having thick succulent leaves. Leaves have a variety of uses in the traditional medicinal system of Bangladesh. They are eaten for diabetes control, used as a diuretic and for dissolving of kidney stones, and taken for respiratory tract infections, as well as applied to wounds, boils, and insect bites (Ghani, 2003). Lagerstroemia speciosa (L.) Pers. (Family: Lythraceae, local name: Jarul) is a medium-sized tree with lilac flowers, and which can also be found throughout most areas of the country. The leaves are used by traditional medicinal practitoners for diabetes, and the bark as a purgative (Ghani, 2003).
Phenolic components (Gaind and Gupta, 1973) and flavonoid glycosides (Gaind and Gupta, 1971) have been reported from Kalanchoe pinnata. The leaf extract of the plant has been shown to significantly delay the onset of leishmaniasis in BALB/c mice when infected with Leishmania amazonensis (Da Silva et al., 1995). Quercitrin (quercetin 3-O-a-L-rhamnopyranoside), obtained from an active aqueous extract of the plant has been identified as the active principle (Muzitano et al., 2006). Effectiveness of an immunomodulatory extract of the plant has also been demonstrated against visceral leishmaniasis in BALB/c mouse model of infection with Leishmania chagasi (Gomes et al., 2010). Other activities reported for the plant or plant components include protection of mice against fatal anaphylactic shock (Cruz et al., 2008), hepatoprotective activity in leaves (Yadav and Dixit, 2003), anti-tumor promoting activity of bufadienolides isolated from leaves of the plant (Supratman et al., 2001) and isolation of insecticidal bufadienolides from leaves of the plant (Supratman et al., 2000).
The anti-diabetic and anti-obesity activity of Lagerstroemia speciosa as well as several phytochemical constituents isolated from the plant has been very well documented (Kakuda et al., 1996; Suzuki et al., 1999; Liu et al., 2001; Hayashi et al., 2002; Judy et al., 2003; Hattori et al., 2003; Liu et al., 2005; Yamada et al., 2008; Klein et al., 2007; Bai et al., 2008; Hou et al., 2009; Sivakumar et al., 2009; Ichikawa et al., 2010). A phytochemical isolated from the plant, orobol 7-O-D-glucoside, has been reported to have inhibitory effects on human rhinoviruses replication (Choi et al., 2010). Free radical scavenging and anti-inflammatory properties have been demonstrated in leaf extracts of the plant (Priya et al., 2008). Ethyl acetate extract of leaves has been shown to ameliorate cisplatin-induced nephrotoxicity in BALB/c mice (Priya et al., 2007). Anti-fungal activity has been demonstrated with hot water as well as methanol extract of the plant against Arthrinium sacchari M001 and Chaetomium funicola M002 strains (Sato et al., 2000). Xanthine oxidase inhibitors (valoneic acid dilactone and ellagic acid) have been isolated from leaves of the plant (Unno et al., 2004). Petroleum ether extract of seeds reportedly showed antibacterial activity against both Gram positive and Gram negative bacteria (Sinhababu et al., 1994).
In our ongoing survey on folk medicinal practices in Bangladesh (Rahmatullah et al., 2009a; 2009b; 2009c), it was observed that leaves of Kalanchoe pinnata were used by the folk medicinal practitioners in several parts of Bangladesh for treatment of rheumatoid arthritis and pain in the bones (unpublished observation). The barks of Lagerstroemia speciosa were also used as an analgesic (unpublished observation). The objective of the present study was to determine the antinociceptive potential of leaves of Kalanchoe pinnata and barks of Lagerstroemia speciosa and determine whether the experimental results validate the two plants' folk medicinal uses.
Materials and Methods
Plant material and extraction
The leaves of Kalanchoe pinnata were obtained from different places in Dhaka, Bangladesh in August, 2009. The plant was taxonomically identified by Mr. Manzur-ul-Kadir Mia, ex-Principal Scientific Officer and Curator of Bangladesh National Herbarium at Dhaka. The dried leaves of Kalanchoe pinnata (leaves were air-dried in the shade for 144 hours) were grounded into a fine powder and were extracted with methanol at a ratio of 1:5 (w/v). After 24 hours, the mixture was filtered; filtrate was collected and the residue was again extracted with methanol at a ratio of 1:3 (w/v) for 24 hrs. Filtrates were combined and evaporated to dryness using rotary evaporator. The bark of Lagerstroemia speciosa was also obtained from Dhaka in November, 2009 and taxonomically identified by Mr. Manzur-ul-Kadir Mia. The bark was air-dried in the shade for 168 hours, grounded into a fine powder and extracted with chloroform at a ratio of 1:3 (w/v). After 24 hours, the mixture was filtered; filtrate was collected and the residue re-extracted with chloroform at a ratio of 1:2 (w/v) for 24 hours. Filtrates were combined and evaporated to dryness using a rotary evaporator.
Chemicals and Drugs
Glacial acetic acid was obtained from Sigma Chemicals, USA; aspirin was obtained from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals were of analytical grade.
In the present study, Swiss albino mice (male), which weighed between 20-25g were used. The animals were obtained from the animal house of International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B). Prior to experiments, all the animals were acclimatized for one week at ambient temperature.
Acetic acid-induced writhing method
Antinociceptive activity of methanol extract of Kalanchoe pinnata leaves and Lagerstroemia speciosa bark was examined using method as previously described (Deb et al., 2010) with minor modifications. In the writhing test, pain was induced through intraperitoneal administration of 1% acetic acid at a dose of 10 ml 1% acetic acid/kg body weight. For experiments with Kalanchoe pinnata leaves, mice were separated into five groups of five mice each. Group-I served as control and was administered vehicle (1% Tween 80 in water, 10 mg/kg body weight). Aspirin was administered to Group-II mice at a dose of 200 mg/kg body weight. Groups-III to V received leaf extract, respectively at (100, 200 and 400 mg/kg body weight) orally 30 minutes before intraperitoneal administration of acetic acid. A period of 5 minutes was given to each animal to ensure bio-availability of acetic acid, following which period, the number of writhings was counted for 10 minutes. For experiments with Lagerstroemia speciosa bark, mice were separated into four groups of four mice each. Group-I served as control and was administered vehicle as described above, Group-II was administered aspirin at a dose of 200 mg/kg body weight. Groups-III and IV were administered, respectively, chloroform extract of the bark at 250 and 500 mg/kg body weight orally 30 minutes before intraperitoneal administration of acetic acid. The rest of the experiment followed as described above for Kalanchoe pinnata methanolic leaf extract.
Experimental values are expressed as Mean [+ or -] SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a P value < 0.05 in all cases.
Results and Discussion
In the acetic acid-induced writhing test model in mice, the crude methanolic extract of leaves of Kalanchoe pinnata demonstrated a significant dose-dependent reduction in the number of writhings. Even at the lowest dose of the crude extract tested (100 mg extract/kg body weight), the extract showed 36.4% inhibition of writhings in mice, which compare favorably with the standard drug, aspirin, given at a dose of 200 mg/kg body weight (38.4% inhibition). Highest inhibition of writhings was observed with the crude methanolic leaf extract at a dose of 400 mg extract/kg body weight. The results are shown in Table 1.
Two doses of crude chloroform extract of bark of Lagerstroemia speciosa were tested for any writhing inhibitory activity in acetic acid-induced pain model in mice. While the crude chloroform bark extract, when administered at a dose of 250 mg extract/kg body weight decreased the number of writhings compared to control mice, the decrease was not statistically significant. However, the extract, when administered at a dose of 500 mg extract/kg body weight, significantly inhibited the number of writhings (50.7%), which was comparable to the inhibition observed with a standard antinociceptive drug, aspirin, given at a dose of 200 mg/kg body weight (54.0%). The results are shown in Table 2.
The present study evaluated the antinociceptive potential of crude methanolic extract of Kalanchoe pinnata leaves and crude chloroform extract of Lagerstroemia speciosa bark in acetic acid-induced writhing model in mice. It has been reported that this test is suitable for detection of both central and peripheral analgesia (Shanmugasundaram and Venkataraman, 2005). When acetic acid (1%) is intraperitoneally administered, it leads to pain and inflammation. The mechanism behind this appears to be production of prostaglandins [mainly prostacyclins (PGI2) and prostaglandin-E (PG-E)], which have been reported to be the responsible factors for excitation of Ad-nerve fibers, leading to sensation of pain (Reynolds, 1982; Rang and Dale, 1993). Therefore, it may be concluded that any agent that lowers the number of writhings will demonstrate analgesia by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition.
Both leaf extract of Kalanchoe pinnata and bark extract of Lagerstroemia speciosa caused significant reductions of writhings induced by intraperitoneal administration of acetic acid in mice in the present study. The results suggest that extracts from both plants contain significant antinociceptive properties. The actual phytochemical constituent(s) responsible for this antinociceptive activity is currently being investigated in our laboratory. However, it may be noted that results obtained in the present study validates the use of both plants in folk medicines of Bangladesh for treatment of pain.
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(1) Ahosan Morshed, (1) Md. Hasibul Hossain, (1) Shahriar Shakil, (1) Khaleda Nahar, (1) Shahnaz Rahman, (1) Dilara Ferdausi, (1) Tozammel Hossain, (1) Ishtiaq Ahmad, (2) Majeedul H. Chowdhury, (1) Mohammed Rahmatullah
(1) Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka, Bangladesh.
(2) New York City College of Technology The City University of New York Broooklyn, NY 11201, USA.
Corresponding Author: Dr. Mohammed Rahmatullah, Pro-Vice Chancello University of Development Alternative House No. 78, Road No. 11A (new) Dhanmondi R/A, Dhaka-1205 Bangladesh
Email: email@example.com Fax: 88-02-8157339
Table 1: Antinociceptive effect of crude methanol extract of Kalanchoe pinnata leaves in the acetic acid-induced gastric pain model mice. Groups Dose (mg/kg Mean no. of Inhibition body weight) writing (%) Control (vehicle) 10 ml 19.8 [+ or -] 1.3 -- Aspirin 200 mg 12.2 [+ or -] 1.0 38.4 * Kalanchoe pinnata 100 mg 12.6 [+ or -] 2.4 36.4 * Kalanchoe pinnata 200 mg 11.4 [+ or -] 1.2 42.4 * Kalanchoe pinnata 400 mg 10.4 [+ or -] 2.0 47.5 * All administrations were made orally. Values are represented as Mean [+ or -] SEM, (n=5); * significant at 5% significance level. Table 2: Antinociceptive effect of crude chloroform extract of Lagerstroemia speciosa bark in the acetic acid-induced gastric pain model mice. Groups Dose (mg/kg Mean no. of body weight) writing Control (vehicle) 10 ml 21.3 [+ or -] 2.1 Aspirin 200 mg 9.8 [+ or -] 0.5 Lagerstroemia speciosa 250 mg 17.8 [+ or -] 2.8 Lagerstroemia speciosa 500 mg 10.5 [+ or -] 0.5 Groups Inhibition (%) Control (vehicle) -- Aspirin 54.0 * Lagerstroemia speciosa 16.4 Lagerstroemia speciosa 50.7 * All administrations were made orally. Values are represented as Mean [+ or -] SEM, (n=4); * significant at 5% significance level.