Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 2, 2006, pp. 115-119

Indian Journal of Pharmacology, Vol. 38, No. 2, March-April, 2006, pp. 115-119

Research Paper

Antiinflammatory activity of leaf extracts of Kalanchoe crenata Andr.

T. Dimo¹ , Agathe L. Fotio¹ , T.B. Nguelefack² , E.A. Asongalem³ , P. Kamtchouing¹

¹ Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde I,B.P. 812 Yaounde, Cameroon.
² Department of Animal Biology, Faculty of Science, University of Dschang, BP 67 Dschang, Cameroon.
³ Department of Physiological. Sciences, Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Cameroon.
Correspondence Address:Théophile Dimo Email: tdimo@uycdc.uninet.cm

Code Number: ph06029

Abstract

Objective: To evaluate the acute and chronic antiinflammatory properties of leaf extracts of Kalanchoe crenata in rats.
Material and methods: The methylene chloride/methanol extract of K. crenata was extracted by using hexane, methylene chloride, ethyl acetate, and n-butanol. The antiinflammatory profile of these extracts was investigated on the basis of paw edema induced by carrageenan. The n-butanol fraction (most potent) was further assessed through acute inflammatory models induced by histamine, serotonin, and formalin. The chronic antiinflammatory and the ulcerogenic activities of the n-butanol fraction were also examined.
Results: The oral administration of n-butanol fraction (600 mg/kg) caused a maximum inhibition of about 45% in paw edema induced by carrageenan. The n-butanol fraction also exhibited acute antiinflammatory activity on paw edema induced by histamine (47.51%), serotonin (54.71%), and formalin-(40.00%) . In the chronic inflammation model, this extract showed maximum inhibition of 61.26% on the ninth day of treatment. The ulcerogenic assessment showed that ulcer indices after oral treatment with n-butanol fraction were zero and 0.4±0.2, for the 300 and 600 mg/kg doses, respectively.
Conclusion: On the basis of these findings, it may be inferred that K. crenata is an antiinflammatory and antiarthritic agent that blocks histamine and serotonin pathways. The results are in agreement with the traditional use of the plant in inflammatory conditions.

Keywords: Antiarthritic, paw edema, ulcer index

Introduction

Kalanchoe crenata Andr. (Crassulaceae), commonly known as "never die" or "Dog′s liver," has been traditionally used for the treatment of ailments, such as, earache, smallpox, headache, inflammation, pain, asthma, palpitations, convulsion, and general debility. Aqueous and alcoholic extracts of K. crenata leaves contain alkaloids and saponins.[1]

In an earlier study, the aqueous and the ethanolic extracts of K. crenata were found to possess antinociceptive activity against acetic acid, formalin, and hot plate, as well as pain models induced by pressure.[2] This work was aimed at the scientific validation of the ethnopharmacological claim about the antiinflammatory property of the leaf extracts.

Materials and methods

Plant material
K. crenata leaves were collected from Dschang (West province, Cameroon) in October 2002 and authenticated by comparison with a voucher specimen number 50103/YA in the National Herbarium, Yaounde, Cameroon. Two kg of the air-dried leaves were blended to a fine powder and extracted with methylene chloride/methanol (CH ₂ Cl ₂ /CH ₃ OH) for 3 days (72 hours). The extract was concentrated using a rotavapor to obtain 234.7g of the CH ₂ Cl ₂ / CH ₃ OH extract, which (212 g) was further extracted successively with hexane, methylene chloride, ethyl acetate and n-butanol. The following fractions were obtained: hexane (92.2 g), CH ₂ Cl ₂ (4.7 g), ethyl acetate (7.1 g), n-butanol (37.0 g), and aqueous residue (23.5 g). The CH ₂ Cl ₂ /CH ₃ OH, CH ₂ Cl ₂ . and n-butanol fractions were dissolved in 2.5 % DMSO and 2.5 % Tween 20, while the hexane and the ethyl acetate fractions were dissolved in 3% DMSO before orally administrating 300 and 600 mg/kg of each fraction to the rats. The preliminary investigations for the antiinflammatory activity of the various fractions on paw edema induced by carrageenan, showed that the n-butanol fraction of the extract was the most potent. Therefore, it was chosen for further investigations.

Phytochemical screening
The extract and its fractions were tested by the Liberman Burchard, Ferric chloride, Magnesium tracings, and Vanillin-sulphuric acid tests to determine the presence of sterols, phenolic compounds, flavonoids, and saponins, respectively.

Chemicals
Indomethacin (Sigma), pyrilamine maleate (Sigma), diclofenac, carrageenan (Sigma), histamine (Fluka), serotonin (5-HT) (Fluka), and formaldehyde (Roth) were used.

Animals
Wistar rats (140-190 g) of both sexes were used for the studies. These rats were obtained from the Department of Animal Biology and Physiology, University of Yaounde I, Cameroon. The animals were housed in cages under standard laboratory conditions (12:12 hour light/dark cycle at 25 ± 2°C). They had free access to standard commercial diet and water. The animals were divided into groups of five and fasted for 12 hours before the experiment. The ethical guidelines for the investigation of animals used in experiments were followed in all tests.

Paw edema induced by carrageenan
0.1 ml of 1% carrageenan in 0.9% NaCl was administered into the plantar surface of the right hind paw of the animals.[3] The experimental groups, negative control group (2.5% DMSO and 2.5% tween 20), and positive control group (10 mg/kg indomethacin) were given either the control drug or test compounds orally, an hour prior to the administration of the carrageenan. Before injection of carrageenan, the average volume (Vo) of the right hind paw of each rat was calculated from 3 readings that did not deviate more than 3%. After injection of the phlogistic agent, readings (Vt) were obtained for each rat at 30, 60, 120, 180, 240, 300 and 360 min, with the aid of a Ugo Basil Plethysmometer (7150). The edema was expressed as an increase in the volume of paw, and the percentage of inhibition for each rat and each group was obtained as follows:

Paw edema induced by histamine and serotonin
In another set of experiments serotonin and histamine were used as the phlogistic agents. The n-butanol fraction of K. crenata extract (experimental groups) and control vehicle (solution of 2.5% DMSO and 2.5 % Tween 20) were administered one hour before the injection of inflammatory mediators. The respective strength of the inflammatory mediators, the volume injected, and the time of determination of volume of edema are indicated in parentheses, serotonin (10 ^-3g/ml, 0.1 mL, 30 min.) and histamine (10 ^-3 g/ml, 0.1 mL, 60 min).[4] Pyrilamine maleate (1 mg/kg) was used as the antagonist of histamine. The volume of paw edema was determined as mentioned previously.

Paw edema induced by formalin
Acute inflammation was induced by subaponeurotic injection of 0.1 ml of 2% formalin one hour after oral administration of n-butanol fraction, diclofenac (5 mg/kg), or vehicle (solution of 2.5% DMSO and 2.5% tween 20). The volume of paw was determined one, two, and four hours following the injection of formalin. For chronic inflammation study, the above animals were further treated with the n-butanol fraction, diclofenac or vehicle, once daily, for 9 consecutive days. A second injection of formalin was given on the third day.[5] The daily changes in the volume of paw were measured plethysmographically.

Ulcerogenic activity
The ulcerogenic potential of the n-butanol fraction (300 and 600 mg/kg), indomethacin (10 mg/kg), or control vehicle (solution of 2.5% DMSO and 2.5% tween 20) was tested on rats that had been fasted for 24 h. Two hours following oral administration of these drugs, the rats were sacrificed by cervical dislocation. The stomach was isolated and opened along the greater curvature. Ulcerated surfaces were measured and scored according to the table described by Martin et al. [6]

Statistical analysis
All values are presented as mean±SEM of five rats. Differences between means were assessed by one-way analysis of variance (ANOVA), followed by Dunnett′s test using Stat-Direct-Software. P< 0.05 was considered significant.

Results

Phytochemical screening
The phytochemical analysis revealed the presence of sterols in the methylene chloride/methanol (CH ₂ Cl ₂ /CH ₃ OH) and its hexane fraction. Sterols, flavonoids, and saponins were found in the methylene chloride and ethyl acetate fractions. Flavonoids and saponins were detected in CH ₂ Cl ₂ /CH ₃ OH extract and its n-butanol fraction.

Paw edema induced by carrageenan
The effects of extracts of K. crenata on paw edema induced by carrageenan are shown in [Table - 1]. The CH ₂ Cl ₂ /CH ₃ OH extract showed (600 mg/kg) a maximum antiinflammatory effect of about 43.47% (30 min), while the hexane, CH ₂ Cl ₂ . ethyl acetate, n-butanol, and aqueous fractions showed maximal antiinflammatory effects of 47.82% (30 min), 38.88% (1 h), 44.44% (1 h), 45.45% (2 h) and 39.13% (30 min), respectively. The antiinflammatory effect of the n-butanol fraction was more potent and significant during the three phases of inflammation, compared with other fractions. The antiinflammatory effect induced by indomethacin progressively increased and reached a maximum (65.82%) at three hours. It was maintained up to six hours.

Paw edema induced by serotonin and histamine
[Table - 2] shows the effect of n-butanol fraction on paw edema induced by serotonin and histamine. At 300 mg/kg dose, the n-butanol fraction significantly reduced the formation of paw edema induced by histamine (41.66%). The percentage of inhibition at the dose of 600 mg/kg was 47.51% and 54.71% for inflammation induced by histamine and serotonin, respectively. Inflammation induced by histamine was inhibited by 58.01% by pyrilamine maleate at the dose of 1 mg/kg.

Paw edema induced by formalin
The n-butanol fraction of extract of K. crenata (300 and 600 mg/kg) and diclofenac (5 mg/kg) significantly inhibited inflammation induced by formalin by 32.85,% 40.00%, and 44.28%, respectively, four hours after administration of formalin. [Table - 3]

The n-butanol fraction showed a significant inhibition of chronic inflammation. An inflammation of 21.37% was observed in rats treated with the extract (600 mg/kg) on the ninth day, presenting a maximum inhibition of 61.26%. [Figure - 1]

Ulcerogenic activity
As shown in [Table - 4], 300 mg/kg of the n-butanol fraction of extract of K. crenata failed to induce gastric ulceration. At the dose of 600 mg/kg, two rats showed blood vessel dilatation, corresponding to an average ulcer index of 0.4. All the animals that received indomethacin presented a significant ulceration of the stomach mucosa (ulcerated area = 6.85 mm ²). Indomethacin as well as n-butanol fraction (300 mg/kg) significantly reduced the mucus weight.

Discussion

The results of this study indicate that the leaf extracts of K. crenata possess acute and chronic antiinflammatory activity against various phlogistic agents. Inflammation induced by carrageenan involves three distinct phases of the release of the mediator, including serotonin and histamine in the first phase (0 - 2 h), kinins in the second phase (3 h), and prostaglandin in the third phase[4] (>4 h). The CH ₂ Cl ₂ /CH ₃ OH extract of K. crenata and the aqueous fraction significantly inhibited paw edema induced by carrageenan in the first phase, suggesting an inhibitory effect on the release of histamine and/or serotonin. The n-butanol fraction showed significant inhibition of the edema in all the three phases. This antiedematous response was also significantly reduced in rats pre-treated with indomethacin, a known cyclooxygenase inhibitor. The n-butanol fraction was chosen for further studies because it was more active, compared with the other fractions. To ascertain the effect of the n-butanol fraction on the activities of the mediator, it was tested on inflammation induced by histamine and serotonin, characterised by increased vascular permeability. It was observed that the n-butanol fraction was capable of inhibiting edema induced by histamine and serotonin. Furthermore, edema induced by formalin was also significantly inhibited by the n-butanol fraction of extract of K. crenata . According to Yuh-Fung et al ,[7] acute inflammation induced by formalin results from cell damage, which provokes the production of endogenous mediators, such as, histamine, serotonin, prostaglandins, and bradykinin.

The n-butanol fraction was further tested on chronic inflammation induced by formalin. It is well known that inhibition of edema induced by formalin in rats is one of the most suitable test procedures to screen antiarthritic and antiinflammatory agents, as it closely resembles human arthritis.[8] Arthritis induced by formalin is a model used for the evaluation of an agent with probable antiproliferative activity.[9] As the n-butanol fraction significantly inhibited this model of inflammation, it can be thought to possess antiproliferative and antiarthritic activities similar to diclofenac, a cyclooxygenase inhibitor.

As results from this study strongly indicate the non-steroidal, antiinflammatory-like activity of the extract, its ulcerogenic effect was tested on fasted animals. Non-steroidal, antiinflammatory drugs are thought to impair the mucosal defense of the stomach and the intestine. They act by inhibition of cyclooxygenase and, therefore, inhibit the production of gastric prostaglandins. This leads to a reduction in production of gastric mucus and an increase in mucosal permeability.[10] The reduced weight of mucus coupled with mild ulceration of the gastric mucosa caused by the n-butanol fraction could be due to inhibition of cyclo-oxygenase. The presence of flavonoids in the n-butanol fraction may account for its observed pharmacological activities. Many compounds from this class have been found to exhibit antiinflammatory effects.[11],[12]

To conclude, the results showed antiinflammatory and antiarthritic activities of the CH ₂ Cl ₂ /CH ₃ OH extract of K . crenata and its fractions. These activities were related to dose and these results corroborate the traditional use of the plant in inflammatory conditions.

References

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