Abstract
The ethanol extract of heartwood of Caesalpinia sappan. Linn (Caesalpiniceae) and three crude fractions [petroleum ether (60–80°C), diethyl ether, and ethyl acetate] were subjected to preliminary qualitative chemical investigation. The ethanol extract and its fractions underwent pharmacological screening for analgesic activity by using acetic acid-induced writhing in albino mice. The ethanol extract and its different fractions of heartwood of Caesalpinia sappan. exhibited significant analgesic activity (p < 0.01). In conclusion, the ethanol extract of heartwood of Caesalpinia sappan. and its three different crude fractions were found to show peripheral analgesic activity.
Introduction
Caesalpina sappan. Linn (Caesalpiniceae) is commonly known as Sappan wood or patang (Hindi). It is a spreading tree or shrub that grows up to 10 m in height and is found in India (West Bengal, Orissa, Kerala) Malaya, China, and Sri Lanka (Kirtikar & Basu, Citation1989). Several triterpenoids, flavonoids, and steroids have been isolated from the heartwood of C. sappan. (Namikoshi et al., Citation1987). The methanol extract of Caesalpinia sappan. has shown anti-inflammatory activity (Wikino et al., Citation1977). However, there are no reports of analgesic activity. Hence, in the present study, the ethanol extract and its different fractions were subjected to pharmacological screening for analgesic activity.
Materials and Methods
The heartwood of Caesalpinia sappan. was collected from the Medicinal Garden of SDM College of Ayurveda, Udupi, and authenticated by Dr. T. Shridhara Bairy who compared it with standard specimens deposited at the Department of Dravaguna, SDM College of Ayurveda, Udupi. Voucher specimens are kept at the NGSM Institute of Pharmaceutical Sciences, Nanthoor, Mangalore, India.
Around 4 kg of shade-dried heartwood C. sappan. was powdered and extracted with 95% ethanol in 13 batches of 300 g each. The extract was concentrated under reduced pressure to yield a blackish brown sticky mass.
The concentrated ethanol extract (200 g) was dispersed in 300 ml of distilled water and subjected to successive fractionation by the solvents petroleum ether (60–80°C), diethyl ether, and ethyl acetate. Each fraction was concentrated and evaporated to dryness.The ethanol extract and its fractions were subjected to qualitative chemical investigation for phytoconstituents such as flavonoids, glycosides, triterpenoids, tannins, steroids, and lipids. The dried ethanol extract and its fractions were stored in a desiccator and subjected to pharmacological studies (Hukkeri et al., Citation2001).
Pharmacological screening
Adult female Wistar albino rats (150–200 g) were used for acute toxicity studies, and swiss albino mice (either sex, 20–25 g) were used for the pharmacological study. Animals were obtained from the animal colony of the NGSM Institute of Pharmaceutical Sciences. They were housed in polypropylene cages in an air-conditioned area at 25°C with a 12 h light and dark cycle. The animals were provided with standard laboratory food and water ad libitum.. The experiment was carried out according to CPCSEA guidelines, and the Institutional Animal Ethics Committee approved the procedure.
Acute toxicity study
The preliminary pharmacological studies were conducted to assess the acute pharmacological effects and LD50 of the ethanol extract and its fractions (petroleum ether, diethyl ether, and ethyl acetate). The ethanol extract and its different fractions were subjected to acute oral toxicity studies in adult female albino rats, according to OECD guideline 423.
Selection of doses
For the assessment of analgesic activity, two dose levels were chosen, i.e., the lower dose was one-tenth of the LD50 cut off value observed, and a high dose was twice that of one-tenth dose (200 and 400 mg/kg, respectively).
Dose preparation
Ethanol extract and its different fractions and aspirin were prepared as suspension using 0.6% w/v carboxy-methyl cellulose as suspending agent.
Experimental design
Animals were randomly divided into ten groups, each containing five animals, and were treated as described below.
Group 1: Served as control, receiving 0.6% w/v of sodium carboxymethyl cellulose (CMC).
Group 2: Served as positive control and received acetyl salicylic acid (aspirin) standard drug (50 mg/kg).
Group 3 & 4: Animals were treated with ethanol extract of heartwood of C. sappan., 200 and 400 mg/kg, respectively.
Group 5 & 6: Animals were treated with petroleum ether fraction of heartwood of C. sappan., 200 and 400 mg/kg, respectively.
Group 7 & 8: Animals were treated with diethyl ether fraction of heartwood of C. sappan., 200 and 400 mg/kg, respectively.
Group 9 & 10: Animals were treated with ethyl acetate fraction of heartwood of C. sappan., 200 and 400 mg/kg, respectively.
The animals in all ten groups were administered 0.1 ml of 0.6% v/v solution of acetic acid by intraperitoneal (i.p.) route 30 min after treatment with extracts and standard drug. The number of writhing episodes of an individual mouse was recorded for 20 min after acetic acid injection (Koster et al., Citation1959).
Statistical analysis
All data are expressed as Mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnet's test. P < 0.01 is considered significant.
Results
Qualitative chemical investigation and acute oral toxicity study
The ethanol extract and its different fractions (petroleum ether, diethyl ether, and ethyl acetate) of C. sappan. heartwood were found to contain flavonoids, triterpenoids, tannins, and sterols.
The ethanol extract and its different fractions do not show any sign of toxicity or mortality. Hence, at dose level 2000 mg/kg, the LD50 cut off value of ethanol extract and its different fractions was found to be infinite.
Analgesic activity
The effect of ethanol extract of Caesalpinia sappan. and its different fractions on acetic acid-induced writhing in mice is summarized in . Acetic acid induced the characteristic writhing response when injected intraperitoneally into Swiss albino mice. Treatment with ethanol extract (200 and 400 mg/kg) significantly inhibited the number of writhings 69.71 and 73.33%, respectively (p < 0.01). Petroleum ether fraction (200 and 400 mg/kg) significantly inhibited the number of writhings 53.16 and 55.16%, respectively (p < 0.01). Diethyl ether fraction (200 and 400 mg/kg) significantly inhibited the number of writhings 48.08 and 49.02%, respectively (p < 0.01). Ethyl acetate fraction (200 and 400 mg/kg) significantly inhibited the number of writhings 62.47 and 68.84%, respectively (p < 0.01). Aspirin (50 mg/kg) significantly inhibited the number of writhings (75.22%) in comparison with the control group (p < 0.01).
Table 1.. Effect of various extracts from Caesalpinia sappan. heartwood on acetic-induced writhings in albino mice.
Discussion
In the present study, the ethanol extract and its different fractions (petroleum ether, diethyl ether, and ethyl acetate) of C. sappan. heartwood were tested for peripheral analgesic activity using acetic acid-induced writhing in mice because acetic acid-induced writhing in mice is a widely used model for screening the compounds for peripheral analgesic activity. Oral administration of the ethanol extract and its different fractions of heartwood of C. sappan. at both dose levels (200 and 400 mg/kg) significantly inhibited acetic acid-induced writhing comparable to the effects of aspirin, indicating that the ethanol extract as well as its fractions had the potential to block the process of pain pathway. Further studies are needed, however, to explore the mechanism by which C. sappan. produces the analgesic action. C. sappan. heartwood contains flavonoids, triterpenoids, and sterols; we speculate that triterpenoids and sterols may be responsible for the observed analgesic activity, since triterpenoids and sterols are known to have anti-inflammatory and analgesic effects.
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