CNS Depressant and Analgesic Activity of a Fraction Isolated from an Ethanol Extract of Curcuma amada Rhizomes

Abstract

In our earlier studies, an ethanol extract of Curcuma amada Roxb. rhizome was reported for anti-inflammatory activity. This ethanol extract was successively partitioned with hexane and ethyl acetate. Each fractionated extract was screened for anti-inflammatory activity, and the most active fraction was purified by column chromatography. This resulted in isolation of the active fraction F, which was characterized by spectral methods. Fraction F was studied for its effect on general behavioral studies in albino mice. Based on these observations, pharmacological investigations were carried out by adopting the following methods in albino mice: effect on exploratory activity, barbiturate sleeping time, acetic acid–induced writhing, and tail-flick test. This fraction showed reduction in exploratory activity and potentiation of barbiturate sleeping time, indicating CNS depressant activity. Further, it also showed reduction in acetic acid–induced writhings, tail-flick response, and carrageenan-induced inflammation, indicating potential antinociceptive and antiphlogistic activity, respectively.

Keywords:

• Analgesic
• anti-inflammatory activity
• CNS activity
• Curcuma amada rhizome
• Fraction F

Introduction

Curcuma amada Roxb., popularly known as mango ginger, belongs to the family Zingiberaceae. It is cultivated in India and Malaysia and is well-known for culinary preparations like pickles due to its exotic aroma. It is used in Ayurveda to treat contusions and sprains.

Chemical investigation of Curcuma amada rhizomes showed the presence of curcumene, a sesquiterpene hydrocarbon, as a major aroma component (Jain & Mishra, 1964). The essential oil of rhizomes of Curcuma amada Roxb. was found to contain α-pinene, car-3-ene and cis-ocimene, where the latter two compounds contributed to the characteristic mango odor of these rhizomes (Gholap & Bandopadhyay, 1984). The analysis of volatile aroma components of C. amada rhizomes by GC-MS led to the identification of a large number of compounds (Rao et al., 1989).

Although the chemical examination of the extract of C. amada rhizomes was initiated in the 1960s, its systematic pharmacological examination is hitherto unknown. Moreover, the therapeutic applications of C. amada as carminative, stomachic (Hussain et al., 1992), and CNS active agent (Bhakuni et al., 1969) suggest its strong potential as a pharmaceutical. In our earlier work, this potential was explored, and the crude ethanol extract was shown to exhibit dose-dependent anti-inflammatory activity in acute and chronic models (Mujumdar et al., 2000s). To locate the active compound(s), it was decided to fractionate and purify the crude extract and screen the fractions for anti-inflammatory activity. The most active fraction F, thus obtained after the above fractionation, was studied for CNS depressant and analgesic activity based on observations in general behavioral studies.

Materials and Methods

Plant material, extraction, and isolation of fraction F

The rhizomes of C. amada (11 kg) were procured in bulk quantity from the Western ghat area of India in the winter season. The rhizomes were authenticated by the Botany Group (voucher specimen no. R044, deposited at Agharkar Herbarium of Maharashtra Association at Agharkar Research Institute, Pune, India) and washed with distilled water to remove soil and then shredded and shade-dried (0.804 kg, 7.31%). It was extracted using a Soxhlet extractor with ethyl alcohol, yielding dark-colored, fragrant, viscous liquid (45.32 g, 5.63%). This active crude extract was successively partitioned with hexane and ethyl acetate to locate the activity. Fractionated extracts thus obtained were screened for anti-inflammatory activity. Based on these results, the most active hexane fraction was purified by column chromatography. The anti-inflammatory activity of each of the seven fractions (A–G) obtained by column chromatography was determined to locate the most active fraction F (F). Preliminary characterization of this fraction was carried out by recording its thin-layer chromatography (TLC) and spectral data.

Experimental animals

The experiments were carried out in Swiss albino mice (20–25 g) and a Wister rats (100–150 g) of either sex. Mice were originally obtained from the National Institute of Virology (Pune, India) and rats (HA strain) were obtained from Hindustan Antibiotics Ltd. (Pimpri, India). They have been inbred at Agharkar Research Institute for several generations at the animal house facility for the last 17 and 6 years, respectively. These animals were reared and housed in polypropylene cages at 25±2°C and 10:14-h light and dark cycle. They were maintained on commercially available Amrut brand animal feed and water ad libitum. The optimum condition for experiments was decided on the basis of pilot experiments. For pharmacological experiments, a group of six animals was used for individual treatment.

Carrageenan-induced rat paw edema

F was administered at 100 or 200 mg/kg or indomethacin 10 mg/kg orally 30 min prior to carrageenan subcutaneous injection in the plantar region of the rat hind paw as a phleogestic agent to induce inflammation (Winter et al., 1962). The paw volume was recorded initially and after 1, 2, and 4 h to measure inflammation by using the plethysmographic method (Harris & Spencer, 1962).

General behavioral observations

F was administered up to a maximum dose of 500 mg/kg orally to albino mice, which were observed for general behavioral changes and mortality, if any, initially for 4 h and then once daily for a week. Based on the observations in these studies CNS depressant and analgesic activity was studied further using various models as follows.

Exploratory behavior

Effect on exploratory behavior was studied by the hole-board method (File & Wardrill, 1975s), using white board of 40 × 40 cm dimension with 4 equidistant holes of 1 cm diameter and 2 cm depth. The mouse was placed on a board, and the animal moved about and dipped its head in the holes. The number of head dips were counted for 7.5 min as an index of exploratory activity. The test was carried out in mice 30 min after treatment of F 50 mg/kg, 100 mg/kg orally and chlorpromazine 1 mg/kg i.m. as a positive control to various groups of albino mice.

Barbiturate sleeping time

Barbiturate sleeping time was measured in albino mice as a time interval between loss and regain of righting reflex after treatment. This study was carried out using chlorpromazine 1 mg/kg as a positive control as follows:

1. Effect of F (50 and 100 mg/kg p.o.) pretreatment on pentobarbitone sodium (35 mg/kg, i.p.) sleeping time.

2. Effect of F on (50 and 100 mg/kg p.o.) pretreatment on pentobarbitone sodium (single dose 35 mg/kg, i.p. for 5 days) sleeping time.

3. Effect of F (50 mg/kg and 100 mg/kg p.o.) pretreatment on barbital sodium (200 mg/kg, i.p.) sleeping time.

Acetic acid — induced writhings

In albino mice, total number of writhings following i. p. administration of 0.6% acetic acid 10 ml/kg were recorded for 20 min, starting 10 min after injection. The animals were pretreated with F 50 and 100 mg/kg 30 min prior to the above treatment, simultaneously using aspirin 165 mg/kg orally as a positive control (Turner, 1965).

Tail flick

The mouse was held firmly to immerse its tail in a water bath maintained at a constant temperature of 58°C. The animals showing typical reaction of violent jerk of tail within 15 s were included in the test. This test was performed 30 min after treatment of F 50 or 100 mg/kg or aspirin 200 mg/kg to various groups of animals for typical reaction of a violent jerk of the tail to assess response to noxious stimulus (Turner, 1965).

Statistical analysis

The statistical analysis of the results was carried out to calculate mean±SEM. Further analysis was carried out by Student's t-test and χ² test to calculate significance of the results. p < 0.05 was considered non-significant.

Results

Fractionation of crude extract

The crude extract (45.32 g) was stirred with hexane (5 × 60 ml) and ethyl acetate (4 × 40 ml) to yield hexane-soluble fraction (30.75 g, 67.8%), ethyl acetate–soluble fraction (2.49 g, 5.49%), and insoluble portion (11.89 g, 26.25%). Screening of these three fractions for anti-inflammatory activity indicated significant action of hexane-soluble fraction, which was obtained in major quantity. This fraction (30 g) was chromatographed over silica gel (1200 g, 60–120 mesh) yielding (i) fraction A, 0.384 g (1.28%); (ii) fraction B, 0.321 g (1.07%); (iii) fraction C, 0.192 g (0.64%); (iv) fraction D, 0.852 g (2.84%); (v) fraction E, 4.05 g (13.5%); (vi) fraction F, 7.44 g (24.8%); and (vii) fraction G, 15.74 g (52.46%). All the fractions were screened for anti-inflammatory activity, out of which fraction F exhibited the significant activity. Examination of fraction F by TLC indicated the presence of closely related spots in the R_f range 0.5–0.75 (hexane: ethyl acetate 10%). The infrared spectrum of this fraction indicated bands at 3400, 1760, 1380, 1700, and 1640 cm⁻¹, indicating the presence of hydroxyl, ester, carbonyl, acid, and olefin functionalities in the constituents. Its PMR spectrum showed prominent signals around 1 δ and 1.5 δ (methyl, methylene, and methine protons) and 5.2 δ (olefinic protons).

Carrageenan-induced rat paw edema

Fraction F as well as indomethacin showed significant reduction in carrageenan-induced inflammatory changes in rat paw edema model up to 4 h, as shown in Table 1.

Table 1 Effect of fraction F on carrageenan-induced rat paw edema.

		% Inflammation±SEM at:
Sr. no.	Treatment	1 h	2 h	4 h
1	Control	59.24 ± 3.44	99.99 ± 6.28	129.41 ± 10.23
2	F 100 mg/kg	23.30 ± 5.03	50.53 ± 5.09	73.39 ± 4.10
3	F 200 mg/kg	21.80±1.89	37.97 ± 3.89	55.12 ± 0.92
4	Indomethacin 10 mg/kg	22.47 ± 3.47	31.37 ± 4.44	56.11 ± 4.56

*Significant as compared to control, p < 0.001.

General behavioral observations

In general, there was reduction in motor activity of mice (+ + + +). They were sitting in the corner with occasional movement, indicating CNS depressant activity. In addition to this, analgesic activity (+ +) was also observed. These observed changes were maximum at 30 min and subsequently they were normal by 4 h. Based on this 30-min pretreatment, 50 mg/kg and 100 mg/kg doses of F were used for further studies.

Exploratory activity

There was significant and dose-dependent reduction due to pretreatment of fraction F at 50 or 100 mg/kg dose levels as compared to the control mice. However, this activity was less as compared to chlorpromazine, as shown in Table 2.

Table 2 Effect of fraction F on exploratory activity of albino mice.

Sr. no.	Pretreatment	Mean number of head dips ± SEM
1.	Control	21.83 ± 0.68
2.	F 50 mg/kg	11.17 ± 0.86
3.	F 100 mg/kg	7.67 ± 0.77
4.	Chlorpromazine HCl 1 mg/kg	7.17 ± 0.28

*Significant as compared to control, p < 0.001.

Effect on barbiturate sleeping time

The pentobarbitone sodium sleeping time was enhanced in normal as well as chronically treated mice significantly by pretreatment with fraction F at 50 or 100 mg/kg or chlorpromazine 1 mg/kg. This pretreatment also potentiated barbitone sodium–induced hypnosis, as shown in Table 3.

Table 3 Effect of fraction F on barbiturate sleeping time in albino mice.

		Pentobarbitone sodium sleeping time in minutes±SEM		Barbitone sodium sleeping time in minutes±SEM
Sr. no.	Pretreatment	Acute	Chronic^a
1	Control	62.67 ± 2.06	12.83 ± 2.89	104.17 ± 5.95
2	F 50 mg/kg	71.17 ± 1.82**	36.17 ± 4.47	139.83 ± 13.43**
3	F 100 mg/kg	103.33±9.03	53.33 ± 2.23	173.83 ± 9.84
4	Chlorpromazine HCl 1 mg/kg	100.33 ± 5.85	45.83 ± 2.25	139.50 ± 6.22

^aPretreatment of single dose of pentobarbitone sodium 35 mg/kg, i.p. (5 days).

*Significant as compared to control, p < 0.001.

**Significant as compared to control, p < 0.02.

Effect on acetic acid–induced writhings

There was significant reduction in acetic acid–induced writhings in mice treated with fraction F at 50 or 100 mg/kg or aspirin 165 mg/kg as compared to the control group, as shown in Table 4.

Table 4 Effect of fraction F on acetic acid–induced writhings.

Sr. no.	Pretreatment	Mean number of writhings±SEM
1	Control	42.50 ± 1.61
2	F 50 mg/kg	31.00 ± 0.74
3	F 100 mg/kg	17.66 ± 0.69
4	Aspirin 165 mg/kg	18.00 ± 1.24

*Significant as compared to control, p < 0.001.

Effect on tail-flick response

The tail-flick response against noxious stimuli was significantly reduced in mice treated with fraction F at 50 or 100 mg/kg or aspirin at 200 mg/kg dose, as shown in Table 5.

Table 5 Effect of fraction F on tail-flick response.

Sr. no.	Pretreatment	% of mice showing analgesia
1	Control	0
2	F 50 mg/kg	50
3	F 100 mg/kg	66.67**
4	Aspirin 200 mg/kg	83.33***

*Significant as compared to control, p < 0.05.

**Significant as compared to control, p < 0.02.

***Significant as compared to control, p < 0.01.

Discussion

The anti-inflammatory activity of the total crude ethyl alcohol extract of C. amada rhizomes was demonstrated by us earlier (Mujumdar et al., 2000s). The fraction F isolated after purifying the crude extract by solvent separation and column chromatography showed dose-dependent significant anti-inflammatory activity up to 4 h. This result indicates activity against early components like histamine, serotonin (5-HT), and the latter components like prostaglandins (Just et al., 1998s). This fraction showed reduction in activity in general behavior studies. In support of this observation there was dose-dependent reduction in exploratory activity of mouse, however, this reduction was less as compared to chlorpromazine pretreatment. Further, it enhanced pentobarbitone sodium–induced hypnosis in normal as well as in chronic treatment. It also enhanced barbitone sodium sleeping time. Usually, pentobarbitone sleeping time is potentiated due to inherent depressant activity or effect on degradation by inhibition of liver microsomal enzyme system by the drug under consideration. Barbitone sodium is known to have central nervous system depressant activity. Moreover, it is not degraded by liver microsomal enzyme system. It is also a well-known fact that after repeated administration of pentobarbitone, there is stimulation of the liver microsomal enzyme system, leading to rapid degradation of pentobarbitone and thereby reduction in sleeping time. In acetic acid–induced writhing model, there was a significant reduction in number of writhings. It is well documented that central nervous system depressants and antihistamines are known to reduce number of writhings (Turner, 1965s). Thus, in light of these observations, fraction F exhibited central nervous system depressant activity in mice. It also showed significant activity in tail-flick response against heat-induced noxious stimuli. The results of acetic acid–induced writhing and tail-flick response exhibited potential antinociceptive action. The observed CNS depressant and antinociceptive activity of fraction F is complementary to antiphlogistic activity, which needs further detailed investigation.