Chemical composition and analgesic activity of Senecio rufinervis essential oil

Abstract

Context: Senecio rufinervis D.C (Asteraceae) is a tall aromatic herb, commonly found in Uttarakhand, India. No investigations on the biological activity of this plant have been published so far. Hence, this plant species became a subject of our scientific interest.

Objective: The aim of the study was to investigate the chemical composition and analgesic activity of Senecio rufinervis essential oil in mice using both thermal and chemical models of pain.

Materials and methods: Essential oil from dried leaves of Senecio rufinervis was extracted by steam distillation and then subjected to GC-MS analysis. Varying doses of essential oil were given to mice, 30 min prior to the induction of abdominal constrictions and determination of mean reaction time in hot-plate maintained at 55° ± 0.5°C.

Results: The main component detected in the essential oil of Senecio rufinervis was germacrene D (40.19%) followed by β-pinene (12.23%), β-caryophyllene (6.21%) and β-longipinene (4.15%). Essential oil exhibited significant and dose-dependent analgesic activity against acetic acid-induced writhing in mice. The percentage inhibition in number of writhes produced by 25, 50 and 75 mg/kg doses was, respectively, 69, 80 and 85%. The oil, at doses 50 and 75 mg/kg, significantly increased the mean latency in the hot-plate after 15 and 30 min of drug administration as compared to the control group.

Discussion and conclusion: The results depicted both central and peripheral analgesic activity of S. rufinervis essential oil which was attributed to the presence of terpenes.

Keywords::

• Germacrene D
• β-pinene
• acetic acid writhing
• hot-plate

Introduction

Senecio is a genus of the daisy family (Asteraceae) that includes ragworts and groundsels. Senecio rufinervis D.C. is a tall aromatic herb, leaves are shortly stalked, ovate, long pointed, toothed sharply and the lower surface is white and tomentose except the nerves. It flowers in small rounded corymbs and the flowers are yellow in color (Gupta, 1968). The plant grows in Uttarakhand, India at an altitude of 1800-3000 m and has no traditional or commercial use. S. rufinervis is an aromatic plant containing essential oil which is produced by many plants and confirms analgesic and anti-inflammatory activities (Santos et al., 1997, 2005; Sayyah et al., 2002, 2003; Hajhashemi et al., 2003; Golshani et al., 2004; Koudou et al., 2005; Lino et al., 2005). The genus Senecio contains many species endowed with pharmacological actions (Perez et al., 1999; Walber et al., 2004; El Hawary et al., 2008; Carro-Juarez et al., 2009); however, no investigations on the biological activity of Senecio rufinervis have been published so far. On the other hand, available synthetic analgesic drugs exert a wide range of side effects and are either too potent or too weak, so the search for new analgesic compounds has been a priority of pharmacologists and pharmaceutical industries. Hence, this plant species became a subject of our scientific interest. The aim of the present study was to screen the essential oil of S. rufinervis for terpenoids and to investigate its effect on animal models of thermal and chemical pain.

Material and methods

Plant

Senecio rufinervis D.C. was collected from Nainital (altitude 2600 m), India in September 2008 and authenticated by Harsh J. Chowdhery, Botanical Survey of India, Dehradun, India. A voucher specimen (no. 112287) was deposited in the Applied Chemistry Department of Birla Institute of Applied Sciences Bhimtal, Nainital, India.

Extraction of essential oil

The dried leaves of S. rufinervis (10 kg) were steam distilled; the distillate was saturated with NaCl and extracted with n-hexane. Anhydrous Na₂SO₄ was then added for drying of the organic phase. This was separated with the help of a separating funnel and finally the solvent was evaporated under reduced pressure (Thakuri et al., 2008). The essential oil (coded as D8) was stored under refrigeration for three days and then used immediately for analysis and evaluation of analgesic activity.

GC and GC/MS

The oil was analyzed using a Nucon 5765 GC (30 m × 0.32 mm, FID) with split ratio 1:48, N₂ flow of 4 kg/cm². GC/MS analysis was performed using a thermoquest (Vienna, Austria) trace GC-2000 interfaced with Finnigen MAT Polaries-Q ion trap mass spectrometer fitted with RTX-5MS (Restek Corporation, Pennsylvania, USA) fused silica capillary column (30 × 0.25 mm, 0.25 µm film coating). The oven temperature was programmed from 60°–210°C at 3°C/min using helium as carrier gas at 1 mL/min. The injector temperature was 210°C; injection volume was 0.1 µL prepared in hexane, split ratio 1:40. Mass spectra were taken at 70 eV Electron Impact (EI) with mass scan range of m/z 40-450 amu with mass scan time 4 s. Identification of the constituents was done on the basis of retention index, library mass search database – National Institute of Standards and Technology (NIST) and Wiley – and by comparing with the mass spectral data (Thakuri et al., 2008).

Animals

Male albino mice of Wistar strain aged 8–10 weeks (25–30 g), procured from the Institutional Animal house of the Birla Institute of Technology, Mesra, Ranchi were used in the study. They were housed under 12:12 h light:dark cycle at controlled temperature (25°C) with free access to food and water. The experimental protocol was approved by the Institutional Animal Ethical Committee of Birla Institute of Technology Mesra, Ranchi (621/02/ac/CPCSEA).

Analgesic activity

Acetic acid writhing test

Animals were divided into five groups containing six animals in each group. Groups 1, 2 and 3 received 25, 50 and 75 mg/kg i.p. doses of the essential oil, D8, prepared in 2% v/v Tween 80. Group 4 served as control and was treated with vehicle (2% v/v Tween 80, 10 mL/kg of body weight). Group 5 served as positive control and received pentazocine (10 mg/kg, i.p.). Pentazocine, a narcotic analgesic was used as standard in both models of pain because it inhibits both types of pain (central and peripheral) at this particular dose (Saraswathy et al., 2008; Mule et al., 2008), while NSAIDS such as paracetamol, aspirin inhibit only the peripheral pain (Elisabetsky et al., 1995; Pal et al., 1999). Half an hour after administration of drugs, each animal received intraperitoneal injection of 1% w/v acetic acid in a volume of 10 mL/kg. Intraperitoneal injection of acetic acid in mice produces a writhing response characterized by abdominal constrictions and hind limb stretching. The number of writhings was counted for a period of 10 min and the percentage inhibition of number of writhing was calculated using the ratio (control mean − treated mean) × 100 ÷ control mean (Kulkarni, 1999).

Hot-plate test

A total of 30 animals divided into five groups were placed on a hot-plate (Techno, India) maintained at a temperature of 55° ± 0.5°C and basal reaction time was noted as hind paw licking or jump response, whichever appeared first. They were then treated with D8 (25, 50, and 75 mg/kg in 2% v/v Tween 80, i.p.) and reaction time was noted after 15, 30 and 60 min. A cut-off period of 15 s was observed to avoid damage to paws (Kulkarni, 1999). Pentazocine (10 mg/kg, i.p.) pretreated animals were used as positive control and the vehicle treated group served as negative control.

Rotarod test

The integrity of motor coordination was assessed with a Rotarod apparatus set at a rotating speed of 16 rpm. A preliminary selection was made on the day of experiment excluding those that did not remain on the Rotarod bar for two consecutive periods of 45 s each. The number of falls from the rod was counted for 45 s, before and 15, 30 and 60 min after the administration of D8 (25, 50, and 75 mg/kg) and vehicle (2% v/v Tween 80) (Pieretti et al., 1999).

Statistical analysis

All the values were expressed as mean ± standard error of mean (SEM) for each group consisting of six animals. Results were analyzed statistically by one-way ANOVA followed by Tukey’s multiple comparison using Sigma Stat software. The difference was considered significant at p <0.05.

Results

GC/MS analysis of essential oil D8

The plant leaves yielded 0.5% (w/w) of essential oil. Eleven components were characterized, representing 78.18% of the total oil components detected (Table 1). Germacrene D (40.19%) was found as the major constituent in D8, followed by β-pinene (12.23%), β-caryophyllene (6.21%) and β-longipinene (4.15%).

Table 1.  Percentage composition of essential oil of the leaves of Senecio rufinervis D.C.

Compounds	RI	Leaf oil (%)	Mode of identification
α-pinene	940	3.24	a,b
β- pinene	981	12.23	a,b
P-cymene	1280	0.87	a,b
δ-elemene	1340	1.60	a,b
β-longipinene	1352	4.15	a,b
β- cubebene	1391	1.9	a,b
β-caryophyllene	1420	6.21	a,b
γ -murrolene	1477	2.3	a,b
germacrene D	1482	40.19	a,b,c
δ-cadinene	1526	2.83	a,b
germacrene D-4-ol	1576	2.66	a,b,c
Total		78.18

a, Mass spectra; b, KI; c, ¹H-NMR; RI, retention indices.

Analgesic activity

Intraperitoneal administration of acetic acid in the control group produced 26 writhes in 10 min. However, D8 significantly and dose-dependently inhibited the acetic acid-induced abdominal twitches. The percentage reduction in number of writhes observed with D8 was 69%, 80% and 85%, respectively, with 25, 50 and 75 mg/kg doses (Figure 1). In this test, pentazocine (10 mg/kg), produced a 72.3% reduction in the number of writhes. The analgesic activity of D8 (50 and 75 mg/kg) was more significant compared to pentazocine.

Figure 1.  Effect of essential oil of Senecio rufinervis (D8) on the nociception induced by intraperitoneal injection of acetic acid. Mean ± SEM. Pentazocine (10 mg/kg, i.p) was used as positive control. *significantly different from control i.e. vehicle treated group, ^asignificantly different from pentazocine treated group (p <0.05, ANOVA followed by Tukey’s test), ^bsignificantly different from D8 (25 mg/kg).

Hot-plate test

In the hot-plate test, D8 (25 mg/kg) did not show any significant increase in the mean reaction time compared to the control group and predrug reaction. However, 50 and 75 mg/kg doses of D8 significantly increased the latency of reaction time 15 and 30 min after the administration of drug. Pentazocine produced 51.2, 56, and 50% increase in reaction time after 15, 30 and 60 min of administration (Table 2). The effect produced by pentazocine in hot-plate was more significant compared to D8. The doses employed in this study were considered non-toxic, since D8 in doses up to 1 g/kg did not cause any behavioral impairment or overt toxicity in mice.

Table 2.  Effect of the essential oil from S. rufinervis (D8 25, 50 and 75 mg/kg, i.p) in hot-plate test in mice.

Group (mg/kg)	Latency (s)
	Predrug	Post drug
		0.25 h	0.5 h	1 h
Vehicle	4.36 ± 1.6	4.48 ± 1.7	4.5 ± 1.3	4.5 ± 1.3
D8 (25)	7.0 ± 0.8	8.8 ± 1.1	8.0 ± 1.0	7.6 ± 0.9
D8 (50)	6.0 ± 1.73	12.6 ± 0.4*^#	9.0 ± 0.9*^#	6.66 ± 0.88
D8 (75)	4.9 ± 0.4	8.8 ± 0.4*^#	7.8 ± 0.6*^#	7.6 ± 1.2
Pentazocine (10)	3.9 ± 0.59	8.6 ± 0.6*^#	8.9 ± 1.0*^#	7.8 ± 0.58*^#

Each group represents mean ± SEM for 6 animals. Pentazocine (10 mg/kg i.p.) was used as positive control and vehicle treated group as negative control. *p <0.05 as compared to vehicle treated group, ^#p <0.05 as compared to predrug reaction (ANOVA followed by Tukey’s test). D8, essential oil of Senecio rufinervis

Rotarod test

D8 did not affect the motor coordination in mice. The number of falls obtained in D8 (25, 50 and 75 mg/kg) treated animals in 45 s, was not statistically different from the control group at 0, 15, 30 and 60 min (Table 3).

Table 3.  Effect of the essential oil of S. rufinervis (D8 25, 50 and 75 mg/kg, i.p) in the Rota rod test.

Group (mg/kg)	Number of falls in 45 s (mean ± SEM)
	0 min	Min after D8 treatment
		15 min	30 min	60 min
D8 (25)	1.9 ± 0.01	1.1 ± 0.017	0.8 ± 0.025	0.9 ± 0.02
D8 (50)	1.86 ± 0.007	1.05 ± 0.023	0.76 ± 0.022	0.87 ± 0.025
D8 (75)	1.91 ± 0.012	1.12 ± 0.015	0.8 ± 0.015	0.86 ± 0.018
Vehicle	1.86 ± 0.012	1.05 ± 0.023	0.76 ± 0.023	0.87 ± 0.015

Each group represents mean ± SEM for 6 animals. Vehicle treated group served as control. D8, essential oil of Senecio rufinervis.

Discussion

The genus Senecio includes many species growing all around the world. According to the literature review there has been no report so far on bioactivity of S. rufinervis. The thermal stimulus in the hot-plate test, and the writhing response of the animals to an intra-peritoneal injection of noxious chemical, are used to screen peripherally and centrally acting analgesic activity. In the present study essential oil D8 of S. rufinervis, demonstrated significant analgesic activity in both acetic acid-induced writhing and the hot-plate model. The acetic acid-induced writhing reaction in mice, described as a typical model for inflammatory pain, has long been used as a screening tool for the assessment of analgesic or anti-inflammatory properties of new agents (Collie et al., 1968; Khandelwal, 2007). This method presents a good sensitivity; however, it shows poor specificity, leaving scope for the misinterpretation of results which can be avoided by complementing the test with other models of nociception and by a performance motor test. On the other hand, the involuntary muscle twitches of the abdomen seen in the acetic acid-induced writhing may be of interest because of their similarity with some of those known in visceral disorders (Vogel & Vogel, 1997; Le Bars et al., 2001). The essential oil of S. rufinervis produced a significant and dose-dependent inhibition of acetic acid-induced writhing, and the effect being more significant than the standard drug pentazocine. The results thus depict significant peripheral analgesic activity of the oil. The hot-plate test was also assayed to characterize the central analgesic activity of the oil. The results depicted that i.p. administration of oil at doses 50 and 75 mg/kg significantly raised the pain threshold at observation times of 15 and 30 min. The hot-plate method is considered to be selective for screening of the compound acting through the opioid receptor, but other centrally acting drugs, including sedatives and muscle relaxants, have also shown activity in this test (Hiruma-Lima et al., 2000). However, unlike sedatives and muscle relaxants, the essential oil did not produce any significant effect on motor coordination of animals when tested in the Rotarod test (Table 3). Thus, it can be inferred that the oil has central analgesic activity and does not have any sedative or muscle relaxant property. From the above results it was found that the oil possesses both peripheral and central analgesic effect. However, the peripheral analgesic effect produced by oil was more pronounced than the central analgesic effect.

The results obtained by GC/MS analysis of the D8 (Table 1) are in agreement with a previous study (Thakuri et al., 2008), though there are slight variations in the percentage of constituents present in the oil. The literature also emphasizes that a variety of geographical and ecological factors, isolation method and the conditions of analysis can lead to qualitative and quantitative differences in the composition of the essential oil (Crabas et al., 2003; Kim & Lee, 2004; Santos-Gomes et al., 2005). At the same time, a number of other factors can influence its composition, such as the developmental stage of the plant, its physiology, the age of leaves and the growing conditions (Bellakhdar et al., 1994; Santos-Gomes et al., 2005). The major constituents present in the essential oil of S. rufinervis are germacrene D (40.19%), β-pinene (12.23%) and β-caryophyllene (6.21%). One of its constituents, β-pinene has been reported to possess analgesic (Liapi et al., 2007) and anti-inflammatory activity (Lorente et al., 1989), while no study confirms analgesic action of germacrene D and β-caryophyllene (Santos et al., 1998). In various studies, the action of volatile oil is attributed to the combined effect of both their active and inactive compounds, and the latter might influence absorption, pharmacokinetics, and bioavailability of the active compounds (Svoboda & Deans, 1995). Hence, it appears that the analgesic effect of D8 is mainly due to the combined effect of terpenes and sesquiterpenes and not only to the majority constituents (Cabo et al., 1986; Santos & Rao, 2000; Ozbek et al., 2006). The mechanism of action could probably be the blockade of the release or the effect of endogeous substances that excite pain nerve endings by the terpenes present in the oil (Lee et al., 2002).

Conclusion

The results of the present study suggest significant central and peripheral analgesic effect of essential oil of Senecio rufinervis. The analgesic effect was attributed to the presence of terpenes, and further studies are required to determine the active chemical constituent(s) responsible and its possible mechanism of action.

Acknowledgements

The authors would like to express their thanks to the staff of the Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi, for their help in conducting experimental studies on animals.

Declaration of interest

The authors are grateful to Birla Institute of Applied Sciences, Bhimtal, Uttarakhand, India for providing financial support.