Chemical Composition , Anti-inflammatory , Analgesic , Antipyretic , Myorelaxant , Antibacterial and Antifungal activity of Rabdosia rugosus Wall . ( Syn . Plectranthus rugosus Wall . )

For the present investigation Rabdosia rugosus Wall. Syn. Plectranthus rugosus Wall. was collected from Pancheshwar, Uttarakhand on the way to Badrinath. The GC and GC-MS analysis, revealed the presence of more than forty compounds out of which 35 compounds were identified amounting to 97.3% of the total oil. The essential oil of R. rugosus was rich in sesquiterpinoids (~90%) and was poor in monoterpenoids (8.1%). α-bisabolol (41.9%) was the major constituent of the oil and the other identified major compounds were germacrene-D (9.7%), β-caryophyllene (7.6%), dehydroabietane (5.2%), ar-curcumene (5.0), trans-ferruginol (3.3%), α-cadinol (3.2%), T-muurolol (2.3%), p-Cymene (3.2%) and γ-terpinene (2.0%). The essential oil of Rabdosia rugosus showed insignificant anti-inflammatory and analgesic activity but shows significant antipyretic, myorelaxant and antimicrobial activity.


INTRODUCTION
The genus Plectranthus consists of about 300 species, belongs to the family Lamiaceae and is widely distributed in Africa, Asia, Australia and some Pacific islands.Many species of Plectranthus are economic and medicinal interest.Several species are cultivated for their edible tubers or as essential oil crops and others are used as food flavouring or fragrance.Some others are also used for medicinal purposes to treat vomiting and nausea, ear infections, respiratory diseases, toothache, headache, sores, and burns or as an antiseptic, purgative, antimicrobial and stimulant [1][2][3][4][5].
Rabdosia rugosus Wall.(Syn.Plectranthus rugosus Wall.), an aromatic shrub is found in Himalayas to Nepal, southeast Arabia (Oman), Afghanistan, Pakistan, and southwest China, very commonly growing on dry mountain slopes at lower altitudes.Plants are much branched, to 1.5 m tall aromatic shrubs.
Stems are erect with rather slender quadrangular branches, leafy, with an indumentum of small stellate dendroid hairs.Leaves are opposite with 2-10 mm long petioles.Leaf lamina is ovate-elliptic, rugose, reticulate and usually dark green on adaxial side and white tomentose with dense stellate trichomes on abaxial side.Flowers are in axillary racemes on terminal leafy panicles.Flowers are white-purplish and pedicellate with up to 6 mm long erect pedicel.Calyx in flower is 2-3.5 mm, indistinctly bilabiate, obliquely campanulate, with simple and/or branched hairs of varying length and density and usually numerous oil globules; calyx enlarging in fruiting stage up to 6 mm.Corolla are bilabiate, upper lip white and spotted with purple, lower lip boat-shaped, entire.Fruit is schizocarp of pale brown to dark brown nutlets.The plant flowers from March-October.According to R. R. Stewart, it is one of the commonest shrubs in the west Himalayas and a good honey source.It is usually a plant of dry rocky slopes, where it can be a dominant species in the community [6,7].

Plant Source
Rabdosia rugosus Wall.Syn.Plectranthus rugosus Wall.was collected from Pancheshwar, Uttarakhand on the way to Badrinath, India and was identified by plant taxonomist Dr. D.S Rawat.The specimens have been deposited in the Department of Chemistry, Pantnagar for future reference.

Isolation of Essential Oil and GC/MS Analysis
Essential oil extraction was carried out by following standard methods [13] as explained previously [30].GC-MS analysis was done [14] as explained before [30].

Experimental Animals
Animals {Swiss albino mice (R)} were procured from Lab animal division, Central Drug Research Institute, Lucknow, India.The mice were divided into four groups of six mice each for the experiments.They were housed in standard cages at a constant temperature of 22±1°C, relative humidity 55±5% with 12 hr light-dark cycle (08:00 to 20:00) for one week at least before the experiment.The experimental protocol was approved by the Committee on Animal Research, (ethical committee) with Registration No. 330/CPCSEA.All tests were conducted under the guidelines of the ethical committee for the study.

Toxicity
The acute toxicity test in mice and rats was carried out as explained previously [30].

Effect of Essential Oil on Isolated Duodenum Smooth Muscles of Wistar Rats
Effect of essential oil on isolated duodenum smooth muscles of Wistar rats was analysed by the method described by Gangwar et al., [31].

Preparation of Essential Oil and Drugs
The test solutions of R. rugosus essential oil (RREO) were freshly prepared along with acetylcholine (Ach), adrenaline, atropine and propanolol in desired concentrations and were used for the experiments.The tissues were allowed to show the maximum response of adrenaline, which took about 1-1.5 minutes.Then test oils as used in previous concentrations were repeated to compare the effect with adrenaline.Then 100 μg of propanolol was added to the organ bath.The tissues were allowed to show the maximum response of propanolol.After the dose of propanolol, adrenaline and essential oil were repeated in the same order.

Statistical Analysis
Data were expressed as Mean±S.E.Results were analysed using one way ANOVA followed by dunetts multiple comparison test and p<0.05 was considered to be statically significant.

Anti-Inflammatory, Analgesic and Antipyretic Activity
The anti-inflammatory effects of the essential oil of R. rugosus (RREO) on carrageenan induced edema in the mice right hind paw are presented in Table 2.There was a gradual increase in edema paw volume of mice in the control and RREO.However, in the ibuprofen treated group a significant reduction (37.19%) in edema was observed at 24 th hr.The inhibitory effect of the RREO recorded with a dose level of 50 and 100 mg/kg b. wt. in 24 h were 6.61% and 6.97%, respectively.RREO could not produce significant reduction in paw volume at doses of 50 and 100 mg/kg b. wt. as compared to the control.In sub-acute anti-inflammatory activity, where arthritis was induced by formaldehyde injection on day zero and the samples were administered orally daily for 10 days.During the investigation the RREO was also found to be insignificant comparatively, ibuprofen (Table 3).
For the determination of centrally acting analgesics, the hot plate test was useful [24] which are known to elevate the pain threshold of mice towards heat.The less reaction time shown by the mice treated with RREO suggests that it is not an effective against centrally acting analgesic.(Table 4).Data recorded in Table 5 on the acetic acid-induced writhing responses in mice are presenting of no analgesic activity of RREO.There was no significant effect of RREO at 50 and 100 mg/kg.b.wt. in decreasing writhing Administration of the yeast to the rats produced significant increase in rectal temperature, 18 h after Sacchromyces cerevisiae injection.RREO showed significant activity less than the paracetamol as compared to control.Maximum inhibition of pyrexia was shown by RREO (72.84%) at dose of 100 mg/kg b. wt. at 6 hr.The observations of the antipyretic activity of the essential oil are presented in Table 6.

Acute Toxicity
RREO administered intraperitoneally and orally at doses of 150, 300, 450 and 600 mg/kg b. wt.The Swiss albino mice were observed during the first two hours for poisonous symptoms and then mortality was recorded for each treated group at 24, 48 and 72 h after essential oil administration.RREO did not cause any behavioral changes and no death was observed, thus it was considered to be practically non-toxic components.

Effect of essential oil, agonists and antagonists on duodenal smooth muscle
Essential oil of R. rugosus (RREO) (2000 μg and 4000 μg) induced least to mild degree of relaxation in the duodenal tissue.However, relaxation disappeared within 15-30 min of washing with Tyrode solution in oils treated duodenal tissue.The normal response of duodenal smooth muscles to ACh (2 μg) did not alter before and after exposure of the oils, and returned to the base line immediately after maximal contraction.Pretreatment with atropine sulphate (2 μg) inhibited the ACh induced contraction in duodenal smooth muscles, however the relaxation induced by the oil (RREO) at the concentrations of 2000 μg and 4000 μg produced same effect as produced before the treatment of atropine sulphate.Though ACh induced tissue response is blocked by the muscarinic receptor antagonist atropine sulphate, the same antagonist did not alter the RREO (2000 μg and 4000 μg) induced relaxation and also did not affect the ACh induced contraction of duodenum smooth muscles.In addition, it is suggested that the oil induced relaxation did not involve blocking of acetylcholine acting muscarinic receptors.The primary action of acetylcholine to produce contraction of smooth muscles occurs through muscarinic receptors by causing depolarization of the cell membrane through increasing the Na + and Ca 2+ conductance [25].
Therefore, these observations suggested that the oil did not affect both the muscarinic receptor response and activity of ACh of the duodenal smooth muscles might be due to the unknown mechanism involving Na + and Ca 2+ ion channels mediated depolarization of the cell membrane.Adrenaline (1 and 2 μg) caused relaxation of tissue which returned to    Propranolol, a non-selective β-blocker, enters to block the site for the adrenaline induced relaxation of GIT smooth muscles i.e. adrenergic receptors.However, propranolol did not block the oil induced duodenal smooth muscle relaxation which suggested that the oil induced relaxation is not mediated through adrenergic receptors [26].In another study, Bazerra, et al., [27] reported GIT smooth muscle relaxation which was not blocked by adrenergic antagonists.Thus, the effect of oil induced adrenergic relaxation occurs probably due to their inhibitory effect on influx of Ca 2+ through cell membrane of mice duodenal smooth muscles.

Antibacterial Activity and Antifungal Activity
The essential oil components act on outer membrane permeability in gram-negative bacteria [28].Zone of inhibition of standard drug (ampicilline), 33.30 ± 0.5 mm against S. aureus and 28.30 ± 0.5 mm against S. enterica enterica.Essential oil of R. rugosus (RREO) showed moderate antibacterial activity against the tested pathogenic bacterial strains.The activity of RREO showed a zone of inhibition, 10.87 ± 0.5 mm against S. aureus and 10.87 ± 0.5 mm against S. enterica enterica (Table 7).
This investigation also reveals that essential oils obtained from R. rugosus exhibit a good antifungal activity in terms of % inhibition produced by the tested sample against three plant pathogen, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii recorded in Table 8.RREO showed maximum % of inhibition of Fusarium oxysporum (84.27%),Rhizoctonia solani (86.38%) and Sclerotium rolfsii (74.34%) at 250 ppm concentration.

CONCLUSION
The study indicates the wide chemical variations in the essential oils of Rabdosia rugosus from previously reported by other researchers in both qualitatively and quantitatively.Rabdosia rugosus essential oil appear to be good and safe natural antimicrobial agent in the control of various human, animal and plant disease and could also be of significance in antipyretic.Further studies should be done to search for new biological active components from essential oil.

Table 7 : Antibacterial activity of the essential oil of Rabdosia rugosus
Values are means of three replications±SE.Means with the same letter are not significantly different at p≤0.05.RREO=Rabdosia rugosus essential oil