Chemical composition and anti-inflamatory, anti-nociceptive and antipyretic activity of rhizome essential oil of Globba sessiliflora Sims. collected from Garhwal region of Uttarakhand

Background & Aim: Family Zingiberaceae is worldwide in distribution. Plants of the zingiberaceae family are used in traditional herbal folk medicine besides their uses in spices, cosmetic, ornamental, food preservatives etc. In Uttarakhand the herbs grow from sub-tropical to temperate region. Globba sessiliflora Sims rhizomes were collected at maturity stage in November from Garhwal region of Uttarakhand, India. In present communication the medicinal use of various zingiberaceous herb provoked us to study the chemical diversity and pharmacological activity determination of this important traditional herb. Experimental: The essential oil was extracted using hydrodistillation method and analyzed by GC-MS. Anti-inflamatory, anti-nociceptive and antipyretic activities of essential oil were experimently determined using mice model. Results: The major compounds identified were β-eudesmol (27.6%), (E)-βcaryophyllene (24.3%), α-humulene (3.0%), (6E)-nerolidol (4.1%), caryophyllene oxide (9.7%), γ-eudesmol (6.4%) and τ-muurolol (8.3%) besides other minor constituents. Essential oil of G. sessiliflora rhizome showed good anti-inflamatory, anti-nociceptive and antipyretic activities at the dose level of 100 mg/kg body weight. The oral administration of the essential oil exhibited no toxicity at 400, 600 and 800 mg/kg b.wt. concentration. Ibuprofen, indomthacin and paracetamol were used as standard drugs for comparison. Recommended applications/industries: G. sessiliflora essential oil can be used as herbal remedy for its nontoxicity anti-inflamatory, anti-nociceptive and antipyretic activities Type: Original Research Topic: Medicinal Plants Received July 24 2016 Accepted December 16 2016


Introduction
The complex biological response by harmful factors like pathogens, irritants and damaged cells results in inflammation.The common symptoms are redness of skin, edema, stiffness, pain and difficulty in of joint function which may be acute or chronic.Inflammation could result in a chronic stage which may indicate a factor of pathogenesis.A self defence mechanism may cause inflammation in primary phase which can be treated with chemotherapic agents to treat and eliminate symptoms and disease (Shailasree et al., 2012).
The nociceptive pathway is described in threeneuron chain which transmits nociceptive information to the cerebral cortex from the periphery (Besson and Chaouch, 1987;Almeida et al., 2004).The dorsal root ganglion has the first order neurons in their hair cell bodies.From here, two axons are projected to the peripheral tissues and the dorsal horn of the spinal cord.From the spinal cord, the second order neurons originate and ascend to the thalamus or the regions of the brainstem.From the thalamus, the third order neurons are ascended to the cerebrocortex (Besson and Chaouch, 1987;Abelson, 2005).
The secondary impact of infection is tissue damage, inflammation, graft rejection, malignancy or other diseased states which results in fever or pyrexia (Chattopadhyay et al., 2005).The body's natural defense system generates in an environment for non survival of the infectious agent or damaged tissues.The enhanced formation of pro-inflammatory mediator's (cytokines like interleukin 1β, α, β and tumor necrosis factor-α) is normally caused by the infected or damaged tissues.The proinflammatory factors results the increase the synthesis of prostaglandin E2 (PGE2) near preoptic hypothalamus area and triggers the hypothalamus area to increase the body temperature (Saper and Breder, 1994).
The clinical treatment of inflammatory diseases is dependent on drugs which belong either to the nonsteroidal or steroidal chemical therapeutic agents.The early steps in the biosynthetic pathway of prostaglandins by inhibition of COX enzymes are affected by the nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin and ibuprofen and reduce the untoward consequences of inflammation (Albert et al., 2002).However NSAIDs cause side effects like gastric injury and ulceration, renal damage and bronchospasm due to their non-selective inhibition of both isoforms of the COX enzymes (Tapiero et al., 2002).The controversy of the use of steroidal drugs as anti-inflammatory agents is because of their multiple side effects (Vanden Worm et al., 2001).Therefore, a demand has arisen for the search of newer antiinflammatory drugs of natural origin with potential activity and with minor side effects as substitutes for chemical therapeutic agents.Some Indian medicinal plants and their active principles which have antiinflammatory activity are documented by Chatterjee and Pal (1984).
The available analgesic drugs in the market are often associated with several adverse effects and are either too potent or too weak.Opiods are known to cause side effects which includes; sedation, respiratory depression, potential for addiction and tolerance whereas NSAIDS are known to cause gastric irritation that may lead to gastric bleeding.There is need for new analgesic compounds (Mattison et al., 1998).Plants of medicinal value in ethnopharmacology are an important source of natural products with potential therapeutic effects (Blumenthal, 2000;Bisset, 2001).Study of plant species that are used in traditional herbal medicine as pain killers, therefore, form a logical search strategy for new analgesic drugs (Farnsworth, 1989;Mattison et al., 1998).
High selectivity of irreversible inhibition of COX-2 expression by antipyretic drugs is the cause of reduction of increase body temperature by possible inhibition of biosynthesis of PGE2.The natural COX-2 inhibitors have lower selectivity with less side effects and less toxic to hepatic cells, glomeruli, cortex of brain and heart muscles.
To combat the proinflammatory mediators, a number of plant extracts have been screened those inhibit leukotriene and prostaglandins synthesis modulating cyclooxygenase pathway by inhibition of COX-1 and COX-2 enzymes (Oj et al., 2007, Alberto et al., 2009).
The members of family Zingiberaceae are currently of interest to phytochemist and pharmacologist all the world-over and work on their chemistry and pharmacology has been intensified recently.The research in family Zingiberaceae has evolked much attention after patent issue on haldi (Curcuma longa L.).Several species of ginger like Zingiber officinale Rosc.Z. capitatum Roxb and Z. roseum Rosc are known to grow in Kumaun and Garhwal region of Uttaranchal (Strachey, 1979).Numbers of plants from this family are used in traditional system of medicine (Hussain et al., 1992).Plants of this family have been attributed to have anti-inflammatory, antiulcer, antioxidant and antimicrobial properties (Jaganath et al., 2000).
Globba (Zingiberaceae-Zingiberales) consists of 100 species of small perennial herbs from East Asia and Malaysia.Flowers of Globba are strinking orange, yellow, purple or white, contrasting with the often green inflorescence bracts (Endress, 1994;Takano and Okada, 2003).Globba L. genus is represented by 17 species in India mainly distributed in the Himalaya, South India and Andman-Nicobar Islands (Karthikeyan et al., 1989).In Uttarakhand the genus is represented by three species G. orixensis Roxb, G. racemosa Smith and G. sessiliflora Sims (Uniyal et al., 2007).G. sessiliflora is erect, pemrennial herbs, with a creeping short rhizome which emits fleshy roots (Babu 1977).
We have earlier studied the mono and sesquiterpene composition of the rhizomes and aerial parts of G. sessiliflora Sims at flowering stage.β-caryophyllene, α-cadinol, selin-11-en-4α-ol, caryophyllene oxide, γeudesmol and myrcene were the major constituents in the rhizome volatile oil.The oil from aerial part revealed the presence of myrcene, β-caryophyllene, selin-11-en-4α-ol, β-longipinene, manool, germacrene D and β-eudesmol as major constituents.The oils showed significant myorelaxant effect on isolated duodenum smooth muscles of Wistar rats.The antioxidant activity of the oils were evaluated using reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and the chelating effect on Fe 2+ .Both rhizome and aerial part oils exhibited good to moderate antioxidant and myorelaxant activity in a dosedependent manner (Kumar et al., 2012).
In present study we worked on the chemical composition of essential oil of mature rhizomes of G. sessiliflora after litter fall collected from Garhwal region of Uttarakhand and anti-inflammatory, antinociceptive and antipyretic activity of rhizome essential oil of G. sessiliflora.

Plant material
G. sessiliflora rhizomes were collected in November, from the Dehradun-Mussoorie highway, Garhwal region of Uttarakhand, India.The

Isolation of essential oil
Essential oil from rhizomes was isolated by hydrodistillation in a Clevenger-type apparatus for 8 hours.The oil was extracted with CH2Cl2 and dried over anhydrous Na2SO4.The solvent was removed under vacuum and the oil was stored at 4°C for further analysis.

GC-MS analysis of essential oil
10% solution of essential oil was prepared in CH2Cl2 and filtered to obtain clear solution.GC-MS analysis of oleoresin solution (0.2 µl) was performed using Autosystem XL (Perkin-Elmer, USA) fitted with Elite-5MS fused silica column (30 m × 0.25 mm; 0.25µm film thickness), with split-splitless injector.Helium flow rate through the column was 1 ml/min in constant flow mode.The initial column temperature was 40ºC rising 325ºC at a rate 3ºC/min.The MS detector temperature was 280ºC.The constituents were detected in the full scan mode from m/z 41 to 620.
A hexane solution of C8-C28 n-alkanes was previously separated under the above conditions, and their retention times were determined.Linear temperature programmed retention indices (LTPRI) were calculated from the results of the separation of the oleoresin and n-alkanes according to eq.: Where tx, tn and tn+1 are the retention times of component x, and n-alkanes with the number of carbon atoms in the molecule n and n+1, respectively.After integration the fraction of each component in the total ion current (TIC) was calculated.a Components of essential oil were identified with the help of NIST II, mass spectra library.Identification was considered reliable if the calculated values of LTPRI confirmed the results of library search at mass spectra library (LTPRI Exp -LTPRI Lit ≤ 10 index units) (Adams, 2007).

Analysis of anti-inflammatory, antinociceptive and antipyretic activity of Essential oil
The investigations of different biological activities of the essential oil were carried out on Swiss strain albino mice weighing 18-23 g and purchased from the Indian Veterinary Research Institute(IVRI), Izatnagar, Bareilly, India.The mice were divided into different experimental groups.Each group consisted of six mice.All the groups were maintained under standard laboratory conditions of food and water.Prior to conduct the experiment, all the mice in each group were weighed individually to calculate the dose of drugs for each group.The animals were maintained in laboratory environment for two weeks to get them acclimatized.All experiments were conducted between 900, 1700 hours.All the animals were sustained under observation for one week, after the completion of experiments to observe acute or sub acute toxicity, if any [Experiments protocols were approved by the institutional animal ethics committee, CPCSEA, New Delhi (Ref: IAEC/Chem/CBSH/118)].

Animal model for anti-inflammatory, antinociceptive and antipyretic activities
Total four different groups of mice with six in each group were taken for conducting various pharmacological activities.The experiments were conducted using two different doses (50 mg/kg and 100 mg/kg b. wt.).These concentrations were selected as per IC50 calculations.50 and 100 mg of oleoresins were separately triturated by the addition of small amount of Tween-20 and saline water was poured to make the final volume of 10 ml.Paracetamol, Ibuprofen and indomethacin were used as positive and saline water as negative control respectively.The essential oil, standard drug and saline water were given at the dose rate of 0.1 mL/10 gm weight of the experimental animals.

Acute Anti-inflammatory assay
Anti-inflammatory activity was determined by carrageenan induced hind paw edema method (Winter et al., 1962).Acute inflammation was produced by subplantar injection of 0.1 mL of 1% suspension of carrageenan.The measurement of paw volume was made by plethysmometer (UGO Basile, Italy) at intervals of 1, 3 and 24 h after the injection of carrageenan.Standard anti-inflammatory drug, ibuprofen suspension was used orally at a dose of 40 mg/kg b.wt.The anti-inflammatory effect was measured as the reduction of volume displacement by hind foot in comparison to control.

Sub-acute Anti-inflammatory assay
The formaldehyde induced arthritis method was used to determine the subacute activity of the rhizome essential oil (Selye, 1949).0.1 ml formaldehyde (1%) solution was injected in the right hind paw of the mice on first day for the experiment.Doses of the oleoresins (50 and 100 mg/kg b. wt.) were administered orally daily in the morning till the end of the experiment study period for 10 days.Whereas control group received only saline water.Ibuprofen at a dose of 10 mg/ kg b.wt.was orally given used as standard antiinflammatory drug.Paw volume of all the mice were measured plethysmometrically in the evening hours for 10 days till the end of the experiment.

Writhing effect
The pain sensation in experimental mice was created by administering Glacial acetic acid intraperitoneally.Initially 0.2 mL of essential oil, standard drug (Ibuprofen -40 mg/kg body wt) and control (saline water) were administered orally to each group of mice.Glacial acetic acid (1% at dose of 0.1 mL/10 g body weight) was administered intraperitoneally after 40 min to each mice.The numbers of writhing per minute were counted for every mice.The inhibition of writhing and percentage of pain protection in mice was calculated as per following formula: % writhing = (T/C) × 100 % inhibition = C-T × 100 C Where: T = treatments (group II-III); C= control saline group (I)

Hot plate method
The standard hot-plate test was used to determine the analgesic response latencies (Langerman et al., 1995).The mice were placed into Perspex cylinder on the heated surface of the hot-plate (maintained at 55.0 ± 0.5 0 C).The time (sec) to discomfort reaction (licking paws or jumping) was recorded to record response latency.The measurements were recorded initially and after 30, 60, 120, and 150 min of oral administration of the essential oil at the dose rate of 50 and 100 mg/kg b.wt.For positive control, standard drug indomethacin (5 mg/kg b. wt.) was used.Saline water (0.2 mL) given orally as a negative control.A latency period of 20 sec.was marketed as complete analgesia and the measurements were stopped if it exceeded the latency period to avoid injury.

Antipyretic assay
To measure the antipyretic activity, pyrexia was induced using yeast as per the method given by Rao et al. (1997) generally being practiced.To record the basal body temperature a thermometer was inserted in to the rectum after restraining the mice.All the experimental groups were given a subcutaneous injection of 10 ml/kg b.wt. of 20% suspension of Brewer's yeast (Sacchromyces cerevisiae) except in control group.The mice were allowed to remain quiet in the cage for 18 hour, to rise in their body temperature.At nineteenth hour, again the rectal temperature was recorded.Immediately, 0.1 ml/10 gm body wt. of the oleoresins in the calculated doses of 50 mg/kg and 100 mg/kg b. wt. and paracetamol (33 mg/kg b. wt.) were given orally.Control group received 0.2 ml normal saline water only.The rectal temperature was recorded at hourly intervals in all the groups up to 3 hours.The percentage reduction in rectal temperature was calculated by considering the total fall in temperature to normal level as 100%.% Reduction = {(B-Cn/B-A) × 100} Where: A=normal temp B= Pyrexia temp C= temp at hourly interval

Assessment of toxicity
The acute toxicity was performed according to OECD guidelines (423, OECD/OCDE, 2001).The Lethality of essential oil of G. sessiliflora was checked by oral administration of essential oil at 400, 600 and 800 mg/kg b.wt.Behavioral changes were recorded for 24 hr.The numbers of casualties were recorded after upto 48 hr.

Statistical analysis
Data were expressed as mean±S.E.Results were analyzed using programme 43 (software) one way ANOVA and p˂0.05 was considered to be statically significant.
It is interesting to note that the essential oil of mature rhizome of G. sessiliflora collected from garhwal region has different chemical makeup, collected from kumaon region (Kumar et al., 2012).In the essential oil collected from both the regions have sabinene, p-cymene, linalool, α-terpineol, βcaryophyllene, α-humulene, caryophyllene oxide and 1-epi-cubenol present in their essential oils.however γeudesmol (6.4%) and β-eudesmol (27.6%) were present in essential oil collected from Garhwal region were as γ-eudesmol (3.0%) and β-eudesmol (4.1%) in the essential oil collected from kumaun region.The eesential oil of Globba sessiliflora from Kumaun region has the presence of selin-11-en-4α-ol (12%), Manool (1.0%) and Sandaracopimara-8(14)-diene (1.5%) which were not present in the oil collected from Garhwal region.However the oil of collected from Garhwal region shows the presence of two diterpines isokaurene (1.0%) and coranorin-E (0.7%).Nootkatone was not detected at maturity stage collected from Garhwal region.Rhizome and aerial part essential oils contain nootkatone which was less than 3% of the oil in earlier stage collection from Kumaun region.The variation in the makeup of essential oils may be because of climatic variations in the two different regions of Uttrakhand.

Acute Anti-inflammatory Assay
The paw volume of carrageenan induced paw edema was remained unchanged in saline treated control.In comparison to the control, the essential oil showed some anti-inflammatory activity.Maximum inhibition (40.06%) was recorded in Ibuprofen treated group and it was almost parallel to essential oil at the dose level 100 mg/kg b. wt.(Table 2).

Sub-acute Anti-inflammatory Assay
In ibuprofen treated mice, significant decrease in the paw volume was noticed from day 3 and value came to normal by day 10.Essential oil (50 mg/kg b.wt.) showed decreased in the sub-acute inflammation from day 1 and 100 mg/kg b. wt.dose of essential oil showed a decrease in the sub-acute inflammation from day 4 and reverted to normal by day 10 (Table 3).
Carrageenan induces acute inflammation method is used for the detection of anti-inflammatory activity in the drugs (Di Rosa and Willoughby, 1971).It has been reported that the edema is developed to carrageenan injection is a biphasic event.In first phase histamine and serotonin are released while in phase two (3-5 hours) is said to be synergised by prostaglandins.The edema is maintained between first and second phase due to kinin.It has also been reported that the second phase edema is sensitive to clinically effective steroids and non steroids anti-inflammatory agents.A variety of clinically ineffective agents can reduce initial phase (Vinegar et al., 1969).
Essential oil of Globba sessiliflora used in this study, showed anti inflammatory activity probably due to the inhibition of the cyclo-oxygenage pathway.It has been reported earlier that the essential oil show antiinflammatory activity due to its interference with the production of prostaglandins (Smith and Willis, 1971;Seibert et al., 1994;Miguel, 2010).In the carrageenan induced paw edema, essential oil showed activity at higher dose level.
For studying sub acute anti-inflammatory activity, arthritis was induced by formaldehyde injection on the first day and the test samples were administered orally every day for 10 days.During present investigation, oleoresins exhibited significant anti-artritis effect.Essential oil had anti-inflammatory effect corresponding to standard drug ibuprofen.

Writhing Effect
Essential oil showed significant analgesic activity with 34.79% inhibition at 50 mg/kg and 43.24% inhibition at the dose level of 100 mg/kg b. wt.As compared to standard drug ibuprofen (43.08 %, inhibition) (Table 4).

Hot Plate Method
Perusal of table 5 reveals that the discomfort reaction time in indomethacin treated mice was maximum at 60 minute while essential oil exhibited a dose dependent manner increase in the paw licking and jumping time.
The abdominal constitution response induced by glacial acetic acid is a sensitive procedure to establish peripherally acting analgesics.This response is thought to involve local peritoneal receptors.Essential oil, significantly attenuated the acetic acid induced squirm.
In this work, we also measured the central nociceptive reactivity to thermal stimuli in mice using the hot plate test.In central analgesic activity, essential oil revealed efficacy against thermal stimulus.

Antipyretic Assay
Globba sessiliflora essential oil produced significant antipyretic effect in a dose dependent manner.Negative control mice did not show any reduction in the body temperature on oral administration of saline.The maximum inhibition was obscured at 3 hr (Table 6).
An appreciable antipyretic effect was noticed by essential oil at dose level of 100 mg/kg b. wt. which was comparable to paracetamol.Previous studies already suggested that intensification of lipid per oxidation at increase body temperature is indicated by pyrexia and is associated by increase in oxidative stress.The use of antioxidants retards the process of lipid per oxidation (Brzezinska-Slebodziniska, 2001).
We have earlier reported that essential oil of G. sessiliflora exhibits antioxidant activity (Kumar et al., 2012).

Toxicity
On oral administration of 400, 600, 800 mg/kg.b.wt. of G. sessiliflora essential oil, no behavioural or physiological change was observed for 24 hr and on subsequent day.None of the treated mice died.All reflexes (pedal and corneal) and rectal temperature as well heart, respiration rates were within normal physiological limits in the extract treated mice.

Conclusion
From the experimental results it can be concluded that the herb can be a good natural source of βeudesmol, (E)-β-caryophyllene, α-humulene (6 E)nerolidol, caryophyllene oxide, γ-eudesmol and τmuurolol etc.The herb exhibited moderate to good pharmacological activity hence the results will be helpful to generate data base on this plant so that it can be exploited scientifically and judiciously for traditional system of medicine besides its academic importance.
identification and authentication of the plant was made by Dr. D. S. Rawat, Assistant Professor and plant taxonomist, Department of Biological Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar.The voucher specimen has been deposited in the Department of Biological Sciences, GBPUAT, Pantnagar for future reference.

Table 1 .
Chemical Composition of rhizome essential oil of G. sessiliflora Sims.

Table 3 .
Effect of rhizomes essential oil of G. sessiliflora on formaldehede induced sub acute inflammation(Mean±SE,  n=6)

Table 6 .
Effect of rhizome essential oil of G. sessiliflora on yeast induced pyrexia in mice (Mean±SE, N=6Percentage reduction in temperature is given within parenthesis, a = significant (p˂0.05) as compared to control, b = significant (p˂0.05) as compared to drug, GSREO: G. sessiliflora rhizome essential oil