ANTINOCICEPTIVE AND GASTRO PROTECTIVE EFFECTS OF INHALED AND ORALLY ADMINISTERED THEVETIA PERUVIANA PERS. K. SCHUM. ESSENTIAL OIL

ANTINOCICEPTIVE AND GASTRO PROTECTIVE EFFECTS OF INHALED AND ORALLY ADMINISTERED THEVETIA PERUVIANA PERS. K. SCHUM. ESSENTIAL OIL

Pragati Kumar ^*1, Prabodh Shukla ², Padmini Shukla ² and Shashi Alok ³

Department of Pharmacognosy ¹, NIMS Institute of Pharmacy, NIMS University Jaipur (R.J.), India.

Department of Pharmacognosy ², Pranveer Singh Institute of Technology, Kanpur, (U.P.), India.

Department of Pharmacognosy ³, Institute of Pharmacy, Bundelkhand University, Jhansi (UP), India.

ABSTRACT: In Present study the antinociceptive and the gastroprotective effects of orally administered or inhaled Thevetia peruviana Pers. K. (Schum) (Oleander) volatile oil, and its principal constituents linalool and 1,8-cineole were evaluated in mice. Either when orally administered (100µl/kg) or inhaled for 60 min Thevetia peruviana volatile oil significantly reduced the acetic acid-writhing response in a naloxone-sensitive manner. In the hot plate test, analgesic activity observed after oil inhalation was inhibited by naloxone and atropine sulphate pretreatment suggesting the involvement of opioidergic as well as cholinergic pathways. Regardless of the administration route and the experimental model used both linalool and 1,8-cineole did not produce significant analgesic response. Oral or inhalatory treatment with analgesic doses of volatile oil did not affect mice spontaneous locomotors activity. Concerning the gastric effects, Oleander oil, linalool and 1,8-cineole oral administration protected against acute ethanol-induced gastric ulcers but did not prevent indomethacin-induced lesions indicating no interference with arachidonic acid metabolic cascade. In conclusion, besides this gastroprotection, Oleander oil reveals an interesting analgesic activity mainly relevant after inhalation, at doses devoid of sedative side effect, suggesting the interest for potential application of this oil in aromatherapy.

Antinociceptive activity, Gastroprotection, Essential oils, Thevetia peruviana, Writhing test, Locomotors Activity, Hot plate test

INTRODUCTION: Volatile oils obtained from different species of Thevetia (T. peruviana, T. nerifolia, Nerium oleander) are frequently used in aromatherapy and massage to obtain many clinical benefits traditionally ascribed to their antibacterial, antifungal, carminative, sedative and antidepressant actions. Several studies have been conducted in order to validate these applications.

In clinical trials oleander volatile oil demonstrated to improve sleep ¹ and to reduce anxiety ².

Furthermore, in animal models, it has been proved to possess anticancer ³ and HIV-1 Reverse Transcriptase and HIV-1 Integrase Inhibitory activity ⁴. Volatile oil showed Anti-inflamatory ⁵ effect on smooth muscle in vitro supporting its use as Antibacterial, antitermite and analgesic agent.

In the recent years, the study of natural products, as Oleander volatile oils, continues to attract researcher’s attention in order to detect possible clinical uses and in particular, the list of oleander biological activities is increasing. Indeed, we have recently highlighted for oleander volatile oil an interesting antiplatelet activity associated with a promising protective effect in an animal model of acute pulmonary thromboembolism ⁶. These whole oil activities are shared by its major components, linalool and 1,8-cineole. Experimental studies conducted with the terpenic alcohol linalool and 1,8-cineole revealed a significant anti-inflammatory activity in carrageenin-induced rat paw oedema ⁵. More recent investigations demonstrated that linalool prevents nociception in different experimental models of thermal, chemical and inflammatory pain ⁷ suggesting analgesic and anti-inflammatory potentials for linalool producing plant species.

Thus, in the current study we investigated the antinociceptive profile of the lavender essential oil, linalool and 1, 8-cineole, orally administered or inhaled in mice, in experimental models of chemical and thermal pain. Furthermore, based on the antiulcer activity previously reported for 1,8-cineole, we assayed also the so far unexplored gastroprotective action of oleander oil in ethanol- and indomethacin-induced gastric ulcers in rats.

MATERIALS AND METHODS:

Plant material and essential oil extraction:

The flowers of Thevetia peruviana were collected from the fields of Jaipur Dist., Rajasthan, India and were collected in the month of July-August 2010 in morning time. The plant was authenticated by a botanist in Department of Botany, University of Rajasthan, Jaipur. A voucher specimen (RUBL20856) has been kept in herbarium in Department of Botany, University of Rajasthan, Jaipur. Organoleptic examination refers to evaluation by means of organ of sense and includes the macroscopic appearance of the drug, its odor and tastes, occasionally the sound or ‘snap’ of its fracture and the feel of the drug to the touch.

The methods used to extract fragrant compounds today are based on the ancient principles of maceration, expression and steam distillation. An "absolute" is an extract obtained by extraction with volatile solvents or by Enfleurage. It is considered the purest perfume material, retaining most of the plant's aromatic constituents. Many modern techniques stem from those of ancient cultures.

Essential oil analysis:

Essential oil samples were analyzed using a “Thermo TRACE GC ULTRA (GC)” gas chromatograph directly coupled to the mass spectrometer system (Thermo DSQ II – MS, Ionization for MS: Electron impact Ionization). Column - TR-5 Phase type: 5% Phenyl Methylpolysiloxane (Dimensions: 30m Length x 0.25mm ID x 0.25um film thickness) was used under the following conditions: oven temperature program from 60°C (5 min) to 250°C at 10°C/min and the final temperature kept for 5 min; injector temperature, 250°C; flow rate 1mL/min; Used oven run time – Yes; No. of Ramps – 1; Post run temp. – Off; Hold time 10 min.; Enable cryogenics- off; Equilibration time – 0.50 min. For the MS Method; Acquisition time – GC run time; Cal Gas – off; Reagent gas – off; Aquire profile – No; Acq. Thresold – 0; Source temp. – 200 ⁰C; Segment – 1; Detector gain – 100000; Chrom filter width – off; Reagent gas flow – off; Scan event – 1; Scan rate – 500.00; Mass defect – 0.00; Scan mode – Full scan; First mass – 50.0; Last mass – 1000.0; Ionization energy 70eV. The constituents of essential oils were identified in comparison with Library calculated in relation to the retention time of a series of alkanes (C - C) ⁸. The oil composition is reported in Table.

 TABLE 1: CHEMICAL COMPOSITION AND RETENTION INDEX OF THE CONSTITUENTS OF THE VOLATILE OIL OF THEVETIA PERUVIANA PERS. K. SCHUM.

Animals:

Experiments were performed on Swiss mice (20–30 g) of either gender, or female Wistar rats (150–200 g). All animals were fasted but had free access to water 18 hours before the experiments.

Drugs:

The following substances were used: linalool, 1, 8-cineole, atropine sulfate, naloxone hydrochloride, mecamylamine hydrochloride, methylcellulose, acetic acid and ethanol, Indomethacinate meglumine.

Oral treatment:

Groups of 6 animals received by gavage Oleander oil (100 mg/kg), linalool (33 mg/kg) and 1, 8-Cineole (36 mg/kg) in a final volume of 1 ml/100 g body weight 1 hour before the experiments. Linalool and 1, 8-Cineole were administered at doses chosen in accordance to the percentages of these constituents in the natural oil as detected by gas chromatography. Control animals received vehicle alone (0.1 % methylcellulose). A 1% aqueous emulsion of Oleander oil in methylcellulose was prepared immediately before use.

Inhalation:

When inhaled, 200 µl of oleander oil, linalool or 1, 8 cineole, contained in a 10 ml glass baker, were positioned at the bottom of plastic cages covered with plastic film in order to saturate the ambient. At saturation, the concentration of the oils in the cage was 2.4 µl /l. Mice introduced into the cage were allowed to inhale oil vapours for controlled time periods (15, 30 and 60 min) prior to performing the final experiments. Control animals were caged in the same conditions but in the absence of the tested oils. All experiments were conducted between 9.00 and 16.00.

Acetic acid writhing test:

The writhing test was performed according to Koster’s method ⁹, briefly, concluded the inhalation time or passed 1 hour from the oral administration of the oils under study or the vehicle, mice were intraperitoneally injected with 0.2 ml of 0.6% acetic acid. After treatment with the algogen agent, mice were placed in observational chambers and the number of writhes of each mouse was counted over a period of 30 min. Different sets of mice were pre-treated with the opioid antagonist naloxone (5 mg/kg i.p.), the muscarinic antagonist atropine (5 mg/kg i.p.) and the nicotinic antagonist mecamylamine (1 mg/kg i.p.) 10 min before the tested oils or vehicle challenge.

Hot plate test:

The hot plate test was performed according to the method described by Eddy and Leimbach ¹⁰. Mice were individually placed on the 55 ⁰C hot plate and the time between the placement and the occurrence of anterior paw licking, shacking or jumping was recorded as Latency Time (s). In order to exclude hypo- or hypersensitive mice, two hours before the final experiment all the animals were tested and those with latency time shorter than 10 sec or longer than 18 sec were eliminated from the study. Basal Latency Time (T₀) was measured before the administration of drugs or vehicle. Forward Latency Times (T₁) were measured starting 1 hour after oral treatment or after 15, 30 and 60 min of exposure to oils vapour with intervals of 15, 30 and 60 min. Different groups of mice were pre-treated with naloxone (5 mg/kg i.p.), atropine (5 mg/kg i.p.) and mecamylamine (1 mg/kg i.p.) 10 min before the tested oils or vehicle administration.

Time of 30 sec was arbitrarily chosen as cut-off time (T₂). Results were expressed as percentage of analgesic effect as follows: % MPE (Percent maximal possible effect) = (T₁-T₀)/(T₂-T₀)X100 (latency time after treatment-basal latency time)/(time of cut off – basal latency time).

Locomotor activity:

Locomotor activity was measured by means of an activity cage. Passed one hour from oral administration of oleander oil or vehicle or at the end of inhalation period times, mice were placed singularly into the activity cage and locomotor activity was recorded every 5 minutes for 90 min. All experiments were conducted from 9.00 to 15.00.

Acute gastrointestinal ulcerogenicity:

Acute gastrointestinal ulcerogenicity was assessed following Rainsford’s method ¹¹. Briefly, rats were treated orally with oleander oil 100 mg/kg. After 5 hours, animals were sacrificed by CO₂ inhalation; the stomachs were removed, fixed in 4% formaldehyde solution and processed for microscopic analysis using an image analyzer system. The total damaged area (mm²) and the number of gastric ulcers were counted for each stomach by an observer unaware of the treatment given to the animals.

Protection against acute indometacin- and ethanol-induced gastric lesions:

Oleander oil, linalool and 1, 8-cineole were tested as potential gastro protective drugs in two different models of acute gastric ulcers. For this purpose, rats were randomly assigned to 4 groups of 6 animals each one. To evaluate the ability of oils to protect against NSAIDs-induced gastric ulcers, animals were treated simultaneously with indomethacin (40 mg/kg i.p.) and oils per os. The animals were killed 5 hours later. The protection against ethanol-induced gastric lesions was tested administering orally 1 ml of 90% ethanol to animals which 1 hour previously had been treated orally with essential oils. The animals were killed 1 hour later. Stomachs were then removed and processed as described before.

Statistical analysis:

All data are expressed as mean ±S.E.M (n=6 observations per group). Results were analyzed statistically using Student’s t-test for unpaired data. P values less than 0.05 or 0.01 were considered as indicative of significance or high significance respectively.

RESULTS:

Chemical composition of the essential oil:

Table 1 show the composition and the relative abundance of the constituents identified in the oleander volatile oil. The major components are linalool (23.07%) and 1, 8-cineole (33.23%).

Acetic acid writhing test:

Administration of oleander oil 100 mg/kg significantly reduced the writhing response to acetic acid treatment to 51% over the control group (P=0.0002) (Fig. 1). This antinociceptive effect was significantly prevented by opioid antagonist naloxone pretreatment but it was completely unaffected by either nicotinic antagonist mecamylamine or muscarinic antagonist atropine administered at doses by themselves unable to modify nociceptive response. When the effects of the two major components, linalool and 1, 8-cineole, were separately considered, a modest antinociception was observed only with linalool oral administration. Inhalation of oleander volatile oil attenuated the writhing numbers in a time dependent manner producing a significant antinociception (61% reduction over control, P<0.0001) (Fig. 1) only after 60 minutes of exposure. In this case, the oleander oil antinociceptive effect was completely prevented by the administration of all the three different antagonists. Linalool and 1, 8-cineole inhalation for 60 minutes caused only a partial reduction of writhing response (Fig. 2).

FIG.1: EFFECT OF ORAL ADMINISTRATION OF OLEANDER OI l (100 mg/kg), l INALOOL (33 mg/kg) AND 1,8 - CINEOLE (36 mg/kg), ON THE ACETIC ACID-INDUCED WRITHINGS AND EFFECT OF PRETREATMENT OF ANIMALS WITH NALOXONE (5 mg/kg/ip), ATROPINE (5 mg/kg/ip) AND MECAMYLAMINE (1 mg/kg/ip) ON OLEANDER ANTINOCICEPTIVE EFFECT. THE NUMBER OF WRITHINGS WAS COUNTED FOR 30 MIN FOL LOWING ACETIC ACID INJECTION. THE VERTICAL BARS INDICATE THE STANDARD ERROR OF THE MEAN. THE NUMBER OF MICE USED FOR EACH GROUP WAS 6. **P < 0.01 COMPARED TO VEHICLE-TREATED MICE.

FIG. 2: EFFECT OF OLEANDER OI L , L INALOOL AND 1, 8 - CINEOLE INHALED FOR 60 MIN ON THE ACETIC ACID-INDUCED WRITHINGS AND EFFECT OF PRETREATMENT WITH NALOXONE (5 mg/kg/ip), ATROPINE (5 mg/kg/ip) AND MECAMYLAMINE (1 mg/kg/ip) ON ANTINOCICEPTIVE EFFECT PRODUCED BY OLEANDER OI L . THE NUMBER OF WRITHINGS WAS COUNTED FOR 30 MIN FOL LOWING ACETIC ACID INJECTION. THE VERTICAL BARS INDICATE THE STANDARD ERROR OF THE MEAN. THE NUMBER OF MICE USED FOR EACH GROUP WAS 6.**P<0.01 COMPARED TO VEHICLE-TREATED MICE.

Hot plate test:

Oral administration of oleander oil 100 mg/kg failed to prolong latency time compared with controls in mice hot plate test. On the other hand, inhalation of oleander oil produced an inhibition of the hotplate response proportional to the time of exposure to oil vapors, yielding a significant delay (P < 0.01) in reaction time after 60 minutes inhalation. This analgesic activity peaked at the suspension of inhalation and progressively diminished disappearing at 60 min. This oleander oil antinociceptive effect was significantly prevented by pretreatment with naloxone, atropine and mecamylamine, administered at doses by themselves unable to modify nociceptive response (Table 2). No analgesia was accounted after 60 minutes inhalation of linalool and 1, 8 - cineole.

TABLE 2: TIME DEPENDENCE OF ANALGESIC EFFECT OF INHALED OLEANDER OIL, LINALOOL AND 1, 8 - CINEOLE IN HOT PLATE TEST IN MICE IN THE ABSENCE AND IN THE PRESENCE OF PRETREATMENT WITH NALOXONE (5 mg/kg/ip), ATROPINE (5 mg/kg/ip) AND MECAMYLAMINE (1 mg/kg/ip).

Results are expressed as mean ± S.E.M of percent Maximal Possible Effect.

* P < 0.05 compared to vehicle-treated mice.

** P < 0.01 compared to vehicle-treated mice

Effect on locomotors activity:

No significant alterations of locomotors activity were observed in mice after treatment with oleander oil either orally administered at the dose of 100 mg/kg (2347 ± 567 counts in 90 min) or inhaled for 60 minutes (2767 ± 756 counts in 90 min) with respect to vehicle-treated animals (2012 ± 497 counts in 90 min).

Gastrolesivity and gastro protection:

Acute oral treatment with oleander oil (100 mg/kg os) did not produce any damage on rat gastric mucosa. Regarding the gastro protection, neither oleander oil oral administration nor oil inhalation protected against indomethacin-induced gastric ulcers being the number and the area of gastric lesions observed in oleander treated rats comparable to vehicle-treated animals (ulcer number 13.7 ± 4.6 and 17.9 ± 4.5 respectively and injured area 8.4 ± 4.7 mm² and 6.7 ± 2.5 mm² respectively).

A significant prevention of acute ethanol-induced gastric lesions was elicited by oleander oil oral administration (100 mg/kg) as it reduced the total injured area by an 87% (P = 0.003) compared with control. A lower but still significant gastro protection was obtained with the administration of linalool (33 mg/kg os) and 1, 8 - cineole (36 mg/kg os) which diminished the hemorrhagic erosion areas of about 54% (P = 0.003) and 47% (P = 0.013) respectively (Fig. 3). Co-administration of the two components did not enhance the gastro protection produced by the oils singularly administered. Essential oil inhalation for 60 min failed to protect gastric mucosa from necrotizing action of ethanol.

FIG. 3: EFFECT OF ORAL ADMINISTRATION OF OLEANDER OI l (100 mg/kg), l INALOOL (33 mg/kg), 1, 8 - CINEOLE (36 mg/kg) AND L INALOOL (33 mg/kg) plus 1, 8 - CINEOLE (36 mg/kg) ON ETHANOL-INDUCED GASTRIC ULCERS. RESULTS ARE EXPRESSED AS TOTAL INJURED AREA. VERTICAL BARS INDICATE THE STANDARD ERROR OF THE MEAN. THE NUMBER OF RATS USED FOR EACH GROUP WAS 6. *P < 0.05 AND **P < 0.01 COMPARED TO VEHICLE-TREATED RATS.

DISCUSSION: In the current study we demonstrate that oral treatment with whole oleander oil produces significant antinociception and gastroprotective activity in animal models. This pharmacological activity could derive from the contribution of various active principles composing the whole oil such as linalool, myrcene and linalyl acetate, previously proved to possess antinociceptive activity. ^{12, 13} Higher analgesic efficacy was exhibited by oleander oil when administered through inhalatory route being the nociceptive responses to chemical (writhing test) and thermal (hot plate test) stimuli significantly reduced. At variance with oleander oil, linalool and 1, 8 – cineole produce only scarce or no analgesic effect in the two pain models here adopted. The different dosage applied in this study with respect to previous investigations ¹² can account for the lack of antinociceptive activity of linalool both in writhing and hot plate tests. The absence of any modification of spontaneous locomotor activity after oral/inhaled administration of antinociceptive doses of whole oil let us to rule out the occurrence of sedative effect confounding analgesia studies. It must be pointed out that in literature the sedative effect of oleander oil upon inhalation in mice has been clearly described.¹⁴

However present findings are not conflicting with previous data describing the sedative effect of oleander oil since in this work we studied locomotor activity by exposing mice to oleander oil lower air concentration than that proved to produce sedative serum oils levels after inhalation.^{14, 15} As for the mechanism underlying oleander oil analgesic action it is noteworthy that opioidergic neurotransmission seems to be primary involved in oral induced analgesia since only naloxone pretreatment prevents oleander effect in writhing test. Also cholinergic system appears to play a significant role in oleander oil analgesia displayed after inhalation since also the blockade of muscarinic and nicotinic receptors prevented antinociception. The involvement of cholinergic transmission could be ascribed to some component terpenes of the oil in addition to linalool, since potent in vitro anticholinesterase activity was reported for terpenoids such as 1,8-cineole, a constituent of various essential oils.¹⁶

As concerns antiulcer activity, interestingly, linalool as well as 1, 8 - cineole demonstrate to contribute to the gastroprotective effect of oleander oil which, orally administered, caused a dramatic reduction of ethanol-induced gastric injury in rats. The involvement also of additional active principles, such as the gastroprotective agent 1, 8-cineole ¹⁷, cannot be ruled out since the antiulcer effect of the co-administration of linalool and 1, 8 - cineole is lower than that of whole oil.

The lack of protective effect against gastric mucosal damage caused by indomethacin led us to hypothesize that gastroprotection afforded by oleander oil cannot be attributed to interference with arachidonic acid metabolic cascade. Actually, we have already described an interesting ability of oleander oil to prevent experimental thrombus formation with an ASA-unlike mechanism of action.⁶ The amelioration of gastric microcirculation could be the mechanism underlying the oleander gastroprotection against ethanol injury which is known to be dependent on microvasculature engulfment in the gastric mucosa.¹⁸

In conclusion the results of this study reveal a remarkable analgesic and gastroprotective activities of oral oleander oil at doses 100–400 fold lower than those proved to be acutely toxic for the main components of the phytocomplex.¹⁵ Furthermore, the effectiveness of oil inhalation in controlling chemical and thermal pain without evidence of central adverse effects supports the interest for potential application of oleander essential oil in aromatherapy.

ACKNOWLEDGEMENTS: Authors are heartily thankful to Chairman, Dr. Balbir S. Tomar and Director, Dr. Shobha Tomar for providing necessary facilities at Institute of Pharmacy, NIMS University, Jaipur, India.

REFERENCES:

1. Graham P H, Browne L, Cox H, Graham J: Inhalation aromatherapy during radiotherapy: results of a placebo controlled double-blind randomized trial. Journal of Clinical Oncology 2003; 21 (12):2372–2376.
2. Dunn C, Sleep J, Collett D: Sensing an improvement: an experimental study to evaluate the use of aromatherapy, massage, and periods of rest in an intensive care unit. Journal of Advanced Nursing 1995; 21 (1):34–40

3. Pathak S, Multani A S, Narayan S, Kumar V, Newman RA: Anvirzel an extract of Nerium oleander, induces cell death in human but not murine cancer cells. Anticancer drugs 2000; 11(6):455-63.
4. Tewtrakul S, Nakamura N, Hattori M, Fujiwara T, Supavita T: Flavanone and Flavonol Glycosides from the Leaves of Thevetia peruvianaand Their HIV-1 Reverse Transcriptase and HIV-1 Integrase Inhibitory Activity. Pharm. Bull 2002; 50(5):630-635.
5. Erdemoglu N, Kupeli E, Yesilada E: Anti-inflammatory and antinociceptive activity assessment of plants used as remedy in Turkish folk medicine. J. Ethnopharmacol 2003; 89(1):123-9.
6. Ballabeni V, Tognolini M, Chiavarini M, Impicciatore M, Bruni R, Bianchi A, Barocelli E: Novel antiplatelet and antithrombotic activities of essential oil from Thevetia peruviana K. Shum. Phytomedicine (In press). 2004; 67(12):234-239.
7. Begum S, Sultana R, Siddiqui B S: Triterpenoids from the leaves of Nerium oleander Phytochemistry 1997; 44(2):329-332.
8. Derwich E, Manar A, Benziane Z, Boukir A: GC/MS Analysis and In vitro Antibacterial Activity of the Essential Oil Isolated from Leaf of Pistacia lentiscus Growing in Morocoo, World Appl. Sci. J. 2010; 8(10):1267-1276.
9. Koster R, Anderson M, De Beer E J: Acetic acid for analgesic screening. Federation Proceedings 1959; 18:412–421.
10. Eddy N B, Leimbach D: Synthetic analgesics. II. Dithienylbutenyl and dithienylbutylamines. Journal of Pharmacology and Experimental Therapy 1953; 107 (2):385–393.
11. Rainsford K D: An analysis of the gastro-intestinal side-effects of non-steroidal anti-inflammatory drugs, with particular reference to comparative studies in man and laboratory species. Rheumatology International 1982; 2 (1):1–10.
12. Peana A T, D’Aquila P S, Panin F, Serra G, Pippia P, Moretti M D: Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 2002; 9 (8):721–726.
13. Rao V S, Menezes A M, Viana G S: Effect of myrcene on nociception in mice. Journal of Pharmacy and Pharmacology 1990; 42 (12):877–878.
14. Buchbauer G, Jirovetz L, Jager W, Plank C, Dietrich H: Fragrance compounds and essential oils with sedative effects upon inhalation. Journal of Pharmaceutical Sciences 1993; 82 (6):660–664.
15. Letizia C S, Cocchiara J, Lalko J, Api A M: Fragrance material review on linalool. Food and Chemical Toxicology 2003; 41 (7):943–964.
16. Savelev S, Okello E, Perry N S, Wilkins R M, Perry E K: Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandulaefolia essential oil. Pharmacology Biochemistry and Behavior 2003; 75 (3):661–668.
17. Santos F A, Rao V S: 1, 8-cineol, a food flavoring agent, prevents ethanol-induced gastric injury in rats. Digestive Disease Science 2001; 46 (2):331–337.
18. Oates P J, Hakkinen J P: Studies on the mechanism of ethanol-induced gastric damage in rats. Gastroenterology 1988; 94 (1):10–21.
    

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    How to cite this article:

    Kumar P, Shukla P, Shukla P and Alok S: Antinociceptive and Gastro Protective Effects of Inhaled and Orally Administered Thevetia Peruviana Pers. K. Schum. Essential Oil. Int J Pharm Sci Res 2015; 6(10): 4496-02.doi: 10.13040/IJPSR.0975-8232.6(10).4496-02.

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