Phytochemical composition , and analgesic and anti-inflammatory properties of essential oil of Chamaemelum nobile ( Asteraceae L All ) in rodents

Purpose: To investigate the in vivo analgesic and anti-inflammatory activities of essential oil of dried flowers of Chamaemelum nobile (Asteraceae L. All) in Swiss mice and Wistar rats, respectively. Methods: The volatile oil of the dried flowers of C. nobile obtained by hydrodistillation was analyzed by gas chromatography-mass spectrometry (GC-MS). Animals were assigned to the following experimental groups: C. nobile (180 mg/kg), ibuprofen (100 mg/kg) and vehicle-treated groups. Chemical and thermal pain models were used for the antinociceptive study in mice while fresh egg albumin-induced acute inflammation model in rats was used for anti-inflammatory study of the essential oil. Results: The most abundant components of the oil were α-bisabolol (50 %) and farnesene (5.35 %). Oral administration of essential oil (180 mg/kg, po) significantly (p < 0.05) reduced pain and prevented inflammation in the different test models used in this study. Conclusion: The essential oil of the dried flowers of C. nobile shows in vivo analgesic and antiinflammatory activities in rodents, and therefore, possesses potentials for development into effective analgesic and anti-inflammatory agent.


INTRODUCTION
Essential oils are complex mixtures of aromatic volatile oily compounds characterized by their odours.These oils are generally less dense compared to water.Although the primary role of essential oil is to protect the plant against bacteria, viruses, fungi and insects, nevertheless, they are known to exert beneficial effects in aromatherapy [1].These plant secondary metabolites have been associated with positive pharmacological properties such as sedative, anxiolytic, antioxidant, antiviral and antimicrobial effects [2][3][4].
Chamomile is known for its therapeutic uses especially for its calming and sleep promoting effects.It is a member of the daisy family (Asteraceae/Compositae) which is represented by two main species: German (Chamomillarecutita) and Roman (Chamaemelum nobile) Chamomile [5].Chamaemelum nobile (C.nobile) is a low-growing perennial plant, with finely divided leaves arranged alternately on the stem.The flower has a yellow central disc surrounded by silvery-white petals which when crushed give an apple-like smell.Of the two common members of this genus, German chamomile has been explored for several biological activities [6], while Roman chamomile (C.nobile) known to be common in South Africa is yet to be extensively studied.Roman chamomile is thought to possess carminative, sedative, antimicrobial, antiemetic and antispasmodic properties [7].Locally, C. nobile is used for treatment of arthritis and other inflammatory conditions.
Traditionally, C. nobile (Asteraceae L. All) is used for the management of pain and inflammation as well as a sedative.The therapeutic properties of C. nobile oil are widely attributed to the presence of farnesene and α-pinene, flavonoids, coumarins, terpenoids and mucilage [8] The aim of this study was to investigate the chemical composition, in vivo safety, analgesic and antiinflammatory properties of essential oil from flowers of C. nobile.To the best of our knowledge, no study on the acute toxicity in vivo analgesic and anti-inflammatory activities of C. nobile essential oil has been reported so far.

EXPERIMENTAL Plant material and chemicals/drugs
C. nobile flower was collected in the Eastern Cape, and was authenticated by Dr Immelman, a taxonomist at KEI Herbarium, Walter Sisulu University, South Africa where a voucher specimen (no.AO2/14024) of the plant material was deposited for future reference.
The following drugs were used for biological studies: Formalin (BDH Chemical Ltd, England) and ibuprofen (Dis-Chem Pharmacies, South Africa).

Essential oil extraction
The plant material was air dried in the laboratory.A Clevenger apparatus was used to extract 300 g of dried C. nobile by hydro-distillation for 4 h.The isolated pale blue essential oil was preserved in a tightly-capped bottle until used for experiments.Dichloromethane was used to dilute oil in a 1: 200 ratio for determination of its composition by gas chromatography and mass spectrometry.

Gas chromatography-mass spectrometeric (GC-MS) analysis
Analysis by GC-MS (gas chromatography-mass spectroscopy) was performed on an Agilent 5973N GC-MS system operating in EI mode at 70 eV, equipped with a HP-5 MS fused silica capillary system with a 5 % phenylmethylsiloxane stationary phase.A 30 m by 0.25 mm capillary column with a film thickness of 0.25 µm was used.The column was heated from 70 C to 240 C at an incremental rate of 5 C/min.Highest temperature attained was 450 C and maintained for 77.25 minutes.The flow rate of the carrier gas, helium, was 1 ml/min at a split ratio of 100:1.Scan time was 78 min with a scanning range of 35 to 450 amu.Kovats indices were determined by the analysis of 1 µl C. nobile diluted in hexane was injected for analysis.A similar procedure was used for n-Alkanes (C 8 to C 30 ).The identification of compounds was also based on the Kovats retention indices which were calculated using n-alkanes C 8 -C 20.Obtained values were then compared with reported values in literature [9,10].

Animals
Wistar rats weighing (150 to 200 g) and Swiss mice weighing 20 to 30 g of both sexes were procured from the South African Vaccine Initiative (Johannesburg).Animals were accommodated in the animal holding facility in the Department of Biological and Environmental Sciences where they had access to food and water ad libitum for two weeks before commencement of tests.Experimental animals were fasted overnight, but allowed access to drinking water before the treatment for proper hydration.Ethical approval for the study was granted by the Walter Sisulu University Research Ethical Committee (Ethical clearance no.DVC (AA & R) DRD/SREC: ref no.03).Studies were carried out in line with the recommendations of the International Guidelines for the use of Animals for Scientific Purposes [11].In this study, essential oil of C. nobile was emulsified with Tween 80 (5 %) before administration to the animals.

Acute toxicity study
Acute toxicity of the essential oil of C. nobile was assessed in mice using the oral route (po) according to Lorke's method [12].This method involves the use of thirteen animals for a rapid and economic LD 50 estimation using oral route.The experimental procedure was divided into 2 phases.In the first phase 9 mice were assigned to 3 groups of 3 rats each for the dose levels of 10, 100 and 1000 mg/kg.In the second phase only one mouse was used per dose level of each of four dose levels: 1000, 1600, 2900 and 5000 mg/kg, respectively.Immediately after treatment, each mouse was placed inside the Plexiglas cage and observed for immediate effects during the first 30 mins and thereafter for 24 h after treatment for lethal effects culminating into death.Animals were monitored for additional 14 days to ascertain the delayed effects of C. nobile.LD 50 of C. nobile essential oil was estimated as the geometric mean of the lowest dose causing death and the highest dose causing no death according to Eq 1.
where A is the maximum dose producing 0 % death and B is the dose that produces 100 % death [15].From the results of LD 50 , the working doses (W) were determined according to Eq 2.

Thermal pain (tail flick) test
In this experiment, mice were randomized into three groups of 6 mice each and treated with: 180 mg/kg of C. nobile, 100 mg/kg ibuprofen and 5% tween 80 respectively 1 h before the thermal challenge.Mice were gently restrained while allowing the posterior third of tail to rest over a glass window slit of the Tail Flick meter (Ugo Basile, model 37360) as described previously by Nkeh-Chungag et al [13].Baseline tail withdrawal latencies from the heat source were determined before oral administration of assigned treatment to animals.Tail withdrawal latencies were again determined 1, 2 and 4 h after drug treatment.

Formalin-induced pain test
Prabhu et al 's [14] method for the formalin test was used.Mice (n = 6 / group) were selected for the study.The control group (group 1) received the vehicle; group 2 received 180 mg/kg essential oil of the C. nobile while group 3 was treated with ibuprofen (positive control) (100 mg/kg) orally, 1h before 0.1 ml of 2.5 % formalin diluted in saline was injected into the mice paws.Animals responded by licking or biting the injected paw.The frequency of licks/bites was computed for the first 5 minutes and then for the next 20 to 30 minutes after formalin injection [15].Pain inhibition (H) was then calculated using Eq 3.

H (%) = {1-(T/C)}100 ……………… (3)
where T represents the number of times treated mice bit or licked the injected paw, and C is the number of times control mice licked/bit the treated paw.

Acute inflammation test (fresh egg albumin model)
Male Wistar rats were divided into 3 groups (n = 6) and treated as follows: group I (control group) -vehicle; group II -with ibuprofen (100 mg/kg, po) and group III -essential oil of C. nobile (180 mg/kg, po).One h later, 0.1 ml fresh egg albumin was injected into the left hind paw of all rats [16].The diameter of injected paws was measured at 0, 1, 2, 3, 4 and 5 h using a Vernier caliper (model YT-7201, Poland) after fresh egg albumin injection.The change in the paw size of the rat was determined by subtracting the paw diameter, at the various times from the baseline measure of the paw diameter.

Statistical analysis
Data was analyzed using Graphpad Instat ® (version 5).ANOVA followed by Dunnett's test.Was performed to compare results from the different treatment groups at indicated times.Results were expressed as mean ± SEM.P < 0.05 was considered statistically significant.1).The rest of the compounds occurred in relatively smaller amounts (< 1 %). Figure 1 shows chemical structures of major compounds identified in the oil.

Acute toxicity study
In the first phase acute toxicity testing using Lorke's method, doses up to 1000 mg/kg did not cause mortality when administered via the oral route.Similarly no mortalities were observed in mice during the second phase when animals received 1000, 1600, 2900 and 5000 mg/kg.Furthermore, no changes were noted in the animals' behavior and physical appearance 30 minutes after oral administration of extracts and up to 14 days later.Thus the LD 50 was estimated as greater than 5000 mg/kg, po (Table 2).

Effect of C. nobile essential oil on thermal pain in mice
Results showed that pre-treatment with 180 mg/kg of C. nobile significantly (p<0.05)increased the tail flick latency in the second hour compared to control.Ibuprofen, however, increased tail flick latency throughout the period of experimentation though these effects were only significant during the first two hours after oral administration of the drug compared to the vehicle group (Figure 2).

Formalin-induced pain
Normally, formalin-induced pain occurs in two phases.The first 5 min period after formalin injection is described as the neurogenic phase while from 20 to 30 min is known as the second/late or inflammatory phase.Neither ibuprofen nor the essential oil of C. nobile affected the neurogenic phase of formalininduced pain compared to control as the number of licks/bites remained relatively high in both groups.In the inflammatory phase however, ibuprofen (7.3±1.15vs 14.7±2.4;p < 0.05) and C. nobile (5.5±0.76 vs 14.7±2.4;p < 0.01) significantly inhibited paw licking/biting compared to the control.

Anti-inflammatory activity
The anti-inflammatory activity of C. nobile was demonstrated by reduction in paw diameter in Wistar rats.As presented in Figure 3, egg albumin induced a time-dependent increase in paw diameter in control animals though this increase was greatly reduced in treated animals.The peak of albumin inflammation was noted at 1h post treatment.The anti-inflammatory effect of C. nobile was very similar to that of ibuprofen.Both C. nobile and ibuprofen significantly (p < 0.01) inhibited the egg induced paw inflammation in rats from the 1 st h to the 5 th h post treatment.

DISCUSSION
A blue color essential oil was isolated from C. nobile harvested in the Eastern Cape of South Africa.This oil showed both analgesic and antiinflammatory properties in experimental models used.The identified major compound (alphabisabolol) has been reported to have antiinflammatory and other therapeutic activities Acute toxicity study gives clues on the range of doses that can be toxic to animals and, could also be used to estimate the therapeutic index of drugs and xenobiotics.Pharmacological substances with LD 50 lower than 5 mg/kg body weight are considered highly toxic; those with LD 50 between 5 mg/kg and 5000 mg/kg body weight are classified as moderately toxic; while those with LD 50 greater than 5000 mg/kg body weight are described as being non-toxic [19].Results from the present study showed that LD 50 of C. nobile essential oil is greater or equal to 5000 mg/kg (LD 50 ≥ 5000 mg/kg), implying that C. nobile essential oil has very low toxicity profile.
The tail flick test is based on the ability of the animal to withdraw its tail due to the increasing intensity of a thermal stimulus, is sensitive to opioid analgesics which act centrally [20] but not NSAIDs.In this study however, the essential oil of C. nobile conferred analgesic protected against thermal pain while the analgesic effects of ibuprofen lasted only during the first two hours of the study.Thermal stimulus is generally transmitted from the periphery to the spinal cord via C 1 fibres [21].This suggests that essential oil of C. nobile may possibly inhibit transmission of pain to the spinal cord by preventing transmission of nerve impulses in the C-fibres.To further confirm the analgesic effect of the essential oil of C. nobile against other pain stimulus, we used formalin-induced pain model.Formalin test which is predominantly used in rat and mice involves a moderate continuous pain generated by tissue injury [22].Animals respond to formalin injection by licking or biting the injected paw.Animal response in this model generally occurs in two phases.The neurogenic or early phase and the second or inflammatory phase of the formalin test are characterized by periods of high frequency of licking and/or biting of injected.The two phases were separated by a 15 min period of relative calm when the animals rested.The neurogenic phase is believed to be caused by the activation of C-fibres due to peripheral stimulation, while inflammatory pain seems to be predominantly caused by the combination of inflammatory reactions in the peripheral tissue and dorsal horn of the spinal cord [15].This study showed that the essential oil of C. nobile inhibited pain induced by formalin in the first phase better than ibuprofen (a known NSAID) thus confirming that fact that the first phase of this model is not sensitive to NSAIDs [23 ] The essential oil of C. nobile demonstrated analgesic effects in the second phase of the formalin test indicating that it may act by inhibiting inflammatory reactions in peripheral tissues or attenuated the functional changes expressed in the dorsal horn of the spinal cord.To elucidate the possible anti-inflammatory effect of C. nobile essential oil, the egg albumin inflammatory test was performed.
Egg albumin induced inflammation is a biphasic process characterized by the initial release of serotonin, histamine and bradykinins between (0-2 h after albumin injection followed by the release of chemical mediators and proinflammatory cytokines IL 1β and TnF-α [24].The release of chemicals and pro-inflammatory cytokines is responsible for the prolonged manifestations of inflammation which may last for up to 6 h [25].The anti-inflammatory effects of C. nobile in this study, was significantly higher from 1 -5 h implying that C. nobile possibly inhibited all the stages of inflammation.

CONCLUSION
The chemical composition, antinociceptive and anti-inflammatory activities of C. nobile have been reported.The three main compounds in C. nobile essential oil were α-bisabolol, farnesene and spathulenol.Biological tests showed that the essential oil of C. nobile is not toxic on acute use and possesses both analgesic and anti-inflammatory properties, thus validating the use of the plant for alleviating inflammation-related diseases in South African traditional medicine.

Figure 1 :
Figure 1: Chemical structures of the major compounds identified in the essential oil of C. nobile

Figure 2 :
Figure 2: Effect of C. nobile essential oil on thermal pain in mice.Each bar represents Mean ± SEM of change in tail flick latency.VEH, C. nobile, and IBU represent vehicle, C. nobile essential oil (180 mg/kg), and ibuprofen (100 mg/kg) respectively.All treatments were by the oral route; *p < 0.05, statistically compared to vehicle.

Table 2 :
Acute toxicity test of the essential oil of C. nobile in mice

Table 1 :
Elution time, Kovat index and composition of essential

Table 3 :
Antinociceptive effect of orally administered C. nobile essential oil on formalin-induced pain