Front. Pharmacol. Frontiers in Pharmacology Front. Pharmacol. 1663-9812 Frontiers Media S.A. 10.3389/fphar.2018.00623 Pharmacology Original Research Chemical Characterization, Analgesic, Antioxidant, and Anticholinesterase Potentials of Essential Oils From Isodon rugosus Wall. ex. Benth Sadiq Abdul 1 * Zeb Anwar 1 Ullah Farhat 1 Ahmad Sajjad 1 Ayaz Muhammad 1 Rashid Umer 2 Muhammad Noor 3 1Department of Pharmacy, University of Malakand, Chakdara, Pakistan 2Department of Chemistry, COMSATS University Islamabad, Abbottabad, Pakistan 3Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Pakistan

Edited by: Lyndy Joy McGaw, University of Pretoria, South Africa

Reviewed by: Kannan R. R. Rengasamy, Alagappa University, India; Abdel-Tawab H. Mossa, National Research Centre, Egypt

 *Correspondence: Abdul Sadiq sadiquom@yahoo.com

This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology

 13 06 2018 2018 9 623 11 06 2017 24 05 2018 Copyright © 2018 Sadiq, Zeb, Ullah, Ahmad, Ayaz, Rashid and Muhammad. 2018 Sadiq, Zeb, Ullah, Ahmad, Ayaz, Rashid and Muhammad

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Isodon rugosus Wall. ex. Benth is an important species and is used in folk medicine for different types of pains such as abdominal pain, earache, toothache, gastric, and generalized body pain. Recently, we also have reported the antinociceptive potential of chloroform fraction of I. rugosus. In this research, we have investigated the antinociceptive, antioxidant and anti-cholinesterase potentials of essential oils from I. rugosus (Ir.EO), and have determined a possible mechanism of anti-nociception. The Ir.EO was subjected to gas chromatography-mass spectroscopy analysis to find out its chemical constituents. The Ir.EO was assayed for analgesic potential following acetic acid induced writhing, formalin test and hot plate method in animal models. The antioxidant activity was conducted against DPPH and ABTS free radicals following spectroscopic analysis. The cholinesterase inhibitory assays were performed using Ellman's assay. The GC-MS analysis of Ir.EO revealed the identification of 141 compounds. Ir.EO demonstrated strong antinociceptive potential in all three in-vivo models. With the use of nalaxone, it was confirmed that the essential oil was acting on the central pathway of nociception. The Ir.EO also exhibited strong free radicals scavenging potential, exhibiting IC50 values of 338 and 118 μg/ml for DPPH and ABTS free radicals respectively. In AChE and BChE inhibitory assays, the observed IC50 values were 93.56 and 284.19 μg/ml respectively. The encouraging antinociceptive, antioxidant and anticholinesterase results revealed that Ir.EO is a rich source of bioactive compounds as obvious from the GC-MS results.

 essential oil GC-MS Isodon rugosus antinociception opioid receptors antioxidant anticholinesterase Introduction

Globally, a large number of medicines are available for the treatment of pain and associated disorders. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the management of pain and inflammation due to their strong efficacy (Zarin et al., 2005). However, their use is associated with severe side effects. Alternatively, the drugs from natural origins are considered to be relatively safe and are associated with fewer unwanted effects. Natural products, especially the plants play a vital role in the discovery of new chemical entities with potential therapeutic values (Rates, 2001; Ayaz et al., 2017a). The traditional use of plants is therefore a logical strategy to find out natural therapeutic agents for different ailments like pain and inflammation (Gupta et al., 2006). Despite the development of therapeutic agents for pain, there is still a demand to search out novel agents which could treat pain and related disorders more efficiently (Calixto et al., 2000).

The reactive oxygen species (ROS) are produced within the body as a result of redox processes and aerobic respiration. These ROS invade lipids, proteins, enzymes, DNA and RNA and ultimately damage the cells. These biomolecules play a vital role in stimulation, propagation and maintenance of inflammatory processes, as well as pain and neurodegenerative disorders (Zhu et al., 2004). These unwanted effects can be reduced by the use of antioxidants which either reduce the production of ROS or diminish them before reaction (Khalil et al., 1999; Cuzzocrea et al., 2001). In this regard, the essential oils isolated from herbal sources may be considered for the management of pain, inflammation, and free radicals scavenging. Several plants have been reported with strong antioxidant potentials against free radicals (Ahmad et al., 2015; Ayaz et al., 2015).

Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by cognitive hypo-function, behavioral turbulence and difficulties in life activities (Ali et al., 2017; Ayaz et al., 2017b). AD is believed to be the major cause of dementia in elder population (Ullah et al., 2016). According to the statistics, 27 million people are affecting globally from Alzheimer and is a major life threat after cancer and cardiovascular diseases (Hebert et al., 2003). AD pathogenesis include synaptic deficiency of essential neurotransmitter (acetylcholine, ACh) which is implicated in the neurotransmission (Sadiq et al., 2015). Other aspects of AD include accumulation of amyloid beta (Aβ), neurofibrillary tangles (NFTs), and free radicals induced neurodegeneration (McLean et al., 1999; Zeb et al., 2014a; Ahmad et al., 2016). The inhibition of cholinesterase is a vital biochemical target involved in the degradation of ACh which increases its accumulation in the synaptic region. Among the five clinically approved anti-Alzheimer drugs, four are cholinesterase inhibitors while the fifth drug memantine is glutametergic system modifier. Despite the fact that several anti-amyloid and anti-NTFs drugs are in clinical trials, but, till date, no one is approved for clinical use. Furthermore, administration of free radical scavengers is also an important strategy, as Aβ is potent generator of free radicals and a mitochondrial poison. Plants are a source of mutli-potent drugs, including anti-AD drugs. Among the currently available anti-AD drugs, physostigmine, and galanthamine are derived from medicinal plants (Ahmad et al., 2016). Furthermore, natural products are free radicals' scavengers and can be effective on multiple pathways (Ayaz et al., 2017c).

Isodon rugosus Wall. ex. Benth. is a well-known species of family Labiateae. The bark of I. rugosus is used ethnomedicinally in the treatment of dysentery and curing of body pain (Shuaib et al., 2015). Folklorically, the fresh leaves' extract of I. rugosus is applied to the effected skin and is also used for earache (Sabeen and Ahmad, 2009). Moreover, the dried leaves of this plant can be used for the treatment of teeth pain (Akhtar et al., 2013). The plant has also been reported to posses potential effectiveness in gastric and abdominal pains (Ahmad et al., 2014). Moreover, other traditional uses of I. rugosus are attributed to its possible use against infectious diseases, pyrexia, blood pressure, rheumatism, and in pain associated with teeth (Khan and Khatoon, 2007; Adnan et al., 2012; Shuaib et al., 2014). The extracts of Isodon rugosus have been previously published to posses certain biological potentials like anti-diarrheal, analgesic, antimicrobial, anticholinesterase, antioxidant, cytotoxic, phytotoxic, hypoglycemic, and as bronchodilator (Sher et al., 2011; Ajmal et al., 2012; Janbaz et al., 2014; Zeb et al., 2014a,b, 2016, 2017).

Based on the ethnomedicinal importance and our previously published work, this piece of research is designed to investigate the in-vivo analgesic mechanism, in-vitro antioxidant, and anti-cholinesterase activities of essential oils of Isodon rugosus.

 Methods Plant sample collection & isolation of essential oil

Isodon rugosus was collected from Dir (L), KP, Pakistan in July. The name Isodon rugosus was confirmed by Dr. Ali Hazrat, Department of Botany, Shaheed Benazir Bhutto University Dir (U), KP, Pakistan. The plant sample was stored for future record at the herbarium with voucher specimen number 1016AZ. The essential oils were extracted by hydrodistillation with the help of a Clevenger type apparatus (Lambert et al., 2001). The isolated essential oils were stored in refrigerator.

 Gas chromatography analysis

The phytocomponents of essential oils were separated using the same GC instrument as we previously reported (Ahmad et al., 2016). A capillary column having dimensions of 30 m × 0.25 mm with film thickness of 0.25 μm in combination with a flame ionization detector was used. The initial temperature was 70°C for 1 min, which was raised gradually to 180°C with 6°C/min increase for 5 min. Finally, the oven temperature was increased to 280°C with 5°C/min increase for 20 min. Temperature of the injector port was 220°C while that of detector was maintained at 290°C. Helium was used as a carrier gas. The sample was diluted in n-pentane (1/1,000, v/v) of 1 μl (Ayaz et al., 2016).

 GC-MS analysis

The GC-MS analysis of essential oil isolated from Isodon rugosus was determined with the previously reported parameters (Ayaz et al., 2015).

 Identification of components

The retention times and spectra of separated compounds by GC-MS were compared with the standard compounds for identifications. The mass spectrum of each separated compound with its fragmentation pattern was compared with the reported compounds (Stein et al., 2002; Adams, 2007).

 Experimental animals

The Swiss albino mice of either sex were used in analgesic experiments which were obtained from research laboratory of National Institute of Health, Islamabad, Pakistan. The animals were used as per the approval of the ethical committee, Department of Pharmacy, University of Malakand, Pakistan according to the animals Bye-Laws 2008 (Scientific Procedure Issue-1).

 Acute toxicity

Swiss albino mice were taken in various groups, having 5 test animals in each group. The essential oil samples were administered to the animals orally in different doses (250–2,000 mg/kg). To increase the aqueous solubility of essential oil, 0.1% v/v tween-80 (Sigma Aldrich)- was used. After administration of the doses, animals were critically observed for 72 h for hypersensitivity, abnormal behavior, and death. The experimental animals were observed for 20 days for sub-chronic effects and lethality (Hosseinzadeh et al., 2000).

 Analgesic activities Acetic acid-induced writhing test

In acetic acid induced writhing test, the essential oil was administered orally (PO) in the same concentrations as mentioned in above section. After 30 min of interval, acetic acid (0.6%, 10 ml/kg) was injected into the mice intra-peritoneally. Tween-80 (0.5%, 3 ml/kg) was administered to Group I animals. The Group I was used as a negative control. The standard drug diclofenac sodium was administered to Group II with a dose of 10 mg/kg. The essential oil samples were administered to Groups III and IV in concentrations of 50 and 100 mg/kg respectively. After administration of acetic acid, the number of writhes were counted for 30 min (Franzotti et al., 2000).

 Formalin test

The formalin-induced licking test of Ir.EO was carried out using Swiss albino mice weighing 25–30 gm. The test was performed in a controlled environmental temperature (23 ± 2°C) with light-dark cycle of 12 h each. Food and water was freely available to the test animals throughout the investigations. The essential oil was administered intraperitonially (I/P) to the experimental animals at various concentrations. After 30 min, 20 μl formalin (2.5%, v/v in distilled water) was injected subcutuneously (S/C) into the plantar surface of the hind paw. Tween-80 (0.5%, 3 ml/kg), a negative control in the experiment was administered to the Group I. Morphine (5 mg/kg), a standard drug, was administered to Group II animals. The animals in Groups III and IV were injected Ir.EO at concentrations of 50 and 100 mg/kg respectively. The nociceptive behavior was designated by formalin-induced licking of paw. The total time taken in the behavioral changes of the mice responses to nociception was recorded, such as licking and/or biting of the injected paw. The time taken was recorded for 30 min. The initial 5 min were considered as early phase, while 2nd period (15–30 min) as the late phase of the response. The early and late phase are termed as neurogenic and inflammatory phase, respectively (Sulaiman et al., 2008).

 Hot plate test

The hot plate test method was assessed for the antinociception potential of essential oil isolated from I. rugosus as per the reported procedure (Zeb et al., 2016). In this method, a heated surface of a hot plate analgesia meter (Ugo Basile, model-7280) was maintained at 55 ± 0.2°C. The animals were kept over a heated surface in a closed glass cylinder. The time of the animals' placement and licking of hind paw or jumping over the heated surface were recorded as response latency. These are the parameters as a result of the thermal reactions. The oil samples, in concentrations of 50 and 100 mg/kg, while morphine 5 mg/kg, i.p., were administered 30 min before the beginning of the assessment. Mice were observed before administration of samples, and then at 30, 60 and 90 min after the samples taken. The cut-off time was 20 s.

 Involvement of opioid receptors

This experiment was carried out to confirm the possible involvement of opioid receptors in the essential oil-induced antinociception. The procedure was evaluated using a hot plate and formalin test method as mentioned earlier. In this method, different groups of experimental mice (n = 6) were pretreated with naloxone (5 mg/kg, S/C), which is a non-selective opioid receptor antagonist. Naloxone was injected 15 min before the administration of Ir.EO and morphine.

 Antioxidant assays DPPH assay

The DPPH free radicals scavenging effect was figured out for Ir.EO as previously published (Shah et al., 2015b). The DPPH solution (0.004%) in methanol was prepared which appeared with a deep violet color. Initially, the stock solution of essential oil with a known concentration of 1,000 μg/mL was prepared in ethanol. Then, this solution was diluted serially to obtain different concentrations from 62.5 to 1,000 μg/mL. Afterwards, 0.1 mL of the serially diluted concentration was added to 3.0 mL of DPPH solutions. This mixture was stored at dark place for 30 min at 23°C. After 30 min, the absorbance of each oil sample was measured by using double beam spectrophotometer at a wavelength of 517 nm. Ascorbic acid served as a positive control. The percent activity of all the samples was recorded as mean ± SEM. The percent radical scavenging potential was figured out using the following formula;

Scavenging effect %=    control absorbance − sample absorbancecontrol absorbance×100 ABTS assay

Antioxidant potential of Ir.EO was also investigated using free radicals of 2, 2-azinobis [3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) (Ullah et al., 2017). Solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were prepared and mixed thoroughly. The prepared solution was stored in a dark place overnight to generate free radicals. The absorbance of this solution was adjusted at 745 nm to 0.7 by addition methanol (50%). ABTS solution 3 mL was added to the test tubes containing samples having volume of 300 μL. The solution was transferred to the sample holder and absorbance was recorded for 6 min by using a double beam spectrophotometer. Ascorbic acid was used as a standard. The percent ABTS free radicals scavenging potential of the oil sample was measured by using the given formula;

Scavenging activity (%)=  Absorbance of control − Absorbance of Ir.EOAbsorbance of control × 100 Anticholinesterase assays

Cholinesterases inhibitory potentials of Ir.EO was evaluated following Ellman's assay (Ellman et al., 1961). This procedure is based on enzymatic breakdown of substrates like acetylthiocholine iodide and butyrylthiocholine iodide by AChE and BChE respectively to form 5-thio-2-nitrobenzoate anions. The resultant anions consequently form a complex with DTNB and are converted into UV detectable yellow color compound. The formation of this compound is quantified in the presence and absence of inhibitor agents. In brief, 5 μL enzyme solution was added to each well of micro plate with subsequent addition of 5 μL DTNB solution. The resulting mixture was incubated for fifteen min at 30°C in water bath, and finally 5 μl substrate solution was added to it. At the end, absorbances were recorded at 412 nm. The control samples were the same as above mentioned but were without inhibitors. The change in absorbance was observed beside reaction time. The activity of enzymes and its inhibitory activities were determined for control as well as test samples from the rate of absorption with change in time as, V = Δ Abs /Δ t, and enzyme inhibition as;

100 × VVmax

Where, Vmax is enzyme activity in the absence of inhibitor agent.

 Estimation of IC<sub>50</sub> values

The median inhibitory concentration (IC50) values of DPPH, ABTS, AChE, and BChE inhibitory assays were find out by linear regression analysis of the percent inhibition versus concentrations of the test samples through MS Excel program (Shah et al., 2015a; Sadiq et al., 2016).

 Statistical data analysis

The values of all the tests were tabulated as mean ± S.E.M. Significant differences of the percent inhibitions of various test samples were analyzed via one way ANOVA following Bonferroni's post-test using GraphPad Prism software in which the P < 0.05 were considered significant.

 Results GC-MS analysis

The essential oil of Isodon rugosus was subjected to GC-MS analysis and total of 141 compounds were identified. On the given GC method, the retention times of the identified compounds were from 6.057 to 81.661 min. The details of all identified compounds are given in Table 1.

List of all the compounds identified in the GC-MS analysis of essential oil of Isodon rugosus.

S. No. Compound label RT Name Formula Hits (DB)
1 Methyl ethyl ketone 6.057 Butanone C4H8O 10
2 2-cyclohexenyl vinyl ether 6.683 Na C8H12O 10
3 Alpha.-Copaene 18.689 Alpha Copaene C15H24 10
4 BETA-bOURBONENE 19.522 BETA. BOURBONENE C15H24 10
5 8-Isopropyl-1-methyl-5-methylene-1,6-cyclodecadiene 19.605 Germacrene D C15H24 10
6 2,6-Dimethylocta-1,4,7-triene 19.643 Cis-Achillene C10H16 10
7 2-Butanone, 4-(2,2-dimethyl-6-methylenecyclohexyl) 19.764 Na C13H22O 10
8 Bicyclo[3,3,1]non-2-ene, 7-oxa-2,8,9-trimethyl-5-acetoxymethyl 19.866 Na C14H22O3 10
9 1,4-Dimethylpent-2-enyl)benzene 19.976 Na C13H18 10
10 Beta.-Caryophyllen 20.312 Caryophyllene C15H24 10
11 1,1,7-TRIMETHYL-4-METHYLENEDECAHYDRO-1H-CYCLOPROPA[E]AZULENE 20.584 AROMADENDRENE C15H24 10
12 Alpha.-Cubebene 20.851 Alpha Cubebene C15H24 10
13 CADINA-1,4-DIENE 20.937 Na C15H24 10
14 3,8-Dimethylundecane 20.98 Na C13H28 10
15 Cycloisolongifolene, 8,9-dehydro- 21.184 Na C15H22 10
16 Epi-bicyclosesquiphellandrene 21.227 Na C15H24 10
17 Alpha.-Amorphene 21.477 Alpha Amorphene C15H24 10
18 Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl 21.549 Ar-Curcumene C15H22 10
19 Cis-(-)-2,4a,5,6,9a-Hexahydro-3,5,5,9-tetramethyl(1H)benzocycloheptene 21.626 Na C15H24 10
20 7-Methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole 21.788 Na C12H14N2O 6
21 Cadina-4,9-diene 21.977 Alpha.-Muurolene C15H24 10
22 1-Hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene 22.452 Germacrene D-4-ol C15H26O 10
23 Calamenene 22.479 Calamenene C15H22 10
24 9-Methyl-S-octahydrophenanathracene 22.603 Na C15H20 10
25 2,5,9,9-Tetramethyl-6,7,8,9-tetrrahydro-5H-benzocycloheptane 22.775 Na C15H22 10
26 5H-Inden-5-one, 1,2,3,3a,4,7a-hexahydro-7a-methyl-, trans- 22.823 Na C10H14O 10
27 1,1,6-trimethyl-1,2-dihydro naphthalene 22.917 CALACORENE C13H16 10
28 3-Heptadecen-5-yne 23.126 Na C17H30 10
29 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl 23.471 Trans-Nerolidol C15H26O 10
30 6-Pentadecen-9-yne 23.885 Na C15H26 10
31 Verbenene 23.923 Verbenene C10H14 10
32 N-Butyl-3-hydroxybutyramide 24.061 Na C8H17NO2 3
33 N-(1-Methylethyl)-2-(1-methylethyl)benzamide 24.073 Na C13H19NO 10
34 Clov-2-ene-9.alpha.-ol 24.145 Na C15H24O 10
35 VIRIDIFLOROL 24.221 Veridiflorol C15H26O 10
36 Ledol 24.319 Ledol C15H26O 10
37 1H-Inden-1-one, octahydro-, cis 24.462 Hydrindan C9H14O 10
38 3-(Hydroxymethyl)-4-hydroxy-5,6,7,8-tetrahydroquinoline 24.531 Na C10H13NO2 3
39 1,2-Naphthalenedione, 3,8-dimethyl-5-(1-methylethyl) 24.561 Mansonone C C15H16O2 10
40 Humulane-1,6-dien-3-ol 24.618 Na C15H26O 10
41 Cedr-8-ene 24.696 Alfa-cedrene C15H24 10
42 Longifolenaldehyde 24.781 Longifolenaldehyde C15H24O 10
43 2,3-Bis[(adamantylcarbonyl)ethynyl]bicyclo[2.2.1]hepta-2,5-diene 24.993 Na C33H36O2 10
44 1,2-Diacetyl-4-methylbenzene 25.134 Na C11H12O2 10
45 1.beta.,10.beta.H-Cadin-4-en-10-ol 25.392 T-Muurolol C15H26O 3
46 4,4-Dimethylpentanenitrile 25.406 Na C7H13N 1
47 Cadin-4-en-10-ol 25.57 Alpha.-Cadinol C15H26O 10
48 5,7-Dimethylquinoline 25.583 Na C11H11N 10
49 Azulene, 1,4-dimethyl-7-(1-methylethyl) 25.686 Azunol C15H18 10
50 1,4-Methanobenzocyclodecene, 1,2,3,4,4a,5,8,9,12,12a-decahydro- 25.864 Na C15H22 5
51 4,4a,5,6,7,8-Hexahydro-4a-methyl-2(3H)-naphthalenone 26.157 Na C11H16O 10
52 7,7-dichlorobicyclo[3.2.0]hept-2-en-6-one 26.262 Na C15H24O 10
53 6-Methylenebicyclo[2.2.1]hept2-en-1-ol 26.348 Na C8H10O 1
54 MUUROLA-4,10(14)-DIEN-3-ONE 26.374 Na C15H22O 10
55 Pentalene, octahydro-1-(2-octyldecyl) 26.513 Na C26H50 10
56 1.beta.,4.beta.H,10.beta.H-Guaia-5,11-diene 26.627 Gamma.-Gurjunene C15H24 10
57 Phosphorochloridic acid, diethyl ester 26.788 Diethyl chlorophosphate C4H10ClO3 10
58 Isoisopulegyl acetate 26.902 Isoisopulegyl acetate C12H20O2 10
59 1,3-dimethyl-3-acetoxymethyl-2-oxabicyclo[2.2.2]octan-5-one 27.314 Na C12H18O4 10
60 Benzyl benzoate 27.873 Benzyl benzoate C14H12O2 10
61 Ttrans-1-(3′′-Cyclopropylidenepropen-1′′-yl)-1-(propen-3′-yl)cyclopropane 28.23 Na C12H16 5
62 Ethyl 3-phenylhexa-2,4-dienoate 29.066 Na C14H16O2 4
63 1-(6′-Methoxy-7′-methyl-1′,2′,3′,4′-tetrahydronaphthalen-1′-yl)ethanol 29.312 Na C14H20O2 2
64 6-(p-Tolyl)-2-methyl-2-heptenol 29.584 Nuciferol C15H22O 10
65 2-Butanol, 4-[2,2,6-trimethylcyclohexyl]-, acetate 30.058 Tetrahydroionyl acetate C15H28O2 10
66 1H-3a,7-Methanoazulene, octahydro-1,4,9,9-tetramethyl 30.327 Patchoulane C15H26 10
67 Trans-Pinocarvyl acetate 30.688 Na C12H18O2 3
68 [1-(R,S),5(R,S)]-3-(2,2-dimethyl-1-(R,S/S,R)-hydroxypropyl)-6-(R,S)-n-octyl-… 31.48 Na C18H32O3 4
69 3-Angelate of felikiol 31.704 Na C20H32O4 1
70 2-thia-6-methyl-7-(2-formylethyl)bicyclo[3.2.0]hept-6-ene-2,2-dioxide 31.795 Na C10H14O3S 3
71 Aphanamol 32.079 Aphanamol C15H24O2 10
72 5,9-Undecadien-2-one, 6,10-dimethyl 32.317 Geranylacetone C13H22O 10
73 4-Chlorobutyric acid, octadecyl ester 32.639 Na C22H43ClO2 10
74 Bicyclo[5.2.0]nonane, 4-methylene-2,8,8-trimethyl-2-vinyl- 32.976 Na C15H24 10
75 2,9-Dimethyl-8-oxatetracyclo[5.4.1.1(3,10).0(5,9)]tridecane-2-endo,7-diol 33.499 Na C14H22O3 10
76 1-(4-Hydroxy-3-isopropenyl-4,7,7-trimethyl-cyclohept-1-enyl)-ethanone 33.918 Na C15H24O2 7
77 1,2-pentanediol, 5-(6-bromodecahydro-2-hydroxy-2,5,5a,8a-tetramethyl-1-napht… 34.228 Na C20H35BrO3 1
78 1-Hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene 35.087 Na C15H26O 1
79 Viridiflorene 35.376 Viridiflorene C15H24 8
80 Dodecylpalmitate 35.978 Dodecylpalmitate C28H56O2 10
81 Sec-Butyl 2,3,4,6-tetra-methyl-.beta.,D-galactopyranoside isomer 36.292 Na C14H28O6 2
82 1,3-EPIMANOYL OXIDE 36.366 Epimanoyl oxide C20H34O 8
83 Pentadecane 36.435 Pentadecane C15H32 10
84 Cyclodecane, octyl 36.599 Octylcyclodecane C18H36 5
85 Labd-14-ene, 8,13-epoxy-, (13R)- 37.211 Na C20H34O 10
86 5.alpha.-allyl-6.alpha.-hydroxy-5.beta.,9.beta.-dimethyl-trans-decalin-1-one 37.726 Na C15H24O2 3
87 Naphthalene, 7-butyl-1-hexyl 38.181 Na C20H28 10
88 1,1,7,12-tetramethyl-8-ethyl-1,2,3,4,9,10,11,12-octahydrophenanthrene 38.718 Na C20H30 10
89 Benzamidine, 4-(4-pentylphenyl)- 39.442 Na C18H22N2 1
90 3-Eicosene 39.652 Na C20H40 10
91 Eicosane 40.011 Eicosane C20H42 10
92 Sulfurous acid, hexyl nonyl ester 40.208 Na C15H32O3S 10
93 2-Cyclohexen-1-ol, 3-methyl-6-(1-methylethyl) 40.372 Na C10H18O 10
94 1-Acetyl-2-amino-3-cyano-7-isopropyl-4-methylazulene 40.546 Na C17H18N2O 1
95 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl 40.682 Phytol C20H40O 10
96 1,1,8,9A-tetramethyl-2,3,5,6,7,9a-hexahydro-1h-benzo[a]cycloheptene 40.915 Na C15H24 10
97 2-n-Heptylcyclopentanone 41.419 Na C12H22O 10
98 7,11,15-TRIMETHYL,3-METHYLENE-1-HEXADECENE 41.645 Neophytadiene C20H38 10
99 Stearic acid 42.294 Stearic acid C18H36O2 10
100 Abietyl alcohol, dehydro 42.844 Abietyl alcohol, dehydro C20H30O 2
101 Oxalic acid, hexadecyl propyl ester 43.093 Na C21H40O4 7
102 1,4a.beta.-Dimethyl-7-isopropyl-2,3,4,4a,9,10-hexahydrophenanthrene 43.238 Na C19H26 10
103 5-Diazo-1-(2′-methyl-4′-nitrophenylazo)-1,3-cyclopentadiene 43.79 Na C12H9N5O2 10
104 (1.alpha.,2a.alpha.,8b.alpha.)-1,2,2a,8b-Tetrahydro-8b-hydroxy-1 44.228 Na C17H13NO 10
105 2-Methyl-5,6-diphenyl-1,2,4-triazin-3(2H)-one 44.27 Na C16H13N3O 1
106 Dehydroabietal 44.302 Dehydroabietal C20H28O 3
107 8-Methoxy-4-methylbenzo[g]quinoline-5,10-dione 44.617 Na C15H11NO3 10
108 Phenol, 4-methoxy-2-[5-(4-methylphenyl)-3-pyrazolyl)- 45.092 Na C17H16N2O2 10
109 Pentatriacontane 45.127 Pentatriacontane C35H72 10
110 Tetracyclo[16.1.0.0(2,9).0(10,17)]nonadeca-2(9),10(17)-diene, 19,19-dimethyl- 45.232 Na C21H32 10
111 1,2-Dihydro-1-methyl-2-trifluoroacetylmethylenequinoline 45.506 Na C13H10F3NO 10
112 Simvastatin 45.671 Simvastatin C25H38O5 3
113 Methyl-9-anthracenemethanamine 45.764 Na C16H15N 7
114 4,8,12,16-Tetramethylheptadecan-4-olide 46.318 Na C21H40O2 2
115 2-Ethyl-3-phenyl-2-butene-1-al 47.678 Na C12H14O 2
116 Hinokione methyl ether 47.827 Hinokione methyl ether C21H30O2 10
117 8-Isopropyl-1,3-dimethylphenanthrene 47.873 Na C19H20 1
118 Cyclohexanone, 2-butyl 48.334 Na C10H18O 10
119 17-Methoxy-d-homo-18-norandrosta-4,8,13,15,17-pentaen-3-one 48.756 Na C20H22O2 10
120 5,8,11,14-Eicosatetraynoic acid, methyl ester 48.899 Na C21H26O2 4
121 MARGOCIN 48.964 MARGOCIN C20H26O2 1
122 Phenanthro[3,2-b]furan-7,11-dione, 1,2,3,4-tetrahydro-4,4,8-trimethyl- 49.337 Na C19H18O3 10
123 1,4-Bis(2-chloro-1,1-dimethylethyl)benzene 49.547 Na C14H20Cl2 2
124 Phenol, 4-methoxy-2-[5-(4-methylphenyl)-3-pyrazolyl)- 49.67 Na C17H16N2O2 10
125 6-Hydroxy-2,2,5,7,8-pentamethyl-4-phenyl-2H-1-benzopyran 50.552 Na C20H22O2 10
126 Phenanthro[3,2-b]furan-7,11-dione, 1,2,3,4-tetrahydro-4,4,8-trimethyl- 51.077 Na C19H18O3 10
127 2-Phosphabicyclo[3.1.0]hex-3-ene, 2,6,6-trimethyl-3,4-diphenyl- 51.481 Na C20H21P 5
128 2,3-(anti)-Epoxy-1,4-dimethyl-1,4,6,13-tetrahydrobenzo[g]pyridazino[1,2-b] 51.743 Na C18H16N2O3 1
129 Benzenamine, N,N-diethyl-4-[2-(4-nitrophenyl)ethenyl]- 52.901 Na C18H20N2O2 10
130 2,6-Bis(1,1-dimethylethyl)-4-phenylmethylenecyclohexa-2,5-dien-1-one 53.235 Na C21H26O 9
131 2H-Pyrazole, 3-amino-5-methyl-2-(4-nitrophenyl)-4-phenyl- 53.378 Na C16H14N4O2 10
132 1-(2-Isopropyl-phenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-indazol-4-one 53.687 Na C19H24N2O 10
133 3-(N,N-Diethylamino)-5-iodoaniline 54.347 Na C10H15IN2 10
134 17-Methoxy-d-homo-18-norandrosta-4,8,13,15,17-pentaen-3-one 54.378 Na C20H22O2 1
135 Heneicosane, 11-(1-ethylpropyl) 56.17 Na C26H54 3
136 2-nitro-5a,6,6-trimethyl-5a,6-dihydro-12h-indolo[2,1-b][1,3]benzoxazine 56.338 Na C18H18N2O3 10
137 6H-Dibenzo[b,d]pyran-1-ol, 6,6,9-trimethyl-3-pentyl- 57.279 Cannabinol C21H26O2 10
138 Sulfurous acid, pentadecyl 2-propyl ester 57.372 Na C18H38O3S 10
139 Hexadecane, 2,6,10,14-tetramethyl 57.927 Na C20H42 10
140 Triacontane 59.093 Triacontane C30H62 10
141 P-Methyl-o-(phenylethynyl)phenol 81.661 Na C15H12O 3
Acute toxicity

No mortality and behavioral change were observed at specified doses to confirm acute toxicity of the samples. According to the assay, dose up to 2,000 mg/kg was considered as safe for essential oil of Isodon rugosus.

 Writhing test

A dose dependent response was observed in acetic acid induced writhing test for the assessment of analgesic activity. The mean writhes of the standard drug at 10 mg/kg, was 21.83 ± 0.60 with 70.29% inhibition. The essential oil sample exhibited mean inhibition of 31.50 ± 1.28 with 57.14% at 100 mg/kg, while, at 50 mg/kg it exhibited mean inhibition of 41.00 ± 0.57 with 44.21%. At 100 mg/kg, Ir.EO and positive control exhibited a response of 57.14 and 70.29% respectively as shown in Table 2.

Percent anti-nociceptive potential of essential oil following acetic acid induced writhing model.

Samples Dose (mg/kg) Mean writhes % Analgesic activity
Negative cont – 73.50 ± 0.61 0.00
Ir.Eo 50 41.00 ± 0.57*** 44.21
Ir.Eo 100 31.50 ± 1.28*** 57.14
Positive cont 10 21.83 ± 0.60*** 70.29

Ir.Eo, Essential oil isolated from Isodon rugosus; Mean writhes are represented as mean ± SEM.

P < 0.001.

 Formalin test

The results obtained from formalin test are shown in the Table 3. The formalin injection (2%, i.p) to the animals revealed a typical biphasic licking response. In the control group, duration of licking was observed as 57.33 ± 0.88 and 67.00 ± 0.93 s for early (0–5 min) and late phase (15–30 min) respectively. Pre-treatment of mice with various concentrations of essential oil (50 and 100 mg/kg) produced a significant effect on the duration of licking in both phases. A dose of 100 mg/kg of Ir.EO brought a significant reduction in paw licking of 54.36 and 43.28% in early and late phase respectively. In comparison, the standard drug morphine (5 mg/kg i.p.) demonstrated overwhelming reduction in both phases, i.e., 79.36% (early phase/neurogenic pain) and 79.59% (late phase/inflammatory pain). The morphine in combination with naloxone exhibited 04.63 and 05.22% activity in early and late phase respectively. In comparison, Ir.EO in combination with naloxone revealed 09.01% (early phase) and 07.95% (late phase) pain inhibitions. So, the naloxone reversed the antinociceptive effect of essential oil considerably at dose of 100 mg/kg in both phases as those of morphine.

Effect of Essential oil of Isodon rugosus on formalin induced pain in mice.

Samples Dose (mg/kg) Total time spent in licking
0–5 min % Inhibition 15–30 min % Inhibition
Negative cont. – 57.33 ± 0.88 – 67.00 ± 0.93 –
Ir.Eo 50 39.50 ± 1.11*** 31.10 49.67 ± 1.92*** 25.86
Ir.Eo 100 26.16 ± 0.94*** 54.36 38.33 ± 0.71*** 43.28
Mor 5 11.83 ± 1.24*** 79.36 13.67 ± 0.67*** 79.59
Mor + Nal 5 + 1 54.67 ± 1.02ns 04.63 63.50 ± 1.17ns 05.22
Ir.Eo + Nal 50 + 1 52.16 ± 0.70** 09.01 61.67 ± 1.14* 07.95

Ir.Eo, Essential oil of Isodon rugosus; Mor, Morphine; Nal, Naloxone; Total time is represented as mean ± SEM.

P < 0.05,

P < 0.01,

P < 0.001.

 Hot plate test

The results obtained in the hot plate assay are shown in Table 4. The Ir.EO revealed a dose dependent increase in the latency time as that of positive control. At 15 min, the mean reaction times for 50 and 100 mg/kg body weights of essential oil were observed as 06.40 ± 0.11 and 08.16 ± 0.08 min respectively. At 90 min, i.e., last interval, the mean reaction times of the same two doses were recorded as 04.40 ± 0.20 and 06.45 ± 0.07 min respectively. In comparison, the standard drug morphine exhibited reaction times of 12.41 ± 0.11 and 09.38 ± 0.08 min at initial and last interval respectively.

Effect of Essential oil of Isodon rugosus on hot plate induced pain in mice.

Samples Dose (mg/kg) Reaction time on hot plate
15 min 30 min 45 min 60 min 90 min
Negative cont. – 04.51 ± 0.10 02.43 ± 0.14 03.55 ± 0.07 02.11 ± 0.15 03.20 ± 0.15
Ir.Eo 50 06.40 ± 0.11 04.18 ± 0.11 05.76 ± 0.08 04.81 ± 0.14 04.40 ± 0.20
Ir.Eo 100 08.16 ± 0.08 06.81 ± 0.13 07.91 ± 0.11 06.80 ± 0.07 06.45 ± 0.07
Mor 5 12.41 ± 0.11 11.63 ± 0.06 11.36 ± 0.08 10.45 ± 0.07 09.38 ± 0.08
Mor + Nal 5 + 1 04.35 ± 0.13 02.80 ± 0.19 04.35 ± 0.07 02.78 ± 0.10 03.56 ± 0.14
Ir.Eo + Nal 50 + 1 05.28 ± 0.13 03.31 ± 0.08 04.70 ± 0.08 03.11 ± 0.15 03.70 ± 0.05

Ir.Eo: Essential oil of Isodon rugosus; Mor: Morphine; Nal: Naloxone; Total time is represented as mean ± SEM.

Moreover, the recorded mean reaction time for Ir.EO with naloxone (50: 1 mg/kg) at 15 min was 05.28 ± 0.13 min. Similarly, for morphine and naloxone (5: 1 mg/kg), the mean reaction time observed was 04.35 ± 0.13 min at initial 15 min. In our experiment, we found a distinct reduction in reaction time with the administration of naloxone.

 Involvement of opioid receptors

In both hot plate and formalin models, we noticed that Ir.EO revealed a similar activity as that of morphine. The potency of Ir.EO was reduced effectively by opioid antagonist naloxone. With the use of naloxone, the decreased in reaction time in hot plate method and reversing the paw licking in formalin assay confirmed the possible involvement of opioid receptors.

 Antioxidant assays

The antioxidant potential of Ir.EO using DPPH and ABTS free radicals scavenging methods are shown in Table 5.

Antioxidant activity of essential oil of Isodon rugosus at various concentrations.

Test sample Free radicals Conc. 62.5 μg/ml Conc. 125 μg/ml Conc. 250 μg/ml Conc. 500 μg/ml Conc. 1,000 μg/ml IC50 μg/ml
EO DPPH 33.00 ± 1.15*** 41.33 ± 0.88*** 46.33 ± 0.33*** 56.67 ± 0.67*** 63.67 ± 1.20*** 338
EO ABTS 39.67 ± 1.76*** 51.00 ± 0.57*** 56.67 ± 1.20*** 62.00 ± 0.57*** 64.33 ± 0.88*** 118
A.A DPPH 77.33 ± 0.88 79.67 ± 0.88 83.00 ± 1.73 86.33 ± 1.45 89.00 ± 1.73 <62.5
A.A ABTS 75.00 ± 0.57 78.00 ± 1.15 81.67 ± 0.67 85.67 ± 0.33 91.33 ± 0.88 <62.5

EO, Essential oil; A.A, Ascorbic acid.

P < 0.001.

 DPPH assay

The observed percent inhibitions for Ir.EO using DPPH free radicals was 63.67 ± 1.20, 56.67 ± 0.67, 46.33 ± 0.33, 41.33 ± 0.88, and 33.00 ± 1.15% at concentrations of 1,000, 500, 250, 125, and 62.5 μg/ml respectively. The calculated IC50 value from the dose response curve was 338 μg/ml. In comparison, the standard drug ascorbic acid exhibited 89.00 ± 1.73, 86.33 ± 1.45, 83.00 ± 1.73, 79.67 ± 0.88, and 77.33 ± 0.88% inhibitions at 1,000, 500, 250, 125, and 62.5 μg/ml respectively with an IC50 value of <0.1 μg/ml.

 ABTS assay

In ABTS assay, Ir.EO attained 64.33 ± 0.88, 62.00 ± 0.57, 56.67 ± 1.20, 51.00 ± 0.57, and 39.67 ± 1.76% inhibitions at 1,000, 500, 250, 125, and 62.5 μg/ml respectively. The calculated IC50 for Ir.EO in scavenging ABTS free radicals was 118 μg/ml. In this assay, ascorbic acid demonstrated 91.33 ± 0.88, 85.67 ± 0.33, 81.67 ± 0.67, 78.00 ± 1.15, and 75.00 ± 0.57% inhibitions at 1,000, 500, 250, 125, and 62.5 μg/ml respectively attaining an IC50 value of <0.1 μg/ml.

 Cholinesterase inhibition assay

In AChE inhibitory assay, Ir.EO exhibited concentration dependent inhibitions against the enzymes (Table 6). Ir.EO showed 67.50 ± 1.04% AChE inhibition at 1.0 mg/ml concentration with IC50 of 93.56 μg/ml. Similarly, the observed inhibitory potential against BChE at the same tested concentration as AChE was 61.33 ± 0.67% with an IC50 of 284.19 μg/ml. In comparison, the standard drug galanthamine exhibited 0.371 and 3.324 μg/ml IC50 against AChE and BChE respectively.

Anticholinesterase activity of essential oil of Isodon rugosus at various concentrations.

Test sample Enzymes Conc. 62.5 μg/ml Conc. 125 μg/ml Conc. 250 μg/ml Conc. 500 μg/ml Conc. 1,000 μg/ml IC50 μg/ml
E.Oil AChE 42.30 ± 0.47 56.46 ± 1.27 57.00 ± 0.57 62.67 ± 0.88 67.50 ± 1.04 93.56
E.Oil BChE 36.56 ± 0.97 41.95 ± 2.01 49.87 ± 1.67 56.00 ± 1.15 61.33 ± 0.67 284.19
Gal AChE 74.00 ± 1.00 79.66 ± 1.85 85.00 ± 1.73 87.46 ± 1.79 94.83 ± 1.92 <62.5
Gal BChE 63.83 ± 0.92 71.16 ± 0.92 77.83 ± 1.09 83.16 ± 1.42 88.00 ± 1.25 <62.5

Data is expressed as Mean±SEM; Gal and E.Oil are abbreviated for Galanthamine and Essential oil respectively.

 Discussion

In our designed work, the essential oil of I. rugosus was evaluated for antinociceptive, antioxidant, and anticholinestease potentials. The essential oils of plants are sources of wide variety of bioactive compounds (Dehpour et al., 2009). The pharmacological potentials of essential oil can be attributed to the hydropholic nature of its components and the same nature of our body cell membranes (Ait-Ouazzou et al., 2011). Various components of essential oils can easily get distributed to different compartments of our body including the central nervous system (Lambert et al., 2001; Vyas et al., 2008). In our current investigational study, the antinociceptive potential of essential oil was recorded with significant results. The possible mechanism of antinociceptive activity of Ir.EO was figured out as the central pathway due to involvement of opioid receptors. Recently, we have also reported the antinociceptive potential of chloroform fraction of I. rugosus following the same mechanism. The antinociceptive potential of essential oil may be due to the presence of large number of bioactive compounds as obvious from its GC-MS analysis. Among the identified compounds, we also observed some of the bioactive compounds previously reported with analgesic potentials. These compounds include α-copaene, germacrene D, β-caryophyllene, α-caryophyllene, aromadendrene, calamenene, viridiflorol, mansonone C, t-muurolol, α-cadinol, azunol, phytol, neophytadiene, and simvastatin. In short, α-copaene has been reported to possess strong analgesic and antioxidant potentials (Him et al., 2008; Chen et al., 2011; Costa et al., 2011). Likewise, germacrene D also possesses analgesic and antioxidant effects (Del-Vechio-Vieira et al., 2009; Victoria et al., 2012). β-Caryophyllene is also reported with its analgesic and antioxidant potentials (Calleja et al., 2013; Klauke et al., 2014). The antinociceptive activity of aromadendrene has also been demonstrated (Cruz et al., 2011). Similarly, α-caryophyllene has been reported for the treatment of body inflammatory pain (Pianowski et al., 2004). The analgesic and antioxidant effects of calamenene have also been demonstrated with significant results (Azevedo et al., 2013; Imam et al., 2014). Viridiflorol, a well-known bioactive compound is also reported to possess analgesic and radical scavenging potentials (Perry et al., 1997; do Amaral et al., 2007). Moreover, Mansonone C (including its reduced form) is also responsible for direct antioxidant activity (Villamil et al., 1990). T-muurolol has been verified for inhibitory activity against DPPH free radicals (Cheng et al., 2004). The analgesic activity of α-cadinol has also been reported with notable results (Boutaghane et al., 2011). The antinociceptive aspects of azunol has also been published previously (Ushiyama et al., 2009). In the same way, ledene has also been reported to possess analgesic activity (Alagammal et al., 2012). A well-known compound, i.e., phytol, is famous for its antioxidant potential along with its antinoceptive potential (Santos et al., 2013). Neophytadiene is also among the famous analgesic and antioxidant candidates (Jayashree et al., 2015). Similarly, simvastatin is also previously reported with its analgesic and antioxidant potentials (Carneado et al., 2002; Chen et al., 2013).

Literature review and the results of our current investigations go parallel with sound correlation. The traditional use of I. rugosus as analgesic is efficiently verified in the current research project, along with the identification of bioactive compounds.

Beside the antioxidant potential of Ir.EO, we also evaluated its AChE and BChE inhibitory potentials. Among other pathological targets of Alzheimer disease, inhibitions of cholinesterase and free radicals are also vital targets. Among the clinically approved anti-Alzheimer drugs, four are cholinesterase inhibitors, which signify the importance of this target in the symptomatic management of the disease. In the current study, we observed a moderate in-vitro cholinesterase inhibitory activity of Ir.EO. In AChE and BChE inhibitory assays, Ir.EO showed concentration dependent inhibitions against the enzymes with IC50 values of 93.56 and 284.19 μg/ml respectively. Though the in-vitro enzyme inhibitory activity of essential oil was low in comparison to galanthamine, yet, we hypothesize that it will have more availability at the target site. However, further studies are required regarding in-vivo efficacy of our tested essential oil.

 Conclusion

Based on the literature survey regarding the medicinal aspects of I. rugosus and the results of current investigational study, it may be deduced that the essential oil of I. rugosus is a good source of natural bioactive compounds containing numerous analgesic and antioxidant agents. Its antioxidant potentials along with cholinesterase inhibitory activity will be potentially effective in the management of Alzheimer's disease patients. It may also be inferred that further exploitation of essential oil of I. rugosus may lead to the development of new analgesic and/or anti-Alzheimer drug candidates.

 Author contributions

AZ and SA carried out experimental work, data collection and literature search under the supervision of AS. FU helped as co-supervision of the research work. MA, NM and UR drafted the manuscript for publication. AS supervise the overall project and make the final version of publication. All the authors have read and approved the final manuscript for publication.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Dr. Ali Hazrat, Department of Botany, Shaheed Benazir Bhutto University, Sheringal Dir (U), KPK, Pakistan for the identification of plant. We are also grateful to Department of Pharmacy, University of Malakand, Pakistan for providing the laboratory facilities to conduct the experiments.

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