Chemical composition and biological activity of Nepeta cilicica

Keywords: Biological activity, Chemical composition, Nepeta cilicica
DOI: 10.3329/bjp.v12i2.32010

Abstract

In the present study, aerial parts of Nepeta cilicica were hydro-distilled for the essential oil. Furthermore, air-dried plant materials were extracted with methanol. According to GC-MS results, 35 compounds were characterized in the oil (totally 92%). Caryophyllene oxide, β-caryophyllene and spathulenol (28.2, 8.9 and 4.2%) were found as main components of the oil. Sixteen compounds were detected by LC/MS, most of which were caffeoylquinic acid, luteolin and apigenin derivatives, in methanolic extract of the N. cilicica. Apart from DPPH radical scavenging activity, inhibitory effects on bacterial and yeast growth of essential oil and the methanolic extract were evaluated. Twenty-four different pathogenic bacterial and Candida strains were tested by M7-A7 and CLSI M27-A2 protocols respectively. The crude extract showed better inhibitory effects against bacteria strains than the essential oil. Both oil and the extract demonstrated strong inhibition on C. tropicalis at a concentration of 47.0 µg/mL (MIC).

Introduction

Nepeta cilicia is a perennial plant with stem size of 22-100 cm. Some Nepeta species are used in the folk medicine for their therapeutic properties such as diaphoretic, diuretic, antiseptic, astringent, antiasthmatic, antitussive, antispasmodic, febrifuge, sedative, emmenagogue and stomachic. Flowering aerial parts of several Nepeta species are used as medicinal teas for their lemony mint odor and flavor (Duda et al., 2015; Shakeri et al., 2014; Formisano et al., 2013; Iscan et al., 2011; Skaltza et al., 2000).

Many compounds have been identified in Nepeta species such as monoterpenes, sesquiterpenes, flavonoids, phenolic acid, steroids and essential oils. The chemical composition of the essential oils derived from Nepeta can be of two categories. Nepetalactone containing and nepetalactoneless group that are generally rich in 1,8-cineole (most abundant), pinenes, β-caryophyllene, β-farnesene, caryophyllene oxide, α-citral and β-citronellol (Asgarpanah et al., 2014; Sharma and Cannoo, 2013; Skaltza et al., 2000). Nepetalactone isomers 4aα,7α,7aα-nepetalactone, 4aβ,7α,7aβ-nepetalactone, 4aβ,7α,7aα-nepetalactone, 4aα,7α,7aβ-nepetalactoneand 4aα,7β,7aα-nepetalactone are considered as biochemical indicator of the nepetalactone-rich group of Nepeta essential oils.

Biological efficacy is usually attributed to isoprene derivatives named nepetalactones especially found in the Nepeta oils (Kökdil et al., 1999; Başer et al., 2000; Skaltza et al., 2000; Nestorović et al., 2010). Even though two studies have been reported on the chemical composition of N. cilicica essential oil. There has not been any report about the chemical composition of the crude extract and antimicrobial activity of essential oil or the extract. According to previous studies sesquiterpenes (β-caryophyllene, spathulenol and β-caryophyllene oxide) and fatty acids have been determined as main compounds of N. cilicica essential oil (Formisano et al., 2013; Kokdil et al., 1997).

In the present study, hydrodistilled essential oil and crude methanolic extracts of flowering aerial parts of N. cilicica were analyzed by chromatospectral techniques. GC-MS with GC-FID and LC-MS systems were used for the determination of essential oil and the extract composition respectively. Both oil and extract were subjected to antimicrobial susceptibility assay against 24 pathogenic bacteria and Candida strains in comparison with the standard antimicrobial agents and also antioxidant assay for the first time.

Materials and Methods

Plant material

N. cilicica was collected from Anamur-Ermenek Road, İçel, Turkey on June 2014 in full flowering stage. Voucher specimen (ESSE: 14795) was kept in the Faculty of Pharmacy Herbarium, Anadolu University, Turkey.

Isolation of essential oil

The flowering aerial parts were air-dried at ambient temperature then subjected to a Clevenger-type apparatus for hydrodistillation for 3 hours according to European Pharmacopoeia 8.0, with a yield of 0.02% (on dried weight basis). The oil was dehydrated over sodium sulfate anhydrous and kept at 4°C until analysis.

Preparation of the extract

Extract of air-dried and grounded plant materials were prepared by the methanol (Merck) in an orbital shaker for 24 hours. After evaporation process at 40°C, dried extract was kept in dark place at 4°C until analysis.

GC-FID and GC-MS analysis

The GC-FID and GC-MS analysis were carried out with innowax FSC columns (60 m x 0.25 mm, 0.25 mm film thickness) with helium (0.8 mL/min) using Agilent 6890N GC and 5975 GC-MSD systems respectively. FID detector temperature was 300°C. To obtain similar retention times between the GC-FID and GC-MS, synchronous automatic duplicated injection was performed using equal column by setting the same operational parameters. Relative percent of the detected compounds were calculated from the FID chromatograms.

Identification of the compounds

Characterization and identification of the N. cilicica essential oil components were fulfilled by comparison of their relative retention time with those of genuine samples or by comparison of their relative retention indices (RRI) to n-alkanes (C6-C16). Software matching against commercial libraries (Wiley GC-MS and Mass Finder 3) (Koenig et al., 2004; McLafferty and Stauffer, 1989) and in-house “Başer Library of Essential Oil Constituents” created by genuine compounds and several constituents of the common essential oils, as well as MS data from previous works (ESO, 2000; Joulain and Koenig, 1998) were used for the identification.

Phenolics analysed by LC-MS/MS method

Applied Biosystems 3200 Q-Trap MS/MS system  coupled to a Shimadzu 20A HPLC system was used in the negative ionization mode for the analyses of the phenolic compounds. Octadecyl silica gel analytical column (ODS 150 x 4,6 mm, i.d., 3 µm particle sizes) for the separation operating at 40°C at a flow rate of 1 mL/min. HPLC elution was carried out using a gradient of the solvent mixture: H2O:CH₃OH:CH2O2 (89:10:1, v/v/v) (solvent A) and CH₃OH:H2O:CH2O(89:10:1, v/v/v) (solvent B). Solvent B composition was increased from 15 to 40% in 15 min, 40 to 45% in 3 min. It was held at 45% concentration for 12 min. Then increased from 45 to 75% in 5 min and increased from 75 to 85% in 3 min. In one minute, it was increased from 85 to 95% and it reached 100% concentration at 40 min.

Determination of total phenolic content

Total phenol contents were calculated as gallic acid equivalents (GAE), which means mg gallic acid/g extract. Total phenolics in the oil and extract were determined using with previously described method (Singleton et al., 1999). 

DPPH radical scavenging activity

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity determined according to the  previous study of Kumarasamy et al. (2007). Serial dilutions of the stock solution of samples and stand arts were prepared as half concentrations of previous one. Equal amounts of DPPH• reactive were added to the diluted solutions. After 30 min UV absorbance was recorded at 517 nm.

Antimicrobial susceptibility tests

Antibacterial and anticandidal properties of the essential oil and methanol extract were evaluated by using partially modified CLSI microdilution broth methods (CLSI-M7-A7 and CLSI-M27-A2). Unlike the reference methods, the essential oil and methanolic extract were diluted between the concentrations of 5.8 to 6000 µg/mL. Ampicillin (Merck), chloramphenicol (Merck), amphotericin B (Sigma-Aldrich) and ketoconazol (Sigma-Aldrich) were used as standard antimicrobial agents. Microorganisms were obtained from ATCC, NRRL and RSSK (Refik Saydam National Type Culture Collection, Turkey).

Results

Phytochemical composition

According to GC/MS results, 35 compounds representing 92% of the essential oil were characterized. The oil was constituted mainly by oxygenated sesquiterpenes (47.5%), sesquiterpene hydrocarbons (25.3%), alkanes (10.2%), oxygenated monoterpenes (3.7%), acycylic-diterpene derivative phytol (2.9%) and fatty acids (2.3%). Caryophyllene oxide (28.2%) and β-caryophyllene (8.9%) were determined as main compounds. Monoterpenes were detected only trace amounts in the essential oil (Table I).

Table I: Composition of the essential oil of N. cilicia

No RRI Compound % Method
1 1032 α-Pinene Trace RRI, MS
2 1118 β-Pinene Trace RRI, MS
3 1132 Sabinene Trace RRI, MS
4 1255 γ-Terpinene Trace RRI, MS
5 1280 p-Cymene Trace RRI, MS
6 1466 α-Cubebene 0.6 MS
7 1497 α-Copaene 2.7 MS
8 1535 β-Bourbonene 1.5 MS
9 1549 β-Cubebene 2.7 MS
10 1611 Terpinen-4-ol 0.3 RRI, MS
11 1612 β-Caryophyllene 8.9 RRI, MS
12 1648 Myrtenal 0.1 MS
13 1670 trans-Pinocarveol 0.1 MS
14 1683 trans-Verbenol 1.5 MS
15 1687 α-Humulene 1.3 RRI, MS
16 1726 Germacrene D 3.0 RRI, MS
17 1755 Bicyclogermacrene 1.5 RRI, MS
18 1773 α-Cadinene 0.9 MS
19 1776 γ-Cadinene 1.0 MS
20 1804 Myrtenol 0.6 MS
21 1849 Calamenene 1.2 MS
22 1900 epi-Cubebol 2.6 MS
23 2001 Isocaryophyllene oxide 3.8 MS
24 2008 Caryophyllene oxide 28.2 RRI, MS
25 2071 Humulene epoxide-II 3.5 MS
26 2144 Spathulenol 4.2 MS
27 2187 T-Cadinol 1.4 MS
28 2239 Carvacrol 1.1 RRI, MS
29 2289 4-Oxo-α-Ylangene 1.7 MS
30 2392 Caryophylla-2(12),6-dien-5 β-ol (=Caryophyllenol II) 2.1 MS
31 2500 Pentacosane 2.5 RRI, MS
32 2622 Phytol 2.9 MS
32 2700 Heptacosane 4.5 RRI, MS
34 2900 Nonacosane 3.2 RRI, MS
35 2931 Hexadecanoic acid 2.3 RRI, MS
    Monoterpene hydrocarbons Trace  
    Oxygenated monoterpenes 3.7  
    Sesquiterpene hydrocarbons 25.3  
    Oxygenated Sesquiterpenes 47.5  
    Fatty acids 2.3  
    Diterpenes 2.9  
    Alkanes 10.2  
    Total 91.9  

RRI  relative retention indices; Trace (<0.1%), %calculated from FID data; Identification method based on the relative retention indices (RRI) of authentic compounds on the HP innowax column; MS, identified on the basis of computer matching of the mass spectra with those of the wiley and mass finder libraries and comparison with literature data

According to the LC-MS/MS analyses, 16 compounds (Figure 1) were determined in the methanol extract of N. cilicica. The main compounds of the extract were determined as 5-caffeoylquinic acid, luteolin glucoside, luteolin glucuronide, apigenin glucoside and apigenin glucuronide. Luteolin and apigenin were also determined as aglycons (Table II).

Figure 1: HPLC chromatogram of Nepeta cilicica methanol extract at 280 nm

Table II: Phenolic compounds of the N. cilicica methanol extract

Rt [M-H]-m/z Fragments Identified as
5.7 353 191, 173 1-caffeoylquinic acid
7.5 315 153 Protocatechuic acid hexoside
9.2 353 191, 179, 173 5-Caffeoylquinic acid
11.5 337 191, 173 5-Coumaroylquinic acid
13.0 448 287, 151, 135 Eriodictiol glucoside
13.2 447 285, 151, 133 Luteolin glucoside
13.6 461 357, 285, 151, 133 Luteolin glucuronide
15.1 431 269, 240, 151, 117 Apigenin glucoside
15.7 445 269, 175, 113 Apigenin glucuronide
16.3 359 197, 161 Rosmarinic acid
18.3 593 323, 269, 151, 117 Apigenin derivative
18.9 487 269, 217, 113 Apigenin derivative
20.0 277 259, 233, 203 Unknown
21.0 285 133 Luteolin
23.4 277 177, 151 Naringenin
23.9 269 151, 117 Apigenin
RRI relative retention indices; Trace (<0.1%), %calculated from FID data; Identification method based on the relative retention indices (RRI) of authentic compounds on the HP innowax column; MS, identified on the basis of computer matching of the mass spectra with those of the wil ey and mass finder libraries and comparison with literature data

Biological activity

The extract demonstrated weak DPPH radical scavenging activity at the concentration of 0.31± 0.05 mg/mL (Gallic acid was 0.0025 ± 0.0002 mg/mL). Total phenol content was determined as 53.17 mg GAE/g extract.

According to antimicrobial assay results essential oil and methanol extract showed weak to strong antimicrobial effects against 24 different pathogenic Candida and Gram positive and negative bacterial strains. The essential oil demonstrated strong activity against food pathogens Gram positive Bacillus subtilis and Listeria monocytogenes at the concentrations of 94 and 187 µg/mL (MIC) respectively (Table III). The strain of ATCC-750 C. tropicalis was inhibited by the essential oil having a MIC value of 47 µg/mL. The essential oil demonstrated moderate to weak inhibitory effects against all other tested pathogens between the concentrations of 3,000 to 375 µg/mL (MIC).

Table III: Antibacterial activity of N. cilicica essential oil and methanol extract (MIC, µg/mL)

Bacteria Sources Nc1 Nc2 St-1 St-2
Pseudomonas aeruginosa ATCC 27853 3000 1500 16 4
Enterobacter aerogenes NRRL 3567 3000 3000 8 1
Proteus vulgaris NRRL B-123 750 94 1 4
Bacillus cereus NRRL B-3711 375 187 2 4
Bacillus subtilis NRRL B-4378 94 94 1 1
Serratiamarcescens NRRL B-2544 3000 3000 16 4
E. coli ATCC 8739 3000 3000 2 1
Salmonella typhimurium ATCC 14028 3000 3000 1 1
Staphylococcus aureus ATCC43300 750 47 1 8
E. coli O157:H7 RSSK 234 3000 3000 1 1
Listeria monocytogenes ATCC 19111 187 90 1 2
S. epidermidis ATCC 14990 750 187 1 1
Nc1: Essential oil of N. cilicica; Nc2: Methanol extract of N. cilicica; St-1: Ampicillin; St-2: Chloramphenicol

According to broth dilution tests, methanol extract obtained from flowering aerial parts of the N. cilicica, demonstrated better antimicrobial effects rather than the essential oil. The methanol extract strongly inhibited the growing of Proteus vulgaris, B. cereus, B. subtilis, Staphylococcus aureus and Listeria monocytogenes at the concentrations of 94, 187, 94, 47 and 90 µg/ml. The essential oil and methanol extract almost showed same effects against Candida strains. Similar to essential oil, C. tropicalis was inhibited by the methanol extract at the concentration of 47 µg/mL (Table IV).

Table IV: Anticandidal activity of N. cilicica essential oil and methanol extract (MIC, µg/mL)

Candida strain Sources Nc1 Nc2 St-1 St-2
C. albicans ATCC 10231 375 750 0.5 0.1
C. utilis NRRL Y-900 375 187 0.3 0.1
C. krusei NRRL Y-7179 750 750 0.5 0.3
C. zeylanoides NRRL Y-1774 750 750 0.5 0.1
C. parapsilosis NRRL Y- 12696 375 187 0.5 0.1
C. glabrata ATCC 2001 750 1500 1 0.1
C. glabrata ATCC 66032 375 1500 1 0.1
C. tropicalis ATCC 1369 750 750 0.5 0.1
C. albicans ATCC® 24433 750 750 0.5 0.1
C. tropicalis ATCC 750 47 47 1 0.1
C. parapsilosis ATCC 22019 750 750 0.5 0.1
C. krusei ATCC 6258 750 750 1 0.3
Nc1: Essential oilof N. cilicica; Nc2: Methanol extract of N. cilicica; St-1: Amphotericin-B; St-2: Ketoconazole

Discussion

Essential oil composition of the Nepeta species has been varying according to the growing region, environmental conditions and soil type, which they are generally divided into two groups nepetalactone containing and nepetalactoneless as reported in literature data. Apart from mono- and sesquiterpenes in essential oils, diterpenes, triterpenes, iridoids and their glucosides, were reported as major constituents of Nepeta species (Asgarpanah et al., 2014; Formisano et al., 2013; Sharma and Cannoo 2013; Başer, 2000; Skaltza et al., 2000).

In accordance with our results, sesquiterpenes were the major compounds (73%) of N. cilicica oil.  According to a few previous works on N. cilica essential oil, sesquiterpenes such as spathulenol (15.1%), hexadecanoic acid (14%) and γ-cadinene (5.5%) were found as main compounds (Formisano et al., 2013). In the other study β-caryophyllene oxide (41%), β-caryophyllene (15.7%) and α-copaene (2.7%) were reported as main components of the N. cilicica essential oil similar to our results (Kökdil et al., 1999).

To the best of our knowledge, this is the first study about phenolic content of N. cilicica. According to LC-MS/MS analyses, 16 compounds were identified. The hydroxybenzoic acid derivatives (compounds 1-4) were identified using the identification keys of Clifford (Clifford et al. 2003). Caffeoylquinic acids are the ester of caffeic acid and (−)-quinic acid. Caffeoylquinic derivatives (chlorogenic acids) present characteristic molecular ion peak at m/z 353 [M - H] ¯. Compounds 1 and 3 were identified as 1-O-caffeoylquinic acid, 5-O-caffeoylquinic acid respectively. Both of the compounds showed a base peak at m/z 191 ([quinic acid-H]−) discrimination were done according to the retention times where 1-O-caffeoylquinic acid elute earlier than 5-O-caffeoylquinic acid. MS fragmentation of compound 4 showed a molecular ion peak at m/z 337 and a base peak ion at m/z 191, being characterized as 5-p-coumaroylquinic acid. Compound 2 showed characteristic MS fragmentation of gentisic acid hexoside and protocatechuic acid hexoside with its pseudo molecular ion peak at m/z 315. Further fragmentation of m/z 315 resulted in an ion at m/z 153 because of the loss of a glucose moiety. Protocatechuic and gentisic acid differing only in their UV spectra characteristic (gentisic acid presents UV max at 330 nm while protocatechuic acid shows UV max at 260nm). Low UV intensity at 330 nm was let us to identify the compound 2 protocatechuic acid hexoside. Compound 14 showed pseudo molecular ion peak at m/z 285 [M-H]- and identified as luteolin. Confirmation of compound 14 with authenthicluteolin standard. Compound 6 and 7 presented an aglycon ion as luteolin. Compound 6 showed a molecular ion peak at m/z 447 [M-H]¯ and its MS/MS spectrum presented deprotonated luteolinaglycon ion at m/z 285 due to loss of a glucose unit (162 amu). Compound 7 presented a pseudo molecular ion peak at m/z 461 which fragmented a product ion at m/z 285 by the loss of a glucuronic acid moiety (-176 amu). So, the compound 6 and 7 were identified as luteolin glucoside and luteolin glucuronide respectively. In a similar way, compound 16 identified as apigenin and compound 8 and compound 9 were identified as apigenin glucoside and apigenin glucuronide respectively. Compound 11 and compound 12 was not fully identified but determined as apigenin derivative. Compound 10 was determined as rosmarinic acid with its molecular ion peak at m/z 359 [M-H]- and further fragmentation was observed at m/z 197 due to the elimination of a caffeoyl moiety. The confirmation of compound 10 was done using the standard rosmarinic acid. Apigenin glucoside, apigenin glucuronide, luteolin glucoside and 5-caffeoylquinic acid were determined as the major compounds of the extract.

Total phenol content was determined as 53.17 mg GAE/g extract. In different study Nepeta total phenol content was determined as 2.1- 23.1 mg GAE/g extract (Kraujalis, et al. 2011). We calculated the highest total phenol content similar to previously calculated in N. catarica, N. bulgaricum, N. transcaucasia (Kraujalis et al. 2011) and N. praetervisa (Fareed et al. 2013).

The essential oil and the crude methanolic extract of N. cilicica were evaluated for their antibacterial and anticandidal properties for the first time here. The methanol extract found more active than essential oil against all tested bacteria species. Except C. tropicalis both essential oil and the methanol extract did not show remarkable effects pathogen Candida panel. According to all antibacterial results, both essential oil and the crude methanolic extract were slightly more active against Gram positive bacteria.

The study mainly focused on the bioactivity of the essential oil and total methanolic extract of N. cilicica used as food as well as medicine. Sesquiterpenes were found as major compounds of the essential oil where the caffeoylquinic acid, luteolin and apigenin derivatives were found in the extract. The essential oil showed strong inhibitory effects especially against food pathogen bacteria B. subtilis and Listeria monocytogenes. The total extract has stronger activity against all tested bacteria species. With the remarkable antimicrobial properties and phenolic content, N. cilicica total extract may be considered as a potential food preservation additive after further analysis.

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Published
2017-06-20

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