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Essential oil composition and

antioxidant activity of different

extracts of Nepeta betonicifolia C.A.

Meyer and Nepeta saccharata BungePeyman Salehi

a, Ali Sonboli

b, Pooneh Khaligh

a& Fateme

Mirzajania

aDepartment of Phytochemistry , Medicinal Plants and Drugs

Research Institute, Shahid Beheshti University , G.C., Evin ,

Tehran 1983963113 , Iranb

Department of Biology , Medicinal Plants and Drugs Research

Institute, Shahid Beheshti University, G.C. , Evin , Tehran

1983963113 , IranPublished online: 13 Oct 2011.

To cite this article: Peyman Salehi , Ali Sonboli , Pooneh Khaligh & Fateme Mirzajani (2012)Essential oil composition and antioxidant activity of different extracts of Nepeta betonicifolia C.A.

Meyer and Nepeta saccharata Bunge, Natural Product Research: Formerly Natural Product Letters,

26:8, 736-743, DOI: 10.1080/14786419.2010.551752

To link to this article: http://dx.doi.org/10.1080/14786419.2010.551752

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functions, are known natural antioxidants in the human body. There is a claim to

find more information concerning the antioxidant potential of plant species. It has

been reminded that the antioxidant activity of plants might be due to their phenolic

compounds. Flavonoids are a group of polyphenolic compounds which show free

radical scavenging activity, inhibition of hydrolytic processes and oxidative enzymes

action (Pourmorad, Hosseinimehr, & Shahabimajd, 2006). Several assays have been

used to investigate the antioxidant activity of plants for clinical studies including

DPPH, ferric reducing antioxidant power (FRAP) and 2,2-azino bis (3-ethyl-

benzothiazoline-6-sulphonic acid) (ABTS) (Thaipong, Boonprakob, Crosby,

Cisneros-Zevallos, & Byrne, 2006). The total phenolic content of plants is measured

using the FolinCiocalteu assay (Marinova, Ribarova, & Atanassova, 2005). The

aims of this study were: (i) analysis the chemical composition of the essential oils of

N. betonicifolia and N. saccharata, (ii) investigation of the antioxidant activity of

their extracts with different polarities, (iii) evaluation of the total phenolic contents

of each extract to find a possible relationship between the presence of these

compounds and observed antioxidant activity.

2. Results and discussion

Essential oils of aerial parts of N. betonicifolia and N. saccharata were both isolated

by hydrodistillation in 0.1% (v/w) yield and analysed by GC-FID and GC-MS.

Thirty-three and 18 components represented 97.9% and 98.2% of the total oils

identified, respectively. The oil of N. betonicifolia was dominated by 62.7% of

oxygenated monoterpenes, 19.1% sesquiterpenes, 6.7% oxygenated sesquiterpenes

5.4% monoterpenes and 4.0% other compounds. The oil of N. saccharata was rich in

68.2% oxygenated monoterpenes and followed by 19.4% sesquiterpenes, 10.3%

monoterpenes and 0.3% oxygenated sesquiterpenes. Results are shown in Table 1

where the compounds are listed in order of their elution from DB-5 and DB-1

columns, respectively. Main components of N. betonicifolia oil were

4a,7,7a-nepetalactone (42.0%), germacrene D (6.0%), triplal (5.2%),

1-nor-bourbonanone (4.0%) and 1,8-cineole (3.2%), whereas the main compounds

ofN. saccharata oil were 4a,7,7a-nepetalactone (66.9%), germacrene D (12.9%),

sabinene (6.5%) and trans-caryophyllene (3.3%). The oil components of

N. betonicifolia from Turkey have already been reported (Baser, Ozek, Demirci, &

Tumen, 2001; Senatore & O zcan, 2003). In the first report on the oil composition of

N. betonicifolia from Turkey caryophyllene oxide (39.2%) and spathulenol (4.7%)were found to be the main compounds. The results of the second report

demonstrated that among 89.6% of the total identified compounds, linalool

(40.5%) and 1,8-cineole (20.8%) were the main constituents. Several methods have

been reported to investigate the antioxidant activity of plants extracts, including;

DPPH, FRAP and ABTS assays (Pourmorad et al., 2006). ABTS assay is based on

the antioxidant ability to react with ABTS produced in the assay system. Whereas

FRAP assay measures the reduction of ferric iron Fe3 to ferrous iron Fe2 in the

presence of antioxidants, which are reductants with half-reaction reduction

potentials above Fe3/Fe2 (Biglari, AlKarkhi, & Easa, 2008).

The DPPH assay is described as a simple, rapid and useful method independentfrom sample polarity for screening of many samples for radical scavenging

Natural Product Research 737

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Table 1. Chemical composition of the essential oils of N. betonicifolia and N. saccharata.

No. CompoundRI (DB-5)

GC Area%RI (DB-1)

GC Area%N. betonicifolia N. saccharata

1 -Pinene 934 0.1 938 tr2 Sabinene 974 0.3 971 6.53 -Pinene 980 2.4 4 -Terpinene 1010 tr5 p-Cymene 1023 0.3 6 -3-Carene 1023 2.47 Limonene 1028 1.8 8 1,8-Cineole 1031 3.2 9 (Z)--Ocimene 1034 1.3

10 (E)--Ocimene 1041 0.5 11 -Terpinene 1084 tr12 -Campholenal 1119 0.5 13 Pinocarvone 1158 1.1

14 Terpinen-4-ol 1161 0.315 Cryptone 1180 0.8 16 Myrtenal 1192 2.0 17 Perilla aldehyde 1199 1.7 18 Triplal 1208 5.2 19 Cumin aldehyde 1234 1.1 20 Carvone 1236 0.4 21 Dihydroedulan 1288 2.0 22 4a,7,7a -Nepetalactone 1357 2.0 1322 0.223 4a,7,7a-Nepetalactone 1331 0.924 4a,7,7a -Nepetalactone 1374 66.925 -Copaene 1377 0.6 1376 tr

26 -Elemene 1385 0.227 4a,7,7a -Nepetalactone 1389 42.0 28 (E)-Caryophyllene 1424 2.7 1405 3.329 (Z)--Farnesene 1437 3.030 (E)--Farnesene 1444 2.9 31 -Humulene 1457 2.7 1452 tr32 Germacrene D 1482 6.0 1479 12.933 Bicyclogermacrene 1497 2.5 34 -Cadinene 1511 tr35 -Cadinene 1518 1.0 36 1-nor-Bourbonanone 1560 4.0 37 1,5-Epoxy salvia-4(14)-ene 1568 1.0 38 Caryophyllene oxide 1586 1.7 1575 2.039 Spathulenol 1597 2.1 40 Patchoulene 1608 0.4 41 -Eudesmol 1622 1.2 42 A-Muurolol 1654 0.4 43 Bulnesol 1659 1.3

Total identified 97.9 98.2Monoterpene hydrocarbons 5.4 10.3Oxygenated monoterpenes 62.7 68.2Sesquiterpene hydrocarbons 19.1 19.4Oxygenated sesquiterpenes 6.7 0.3Others 4.0

Note: RI: retention indices on capillary columns relative to C6-C24 n-alkanes.

738 P. Salehi et al.

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activity (Marxen et al., 2007). Table 2 shows results of DPPH, FRAP and ABTS

assays for extracts. Results of N. betonicifolia demonstrated that in all antioxidant

assay methods the activity of the extracts followed the order as buta-

nol4water4methanol4chloroform. The results signified that in all antioxidant

assays, the mechanisms were mainly electron transfer and not hydrogen transfer,

because the butanolic subfraction, that did not have phenolic compounds, showed

higher antioxidant activity and also the antioxidant activity of other extracts were

not directly related to their phenolic content (Pourmorad et al., 2006). It can behypothesised that other components than phenolic compounds like nepetalactones,

terpenes, carotenoids and minerals are responsible for antioxidant activity (Suhaj,

2006). In the case of N. betonicifolia our studies demonstrated that the butanolic

subfraction had the highest antioxidant activity, except for DPPH assay.

3. Experimental

3.1. Plant material

The aerial parts of N. betonicifolia were collected from Iran; West AzarbaijanProvince, Khoy, Qotour, North of Habashe bala, Turgan on 3 June 2008. The aerial

parts of N. saccharata were collected from the TehranGazvin highway, Kavandaj

ShekarnabHajiabad road on 24 May 2006. Voucher specimens were deposited at

the Medicinal Plants and Drugs Research Institute Herbarium of Shahid Beheshti

University (MPH). Herbarium number of N. betonicifolia and N. saccharata are

MPH-1310 and MPH-1045, respectively.

3.2. Isolation of the essential oils

Dried aerial parts of each plant (100g) were hydrodistilled for 3 h using aClevenger-type glass apparatus.

Table 2. Antioxidant activity and total phenol content of N. betonicifolia and N. saccharata.

Extract 0.001 mg mL1DPPH

IC50 mg mL1

FRAPmmol Fe2/g extract

ABTS mmolTorolox /g

extract

Total phenolmg gallic

acid /g extract

N. betonicifoliaButanol 20.1 2.7 1344.6 1.5 356.2 9.3 Water 40.3 2.1 348.8 13.8 353.8 10.0 19.3 1.7Methanol 53.1 1.0 343.8 8.1 236.7 6.8 25.3 4.8Chloroform 269.2 36.5 153.3 3.3 136.8 9.1

N. saccharataButanol 85.7 0.9 200.5 3.8 406.4 27.0 36.1 4.1Water 76.5 2.1 113.8 0.1 330.8 5.0 26.5 0.9Methanol 179.6 7.7 29.1 1.9 277.1 8.5 9.0 1.8Chloroform 324.7 123.7 25.9 4.6 307.3 16.3 BHT 18.4 1.0

Note: Results are as Mean SD of three repetitions.


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3.3. Preparation of plant extracts

Twenty grams of the dried powdered plants materials (aerial parts of N. betonicifolia

and N. saccharata) were extracted using 200 mL methanol for two days. The extracts

were filtered and concentrated under reduced pressure at 40C. Water was added and

the whole was partitioned using chloroform and n-butanol, consecutively. Thusmethanol extract (M) and chloroform (C), butanol (B) and water (W) subfractions

were obtained, respectively. In each partitioning process 10 mL of solvents were used

and each extraction was repeated five times. Eight extracts from N. betonicifolia and

N. saccharata were concentrated under reduced pressure and stored at 4C.

3.4. GC and GC-MS analyses

GC analysis was performed on a Thermoquest-Finnigan Trace GC instrument

equipped with a capillary DB-1 and DB-5 fused silica column (30 m 0.25 mm i. d.,film thickness 0.25 mm). The oven temperature was raised from 60C to 250C at a

rate of 5Cmin1, then held at 250C for 10 min. Nitrogen was used as the carrier gas

at a flow rate of 1.1 mL min1. Split ratio was adjusted at 1/50. The injector and

detector (FID) temperatures were kept at 250C and 280C, respectively. GC-MS

analysis was performed on a Thermoquest-Finnigan Trace GC-MS instrument

equipped with a DB-1 and DB-5 fused silica capillary column (60 m 0.25 mm i.d.,

film thickness 0.25mm). The temperature program was the same as GC. Transfer line

temperature was 250C. Helium was used as the carrier gas at a flow rate of

1.1 mL min1. A quadrupole mass spectrum was scanned over 45465 amu with an

ionising voltage of 70 eV and an ionising current of 150 A.

3.5. DPPH assay

The hydrogen atoms or electrons transfer ability of the corresponding extracts and

some pure compounds were measured from bleaching of purple coloured DPPH

solution. The effect of each extract and subfractions on DPPH radical were

investigated according to the method described elsewhere (Gulluce et al., 2007). In

brief, various concentrations of extracts were added to 4 mL solution of DPPH

(90mM). The mixtures were shaken for 1 h, and the absorbance of resulting solutionswere measured at 517 nm using a Shimadzu 2501 PC UV-Vis spectrophotometer.

Radical scavenging capacity (RSC) was calculated using the following equation;

(Foti, Daquino, & Geraci, 2004; Mimica-Dukic, Bozin, Sokovic, & Simin, 2004).

RSC % 100Ablank Asample

Ablank

1

where Ablank is the absorbance of the control reaction (containing all reagents except

for the test compound), and Asample is the absorbance of the test compound. IC50value is the effective concentration at which DPPH radicals were scavenged by 50%

and was obtained by interpolation from linear regression analysis. BHT was used asa control (Gulluce et al., 2007).


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3.6. FRAP assay

FRAP assay was based on the reduction of yellow Fe3- TPTZ (2,4,6-tri-2-pyridyl-

1,3,5-triazine) to a blue coloured Fe2- TPTZ. The antioxidant potential of the

extracts were investigated against a standard curve of ferrous sulphate (10, 30, 60, 90,

120 ppm). The FRAP reagent was freshly prepared by mixing 100 mL of acetate

buffer (300 mM, pH 3.6), 10 mL of TPTZ solution (10 mM TPTZ in 40 mM HCl),

and 10 mL of FeCl3.6H2O (20mM) in a ratio of 10:1:1, at 37C. To perform the

assay, 2 mL of FRAP reagent, was added to various concentrations of samples or

standard, and incubated at 37C for 5 min. The absorbance of resulting solutions was

measured at 593 nm. The amount of relative absorbance should be within the range

02.0, otherwise, the sample should be diluted. The antioxidant potential of

samples were investigated from a standard curve of FeSO4.7H2O (Kubola &

Siriamornpun, 2008).

3.7. ABTS radical scavenging assay

Using ABTS radical cation is one of the spectrophotometric methods for the

investigation of antioxidant activity. ABTS was produced by reacting ABTS stock

solution (7 mM in water) with 2.45 mM potassium persulphate at a ratio of 1 : 1, in

dark place and room temperature for 1216 h. The resulting solution was diluted

with ethanol to reach pH 7.4, and an absorbance of 0.7(0.02) at 734 nm. A mixture

of 3 mL of this reagent and various concentrations of each extract was added to test

tubes and after 6 min their absorbances were measured at 734 nm. The antioxidant

potential of samples was calculated from a standard curve of Trolox. Results are

reported as mm Trolox/g of extract (Re et al., 1999).

3.8. Total phenol assay

The total phenolic content was investigated using the Folin-Ciocalteu method. 20 mL

of plant extracts (10 g L1) were mixed with 2 mL of distilled water and 100 mL of

Folin-Ciocalteu reagent. 300mL of Na2CO3 solution (7%) was added to the test

tubes after 3 min and thoroughly shaken for 2 h. The resulting solutions absorbances

were measured at 765 nm, using a UV-Vis spectrophotometer. Total phenolic content

was calculated from a standard curve using gallic acid (Slinkard & Singleton, 1977).

4. Conclusions

The essential oils of N. betonicifolia and N. saccharata were analysed by GC and

GC-MS. Although the oil composition of N. betonicifolia has already been reported

from Turkey, the oil of Iranian species shows fundamental differences. The essential

oil analysis of N. saccharata is reported for the first time. Both oils were dominated

by nepetalactones and could be used as a new source of these valuable compounds.

The antioxidant activity of the extracts ofN. betonicifolia and N. saccharata with

different polarities showed that the butanol subfraction of the former had a high

radical scavenging activity near to BHT as a synthetic commercially availableantioxidant. The radical scavenging power of the extracts were not directly related to


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the total phenolic content of the samples and showed that other group of compounds

may be responsible for the observed activities.

Acknowledgement

We are grateful to Shahid Beheshti Universtiy Research Council for financial support ofthis work.

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