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Natural Product Research: Formerly Natural Product Letters


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Essential oil composition and antioxidant activity of different extracts of Nepeta betonicifolia C.A. Meyer and Nepeta saccharata Bunge
Peyman Salehi , Ali Sonboli , Pooneh Khaligh & Fateme Mirzajani
a a a b a

Department of Phytochemistry , Medicinal Plants and Drugs Research Institute, Shahid Beheshti University , G.C., Evin , Tehran 1983963113 , Iran
b

Department of Biology , Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. , Evin , Tehran 1983963113 , Iran Published 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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Natural Product Research Vol. 26, No. 8, April 2012, 736743

Essential oil composition and antioxidant activity of different extracts of Nepeta betonicifolia C.A. Meyer and Nepeta saccharata Bunge
Peyman Salehia*, Ali Sonbolib, Pooneh Khaligha and Fateme Mirzajania
a Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran 1983963113, Iran; bDepartment of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran 1983963113, Iran

(Received 11 July 2010; final version received 30 December 2010)

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Aerial parts essential oil of Nepeta betonicifolia and N. saccharata were obtained by hydrodistillation and analysed by GC-FID and GC-MS. Thirty-three and eighteen components represented 97.9% and 98.2% of the total oils identified, respectively. Main compounds of the oil of N. betonicifolia 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%). The principal constituents of the essential oil of N. saccharata were found to be 4a ,7 ,7a -nepetalactone (66.9%), germacrene D (12.9%), sabinene (6.5%) and trans-caryophyllene (3.3%). The radical scavenging capacity (RSC) of methanol extracts and chloroform, butanol and water subfractions of aerial parts of N. betonicifolia and N. saccharata were evaluated by using DPPH, FRAP and ABTS assays. TPC of each extract was measured using FolinCiocalteau. The antioxidant activity of the butanolic subfractions of both plants was higher than other extracts examined. Keywords: N. betonicifolia C.A. Meyer; N. saccharata Bunge; essential oil; antioxidant activity

1. Introduction Nepeta is a genus of Lamiaceae family with 250 species which are found in Asia, Europe and North Africa. The genus is represented by 67 species in flora of Iran (Sajjadi, 2005). Essential oils of Nepeta species mainly contain different isomers of nepetalactones as the main components. Antibacterial, antifungal, antiviral and opioid analgesic activities have been attributed to nepetalactones (Ghannadi, Aghazari, Mehrabani, Mohagheghzadeh, & Mehregan, 2003). Some Iranian Nepeta species have been used in Iranian folk medicines (Ibrahim & Ali, 2007) and used for treatment of various sicknesses, such as nervous, respiratory and gastrointestinal diseases (Ghannadi et al., 2003). The oxidation process is one of the most important routes for producing free radicals in food, drugs and living systems. Catalase and hydroperoxidase enzymes which convert hydrogenperoxide and hydroperoxides into non-radical forms and
*Corresponding author. Email: p-salehi@sbu.ac.ir

ISSN 14786419 print/ISSN 14786427 online 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2010.551752 http://www.tandfonline.com

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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-ethylbenzothiazoline-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 of N. 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, & zcan, 2003). In the first report on the oil composition of Tumen, 2001; Senatore & O 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 independent from sample polarity for screening of many samples for radical scavenging

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Table 1. Chemical composition of the essential oils of N. betonicifolia and N. saccharata. RI (DB-5) RI (DB-1) N. betonicifolia GC Area% N. saccharata GC Area% 934 974 980 1023 1028 1031 1041 1119 1158 1180 1192 1199 1208 1234 1236 1288 1357 1377 1389 1424 1444 1457 1482 1497 1518 1560 1568 1586 1597 1608 1622 1654 1659 0.1 0.3 2.4 0.3 1.8 3.2 0.5 0.5 1.1 0.8 2.0 1.7 5.2 1.1 0.4 2.0 2.0 0.6 42.0 2.7 2.9 2.7 6.0 2.5 1.0 4.0 1.0 1.7 2.1 0.4 1.2 0.4 1.3 97.9 5.4 62.7 19.1 6.7 4.0 938 971 1010 1023 1034 1084 1161 1322 1331 1374 1376 1385 1405 1437 1452 1479 1511 1575 tr 6.5 tr 2.4 1.3 tr 0.3 0.2 0.9 66.9 tr 0.2 3.3 3.0 tr 12.9 tr 2.0 98.2 10.3 68.2 19.4 0.3

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Compound -Pinene Sabinene -Pinene -Terpinene p-Cymene -3-Carene Limonene 1,8-Cineole (Z)- -Ocimene (E)- -Ocimene -Terpinene -Campholenal Pinocarvone Terpinen-4-ol Cryptone Myrtenal Perilla aldehyde Triplal Cumin aldehyde Carvone Dihydroedulan 4a ,7 ,7a -Nepetalactone 4a ,7 ,7a -Nepetalactone 4a ,7 ,7a -Nepetalactone -Copaene -Elemene 4a ,7 ,7a -Nepetalactone (E)-Caryophyllene (Z)- -Farnesene (E)- -Farnesene -Humulene Germacrene D Bicyclogermacrene -Cadinene -Cadinene 1-nor-Bourbonanone 1,5-Epoxy salvia-4(14)-ene Caryophyllene oxide Spathulenol Patchoulene -Eudesmol A-Muurolol Bulnesol Total identified Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others

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

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Table 2. Antioxidant activity and total phenol content of N. betonicifolia and N. saccharata. ABTS m mol Total phenol mg gallic Torolox /g acid /g extract extract 356.2 9.3 353.8 10.0 236.7 6.8 136.8 9.1 406.4 27.0 330.8 5.0 277.1 8.5 307.3 16.3 19.3 1.7 25.3 4.8 36.1 4.1 26.5 0.9 9.0 1.8

Extract 0.001 mg mL1 N. betonicifolia Butanol Water Methanol Chloroform N. saccharata Butanol Water Methanol Chloroform BHT

DPPH FRAP IC50 mg mL1 m mol Fe2/g extract 20.1 2.7 40.3 2.1 53.1 1.0 269.2 36.5 85.7 0.9 76.5 2.1 179.6 7.7 324.7 123.7 18.4 1.0 1344.6 1.5 348.8 13.8 343.8 8.1 153.3 3.3 200.5 3.8 113.8 0.1 29.1 1.9 25.9 4.6

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Note: Results are as Mean SD of three repetitions.

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 butanol4water4methanol4chloroform. 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 be hypothesised 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 Azarbaijan Province, 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 (100 g) were hydrodistilled for 3 h using a Clevenger-type glass apparatus.

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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 40 C. Water was added and the whole was partitioned using chloroform and n-butanol, consecutively. Thus methanol 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 4 C.

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 60 C to 250 C at a rate of 5 C min1, then held at 250 C 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 250 C and 280 C, 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 0.25 mm). The temperature program was the same as GC. Transfer line temperature was 250 C. 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.

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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 (90 mM). The mixtures were shaken for 1 h, and the absorbance of resulting solutions were 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).   Ablank Asample 1 RSC % 100 Ablank 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. IC50 value is the effective concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis. BHT was used as a control (Gulluce et al., 2007).

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FRAP assay was based on the reduction of yellow Fe3 - TPTZ (2,4,6-tri-2-pyridyl1,3,5-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 (20 mM) in a ratio of 10 : 1 : 1, at 37 C. To perform the assay, 2 mL of FRAP reagent, was added to various concentrations of samples or standard, and incubated at 37 C 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).

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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. 300 mL 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 of N. 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 available antioxidant. 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 of this work.

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