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SUPPLEMENTARY MATERIAL

Chemical composition of essential oils and hydrosols from fresh flowers of Cerasus subhirtella and Cerasusserrulatafrom East China

Gaoming Leia, LonghuWangb*, XuesongLiub and AnyunZhanga*

aDepartment of Chemical and Biological Engineering, Zhejiang University, No. 38 Zheda Road, Hangzhou 310027, China

bDepartment of Chinese Medicine Sciences and Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, China

*Corresponding authors. E-mail: wang2000@zju.edu.cn; zhangay@zju.edu.cn

Essential oils and hydrosols from fresh flowers of Cerasus subhirtella (Miq.)Sok.andCerasusserrulata (Lindl.) London fromEast China were analysed by gas chromatography and gas chromatography-mass spectrometry for the first time. The major components of essential oils fromC. subhirtella and C.serrulata were benzaldehyde (31.2% and 42.1%, respectively), tricosane (23.1% and 27.7%, respectively) and pentacosane (23.2% and 19.0%, respectively). The main constituents of hydrosol volatiles from C. subhirtella and C.serrulata were benzaldehyde (67.5% and 64.3%, respectively) and mandelonitrile (12.5% and 12.4%, respectively). Benzaldehyde was the key component of the essential oils, while benzaldehyde as well as mandelonitrile was the principal compound of the hydrosols.

Keywords:Cerasus subhirtella; Cerasusserrulata; essential oil; hydrosol; benzaldehyde; mandelonitrile

1. Experimental

1.1.Plant materials and chemicals

Fresh flowers of Cerasus subhirtella (Miq.)Sok.andCerasusserrulata (Lindl.) London were collected during late March and early April2013, from the campus of Zhejiang University, Hangzhou, China (7 m altitude, 3018Nlatitude and 12005Elongitude). The species were identified by Associate Professor JuanhuaXu, Department of Chinese Medicine Sciences and Engineering, Zhejiang University. Voucher specimens (HZU60009157 for C. subhirtella; HZU60036168 for C.serrulata) were deposited at Herbarium of Zhejiang University.After collection, the fresh flowers were stored at 5C for a few hours prior to hydrodistillation.The moisture content of fresh materials was 80% as determined by toluene distillation (Chinese Pharmacopoeia Commission 2010).

Alkane standard solution (C7-C30) was supplied bySupelco Analytical (Bellefonte, PA, USA). Other reagents were of analytical grade.

1.2. Isolation of essential oils

Fresh flowers(1000 g)were soaked in aqueous sodium chloridesolution(2000 mL)and hydrodistilled in a Clevenger-type apparatus (Clevenger 1928) for 3 h to obtain the essential oil. The oil ratesoffresh flowers of C. subhirtella and C.serrulata were 0.035% and 0.030% (v/w, based on fresh weight), respectively.The essential oil was diluted with hexane, dried over anhydrous sodium sulphate and filtered. It was then stored at -5C prior to analysis.

1.3. Isolation of hydrosol volatiles

Fresh flowers (500 g) immersed in aqueous solution of sodium chloride (1250 mL), were hydrodistilled to yieldthe hydrosol (500 mL). The experiment was carried out in duplicate and hydrosols obtained were combined (1000 mL). The hydrosol was then extracted with diethyl ether in presence of sodium chloride. The ether layer collected was dried over anhydrous sodium sulphate, filtered and concentrated to obtain the volatile oil. The oil contents of hydrosols of C. subhirtella and C.serrulata were 0.064% and 0.058% (w/v), respectively.The volatile oil of hydrosol was then stored at -5C and diluted with diethyl ether prior to analysis.

1.4. Gas chromatography and gas chromatography-mass spectrometry

Gas chromatography (GC) was performed using an Agilent 6890N GC equipped with a flame ionization detector (FID). The chromatograph was fitted with a HP-5ms column (30 m 0.32 mm, 0.25 m film thickness, Agilent technologies, USA). The oven temperature was kept at 60C for 2 min, raised to 240C at 4C/min, and maintained at 240C for 10 min. Temperatures of the injector and the detector were 260C and 290C, respectively. The injection volumewas 1.0 L with a split ratio of 20:1. The carrier gas was helium at a flow rate of 1 mL/min.

Gas chromatography-mass spectrometry (GC-MS) was carried out using an Agilent 6890N GC coupled with a 5975 mass selective detector (MSD) and fitted with a HP-5ms column (30 m0.32 mm, 0.25 m film thickness, Agilent). The temperature program was the same as that for GC-FID. Injector temperature,260C; injection volume, 1.0 L; split ratio, 20:1;carrier gas, helium; flow rate, 1 mL/min. MSD conditions: transfer line temperature, 290C; ion source temperature, 230C;electron energy, 70 eV; mass scan range, 50-550 amu.

1.5. Component identification

Identification was based on mass spectra and retention indices (Van den Dool&Dec Kratz 1963). The mass spectra were matched with the NIST 05a library (on Agilent MSD ChemStation). Retention indices determined relative to a series of n-alkanes were compared with literature data (NIST 2011). The percentage amounts of individual components were calculated based on GC-FID peak areas.Chemical composition and chemical classes were given in Table S1 and Figure S1, respectively.

Compounds 1 and 9 were recognised as key aroma and active components of the essential oils and hydrosols, respectively. Compound 1, RI 958 (HP-5ms);GC-MS, 70 eV, m/z (%): 106 (100, M+), 105 (100, [M-H]+), 77 (100, C6H5+), 51 (57, C4H3+). The mass spectrum was shown in Figure S2. It was identified as benzaldehyde (Mori et al. 1994).Compound 9, RI 1323 (HP-5ms); GC-MS, 70 eV, m/z (%): 133 (80, M+), 132 (56, [M-H]+), 116 (29, [M-OH]+), 115 (41, [M-H2O]+), 106 (65, [M-HCN]+), 105 (100, C6H5CO+), 77 (91, C6H5+), 51 (43, C4H3+). The mass spectrum was given in Figure S3. This compound was identified as mandelonitrile (Mori et al. 1994).

References

Chinese Pharmacopoeia Commission. 2010. Chinese Pharmacopoeia. Vol. 1.Beijing: China Medical Science Press; Appendix 52-53.

ClevengerJF.1928.Apparatus for the determination of volatile oil. J Am Pharm Assoc. 17: 345-349.

Mori N, Kuwahara Y, Yoshida T, Nishida R. 1994. Identification of benzaldehyde, phenol and mandelonitrile from Epanerchodusjaponicus Carl(Polydesmida: Polydesmidae) as possible defense substances. ApplEntomol Zool. 29: 517-522.

NIST: Chemistry WebBook [Internet]. 2011. Gaithersburg: National Institute of Standards and Technology;[Cited 2014 Mar15]. Available from: http://webbook.nist.gov/chemistry/

Van den Dool H, Dec Kratz P. 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J Chromatogr A. 11: 463-471.

Captions

Table S1. Chemical composition (%) of essential oils and hydrosols fromC. subhirtella and C.serrulata fresh flowers.

Figure S1. Major chemical classes of essential oils and hydrosolsfromC. subhirtella and C.serrulata (EO, essential oil; Hds, hydrosol).

Figure S2.Mass spectrum of compound 1 (GC-MS, 70 eV).

Figure S3.Mass spectrum of compound 9 (GC-MS, 70 eV).

Table S1. Chemical composition (%) of essential oils and hydrosols fromC. subhirtella and C.serrulata fresh flowers.

No.a

Componentsb

RIexpc

RIlitd

Essential oils

Hydrosols

C. subhirtella

C.serrulata

C. subhirtella

C.serrulata

1

benzaldehyde

958

961

31.2

42.1

67.5

64.3

2

decane

1000

1000

0.7

-

-

-

3

benzyl alcohol

1030

1035

-

-

1.2

1.3

4

linalool

1100

1098

-

-

0.5

0.3

5

nonanal

1104

1102

1.7

tr

1.0

0.7

6

coumaran

1218

1223

-

-

3.7

1.8

7

undecanal

1307

1305

2.0

tr

1.0

1.0

8

4-vinylguaiacol

1310

1314

1.6

tr

3.8

2.7

9

mandelonitrile

1323

-

-

-

12.5

12.4

10

methyl cinnamate

1379

1378

-

-

-

2.5

11

coumarin

1428

1432

-

-

4.4

1.8

12

4,11-dimethyltetradecane

1463

1462

2.1

tr

1.4

1.5

13

tridecanal

1510

1510

tr

-

-

-

14

hexahydrofarnesyl acetone

1843

1845

1.0

0.9

0.4

0.4

15

nonadecane

1900

1900

tr

0.6

-

-

16

heneicosane

2100

2100

1.1

1.7

-

0.4

17

tricosane

2300

2300

23.1

27.7

1.5

4.9

18

tetracosane

2400

2400

1.2

1.1

-

-

19

pentacosane

2500

2500

23.2

19.0

1.0

3.1

20

heptacosane

2700

2700

8.8

5.2

-

0.7

21

methyl lignocerate

2725

2731

tr

tr

-

-

22

nonacosane

2900

2900

2.2

1.2

-

-

total identified

99.9

99.5

99.9

99.8

group composition

hydrocarbons

62.4

56.5

3.9

10.6

oxygenate