Bangladesh J Pharmacol. 2012; 7: 114-119.

DOI:10.3329/bjp.v7i2.10815

| Research | Article |

Comparison of terpene components from flowers of Artemisia annua

Zhan-nan Yang1,2, Shi-qiong Zhu1 and Zheng-wen Yu1

1School of Life Sciences, Guizhou Normal University, Guiyang, Guizhou, 550 001, Peoples Republic of China; 2Key Laboratory for Information System of Mountainous Area and Protection of Ecological Environment of Guizhou Province, Guizhou Normal University, Guiyang, Guizhou 550 001, Peoples Republic of China.

Principal Contact

Abstract

Terpene constituents of essential oils obtained by steam distillation from Artemisia annua flowers at the pre-, full- and post-flowering stage was investigated by gas chromatography(GC) and gas chromatography-mass spectrometric detector (GC-MS). The aim was to evaluate change the biosynthesis pathway of terpenes at different flowering stages. The samples studied showed that main components of essential oils were monoterpenes hydrocarbons (48.11%) and oxygenated monoterpenes (41%) in the pre-flowerig, oxygenated monoterpenes (35.59%) and sesquiterpenes hydrocarbons (49.99%) in the full-flowerig, and oxygenated monoterpenes (29.59%), sesquiterpenes hydrocarbons (32.18%) and oxygenated sesquiterpenes (25.27%) in the post-flowering, respectively. The relative content of monoterpenes decreased from pre-flowerig to post-flowerig, while that of sesquiterpenes increased. The results indicated that the biosynthesis pathway of terpenes might be changed at different flowering at stages, while the change of content and composition of terpenes might be a self-adaptation of A. annua.


Introduction

Artemisia annua L. is an annual herb native of China, where it has been used in the treatment of fever and malaria for many centuries. Many secondary metabolites of terpene peroxides were isolated from the plant, such as artemisia ketone, artemisinic alcohol, arteannuin B and myrcene hydroperoxide (Bertea et al.,2005; Brown et al., 2003). The most famous terpene peroxide is Artemisinin, which chemical structure is an amorphane-type sesquiterpene endoperoxide, and it has become an important plant-derived compound in the treatment of the chloroquine-resistant and cerebral malarias (Klayman, 1985). The essential oils, another important composition of A. annua, have been subjected to extensive chemical study. It was reported that the oils contained artemisia ketone, 1,8-cineole and camphor as key components. Significant variations in the percentage occurrence of different constituents have also been reported. The percentage of artemisia ketone, 1, 8-cineole and camphor were reported to vary from 0.0-63.0%, 1.5-31.5% and 5.0-20.0%, respectively, Other major components reported were α-, β-pinene, borneol, carvacrol, thymol, myrcene, limonene, camphene, copaene, β-caryophyllene, α-terpineol, α-, β- and γ-elemene, sabinine, α-guaiene, caryophyllene, caryophyllene oxide, germacrene-D and so on (Fabien et al.,2002; Neetu et al., 2002; Perazzo et al., 2003; Soylu et al., 2005; Rasooli et al., 2003; Flora et al., 2004; Ma, et al.,2007; Divya et al.,2007; Hashemi et al., 2007).

Chongqing, China, shares eighty percent of A. annua around the world, where an A. annua GAP Cultivation Demonstration Site has been built for three years. But systemic study about the essential oils of the herb has not been previously mentioned in literature. The effect of flowering on terpenes content of A.annua flower essential oils at pre-,full- and post-flowering stage was investigated. In the present paper we report the analytical results of the essential oils at different flower developing stages.


Materials and Methods

A. annua cultivar Wuling-3938 plants were grown in the Artemisia GAP Cultivation Demonstration Site of Holleypharm, Chongqing, China. The flowers were harvested at pre-, full- and post-flowering stage in September to November 2007 on a same plant. The flowers were separated from other capitula organs, leaves and stem of A. annua, and identified by Mr Rongchang Luo of Holley Natural Resource Exploiture Co. Ltd, Chongqing, China and deposited in the Herbarium, College of Bioengineering, Chongqing University, Chongqing, China.

Shade-dried plant materials (50.0 g) were hydro-distilled separately in Clevenger-type equipment for 4h. The oils were collected and dried over anhydrous sodium sulfate and stored in a refrigerator at 4ºC for analysis. GC analyses were performed using a Shimadzu GC-2010 gas chromatograph equipped with an FID and an HP-5 fused silica column (30 m×0.32 mm i.d., 0.25 μm film thickness) with a 5% phenyl-substituted methylpolysiloxane phase. The oven temperature was programmed at 40ºC for 4 min and then increased to 240ºC at a rate of 4ºC /min. Injector and detector temperatures were 250ºC and 265ºC, respectively. The carrier gas, helium (99.999%), was adjusted to a linear velocity of 43 cm/sec. The essential oil samples were diluted 5-fold, and 1 μL of a diluted solution was injected into the GC/MS in the split mode with a split ratio of 1/20.

MS analyses were performed using a Shimadzu MS-QP2010 with ionization energy of 70 eV, a scan time of 0.5 sec and a mass range of 33–450 amu. The percentages of compounds were calculated by the area normalization method without considering response factors. The components of the oil were identified by comparison of their mass spectra with those of the spectrometer database using the NIST147 mass spectral database and also with those of authentic compounds. The identifications were confirmed by comparison of the fragmentation patterns and Retention index with those reported in the literature (Divya, et al.2007a; Flora, et al., 2007; Divya, et al., 2007b). The retention index was found with a standard mixture of C8 to C22 compounds under chromatography conditions, consistent with those of the chromatography conditions of the samples analyzed.


Results and Discussion

The flower essential oils were obtained from A. annua at pre-,full-and post-flowering stage with 2.21%, 1.42% and 1.25% yield (relative to dried weight),respectively. Three oils were pale yellow, and the results of the analysis of the essential oils are given in Table I.

A total of sixty-three compounds were identified in three essential oil samples. The oils contain mainly monoterpenes and sesquiterpenes, representing 96.21%, 98.88% and 99.27% of the oils in A. annua at pre-, full- and post-flowering stage, respectively. onoterpenes hydrocarbons and oxygenated monoterpenes content was greater in the preliminary florescence oil, they were 48.11% and 41.00%. The main compounds in the oil were β-myrcene (37.71%), 1,8-cineole (16.11%) and camphor (14.97%). The oxygenated monoterpenes and sesquiterpenes hydrocarbons were dominants in the flourishing florescence oil (35.59% and 49.99%). The oil contained predominantly caryophyllene (19.40%), germacrene D (18.10%), camphor (15.84%), 1,8-cineole (10.60%),(Z)-β-farnesene (9.43%). The terminal florescence oil contains mainly oxygenated monoterpenes (29.59%), sesquiterpenes hydrocarbons (32.18%) and oxygenated sesquiterpenes (25.27%). The major onstituents identified in the oil were camphor (16.62%), caryophyllene (16.27%), β-caryophyllene oxide (15.84%), β-farnesene (9.05%),(-)-spathulenol(7.21%). A significant difference was the absence of oxygenated sesquiterpenes in preliminary florescence, while the monoterpenes hydrocarbons were lacked in the terminal florescence oil.

It is reported (Liu et al.,1988) that the inflorescence oil of A. annua from Changchun, China, contained Artemisia ketone (63.10%), 1,8-cineole (1.50%), β-pinene (1.50%) and caryophyllene (1.92%). Another literature (Divya Goel et al. 2007) reported the major compounds of the cultivar A. annua petal oil were trans-sabinol (10.20%), paramentha-1, 4 (8)-dien-3-ol (10.10%) and 1,8-cineole (6.80%). The influence of transplanting time on inflorescence essential oil yield and composition has been studied (Flora Haider et al. 2007). Their result shows that oil yield was found to range from 0.5 to 1.6% (w/v). Camphor (23.30-57.00%), 1,8-cineole (5.60-21.40%) and β-caryophyllene (2.54-8.69%) were key compounds. The percentage occurrence of rest of the compounds was found to vary with different transplanting time. Our results were more similar to that of Flora Haider.

The content of the monoterpenes hydrocarbons were decreased sharply with flower developing, especially for β-myrcene, it from 37.71% in the preliminary florescence oil to 0.20% in the flourishing florescence oil, the compound was lacked in the terminal florescence oil (Figure 1a). The oxygenated sesquiterpenes were increased quickly with flower

Table I
Chemical composition of the flower essential oils at pre-, full- and post-flowering stage of A.annua

No.

TR

RIa

Components

Content ( % )

Pre-flowering

Full-flowering

Post-flowering

1

5.033

816

(3-Methyl-2-oxiranyl)methanol

0.48

2

5.111

820

2-Ethoxypropane

1.70

0.51

3

7.275

928

Origanene

0.25

4

7.45

937

α-Pinene

0.87

5

7.798

955

Camphene

3.05

0.40

6

8.216

976

Sabinene

3.82

0.42

7

8.302

981

2,2-Dimethylhexanal

0.15

8

8.342

983

β-Pinene

1.53

9

8.514

991

β-Myrcene

37.71

0.20

10

8.578

995

Yomogi alcohol

0.66

11

8.59

995

2,3-Dehydro-1,8-cineole

0.56

12

9.09

1019

(+)-4-Carene

0.13

-

13

9.123

1021

No

0.09

14

9.286

1029

No

0.08

15

9.356

1032

Limonene

0.47

16

9.455

1037

1,8-cineole

16.11

10.57

0.28

17

9.881

1057

Artemisia ketone

0.1

0.20

2.43

18

9.945

1060

Tricyclene

0.28

19

9.984

1062

γ-Terpinen

0.34

20

10.27

1076

cis-β-Terpineol

0.44

0.53

21

10.369

1081

No

0.48

22

10.603

1092

5-(2-Methylenecyclopropyl)-1-pentanol

0.70

23

10.79

1100

(3E,5E)-2,6-Dimethyl-3,5,7-octatrien-2-ol

3.99

1.35

2.61

24

10.87

1104

No

0.23

25

10.91

1106

Nonanal

0.39

26

10.93

1107

Plinol C

0.59

0.63

27

11.42

1128

trans-p-Mentha-2,8-dienol

0.24

28

11.69

1140

ND

0.33

0.33

0.44

29

11.77

1143

Ipsdienol

0.36

30

11.9

1149

Pinocarveol

0.33

0.16

0.35

31

11.98

1152

Berbenol

0.23

32

12.08

1157

Camphor

14.97

15.84

16.62

33

12.26

1165

Nerol

0.33

34

12.27

1165

Lavandulol

0.39

35

12.31

1167

(-)-cis-Myrtanol

0.22

36

12.33

1168

Isogeraniol

0.45

0.23

37

12.39

1171

ND

0.21

38

12.52

1176

Myrcenol

0.19

0.44

39

12.62

1180

Borneol

0.46

1.12

3.93

40

12.77

1187

4-Terpineol

0.62

1.16

0.99

41

12.94

1194

iso-Amyl tiglate

0.47

0.48

0.36

42

13.04

1199

1,5-Menthadien-7-ol

0.14

43

13.11

1201

α-Terpineol

1.34

0.33

0.23

Table II
Chemical composition of the flower essential oils at pre-, full- and post-flowering stage of A. annua (Cont.)

No.

TR

RIa

Components

Content ( % )

Pre-flowering

Full-flowering

Post-flowering

44

13.16

1204

Myrtenol

0.48

0.25

45

13.52

1217

trans-3(10)-Caren-2-ol

0.25

0.29

0.51

46

13.95

1234

(E)-3(10)-Caren-4-ol

0.20

47

14.29

1247

(2E)-2,7-Dimethyl-2,6-octadien-1-ol

0.10

0.15

48

14.31

1248

ND

0.20

49

14.59

1259

4,6,6-Trimethylbicyclo[3.1.1]hept-3-en-2- yl acetate

1.60

50

15.12

1279

Nerol acetate

0.21

0.42

51

16.04

1310

Hydroxy-α-terpenyl acetate

0.57

52

18.65

1377

Copaene

1.09

1.44

53

19.21

1392

β-Elemen

1.46

54

20.78

1420

β-Caryophyllene

2.32

19.41

16.27

55

22.36

1445

β-Farnesene

2.57

9.43

9.05

56

22.94

1454

α-Caryophyllene

1.05

57

23.89

1470

Chamigren

1.76

58

24.46

1479

Germacrene D

1.90

18.13

3.96

59

25.49

1495

γ-Elemene

0.31

60

25.68

1498

Germacrene B

0.88

61

33.1

1570

(-)-Spathulenol

1.81

7.21

62

33.63

1575

β-Caryophyllene oxide

2.99

15.84

63

47.66

1681

Aromadendrene oxide-(2)

2.85

2.22

64

62.86

1904

ND

1.35

65

62.97

1907

δ-Cadinol

1.09

66

63.47

1919

(10Z,12Z)-9-Methyl-10,12-hexadecadienyl acetate

1.13

67

64.41

1942

ND

0.45

68

65.18

1961

ND

0.75

69

65.43

1967

n-Hexadecanoic acid

4.37

70

65.85

1977

ND

0.34

71

65.94

1979

9,12,15-Octadecatrienal

0.26

72

70.57

2106

trans-Phytol

0.37

73

71.39

2131

Stearolic acid

0.47

74

76.91

2297

2,6,10,14-Tetramethylheptadecane

0.62

75

Total identified

98.88

99.27

96.21

76

Monoterpenes hydrocarbons

48.11

1.36

77

Oxygenated monoterpenes

41

35.59

29.59

78

Sesquiterpenes hydrocarbons

8.86

49.99

32.18

79

Oxygenated sesquiterpenes

8.74

25.27

80

Fatty acids and aliphatic esters

7.22

TR = retention time; ND = not identified; (-) Not detected; aRI is the Retention index relative to C8-C22 n-alkanes on the HP-5ms column

developing, the(-)-spathulenol and β-caryphyllene oxide were nice examples, both the compounds weren’t detected in the preliminary florescence oil, there were only 1.81% and 2.99% in the flourishing florescence oil, they were added rapidly to 7.21% and 15.84% in the terminal florescence oil (Figure 1).

In general, the oxygenated monoterpenes were declined slowly with flower developing, among major compounds, artemisia ketone, camphor, borneol and trans-3(10)-Caren-2-ol were raised tendency, while 1,8- cineole was declined (Figure 1c). Interestingly, the sesquiterpenes hydrocarbons increased sharply from 8.86% at pre-flowering stage to 49.99% (at the top of content) at full-flowering stage, followly by, which decreased slowly to 32.18% at post-flowering stage. Among of these compounds, the change of β- caryophyllene and β-farnesene was good examples. The content of former from 2.32% added to 19.41%, then decreased to 16.27%; and that of the later from 2.57% added to 9.43%, then decreased to 9.05% (Figure 1d).

As to the change of all terpenes in flower oils of A. annua at different flowering stage, the content of the monoterpenes hydrocarbons and oxygenated monoterpenes were the highest at the pre-flowering stage, and the sesquiterpenes hydrocarbons arrive to the top at the full-flowering stage, while the oxygenated sesquiterpenes in flower oils were peak at the post-flowering phase (Figure 1e).

The present results have shown that the content of terpenes in the A annua oils has close relationship with flower developing. The flowering effect on monoterpenes content of plant oil has been reported elsewhere (Dudai, et al.,1992). So flowering may be change the biosynthesis pathway of terpenes in the A annua flower oil, in turn, the change of content of terpenes in the oils may be the self-adaptation for the physiological phenomenon of flowering.

Click to see

Acknowlegement

This work was financially supported by grants from the National Natural Science Foundation of P.R. China (No.31060056).


References

Bertea CM, F reije JR, Van DWH, Verstappen FW, Perk L, Marquez V, De Kraker JW, Posthumus MA, Jansen BJ, de Groot A, Franssen MC, Bouwmeester HJ. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.Planta Med. 2005; 71: 40-47.

Brown GD, Liang GY, Sy LK. Terpenoids from the seeds of Artemisia annua L. Phytochemistry 2003; 64: 303-23.

Dudai N, Putievsky E, Ravid U, Palevitch D, Halevy AH. Monoterpene content in Origanum syriacum as effected by environmental conditions and flowering. Phys Plant 1992;84: 453–59.

Goel D, Singh V, Ali M, Mallavarupu GR, Kumar S. Essential oils of petal, leaf and stem of the antimalarial plant Artemisia annua. J Nat Med. 2007a; 61: 187-91.

Goel D, Singh V, Ali M, Mallavarupu GR, Kumar S Composition of the essential oil from the root of Artemisia annua. J Nat Med. 2007b; 61: 458-61.

Haider F, Dwivedi P, S Singh, Naqvi AA, Bagchi G. Influence of transplanting time on essential oil yield and composition in Artemisia annua plants growth under the climatic conditions of sub-tropical north India. Flavour Fragrance J. 2004; 19: 51-53.

Hashemi P, Abolghasemi MM, Fakhari AR, Ebrahimi SN, Ahmadi S Hydrodistillation-solvent microextraction and GC–MS identification of volatile components of Artemisia aucheri. Chromatographia 2007; 66: 283-86.

Iraj Ri, Rezaee MB, Moosavi ML, Kamkar J. Microbial sensitivity to and chemical properties of the essential oil of artemisia annua L. J Essent Oil Res. 2003; 15: 59-62.

Jain N, Srivastava SK, Aggarwal KK, Kumar S, Syamasundar KV, Jain N, Kumar S. Essential oil composition of Artemisia annua L. 'Asha' from the plains of Northern India. J Essent Oil Res. 2002; 14: 305-07.

Juteau F, Masotti V, Bessière JM, Dherbomez M, Viano J Antibacterial and antioxidant activities of Artemisia annua essential oil. Fitoterapia 2002; 73: 532-35.

Klayman DL. Qinghaosu (Artemisinin): An antimalarial drug from China. Science 1985; 228: 1049-55.

Liu Q, Yang ZY, Deng ZB, Sa GH, Wang XJ. Preliminary analysis on chemical constituents of essential oil from inflorescence of A. annua L. Acta Botanica Sinica. 1988; 30: 223-25

Ma CF, Wang HH, Lu X, Li HF, Liu B, Xu GW. Analysis of Artemisia annua L. volatile oil by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. J Chromatogr. A 2007; 1150: 50–53.

Perazzo FF, Carvalho JCT, Carvalho JE, Rehder VLG. Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L. Pharmacol Res. 2003;48: 497-502.

Soylu EM, Yigitbas H, Tok FM, Soylu S, Kurt S, Baysal Ö, Kaya AD. Chemical composition and antifungal activity of the essential oil of Artemisia annua L. against foliar and soil- borne fungal pathogens, Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz. 2005; 112: 229-39.