Z LebensmUnters Forsch (1992) 195:99-103
Zeitschrift for
9 Springer-Verlag 1992
Original paper Antioxidative constituents of RosmaHnus officinalis and Salvia officinalis II. Isolation of carnosic acid and formation of other phenolic diterpenes Karin Schwarz and Waldemar Ternes Institute of Food Science, University ofHannover, Wunstorfer Strasse 14, W-3000 Hannover 91, Federal Republic of Germany Received February 25, 1992 Received February 25, 1992
Antioxidativ wirksame Inhaltsstoffe aus Rosmarinus officinalis und Salvia officinalis II. Isolierung yon Carnosolsiiure und Bildung anderer phenolischer Diterpene Zusammenfassung. Das phenolische Diterpen Carnosolsfiure ist die Hauptsubstanz, aus der sich durch Oxidation Artefakte mit ?- und 6-Lactonstruktur in Extrakten yon Rosmarinus offieinalis und Salvia officinalis bilden. Bisher war es nur m6glich, Carnosolsfiure durch hydrierende Lactonspaltung von Carnosol zu gewinnen. Es wurde eine semipr~iparative HPLC-Methode entwickelt, die es erm6glicht, Carnosols/iure neben anderen phenolischen Diterpenen zu isolieren. Die isolierten Substanzen wurden anhand von 13C_Kernresonanz (NMR)-, 1H-NMR-, Massen- und IR-Spektroskopie identifiziert. Die Umwandlung von Carnosols/iure und Carnosol zu anderen phenolischen Diterpenen wurde mittels HPLC untersucht. Summary. The phenolic diterpene carnosic acid appears to be the main substance for general oxidation leading to artifacts with 7- or 6-1actone structure in extracts of Rosmarinus officinalis and Salvia officinalis. Until now it was only possible to prepare carnosic acid by hydrogenolysis of carnosol. A semipreparative HPLC method has been developed isolating carnosic acid among other phenolic diterpenes. The separated substances were identified by 13C-nuclear magnetic resonance (NMR), 1H-NMR, mass and IR spetroscopy. Conversion of carnosic acid and carnosol to other phenolic diterpenes was investigated by HPLC.
ble for the strong antioxidative properties of rosemary (Rosmarinus officinalis) and sage (Salvia officinalis), as compared to other herbs [1, 2] (Fig. 1). So far, only ,derivatives of carnosic acid have been isolated since this compound is considered rather unstable. To obtain carnosic acid the lactone ring of carnosol is cleaved using catalytically excited hydrogen and the hydroxyl group at C-7 is reduced [3]. Carnosic acid, constituting 0.35% of the dry matter of rosemary leaves [4], has been postulated to be the precursor of phenolic diterpenes with 6- or ?-lactone structure [4]. In the present paper substances separated by means of HPLC [5] are characterized, demonstrating that most of the phenolic diterpenes known for rosemary and sage could be separated using this method. Establishing patterns of degradation of carnosic acid and carnosol in polar and non-polar system by means of HPLC rendered possible an addition to the mechanisms of the reactions postulated [4].
.oJL . NO 1
/
,16
12
HOOCZ~ I. II Cornosic ocid
Rosrnonol
Ho.~O~z. O~ Cornoso[
7-Methyl-epirosmanol N'~
HO OH
Introduction Above all, the phenolic diterpenes carnosol, carnosic acid, rosmanol, epi- and isorosmanol are held responsiCorrespondence to: W. Ternes
Epirosrnonol Fig. ]. Formation of phenolic diterpenes with 7- and (%lactone structures
100
Materials and methods Chemicals. These were all obtained from Merck (Darmstadt, FRG): methanol (no. 6009), double distilled water, citric acid monohydrate (no. 244), sodiumdihydrogenphosphate (NaHePO~ x HzO; no. 6346), and potassiumdihydrogenphosphate (KH2PO4; no. 4873). Isolation of phenolic diterpenes from extracts. The extract was dissolved in methanol/water (8:2) and filtered followed by semipreparative HPLC using: two pumps (Type 64, Knauer, Bad Homburg, FRG), 2.5 ml/min; injection volume, 500 Ixl; column, 250 m m x 8 mm ID, ODS Hypersil 10 I~m (Knauer); UV detection at 230 nm; a spectrophotometer Uvikon 720 LC (Kontron, Hamburg, FRG) eluent A, methanol/water/2 tool citric acid (35:65:0,5); eluent B, methanol/2 mol citric acid (100:0,5). The gradient used is shown in Table 1. Fractions of approx. 4 ml eluent were collected in 2 m l 0.2mol phosphate buffer (NaH2PO 4 x H 2 0 / K H 2 P O 4 ; 16.2:83.8) and with 5 ml distilled water was added. Eluent separation and fraction purification was done using RP-18 cartridges (Macherey & Nagel, Diiren, FRG) and included conditioning of the cartridge with 5 ml distilled water, rinsing twice with 2.5 ml distilled water after separation of the eluent and collecting of the fraction in methanol (3 x 1 ml). The whole procedure was repeated for purifying individual fractions. Methanol was then evaporated at 50 ~ C under vacuum and the remaining solvent was removed by lyophilization at - 4 0 ~ C and a vacuum of < i mbar.
Determination of the phenolic diterpenes. Method noted in [5]; using UV detection at 230 nm.
Extraction of fresh leaves of rosemary and sage. Comminuted leaves of rosemary or sage (0.15 g) were extracted in a centrifuge tube in 1.5 ml methanol using a homogenizer (Ultra Turrax; Janke & Kunkel, Staufen, FRG). The extract was centrifuged for 1 min at 3000 rpm (Centrifuge Labofuge I; Heraeus, Osterode, FRG) and the supernatant diluted with methanol (1:10) before analysis by means of HPLC.
Spectrometry. Mass spectra were obtained using a Finnigan MAT 44 S (Bremen, FRG) instrument. 1H-nuclear magnetic resonance (NMR) spectra were measured in methanol and acetone using a Comp. Aspect 3000 Cryomagnet at 300 MHz (Bruker, Reinstetten, FRG). ~aC-NMR spectra were measured in methanol using a
Table 1. Elution gradient programme
Time (rain)
Eluent A (%)
Eluent B (%)
0-2.5 4-6.5 7.5 9.5
50 28 58 50
50 72 42 50
Table 2. 1H-nuclear magnetic resonance
(NMR) data of rosmanol, epirosmanol, 7-methyl-epirosmanol, carnosol and earnosic acid; n.d., Position of the signal was not determined; m, multiplet; d, doublet; s, singlet; dd, double doublet; br, broad
Comp. Aspect 3000 Cryomagnet at 75 MHz (Bruker). IR spectra were measured in Nujol using a Perkin Elmer 1420 (Oberlingen, FRO).
Results and discussion Seven substances were isolated using semipreparative HPLC followed by a solid phase extraction to separate the solvent and purify the compounds.
Substances 1 and 2 By correlating the values obtained with those cited in literature [6, 7] substances 1 and 2 were identified as rosmanol and epirosmanol, respectively. Both substances revealed molecular ion peaks at m/z 346 and fragments at m/z 330 (+M-O), 314 (330-0), 300 (314-CH2), 284 and 269 in their mass spectra. A maximum at 1755 cm- 1 in the IR spectra of substances 1 and 2 implies the presence of 7-1actone structures. Substance 1 revealed two doublets at 6 4.52 and 4.59 in the 1H-NMR spectrum (Table 2), with a coupling constant of J = 3.3 Hz, which are to be assigned to the C-7 and C-6 protons, while protons H-7 and H-6 in epirosmanol correspond to a broad singlet at ~ 4.71 (2H) [7]. Data of the ~3C-NMR spectra are listed in Table 3.
Substance 3 On the basis of a maximum at 1750 cm- ~ in the IR spectrum a 7-1actone structure may also be assumed for substance 3. The mass spectrum of substance 3 showed a molecular ion peak at m/z 360 and fragments at m/z 328 (~M-O-CH3) , 284 (328-CO2) and 269 (284-CH3). The singlet at 6 3.63 for a methoxy group, two doublets at 4.81 and 4.38 for protons H-6 and H-7 (J = 3.2 Hz), and a singlet at 6 6.72 for the aromatic proton H-14 in the 1HNMR spectrum correlated with the data for a 7-t-ether of the 7-1actone of carnosic acid, as described by Brieskorn and D6mling [3], which is the equivalent of 7methyl-epirosmanol. The 13C-NMR spectrum showed clear correlations with the spectra of rosmanol and epirosmanol, differing only in a signal of the 7-methoxy group a 6 58.5.
Proton No.
Rosmanol
Epirosmanol
7-Methylepirosmanol
Carnosol
Carnosic acid
H - 1 (a) H - 1 (fl) H- 5 H - 6 (~) H - 6 (fl) H - 7 (c0 H - 7 (fl) H-14 H-15 O-Me
2.0 m (n. d.) 2.26 s 4.52 d -
(n. d.) (n. d.) 2.04 4.71 s (br) 4.71 s (br) 6.96 3.30 m -
(n. d.) (n. d.) 2.20 4.38 d 4.81 d 6.72 s 3.21 m 3.63 s
2.41 2.65 1.56 2.06 1.67 5.30 _ 6.55 3.08 -
2.03 3.53 d 1.78 1.50 2.40 m t ~2"78 dd 6.45 s 3.20 m -
4.59 d 6.85 s 3.21 m -
dd m m d s m
101 Table 3. 13C-NMR data of rosmanol, epirosmanol, 7-methyl-epirosmanol, carnosol and carnosic acid; n.d., position of the signal was not determined due to interference from the solvent; ( )*, position of signal from Inatani and Nakatani [6]
Carbon No.
Rosmanol
Epirosmanol
7-Methylepirosmanol
Carnosol
Carnosic acid
C- I C- 2 C- 3 C- 4 C- 5 C- 6 C- 7 C- 8 C- 9 C-10 C-11 C 12 C-13 C-14 C-15 (2-16 C-17 C-18 C-19 C-20 Me-O
28.7 20.2 39.5 32.3 51.7 79.9 69.2 129.4 125.0 (n. d.; 47.7)* 145.4 143.5 137.6 120.5 27.9 22.9 23.2 31.8 22.5 181.0 -
28.5 20.1 39.1 32.6 (n. d.) 81.0 70.9 130.5 124.5 (n. d.) 145.1 143.3 137.5 118.9 28.0 22.9 23.2 31.9 22.3 180.9 -
28.6 20.2 39.4 32.4 52.3 79.7 76.1 127.6 125.1 (n. d.) 145.3 143.8 137.4 120.9 27.9 22.9 23.1 32.0 22.4 180.9 58.5
30.1 20.0 42.2 35.4 (n. d.; 47.0)* 30.1 79.7 133.3 123.0 (n. d.; 49)* 144.6 144.2 136.1 112.5 27.9 23.1 23.2 32.1 20.1 179.2 -
36.6 20.45 43.5 35.1 50.9 56.0 32.9 13(I.8 127.2 (n. d.) 145.2 142.0 133.9 118.6 28.0 23.0 23.1 33.3 21.9 181.7 -
a
Substance 4 Substance 4 h a d the same retention time in the c h r o m a t o g r a m as carnosol, which was used as a reference. A maxim u m at 1710 c m - 1 , typical for 6-1actones, and the mass spectrum with the molecular ion p e a k at m/z 330 and fragments at m/z 286 ( + M - C O z ) , 284 (286-Hz), 271 (286CH3) and 269 (284-CH3) were in g o o d correlation with the d a t a f o u n d in literature for carnosol [6,8]. In the 1HN M R spectrum o f carnosol (see Fig. 3a) a doublet appeared at 6 5.3 (2H) for H-7 whereas the signals o f H-6 (~) a n d H-6(fl) were shifted to a higher field a n d a p p e a r at 6 2.06 and 6 1.67, respectively [6]. In c o n t r a s t to r o s m a nol, epirosmanol and 7-methyl-epirosmanol the 13CN M R spectrum o f substance 4, the d a t a o f which corres p o n d completely with those obtained for carnosol [5], showed a shift o f the C-6 to 6 30.1, while C-7 emerged in tile same area (6 79.7).
l m
.
C i
Substance 5 Substance 5 was identified as carnosic acid. A molecular ion peak at m/z 332 and fragments at m/z 286 and 271 suggest that CO, H z O a n d CH3 are cleaved off (Fig. 2). I n the I R spectrum o f carnosic acid a m a x i m u m in the
lOO
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2 o
L~.U .r
'
i6
. . . . . . . . . . .
........... 7.0
332 100
1,50
200
250
300
Fig. 2. Mass spectrum of carnosic acid; (INT) intensity
m/z
350
6.0
,
. . . . . .
5.0
4.0
5.0
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ii
I t . . . .
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1
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t
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I,.4.,..I,,i........,....,....,...., ! 2.0 1.0 O~
ppm
i O' I I 13.
Fig.3a-c. 1H-Nuclear magnetic resonance (NMR) of carnosol (a), carnosic acid (b) and two-dimensional 1H-NMR ofcarnosic acid (c)
102 area of lactones (1710-1750 cm -1) is missing, instead there is a maximum at 1645 cm- 1 for a C = O group. The maximum at 3540 cm-1 is to be assigned to C-20 OHgroup [9]. In virtue of the missing lactone structure signals of protons H-6 and H-7 in the IH-NMR spectrum (Fig. 3b) are shifted to higher field [9, 4]. The two dimensional 1H-NMR of diacetylcarnosol represents a coupling of the H-5 (5 1.78) with H-7 and H-6, the signals of which are within the range of 5 2.3 to 2.8 [10]. In the two dimensional 1H-NMR spectrum for carnosic acid (Fig. 3b, c) H-6(/?), which is deshielded due to three dimensional proximity of the C = O group (C-10), appears as a multiplet at 6 2.4, which couples with the signal at 5 1.5 for H-6 (~) and H-5 (5 1.78). H-7 (~, #) appear as a double doublet at 5 2.78 (2H), coupling with H-5 and H6 (#), in the ~H-NMR spectrum. A doublet at 5 3.53 (1H) corresponds with H-l(/?), the double doublet at 5 2.8 with the protons at the C-7 position, the multiplet at 5 2.4 with H-6(e) and the doublet at 5 1.78 corresponds with H-5. One multiplet, at 5 3.3, originates from H-15 and the singlet at 5 6.45 from an aromatic proton on the C-14. The double doublet at 5 3.53 (1H) probably corresponds to H-l(/?), which is deshielded by the carboxyl group and couples with signals at 5 1.1, 1.5 and 5 2.05.
The 13C-NMR spectra (Fig. 4) of carnosol and carnosic acid exhibit strong similarities. The different position of the signal of C-7 is obvious. In carnosol C-7 appears at 5 79.7 with almost a higher intensity than C-20, whereas in carnosic acid C-7 is shifted because of the missing lactone structure to 5 33, obtained by C-Hcorrelated spectrum. The relatively weak signal at 5 79.7 may be interpreted as impurity or the appearing of the first products of lactonization. That carnosic acid may be isolated in its native form under the conditions described above is verified by the data obtained for substance 5.
Substances 6 and 7 For substances 6 and 7 the mass spectrum showed molecular ion peaks at m/z 318 and m/z 346, respectively. A detailed analysis of structure of the compounds has not yet been completed. The behaviour of these substances under different thermal conditions [11] and the presence in extracts of rosemary and sage (Fig. 5) justifies a separate report on the structures.
Raw rosemary extract a 7 'ft~
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To investigate which phenolic diterpenes were present in leaves of rosemary and sage, respectively, comminuted leaves were extracted in methanol for 30 s, centrifuged and analysed immediately. As shown in Fig. 5, carnosic acid represents the main constituent of the extract. Isolated carnosic acid is soluble in methanol, pentane or hexane and may thus be extracted from the leaves of rosemary and sage using a number of solvents of different polarities. The amount of carnosol was approx. 10% of the content of carnosic acid, and the 5-1actones rosmanol, epirosmanol and 7-methyl-epirosmanol were present in minute quantities only, suggesting that phenolic diterpenes with a lactone structure are formed upon contact with air during extraction. This correlates with observations reported on by other authors, isolating carnosic acid as the only phenolic diterpene from acetylated rosemary extract [4].
Fig. 4a, b. t3C-NMR ofcarnosol (a) and carnosicacid (b) Conversion of carnosic acid
Ext (X=230n~
i
2
i
4
i
i
6
i
I
8
J
i
10
i
I
12
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i
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D
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Fig. 5. Chromatogram of methanolic rosemary extract; 3 7-methylepirosmanol; 4 carnosol; 5 carnosic acid; 6 substance 6; 7 substance
7
Wenkert et al. put forward the hypothesis that 6- and ?lactones are formed via the tautomerism of benzoquinone to the semiquinone of carnosic acid [4]. Brieskorn and D6mling observed that carnosic acid changed to carnosol in methanol, and 7-methyl-epirosmanol cristallized from methanol after complete separation of carnosol [3]. The degradation of isolated carnosic acid in methanol is shown in Fig. 6. The amount ofcarnosic acid decreased while carnosol was formed, which in turn decomposed to form 7-1actones, rosmanol, epirosmanol and 7-methylepirosmanol.
103 Area (%)
the same extent in freshly prepared raw extracts and those commercially available [11] implies the natural abundance of both compounds in leaves of rosemary and sage.
100
80
Influence of a i r and daylight
1
2
3
4
5
6
7
Days
Fig. 6. Degradation ofcarnosic acid in methanol. (11)Rosmanol; (N) carnosol; (N) epirosmanol; (@)carnosic acid; (t~)7-methyl-epirosmanol A r e a (%) 120
ijj
100 8o
6O 4.0 20 0
[
] I ] i
]L
1
2
3
4
5
For another experiment isolated carnosic acid was dissolved in methanol and exposed to daylight. Nitrogen was passed through the solution to exclude the presence of oxygen (02) from air. A parallel control solution was saturated with air in the dark. Analysis after 5 h revealed that carnosic acid remained stable under exposure to light without Oz, while addition of air led to the formation of carnosol, rosmanol, epirosmanol and 7-methylepirosmanol. These observations show that the degradation of carnosic acid to 6- and 7-1actones is caused by the presence of O2. Using HPLC, however, it is possible to restrict the time samples are exposed to air to an extent allowing the isolation of carnosic acid sensitive to O2 directly, together with lactones.
Li
B 6
Acknowledgements. The authors are grateful to H.-C. Krebs, W.A. _ L
7
9 8
9
Day8
Heidmann, A. Btithe, Chemisches Institut of the Tier~irztliche Hochschule Hannover, for measuring NMR spectra and mass spectra. They are also indebted to P. Hammann, H6chst, and L. Busam, Procter and Gamble, for valuable discussions.
Fig. 7. Degradation of carnosol in methanol. (I) Rosmanol; (@Icarnosol; (N) epirosmanol; ([]) 7-methyl-epirosmanol References
Conversion of carnosol Looking at the degradation of isolated carnosol in methanol (Fig. 7) it becomes clear that the 7-1actones, rosmanol, epirosmanol and 7-methyl-epirosmanol are formed from carnosol, and not via the intermediates quinone and semiquinone of the-carnosic acid (see Fig. 1). The formation of rosmanol from carnosol was also observed in an N a O H solution containing methanol [10]. The formation of 7-1actones carrying a hydroxyl group at C-7, from carnosol remains unsolved for the time being. Substances 6 and 7 appeared neither on the degradation of carnosol nor of carnosic acid, suggesting that these compounds are not the residues of degradation of either. The fact that substances 6 and 7 are present to
1. Inatani R, Nakatani N, Fuwa H (1983) Agric Biol Chem 47:521-528 2. Brieskorn CH, D6mling HJ (1969) Z Lebensm Unters Forsch 141:10-16 3. Brieskorn CH, D6mling HJ (1969) Arch Pharm 302:641-649 4. Wenkert E, Fuchs A, McChesney JD (1965) J Org Chem 30:2931-2940 5. Sehwarz K, Ternes W (1992) Z Lebensm Unters Forsch 195:95-98 6. Inatani R, Nakatani N, Fuwa H, Seto H (1982) Agric Biol Chem 46:1661-1666 7. Nakatani N, Inatani R (1984) Agric Biol Chem 48:2081--2085 8. Wu JW, Lee MH, Ho CT, Chang SS (1982) JAOCS 59:339345 9. D6mling HJ (1968) Dissertation, Universit/it Wiirzburg 10. Weinreich B (1989) Dissertation, Universit/it Mtinchen 1). Schwarz K, Ternes W, Schmauderer E (1992) Z Lebensm Unters Forsch 195:104-107
Zeitschrift for
9 Springer-Verlag 1992
Original paper Antioxidative constituents of RosmaHnus officinalis and Salvia officinalis II. Isolation of carnosic acid and formation of other phenolic diterpenes Karin Schwarz and Waldemar Ternes Institute of Food Science, University ofHannover, Wunstorfer Strasse 14, W-3000 Hannover 91, Federal Republic of Germany Received February 25, 1992 Received February 25, 1992
Antioxidativ wirksame Inhaltsstoffe aus Rosmarinus officinalis und Salvia officinalis II. Isolierung yon Carnosolsiiure und Bildung anderer phenolischer Diterpene Zusammenfassung. Das phenolische Diterpen Carnosolsfiure ist die Hauptsubstanz, aus der sich durch Oxidation Artefakte mit ?- und 6-Lactonstruktur in Extrakten yon Rosmarinus offieinalis und Salvia officinalis bilden. Bisher war es nur m6glich, Carnosolsfiure durch hydrierende Lactonspaltung von Carnosol zu gewinnen. Es wurde eine semipr~iparative HPLC-Methode entwickelt, die es erm6glicht, Carnosols/iure neben anderen phenolischen Diterpenen zu isolieren. Die isolierten Substanzen wurden anhand von 13C_Kernresonanz (NMR)-, 1H-NMR-, Massen- und IR-Spektroskopie identifiziert. Die Umwandlung von Carnosols/iure und Carnosol zu anderen phenolischen Diterpenen wurde mittels HPLC untersucht. Summary. The phenolic diterpene carnosic acid appears to be the main substance for general oxidation leading to artifacts with 7- or 6-1actone structure in extracts of Rosmarinus officinalis and Salvia officinalis. Until now it was only possible to prepare carnosic acid by hydrogenolysis of carnosol. A semipreparative HPLC method has been developed isolating carnosic acid among other phenolic diterpenes. The separated substances were identified by 13C-nuclear magnetic resonance (NMR), 1H-NMR, mass and IR spetroscopy. Conversion of carnosic acid and carnosol to other phenolic diterpenes was investigated by HPLC.
ble for the strong antioxidative properties of rosemary (Rosmarinus officinalis) and sage (Salvia officinalis), as compared to other herbs [1, 2] (Fig. 1). So far, only ,derivatives of carnosic acid have been isolated since this compound is considered rather unstable. To obtain carnosic acid the lactone ring of carnosol is cleaved using catalytically excited hydrogen and the hydroxyl group at C-7 is reduced [3]. Carnosic acid, constituting 0.35% of the dry matter of rosemary leaves [4], has been postulated to be the precursor of phenolic diterpenes with 6- or ?-lactone structure [4]. In the present paper substances separated by means of HPLC [5] are characterized, demonstrating that most of the phenolic diterpenes known for rosemary and sage could be separated using this method. Establishing patterns of degradation of carnosic acid and carnosol in polar and non-polar system by means of HPLC rendered possible an addition to the mechanisms of the reactions postulated [4].
.oJL . NO 1
/
,16
12
HOOCZ~ I. II Cornosic ocid
Rosrnonol
Ho.~O~z. O~ Cornoso[
7-Methyl-epirosmanol N'~
HO OH
Introduction Above all, the phenolic diterpenes carnosol, carnosic acid, rosmanol, epi- and isorosmanol are held responsiCorrespondence to: W. Ternes
Epirosrnonol Fig. ]. Formation of phenolic diterpenes with 7- and (%lactone structures
100
Materials and methods Chemicals. These were all obtained from Merck (Darmstadt, FRG): methanol (no. 6009), double distilled water, citric acid monohydrate (no. 244), sodiumdihydrogenphosphate (NaHePO~ x HzO; no. 6346), and potassiumdihydrogenphosphate (KH2PO4; no. 4873). Isolation of phenolic diterpenes from extracts. The extract was dissolved in methanol/water (8:2) and filtered followed by semipreparative HPLC using: two pumps (Type 64, Knauer, Bad Homburg, FRG), 2.5 ml/min; injection volume, 500 Ixl; column, 250 m m x 8 mm ID, ODS Hypersil 10 I~m (Knauer); UV detection at 230 nm; a spectrophotometer Uvikon 720 LC (Kontron, Hamburg, FRG) eluent A, methanol/water/2 tool citric acid (35:65:0,5); eluent B, methanol/2 mol citric acid (100:0,5). The gradient used is shown in Table 1. Fractions of approx. 4 ml eluent were collected in 2 m l 0.2mol phosphate buffer (NaH2PO 4 x H 2 0 / K H 2 P O 4 ; 16.2:83.8) and with 5 ml distilled water was added. Eluent separation and fraction purification was done using RP-18 cartridges (Macherey & Nagel, Diiren, FRG) and included conditioning of the cartridge with 5 ml distilled water, rinsing twice with 2.5 ml distilled water after separation of the eluent and collecting of the fraction in methanol (3 x 1 ml). The whole procedure was repeated for purifying individual fractions. Methanol was then evaporated at 50 ~ C under vacuum and the remaining solvent was removed by lyophilization at - 4 0 ~ C and a vacuum of < i mbar.
Determination of the phenolic diterpenes. Method noted in [5]; using UV detection at 230 nm.
Extraction of fresh leaves of rosemary and sage. Comminuted leaves of rosemary or sage (0.15 g) were extracted in a centrifuge tube in 1.5 ml methanol using a homogenizer (Ultra Turrax; Janke & Kunkel, Staufen, FRG). The extract was centrifuged for 1 min at 3000 rpm (Centrifuge Labofuge I; Heraeus, Osterode, FRG) and the supernatant diluted with methanol (1:10) before analysis by means of HPLC.
Spectrometry. Mass spectra were obtained using a Finnigan MAT 44 S (Bremen, FRG) instrument. 1H-nuclear magnetic resonance (NMR) spectra were measured in methanol and acetone using a Comp. Aspect 3000 Cryomagnet at 300 MHz (Bruker, Reinstetten, FRG). ~aC-NMR spectra were measured in methanol using a
Table 1. Elution gradient programme
Time (rain)
Eluent A (%)
Eluent B (%)
0-2.5 4-6.5 7.5 9.5
50 28 58 50
50 72 42 50
Table 2. 1H-nuclear magnetic resonance
(NMR) data of rosmanol, epirosmanol, 7-methyl-epirosmanol, carnosol and earnosic acid; n.d., Position of the signal was not determined; m, multiplet; d, doublet; s, singlet; dd, double doublet; br, broad
Comp. Aspect 3000 Cryomagnet at 75 MHz (Bruker). IR spectra were measured in Nujol using a Perkin Elmer 1420 (Oberlingen, FRO).
Results and discussion Seven substances were isolated using semipreparative HPLC followed by a solid phase extraction to separate the solvent and purify the compounds.
Substances 1 and 2 By correlating the values obtained with those cited in literature [6, 7] substances 1 and 2 were identified as rosmanol and epirosmanol, respectively. Both substances revealed molecular ion peaks at m/z 346 and fragments at m/z 330 (+M-O), 314 (330-0), 300 (314-CH2), 284 and 269 in their mass spectra. A maximum at 1755 cm- 1 in the IR spectra of substances 1 and 2 implies the presence of 7-1actone structures. Substance 1 revealed two doublets at 6 4.52 and 4.59 in the 1H-NMR spectrum (Table 2), with a coupling constant of J = 3.3 Hz, which are to be assigned to the C-7 and C-6 protons, while protons H-7 and H-6 in epirosmanol correspond to a broad singlet at ~ 4.71 (2H) [7]. Data of the ~3C-NMR spectra are listed in Table 3.
Substance 3 On the basis of a maximum at 1750 cm- ~ in the IR spectrum a 7-1actone structure may also be assumed for substance 3. The mass spectrum of substance 3 showed a molecular ion peak at m/z 360 and fragments at m/z 328 (~M-O-CH3) , 284 (328-CO2) and 269 (284-CH3). The singlet at 6 3.63 for a methoxy group, two doublets at 4.81 and 4.38 for protons H-6 and H-7 (J = 3.2 Hz), and a singlet at 6 6.72 for the aromatic proton H-14 in the 1HNMR spectrum correlated with the data for a 7-t-ether of the 7-1actone of carnosic acid, as described by Brieskorn and D6mling [3], which is the equivalent of 7methyl-epirosmanol. The 13C-NMR spectrum showed clear correlations with the spectra of rosmanol and epirosmanol, differing only in a signal of the 7-methoxy group a 6 58.5.
Proton No.
Rosmanol
Epirosmanol
7-Methylepirosmanol
Carnosol
Carnosic acid
H - 1 (a) H - 1 (fl) H- 5 H - 6 (~) H - 6 (fl) H - 7 (c0 H - 7 (fl) H-14 H-15 O-Me
2.0 m (n. d.) 2.26 s 4.52 d -
(n. d.) (n. d.) 2.04 4.71 s (br) 4.71 s (br) 6.96 3.30 m -
(n. d.) (n. d.) 2.20 4.38 d 4.81 d 6.72 s 3.21 m 3.63 s
2.41 2.65 1.56 2.06 1.67 5.30 _ 6.55 3.08 -
2.03 3.53 d 1.78 1.50 2.40 m t ~2"78 dd 6.45 s 3.20 m -
4.59 d 6.85 s 3.21 m -
dd m m d s m
101 Table 3. 13C-NMR data of rosmanol, epirosmanol, 7-methyl-epirosmanol, carnosol and carnosic acid; n.d., position of the signal was not determined due to interference from the solvent; ( )*, position of signal from Inatani and Nakatani [6]
Carbon No.
Rosmanol
Epirosmanol
7-Methylepirosmanol
Carnosol
Carnosic acid
C- I C- 2 C- 3 C- 4 C- 5 C- 6 C- 7 C- 8 C- 9 C-10 C-11 C 12 C-13 C-14 C-15 (2-16 C-17 C-18 C-19 C-20 Me-O
28.7 20.2 39.5 32.3 51.7 79.9 69.2 129.4 125.0 (n. d.; 47.7)* 145.4 143.5 137.6 120.5 27.9 22.9 23.2 31.8 22.5 181.0 -
28.5 20.1 39.1 32.6 (n. d.) 81.0 70.9 130.5 124.5 (n. d.) 145.1 143.3 137.5 118.9 28.0 22.9 23.2 31.9 22.3 180.9 -
28.6 20.2 39.4 32.4 52.3 79.7 76.1 127.6 125.1 (n. d.) 145.3 143.8 137.4 120.9 27.9 22.9 23.1 32.0 22.4 180.9 58.5
30.1 20.0 42.2 35.4 (n. d.; 47.0)* 30.1 79.7 133.3 123.0 (n. d.; 49)* 144.6 144.2 136.1 112.5 27.9 23.1 23.2 32.1 20.1 179.2 -
36.6 20.45 43.5 35.1 50.9 56.0 32.9 13(I.8 127.2 (n. d.) 145.2 142.0 133.9 118.6 28.0 23.0 23.1 33.3 21.9 181.7 -
a
Substance 4 Substance 4 h a d the same retention time in the c h r o m a t o g r a m as carnosol, which was used as a reference. A maxim u m at 1710 c m - 1 , typical for 6-1actones, and the mass spectrum with the molecular ion p e a k at m/z 330 and fragments at m/z 286 ( + M - C O z ) , 284 (286-Hz), 271 (286CH3) and 269 (284-CH3) were in g o o d correlation with the d a t a f o u n d in literature for carnosol [6,8]. In the 1HN M R spectrum o f carnosol (see Fig. 3a) a doublet appeared at 6 5.3 (2H) for H-7 whereas the signals o f H-6 (~) a n d H-6(fl) were shifted to a higher field a n d a p p e a r at 6 2.06 and 6 1.67, respectively [6]. In c o n t r a s t to r o s m a nol, epirosmanol and 7-methyl-epirosmanol the 13CN M R spectrum o f substance 4, the d a t a o f which corres p o n d completely with those obtained for carnosol [5], showed a shift o f the C-6 to 6 30.1, while C-7 emerged in tile same area (6 79.7).
l m
.
C i
Substance 5 Substance 5 was identified as carnosic acid. A molecular ion peak at m/z 332 and fragments at m/z 286 and 271 suggest that CO, H z O a n d CH3 are cleaved off (Fig. 2). I n the I R spectrum o f carnosic acid a m a x i m u m in the
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200
250
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m/z
350
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Fig.3a-c. 1H-Nuclear magnetic resonance (NMR) of carnosol (a), carnosic acid (b) and two-dimensional 1H-NMR ofcarnosic acid (c)
102 area of lactones (1710-1750 cm -1) is missing, instead there is a maximum at 1645 cm- 1 for a C = O group. The maximum at 3540 cm-1 is to be assigned to C-20 OHgroup [9]. In virtue of the missing lactone structure signals of protons H-6 and H-7 in the IH-NMR spectrum (Fig. 3b) are shifted to higher field [9, 4]. The two dimensional 1H-NMR of diacetylcarnosol represents a coupling of the H-5 (5 1.78) with H-7 and H-6, the signals of which are within the range of 5 2.3 to 2.8 [10]. In the two dimensional 1H-NMR spectrum for carnosic acid (Fig. 3b, c) H-6(/?), which is deshielded due to three dimensional proximity of the C = O group (C-10), appears as a multiplet at 6 2.4, which couples with the signal at 5 1.5 for H-6 (~) and H-5 (5 1.78). H-7 (~, #) appear as a double doublet at 5 2.78 (2H), coupling with H-5 and H6 (#), in the ~H-NMR spectrum. A doublet at 5 3.53 (1H) corresponds with H-l(/?), the double doublet at 5 2.8 with the protons at the C-7 position, the multiplet at 5 2.4 with H-6(e) and the doublet at 5 1.78 corresponds with H-5. One multiplet, at 5 3.3, originates from H-15 and the singlet at 5 6.45 from an aromatic proton on the C-14. The double doublet at 5 3.53 (1H) probably corresponds to H-l(/?), which is deshielded by the carboxyl group and couples with signals at 5 1.1, 1.5 and 5 2.05.
The 13C-NMR spectra (Fig. 4) of carnosol and carnosic acid exhibit strong similarities. The different position of the signal of C-7 is obvious. In carnosol C-7 appears at 5 79.7 with almost a higher intensity than C-20, whereas in carnosic acid C-7 is shifted because of the missing lactone structure to 5 33, obtained by C-Hcorrelated spectrum. The relatively weak signal at 5 79.7 may be interpreted as impurity or the appearing of the first products of lactonization. That carnosic acid may be isolated in its native form under the conditions described above is verified by the data obtained for substance 5.
Substances 6 and 7 For substances 6 and 7 the mass spectrum showed molecular ion peaks at m/z 318 and m/z 346, respectively. A detailed analysis of structure of the compounds has not yet been completed. The behaviour of these substances under different thermal conditions [11] and the presence in extracts of rosemary and sage (Fig. 5) justifies a separate report on the structures.
Raw rosemary extract a 7 'ft~
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To investigate which phenolic diterpenes were present in leaves of rosemary and sage, respectively, comminuted leaves were extracted in methanol for 30 s, centrifuged and analysed immediately. As shown in Fig. 5, carnosic acid represents the main constituent of the extract. Isolated carnosic acid is soluble in methanol, pentane or hexane and may thus be extracted from the leaves of rosemary and sage using a number of solvents of different polarities. The amount of carnosol was approx. 10% of the content of carnosic acid, and the 5-1actones rosmanol, epirosmanol and 7-methyl-epirosmanol were present in minute quantities only, suggesting that phenolic diterpenes with a lactone structure are formed upon contact with air during extraction. This correlates with observations reported on by other authors, isolating carnosic acid as the only phenolic diterpene from acetylated rosemary extract [4].
Fig. 4a, b. t3C-NMR ofcarnosol (a) and carnosicacid (b) Conversion of carnosic acid
Ext (X=230n~
i
2
i
4
i
i
6
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8
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10
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12
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Fig. 5. Chromatogram of methanolic rosemary extract; 3 7-methylepirosmanol; 4 carnosol; 5 carnosic acid; 6 substance 6; 7 substance
7
Wenkert et al. put forward the hypothesis that 6- and ?lactones are formed via the tautomerism of benzoquinone to the semiquinone of carnosic acid [4]. Brieskorn and D6mling observed that carnosic acid changed to carnosol in methanol, and 7-methyl-epirosmanol cristallized from methanol after complete separation of carnosol [3]. The degradation of isolated carnosic acid in methanol is shown in Fig. 6. The amount ofcarnosic acid decreased while carnosol was formed, which in turn decomposed to form 7-1actones, rosmanol, epirosmanol and 7-methylepirosmanol.
103 Area (%)
the same extent in freshly prepared raw extracts and those commercially available [11] implies the natural abundance of both compounds in leaves of rosemary and sage.
100
80
Influence of a i r and daylight
1
2
3
4
5
6
7
Days
Fig. 6. Degradation ofcarnosic acid in methanol. (11)Rosmanol; (N) carnosol; (N) epirosmanol; (@)carnosic acid; (t~)7-methyl-epirosmanol A r e a (%) 120
ijj
100 8o
6O 4.0 20 0
[
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1
2
3
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For another experiment isolated carnosic acid was dissolved in methanol and exposed to daylight. Nitrogen was passed through the solution to exclude the presence of oxygen (02) from air. A parallel control solution was saturated with air in the dark. Analysis after 5 h revealed that carnosic acid remained stable under exposure to light without Oz, while addition of air led to the formation of carnosol, rosmanol, epirosmanol and 7-methylepirosmanol. These observations show that the degradation of carnosic acid to 6- and 7-1actones is caused by the presence of O2. Using HPLC, however, it is possible to restrict the time samples are exposed to air to an extent allowing the isolation of carnosic acid sensitive to O2 directly, together with lactones.
Li
B 6
Acknowledgements. The authors are grateful to H.-C. Krebs, W.A. _ L
7
9 8
9
Day8
Heidmann, A. Btithe, Chemisches Institut of the Tier~irztliche Hochschule Hannover, for measuring NMR spectra and mass spectra. They are also indebted to P. Hammann, H6chst, and L. Busam, Procter and Gamble, for valuable discussions.
Fig. 7. Degradation of carnosol in methanol. (I) Rosmanol; (@Icarnosol; (N) epirosmanol; ([]) 7-methyl-epirosmanol References
Conversion of carnosol Looking at the degradation of isolated carnosol in methanol (Fig. 7) it becomes clear that the 7-1actones, rosmanol, epirosmanol and 7-methyl-epirosmanol are formed from carnosol, and not via the intermediates quinone and semiquinone of the-carnosic acid (see Fig. 1). The formation of rosmanol from carnosol was also observed in an N a O H solution containing methanol [10]. The formation of 7-1actones carrying a hydroxyl group at C-7, from carnosol remains unsolved for the time being. Substances 6 and 7 appeared neither on the degradation of carnosol nor of carnosic acid, suggesting that these compounds are not the residues of degradation of either. The fact that substances 6 and 7 are present to
1. Inatani R, Nakatani N, Fuwa H (1983) Agric Biol Chem 47:521-528 2. Brieskorn CH, D6mling HJ (1969) Z Lebensm Unters Forsch 141:10-16 3. Brieskorn CH, D6mling HJ (1969) Arch Pharm 302:641-649 4. Wenkert E, Fuchs A, McChesney JD (1965) J Org Chem 30:2931-2940 5. Sehwarz K, Ternes W (1992) Z Lebensm Unters Forsch 195:95-98 6. Inatani R, Nakatani N, Fuwa H, Seto H (1982) Agric Biol Chem 46:1661-1666 7. Nakatani N, Inatani R (1984) Agric Biol Chem 48:2081--2085 8. Wu JW, Lee MH, Ho CT, Chang SS (1982) JAOCS 59:339345 9. D6mling HJ (1968) Dissertation, Universit/it Wiirzburg 10. Weinreich B (1989) Dissertation, Universit/it Mtinchen 1). Schwarz K, Ternes W, Schmauderer E (1992) Z Lebensm Unters Forsch 195:104-107
