Identification of New Compounds from Sage Flowers (Salvia

Article Cite This: J. Agric. Food Chem. 2018, 66, 1843−1853

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Identification of New Compounds from Sage Flowers (Salvia of ficinalis L.) as Markers for Quality Control and the Influence of the Manufacturing Technology on the Chemical Composition and Antibacterial Activity of Sage Flower Extracts Sebastian Gericke,*,† Tilo Lübken,‡ Diana Wolf,∥ Martin Kaiser,§ Christian Hannig,⊥ and Karl Speer*,† †

Food Chemistry, ‡Organic Chemistry, and §Inorganic Chemistry, Technische Universität Dresden, Bergstrasse 66, D-01069 Dresden, Germany ∥ Institute of Microbiology, Technische UniCversität Dresden, Zellescher Weg 20b, D-01217 Dresden, Germany ⊥ Clinic of Operative and Pediatric Dentistry, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstr. 74, D-01307 Dresden, Germany S Supporting Information *

ABSTRACT: Parts of Salvia species such as its flowers and leaves are currently used as a culinary herb and for some medicinal applications. To distinguish the different sage extracts it is necessary to analyze their individual chemical compositions. Their characteristic compounds might be established as markers to differentiate between sage flowers and leaf extracts or to determine the manufacturing technology and storage conditions. Tri-p-coumaroylspermidine can be detected only in flowers and has been described here for Salvia and Lavandula species for the first time. Markers for oxidation processes are the novel compounds salviquinone A and B, which were generated from carnosol by exposure to oxygen. Caffeic acid ethyl ester was established as an indirect marker for the usage of ethanol as extraction solvent. The compounds were identified by LC-QTOF-HRESIMS, LC-MS, NMR, IR, and single-crystal X-ray diffraction after isolation by semipreparative HPLC. Furthermore, sage flower resin showed interesting antibacterial in vitro activities against Gram-positive and Gram-negative bacteria. KEYWORDS: salvia, flowers, sage flower resin, tri-p-coumaroylspermidine, salviquinone A, salviquinone B, caffeic acid ethyl ester, manufacturing technology, marker, antibacterial activities

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The first study focusing on the medicinal benefit of sage flowers was published in 1778 by Johann Hill. He described that the flowers of sage plants possess a special gummy and resinous material that can be extracted by maceration with an ethanol/water mixture.21 The ethanolic extracts obtained from the flowers are called fluid extracts and, nowadays, constitute the basis of several sage flower products manufactured in Germany. A distillation of the fluid extract is necessary to remove the alcohol and to separate the resinous part by precipitation. After the decantation of the aqueous part, sage resin is dried to make it unperishable. In 1956, sage flower resin was physically and chemically analyzed for the first time. It has been shown that sage flower resin contains almost no essential oil but many phenolic substances. Additionally, some antibacterial activities of sage flower resin have been reported.22 Until today, this sage flower resin has not been used medically. Presently, lipophilic plant extracts are usually processed by supercritical carbon dioxide extraction to obtain the natural compounds unmodified. The antibacterial activity of sage flower CO2 extracts was shown first in 2000, and the application as a deodorant was established.23 The antioxidant

INTRODUCTION

Sage (Salvia off icinalis L.) is a perennial, woody subshrub native to the Mediterranean region and is cultivated in Europe and North America. Usually, sage is a culinary herb or spice, used for flavoring and also as a tea or tincture for purported benefits in promoting health and treatment of ailments. The genus Salvia includes more than 900 species and belongs to the mint family (Labiatae). In addition to S. off icinalis the species S. sclarea, S. miltiorrhiza, and S. triloba are known as medicinal herbs. Extracts isolated from common sage (S. off icinalis) include sage infusions, essential oils, alcoholic extracts, and supercritical carbon dioxide extracts. They can be isolated from the leaves, roots, or flowers of the sage plant. The leaves are the most studied part of the Salvia species, and they are the origin of a major number of applications.1−3 The pharmacological properties of sage (S. of f icinalis) are also well-known today due to their leaf extracts. Characteristic compounds of sage such as carnosol, carnosic acid, rosmarinic acid, flavonoids, and polysaccharides have been identified as being particularly relevant for many pharmacological activities including high antioxidant capacity,4,5 enzyme inhibition,6 tumor inhibition,7,8 antidiabetic activity,9,10 BZD-receptor binding activity,11 antiviral,12,13 antifungal,14,15 and antibacterial activity,16,17 gastroprotective,18 neuroprotective,19 and anti-inflammatory6 properties, and immunomodulatory activities.20 © 2018 American Chemical Society

Received: Revised: Accepted: Published: 1843

January 31, 2018 February 14, 2018 February 16, 2018 February 16, 2018 DOI: 10.1021/acs.jafc.8b00581 J. Agric. Food Chem. 2018, 66, 1843−1853

Article

Journal of Agricultural and Food Chemistry

(Schwerte, Germany). Methanol-d4 (99.5%), n-pentane, xylol isomers (≥99%), n-hexane, and acetone (HPLC grade) were supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and 96% ethanol (v/v) and deionized water for extraction by Bombastus-Werke AG (Freital, Germany). Analytical standards such as rosmarinic acid (98%), luteolin-7-O-glucuronide (93%), and trans-caffeic acid ethyl ester (93%) were acquired from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Na-danshensu (99%) by Cfm Oskar Tropitzsch (Marktredwitz, Germany), carnosol (98.3%), and carnosic acid (97.3%) came from Apin Chemicals Ltd. (Milton, UK), and caffeic acid (100%), luteolin (100%), and apigenin (100%) were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). pCoumaric acid (≥98.0%) came from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). All of the chemicals were of analytical grade, and 0.2% chlorhexidine-digluconate solution (Meridol med CHX 0.2%) (CP GABA GmbH, Hamburg, Germany) was obtained from a pharmacy. Propylene glycol (Ph. eur.) for in vitro experiments was acquired from Bombastus-Werke AG. Nitrogen was produced with a nitrogen generator. GC-MS Analysis. For the GC analysis, a model 6890 gas chromatograph, coupled with a model 5973 MSD quadrupole mass spectrometer (Agilent, Waldbronn, Germany), was used. GC parameters: as capillary column was used a 25 m × 200 μm i.d., 0.33 μm, HP-1 methyl siloxane (Agilent). The pressure was set at 23.20 psi, the initial flow at 1.2 mL/min, and the average velocity at 45 cm/s. The injection volume was 1.0 μL. The inlet was used in the split mode with the split ratio at 50:1, the initial temperature at 220 °C, pressure at 12 psi, split flow at 31.5 mL/min, and total flow at 35.0 mL/min. The carrier gas was helium. The oven temperature was set at 70 °C with an equilibration time of 30 s (initial time: 13.0 min). After 5 min the oven temperature was increased with a rate of 6 °C/min to 280 °C and maintained at that temperature for 53 min. The data rate (MS quadrupole) was set at 20 Hz. HPLC-PDA-MS/MS Analysis. A model 1200 analytical HPLC system with DAD detection (Agilent, Waldbronn, Germany) coupled with a TSQ Quantum Assess MAX triple quadrupole mass spectrometer (Thermo Fisher, Waltham, MA) was used to identify and quantitate the included compounds. A second HPLC consisting of a Chromaster system and ELSD90 evaporative light-scattering detector (VWR International, Darmstadt, Germany) was used to analyze the purity of isolated compounds of the extracts. The extracts were chromatographed using a 250 mm × 4.6 mm i.d., 2 μm, Chromolith RP18e column, with a 10 mm × 4.6 mm i.d. guard column of the same material (VWR International) for Method A. Since analytical Chromolith columns are not commercially available in this length, the required length was realized by coupling three columns (one 50 mm and two 100 mm) from VWR International. Method B was carried out by using a 250 mm × 3 mm i.d., 4 μm, Synergy Polar RP18 column, with a 10 mm × 3 mm i.d., guard column of the same material (Phenomenex, Aschaffenburg, Germany). The oven temperature was set at 45 °C, and the mobile phase was 0.35% formic acid (Eluent A) and acetonitrile/methanol (65:35, v/v) including 0.35% formic acid (Eluent B) with a flow rate of 1.2 mL/min for Method A and 0.5 mL/min for Method B. The LC-MS analysis was carried out by Method B. The following gradient was used for both methods: 0− 3.0 min, 12% B; 3.0−14.3 min, 12−23% B; 14.3−28.0 min, held isocratically; 28−38 min, 23−37% B; 38−43 min, held isocratically; 43−70 min, 37−60% B; 70−89 min, 60−100% B; 89−110 min, held isocratically; decreasing to initial conditions in 0.1 min, and column equilibrated for 6 min. The injection volume was 10 μL. The temperature of the ELSD90 was set at 50 °C and the N2-pressure at 3.5 bar. The diode array detector (DAD) was set at 285 nm. Liquid Extraction and Semipreparative HPLC for the Fractionation of Unknown Compounds from Sage and Lavender Flowers. 10 g of sage flower resin was extracted with 100 mL of methanol by Soxhlet and subsequently filtered using a 0.45 μm nylon membrane filter. Afterward, time-based fraction collection mode was used to split the resin extract in eight prefractions including unknown compounds by a semipreparative 1200 HPLC system (Agilent) combined with an analytical fraction collector and funnel

potential was also tested and shown to be concentration dependent.24 On the basis of these properties, sage flower CO2 extracts are currently used in some cosmetic products such as tooth paste or skin cream. For the characterization of the previously rarely examined sage flower resin, we used modern analytical methods to analyze the chemical composition in more detail using semipreparative HPLC for separation and concentration for the subsequent elucidation by high-resolution mass spectrometry, NMR, IR, and single-crystal X-ray diffraction. Moreover, the antibacterial activities of sage flower resin in comparison to other sage extracts have been investigated, especially against the caries pathogen Streptococcus mutans.

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MATERIALS AND METHODS

Sage Samples. A variety of sage samples for analytical and antibacterial characterization were prepared. The seeds and already mature sage plants were acquired from certified institutions. The Bombastus Company (Freital, Germany) received their sage plants Salvia of ficinalis, S. lavandulifolia, and S. sclarea from Pharmaplant GmbH (Artern, Germany). The mature flowering plants S. cadmica, S. nemorosa, and S. tomentosa were a gift from the Botanical Garden (TU Dresden, Germany). Preparation procedures of samples were as follows. Fresh Sage Flower Samples for HPLC. Fresh sage flowers from different species of Salvia (S. of f icinalis, S. lavandulifolia, S. sclarea, S. cadmica, S. nemorosa, and S. tomentosa) were harvested at the height of their blooming period, cut, and extracted with 80% methanol/water (v/v) in a three-stage hot extraction (90 °C in water bath) under reflux for 10 min each to obtain an extract of 100 mg/mL. Each extract was filtered using a 0.45 μm nylon membrane filter. The flowers from S. of f icinalis were additionally separated into petals and sepals. The extraction procedure was the same as described above. Sage Flower Resin, Sage Leaf Resin, and Sage Flower CO2 Extract for HPLC. 15.0 mg of the powdered resin or stirred CO2 extract was dissolved in 5.0 mL of methanol, placed in an ultrasonic bath for 10 min, and filtered using a 0.45 μm nylon membrane filter. The CO2 extract was produced in 2010 by Flavex Naturextrakte GmbH (Rehlingen, Germany) at 300 bar and 35 °C. Sage flower resins were manufactured in the years 2009, 2010, and 2014 as well as sage leaf resin in 2010 by Bombastus-Werke AG (Freital, Germany). The flowers were collected during the months May and June and the leaves in July. Essential Oils for GC. Essential oils from sage plants and sage flower CO2 extracts (S. of f icinalis) were obtained by steam distillation. For this, a steam-distillation apparatus from WEPA Apothekenbedarf GmbH & Co KG (Hillscheid, Germany) was used. 40 g of fresh plant material and 1.1 g of sage flower CO2 extract were chosen. The method for obtaining the essential oil was carried out according to European Pharmacopoeia 2.8.12 and GC-MS analysis according to 2.2.28.25 Dry Weight Determination. To obtain the absolute dry weight of the samples, method 2.2.32 (fresh plant material was dried for 4 h at 105 °C) was used.25 Antibacterial in Vitro Assays. The sage extracts were dissolved in noninhibiting solvents. Sage resin solutions with propylene glycol were used against S. mutans and sage resin solutions with propylene glycol/ water (15.8:84.2, v/v) against other bacteria. Sage flower CO2 extracts were dissolved in n-hexane. The dissolved extracts were filtered using 0.45 μm nylon membrane filters. All solvents used were tested against bacteria to avoid false positive results. Lavender Samples. Dried lavender flowers (Lavandulifolia augustifolia MILL.) were acquired from Bombastus-Werke AG (Freital, Germany). Chemicals. Methanol and water (HPLC grade) were acquired from VWR International (Darmstadt, Germany) and acetonitrile from Th. Geyer GmbH & Co. KG (Renningen, Germany). Methanol and acetonitrile (LC-MS grade) were purchased from Fisher Scientific 1844

DOI: 10.1021/acs.jafc.8b00581 J. Agric. Food Chem. 2018, 66, 1843−1853

Article


Figure 1. HPLC-PDA profiles (Method A): comparison of sage flower extracts: (A) sage flower CO2 extract 2010, (B) sage flower resin 2014, and (C) oxidized sage flower resin 2009 at 285 nm (each 3.0 mg/mL in MeOH). Identification of peaks corresponds to structures shown in Figure 2. gas flow, 0.1 L/min; capillary voltage, 4000 V; and nozzle voltage 65 V. MS/MS experiments were carried out by setting CID between 15 and 30. A mixture of MeOH/H2O (1:1, v/v) with 0.1% HCOOH was used as eluent. Compound 8: injection volume, 1 μL; m/z 584.27546 [M + H]+ (calculated for C34H37N3O6, m/z 583.26824, error 0.26 ppm). Compound 10: injection volume, 0.1 μL; m/z 377.15920 [M + H]+ (calculated for C20H24O7, m/z 376.1522, error 0.32 ppm). Compound 13b: injection volume, 0.5 μL; m/z 389.15937 [M + H]+ (calculated for C21H24O7, m/z 388.1522, error 2.34 ppm). NMR Analysis. The NMR data were recorded at ambient temperature on an Avance AV-III 600 spectrometer (Bruker Bio Spin GmbH, Rheinstetten, Germany) operating at 600 MHz for 1H and at 151 MHz for 13C with standard Bruker pulse programs. Chemical shifts δ are given in ppm relative to TMS. The solvent signals were used as reference (1H: δH 3.38 ppm residual CHD2OD in CD3OD, 13C: δC 49.00 ppm). Coupling constants J are given in Hertz and were determined assuming first-order spin−spin coupling. Twodimensional NMR spectra included HSQC, HMBC, and NOESY. IR Analysis. An Alpha-FT-IR spectrometer with Platinum ATR (Bruker, Billerica, MA) was used to analyze functional groups of the isolated compound 8. Single-Crystal X-ray Diffraction. Compound 10 was crystallized at room temperature from methanol/water (1:1, v/v), and an appropriate well-shaped single-crystal was selected for the experiment. The crystal was glued to a glass fiber. Single-crystal X-ray diffraction was measured on a four-circle Kappa APEX II CCD diffractometer (Bruker, Karlsruhe, Germany) with a graphite(002)-monochromator and a CCD-detector at T = 170(2) K. Mo Kα radiation (λ = 71.073 pm) was used. A multiscan absorption was applied.26 The structure was solved with direct methods and refined against F02.27,28 CCDC 1536926 contains the supplementary crystallographic data for 10 × H2O. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.29 Measured Analytical Data of Isolated Compounds. Compound 8. UV (mobile phase at 40.8 min, Method A, 37% Eluent B (v/ v)): λmax (nm) 228, 295, 306. Positive HRESIMS: m/z 606.25756 [M + Na]+, 584.27546 [M + H]+, calculated for C34H37N3O6, 583.26824. Positive HRESIMS2: m/z 584.27546 ([M + H]+, 7), 464.2175 (0), 438.2388 (100), 420.2279 (16), 275.1754 (6), 204.1021 (11), 147.0441 (10). Negative ESI-MS2: m/z 582 ([M − H]−, 100), 462

trays. These prefractions were dried by evaporation. Again, all the dried extract fractions were dissolved in methanol (100 mg/mL). The samples were used again for semipreparative chromatography. The concentrated extracts were chromatographed employing a 250 mm × 10 mm i.d., 4 μm, Synergi Hydro RP18 column, with 10 mm × 10 mm guard column of the same material (Phenomenex, Aschaffenburg, Germany). The gradient was similar to the analytical analysis method and varied in dependence of time-based or peak-based fraction collection mode. The flow rate was 3.5 mL/min, and the injection volume was set at 200 μL. The collected and purified fractions 5, 8, 10, 11, 12, and 13b (Figure 1) were evaporated to dryness. To separate more easily and in high yield very nonpolar compounds 15, 16, and 17 (Figure 1) of the sage flower resin, 30 g of the resin was extracted with 300 mL of n-pentane using a Soxhlet apparatus. The obtained extract was evaporated to dryness and subsequently dissolved in methanol to obtain a concentration of 100 mg/mL. After separation of single compounds by peak-based fraction collection, the purified fractions were evaporated to dryness and used for further investigations. An amount of 40 g of dried lavender flowers was extracted with 400 mL of 80% aqueous methanol (v/v) for 30 min under reflux at 90 °C. Subsequently, the extract was evaporated to dryness. The remaining residue was resuspended in 100 mL of water, shaken, and centrifuged (6 min at 4000 rpm) to separate very polar compounds. Then, the aqueous phase (red color) was removed. The insoluble residue including peak 8 was extracted twice with 40 mL of n-pentane and centrifuged to remove very nonpolar compounds such as fatty acids and waxes. The new residue was dissolved in 10 mL of methanol and filtered using a 0.45 μm nylon membrane filter. This extract was used for semipreparative HPLC to obtain peak 8 in high yield and purity. The fractions were analyzed by HPLC-DAD-ESI-MS, HRESIMS, IR, single-crystal X-ray diffraction, and after dissolving in deuterated methanol, by NMR. HPLC-QTOF-ESI-MS Analysis. The high-resolution mass of the isolated unknown compounds was determined by a 6538 QTOF (Agilent, Waldbronn, Germany) analysis under the following parameters: the fragmentor voltage was set at 160 V ranging from m/z 60 to 1000 in the positive mode with an acquisition rate of 2 spectra/s; drying gas temperature, 300 °C; drying gas flow rate, 10 L/ min; nebulizer pressure, 50 psi; sheath gas temperature, 30 °C; sheath 1845


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Table 1. Major Constituents of Used Sage Extracts (Salvia of f icinalis) by LC Analysis with Method A [mean (mg/g); n = 2]a compound

tR (min)

1 2 3 4 5 6 7 8

4.1 7.5 16.4 20.0 27.3 29.9 38.4 40.8

9 10 11 12 13 13b 14 15 16

45.0 51.2 62.0 63.5 66.9 67.0 77.8 78.9 80.3

17 85.0 essential oil Σ f lavonoids

compound name danshensu caffeic acid luteolin-7-O-glycosidsc rosmarinic acid caffeic acid ethyl ester luteolin apigenin tri-pcoumaroylspermidine rosmanol isomere salviquinone A salviquinone Bd galdosol camosol unknown compoundd camosic acid 20-hydroxyfemiginole 12-O-methyl camosic acid ferruginole

Σ phenolic acids (derivatives) Σ abietane diterpenes

sage leaf resin 2010

sage flower resinb 2010

sage flower resinb 2014

sage flower resinb ox

sage flower CO2 extractb

1.7 ± 0.1 0.9 ± 0.0(4) 14.3 ± 0.0(3) 35.3 ± 1.4 1.7 ± 0.1 1.5 ± 0.1 1.1 ± 0.0(4) n.d.

0.6 ± 0.0(03) 0.6 ± 0.0(04) 0.3 ± 0.0(2) 9.2 ± 0.1 5.6 ± 0.0(2) 4.6 ± 0.0(4) 3.9 ± 0.0(01) 25.2 ± 0.1

0.6 ± 0.0(02) 0.6 ± 0.0(03) 2.8 ± 0.0(4) 30.0 ± 0.1 5.9 ± 0.0(1) 5.2 ± 0.0(3) 3.9 ± 0.0(02) 15.1 ± 0.1

1.0 ± 0.1 1.2 ± 0.0(3) 1.3 ± 0.0(3) 7.2 ± 0.0(4) 11.5 ± 0.5 4.7 ± 0.8 4.7 ± 0.8 19.5 ± 1.1

n.d n.d. n.d. n.d. n.d. n.d. n.d. n.d.

8.8 ± 0.4 n.d. n.d. 21.9 ± 0.7 3.2 ± 0.1 n.d. 4.0 ± 0.1 0.7 ± 0.0(3) 12.8 ± 0.5

21.6 ± 0.1 4.3 ± 0.0(1) n.d. 41.2 ± 0.1 78.7 ± 0.1 n.d. n.d. 9.1 ± 0.0(3) 67.0 ± 0.0(3)

20.6 ± 0.0(4) 5.2 ± 0.0(3) n.d. 42.3 ± 0.1 82.7 ± 0.1 n.d. n.d. 9.8 ± 0.0(2) 67.6 ± 0.3

39.9 ± 0.8 67.1 ± 0.8 1.3 ± 0.0(4) 14.9 ± 0.0(3) n.d. 8.7 ± 0.2 n.d. 6.1 ± 0.2 47.9 ± 0.2

n.d. n.d. n.d. n.d. n.d. n.d. 61.9 ± 0.1 8.9 ± 0.0(4) 36.3 ± 0.2

0.30 ± 0.0(1) n.a. approximately 16.9 approximately 39.6 approximately 51.7

7.2 ± 0.4 n.a. approximately 8.8

5.8 ± 0.1 n.a. approximately 11.9

0.2 ± 0.0(1) n.a. approximately 10.7

12.9 ± 0.1 299 ± 15 n.d.

approximately 41.2

approximately 52.2

approximately 40.4

n.d.

approximately 229., 1

approximately 234.0

approximately 186.1

approximately 120.0

a n.d., not detected; n.a., not analyzed; ox, oxidized. bInflorescences. cCalcd as luteolin-7-O-glucuronide. dCalcd as salviquinone A. eCalcd as carnosol. All other compounds were calibrated with analytical standards. Calibration curves with standard reference compounds purities 95%; Method A and B) with UV maxima of 212, 277, and 395 nm. A complete identification of compound 13b failed, but it probably represents a derivative of 11 with an additional double bond within the molecule due to its molecular weight of 388.1522 Da and calculated chemical formula C21H24O7. Analysis of Fresh Plant Material. After the identification of the compounds in sage flower resin, it was ascertained that not all the compounds were known in the literature regarding the genus Salvia. Most of the sage compounds were identified in leaf extracts in the past. Therefore, the resin from the flowers was compared with a resin from the leaves. However, sage resin from the leaves does not include compound 8 which was identified as tri-p-coumaroylspermidine, 8 (Figure 4). Therefore, 8 must be a compound which is typical for flowers of the Salvia species. This supposition was verified by analyzing 8 in the flowers of some more Salvia species. Additionally, the flowers of lavender plants, which also belong to the mint family, were analyzed, and 8 was identified also. These results confirm 1849


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storage of oxygen-free sage flower fluid extracts at room temperature results in a transition of carnosol to rosmanol and galdosol. However, only the storage of a half-filled bottle at room temperature leads to the formation of salviquinone A. A second experiment was conducted over one month with opened vessels. The result was an increase in rosmanol, galdosol, salviquinone A, salviquinone B, and unknown 13b, whereas carnosol decreases (Figure 7). In summary, these experiments indicate that the contact of the extracts with oxygen is the main reason for the transformation of the compounds, particularly for the generation of the new elucidated compound salviquinone A. Possible Pathway of the Oxidation Process. The transformation from carnosol into galdosol is well-known and has already been described.47 The pathway shown in Figure 8 hypothesizes a mechanism for formation of salviquinone A from carnosol. Potential Markers for Quality Control. Tri-p-coumaroylspermidine, 8, might be a quality marker for the application of sage flowers and can provide information about the proportion of the flowers. The ratio of the weight proportion of the flowers to the inflorescences is a further important quality parameter for the Bombastus Company and its sage flower products. Caffeic acid ethyl ester, 5, indicates the use of ethanol in sage flower extraction. Salviquinone A, 10, also shows potential as a marker for quality control, due to the fact that 10 is generated from carnosol only by exposure to oxygen (Figure 7). Kinetic experiments will be needed to determine the rate of the transformation from carnosol to salviquinone A. Antibacterial Properties of Sage Flower Extracts. In this study we investigated the antimicrobial effect of different sage extracts against Gram-positive and Gram-negative bacteria (Table 3, Figure 9). We used inhibition zone experiments due to their robustness and easy performance. This method is wellsuited for first checking an antimicrobial effect. As a result, we were able to confirm the findings of Walther22 concerning the antimicrobial activity of sage flower resin against Gram-positive bacteria.22 Moreover, sage flower resin also causes growth inhibition of the Gram-negative bacterium E. coli. Sage flower CO2 extract showed a growth inhibition against S. aureus and B. subtilis. The results clearly showed no antibacterial activity of sage leaf resin against Gram-positive and Gram-negative bacteria in the analyzed concentration. These experiments indicate that sage resin from the flowers has antibacterial activities against Gram-positive and Gram-negative bacteria and

Figure 5. Amount of tri-p-coumaroylspermidine, 8 (d.w.), in fresh sage flowers (Salvia of f icinalis L.) over the blooming period between May and July in 2011, 2012, and 2013. The amount was calculated by means of the isolated compound. [Mean ± RSD ≤ 10%, mg/g, d.w., n = 4].

might explain this observed behavior in sage flowers due to the location of 8 in the petals. Analysis of Manufacturing Technology of Sage Flower Resin. Sage flower resin contains mainly nonpolar compounds and includes natural compounds and for sage also unknown artifacts in high concentrations. Interesting interconversions of some of the compounds included in sage flower resin could already be observed during the production process. In an alcoholic solution, carnosol extraction increases over time from plant material. At the same time, carnosol appears to be transformed into rosmanol, galdosol, and salviquinone A. Some caffeic acid is converted into its ethyl ester during the maceration in ethanol, and rosmarinic acid dissociates into caffeic acid and danshensu. The curves in Figure 6 are the result of complex processes between the extraction of the compounds and its transformation into other compounds. Moreover, the storage of alcoholic sage flower extracts after the maceration process results in a decreasing amount of carnosol and an increase in other compounds. Thus, salviquinone A appears as a dominant peak in oxidized sage flower resin (Figure 1C). The content is more than 13 times higher than in native sage flower resin (Table 1). However, an analysis with oxygen-free sage flower fluid extracts (full bottle filling without gas phase) stored over 8 months at 4 °C reveals less decrease in carnosol. The

Figure 6. Mean amounts of (A) cinnamic acid derivatives and (B) diterpenes in sage flower resin after different extraction times of fresh plant material collected in 2013 and macerated in ethanol/water. [Mean in weighted sample, mg/g, n = 2]. 1850


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Figure 7. HPLC PDA profiles (Method A): Storage experiments of sage flower fluid extract (285 nm). (A) Before storing and (B) after storage in opened vessels after one month. Loss of solvent was corrected before analysis. Vessels were stored in the dark.

Figure 8. Possible pathway of oxidation from carnosol, 13, to the novel compound salviquinone A, 10. Its methyl ester, 11, could not be detected in all sage flower extracts. The route to its formation is unknown.

known.48 S. mutans belongs to the Gram-positive bacteria and is the cause of dental caries. Sage flower CO2 extracts are used in tooth paste, today. Its medicinal effect against S. mutans is not scientifically proven. However, sage flower resin is not yet applied as a medicinal, active substance but has the potential as a new application for fighting dental caries. The experiments were carried out using sage flower resin (from the years 2010 and 2014 and one oxidized resin from 2009) and a sage flower CO2 extract from 2010. The antibacterial in vitro activity of sage flower resin against S. mutans was proven here for the first time. The potency was nearly 80% in comparison to chlorhexidinedigluconate in the same concentration (0.2%) and showed a weak concentration-dependent behavior between 0.1 and 0.3% (Figure 9). The increases of the inhibition zones are not proportional to the concentration due to the saturation concentration of the sage flower resin. Higher concentrations do not necessarily cause higher inhibition zones. Chlorhexidine is a commercially available substance, active against oral pathogens, which is included in mouth rinses. However, sage flower CO2 extract caused no effects at all. Chemical variations in the composition of sage flower resin had no negative effect on the high antibacterial activity against S. mutans.

Table 3. Antibacterial Activity by Zone of Inhibition Test with Sage Extracts and Gram-Negative and Gram-Positive Bacteriaa sage extract (0.2%) Salvia off icinalis sage leaf resin (2010) sage flower resin (2010) sage flower CO2 extract (2010)

Pseudomonas aeruginosa Gram-negative

Escherichia coli Gramnegative

Bacillus subtilis Grampositive

[Mean diameter (cm), n = 2, 100 μL] n.d. n.d. n.d.

Staphylococcus aureus Grampositive n.d.

n.d.

2.0 ± 0.0

1.8 ± 0.0

1.4 ± 0.0

n.d.

n.d.

1.5 ± 0.1

1.5 ± 0.0

a

The solvents of sage extracts had no antibacterial activity; n.d., no detected inhibition zone.

is an interesting extract for antiseptic applications. In ancient times, fresh sage leaves were used for rubbing on the teeth for cleaning them and strengthening the gums, so the antibacterial effect of sage leaf extracts on Streptococcus mutans was already 1851


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Figure 9. Antibacterial in vitro activity of sage flower extracts determined by zone of inhibition test with Streptococcus mutans. 0.2% chlorhexidinedigluconate solution was used as positive control. Tested sage flower CO2 extract showed no antibacterial effects. [Mean ± SD, * SD = 0, cm, n = 3, 80 μL].

In summary, sage flower resin (SFR) has a specific high antibacterial in vitro activity, especially against S. mutans, and includes some new compounds which have their origin in the flowers of the plant and in the manufacturing process. These compounds have potential as new markers for the quality control of sage flower resin. Nevertheless, possible applications in dentistry are supported by other ingredients such as flavonoids, hydroxycinnamonic acids, and phenolic diterpenes which have antioxidant and anti-inflammatory effects.4,49

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely thank the Institute of Bioanalytical Chemistry, Dr. Susann Machill (TU Dresden, Germany), for the QTOF-MS measurements, the Bombastus-Werke AG, Dr. Christoph Grunert (Freital, Germany), and the Botanical Garden, Dr. Barbara Ditsch (TU Dresden, Germany), for the provision of the sage plants.

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ASSOCIATED CONTENT

* Supporting Information S

ABBREVIATIONS USED DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen; PDA, photodiode array; SF-CO2-extract, sage flower CO2 total extract; SFR, sage flower resin; SFR-ox, oxidized sage flower resin

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00581. 1 H and 13C NMR data of isolated compounds; X-ray crystallographic data of salviquinone A; further measured data of isolated compounds; storage experiments of sage flower fluid extract at different conditions; HPLC-PDAMS data of sage and lavender flower extracts; HPLC profiles from petals and sepals; correlation between the yield of sage flower resin and the part of flowers on inflorescences; HPLC semipreparative profile of prefractioning; GC-MS analysis of essential oils from sage leaves, sage flowers, and sage flower CO2 extract (PDF)

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REFERENCES

(1) Brand, N. Salvia. In Hagers Enzyklopädie der Arzneipflanzen und Drogen, 6 ed.; Blaschek, W., Ebel, S., Hackenthal, E., Holzgrabe, U., Keller, K., Reichling, J., Schulz, V., Eds.; Springer Wissenschaftliche Verlagsgesellschaft mbH Stuttgart: Stuttgart, Germany, 2007; Vol. 14, 124−170. (2) Lu, Y.; Foo, L. Y. Polyphenolics of Salvia - a review. Phytochemistry 2002, 59, 117−140. (3) Kintzios, S. E., Ed. Sage. The genus Salvia. In Medicinal and aromatic plants - industrial profiles; Hardman, R., Series Ed.; Harwood Academic Publishers: Amsterdam, Netherlands, 2000; Vol. 14. (4) Lu, Y.; Foo, L. Y. Antioxidant activities of polyphenols from sage (Salvia of f icinalis). Food Chem. 2001, 75, 197−202. (5) Schwarz, K.; Ternes, W. Antioxidative constituents of Rosmarinus of f icinalis and Salvia of f icinalis. I. Determination of phenolic diterpenes with antioxidative activity amongst tocochromanols using HPLC. Z. Lebensm.-Unters. Forsch. 1992, 195, 95−98. (6) Bauer, J.; Kuehnl, S.; Rollinger, J. M.; Scherer, O.; Northoff, H.; Stuppner, H.; Werz, O.; Koeberle, A. Carnosol and carnosic acids from

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Corresponding Authors

*(S.G.) Phone: +49 351 276 99442. Fax: +49 351 463 33132. E-mail: [email protected]. *(K.S.) Phone: +49 351 46333132. Fax: +49 351 463 33132. Email: [email protected]. ORCID

Sebastian Gericke: 0000-0002-9369-6187 Tilo Lübken: 0000-0003-3383-9518 1852


Article

Journal of Agricultural and Food Chemistry Salvia of f icinalis inhibit microsomal prostaglandin E2 synthase-1. J. Pharmacol. Exp. Ther. 2012, 342, 169−176. (7) Ito, H.; Miyake, M.; Nishitani, E.; Mori, K.; Hatano, T.; Okuda, T.; Konoshima, T.; Takasaki, M.; Kozuka, M.; Mukainaka, T.; Tokuda, H.; Nishino, H.; Yoshida, T. Anti-tumor promoting activity of polyphenols from Cowania mexicana and Coleogyne ramosissima. Cancer Lett. 1999, 143, 5−13. (8) Johnson, J. J. Carnosol: a promising anti-cancer and antiinflammatory agent. Cancer Lett. 2011, 305, 1−7. (9) Behradmanesh, S.; Derees, F.; Rafieian-Kopaei, M. Effect of Salvia of f icinalis on diabetic patients. J. Renal Inj. Prev. 2013, 2, 51−54. (10) Christensen, K. B.; Jorgensen, M.; Kotowska, D.; Petersen, R. K.; Kristiansen, K.; Christensen, L. P. Activation of the nuclear receptor PPARgamma by metabolites isolated from sage (Salvia of f icinalis L.). J. Ethnopharmacol. 2010, 132, 127−133. (11) Kavvadias, D.; Monschein, V.; Sand, P.; Riederer, P.; Schreier, P. Constituents of sage (Salvia of f icinalis) with in vitro affinity to human brain benzodiazepine receptor. Planta Med. 2003, 69, 113−117. (12) Smidling, D.; Mitic-Culafic, D.; Vukovic-Gacic, B.; Simic, D.; Knezevic-Vukcevic, J. Evaluation of antiviral activity of fractionated extracts of sage Salvia of f icinalis L. (Lamiaceae). Arch. biol. sci. (Beogr.) 2008, 60, 421−429. (13) Tada, M.; Okuno, K.; Chiba, K.; Ohnishi, E.; Yoshii, T. Antiviral diterpenes from. Phytochemistry 1994, 35, 539−541. (14) Bozin, B.; Mimica-Dukic, N.; Samojlik, I.; Jovin, E. Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus of ficinalis L. and Salvia of f icinalis L., Lamiaceae) essential oils. J. Agric. Food Chem. 2007, 55, 7879−7885. (15) Kanetis, L.; Exarchou, V.; Charalambous, Z.; Goulas, V. Edible coating composed of chitosan and Salvia f ruticosa Mill. extract for the control of grey mould of table grapes. J. Sci. Food Agric. 2017, 97, 452− 460. (16) Masterova, E.; Misikova, L.; Sirotkova, S.; Vaverkova, K. Royleanone in der Wurzel der einheimischen Salvia of f icinalis L. und ihre antimikrobielle Aktivität. Tschech. und Slowak. Pharmazie 1996, 45, 242−245. (17) Moujir, L.; Gutiérrez-Navarro, A. M.; San Andrés, L.; Luis, J. G. Structure - antimicrobial activity relationships of abietane diterpenes from Salvia species. Phytochemistry 1993, 34, 1493−1495. (18) Mayer, B.; Baggio, C. H.; Freitas, C. S.; dos Santos, A. C.; Twardowschy, A.; Horst, H.; Pizzolatti, M. G.; Micke, G. A.; Heller, M.; dos Santos, E. P.; Otuki, M. F.; Marques, M. C. A. Gastroprotective constituents of Salvia of f icinalis L. Fitoterapia 2009, 80, 421−426. (19) Fischedick, J. T.; Standiford, M.; Johnson, D. A.; Johnson, J. A. Structure activity relationship of phenolic diterpenes from Salvia of f icinalis as activators of the nuclear factor E2-related factor 2 pathway. Bioorg. Med. Chem. 2013, 21, 2618−2622. (20) Capek, P.; Hr̆íbalová, V.; Švandová, E.; Ebringerová, A.; Sasinková, V.; Masarová, J. Characterization of immunomodulatory polysaccharides from Salvia of f icinalis L. Int. J. Biol. Macromol. 2003, 33, 113−119. (21) Hill, J. Das heilige Kraut, oder die Kräfte der Salbei zur Verlängerung des menschlichen Lebens; Aurum Verlag GmbH & Co. KG: Freiburg bei Breisgau, Germany, 1778. (22) Walther, H.-J. Chemische und bakteriologische Untersuchungen des Harzkö rpers von Salvia officinalis L. Ph.D. Dissertation. Technische Universität Dresden, Dresden, Germany, 1956. (23) Reinhard, M.; Geissler, J. Use of extracts from sage for producing a deodorizing preparation. U.S. Patent 6,139,825, October 31, 2000. (24) Zerm, H. Antioxidative Eigenschaften verschiedener Extrakte aus Salvia officinalis L. Ph.D. Dissertation. Technische Universität Dresden, Dresden, Germany, 2000. (25) European Pharmacopoeia 8.0, 2014. https://www.edqm.eu/ medias/fichiers/index_english.pdf (accessed Dec 10, 2017). (26) Sheldrick, M. SADABS Area-Detector Absorption Correction, version 2014/5; Bruker AXS: Madison, WI, USA, 2014.

(27) Sheldrick, G. M. SHELX2014, Programs for Crystal Structure Determination; Georg-August-Universität Göttingen: Göttingen, Germany, 2014. (28) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112−122. (29) The Cambridge Crystallographic Data Centre, 2017; http:// www.ccdc.cam.ac.uk/structures (accessed Mar 13, 2017). (30) Santos-Gomes, P. C.; Fernandes-Ferreira, M. Organ- and season-dependent variation in the essential oil composition of Salvia of f icinalis L. cultivated at two different sites. J. Agric. Food Chem. 2001, 49, 2908−2916. (31) Oelschlaegel, S.; Quaas, S.; Speer, K. The essential oil content and composition of selected Salvia species, sage teas, and sage drops in food chemistry. J. Food Sci. Eng. 2012, 2, 427−436. (32) Bokern, M.; Witte, L.; Wray, V.; Nimtz, M.; Meurer-Grimes, B. Trisubstituted hydroxycinnamic acid spermidines from Quercus dentata pollen. Phytochemistry 1995, 39, 1371−1375. (33) González, A. G.; Fraga, B. M.; Luis, J. G.; Ravelo, A. G. Constituents of labiatae XIX. Structure of galdosol, a new diterpene from Salvia canariensis L. Experientia 1973, 29, 1471. (34) Djarmati, Z.; Jankov, R. M.; Csanadi, J.; Djordjević, A.; Ribar, B.; Lazar, D.; Engel, P. NMR studies and X-ray crystallography of galdosol 5-methyl ether from Salvia of f icinalis L. J. Crystallogr. Spectrosc. Res. 1992, 22, 585−590. (35) Son, K.-H.; Oh, H.-M.; Choi, S.-K.; Han, D. C.; Kwon, B.-M. Anti-tumor abietane diterpenes from the cones of Sequoia sempervirens. Bioorg. Med. Chem. Lett. 2005, 15, 2019−2021. (36) Aydogmus, Z.; Yesilyurt, V.; Topcu, G. Constituents of Salvia microphylla. Nat. Prod. Res. 2006, 20, 775−781. (37) Eghtesadi, F.; Moridi Farimani, M.; Hazeri, N.; Valizadeh, J. Abietane and nor-abitane diterpenoids from the roots of Salvia rhytidea. SpringerPlus 2016, 5, 1068. (38) Ma, C.-M.; Nakamura, N.; Hattori, M. Inhibitory effects on HIV-1 protease of tri-p-coumaroylspermidine from Artemisia caruifolia and related amides. Chem. Pharm. Bull. 2001, 49, 915−917. (39) Sobolev, V. S.; Sy, A. A.; Gloer, J. B. Spermidine and flavonoid conjugates from peanut (Arachis hypogaea) flowers. J. Agric. Food Chem. 2008, 56, 2960−2969. (40) Walters, D.; Meurer-Grimes, B.; Rovira, I. Antifungal activity of three spermidine conjugates. FEMS Microbiol. Lett. 2001, 201, 255− 258. (41) Steiner, T. The hydrogen bond in the solid state. Angew. Chem., Int. Ed. 2002, 41, 48−76. (42) Martin-Tanguy, J.; Cabanne, F.; Perdrizet, E.; Martin, C. The distribution of hydroxycinnamic acid amides in flowering plants. Phytochemistry 1978, 17, 1927−1928. (43) Meurer, B.; Wray, V.; Grotjahn, L.; Wiermann, R.; Strack, D. Hydroxycinnamic acid spermidine amides from pollen of Corylus avellana L. Phytochemistry 1986, 25, 433−435. (44) Slocum, R. D.; Kaur-Sawhney, R.; Galston, A. W. The physiology and biochemistry of polyamines in plants. Arch. Biochem. Biophys. 1984, 2, 235−283. (45) Oppermann, Y. Regulation des Methioninzyklus durch die Polyaminbiosynthese und den Ethylensignalweg in Arabidopsis thaliana. Ph.D. Dissertation. Christian-Albrechts-Universität zu Kiel, Kiel, Germany, 2013. (46) Kandil, M. M.; Ibrahaim, S. M.M.; El-Hanafy, S. H. Effect of putrescine and uniconazole on some flowering characteristics, and some chemical constituents of Salvias splendens F. plant. Int. J. ChemTech Res. 2015, 8, 174−186. (47) British Herbal Medicine Association. Sage leaf. Salviae officinalis folium 2012 http://bhma.info/wp-content/uploads/2012/12/ SalviaMonograph1.pdf (accessed Jul 10, 2017). (48) Smullen, J.; Finney, M.; Storey, D. M.; Foster, H. A. Prevention of artificial dental plaque formation in vitro by plant extracts. J. Appl. Microbiol. 2012, 113, 964−973. (49) González, A. G.; Andrés, L. S.; Aguiar, Z. E.; Luis, J. G. Diterpenes from Salvia mellifera and their biogenetic significance. Phytochemistry 1992, 31, 1297−1305. 1853


Identification of New Compounds from Sage Flowers (Salvia

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