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Identification of New Compounds from Sage Flowers (Salvia officinalis 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00581 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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Journal of Agricultural and Food Chemistry

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Identification of New Compounds from Sage Flowers (Salvia officinalis L.) as

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Markers for Quality Control and the Influence of the Manufacturing Technology

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on the Chemical Composition and Antibacterial Activity of Sage Flower Extracts

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Sebastian Gericke,*,† Tilo Lübken,‡ Diana Wolf,§ Martin Kaiser,# Christian Hannig,┴ and Karl

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Speer*,†

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8 9 10 11 12



Food Chemistry,



Organic Chemistry,

#

Inorganic Chemistry, Technische Universität Dresden, Bergstrasse 66, D-01069 Dresden,

Germany, §

Institute of Microbiology, Technische Universität Dresden, Zellescher Weg 20b, D-01217

Dresden, Germany ┴

Clinic of Operative and Pediatric Dentistry, Medical Faculty Carl Gustav Carus, TU Dresden,

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Fetscherstr. 74, D-01307 Dresden, Germany

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*Corresponding Author (Tel: +49 351 276 99442; Fax: +49 351 463 33132;

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E-Mail: [email protected])

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ABSTRACT:

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Parts of Salvia species such as its flowers and leaves are currently used as a culinary herb and for

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some medicinal applications. To distinguish the different sage extracts it is necessary to analyze

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their individual chemical compositions. Their characteristic compounds might be established as

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markers to differentiate between sage flowers and leaf extracts or to determine the manufacturing

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technology and storage conditions. Tri-p-coumaroylspermidine can be detected only in flowers

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and has been described here for Salvia and Lavandula species for the first time. Markers for

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oxidation processes are the novel compounds salviquinone A and B, which were generated from

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carnosol by exposure to oxygen. Caffeic acid ethyl ester was established as an indirect marker for

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the usage of ethanol as extraction solvent. The compounds were identified by LC-QTOF-

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HRESIMS, LC-MS, NMR, IR, and single-crystal X-ray diffraction after isolation by

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semipreparative HPLC. Furthermore, sage flower resin showed interesting antibacterial in vitro

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activities against Gram-positive and Gram-negative bacteria.

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KEYWORDS: Salvia, flowers, sage flower resin, tri-p-coumaroylspermidine, salviquinone A,

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salviquinone B, caffeic acid ethyl ester, manufacturing technology, marker, antibacterial

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activities

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INTRODUCTION

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Sage (Salvia officinalis L.) is a perennial, woody sub-shrub native to the Mediterranean region

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and is cultivated in Europe and North America. Usually, sage is a culinary herb or spice, used for

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flavoring, and also as a tea or tincture for purported benefits in promoting health and treatment of

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ailments. The genus Salvia includes more than 900 species and belongs to the mint family

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(Labiatae). In addition to S. officinalis the species S. sclarea, S. miltiorrhiza and S. triloba are

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known as medicinal herbs. Extracts isolated from common sage (S. officinalis) include sage

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infusions, essential oils, alcoholic extracts, and supercritical carbon dioxide extracts. They can be

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isolated from the leaves, roots or flowers of the sage plant. The leaves are the most studied part of

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the Salvia species, and they are the origin of a major number of applications.1–3 The

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pharmacological properties of sage (S. officinalis) are also well known today due to their leaf

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extracts. Characteristic compounds of sage such as carnosol, carnosic acid, rosmarinic acid,

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flavonoids and polysaccharides have been identified as being particularly relevant for many

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pharmacological activities including high antioxidant capacity,4,5 enzyme inhibition,6 tumor

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inhibition,7,8

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antifungal,14,15 and antibacterial activity,16,17 gastroprotective,18 neuroprotective,19 and anti-

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inflammatory6 properties and immunomodulatory activities20.

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The first study focusing on the medicinal benefit of sage flowers was published in 1778 by

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Johann Hill. He described that the flowers of sage plants possess a special gummy and resinous

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material that can be extracted by maceration with an ethanol/water mixture.21 The ethanolic

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extracts obtained from the flowers are called fluid extracts and, nowadays, constitute the basis of

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several sage flower products manufactured in Germany. A distillation of the fluid extract is

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necessary to remove the alcohol and to separate the resinous part by precipitation. After the

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decantation of the aqueous part, sage resin is dried to make it unperishable. In 1956, sage flower

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resin was physically and chemically analyzed for the first time. It has been shown that sage

antidiabetic

activity,9,10

BZD-receptor

binding

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activity,11

antiviral,12,13

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flower resin contains almost no essential oil but many phenolic substances. Additionally, some

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antibacterial activities of sage flower resin have been reported.22 Until today, this sage flower

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resin has not been used medically. Presently, lipophilic plant extracts are usually processed by

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supercritical carbon dioxide extraction to obtain the natural compounds unmodified. The

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antibacterial activity of sage flower CO2 extracts was shown first in 2000 and the application as a

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deodorant was established.23 The antioxidant potential was also tested and shown to be

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concentration dependent.24 On the basis of these properties, sage flower CO2 extracts are

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currently used in some cosmetic products such as tooth paste or skin cream.

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For the characterization of the previously rarely examined sage flower resin, we used modern

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analytical methods to analyze the chemical composition in more detail using semipreparative

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HPLC for separation and concentration for the subsequent elucidation by high resolution mass

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spectrometry, NMR, IR, and single-crystal X-ray diffraction. Moreover, the antibacterial

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activities of sage flower resin in comparison to other sage extracts have been investigated,

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especially against the caries pathogen Streptococcus mutans.

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MATERIALS AND METHODS

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Sage Samples. A variety of sage samples for analytical and antibacterial characterization were

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prepared. The seeds and already mature sage plants were acquired from certified institutions. The

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Bombastus Company (Freital, Germany) received their sage plants Salvia officinalis, S.

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lavandulifolia and S. sclarea from Pharmaplant GmbH (Artern, Germany). The mature flowering

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plants S. cadmica, S. nemorosa and S. tomentosa were a gift from the Botanical Garden (TU

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Dresden, Germany). Preparation procedures of samples were as follows:

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Fresh Sage Flower Samples for HPLC. Fresh sage flowers from different species of Salvia (S.

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officinalis, S. lavandulifolia, S. sclarea, S. cadmica, S. nemorosa and S. tomentosa) were

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harvested at the height of their blooming period, cut, and extracted with 80% methanol/water ACS Paragon Plus Environment

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(v/v) in a three-stage hot extraction (90 °C in water bath) under reflux for 10 min each to obtain

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an extract of 100 mg/mL. Each extract was filtered using a 0.45 µm nylon membrane filter. The

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flowers from S. officinalis were additionally separated into petals and sepals. The extraction

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procedure was the same as described above.

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Sage Flower Resin, Sage Leaf Resin and Sage Flower CO2 Extract for HPLC. 15.0 mg of the

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powdered resin or stirred CO2 extract were dissolved in 5.0 mL methanol, placed in an ultrasonic

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bath for 10 min, and filtered using a 0.45 µm nylon membrane filter. The CO2 extract was

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produced in 2010 by Flavex Naturextrakte GmbH (Rehlingen, Germany) at 300 bars and 35 °C.

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Sage flower resins were manufactured in the years 2009, 2010 and 2014 as well as sage leaf resin

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in 2010 by Bombastus-Werke AG (Freital, Germany). The flowers were collected during the

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months May and June and the leaves in July.

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Essential Oils for GC. Essential oils from sage plants and sage flower CO2 extracts (S. officinalis)

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were obtained by steam distillation. For this, a steam distillation apparatus from WEPA

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Apothekenbedarf GmbH & Co KG (Hillscheid, Germany) was used. 40 g of fresh plant material

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and 1.1 g of sage flower CO2 extract were chosen. The method for obtaining the essential oil was

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carried out according to European Pharmacopoeia 2.8.12 and GC-MS analysis according to

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2.2.28.25

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Dry Weight Determination. To obtain the absolute dry weight of the samples, method 2.2.32

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(fresh plant material was dried for 4 h at 105 °C) was used.25

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Antibacterial in vitro Assays. The sage extracts were dissolved in non-inhibiting solvents. Sage

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resin solutions with propylene glycol were used against S. mutans and sage resin solutions with

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propylene glycol/water (15.8:84.2, v/v) against other bacteria. Sage flower CO2 extracts were

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dissolved in n-hexane. The dissolved extracts were filtered using 0.45 µm nylon membrane

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filters. All solvents used were tested against bacteria to avoid false positive results. ACS Paragon Plus Environment

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Lavender Samples. Dried lavender flowers (Lavandulifolia augustifolia MILL.) were acquired

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from Bombastus-Werke AG (Freital, Germany).

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Chemicals. Methanol and water (HPLC grade) was acquired from VWR International

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(Darmstadt, Germany) and acetonitrile from Th. Geyer GmbH & Co. KG (Renningen, Germany).

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Methanol and acetonitrile (LC-MS grade) was purchased from Fisher Scientific (Schwerte,

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Germany). Methanol-d4 (99.5%), n-pentane, xylol isomers (≥ 99%), n-hexane and acetone

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(HPLC grade) was supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany), 96% ethanol

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(v/v) and deionized water for extraction by Bombastus-Werke AG (Freital, Germany). Analytical

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standards such as rosmarinic acid (98%), luteolin-7-O-glucuronide (93%), and trans-caffeic acid

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ethyl ester (93%) were acquired from PhytoLab GmbH & Co. KG (Vestenbergsgreuth,

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Germany), Na-danshensu (99%) by Cfm Oskar Tropitzsch (Marktredwitz, Germany), carnosol

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(98.3%) and carnosic acid (97.3%) came from Apin Chemicals Ltd. (Milton, UK) and caffeic

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acid (100%), luteolin (100%), apigenin (100%) were purchased from Carl Roth GmbH & Co. KG

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(Karlsruhe, Germany) and p-coumaric acid (≥ 98.0%) came from Sigma-Aldrich Chemie GmbH

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(Taufkirchen, Germany). All of the chemicals were of analytical grade. 0.2% Chlorhexidine-

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digluconate solution (Meridol med CHX 0.2%) (CP GABA GmbH, Hamburg, Germany) was

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obtained from a pharmacy. Propylene glycol (Ph.Eur.) for in vitro experiments was acquired

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from Bombastus-Werke AG. Nitrogen was produced with a nitrogen generator.

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GC-MS Analysis. For the GC analysis, a model 6890 gas chromatograph, coupled with a model

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5973 MSD quadrupole mass spectrometer (Agilent, Waldbronn, Germany) was used. GC-

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Parameters: as capillary column was used a 25 m x 200 µm i.d., 0.33 µm, HP-1 methyl siloxane

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(Agilent). The pressure was set at 23.20 psi, the initial flow at 1.2 mL/min, and the average

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velocity at 45 cm/s. The injection volume was 1.0 µL. The inlet was used in the split mode with

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the split ratio at 50:1, the initial temperature at 220 °C, pressure at 12 psi, split flow at 31.5

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mL/min and total flow at 35.0 mL/min. The carrier gas was helium. The oven temperature was ACS Paragon Plus Environment

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set at 70 °C with an equilibration time of 30 s (initial time: 13.0 min). After 5 min the oven

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temperature was increased with a rate of 6 °C/min to 280 °C and maintained at that temperature

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for 53 min. The data rate (MS quadrupole) was set at 20 Hz.

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HPLC-PDA-MS/MS Analysis. A model 1200 analytical HPLC system with DAD-detection

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(Agilent, Waldbronn, Germany) coupled with a TSQ Quantum Assess MAX triple quadrupole

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mass spectrometer (Thermo Fisher, Waltham, MA) was used to identify and quantitate the

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included compounds. A second HPLC consisting of a Chromaster system and ELSD90

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evaporative light-scattering detector (VWR International, Darmstadt, Germany) was used to

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analyze the purity of isolated compounds of the extracts. The extracts were chromatographed

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using a 250 mm x 4.6 mm i.d., 2 µm, Chromolith RP18e column, with a 10 mm x 4.6 mm i.d.

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guard column of the same material (VWR International) for Method A. Since analytical

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Chromolith columns are not commercially available in this length, the required length was

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realized by coupling three columns (one 50 mm and two 100 mm) from VWR International.

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Method B was carried out by using a 250 mm x 3 mm i.d., 4 µm, Synergy Polar RP18 column,

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with a 10 mm x 3 mm i.d., guard column of the same material (Phenomenex, Aschaffenburg,

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Germany). The oven temperature was set at 45 °C and the mobile phase was 0.35% formic acid

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(Eluent A) and acetonitrile/methanol (65:35, v/v) including 0.35% formic acid (Eluent B) with a

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flow rate of 1.2 mL/min for Method A and 0.5 mL/min for Method B. The LC-MS analysis was

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carried out by Method B. The following gradient was used for both methods: 0-3.0 min, 12% B;

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3.0-14.3 min, 12-23% B; 14.3-28.0 min, held isocratically; 28-38 min, 23-37% B; 38-43 min,

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held isocratically; 43-70 min, 37-60% B; 70-89 min, 60-100% B; 89-110 min, held isocratically;

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decreasing to initial conditions in 0.1 min, and column equilibrated for 6 min. The injection

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volume was 10 µL. The temperature of the ELSD90 was set at 50 °C and the N2-pressure at 3.5

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bars. The diode array detector (DAD) was set at 285 nm.

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Liquid Extraction and Semipreparative HPLC for the Fractionation of Unknown Compounds

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from Sage and Lavender Flowers. 10 g of sage flower resin were extracted with 100 mL

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methanol by Soxhlet and subsequently filtered using a 0.45 µm nylon membrane filter.

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Afterwards, time-based fraction collection mode was used to split the resin extract in eight pre-

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fractions including unknown compounds by semipreparative 1200 HPLC system (Agilent)

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combined with an analytical fraction collector and funnel trays. These pre-fractions were dried by

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evaporation. Again, all the dried extract fractions were dissolved in methanol (100 mg/mL). The

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samples were used again for semipreparative chromatography. The concentrated extracts were

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chromatographed employing a 250 mm x 10 mm i.d., 4 µm, Synergi Hydro RP18 column, with

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10 mm x 10 mm, guard column of the same material (Phenomenex, Aschaffenburg, Germany).

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The gradient was similar to the analytical analysis method and varied in dependence of time-

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based- or peak-based fraction collection mode. The flow rate was 3.5 mL/min, and the injection

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volume was set at 200 µL. The collected and purified fractions 5, 8, 10, 11, 12, 13b (Figure 1)

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were evaporated to dryness.

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To separate more easily and in high yield very nonpolar compounds 15, 16, 17 (Figure 1) of the

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sage flower resin, 30 g of the resin was extracted with 300 mL n-pentane using a Soxhlet

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apparatus. The obtained extract was evaporated to dryness and subsequently dissolved in

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methanol to obtain a concentration of 100 mg/mL. After separation of single compounds by

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peak-based fraction collection, the purified fractions were evaporated to dryness and used for

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further investigations.

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40 g dried lavender flowers were extracted with 400 mL of 80% aqueous methanol (v/v) for 30

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min under reflux at 90 °C. Subsequently, the extract was evaporated to dryness. The remaining

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residue was resuspended in 100 mL water, shaken and centrifuged (6 min at 4000 rpm) to

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separate very polar compounds. Then, the aqueous phase (red color) was removed. The insoluble

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residue including peak 8 was extracted twice with 40 mL n-pentane and centrifuged to remove ACS Paragon Plus Environment

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very nonpolar compounds such as fatty acids and waxes. The new residue was dissolved in 10

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mL methanol and filtered using a 0.45 µm nylon membrane filter. This extract was used for

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semipreparative HPLC to obtain peak 8 in high yield and purity.

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The fractions were analyzed by HPLC-DAD-ESI-MS, HRESIMS, IR, single-crystal X-ray

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diffraction, and after dissolving in deuterated methanol, by NMR.

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HPLC-QTOF-ESI-MS Analysis. The high resolution mass of the isolated unknown compounds was

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determined by a 6538 QTOF (Agilent, Waldbronn, Germany) analysis under the following

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parameters: the fragmentor voltage was set at 160 V ranging from m/z 60-1000 in the positive

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mode with an acquisition rate of 2 spectra/s; drying gas temperature, 300 °C; drying gas flow

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rate, 10 L/min; nebulizer pressure, 50 psi; sheath gas temperature, 30 °C; sheath gas flow, 0.1

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L/min; capillary voltage, 4000 V; and nozzle voltage 65 V. MS/MS experiments were carried out

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by setting CID between 15 and 30. A mixture of MeOH/H2O (1:1, v/v) with 0.1% HCOOH was

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used as eluent. Compound 8: injection volume, 1 µL; m/z 584.27546 [M+H]+ (calculated for

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C34H37N3O6, m/z 583.26824, error 0.26 ppm). Compound 10: injection volume, 0.1 µL; m/z

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377.15920 [M+H]+ (calculated for C20H24O7, m/z 376.1522, error 0.32 ppm). Compound 13b:

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injection volume, 0.5 µL; m/z 389.15937 [M+H]+ (calculated for C21H24O7, m/z 388.1522, error

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2.34 ppm).

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NMR Analysis. The NMR data were recorded at ambient temperature on an Avance AV-III 600

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spectrometer (Bruker Bio Spin GmbH, Rheinstetten, Germany) operating at 600 MHz for 1H and

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151 MHz for

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relative to TMS. The solvent signals were used as reference (1H: δH 3.38 ppm residual CHD2OD

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in CD3OD,

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assuming first-order spin-spin coupling. Two-dimensional NMR spectra included HSQC,

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HMBC, and NOESY.

13

13

C with standard Bruker pulse programs. Chemical shifts δ are given in ppm

C: δC 49.00 ppm). Coupling constants J are given in Hertz and were determined

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IR Analysis. An Alpha-FT-IR spectrometer with Platinum ATR (Bruker, Billerica, MA) was used

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to analyze functional groups of the isolated compound 8.

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Single-Crystal X-ray Diffraction. Compound 10 was crystallized at room temperature from

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methanol/water (1:1, v/v), and an appropriate well-shaped single-crystal was selected for the

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experiment. The crystal was glued to a glass fiber. Single-crystal X-ray diffraction was measured

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on a four-circle Kappa APEX II CCD diffractometer (Bruker, Karlsruhe, Germany) with a

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graphite(002)-monochromator and a CCD-detector at T = 170(2) K. MoKa radiation (λ = 71.073

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pm) was used. A multiscan absorption was applied.26 The structure was solved with direct

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methods and refined against Fo2.27,28 CCDC 1536926 contains the supplementary crystallographic

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data for 10 x H2O. This data can be obtained free of charge from The Cambridge Crystallographic

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Data Centre.29

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Measured Analytical Data of Isolated Compounds. Compound 8: UV (mobile phase at 40.8 min,

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Method A, 37% Eluent B (v/v)) λmax (nm) 228, 295, 306. Positive HRESIMS: m/z 606.25756

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[M+Na]+, 584.27546 [M+H]+, calculated for C34H37N3O6, 583.26824. Positive HRESIMS2: m/z

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584.27546 ([M+H]+, 7), 464.2175 (0), 438.2388 (100), 420.2279 (16), 275.1754 (6), 204.1021

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(11), 147.0441 (10). Negative ESI-MS2: m/z 582 ([M-H]-, 100), 462 (45), 436 (10), 342 (38), 316

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(4). IR (ATR): νmax (1/cm) 3231 (OH), 1643 (CONH–), 1580, 1510, 1436 (benzene ring), 1215,

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1167, 976, 826, 513.

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Compound 10: UV (mobile phase at 51 min, Method A, 44% Eluent B (v/v)) λmax (nm) 213, 272,

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400. Positive HRESIMS: m/z 399.141106 [M+Na]+, 377.15920 [M+H]+, calculated for C20H24O7,

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376.1522. Positive HRESIMS2: m/z 377.1593 ([M+H]+, 7), 331.1543 (100), 313.1421 (3),

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303.1591 (70), 285.1484 (49), 275.1607 (27), 247.0966 (37), 219.1016 (14), 181.0861 (7).

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Negative ESI-MS2: m/z 375 ([M-H]-, 41), 331 (9), 303 (3), 287 (7), 275 (9). 1H NMR (600 MHz,

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CD3OD) δ 1.64 (C-1α; ddd (J = 14.1, 11.1 and 4.7 Hz)), 2.54 (C-1ß; ddd (J = 14.1, 4.3 and 4.2

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Hz), 1.70 (C-2α; dquin (J = 14.2 and 4.5 Hz)), 2.18 (C-2ß; dddt (J = 14.2, 11.9, 11.1 and 4.0 ACS Paragon Plus Environment

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Hz)), 1.61 (C-3α; ddd (J = 13.6, 11.9 and 3.9 Hz)), 1.45 (C-3ß; dddt (J = 13.6, 4.7, 3.8 and 1.1

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Hz)), 3.09 (C-5; br d (J = 8.5 Hz)), 4.90 (C-6α; d (J = 8.3 Hz)), 6.52 (C-14; d (J = 1.3 Hz)), 3.07

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(C-15; sept d (J = 6.9 and 1.3 Hz)), 1.22 (C-16; d (J = 6.9 Hz)), 1.22 (C-17; d (J = 6.9 Hz)), 0.94

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(C-18; s), 0.98 (C-19; s); 13C NMR (151 MHz, CD3OD) δ 29.6 (C-1), 20.6 (C-2), 35.1 (C-3), 31.8

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(C-4), 53.1 (C-5), 78.3 (C-6), 172.5 (C-7), 188.5 (C-8), 120.4 (C-9), 48.8 (C-10), 156.3 (C-11),

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184.1 (C-12), 152.3 (C-13), 133.8 (C-14), 27.9 (C-15), 21.5 (C-16), 21.3 (C-17), 28.6 (C-18),

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28.4 (C-19), 181.7 (C-20). Single-crystal X-ray diffraction. Compound 10 crystallizes in the

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polar orthorhombic space group P212121 (no. 19) with a = 7.5609(3) Å, b = 11.8725(5) Å, and c

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= 21.8352(8) Å.

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Compound 11: UV (mobile phase at 62 min, Method A, 50% Eluent B (v/v)) λmax (nm) 213, 271,

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400. Negative ESI-MS: m/z 389 [M-H]-. 1H NMR (600 MHz, CD3OD) δ 1.62 (C-1α; HMBC),

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2.54 (C-1ß; dt (J = 13.7 and 4.0 Hz)), 1.69 (C-2α; dquin (J = 13.7 and 4.6 Hz)), 2.19 (C-2ß;

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HMBC), 1.59 (C-3α; HMBC), 1.44 (C-3ß; dt (J = 13.3 and 4.2 Hz)), 3.11 (C-5; br d (J = 8.3

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Hz)), 4.95 (C-6α; d (J = 8.4 Hz)), 6.48 (C-14; br s), 3.05 (C-15; sept (J = 6.8 Hz)), 1.21 (C-16; d

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(J = 6.8 Hz)), 1.21 (C-17; d (J = 6.8 Hz)), 0.88 (C-18; s), 0.98 (C-19; s), 3.84 (C-21; s); 13C NMR

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(151 MHz, CD3OD) δ 29.6 (C-1, HSQC), 20.6 (C-2; HSQC), 35.1 (C-3; HSQC), 31.8 (C-4;

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HMBC), 53.1 (C-5; HSQC), 77.9 (C-6; HSQC), 171.6 (C-7; HMBC), 188.0 (C-8), 119.5 (C-9;

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HMBC), 48.8 (C-10; HMBC), n.d. (C-11), 184.7 (C-12; HMBC), 152.1 (C-13; HMBC), 134.3

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(C-14; HSQC), 27.9 (C-15; HSQC), 21.6 (C-16; HSQC), 21.4 (C-17; HSQC), 28.3 (C-18;

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HSQC), 28.6 (C-19; HSQC), 181.8 (C-20; HMBC), 53.2 (C-21; HMBC).

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Compound 13b: UV (mobile phase at 69 min, Method A, 59% Eluent B (v/v)) λmax (nm) 212,

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277, 395. Positive HRESIMS: m/z 411.1416 [M+Na]+, 389.15937 [M+H]+, calculated for

250

C21H24O7, 388.1522. Positive HRESIMS2: m/z 389.1596 ([M+H]+, 100), 371.1486 (1), 358.1393

251

(3), 331.1543 (3), 311.1274 (11), 301.1435 (6), 283.1328 (28), 273.1484 (55), 255.1376 (33),

252

231.1010 (21), 213.0904 (21), 179.0785 (9), 159.0785 (18), 151.0751 (5).

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Antibacterial in vitro Assays. Bacterial test strains Pseudomonas aeruginosa ATCC27853,

254

Escherichia coli DSM30083, Bacillus subtilis W168 (BGSC) and Staphylococcus aureus

255

(clinical isolate from Medical Microbiology, TU Dresden, strain list number B15) were grown

256

overnight in the Luria-Bertani (LB) medium (1% tryptone, 1% sodium chloride, and 0.5% yeast

257

extract) under aeration at 37 °C. S. mutans DSM6178 was grown in the trypticase soy yeast

258

extract medium (medium 92, DSMZ). The next day, 100 µL of each culture was plated onto LB

259

agar plates. Streptococcus mutans DSM6178 was plated onto trypticase soy yeast extract agar

260

plates. A sterile cork borer was used to stamp an 8 mm hole out of the agar. 70 – 100 µL of the

261

appropriate test reagents were pipetted into the agar hole. Subsequently, the plates were incubated

262

overnight at 37 °C for 25 h, and the diameter of the growth inhibition halos was determined

263

accordingly. As reference substances we used a chlorhexidine solution for the positive control

264

against S. mutans and the solvents propylene glycol and n-hexane for the negative control against

265

all tested bacteria.

266

RESULTS AND DISCUSSION

267

In this study, different sage extracts from the areal parts were characterized by HPLC and GC.

268

The HPLC-PDA profiles showed strong differences between sage flower CO2 extract and sage

269

flower resin extract (Figure 1). Only compounds 15 and 16 were included in all analyzed sage

270

flower extracts and compound 16 showed a good response in all the sage flower samples. Sage

271

resin from the flowers contained between 1.5-2 times more abietane diterpenes than sage flower

272

CO2 extract. Sage leaf resin differed even more drastically in the composition of sage flower

273

extracts. There were, in summary, more flavonoids and significantly fewer abietane diterpenes

274

included. Furthermore, sage flower CO2 extract contained approx. 30% of essential oil (Table 1),

275

whereas sage resin included only traces of essential oil.22 The differences in the composition of

276

sage flower CO2 extract and sage flower resin were mostly based on the different production

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technology as the plant material was the same. The respective technology applied also affected ACS Paragon Plus Environment

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the yields of sage extracts significantly. Thus, the production of 1 kg of sage flower CO2 extract

279

or sage flower resin required 375 kg or 130 kg of fresh sage flowers (inflorescences),

280

respectively. These data also illustrated that the yield of sage flower resin was approx. three times

281

higher than the yield of sage flower CO2 extracts in relation to the fresh plant mass used.

282

Moreover, the yield of sage flower resin was also influenced by the parts of the plant which were

283

used. Inflorescences contain parts of flowers, leaves, and stems. The proportion depended on the

284

growth and the cutting height of the plants. Stems contained fewer resinous compounds and

285

drastically decrease the high quality of sage flower resin. The correlation (R2 ≈ 0.9) between the

286

sage flower parts on the inflorescences used and the ratio (amount in kg of fresh plant mass in

287

relation to 1 kg of sage flower resin) underpins the hypothesis21 that the flowers contain the most

288

of the resinous parts of the sage plant.

289

Sage compounds (S. officinalis) are generally well known today.2,30 Each utilized sage extract

290

contains a special composition of these known compounds due to showing different effects.31

291

Furthermore, sage flower resin also includes some novel compounds. These compounds are

292

located in the flowers of the plant or become partly transformed by the manufacturing processes.

293

GC-MS Analysis. Essential oils from the sage flowers had less content of thujone than the leaves.

294

Due to the current discussion on the toxicity of thujone, the use of sage flowers has a distinct

295

advantage regarding the low content of thujone. Thus, the remaining residues were also lower.

296

Identification of Compounds. Due to missing reference standards many peaks could only be

297

elucidated after isolating the compounds from the sage flower resin. In the case of peak 8 the

298

compound was isolated from lavender flowers, a plant that also belongs to the mint family. The

299

extracts were less complex in comparison to those of the sage flower resin, allowing peak 8 to be

300

isolated in sufficiently high yield and purity for the subsequent elucidation by HRESIMS, UV-

301

and IR-spectroscopy. A first NMR analysis failed due to the fact that the compound is light-

302

unstable. The obtained NMR-signals were not interpretable. After identification, a reanalysis was ACS Paragon Plus Environment

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not possible due to the small fractionated amount. A cis-trans isomerization has already been

304

described in the literature.32 Since the compound was still unknown at the time of NMR

305

measurement, this particular property would be unpredictable. To ensure that the isolated

306

compounds from sage and lavender flowers are the same, we compared the retention times of the

307

peaks with two different HPLC methods (A and B) and various detectors as well as their mass-

308

and UV spectra. Further compounds were identified by their mass spectra via LC-MS/MS and in

309

comparison with references described in the literature. All other compounds could be associated

310

with reference standards by comparing the retention times and UV-spectra via HPLC-PDA.

311

Measured data for already known compounds in Salvia species (5, 12,33,34 15,35 16,10,36 and 1737)

312

isolated from sage flower resin agree with literature data. The peaks are shown in Figure 1 and

313

their structures in Figure 2.

314

Compound 8 was obtained as white-beige amorphous plates (6 mg, purity by HPLC-ELSD:

315

98.6%; Method A and B) with UV maxima of 228, 295 and 306 nm and an exact molecular

316

weight of 583.26824 Da with the calculated chemical formula C34H37N3O6. The compound was

317

identified in comparison with HRMS-, UV- and IR-data from the literature as tri-p-

318

coumaroylspermidine, 8.38–40

319

Compound 10 was obtained as orange crystalline compound, m.p. 96-97 °C (8.7 mg, purity by

320

HPLC-ELSD: 99%; Method A and B) with UV maxima of 213, 272 and 400 nm and an exact

321

molecular weight of 376.15220 Da with the calculated chemical formula C20H24O7. The UV

322

maximum at 400 nm indicates a quinone system in the molecule. The different NMR analyses

323

(1H; 13C{1H}; DEPT 135; 1H/13C-HSQC; 1H/13C-HMBC; NOESY) indicated a structure with 20

324

carbons in the molecule including a quinone, lactone and carbon acid group. The isopropyl group

325

at C-13 (δ 152.3 ppm) and the two methyl groups at C-4 (δ 31.8 ppm) have the same chemical

326

shifts as in galdosol. The chemical shift at C-7 from δ 191.8 ppm in galdosol, 12, to 172.5 ppm in

327

salviquinone, 10, occurs due to the additional adjacent oxygen atom. Also C-8 has a different

328

chemical shift due to the new chemical vicinity. The crystal structure is prone to inversion ACS Paragon Plus Environment

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twinning which was taken into account in the course of structure refinements. C20H24O7

330

molecules (salviquinone A) and water of crystallization constitute the crystal structure of 10. The

331

salviquinone A molecules are densely packed via moderate intermolecular hydrogen bonds with

332

the water of crystallization.41 NMR- and single-crystal X-ray diffraction analyses independently

333

led to the same molecular structure, supported by HRESIMS analysis. The new compound was

334

identified

335

oxooctahydroisobenzofuran-1-carboxylic acid (Figure 2 and 3). Due to the fact that the identified

336

compound has not yet been described, it was named salviquinone A.

337

Compound 11 was also obtained as orange crystalline compound (7 mg, purity by HPLC-

338

ELSD: 100%; Method A and B) with UV maxima of 213, 271 and 400 nm. The similar UV

339

spectrum of 11 indicates a chemical relationship with a quinone structure in comparison to

340

compound 10. Furthermore, compound 11 was also produced in alcoholic sage flower extracts by

341

contact with oxygen. The HSQC showed that C-21 belongs to the methyl ester of 10 that is also

342

indicated due to the protons (δ 3.844 ppm, s) of the methyl group, which is bonded to the

343

carboxylic acid hydroxyl group. Using the HRMS- and NMR-spectra from compound 10, the

344

compound 11 was identified as its methyl ester with the IUPAC name (2-hydro-4-isopropyl-3,6-

345

dioxocyclohexa-1,4-dien-1-yl)-7-7-dimethyl-3-oxooctahydroisobenzofuran-1-carboxylic

346

methyl ester (C21H26O7). This previously unreported compound was named salviquinone B. It

347

was not possible to establish the stereochemistry for salviquinone B.

348

Compound 13b was obtained also as orange crystalline compound (1 mg, purity by HPLC-

349

ELSD: > 95%; Method A and B) with UV maxima of 212, 277 and 395 nm. A complete

350

identification of compound 13b failed but it probably represents a derivative of 11 with an

351

additional double bond within the molecule due to its molecular weight of 388.1522 Da and

352

calculated chemical formula C21H24O7.

353

Analysis of Fresh Plant Material. After the identification of the compounds in sage flower resin,

354

it was ascertained that not all the compounds were known in the literature regarding the genus

as

(2-hydro-4-isopropyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-7-7-dimethyl-3-

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Salvia. Most of the sage compounds were identified in leaf extracts in the past. Therefore, the

356

resin from the flowers was compared with a resin from the leaves. However, sage resin from the

357

leaves does not include compound 8 which was identified as tri-p-coumaroylspermidine, 8

358

(Figure 4). Therefore, 8 must be a compound which is typical for flowers of the Salvia species.

359

This supposition was verified by analyzing 8 in the flowers of some more Salvia species.

360

Additionally, the flowers of lavender plants, which also belong to the mint family, were analyzed

361

and 8 identified also. These results confirm that spermidine conjugates are typical for the flowers.

362

Spermidine conjugates have already been identified in plant flowers. Cinnamoyl spermidine

363

conjugates were identified in some angiosperms for the first time in 1978.42 These compounds

364

belong to the polyamine class and are biosynthesized from putrescine. Compound 8 isolated from

365

the pollen of Corylus avellana L. was first reported in 1986.43 Compound 8 was also detected in

366

the flowers of other plants such as Artemisia caruifolia or Arachis hypogaea in the past.38,39 We

367

have now identified 8 in the flowers of Salvia officinalis, S. tomentosa, S. nemorosa, S. cadmica,

368

S. sclarea and S. lavandulifolia (Table 2) and in flowers of Lavandula augustifolia for the first

369

time.

370

An analytical observation of the blooming period and the flower development of S. officinalis

371

showed an increasing correlation between flowering and the biosynthesis of 8 in the plant (Figure

372

5). During the development processes of plant organs, 8 can be detected in sage flowers only in a

373

special time window. In this period, the development of petals advances. In the budding and

374

fading or seed ripening period, 8 concentrations are very low. Only at the peak of flowering were

375

the amounts of 8 high. In sage (S. officinalis), 8 is mainly located in the blue-violet colored petals

376

of the flower. Polyamine substances are involved in growth responses and development

377

processes. Furthermore, a function of the polyamines as protection against pathogens, cell

378

division, and flowering induction or sexual differentiation has been investigated.39,44–46 These

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plant physiological properties might explain this observed behavior in sage flowers due to the

380

location of 8 in the petals.

381

Analysis of Manufacturing Technology of Sage Flower Resin. Sage flower resin contains mainly

382

nonpolar compounds and includes natural compounds and for sage also unknown artefacts in

383

high concentrations. Interesting interconversions of some of the compounds included in sage

384

flower resin could already be observed during the production process. In an alcoholic solution,

385

carnosol extraction increases over time from plant material. At the same time, carnosol appears to

386

be transformed into rosmanol, galdosol, and salviquinone A. Some caffeic acid is converted into

387

its ethyl ester during the maceration in ethanol and rosmarinic acid dissociates into caffeic acid

388

and danshensu. The curves in Figure 6 are the result of complex processes between the extraction

389

of the compounds and its transformation into other compounds. Moreover, the storage of

390

alcoholic sage flower extracts after the maceration process results in a decreasing amount of

391

carnosol and an increase in other compounds. Thus, salviquinone A appears as dominant peak in

392

oxidized sage flower resin (Figure 1C). The content is more than 13 times higher than in native

393

sage flower resin (Table 1). However, an analysis with oxygen-free sage flower fluid extracts

394

(full bottle filling without gas phase) stored over 8 months at 4 °C reveals less decrease in

395

carnosol. The storage of oxygen-free sage flower fluid extracts at room temperature results in a

396

transition of carnosol to rosmanol and galdosol. But only the storage of a half-filled bottle at

397

room temperature leads to the formation of salviquinone A. A second experiment was conducted

398

over one month with opened vessels. The result was an increase in rosmanol, galdosol,

399

salviquinone A, salviquinone B, and unknown 13b, whereas carnosol decreases (Figure 7). In

400

summary, these experiments indicate that the contact of the extracts with oxygen is the main

401

reason for the transformation of the compounds, particularly for the generation of the new

402

elucidated compound salviquinone A.

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Possible Pathway of the Oxidation Process. The transformation from carnosol into galdosol is

404

well known and has already been described.47 The pathway shown in Figure 8 hypothesizes a

405

mechanism for formation of salviquinone A from carnosol.

406

Potential Markers for Quality Control. Tri-p-coumaroylspermidine, 8, might be a quality marker

407

for the application of sage flowers, and can provide information about the proportion of the

408

flowers. The ratio of the weight proportion of the flowers to the inflorescences is a further

409

important quality parameter for the Bombastus Company and its sage flower products. Caffeic

410

acid ethyl ester, 5, indicates the use of ethanol in sage flower extraction. Salviquinone A, 10, also

411

shows potential as a marker for quality control, due to the fact that 10 is generated from carnosol

412

only by exposure to oxygen (Figure 7). Kinetic experiments will be needed to determine the rate

413

of the transformation from carnosol to salviquinone A.

414

Antibacterial Properties of Sage Flower Extracts. In this study we investigated the antimicrobial

415

effect of different sage extracts against Gram-positive and Gram-negative bacteria (Table 3,

416

Figure 9). We used inhibition zone experiments due to their robustness and easy performance.

417

This method is well-suited for first checking an antimicrobial effect. As a result, we were able to

418

confirm the findings of Walther22 concerning the antimicrobial activity of sage flower resin

419

against Gram-positive bacteria.22 Moreover, sage flower resin also causes growth inhibition of

420

the Gram-negative bacterium E. coli. Sage flower CO2 extract showed a growth inhibition against

421

S. aureus and B. subtilis. The results clearly showed no antibacterial activity of sage leaf resin

422

against Gram-positive and Gram-negative bacteria in the analyzed concentration. These

423

experiments indicate that sage resin from the flowers has antibacterial activities against Gram-

424

positive and Gram-negative bacteria and is an interesting extract for antiseptic applications. In

425

ancient times, fresh sage leaves were used for rubbing on the teeth for cleaning them and

426

strengthening the gums, so the antibacterial effect of sage leaf extracts on Streptococcus mutans

427

was already known.48 S. mutans belongs to the Gram-positive bacteria and is the cause of dental ACS Paragon Plus Environment

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caries. Sage flower CO2 extracts are used in tooth paste, today. Its medicinal effect against S.

429

mutans is not scientifically proven. However, sage flower resin is not yet applied as a medicinal,

430

active substance but has the potential as a new application for fighting dental caries. The

431

experiments were carried out using sage flower resin (from the years 2010 and 2014 and one

432

oxidized resin from 2009) and a sage flower CO2 extract from 2010. The antibacterial in vitro

433

activity of sage flower resin against S. mutans was proven here for the first time. The potency

434

was nearly 80% in comparison to chlorhexidine-digluconate in the same concentration (0.2%)

435

and showed a weak concentration-dependent behavior between 0.1-0.3% (Figure 9). The

436

increases of the inhibition zones are not proportional to the concentration due to the saturation

437

concentration of the sage flower resin. Higher concentrations do not necessarily cause higher

438

inhibition zones. Chlorhexidine is a commercially available substance, active against oral

439

pathogens, which is included in mouth rinses. However, sage flower CO2 extract caused no

440

effects at all. Chemical variations in the composition of sage flower resin had no negative effect

441

on the high antibacterial activity against S. mutans.

442

In summary, sage flower resin (SFR) has a specific high antibacterial in vitro activity, especially

443

against S. mutans, and includes some new compounds which have their origin in the flowers of

444

the plant and in the manufacturing process. These compounds have potential as new markers for

445

the quality control of sage flower resin. Nevertheless, possible applications in dentistry are

446

supported by other ingredients such as flavonoids, hydroxycinnamonic acids and phenolic

447

diterpenes which have antioxidant and anti-inflammatory effects.4,49

448

ASSOCIATED CONTENT

449

Supporting Information

450

This material is available free of charge via the Internet at http://pubs.acs.org.

451 452

1

H and

13

C 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 ACS Paragon Plus Environment

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different conditions; HPLC-PDA-MS data of sage and lavender flower extracts; HPLC profiles

454

from petals and sepals; correlation between the yield of sage flower resin and the part of flowers

455

on inflorescences; HPLC semipreparative profile of pre-fractioning; GC-MS analysis of essential

456

oils from sage leaves, sage flowers and sage flower CO2 extract (PDF)

457

AUTHOR INFORMATION

458

Corresponding Authors

459

*(S.G.) Phone: +49 351 276 99442. Fax: +49 351 463 33132. E-Mail: [email protected]

460

*(K.S.) Phone: +49 351 46333132. Fax: +49 351 463 33132. E-Mail: [email protected]

461

dresden.de

462

ORCID

463

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

464

Notes

465

The authors declare no competing financial interest.

466

ABBREVIATIONS USED

467

DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; PDA, Photo Diode Array;

468

SF-CO2-extract, sage flower CO2 total extract; SFR, sage flower resin; SFR-ox, oxidized sage

469

flower resin.

470

ACKNOWLEDGMENTS

471

We sincerely thank the Institute of Bioanalytical Chemistry, Dr. Susann Machill (TU Dresden,

472

Germany), for the QTOF-MS measurements, the Bombastus-Werke AG, Dr. Christoph Grunert

473

(Freital, Germany) and the Botanical Garden, Dr. Barbara Ditsch (TU Dresden, Germany) for the

474

provision of the sage plants.

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REFERENCES (1) Brand, N. Salvia. In Hagers Enzyklopädie der Arzneipflanzen und Drogen, edition 6; 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 officinalis). Food Chem. 2001, 2, 197–203. (5) Schwarz, K.; Ternes, W. Antioxidative constituents of Rosmarinus officinalis and Salvia officinalis. I. Determination of phenolic diterpenes with antioxidative activity amongst tocochromanols using HPLC. Z Lebensm Unters Forch 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 Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1. J. Pharm. 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 anti-inflammatory agent. Cancer Lett. 2011, 305, 1–7. (9) Behradmanesh, S.; Derees, F.; Rafieian-Kopaei, M. Effect of Salvia officinalis 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 officinalis L.). J. Ethnopharmacol. 2010, 132, 127–133.

ACS Paragon Plus Environment

21

Journal of Agricultural and Food Chemistry

Gericke et al. 2017 (11)

Kavvadias, D.; Monschein, V.; Sand, P.; Riederer, P.; Schreier, P. Constituents of sage (Salvia

officinalis) 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 officinalis L. (Lamiaceae). Arch. biol. sci. (Beogr.) 2008, 60, 421–429. (13)

Tada, M.; Okuno, K.; Chiba, K.; Ohnishi, E.; Yoshii, T. Antiviral diterpenes from Salvia

officinalis. Phytochemistry 1994, 35, 539–541. (14)

Bozin, B.; Mimica-Dukic, N.; Samojlik, I.; Jovin, E. Antimicrobial and antioxidant properties of

rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis 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 fruticosa 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 officinalis 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 officinalis 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 officinalis as activators of the nuclear factor E2-related factor 2 pathway. Bioorg. Med. Chem. 2013, 21, 2618–2622.

ACS Paragon Plus Environment

22

Page 22 of 40

Page 23 of 40

Journal of Agricultural and Food Chemistry

Gericke et al. 2017 (20)

Capek, P.; Hr̆ıb́ alová, V.; Švandová, E.; Ebringerová, A.; Sasinková, V.; Masarová, J.

Characterization of immunomodulatory polysaccharides from Salvia officinalis 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 Harzkörpers 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 officinalis 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.

ACS Paragon Plus Environment

23

Journal of Agricultural and Food Chemistry

Gericke et al. 2017 (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 officinalis 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, 283–303. ACS Paragon Plus Environment

24

Page 24 of 40

Page 25 of 40

Journal of Agricultural and Food Chemistry

Gericke et al. 2017 (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.

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Figure captions 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. Figure 2. Chemical structures of identified compounds. Figure 3. (A) ORTEP diagram and (B) molecule structure of (2-hydro-4-isopropyl-3,6dioxocyclohexa-1,4-dien-1-yl)-7-7-dimethyl-3-oxooctahydroisobenzofuran-1-carboxylic acid x H2O (salviquinone A), 10. Figure 4. Comparison of HPLC-PDA profiles (285 nm) for (A) sage flower resin and (B) sage leaves resin (both 3.0 mg/mL in MeOH, Method A). Figure 5. Amount of tri-p-coumaroylspermidine, 8 (d.w.) in fresh sage flowers (Salvia officinalis 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]. 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]. Figure 7. HPLC PDA profiles (Method A): Storage experiments of sage flower fluid extract (285 nm). (A) Before storing; (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. ACS Paragon Plus Environment

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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% chlorhexidine-digluconate solution was used as positive control. Tested sage flower CO2 extract showed no antibacterial effects. [Mean ± SD, * SD = 0, cm, n = 3, 80 µL].

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Tables Table 1. Major Constituents of Used Sage Extracts by LC Analysis with Method A

n.d., not detected; n.a., not analyzed; ox, oxidized; ∆ inflorescences; + calc. as luteolin-7-O-glucuronide; * calc. as salviquinone A; # calc. as carnosol. All other compounds were calibrated with analytical standards. Calibration curves with standard reference compounds purities < 95% were corrected with multiplication of each concentration point with the purity of the compound. Calibration correlations (R²) were ≥ 0.990.

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Table 2. Amount of Tri-p-coumaroylspermidine, 8, Measured in Fresh Flowers and Calculated on Dry Weight of some Salvia Species Salvia species harvest year

S. officinalis 2014

S. sclarea 2014

S. lavandulifolia 2014

S. tomentosa 2013

S. nemorosa 2013

S. cadmica 2013

3.2 ± 0.0(2)

3.8 ± 0.3(2)

[Mean ± SD, mg/g, d.w., n = 4] compound 8

2.1 ± 0.4(0)

0.9 ± 0.0(1)

0.9 ± 0.0(4)

5.1 ± 0.2(6)

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Table 3. Antibacterial Activity by Zone of Inhibition Test with Sage Extracts and Gram-negative and Gram-positive Bacteria

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

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Figures Figure 1

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Figure 2 OH OH HO

O

OH O HO

OH

danshensu, 1

O

O

HO

caffeic acid, 2

luteolin-7-O-glucuronide, 3

OH O

OH

OH

O

O

HO

HO

O

O

HO

HO

rosmarinic acid, 4

caffeic acid ethyl ester, 5

apigenin, 7 16

tri-p-coumaroylspermidine, 8 16

17

15

13 14

14

12

8

11

16

8

11

9

9

3

4

20

10 5

2 3

6

1

4

10

20

5

6

7 18

19

2 7

18

3

1

4

10

9

17

14 8 7

5 6

19

18

salviquinone A, 10

13

20

21

15

12

11 1

rosmanol isomer, 9

17

15

13 12

2

luteolin, 6

salviquinone B, 11

19

galdosol, 12

carnosol, 13 OH

carnosic acid, 14

20-hydroxyferruginol, 15 12-O-methyl carnosic acid, 16 ACS Paragon Plus Environment

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ferruginol, 17

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Figure 3 A

B

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Figure 4

38

39

40

41

42

min

38

39

40

41

42

min

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Figure 5

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Figure 6

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Figure 7

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Figure 8 O

H

ring-opening H O and oxidation

O O O

?

O R O

carnosol, 13

rosmanol, 9

galdosol, 12

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salviquinone, 10/11

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Figure 9

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Table of Contents Graphic

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