Morphology and Phytochemistry of Sanguisorba Officinalis L. Seeds (Rosaceae)
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Journal of Applied Botany and Food Quality 94, 92 - 98 (2021), DOI:10.5073/JABFQ.2021.094.011 1Department of Analytical Development and Research, Section Phytochemical Research, WALA Heilmittel GmbH, Bad Boll/Eckwälden, Germany 2Department of Plant Systems Biology, Hohenheim University, Stuttgart, Germany Morphology and phytochemistry of Sanguisorba offcinalis L. seeds (Rosaceae) Marek Bunse1,2, Florian Conrad Stintzing1, Dietmar Rolf Kammerer1,* (Submitted: October 15, 2020; Accepted: April 29, 2021) Summary grow 10 to 200 cm high, with further leaves arranged alternately up Great burnet (Sanguisorba offcinalis) has been used as medicinal the stem. The leaves are pinnate with serrated margins. Flowers are plant for more than 2000 years. However, little is known about the small, tetramerous or trimerous and often unisexual, and they lack morphology and the secondary metabolites of its seeds. The inves- petals (WANG et al., 2020). The stamina have long flaments, and gy- tigations reported here focus on the morphology and the characteri- noecia consist of a single carpel topped with a feathery style (KALK- zation of phenolics and fatty acids in S. offcinalis seeds. For this MAN, 2004). The fowers are small, dense clusters or spikes with a purpose, dried seeds were investigated using scanning electron length of 1 to 7 cm (BLAscHEK et al., 2018; UCHIDA and OHARA, microscopy to clarify their compartment structures. Furthermore, 2018). The fowering stage ranges from June to September and the fruit phases are usually from August to November. The fruits of San- the seeds were extracted with CH2Cl2 and MeOH to characterize the fatty acids and to assess the secondary metabolite profle. The guisorba species belong to the nuts. The nuts of great burnet (S.f o - seed structure consists of a foral cup, a brown pericarp with cal- fcinalis) are narrowly winged (Fig. 1), with smooth surfaces, oval cium oxalate crystals, a fbre layer and the seed kernel with its seed or broadly triangular in cross-section; not heteromorphic. The size coat. Individual compounds were characterized by high performance ranges from 2.6 × 1.4 × 1.4 mm to 3.5 × 2.2 × 2.1 mm (VERBAND liquid chromatography and gas chromatography coupled with mass BOTANIscHER GÄRTEN, 2020). S. offcinalis is a typical traditional n Chinese medicinal plant. Especially the roots and herbal parts have spectrometric detection (HPLC-DAD-MS and GC/MS). CH2Cl2 extraction and GC investigations revealed the occurrence of fatty been used to treat burns, hematemesis, asthma, intestinal infec- acids (29 % of the seed dry weight), with linoleic acid, linolenic acid, tions and dermatitis (LEE et al., 2010; YANG et al., 2015; YOKOZAWA and oleic acid as major compounds. In addition, MeOH extracts were et al., 2002; YU et al., 2011; ZHANG et al., 2018). The primary bio- analyzed by LC/MSn, which revealed the occurrence of favonoids logically active constituents belong to the terpenoids, tannins and fa- (quercetin, catechin, epicatechin), ellagitannins and caffeic acid de- vonoids, which are associated with antioxidant, anti-infammatory, rivatives. antiviral, antifungal, hemostatic and cytotoxic activities (CAI et al., 2012; KIM et al., 2008; LIANG et al., 2013; SUN et al., 2012; ZHANG et al., 2013; ZHANG et al., 2012). There is an increasing interest of Introduction fnding essential fatty oils needed for human health and well-being, for pharmaceuticals, nutritional foods or cosmetics. As an example, Members of the Rosaceae family are generally subdivided into three the seed oil of Borago offcinalis L. is rich in gamma-linolenic acid subfamilies: Rosoideae, Amygdaloideae, Dryadoideae. This family and used as dietary food supplement and ingredient for cosmetic of fowering plants includes about 4,828 known species in 91 genera, preparations (ASADI-SAMANI et al., 2014). Many traditional medi- which may grow as trees, shrubs, or herbaceous plants. This family cinal plants contain phenolic fatty oil in their seeds, which could is characterized by exceptional horticultural signifcance with eco- be of interest for exactly these purposes. But there are only a few nomically important fruit-bearing plants. The fruits occur in many studies on almost forgotten medicinal plants and their primary and varieties and were once considered the main characters for the def- secondary metabolites of the seeds. Since studies on S. offcinalis nition of the aforementioned subfamilies. The fruits are also charac- seeds are still rare, the present study aimed at a profound characteri- terized by great diversity and may occur as follicles, capsules, nuts, zation of the phenolic compounds and reports the detailed structure achenes, drupes and accessory fruits, like the pome of an apple, or of the seeds for the frst time. the hip of a rose. Well-known examples of the three subfamilies are as follows: Amygdaloideae: plums, cherries, peaches, apricots, al- monds, apples, pears, quince, ornamental shrubs such as Exochorda, Materials and methods Sorbaria and Physocarpus; Dryadoideae: Cercocarpus, Chamae- batia, Dryas, Purshia; Rosoideae: strawberries, blackberries and Seeds of Sanguisorba offcinalis L. (Year of harvest: 2015; Location: raspberries, roses and other ornamental and medicinal plants such Germany; voucher number: HOH-022713; thousand-seed weight COP as Geum, Potentilla, Alchemilla and Sanguisorba (CHALLICE, 1974; (TSW) = 2.04082 g), Sanguisorba minor S . (Year of harvest: un- MCNEILL, 2012; SIMPSON, 2018). known; Location: Germany; voucher number: HOH-022757; TSW = The genus Sanguisorba is comprised of approximately 18 to 34 spe- 8.33333 g), Sanguisorba tenuifolia FIscH. EX LInk (Year of harvest: cies and subspecies, which are widely distributed in the Northern unknown; Location: Germany; voucher number: HOH-022755; hemisphere of Eurasia and North America (GBIF Secretariat, 2019; TSW = 1.33333 g), Sanguisorba parvifora TAKEDA (S. tenuifolia var. KURTTO, 2009). The common name of Sanguisorba in western coun- parvifora; Year of harvest: unknown; Location: Germany; voucher tries is burnet, and the plant is known to have hemostatic properties. number: HOH-022756; TSW = 1.0989 g) were acquired from Jelitto The plants are perennial herbs with leaf rosettes, the stems of which Perenial Seeds GmbH (Schwarmstedt, Germany). The species were identifed by Dr. R. Duque-Thüs and voucher specimens were de- posited with the Herbarium of the Institute of Botany, Hohenheim * Corresponding author University. Seeds of Sanguisorba offcinalis L. 93 Dry seeds were cut with a razor blade and were photographed with a Sony alpha 6000 (SIGMA 70 mm 1:2.8 DG MACRO + Macro lens). Seeds of S. offcinalis were mounted on adhesive carbon tabs on aluminium stubs and sputter-coated with gold-palladium (20/80; 14-15 mA; 0.1 - 0.15 mbar; SCD 040, Balzer Union, Wallruf, Germa- ny) and investigated using a scanning electron microscope DSM 940 (Zeiss, Oberkochen, Germany) at 5 kV (LORENZ et al., 2018). More- over, seeds of S. offcinalis were photographed at different develop- mental stages, i.e. after pre-soak in water (5 days), after germination (8 days) and as seedling (14 days). Furthermore, seeds of S. offci- nalis (20.0 g) were immersed in CH2Cl2 (180 ml) and comminuted by Ultra-Turrax treatment (3 min; 21000 rpm, IKA Werke GmbH & Co. KG, Staufen, Germany), under external ice cooling. After mace- ration overnight at 4 °C the seeds were fltered off over Celite by vacuum suction and extracted a second time in the same manner. An oil fraction (5.74 g; 28.7% of the seed weight) was recovered from the combined CH2Cl2 extracts by vacuum rotoevaporation of the solvent. Subsequently, the degreased seeds were extracted twice with MeOH (180 ml) overnight (+ 4 °C), fltered off and MeOH was removed in vacuo to yield a crude extract. For GC analyses, fatty acid methyl esters were prepared by on-column derivatization with trimethylsulfonium hydroxide (TMSH, 0.25 M in MeOH). Briefy, 10 mg of viscous oil sample (CH2Cl2 residue) were dissolved in 2000 μl TBME. Aliquots of 10 μl of this test solution were mixed with 170 μl of TBME followed by 60 μl of TMSH. Subsequently, the mixture was directly injected into the GC/MS system (PerkinElmer Clarus 500; (HEINRICH et al., 2017)). For phenolic compound analysis, the previously mentioned MeOH crude extract (3-5 mg/ml) was redissolved in MeOH/H2O (50/50, v/v). Liquid chromatographic analyses were carried out on an Agilent 1200 HPLC system (Agilent Technologies, Inc., Palo Alto, USA), equipped with a binary pump, a micro vacuum degasser, an auto- sampler, a thermostatic column compartment and a UV/VIS diode array detector. A Kinetex® C18 reversed-phase column (2.6 μm par- ticle size, 150 × 2.1 mm i.d., Phenomenex Ltd., Aschaffenburg, Ger- many) was used for chromatographic separation. The LC system was coupled to an HCTultra ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) with an ESI source operating in the nega- tive ion mode (BUnsE et al., 2020). For characterizing the compound profles, three biological replicates (n = 3) of all samples were pre- Fig. 1: Overview of seed shapes and their morphological variations of S. of- pared. In addition, technical replicates were prepared to obtain the fcinalis (A), S. minor (B), S. tenuifolia (C) and S. parvifora (D). graphs, which allowed the calculation of standard deviations. Scale bare = 1 mm. Results cotyledons, the epicotyl, the hypocotyl and the radicle covered by Seed morphology the seed coat. The nuts of Sanguisorba reveal species-dependent variations. Natu- Beside the aforementioned morphological structures, the longitudi- rally, also nuts within a species reveal variations of their individual nal section of pre-swollen seeds (5 days) of great burnet (Fig. 4, A) appearance (Fig. 1). Fig. 2 shows seeds and their cross sections of showed the white endosperm of the cotyledons. Furthermore, SEM S. offcinalis (A), S. minor (B), S. tenuifolia (C), and S. parvifora (D). analyses revealed the epicotyl (e) of the embryo with the base of The species A, C, D are characterized by similar narrowly winged plumule.