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 officinalis L. seeds (Rosaceae) Marek Bunse1,2, Florian Conrad Stintzing1, Dietmar Rolf Kammerer1,* (Submitted: October 15, 2020; Accepted: April 29, 2021) * Corresponding author Summary Great burnet (Sanguisorba officinalis) has been used as medicinal plant for more than 2000 years. However, little is known about the morphology and the secondary metabolites of its seeds. The inves- tigations reported here focus on the morphology and the characteri- zation of phenolics and fatty acids in S. officinalis seeds. For this purpose, dried seeds were investigated using scanning electron microscopy to clarify their compartment structures. Furthermore, the seeds were extracted with CH2Cl2 and MeOH to characterize the fatty acids and to assess the secondary metabolite profile. The seed structure consists of a floral cup, a brown pericarp with cal- cium oxalate crystals, a fibre layer and the seed kernel with its seed coat. Individual compounds were characterized by high performance liquid chromatography and gas chromatography coupled with mass spectrometric detection (HPLC-DAD-MSn and GC/MS). CH2Cl2 extraction and GC investigations revealed the occurrence of fatty acids (29 % of the seed dry weight), with linoleic acid, linolenic acid, and oleic acid as major compounds. In addition, MeOH extracts were analyzed by LC/MSn, which revealed the occurrence of flavonoids (quercetin, catechin, epicatechin), ellagitannins and caffeic acid de- rivatives. Introduction Members of the Rosaceae family are generally subdivided into three subfamilies: Rosoideae, Amygdaloideae, Dryadoideae. This family of flowering plants includes about 4,828 known species in 91 genera, which may grow as trees, shrubs, or herbaceous plants. This family is characterized by exceptional horticultural significance with eco- nomically important fruit-bearing plants. The fruits occur in many varieties and were once considered the main characters for the defi- nition of the aforementioned subfamilies. The fruits are also charac- terized by great diversity and may occur as follicles, capsules, nuts, achenes, drupes and accessory fruits, like the pome of an apple, or 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, Sorbaria and Physocarpus; Dryadoideae: Cercocarpus, Chamae- batia, Dryas, Purshia; Rosoideae: strawberries, blackberries and raspberries, roses and other ornamental and medicinal plants such as Geum, Potentilla, Alchemilla and Sanguisorba (ChalliCe, 1974; MCNeill, 2012; SiMpSoN, 2018). The genus Sanguisorba is comprised of approximately 18 to 34 spe- cies and subspecies, which are widely distributed in the Northern hemisphere of Eurasia and North America (GBIF Secretariat, 2019; Kurtto, 2009). The common name of Sanguisorba in western coun- tries is burnet, and the plant is known to have hemostatic properties. The plants are perennial herbs with leaf rosettes, the stems of which grow 10 to 200 cm high, with further leaves arranged alternately up the stem. The leaves are pinnate with serrated margins. Flowers are small, tetramerous or trimerous and often unisexual, and they lack petals (WaNg et al., 2020). The stamina have long filaments, and gy- noecia consist of a single carpel topped with a feathery style (KalK- MaN, 2004). The flowers are small, dense clusters or spikes with a length of 1 to 7 cm (BlaSCheK et al., 2018; uChida and ohara, 2018). The flowering stage ranges from June to September and the fruit phases are usually from August to November. The fruits of San- guisorba species belong to the nuts. The nuts of great burnet (S. of- ficinalis) are narrowly winged (Fig. 1), with smooth surfaces, oval or broadly triangular in cross-section; not heteromorphic. The size ranges from 2.6 × 1.4 × 1.4 mm to 3.5 × 2.2 × 2.1 mm (VerBaNd BotaNiSCher gärteN, 2020). S. officinalis is a typical traditional Chinese medicinal plant. Especially the roots and herbal parts have been used to treat burns, hematemesis, asthma, intestinal infec- tions and dermatitis (lee et al., 2010; YaNg et al., 2015; YoKozaWa et al., 2002; Yu et al., 2011; zhaNg et al., 2018). The primary bio- logically active constituents belong to the terpenoids, tannins and fla- vonoids, which are associated with antioxidant, anti-inflammatory, 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 finding essential fatty oils needed for human health and well-being, for pharmaceuticals, nutritional foods or cosmetics. As an example, the seed oil of Borago officinalis L. is rich in gamma-linolenic acid and used as dietary food supplement and ingredient for cosmetic preparations (aSadi-SaMaNi et al., 2014). Many traditional medi- cinal plants contain phenolic fatty oil in their seeds, which could be of interest for exactly these purposes. But there are only a few studies on almost forgotten medicinal plants and their primary and secondary metabolites of the seeds. Since studies on S. officinalis seeds are still rare, the present study aimed at a profound characteri- zation of the phenolic compounds and reports the detailed structure of the seeds for the first time. Materials and methods Seeds of Sanguisorba officinalis l. (Year of harvest: 2015; Location: Germany; voucher number: HOH-022713; thousand-seed weight (TSW) = 2.04082 g), Sanguisorba minor SCop. (Year of harvest: un- known; Location: Germany; voucher number: HOH-022757; TSW = 8.33333 g), Sanguisorba tenuifolia FiSCh. ex liNK (Year of harvest: unknown; Location: Germany; voucher number: HOH-022755; TSW = 1.33333 g), Sanguisorba parviflora taKeda (S. tenuifolia var. parviflora; Year of harvest: unknown; Location: Germany; voucher number: HOH-022756; TSW = 1.0989 g) were acquired from Jelitto Perenial Seeds GmbH (Schwarmstedt, Germany). The species were identified by Dr. R. Duque-Thüs and voucher specimens were de- posited with the Herbarium of the Institute of Botany, Hohenheim University. Seeds of Sanguisorba officinalis 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. officinalis 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. officinalis 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. offici- 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 filtered 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), filtered 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). Briefly, 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 profiles, three biological replicates (n = 3) of all samples were pre- pared. In addition, technical replicates were prepared to obtain the graphs, which allowed the calculation of standard deviations. Results Seed morphology The nuts of Sanguisorba reveal species-dependent variations. Natu- rally, also nuts within a species reveal variations of their individual appearance (Fig. 1). Fig. 2 shows seeds and their cross sections of S. officinalis (A), S. minor (B), S. tenuifolia (C), and S. parviflora (D). The species A, C, D are characterized by similar narrowly winged seed shapes and contained only one seed kernel (achene), whereas the nuts of S. minor (B) usually contained 1-3 seed kernels per nut, and the hypanthium showed serrated netting strips. For a more detailed investigation of the seed morphology, SEM images of the seed structures of S. officinalis were taken. The dried fruit (seed) of S. officinalis consists of several layers (Fig. 3). The floral cup or hypanthium forms the outer layer, the surface of the fruit. The pericarp is located below the perigynous hypanthium and encloses the inner seed kernel. The pericarp shows an outer layer with cells containing crystal structures. The cells located inside show a fibrous structure with thickened inner walls and extended cells. The pericarp converges at the apex of the seed and forms the stylus, which ends with the stigma in the flowering stage. The seed kernel is located inside the pericarp. It consists of the embryo with its two cotyledons, the epicotyl, the hypocotyl and the radicle covered by the seed coat. Beside the aforementioned morphological structures, the longitudi- nal section of pre-swollen seeds (5 days) of great burnet (Fig. 4, A) showed the white endosperm of the cotyledons. Furthermore, SEM analyses revealed the epicotyl (e) of the embryo with the base of plumule. Fig. 4 B shows the primary root of a seedling 8 days after germination with a piece of seed coat at its tip. After 6 further days, a seedling of S. officinalis (Fig. 4, C) has developed a root (r), an elon- gated hypocotyl (h), green cotyledons (co), and the epicotyl shows primary leaves (pl, leaf bud) at an early developmental stage. Lipid constituents For investigating the lipid constituents, a CH2Cl2 extract from dried seeds of S. officinalis was prepared to yield a fatty oil (29% of dry weight of the seeds). GC/MS analyses revealed a complex peak pro- file. By comparing the mass spectra of individual components with those of reference compounds, a number of fatty acids with carbon- chain lengths of C16 - C22 were assigned (Fig. 5). Unsaturated fatty acids: 39% linolenic acid (C18:3), 36% linoleic acid (C18:2), 17% Fig. 1: Overview of seed shapes and their morphological variations of S. of- ficinalis (A), S. minor (B), S. tenuifolia (C) and S. parviflora (D). Scale bare = 1 mm. 94 M. Bunse, F.C. Stintzing, D.R. Kammerer A B C D 238 Fig. 3: SEM micrographs of the fruit (seed) of Sanguisorba officinalis. A, Longitudinal section. B, Longitudinal 239 section of the perygynous hypanthium and the pericarp with calcium oxalate crystals. C, Inner layer of the pericarp 240 with elongated cells. D, Surface of the fruit. E, Surface of the seed kernel (testa, seed coat). e: epicotyl; h: hypocotyl; 241 r: radicle. Scale bars: A, D = 500 µm, B = 20 µm, C = 50 µm, E = 200 µm. 242 243 hypanthium pericarp stylus embryo cotyledons h r e testa hypanthium pericarp calcium oxalate A B C D E Fig. 3: SEM micrographs of the fruit (seed) of Sanguisorba officinalis. A, Longitudinal section. B, Longitudinal section of the perygynous hypanthium and the pericarp with calcium oxalate crystals. C, Inner layer of the pericarp with elongated cells. D, Surface of the fruit. E, Surface of the seed kernel (testa, seed coat). e: epicotyl; h: hypocotyl; r: radicle. Scale bars: A, D = 500 μm, B = 20 μm, C = 50 μm, E = 200 μm. Fig. 2: Seed shapes and cross sections of S. officinalis (A), S. minor (B), S. tenuifolia (C) and S. parviflora (D). Scale bare = 2 mm. 244 Fig. 4: A, longitudinal section of a S. officinalis seed after pre-soak in water for 5 days. B, germinated seed 245 (S. officinalis) with primary root (r) after 8 days. C, seedling 14 days after germination. 246 co: cotyledons, e: epicotyl, h: hypocotyl, pl: primary leaves, r: radicle/ root. Scale bars = 1 mm. 247 248 A C testa cotyledons h r e pericarp hypanthium B r co e h r pl Fig. 4: A, longitudinal section of a S. officinalis seed after pre-soak in water for 5 days. B, germinated seed (S. officinalis) with primary root (r) after 8 days. C, seedling 14 days after germination. co: cotyledons, e: epicotyl, h: hypocotyl, pl: primary leaves, r: radicle/ root. Scale bars = 1 mm. oleic acid (C18:1) and < 0.5% eicosenoic acid (C20:1); saturated fatty acids: 4% palmitic acid (C16:0), 2% stearic acid (C18:0), < 2% ara- chidic acid (C20:0) and behenic acid (C22:0). Thus, linolenic acid, linoleic acid and oleic acid were the predominant fatty acids. Phenolic constituents Methanolic crude extracts of the previously defatted seeds of S. of- ficinalis were investigated by LC/MSn. In summary, 18 compounds were tentatively assigned in this fraction based on their retention time (tR), UV spectra, mass-to-charge ratios (negative ionization mode), as well as their specific fragmentation patterns and corresponding bibliographic references. The chromatogram (Fig. 6) and mass spectra revealed the occurrence of compounds belonging to differ- ent substance classes. Among these, hydroxybenzoic acids (methyl- gallate-hexoside), tannins (ellagic acid), flavonoids (quercetin-pento- side, catechin, procyanidins) were assigned, but also L-tryptophan, being an aromatic representative of amino acids (Tab. 1). Further- more, the relative abundance [%] of the assigned compound is shown in Fig. 7. A B C D A B C D Seeds of Sanguisorba officinalis L. 95 249 Fig. 5: Fatty acid composition of S. officinalis seeds illustrated as mean value of the relative abundance of individual 250 compounds [%] including standard deviation (n=3). Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), 251 linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), eicosanoic acid (C20:1), behenic acid (C22:0). 252 253 254 255 256 Fig. 6: LC/MSn chromatogram (UV detection at 280 nm) of phenolic compounds in a MeOH seed extract of 257 Sanguisorba officinalis. For compound characterization, cf. Table 1. 258 259 0 10 20 30 40 50 re la ti ve a bu nd an ce [% ] UV 280 nm 0 5 10 15 20 25 In te ns iti y [m A U ] 20 30 40 50 Time [min]0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 249 Fig. 5: Fatty acid composition of S. officinalis seeds illustrated as mean value of the relative abundance of individual 250 compounds [%] including standard deviation (n=3). Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), 251 linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), eicosanoic acid (C20:1), behenic acid (C22:0). 252 253 254 255 256 Fig. 6: LC/MSn chromatogram (UV detection at 280 nm) of phenolic compounds in a MeOH seed extract of 257 Sanguisorba officinalis. For compound characterization, cf. Table 1. 258 259 0 10 20 30 40 50 re la ti ve a bu nd an ce [% ] UV 280 nm 0 5 10 15 20 25 In te ns iti y [m A U ] 20 30 40 50 Time [min]0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fig. 5: Fatty acid composition of S. officinalis seeds illustrated as mean value of the relative abundance of individual compounds [%] including standard deviation (n=3). Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), ara- chidic acid (C20:0), eicosenoic acid (C20:1), behenic acid (C22:0). Fig. 6: LC/MSn chromatogram (UV detection at 280 nm) of phenolic com- pounds in a MeOH seed extract of Sanguisorba officinalis. For com- pound characterization, cf. Tab. 1. 260 Fig. 7: Phenolic composition of S. officinalis seeds illustrated as mean value of the relative abundance of individual 261 compounds [%] (n=2). For compound assignment, see table 1. 262 263 Tables 264 265 Tab. 1: Compound assignment of metabolites detected in MeOH extracts of S. officinalis seeds using HPLC-DAD-266 ESI-MSn (negative ionization mode) 267 268 No.a Constituent tR [min] llmax, UV [nm] [M-H]- [m/z] Fragmentation ions [m/z] Reference 1 L-tryptophan 20.8 218, 280 203 186, 159, 142, 116 b 2 1-O-caffeoylquinic acid 24.2 324, 374 353 345, 323, 289, 191, 179, 171, 135, 127 (Plazonić et al., 2009) 3 methyl-gallate-hexoside 26.1 214, 270 345 183, 168, 124 (Abu-Reidah et al., 2015) 4 procyanidin B1/B2 27.9 324, 374 578 559, 425, 407, 289, 285, 257 (Bunse et al., 2020) 5 trans-3-p-coumaroylquinic acid 29.2 204, 230sh, 312 337 311,289, 191, 163, 119 b(Makita et al., 2017) 3.2 % 0.5 % 7.0 % 0.1 % 1.7 % 33.5 % 14.9 % 1.1 % 5.6 % 0.4 % 7.6 % 6.1 % 4.0 % 0.3 % 2.9 % 1.6 % 0.7 % 1.2 % 2.5 % 5.1 % 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fig. 7: Phenolic composition of S. officinalis seeds illustrated as mean value of the relative abundance of individual compounds [%] (n=2). For compound assignment, see Tab. 1. Discussion The dry fruits of Sanguisorba form achenes, which persist in dense spikes until late autumn when they shatter, scattering most seeds within a 1 m2 area around the plant. When harvested, a dry, papery calyx hull surrounds the achenes (holloWaY and MatheKe, 2003). The fruits of S. officinalis, S. tenuifolia, and S. parviflora (Fig. 1) are ellipsoid to globose, 4-angled, 4-winged with smooth surfaces and one achene. In contrast, the fruits of S. minor have serrated netting strips. The serrated surface of S. minor appears to be an adaption aiming at the distribution of seeds via the fur of mammals. Large numbers of seeds are distributed by passive attachment to the fur of mammals. As mammals walk through the vegetation, seeds of dif- ferent sizes, rough surfaces and distributions of hooks are dislodged from the parent plants and attached to the fur (StileS and FeNNer, 2000). S. officinalis has short, erect botryoids with several single flowers on it. The gynoecium, the female part of a flower, is unicarpellate and includes a stigma, a stylus and a unilocular ovary. It is surrounded by a perigynous hypanthium. Within the locule, a single anatropous unitegmic ovule is formed. After successful fertilization of an inflo- rescence, the seed (embryo) is formed inside the gynoecium. Dur- ing that process the inflorescence begins to wither, the bracts and the stamina at the apex of the hypanthium and the stigma dry out and fall off. The fruit (seed, nut) is formed with the dried hypan- thium still serving as protection. The gynoecium wall becomes the pericarp during fructification. The inner epidermis of the pericarp is called the endocarp, the outer epidermis is referred to as exocarp, with the mesocarp in between. In the case of nuts, these three layers are equally lignified. The outer layer of S. officinalis pericarp, the exocarp, appears to be less lignified, but at the same time containing calcium oxalate crystals (Weddellit, Ca(C2O4) · 2 H2O, (hartl et al., 2007)). Calcium oxalate widely occurs in plants and may account for 3-80% of plant dry weight (liBert and FraNCeSChi, 1987). As much as 90% of total calcium in a plant may be found as its oxalate salt (gallaBer, 1975). Calcium oxalate is generally considered an end product, and thus its formation reduces the availability of cal- cium, although some studies have also shown calcium oxalate to be a reversible product (FraNCeSChi and horNer, 1980; FraNCeS- Chi, 1989; huaNg et al., 2015). The occurrence of calcium oxalate crystals in flowering plants is more or less widespread in different plant parts, such as leaves, stems, roots, floral parts, fruits, seeds and pericarps (MuKherjee and jaNa, 2014). Calcium oxalate plays an important role in plant defense. Among others, it is an effective de- terrent to herbivores. The embryo is surrounded by a thin seed coat, the testa. The two cotyledons serve as nutritional tissue for germination. To monitor seed germination and development into a seedling, nuts of S. offici- nalis were pre-swollen in water and scattered on soil. After 5 days the cotyledons of the embryo seemed to bulge, and the epicotyl, hypocotyl and radicle started to be developed. After 8 days the radi- cle of the first seedlings broke off the seed coat, and 14 days after germination small seedlings were developed. Seeds of great burnet (S. officinalis) germinate most rapidly at ca. 25 °C after 6 months of dry storage at 4 °C. The cold period is necessary to break seed dormancy. The seeds also germinate without stratification, but then only very sporadically and over a longer period of time (holloWaY and MatheKe, 2003). Starch, sugars and fats of the cotyledons provide the energy for ger- mination. The seeds were analyzed by GC/MS to assess their fatty acid profile revealing an average composition typical of members of the Rosaceae family. The study of MatthauS and ÖzCaN (2014) showed that oil contents of 26 varieties of Rosaceae seeds ranged from 3.5 to 46.2 g/100 g. These oils were composed of 3.25-9.17% palmitic, 1.19-4.27% stearic, 6.50-67.11% oleic, 22.08-68.62% lino- leic and 0.10-61.59% eicosenoic acids (MatthauS and ÖzCaN, 2014). Based on these wide ranges it is not surprising that the fatty acid profile of S. officinalis seeds is within the aforementioned mar- gins (unsaturated fatty acids: 39% linolenic acid (C18:3), 36% lino- leic acid (C18:2), 17% oleic acid (C18:1) and < 0.5% eicosenoic acid 96 M. Bunse, F.C. Stintzing, D.R. Kammerer Tab. 1: Compound assignment of metabolites detected in MeOH extracts of S. officinalis seeds using HPLC-DAD-ESI-MSn (negative ionization mode). No.a Constituent tR λmax, UV [M-H]- Fragmentation Reference [min] [nm] [m/z] ions [m/z] 1 L-tryptophan 20.8 218, 280 203 186, 159, 142, 116 b 2 1-O-caffeoylquinic acid 24.2 324, 374 353 345, 323, 289, 191, (Plazonić et al., 2009) 179, 171, 135, 127 3 methyl-gallate-hexoside 26.1 214, 270 345 183, 168, 124 (aBu-reidah et al., 2015) 4 procyanidin B1/B2 27.9 324, 374 578 559, 425, 407, 289, (BuNSe et al., 2020) 285, 257 5 trans-3-p-coumaroylquinic acid 29.2 204, 230sh, 312 337 311, 289, 191, b(MaKita et al., 2017) 163, 119 6 procyanidin 30.3 202, 230sh, 280 577 559, 451, 425, 407, (BuNSe et al., 2020) 389, 289, 285, 257, 213 7 catechin / epicatechin 31.9 204, 230sh, 280 289 245, 227, 203, 187, (zhao et al., 2013) 175, 161 8 caffeoylquinic acid 32.4 202, 232, 324 353 289, 250, 191, 173, b 127, 85 9 procyanidin trimer 33.1 202, 230, 280 865 847, 739, 695, 677, (roCKeNBaCh et al., 2012) 577 543, 525, 391 10 5,6,7-trihydroxy-2,3-dihydrocyclopenta[b] 36.5 280, 358, 522 291 247, 219, 203, 191, (FraterNale et al., 2015) chromene-1,9-dione-3-carboxylic acid 175 11 p-coumaroylquinic acid 39.3 232, 312 337 191, 173, 155, 127, (BuNSe et al., 2020) 111, 93, 85 12 procyanidin dimer 40.7 202, 230, 282 577 451, 425, 407, 389, (BuNSe et al., 2020) 289, 285, 257, 213 13 unidentified gallic acid derivative 42.9 202, 224, 278 497 465, 345, 183, 168, (hoFMaNN et al., 2016) 124 14 trigalloyl hexose 43.9 280, 360 635 465, 313, 285, 241, (aBu-reidah et al., 2015) 221, 193, 169 15 unidentified gallic acid derivative 45.3 234, 274, 364 497 463, 345, 313, 183, (hoFMaNN et al., 2016) 168, 124 16 unidentified 46.0 280, 374 537 519, 339, 195, 179, 161, 143, 119, 89 17 unidentified 51.7 234, 280, 314 662 644, 602, 516, 456, 438, 324, 307, 248, 205, 163, 145 18 quercetin-3-O-arabinoside / Dxyloside 53.1 240, 258, 362 433 345, 301, 257, 229, (ReSpect for Phytochemicals, 185 2020.000Z) 19 ellagic acid 54.7 254, 362 301 284, 257, 245, 229, b 201, 185, 165 20 isoquercetrin 56.5 238, 268, 360 463 301, 271, 255, 229, (iBrahiM et al., 2015) 193, 179, 151, 107 a For peak assignment see Fig. 5. b Reference spectra (MoNa, 2020). (C20:1); saturated fatty acids: 4% palmitic acid (C16:0), 2% stearic acid (C18:0), < 2% arachidic acid (C20:0) and behenic acid (C22:0)). In addition, the phenolic profile of S. officinalis seeds is typical of the rose family (Fahad al juhaiMi et al., 2016). Hydroxybenzoic acids, tannins and flavonoids belong to the main classes of phenols detected in great burnet herb, roots and flowers (BuNSe et al., 2020). Most of these phenolics are bioactive constituents and are exploited for pharmacological purposes as mentioned before. In contrast, the role of these compounds in seeds of S. officinalis is still largely unknown and needs further elucidation. However, based on their activity pro- file, protection of the seeds from bacterial or fungal growth and rot appears conclusive. Conclusion This study aimed at a profound characterization of the morphology of Sanguisorba officinalis seeds and of their primary and secondary metabolites. For the first time the structure of great burnet seeds was elucidated by scanning electron microscopy. The seed is enclosed by a thin seed coat, which is surrounded by the pericarp and the floral Seeds of Sanguisorba officinalis L. 97 cup. As a special feature, calcium oxalate crystals were discovered in the outer layer of the lignified pericarp. The occurrence of calcium oxalate crystals in flowering plants is more or less widespread in dif- ferent plant parts and plays an important role in plant defense. In addition to these morphological studies, the fatty acid and phenolic compound profiles of S. officinalis seeds were characterized for the first time. The genus Sanguisorba is an important member of the Ro- saceae family, the natural habitat of which is becoming increasingly rare. This short study provides a first insight into the seed structure of great burnet and complements previous studies on the flower deve- lopment of different Sanguisorba species (WaNg et al., 2020). In the future, further Sanguisorba species should be investigated regard- ing their morphology and phytochemistry to allow direct comparison of taxonomically related species. Furthermore, more detailed infor- mation on different compartment structures also considering their specific secondary metabolites may help to deduce the functionality of the latter in the plant. This may also contribute to a better chemo- taxonomic differentiation between closely related species or hybrids. In addition, new sources of healthy fatty oils, especially needed for human health and well-being, but also cosmetic applications, are in- creasingly important. For example, they can supply the body with essential fatty acids or support it in healing processes, e.g. inflam- matory skin regions. In the future there could be a greater focus on indigenous medicinal plants cultivated locally, that contain high- quality oils as deduced from their compound profile being abundant in unsaturated fatty acids and revealing the occurence of biologically active phenolics. Acknowledgement The authors are grateful to Prof. Dr. Waltraud X. Schulze (Depart- ment of Plant Systems Biology, Hohenheim University) for the sup- port in creating the manuscript. The authors also gratefully acknow- ledge Dr. Rhinaixa Duque-Thüs (Institute of Botany, Hohenheim University) for identification of the plant specimens and we would like to thank Dr. Annerose Heller and her team (Institute of Botany, Hohenheim University) for the great support in teaching microscopy and botany over the last years. Conflict of interest The authors reported no potential conflict of interest. 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DOI: 10.1093/chromsci/bms138 ORCID Marek Bunse https://orcid.org/0000-0003-2563-6534 Dietmar Rolf Kammerer https://orcid.org/0000-0003-1126-3431 Florian Conrad Stintzing https://orcid.org/0000-0001-7006-6804 Address of the corresponding authors: Prof. Dr. Dietmar Rolf Kammerer, Department of Analytical Development and Research, Section Phytochemical Research, WALA Heilmittel GmbH, Dorfstraße 1, 73087 Bad Boll/Eckwälden, Germany E-mail: dietmar.kammerer@wala.de © The Author(s) 2021. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1186/s40529-014-0048-4 http://dx.doi.org/10.3390/molecules14072466 http://dx.doi.org/10.1016/j.foodres.2012.07.001 http://dx.doi.org/10.3390/molecules17077629 http://dx.doi.org/10.1111/1442-1984.12195 http://dx.doi.org/10.1093/botlinnean/boaa009 http://dx.doi.org/10.1016/j.phymed.2015.09.006 http://dx.doi.org/10.1016/S0006-2952(01)00930-3 http://dx.doi.org/10.1016/j.jep.2010.08.060 http://dx.doi.org/10.1016/j.ijbiomac.2018.01.214 http://dx.doi.org/10.1016/S1995-7645(13)60117-0 http://dx.doi.org/10.3390/molecules171213917 http://dx.doi.org/10.1093/chromsci/bms138 https://orcid.org/0000-0003-2563-6534 https://orcid.org/0000-0001-7006-6804 https://orcid.org/0000-0003-1126-3431