Sesquiterpenes from Myrrh and Their ICAM-1 Inhibitory Activity In Vitro

By using various chromatographic steps (silica flash, CPC, preparative HPLC), 16 sesquiterpenes could be isolated from an ethanolic extract of myrrh resin. Their chemical structures were elucidated by 1D and 2D NMR spectroscopy and HRESIMS. Among them, six previously unknown compounds (1–6) and another four metabolites previously not described for the genus Commiphora (7, 10, 12, 13) could be identified. Sesquiterpenes 1 and 2 are novel 9,10-seco-eudesmanes and exhibited an unprecedented sesquiterpene carbon skeleton, which is described here for the first time. New compound 3 is an 9,10 seco-guaian and the only peroxide isolated from myrrh so far. Compounds 1, 2, 4, 7–9, 11, 13–16 were tested in an ICAM-1 in vitro assay. Compound 7, as well as the reference compound furanoeudesma-1,3-diene, acted as moderate inhibitors of this adhesion molecule ICAM-1 (IC50: 44.8 and 46.3 μM, respectively). These results give new hints on the activity of sesquiterpenes with regard to ICAM-1 inhibition and possible modes of action of myrrh in anti-inflammatory processes.


Introduction
Myrrh is a gum resin produced by plants of the genus Commiphora (Burseraceae), but is mainly obtained from Commiphora myrrha (NEES) ENGL. a spinescent tree that is native to northeastern Africa, southern Arabia and India [1,2]. Besides its traditional use as incense, embalming ointment or perfume, it always had relevance as a medicinal plant and was often administered for diseases related to infection and inflammation [1,3].
In modern times, it has been shown that myrrh has antimicrobial, analgesic and anti-inflammatory activities in vitro and in vivo [1]. For instance, it is a potent inhibitor of many chemokines, cytokines and prostaglandine in vitro, which act as pro-inflammatory mediators. The essential oil of myrrh can, for example, inhibit the production of IL-1bstimulated IL-6 and IL-8 in human gingival fibroblasts [4]. Further, ethanolic extracts were able to reduce CXCL13 and TNFα levels in activated human macrophages [5] or IL-6, IL-8, PGE 2 , MCP-1 and TNFα in a co-culture cell model of the intestinal mucosa [6], whereas an aqueous extract inhibited LPS-induced production of NO in peritoneal macrophages [7]. These effects seem to be not limited to cell culture models and have also been confirmed in vivo. It has been shown that myrrh reduces inflammatory mediators as well as the numbers of neutrophils and macrophages during cecal ligation and puncture-induced sepsis in mice and thus can increase the survival rate compared to a control group [7]. Furthermore, in an acetic acid-induced ulcerative colitis (UC) in rats, myrrh was able to decrease NO and PGE 2 levels significantly and attenuate inflammatory processes in a concentration dependent manner [8].

Results and Discussion
By alternating maceration and percolation an ethanolic myrrh extract was prepared from the resin and portioned by liquid-liquid partition between methanol and n-heptane. Subsequently the n-heptane fraction was further separated by subsequent silica-flash, centrifugal partition chromatography (CPC) and preparative HPLC on a biphenyl column to obtain 16 sesquiterpenes (Figure 1). Compounds 1-6 are described here for the first time and four substances (7, 10, 12, 13) were previously unknown for the genus Commiphora. The isolation of compounds 9 and 14 was published before [6] as part of a characterization of a myrrh extract. Now these two substances together with recently obtained compounds and two reference substances, namely, furanoeudesma-1,3-diene (FUR) and curzerenone (CUR), are examined in an ICAM-1 in vitro assay.
For several sesquiterpenes the biosynthetic cleavages of C-C bonds is described resulting in seco-compounds. Thus, for example elemanes derive from eudesmanes by bond cleavage between pos. 2 and 3, but there also exist references for other seco-sesquiterpenes, which originate for example from eudesmanes [20][21][22], cadinanes [23], germacranes [23] or guaianes [24][25][26]. Thus, compounds 1 und 2 could derive from eudesmanes like 8 by cleavage of the C-C bond between pos. 9 and 10. To the best of our knowledge, this is the first report for the isolation of this unique carbon skeleton and reflecting trivial names of known sesquiterpenoides we suggest the names 9-nor-9,10-seco-isolindestrenolide (1) and 9,10-seco-isohydroxylindestrenolide (2).   (Table 1). Subsequent analysis of HMBC and COSY spectra revealed that 3 also possess an α,β-unsaturated γ-lactone like compound 1, which here is linked to a cyclopentane ring. Due to HMBC signals to both ring systems, pos. 6 seemed to form the conjunction between these two structure elements. Furthermore, a methyl and an isopropenyl group could be established as substituents of the cyclopentyl ring and the position of the oxygenated carbons was ascertained in pos. 4 and 6. Nevertheless, the formula determined by HRESIMS showed one more degree of unsaturation and due to the fact that all other atoms could be unambiguously assigned, a closure of the cyclic peroxide from pos. 4 to 6 could be implied ( Figure 3a). Additionally, the relative configuration was determined by investigation of NOESY spectra, which showed signals between the methyl group (H3-15) and the protons H-5 and H2-8. Thus, it could be assumed that these elements are located at one side of the cyclic oxide ( Figure 3b). Furthermore, correlations between pos. 5, 15 and the isopropyl protons (H-9, -14) could also be observed and indicated an identical orientation in pos. 1 (Figure 3c), which implied a rel-1R,4R,5R,6S configuration. Compound 2 (2.1 mg) was obtained as a colorless oil and the molecular formula, which was calculated by HRESIMS as C 15 3 and one oxygen in comparison to 1. A first analysis of the NMR data showed similarities to compound 1 except of a missing signal for the sp 3 methylene C-8. Instead, an oxygenated tertiary carbon (δ H 105.3 (C-8)) and a tertiary methyl (δ H 1.58 (s, H 3 -9); δ C 24.3) (Table 1) was observed, which indicated a different substitution pattern. Due to HMBC signals the proximity of the additional CH 3 group and pos. 9 could be demonstrated adding a methyl and a hydroxyl group at the α,β-unsaturated γ-lactone ring ( Figure 2).
For several sesquiterpenes the biosynthetic cleavages of C-C bonds is described resulting in seco-compounds. Thus, for example elemanes derive from eudesmanes by bond cleavage between pos. 2 and 3, but there also exist references for other seco-sesquiterpenes, which originate for example from eudesmanes [20][21][22], cadinanes [23], germacranes [23] or guaianes [24][25][26]. Thus, compounds 1 und 2 could derive from eudesmanes like 8 by cleavage of the C-C bond between pos. 9 and 10. To the best of our knowledge, this is the first report for the isolation of this unique carbon skeleton and reflecting trivial names of known sesquiterpenoides we suggest the names 9-nor-9,10-seco-isolindestrenolide (1) and 9,10-seco-isohydroxylindestrenolide (2 (Table 1). Subsequent analysis of HMBC and COSY spectra revealed that 3 also possess an α,β-unsaturated γ-lactone like compound 1, which here is linked to a cyclopentane ring. Due to HMBC signals to both ring systems, pos. 6 seemed to form the conjunction between these two structure elements. Furthermore, a methyl and an isopropenyl group could be established as substituents of the cyclopentyl ring and the position of the oxygenated carbons was ascertained in pos. 4 and 6. Nevertheless, the formula determined by HRESIMS showed one more degree of unsaturation and due to the fact that all other atoms could be unambiguously assigned, a closure of the cyclic peroxide from pos. 4 to 6 could be implied ( Figure 3a). Additionally, the relative configuration was determined by investigation of NOESY spectra, which showed signals between the methyl group (H 3 -15) and the protons H-5 and H 2 -8. Thus, it could be assumed that these elements are located at one side of the cyclic oxide ( Figure 3b). Furthermore, correlations between pos. 5, 15 and the isopropyl protons (H-9, -14) could also be observed and indicated an identical orientation in pos. 1 (Figure 3c), which implied a rel-1R,4R,5R,6S configuration. Endoperoxid substructures have been rarely found among secondary plant metabolites, but are also present in other sesquiterpenes like artemisinine. Furthermore, the biosynthetic origin of the carbon skeleton of compound 3 can be explained by a C-C cleavage between pos. 8 and 9 in a guaiane type sesquiterpene. In the literature, other seco-guaianolides were previously described mostly in the genus Tanacetum and Artemisia. The majority of these compounds belong to the 1,10-seco-type [24,25,[27][28][29], but also 4,5-seco-molecules do occur [26,30] whereas a 8,9 C-C cleavage has only once been reported for Curcuma wenyujin [31]. Thus, describing a peroxide in myrrh for the first time, we suggest the name myrrhanoperoxide.   (Table 2). Secondary analyses of 2D data suggested that 4 is a guaiane type sesquiterpene with an olefinic double bond between C-10 and C-14, a hydroxylation at C-4 and an acetylation at C-11. Key HMBC and COSY correlations are shown in Figure 4a. Due to NOESY signals between H-1, H-15, H-6a (δH 1.51) and H-7 these protons could be located on one side of the ring level, whereas H-6b (δH 1.64) and the acetylated isopropyl structure (H-12/13) form a signal on the other side. Thus, the relative configuration 1S,4R,7S was established while the relative stereochemistry at pos. 5 could not be determined due to interference of H-5 with other protons. Literature research showed that a structure with the same constitution has been previously isolated [32] whereby a 1R,4R,5R configuration was postulated for a [α]D = +20 Although the few published NMR shifts match those of 4, it is excluded that the two compounds are identical because of distinct NOESY signals and a deviating [α] (+38.2). Therefore, reflecting trivial names of known guaianes, we suggest the name rel-(+)-(1S,4R,7S)-11-acetyl-guai-10(14)-en-4,11-ol for 4.
Compound 5 (4.1 mg) is an isomer of 4 with the same molecular formula C17H28O3 (HRESIMS, m/z 303.1930 [M + Na] + , calcd. for 303.1931) and was also obtained as a colorless oil. A review of NMR data showed similar characteristics and shifts except for the sp 2 methylene (C-14), which was missing and replaced by an additional tertiary methyl (δH 1.55 (s, H3-14); δC 20.9) ( Table 2). This indicated a shift of the double bond from pos. 10/14 to 1/10, which could be confirmed by HMBC and COSY correlations ( Figure 4b).
Additionally, the position of the acetyl group could be established by a NOESY correlation between H3-2′ and the two methyl groups in pos. 12/13. Furthermore, in contrast to 4, the complete relative configuration could be determined by NOESY signals above the ring level from H-6a (δH 1.51) to H-7 and -15 and below from H-5 to H-6b (δH 1.72) and the two methyl groups H-12/13 ( Figure 4c). Thus, a relative stereochemistry of 4R,5R,7S can Endoperoxid substructures have been rarely found among secondary plant metabolites, but are also present in other sesquiterpenes like artemisinine. Furthermore, the biosynthetic origin of the carbon skeleton of compound 3 can be explained by a C-C cleavage between pos. 8 and 9 in a guaiane type sesquiterpene. In the literature, other seco-guaianolides were previously described mostly in the genus Tanacetum and Artemisia. The majority of these compounds belong to the 1,10-seco-type [24,25,[27][28][29], but also 4,5seco-molecules do occur [26,30] whereas a 8,9 C-C cleavage has only once been reported for Curcuma wenyujin [31]. Thus, describing a peroxide in myrrh for the first time, we suggest the name myrrhanoperoxide.
Additionally, the position of the acetyl group could be established by a NOESY correlation between H 3 -2 and the two methyl groups in pos. 12/13. Furthermore, in contrast to 4, the complete relative configuration could be determined by NOESY signals above the ring level from H-6a (δ H 1.51) to H-7 and -15 and below from H-5 to H-6b (δ H 1.72) and the two methyl groups H-12/13 (Figure 4c). Thus, a relative stereochemistry of 4R,5R,7S can be implied and we suggest the name rel-(+)-(4R,5R,7S)-11-acetyl-guai-1(10)-en-4,11-ol in analogy to 4.
Compound 6 (0.9 mg) was isolated as a colorless oil and assigned a molecular formula of C 18 H 22 O 5 by HRESIMS (m/z 341.1359 [M + Na] + , calcd. 341.1359). According to NMR data, the structure contained an aromatic system consisting of five quaternary carbons (δ C 124.0 (C-7), 131.7 (C-1), 133.0 (C-6), 140.3 (C-10), 153.4 (C-8)) and a sp 2 methine (δ H 6.96 (s, H-9); δ C 112.9) as well as a methoxy (δ H 3.45 (s, H 3 -1 ); δ C 56.4) and an acetyl substituent (δ H 2.17 (s, H 3 -2 ); δ C 20.9 (C-2 ) and 171.1 (C-1 )). Additionally, three tertiary methyls ((δ H 1.09 (d, H 3 -15 1, 74.9, respectively) and a carbonyl carbon (δ C 178.3 (C-12)) were found ( Table 3). The structure showed similarities to previously isolated commiterpene B from C. myrrha [33] including the substitution pattern and relative configuration, but the signals of H-11 and H-13 were split to a quartet and a doublet and thus shared a coupling constant of 7.4 Hz. This indicated that here the furan ring of commiterpene B is oxygenated to a γ-lactone as in case of other cadinane type sesquiterpenlactones from Chloranthus henryi [34] (Figure 5a). To determine the orientation of the so emerged additional stereo center in pos. 11, the NOESY signals of the substituents were compared with those of pos. 5. An R configuration could be implied due to the strong correlation between the H-11 and the acetyl group as well as H-5 to H-13 ( Figure 5b). Referring to the nomenclature started by Xu et al. [33], we suggest the name commiterpene D for compound 6.  Compound 7 (6.6 mg) was obtained as a colorless oil and assigned a molecular formula of C15H18O2 by HRESIMS (m/z 231.1382 [M + H] + , calcd. for 231.1380). According to NMR data 7, was identified as previously isolated lindestrenolide [35], which was described 1964 by Takeda et al. as a constituent of Lindera strychnifolia VILL. Due to the fact that the characterization of this compound in the literature is insufficient, a complete set of NMR data is here reported for the first time (shown in Figure 6 and Table 3). Key to structure elucidation were HMBC correlations and three independent spin systems, which indicated a typical eudesmanolide. Additional NOESY signals between H-9a (δH 2.37) and -5 as well as between H-8 and H-14 revealed a 5S,8S,10S configuration as described in the literature.  ). According to NMR data 7, was identified as previously isolated lindestrenolide [35], which was described 1964 by Takeda et al. as a constituent of Lindera strychnifolia VILL. Due to the fact that the characterization of this compound in the literature is insufficient, a complete set of NMR data is here reported for the first time (shown in Figure 6 and Table 3). Key to structure elucidation were HMBC correlations and three independent spin systems, which indicated a typical eudesmanolide. Additional NOESY signals between H-9a (δ H 2.37) and -5 as well as between H-8 and H-14 revealed a 5S,8S,10S configuration as described in the literature. In the literature the diastereomeres 15 and 16 were isolated in a 1:1 mixture and characterized as 2-methoxyisogermafurenolide and 8-epi-2-methoxyisogermafurenolid with a configuration of rel-5S,8R,10R and rel-5S,8S,10R, respectively [36]. Now thes compounds could be separated for the first time by preparative HPLC on a bipheny column, which allowed a more precise assessment of their stereochemistry by NOESY spectra. Thus, the configuration of 16 could be confirmed, while the new data indicated different relative stereochemistry for 15. This was suggested by correlations under th ring level from H-5 to -9a and the methyl group at pos. 14 as well as above between H-9 and -1, -8 and -15 ( Figure 7). In addition, signals from one side of the ring to the othe were missing or much weaker. Therefore, a rel-5S,8S,10S configuration for 15 seem much more likely, defining this compound as 10-epi isomer of 16, namely 10-epi-2-methoxyisogermafurenolide. In the literature the diastereomeres 15 and 16 were isolated in a 1:1 mixture and characterized as 2-methoxyisogermafurenolide and 8-epi-2-methoxyisogermafurenolide with a configuration of rel-5S,8R,10R and rel-5S,8S,10R, respectively [36]. Now these compounds could be separated for the first time by preparative HPLC on a biphenyl column, which allowed a more precise assessment of their stereochemistry by NOESY spectra. Thus, the configuration of 16 could be confirmed, while the new data indicated a different relative stereochemistry for 15. This was suggested by correlations under the ring level from H-5 to -9a and the methyl group at pos. 14 as well as above between H-9e and -1, -8 and -15 ( Figure 7). In addition, signals from one side of the ring to the other were missing or much weaker. Therefore, a rel-5S,8S,10S configuration for 15 seems much more likely, defining this compound as 10-epi isomer of 16, namely, 10-epi-2methoxyisogermafurenolide. different relative stereochemistry for 15. This was suggested by correlations under the ring level from H-5 to -9a and the methyl group at pos. 14 as well as above between H-9e and -1, -8 and -15 (Figure 7). In addition, signals from one side of the ring to the other were missing or much weaker. Therefore, a rel-5S,8S,10S configuration for 15 seems much more likely, defining this compound as 10-epi isomer of 16, namely 10-epi-2-methoxyisogermafurenolide. Other compounds were identified according to literature as lindestrenolide (7) [35] isohydroxylindestrenolide (8) [36], hydroxylindestrenolide (9) [35,37], atractylenolide III (10) [38], commiphorane E3 (11) [39], 4β-hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione (12) [40], isogermafurenolide (13) [35], hydroxyisogermafurenolide (14) [35,37] and 2-methoxyiso-germafurenolide (15) [36].

Purity and ICAM-1 Inhibition
To examine the activity of myrrh, the ethanolic extract and an HEP fraction (n-heptane fraction) were tested in an in vitro assay to monitor the TNFα dependent expression of ICAM-1 in HMEC-1 cells. The assay has been performed as previously described [41]. In brief, the assay was carried out using an untreated control (u.c.), a negative control with TNFα (10 ng/mL), referred to as 100% value and a positive control with parthenolide (5 µM). Whereas the extract showed a moderate effect, the HEP fraction was able to cause a significant inhibition of ICAM-1 expression in a concentration depended manner ( Figure 8). All tested concentrations were also investigated in a MTT assay for their cytotoxic effect on HMEC-1 cells and the mean viability was within a range of 95-105% ( Figure S1). tested concentrations were also investigated in a MTT assay for their cytotoxic effect on HMEC-1 cells and the mean viability was within a range of 95-105% ( Figure S1). Compounds that could be obtained in sufficient purity (>90%) and amount, 1, 2, 4, 7-9, 11, 13-16 as well as two reference substances (FUR and CUR, Figure 1) from C. myrrha were also tested in the same assay. Despite of the structural resemblance of all the tested substances, only two of them were able to reduce the ICAM-1 expression in a significant manner. Compound 7 as well as furanoeudesma-1,3-diene showed a concentration dependent effect in this test with IC50 values of 44.8 and 46.3 μM, respectively (Figure 9). All tested concentrations were investigated in an MTT assay for their effect on HMEC-1 cells Figure 8. Influence of the ethanolic extract and the n-heptane fraction (HEP) on ICAM-1 expression in HMEC-1 cells. The test was performed with pure medium (u.c.), with TNFα (10 ng/mL) and parthenolide + TNFα (par, 5 µM) as positive control. Substance concentrations between 12.5-50 µg/mL were applied. Data are presented as mean ± SD; # p < 0.001 vs. u.c.; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNFα (n = 3).
Compounds that could be obtained in sufficient purity (>90%) and amount, 1, 2, 4, 7-9, 11, 13-16 as well as two reference substances (FUR and CUR, Figure 1) from C. myrrha were also tested in the same assay. Despite of the structural resemblance of all the tested substances, only two of them were able to reduce the ICAM-1 expression in a significant manner. Compound 7 as well as furanoeudesma-1,3-diene showed a concentration dependent effect in this test with IC 50 values of 44.8 and 46.3 µM, respectively ( Figure 9). All tested concentrations were investigated in an MTT assay for their effect on HMEC-1 cells and the mean viability was within a range of 95-105% ( Figure S1).
Several sesquiterpenlactones like helenalin [42] and parthenolide [43] are known for their ability to inhibit the central transcription factor NF-κB, which is linked to TNFα. The mechanism of this interaction has been intensely investigated and affiliated to α,βunsaturated carbonyl structures, for example α-methylene-γ-lactones or α,β-unsubstituted cyclopentenones which can cause a Michael like addition of sesquiterpene lactones to SH-groups of NF-κB [44][45][46]. This alkylation prevents the transcription factor of binding to the DNA and enhance the production of proinflammatory mediators and effectors like ICAM-1 [47].
All compounds tested didn't contain α-methylene-γ-lactones and from the α,βunsubstituted cyclopentenone substructures in 11 and 12 a potent Michael activity cannot be expected [48]. Thus, activity of 7 and FUR is somewhat surprising. Remarkably, all tested substances exhibiting a hydroxyl group in pos. 8 such as 8, 9 and 14 did not show any activity in the assay although other structural elements remained unaltered. A similar observation was made in a quantitative structure-activity relationship study, which showed that the number of hydroxyl groups in a sesquiterpene lactone has a negative effect on its inhibitory effect on NF-κB [49]. Weather this is the result of an interference with the mode of action or the hydroxyl groups just prevents the molecule of reaching the cytosol through lipophilic membranes remains unclear. Furthermore, cleavages between pos. 9 and 10 (seco-eudesmanes 1 and 2) or 2 and 3 (elemanes 13-16) seem to result in a loss of activity even though the lipophilicity remains alike.
In contrast to compound 7, furanoeudesma-1,3-diene is missing the lactone ring, which is replaced by a 3-methylfuran. Therefore, an alkylation of NF-κB as mode of action of furanoeudesma-1,3-diene seems also unlikely. Curzerenone also did not show any activity due to the fact, that the carbonyl structure is located next to the furan ring and therefore not accessible for a Michael like addition.
To our knowledge, this is the first report showing an activity of myrrh compounds towards an ICAM-1 inhibition. ICAM-1 expression is known to be upregulated in IBD and considered as a possible target for UC [19]. UC aetiology is not fully understood but the current understanding is that different factors lead to an inadequate immune response and intestinal barrier impairment [11][12][13]. Myrrh extracts have already shown activity in in vitro and in vivo tests aiming towards this inadequate immune response and intestinal barrier impairment [5,6,8,50]. This study hints towards a new possible mechanism for myrrh or myrrh constituents in IBD emphasizing a multimodal activity of myrrh.

Plant Material and Extraction
Powdered myrrh resin of C. myrrha (Myrrha, Ph. Eur. 2016) was provided by Lomapharm ® (lot NM0160, Rudolf Lohmann GmbH KG, Emmerthal, Germany). 3 kg powdered resin was mixed with 4.5 kg sea sand and extracted over seven days by alternating percolation and maceration with 26 L ethanol 96% (v/v). The extract was dried by evaporation and lyophilisation yielding a total amount of 761.65 g (DER: 3.9:1) and was stored light protected at −20 • C.

Liquid-liquid Partition
Four portions (100 g) of ethanolic extract were solved in 1 L methanol each and partitioned eight times with 0.5 L n-heptane in a separatory funnel. Subsequently the combined methanol (MeOH) and n-heptane (HEP) portions were dried by evaporation and lyophilisation and were stored light protected at −20 • C. The total amounts gained during this process add up to 328.13 g (MeOH) and 69.99 g (HEP). were further investigated. The process was repeated ten times and the fractions pooled and dried by evaporation to gather 2.4905 g F5, 1.1153 g F6 and 1.6045 F7.

CPC (Centrifugal Partition Chromatography)
CPC was performed on a Spot CPC device with a 250 mL rotor (Armen Instrument, Paris, France), a 510 HPLC pump (Waters GmbH, Eschborn, Germany) and a 2111 Multirac Fraction Collector (LKB-Produkter AB, Bromma, Sweden). Prior to separation, a solvent system consisting of n-hexane, acetonitrile and methanol (40/25/10 v/v/v) [51] was equilibrated in a separatory funnel and the two phases were separated before analysis and degassed for 10 min. Subsequently the rotor was filled with lower phase (LP) and then loaded with upper phase (UP) in ascending mode (ASC) with a rotation speed of 1000 rpm and a flow of 5 mL/min. 1.0-1.5 g of F5-7 were solved in a mixture of UP/LP (50/50, v/v), injected in the equilibrated system and fractions of 5 mL were collected. After 800 mL the mode was switched to descending mode (DSC) and the system purged with 200 mL (LP). For F5 the process war repeated once. Following, subfractions (F5C1-6, F6C1-8 and F7C1-8) were formed according to TLC control and dried by evaporation for further use.

TLC (Thin Layer Chromatography)
Fraction control by TLC was carried out for Flash chromatography and CPC on silica gel 60 F254 (Merck, Darmstadt, Germany) with a mobile phase consisting of toluene and ethylacetate (95/5, v/v). Plates were derivatized with anisaldehyde reagent R and a Camag TLC visualizer was used for documentation (Camag AG, Muttenz, Switzerland).

Preparative HPLC (High-Performance Liquid Chromatography)
An preparative HPLC equipped with a 1260 Infinity binary pump, a 1260 Infinity manual injector, a 1260 Infinity fraction collector, a 1260 Infinity diode array detector (all Agilent Technologies, Santa Clara, CA, USA) and a Kinetex ® column (Biphenyl, 100 Å, 5 µm, 21.2 × 250 mm, Phenomenex, Aschaffenburg, Germany) was used. Samples were solved in acetonitrile and portions of 0.2-5 mg were injected manually following a separation with acetonitrile (A) / water (B) and a flow of 21 mL/min. Thereby, peaks were detected at 200 nm, collected manually and pooled. After elimination of acetonitrile via evaporation the water fractions were partitioned four times with diethyl ether and the organic phases were dried in a nitrogen flow. For separation, the following gradients were used to collect the mentioned isolates:  (10)).

Optical Methods
All optical data were determined using solutions in methanol. Specific optical rotations were recorded at an UniPol L 1000 polarimeter (Schmidt + Haensch GmbH & Co., Berlin, Germany) using a micro tube (50 mm, 550 µL) at 589 nm. UV-spectra were measured on a Cary 50 Scan UV-spectrophotometer (Varian Deutschland GmbH, Darmstadt, Germany) in a quartz cuvette (QS, 1.0 cm, Hellma GmbH and Co. KG, Müllheim, Germany) in a range of 200-800 nm. For CD spectra a J-715 spectropolarimeter (JASCODeutschland GmbH, Gross-Umstadt, Germany) was used with a 0.1 cm quartz cuvette (Type: 100-QSQ, Hellma GmbH and Co. KG). Each measurement was repeated ten times at 22 • C from 190-300 nm with a scanning rate of 200 nm/min in 0.5 nm steps. Savitzky-Golay algorithm was used for spectra smoothing (convolution width: 15).

Purity
The purity of isolates was determined by HPLC-DAD (190-400 nm) analysis using an Elite LaChrom system consisting of an autosampler L-2200, a pump L-2130, an column oven L-2350, a diode array detector L-2455 (all Hitachi, Tokyo, Japan) and a Kinetex ® biphenyl column (100 Å, 5 µm, 4.6 × 250 mm, Phenomenex, Aschaffenburg, Germany). The gradients described in Section 3.3.6 were used to analyze 5 µL (acetonitrile, 1 mg/mL) with a flow of 1 mL/min and the chromatograms processed with EZChrom Elite 3.1.7 (Hitachi). Thus, the purity was calculated as the proportion of the integral of the main peak in the chromatogram using the maxplot (adjusted by a blank).

Statistics
Significance levels of ICAM-1 expressions were calculated in a one-way Anova followed by Tukey-HSD test using SPSS 26 (IBM, Armonk, NY, USA). IC 50 levels were obtained using non-linear regression by GraphPad Prism 8.0.0 (GraphPad Software, San Diego, CA, USA).

Data Availability Statement:
The data presented in this study are available on request from J.H. and K.K.