Pseudosterins A–C, Three 1-Ethyl-3-formyl-β-carbolines from Pseudostellaria heterophylla and Their Cardioprotective Effects

Pseudostellaria heterophylla is used in China not only as a functional food but also as an herb to tonify the spleen, enhance immunity, and treat palpitation. Our previous investigation showed that a fraction enriched in glycosides obtained from the roots of P. heterophylla possessed pronounced protective effects on H9c2 cells against CoCl2-induced hypoxic injury. However, the active compounds responsible for the observed effects were still unknown. In the current investigation, pseudosterins A–C (1–3), three new alkaloids with a 1-ethyl-3-formyl-β-carboline skeleton, together with polydatin, have been isolated from the active fraction. Their structures were elucidated on the basis of spectroscopic analysis and quantum chemical calculations. The four compounds showed cardioprotective effects against sodium hydrosulfite-induced hypoxia-reoxygenation injury in H9c2 cells, with the three alkaloids being more potent. This is also the first report of alkaloids with a β-carboline skeleton isolated from P. heterophylla as cardioprotective agents.


Introduction
Cardiovascular diseases (CVDs) are a leading cause of death and disability worldwide, with an estimate of 17.9 million deaths every year [1]. As one of the most serious CVDs, myocardial infarction (MI), which is defined by pathology as myocardial cell death due to prolonged ischemia, was reported with over 700,000 deaths every year in China [2,3]. Effective treatment of MI (e.g., reperfusion therapy) generally involves procedures to promote the return of blood flow to the ischemic zone of the myocardium. However, reperfusion itself may aggravate myocardial damage and may lead to further irreversible myocardial cell death (i.e., lethal myocardial reperfusion injury) [4,5]. Protection of myocardium against ischemia/reperfusion (I/R) injury is, therefore, crucial in the process of reperfusion. Unfortunately, no effective therapy is currently available for combined I/R injury on the market, yet clinical trials in the past 10 years demonstrated that some chemical entities (e.g., cyclosporine A and metoprolol) were effective in ameliorating the myocardial damage [6][7][8]. Therefore, the development of powerful cardioprotective agents or functional foods to limit the extent of infarcted tissue caused by I/R injury is of great clinical importance. 2 of 11 The roots of Pseudostellaria heterophylla (Taizisheng in Chinese) were used not only as an herb to tonify the spleen, enhance immunity, and treat palpitation in Chinese herbal medicine (CHM) and local ethnic medicines [9][10][11][12], but also as a tonic food. A number of functional foods prepared from P. heterophylla (e.g., TaizishengHuangjing capsule ® and Taiziseng Tea (Hubei Zhenhao Biological Engineering Co., LTD, Yixing, China)) have been put into the market. Studies showed that P. hetetophylla is rich in a variety of chemical components, including polysaccharides, glycosides, cyclic peptides, sterols, oils, and other volatile oily substances with a wide range of bioactivities [13][14][15]. Previous pharmacological investigations showed that the extracts of P. heterophylla possessed cardioprotective effects [16][17][18][19]. Recently, we found that pretreatment of H9c2 cells with a fraction enriched in glycosides markedly protected the cells from CoCl 2 -induced hypoxic injury with effects comparable to that of the positive control N-Acetyl-L-Cysteine (NAC), and that the fraction may protect the cardiomyocytes from oxidative injury by preventing the increase of oxidative stress [20]. However, the active compounds responsible for the observed effects were still unknown. In the current investigation, three new alkaloids (1-3, Figure 1) with a 1-ethyl-3-formyl-β-carboline skeleton have been isolated from the active fraction. Their structures were elucidated on the basis of spectroscopic analysis and quantum chemical TDDFT calculations. Biological evaluations indicated that the three alkaloids showed more potent protective effects against sodium hydrosulfite-induced oxidative injury in H9c2 cells than that of polydatin, which was justified as an active principle in many fruits and vegetables [21][22][23]. These β-carboline alkaloids may be a promising type of compounds for the intervention of CVDs.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 functional foods to limit the extent of infarcted tissue caused by I/R injury is of great clinical importance. The roots of Pseudostellaria heterophylla (Taizisheng in Chinese) were used not only as an herb to tonify the spleen, enhance immunity, and treat palpitation in Chinese herbal medicine (CHM) and local ethnic medicines [9][10][11][12], but also as a tonic food. A number of functional foods prepared from P. heterophylla (e.g., TaizishengHuangjing capsule ® and Taiziseng Tea (Hubei Zhenhao Biological Engineering Co., LTD, Yixing, China)) have been put into the market. Studies showed that P. hetetophylla is rich in a variety of chemical components, including polysaccharides, glycosides, cyclic peptides, sterols, oils, and other volatile oily substances with a wide range of bioactivities [13][14][15]. Previous pharmacological investigations showed that the extracts of P. heterophylla possessed cardioprotective effects [16][17][18][19]. Recently, we found that pretreatment of H9c2 cells with a fraction enriched in glycosides markedly protected the cells from CoCl2-induced hypoxic injury with effects comparable to that of the positive control N-Acetyl-L-Cysteine (NAC), and that the fraction may protect the cardiomyocytes from oxidative injury by preventing the increase of oxidative stress [20]. However, the active compounds responsible for the observed effects were still unknown. In the current investigation, three new alkaloids (1-3, Figure 1) with a 1-ethyl-3-formyl-β-carboline skeleton have been isolated from the active fraction. Their structures were elucidated on the basis of spectroscopic analysis and quantum chemical TDDFT calculations. Biological evaluations indicated that the three alkaloids showed more potent protective effects against sodium hydrosulfite-induced oxidative injury in H9c2 cells than that of polydatin, which was justified as an active principle in many fruits and vegetables [21][22][23]. These β-carboline alkaloids may be a promising type of compounds for the intervention of CVDs.
Pseudosterin B (2) was obtained as a gum and the HR-ESI-MS peak at m/z 417.1302 [M−H] − (calcd 417.1303) (see Figure S18) suggested its molecular formula to be C 20 H 22 N 2 O 8 with 11 degrees of unsaturation. Its UV and IR spectra (see Figures S19 and S20) bore a resemblance to those of 1, indicating the presence of similar functionalities. Except signals for the glutamic acid moiety, the 13 C-NMR data of 2 are very close to those of 1 (see Table 1 and Figures S22-S23), suggesting a possible β-carboline skeleton and a hexose moiety for 2. However, the poor solubility of 2 in CD 3 OD gave poor peak resolution, which retarded the full assignments of the NMR data, whereas 1 H-NMR data recorded in pyridine-d 5 are well-resolved and the structure was established by detailed 2D NMR ( 1 H-1 H COSY, HSQC, and HMBC) data analysis (see Figures S21, S24-S26). As described for 1, a 1-ethyl-3-formyl-β-carboline skeleton can be easily identified by 1 H-1 H COSY, HSQC, and HMBC correlation analyses (see Figure 2). Coupling constants between the 1,3-diaxial protons (H-2 /H-4 and H-1 /H-3 /H-5 ) suggested the presence of a β-glucopyranosyl group, the configuration of which was also consistent with the coupling constant (δ H 5.19, 1H, d, J = 7.3 Hz) of the anomeric proton. HMBC correlation between H-1 and C-11 placed the β-glucopyranosyl group at C-11. Chemical shift (δ C 168.8), molecular formula, and IR analysis suggested a carboxylic acid group for C-10. The structure of 2, except the configuration of C-11, was, therefore, established.   Figure S18) suggested its molecular formula to be C20H22N2O8 with 11 degrees of unsaturation. Its UV and IR spectra (see Figures S19 and S20) bore a resemblance to those of 1, indicating the presence of similar functionalities. Except signals for the glutamic acid moiety, the 13 C-NMR data of 2 are very close to those of 1 (see Table  1 and Figures S22-S23), suggesting a possible β-carboline skeleton and a hexose moiety for 2. However, the poor solubility of 2 in CD3OD gave poor peak resolution, which retarded the full assignments of the NMR data, whereas 1 H-NMR data recorded in pyridine-d5 are well-resolved and the structure was established by detailed 2D NMR ( 1 H-1 H COSY,  Figure S27), indicating 11 degrees of unsaturation. The IR absorptions suggested the presence of hydroxy or/and amino (3443-3210 cm −1 ), -NH 2 (1649 and 1562 cm −1 ), and aromatic (1625 and 1508 cm −1 ) groups (see Figure S29). The 1 H-NMR data (see Table 1 and Figure S30) of 3 was very close to those of 2 in DMSO-d 6 (due to poor coupling splitting in pyridine-d 5 , 3 was detected in DMSO-d 6 ); these observations suggested that compound 3 also possessed a similar 11-βglucopyranosyl-β-carboline structure, which was also supported by a UV, 13  COSY, HSQC, and HMBC correlation (see Figure 2 and Figures S28, S31-S35). HMBC correlation between -NH 2 (δ H 7.98, 1H, d, J = 2 Hz; δ H 7.45, 1H, d, J = 2 Hz) and C-10 (δ C 166.9), as well as the IR analysis (1643 and 1562 cm −1 ), indicated the presence of a CO-NH 2 group at C-10. Therefore, the planar structure of 3 was established with only the configuration of C-11 to be determined.
The absolute configurations of C-11 in pseudosterins A-C (1-3) were determined by comparing their experimental ECD spectra with the quantum chemical TDDFT calculationproduced ECD spectra [27] of model compounds 1a and 2a (see Figure 3, Figure S1 and Table S1) that possess the chemical features of the chromophore and adjacent chiral carbons of the three compounds. In the experimental ECD spectra, three compounds showed a small positive first Cotton effect around 320 nm (see Figure 3, Tables S2 and S3), two negative ones around 270 and 235 nm, and ended by a positive one near 200 nm. Similarly, the calculated ones for the corresponding model compounds exhibited a positive first, negative second and third, and positive fourth Cotton effect. Since the glucose has already been determined to be D-glucose with GC analysis, NMR and optical rotation data all supported the assignment of compounds 1-3 to be a single compound but not a mixture of enantiomers, the theoretical and experimental ECD data qualitatively allowed the determination of the absolute configuration at C-11 of pseudosterins A-C (1-3) as R.
The absolute configurations of C-11 in pseudosterins A-C (1-3) were determined by comparing their experimental ECD spectra with the quantum chemical TDDFT calculation-produced ECD spectra [27] of model compounds 1a and 2a (see Figure 3, Figure S1 and Table S1) that possess the chemical features of the chromophore and adjacent chiral carbons of the three compounds. In the experimental ECD spectra, three compounds showed a small positive first Cotton effect around 320 nm (see Figure 3, Tables S2 and S3), two negative ones around 270 and 235 nm, and ended by a positive one near 200 nm. Similarly, the calculated ones for the corresponding model compounds exhibited a positive first, negative second and third, and positive fourth Cotton effect. Since the glucose has already been determined to be D-glucose with GC analysis, NMR and optical rotation data all supported the assignment of compounds 1-3 to be a single compound but not a mixture of enantiomers, the theoretical and experimental ECD data qualitatively allowed the determination of the absolute configuration at C-11 of pseudosterins A-C (1-3) as R.

Hypoxia/Reoxygenation Injury Protective Activity
Since these compounds were obtained from the cardioprotective fraction, they were further evaluated for cardioprotective effects in H9c2 cells according to the method reported [28]. All the compounds showed no cytotoxicity at concentrations up to 500 µM (see Figure S2), whereas compounds 1-3 exhibited protective effects against Na 2 S 2 O 4 -induced hypoxia-reoxygenation injury in a concentration-dependent manner in the range of 100-400 µM (Figure 4), with a potency better than that of polydatin, a well-known natural cardioprotective agent that significantly decreased apoptosis rate in ischemia/reperfusion (I/R)-induced myocardial injury of rats [29,30] and that was also isolated as an active compound in the current investigation. The structure-activity relationship analysis showed that compounds 2 and 3, which carry a free carboxylic or a primary amide group at C-3, are more active than compound 1, which carries a secondary amide group. However, a negligible effect was observed when changing a free carboxylic group (as in 2) to a primary amide one (as in 3). ural cardioprotective agent that significantly decreased apoptosis rate in ischemia/reperfusion (I/R)-induced myocardial injury of rats [29,30] and that was also isolated as an active compound in the current investigation. The structure-activity relationship analysis showed that compounds 2 and 3, which carry a free carboxylic or a primary amide group at C-3, are more active than compound 1, which carries a secondary amide group. However, a negligible effect was observed when changing a free carboxylic group (as in 2) to a primary amide one (as in 3). Myocardial ischemia/reperfusion (I/R) injury is a highly paradoxical injury in cardiovascular disease and its severity is proportional to the duration of ischemia. Reducing reperfusion injury would decrease the incidence of heart failure [31]. Myocardial cells exposed to hypoxia and reoxygenation (H/R) have been proved to be a useful tool for determining the biochemical responses of myocardial cells to I/R [32,33]. Sodium hydrosulfite is known as an oxygen scavenger, which was usually used as a chemical agent for establishing the myocardial cell model of hypoxia/reoxygenation [28,34]. In this study, pseudosterins A-C (1-3) effectively protected H9c2 cells from H/R injury induced by sodium hydrosulfite better than polydatin, which was proved to markedly alleviate the ischemic Myocardial ischemia/reperfusion (I/R) injury is a highly paradoxical injury in cardiovascular disease and its severity is proportional to the duration of ischemia. Reducing reperfusion injury would decrease the incidence of heart failure [31]. Myocardial cells exposed to hypoxia and reoxygenation (H/R) have been proved to be a useful tool for determining the biochemical responses of myocardial cells to I/R [32,33]. Sodium hydrosulfite is known as an oxygen scavenger, which was usually used as a chemical agent for establishing the myocardial cell model of hypoxia/reoxygenation [28,34]. In this study, pseudosterins A-C (1-3) effectively protected H9c2 cells from H/R injury induced by sodium hydrosulfite better than polydatin, which was proved to markedly alleviate the ischemic injury of cardiomyocytes from an animal model (30 mg/kg rat body weight, i.p.) [35]. Although extensive in vivo experiments of the three compounds are still needed for evaluating their cardioprotective effect, the current results suggested that they were possibly potent cardioprotective agents against myocardial I/R. Multiple mechanisms have been proved to be related to myocardial I/R injury. Reactive oxygen species (ROS) [36] and mitochondria [37,38] have been reported to be involved in myocardial I/R. Naturally occurring β-carbolines existing in foods (e.g., soy sources) and plants (e.g., Peganum harmala) are reported to be effective hydroxyl radical scavengers and antioxidants [39][40][41]. Therefore, the mechanisms of compounds 1-3 against Na 2 S 2 O 4 -induced H/R injury may be associated with ROS elimination. However, further research in this regard is needed.

Plant Material
The roots of Pseudostellaria heterophylla (Miq.) Pax were collected in October 2011 from the Radix Pseudostellariae cultivation base, Shibing County, Guizhou province, China and were identified by Prof. Qing-De Long of Guizhou Medical University. A voucher specimen (accession No. 20111101) was deposited at the Herbarium of the School of Pharmacy, Guizhou Medical University.

Acid Hydrolysis, Derivatization, and GC Analysis
Pseudosterins A-C (1-3), 4.0, 3.0, and 3.0 mg, respectively) were hydrolyzed by 2 N HCl at 80 • C in wedge bottles. The sugar solutions were obtained after removal of impurity with dichloromethane, and the sugar was subsequently purified by the Sephadex LH-20 column. The obtained sugars were determined to be D-glucose by direct optical rotation comparison and co-TLC (BuOH/acetone/H 2 O, 4/3/1, Rf = 0.45) with that of an authentic D-glucose. Further derivatization and identification were conducted according to previous procedures [43,44]. Briefly, 1.2 mg hydroxylamine-HCl and 0.4 mg sugar samples (or standard D-glucose and D-mannose) were dissolved with 2 mL pyridine in batches in wedge bottles to react for 30 min at 90 • C. The solutions were then cooled to room temperature. After addition of 1 mL of acetic anhydride to react at 90 • C for 30 min, the mixture was cooled to room temperature. Chloroform (1 mL) was then added and the organic phase was washed twice with 1 mL of water. The obtained organic phase was dried with rotary evaporator, and then the residue was dissolved in 100 µL of ethyl acetate-hexane (1:1, v/v) for GC analysis. The GC analysis was performed on a 7890-5975C system equipped with flame ionization detector (FID) (Agilent Technologies Inc., Santa Clara, CA, USA) and HP-5 capillary column (30 m × 320 µm i.d., 0.25 µm film thickness). Samples (1 µL) were injected with a split ratio of 60:1 by the Agilent auto-injector. Helium was used as the carrier gas at a constant flow rate of 2 mL/min. The temperature program was set as follows: the initial column temperature of 180 • C was increased at 2 • C min −1 to 250 • C, and held for 2 min. The inlet temperature was 240 • C and the temperature of detector was 280 • C. All the sugar substructures of pseudosterins A-C (1-3) were finally identified as D-glucose via a comparison with an authentic substance.

Acid Hydrolysis, Derivatization, and LC-ESI MS Analysis
Compound 1 (1 mg) with 6 N HCl (200 µL) were heated at 115 • C for 16 h according to report to release glutamic acid [45]. This solution was evaporated to dryness, and the residue was dissolved in 50 µL of water. To the glutamic acid-containing solution was added 40 µL of 1 M NaHCO 3 and then 120 µL of 1% L-FDLA in acetone. The solution was stirred and incubated at 40 • C for 2 h, then quenched by 40 µL of 1 N HCl. Finally, the solution was evaporated and re-dissolved with 500 µL of HPLC grade methanol for LC-ESI MS analysis. The corresponding L-and D-glutamic acids were modified in the same way. LC-ESI MS analysis was performed with an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a TSQ endure mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic column used was Agilent Eclipse Plus C18 (2.10 mm × 100 mm, 1.8 µm) maintained at 40 • C. Acetonitrile-0.1% formic acid aqueous solution was used as mobile phase under a linear gradient elution program (acetonitrile, 20%-40%) for 45 min with photodiode array detection. The flow rate was 0.2 mL/min and the injection volume was 5 µL. The following MS parameters were employed: the capillary voltage was set at 2.5 kV with the sheath and auxiliary nebulizing gas (N 2 ) pressure set at 2 psi, respectively, and the vaporizer temperature was 275 • C. The scan range was set at m/z 50-1000. MS analysis was operated in the negative ion mode of electrospray ionization.

Hypoxia/Reoxygenation (H/R) Model and Experimental Protocols
The H/R model was established according to literature with minor modification [34]. Briefly, H9c2 cells were cultured in high glucose DMEM media supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), including penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells grown at logarithmic growth stage were inoculated in a 96-well plate with a density of 4 × 10 4 cells/mL (100 µL for each well) and cultured in an incubator with 5% CO 2 at 37 • C for 24 h. The cells were then randomly divided into 14 groups: control group (with culture medium only), model group (with 20 mM of sodium hydrosulfite in the culture medium), and test groups (each tested compound had three groups with concentrations of 100, 200, and 400 µM of the compound in the culture medium, respectively). After removal of the supernatant, 100 µL of culture medium without (for blank and model groups) or with the tested compounds (for groups of polydatin and pseudosterins A-C, all purities above 95%) were added, and the mixtures were then cultured in an incubator for 24 h. Except for the blank group, the supernatant was removed and the cells were washed twice with sugar-free DMEM medium. The Na 2 S 2 O 4 solution (20 mM) prepared with sugar-free DMEM medium was then added to the cells and incubated for 15 min to induce hypoxic condition in vitro. After removal of the Na 2 S 2 O 4 solution from cells, high glucose DMEM medium was added and the cells were cultured for another 15 min of reoxygenation to mimic reperfusion. Cell viability was then determined by the MTS method. The OD values were measured with microplate reader at a wavelength of 490 nm. The cell survival rate was calculated as OD test /OD control × 100%.

Statistical Analysis
Experimental data were shown as mean ± SD. The experiments were repeated three times. The difference between the mean values of two groups was assessed by the Student's t-test. Multiple group comparisons were performed using a Dunnett's test. The accepted level of significance for the test was p < 0.05. All statistical tests were carried out using the SPSS 19.0 for Windows.

Conclusions
In the current investigation, three β-carbolines with a 1-ethyl-3-formyl-β-carboline skeleton have been isolated as cardioprotective agents from P. heterophylla in the treatment of cardiovascular diseases and in its use as a tonic food. β-Carbolines are an important subclass of carboline alkaloids with prominent biological effects. Diverse bioactivities, including antimicrobial, antitumor, antiparasitic, anticonvulsant, and vasorelaxant activities, have intrigued chemists and pharmacologists over centuries since the isolation of harmalin in 1841, and nine β-carboline drugs have been commercialized [46][47][48][49]. Thus, the discovery of 1-ethyl-3-formyl-β-carbolines as a new type of cardioprotective agent would not only stimulate efforts toward their structure-activity relationship, pharmacological application, and/or chemical synthesis, but also promote the use of P. heterophylla as a functional food for patients with cardiovascular diseases.
Supplementary Materials: Supplementary Materials are available online. Tables S1-S3, and Figure  S1: ECD calculation details; Table S4: Optical rotation, UV and IR data of compounds 1-3; Figure S2