Phytochemicals, Two New Sulphur Glycosides and Two New Natural Products, from Shepherd’s Purse Seed and Their Activities

Two new sulfur glycosides, bursapastoris A–B (3–4), were extracted and isolated from shepherd’s purse seed, along with two new natural products, 11-(methylsulfinyl)undecanoic acid (2) and 10-(methylsulfinyl)decanoic acid (1). Their structures were determined though infrared spectroscopy, one-dimensional nuclear magnetic resonance (1H and 13C), and electrospray ionization mass spectrometry. Additionally, the structures of 3–4 were further identified by two-dimensional nuclear magnetic resonance (HMBC, HSQC, 1H-1H COSY, and NOESY). Compounds 1–4 showed relatively favorable docking to NF-κB. Unfortunately, we only discovered that compound 1–4 had weak anti-radiation activity at present. Therefore, further research regarding the biological activity of these organosulfur compounds is required at a later stage.


Introduction
Capsella bursa-pastoris (shepherd's purse), which belongs to the cruciferae family, is distributed all over the world and is used as a vegetable as well as a folk medicine [1].Shepherd's purse has been used as a remedy for centuries and has garnered attention for its various medicinal properties, including antihemorrhoidal [2], anti-inflammatory [3], antimicrobial [4], and hemostatic [5,6] activities, among others.In traditional Chinese medicine, it is used for dysentery and eye disorders, with people chewing the seeds of shepherd's purse to improve vision.The seeds not only have high nutritional value but also possess multifarious medicinal benefits, such as improving visual acuity, alleviating ocular discomfort, tonifying the digestive system, and enhancing vision.
Organosulfur compounds play a crucial role as therapeutic agents in medicinal chemistry, holding the key to the health-promotion benefits as they possess biological activities, including antihypertensive [7], antiatherosclerotic [8], antifungal [9], antibacterial [10], and anticancer effects [11], among others.Sulfur exhibits unique chemical properties, including a wide range of oxidation states and versatility in reactions that facilitate essential biological processes and redox biochemistry [12].The presence of sulfur is responsible for the distinctive characteristics of organosulfur compounds, which have been harnessed Molecules 2024, 29, 4145 2 of 10 in the treatment of diseases mediated by oxidative stress.These compounds are mainly present in fruits, vegetables, and edible mushrooms, and undergo slow biotransformation into various metabolites such as thiols, sulfides, and peptides during biosynthesis [13].However, the distinctive biological activity of these metabolites remains largely unexplored.As such, there is a need for further development of organic sulfur species.
Previously, we identified four organosulfur compounds in shepherd's purse seeds [14].Our current research is focused on the exploration of additional organosulfur compounds present in the shepherd's purse seed, which has prompted us to continue conducting a comprehensive phytochemical investigation on these seeds [15].In this study, we have discovered four more organosulfur compounds, including two new sulfur glycosides (3)(4) and two new natural products (1-2) (Figure 1).Then, the molecular docking method was used to screen the compounds, and it was found that compounds 1-4 showed relatively favorable docking to NF-κB.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 10 have been harnessed in the treatment of diseases mediated by oxidative stress.These compounds are mainly present in fruits, vegetables, and edible mushrooms, and undergo slow biotransformation into various metabolites such as thiols, sulfides, and peptides during biosynthesis [13].However, the distinctive biological activity of these metabolites remains largely unexplored.As such, there is a need for further development of organic sulfur species.Previously, we identified four organosulfur compounds in shepherd's purse seeds [14].Our current research is focused on the exploration of additional organosulfur compounds present in the shepherd's purse seed, which has prompted us to continue conducting a comprehensive phytochemical investigation on these seeds [15].In this study, we have discovered four more organosulfur compounds, including two new sulfur glycosides (3)(4) and two new natural products (1-2) (Figure 1).Then, the molecular docking method was used to screen the compounds, and it was found that compounds 1-4 showed relatively favorable docking to NF-κB.

Structure Elucidation
Compound  13 C NMR spectrum revealed that the glucose fragment exhibited an anomeric carbon signal at δ C 90.3, indicating its connection with a sulfur atom [16].Note: s (a single peak), d (a double peak), t (a triple peak), q (a quadruple peak), dd (doublet of doublets) and m (a multiple peak).
The glucose was obtained and analyzed through acid hydrolysis and HPLC analysis of 3.Meanwhile, the negative optical rotation and the small coupling constant (5.8 Hz) of the anomeric proton at δ H 5.43 (H-1 ′ ) implies that the glucose is in the α-D-configuration [17].Eventually, the assignment of protons and carbons were reached by the HMBC, HSQC, and 1 H-1 H COSY spectra (Figure 2A).The nuclear overhauser effect (NOE) correlations were observed (Figure 2B).Therefore, its structure was elucidated (Figure 1) and designated as bursapastoris A. [17].Eventually, the assignment of protons and carbons were reached by the HMBC, HSQC, and 1 H-1 H COSY spectra (Figure 2A).The nuclear overhauser effect (NOE) correlations were observed (Figure 2B).Therefore, its structure was elucidated (Figure 1) and designated as bursapastoris A. Its NMR spectra (Table 1, Figures S16 and S17) closely resembled those of compound 3, except for the distinctive anomeric proton signal (4.12, 1H, d, 9.36 Hz, H-1′).Acid hydrolysis combined with HPLC analysis confirmed the presence of glucose in compound 4. In addition, the negative optical rotation and the large coupling constant (9.36 Hz) of the anomeric proton at δH 4.12 (H-1′) indicate that the glucose is in the β-D-configuration [16].
The assignment of carbons and protons were ultimately achieved through the analysis of HMBC, HSQC, and 1 H-1 H COSY spectra (Figure 3A).The nuclear overhauser effect (NOE) correlations were observed in the NOESY experiment (Figure 3B).The structure of 4 was elucidated (Figure 1) and designated as bursapastoris B based on the aforementioned evidence.).Its NMR spectra (Table 1, Figures S16 and S17) closely resembled those of compound 3, except for the distinctive anomeric proton signal (4.12, 1H, d, 9.36 Hz, H-1 ′ ).Acid hydrolysis combined with HPLC analysis confirmed the presence of glucose in compound 4. In addition, the negative optical rotation and the large coupling constant (9.36 Hz) of the anomeric proton at δ H 4.12 (H-1 ′ ) indicate that the glucose is in the β-Dconfiguration [16].
The assignment of carbons and protons were ultimately achieved through the analysis of HMBC, HSQC, and 1 H-1 H COSY spectra (Figure 3A).The nuclear overhauser effect (NOE) correlations were observed in the NOESY experiment (Figure 3B).The structure of 4 was elucidated (Figure 1) and designated as bursapastoris B based on the aforementioned evidence.

Proposed Biosynthetic Pathway
A plausible biogenetic pathway [18] was proposed for the formation of these organosulfur compounds, involving the following key steps (Figure 4

Proposed Biosynthetic Pathway
A plausible biogenetic pathway [18] was proposed for the formation of these organosulfur compounds, involving the following key steps (Figure 4

Anti-Radiation Activity
In the investigation of the activity of compounds 1-4, it was observed that they did not exhibit any significant anti-inflammatory, anti-tumor, or anti-oxidation properties.However, organosulfur compounds have been known to demonstrate promising effects in protecting against radiation damage, such as diallyl sulfide [19], cysteamine [20], amifostine [21,22], and others.Therefore, it is intriguing to explore whether compounds 1-4 (four natural organic sulfur compounds) possess potential for mitigating radiationinduced damage.The anti-radiation activities of compounds 1-4 were evaluated using Xray irradiated AHH-1 cells as a model, and the result showed that the four organosulfur compounds had weak radioprotective effects (Figure 5).

Molecular Docking Analysis
Although there was no direct evidence of biological activity for these compounds from the experiment, we sought to investigate potential clues through molecular docking.This is based on the consideration that these four organosulfur compounds may possess some level of biological activity.The nuclear transcription factor Nuclear Factor kappa-B (NF-κB) plays a crucial role in regulating the expression of numerous genes [23].NF-κB is considered to be a component of the stress response, as it can be induced by various stimuli including UV radiation, pharmacological agents, and stressful conditions [24][25][26]

Anti-Radiation Activity
In the investigation of the activity of compounds 1-4, it was observed that they did not exhibit any significant anti-inflammatory, anti-tumor, or anti-oxidation properties.However, organosulfur compounds have been known to demonstrate promising effects in protecting against radiation damage, such as diallyl sulfide [19], cysteamine [20], amifostine [21,22], and others.Therefore, it is intriguing to explore whether compounds 1-4 (four natural organic sulfur compounds) possess potential for mitigating radiation-induced damage.The anti-radiation activities of compounds 1-4 were evaluated using X-ray irradiated AHH-1 cells as a model, and the result showed that the four organosulfur compounds had weak radioprotective effects (Figure 5).

Anti-Radiation Activity
In the investigation of the activity of compounds 1-4, it was observed that they did not exhibit any significant anti-inflammatory, anti-tumor, or anti-oxidation properties.However, organosulfur compounds have been known to demonstrate promising effects in protecting against radiation damage, such as diallyl sulfide [19], cysteamine [20], amifostine [21,22], and others.Therefore, it is intriguing to explore whether compounds 1-4 (four natural organic sulfur compounds) possess potential for mitigating radiationinduced damage.The anti-radiation activities of compounds 1-4 were evaluated using Xray irradiated AHH-1 cells as a model, and the result showed that the four organosulfur compounds had weak radioprotective effects (Figure 5).

Molecular Docking Analysis
Although there was no direct evidence of biological activity for these compounds from the experiment, we sought to investigate potential clues through molecular docking.This is based on the consideration that these four organosulfur compounds may possess some level of biological activity.The nuclear transcription factor Nuclear Factor kappa-B (NF-κB) plays a crucial role in regulating the expression of numerous genes [23].NF-κB is considered to be a component of the stress response, as it can be induced by various stimuli including UV radiation, pharmacological agents, and stressful conditions [24][25][26].The docking energies of compounds 1-4 with NF-κB are −4.76,−4.20, −3.28, and −4.04

Molecular Docking Analysis
Although there was no direct evidence of biological activity for these compounds from the experiment, we sought to investigate potential clues through molecular docking.This is based on the consideration that these four organosulfur compounds may possess some level of biological activity.The nuclear transcription factor Nuclear Factor kappa-B (NF-κB) plays a crucial role in regulating the expression of numerous genes [23].NF-κB is considered to be a component of the stress response, as it can be induced by various stimuli including UV radiation, pharmacological agents, and stressful conditions [24][25][26].The docking energies of compounds 1-4 with NF-κB are −4.76,−4.20, −3.28, and −4.04 KCal/mol, respectively.These values indicate that compounds 1-4 exhibited relatively favorable docking to NF-κB.
The binding site of NF-κB with compounds 1-4 includes hydrophobic pockets surrounded by GLY-130, LYS-122, ARG-132, GLY-53, GLU-20, ASP-21, ASN-50, ARG-131, GLU-14, ASN-42, GLU-14, SER-45, and LYS-122 (Figure 6).Take compound 3 as an example: the docked pose of compound 3 with NF-κB, exhibiting the lowest energy, demonstrates significant interactions with GLU-14 and ASN-42.The docked compound 3 is near to GLU-14 with closest distance 1.9 Å.Although compounds 1-4 have exhibited highly favorable docking scores with NF-κB, it is important to note that NF-κB is associated with a wide range of biological activities.The specific biological activities of compounds 1-4 in relation to NF-κB are not yet clear, and further research is required to explore the potential biological activity of these four organosulfur compounds in future experiments.
The binding site of NF-κB with compounds 1-4 includes hydrophobic pockets surrounded by GLY-130, LYS-122, ARG-132, GLY-53, GLU-20, ASP-21, ASN-50, ARG-131, GLU-14, ASN-42, GLU-14, SER-45, and LYS-122 (Figure 6).Take compound 3 as an example: the docked pose of compound 3 with NF-κB, exhibiting the lowest energy, demonstrates significant interactions with GLU-14 and ASN-42.The docked compound 3 is near to GLU-14 with closest distance 1.9 Å.Although compounds 1-4 have exhibited highly favorable docking scores with NF-κB, it is important to note that NF-κB is associated with a wide range of biological activities.The specific biological activities of compounds 1-4 in relation to NF-κB are not yet clear, and further research is required to explore the potential biological activity of these four organosulfur compounds in future experiments.

Plant Materials
The seeds of Capsella bursa-pastoris were obtained from the Bozhou Traditional Chinese Medicine Trading Center, and their producing area is located at a geographical coordinate of 114

Anti-Radiation Assay
To investigate the radiation protection effects of compounds 1-4 against apoptosis induced by X-ray radiation in the AHH-1cells [29], AHH-1 cells were seeded in 96-well plates at a density of 2.6 × 10 4 cells per well and incubated overnight at 37 • C under 5% CO 2 .Then, the cells were treated with compounds 1-4 at a concentration of 100 µM for 2 h followed by radiation.For the radiation, cells preincubated with compounds 1-4 were exposed to X-ray beams with a dose of 8 Gy in total.The cells with radiant inducement were used as an irradiation group, and recilisib (Ex-RAD), a radioprotectant, was used as a positive control group at a concentration of 100 µM.After incubating under the same conditions for 24 h, cell viability was monitored by CCK8, and the absorbance of each well at 450 nm was measured on a microplated reader (Multiskan MK-3, Thermo, Waltham, MA, USA).

Molecular Docking Assay
The docking study was conducted on pertinent protein targets associated with inflammation utilizing the AutoDock 4.2.6 and AutoDockTools 1.5.7 program [30].

Conclusions
In summary, we have isolated, purified, and identified two new sulfur glycosides and two new natural products from shepherd's purse seed.The structures were elucidated through the utilization of NMR experiments as well as mass spectrometry.Despite receiving less research attention in the past, the seeds of shepherd's purse contain natural organic sulfur compounds that warrant further exploration.We are confident that additional new organic sulfur compounds can be obtained from it to enrich the types of organosulfur compounds and sulfur glycosides in plants.Molecular docking studies have shown that compounds 1-4 exhibited relatively favorable docking to NF-κB; however, these four organosulfur compounds demonstrated weak radioprotective effects.This may be attributed to the fact that their outstanding biological activity has not been identified, thus necessitating further screening and verification through additional experiments in our later stages.

Compound 1
had the chemical formula of C 11 H 22 O 3 S as its MS m/z 233.1207 [M-H] − (calcd.for C 11 H 21 O 3 S − , 233.1211).Its 1 H NMR spectrum (Figure S1 and
):(1) an oxidation reaction converted fatty acids to 10P1 and 11P1; (2) the enzymatic catalytic reaction of 10P1 or 11P1 and methionine to 10P2 or 11P2 was catalyzed by sulfurtransferase; (3) the thioether was easily oxidized into compounds 1 and 2 by oxygenase; (4) P3 was derived from compound 2 through a reaction with a hydroxyamine unit; (5) protonation of P3 leads to a proposed intermediate P4; (6) P5 was derived from glucose-1-phosphate through a reaction with an L-cysteine unit; (7) compounds 3 and 4 were derived from the combination of P4 with P5 by the leaving of H2O.
): (1) an oxidation reaction converted fatty acids to 10P1 and 11P1; (2) the enzymatic catalytic reaction of 10P1 or 11P1 and methionine to 10P2 or 11P2 was catalyzed by sulfurtransferase; (3) the thioether was easily oxidized into compounds 1 and 2 by oxygenase; (4) P3 was derived from compound 2 through a reaction with a hydroxyamine unit; (5) protonation of P3 leads to a proposed intermediate P4; (6) P5 was derived from glucose-1-phosphate through a reaction with an L-cysteine unit; (7) compounds 3 and 4 were derived from the combination of P4 with P5 by the leaving of H 2 O.

CD 3 OD) Compond 4 (DMSO-d 6 ) δ H (J in Hz) δc δ H (J in Hz) δc
• 52 ′ -115 • 31 ′ E, 32 • 35 ′ -33 • 08 ′ N, with an approximate altitude of 40 m in Anhui province, China, in October 2021.The identification was performed by Professor Bin Li from the Department of Pharmaceutical Chemistry at Beijing Institute of Radiation Medicine.The voucher specimen (No. 2021-1006) has been deposited in the specimen cabinet at the Beijing Institute of Radiation Medicine.