Phytochemical Constituents from the Seeds of Capsella bursa-pastoris and Their Antioxidant Activities

Phytochemical investigation of 70% EtOH extract of the seeds of Capsella bursa-pastoris led to the isolation of a new cyclobutane organic acid (1), and fourteen known compounds, including two organosulfur compounds (2, 3), two quinonoids (4, 5), five flavonoids (6-10), three sterols (11-13) and two other types (14, 15). The structures of the compounds were elucidated by extensive spectroscopic analyses as well as comparison of their spectroscopic data with those reported in the literature. The antioxidant capacities of all compounds and extractive fractions were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging test and ferric reducing antioxidant power (FRAP) assay. Then the antioxidative substances were evaluated for their neuroprotective effects against H2O2-induced HT22 cell injury. The results indicated the strong scavenging ability to free radical of the extractive fractions and compounds 1-3, 8-10 and 13, and the ferric reducing antioxidant power of the extractive fractions and compounds 1-3, 8 and 10, which were close to or higher than that of the positive control trolox. The EtOAc fraction, n-BuOH fraction, and compounds 1, 3 and 8 can protect HT-22 cells from oxidative damage.


Introduction
With increasing human life expectancy, aging population and increasing social pressure, the prevalence of brain diseases is increasing year by year [1][2][3].When neurons in the brain, spinal cord, and/or peripheral nervous system start to degrade or function improperly, neurodegenerative disorders result.People may at first experience relatively minor symptoms associated with neuronal degeneration, such as difficulties with coordination or name memory, but as more neurons are impacted, the symptoms may intensify and eventually some patients will lose their ability to walk or live independently [4].As a result, degenerative disorders of the central nervous system have grown to be a serious issue that has an impact on people's health and quality of life.
The term "central nervous system degenerative diseases" refers to a set of conditions caused by persistent and progressive deterioration of the central nervous system tissue, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis.Many central nervous system diseases often cause varying degrees of dysfunction.At present, traditional clinical intervention methods are difficult to cure, and the intervention effect is Ti-Qiang Zhou and Zhen-Zhen Wei contributed equally to this work.

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limited for conditions such as myelitis, Guillain Barre Syndrome, and multiple sclerosis.These conditions have a poor prognosis, high disability rate, and heavy family and social burden.Therefore, it is urgent to study effective methods to intervene in neurological diseases.
In China, traditional medicinal plants have been utilized for a very long time to treat a variety of illnesses.Secondary metabolites are viewed as significant sources of natural substances for combating nerve injury illnesses due to the astonishing diversity of their chemical structures and biological activity.Natural products have received a lot of attention in research since they are thought to be more cost-effective and safer than wholly synthetic medications.
Capsella bursa-pastoris (L.) Medik commonly known as shepherd's purse is a cruciferous plant that is often used for both its edible and medicinal properties [5].This family of plants has gained widespread attention due to their nutritional and medicinal value.They are often used as edible vegetables, oil-bearing crops, condiments, ornamental plants, herbs to treat various diseases, and so on.Seeds from these plants, including rape seeds [6], turnip seeds [7], white mustard seeds [8], semen lepidii [9], and orychophragmus violaceus seeds [10], exhibit anti-cancer, anti-bacterial, antiinflammation, anti-oxidation, anti-radiation and other beneficial biological activities.The 'Compendium of Materia Medica' documents that shepherd's purse seed is gentle and non-toxic, and has the ability to improve eyesight, relieve eye pain, and supplement the deficiency of entrails such as the heart, liver, spleen, lung, and kidney.Despite numerous studies on shepherd's purse [11][12][13], the chemical constituents and biological activities of its seeds have not been systematically investigated.
Cellular oxidative stress and inflammation play a vital role in the pathological process of neural damage.Oxidative stress occurs in the vast majority of degenerative diseases of the nervous system and pathological process of neural damage [14,15].It is essential to develop diets and drugs that target inflammation and oxidative stress in the nervous system.It is well known that cruciferous plants have a wide range of antioxidant compounds [16].Antioxidant capacity is one of the key indicators of the nutritional and therapeutic potential of cruciferous plants.In this study, fifteen components of shepherd's purse seed and their antioxidant activities were investigated.As a part of our ongoing research [17], a new cyclobutane organic acid (1) and fourteen known compounds, including two organosulfur compounds (2, 3), two quinonoids (4, 5), five flavonoids (6-10), three sterols (11)(12)(13) and two other types (14,15) were isolated from the seed of C. bursa-pastoris.The antioxidant capacities of the extractive fractions and all the compounds were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging test and ferric reducing antioxidant power (FRAP) assay.Then the neuroprotective effects of the antioxidative substances were tested against H 2 O 2 -induced HT22 cell injury.The goal of the current study was to offer a theoretical foundation for the further development and use of this plant.

Materials and Methods
The materials and methods section is presented as supplementary material.

Structure Elucidation
Compound 1 was obtained as white amorphous powder.S1 in the supplementary material), the molecular formula was deduced to be C 22 H 24 O 10 , indicating eleven degrees of unsaturation.The UV spectrum (Fig. S2 and S3) exhibited the characteristic absorption peaks at 240 nm and 360 nm.The IR spectrum, as depicted in Fig. S4, confirmed the presence of benzene ring (1610, 1521, 1463, 839 and 803 cm −1 ), C=O (1686 cm −1 ), and OH (3422 cm −1 ).
Ferric Reducing Antioxidant Power Besides, the ferric reducing antioxidant power assay was performed to evaluate the antioxidant capacities of the fractions and all the compounds isolated, and the results are shown in Table S2 and Fig. 4. The FeSO 4 equivalent values of the EtOAc, DCM, n-BuOH and total fractions were 2.53 ± 0.15, 1.32 ± 0.15, 1.31 ± 0.04 and 1.23 ± 0.09, respectively, indicating that the EtOAc fraction had a much stronger ferric reducing antioxidant power than the other fractions.Compound 10 showed potent ferric reducing antioxidant power with FeSO 4 equivalent value of 4.03 ± 0.01, which was evidently higher than that of positive control group trolox.Compounds 1, 2, 3 and 8 showed moderate ferric reducing antioxidant powers, and their FeSO 4 equivalent values were in the range of 1.70 ± 0.02 to 1.94 ± 0.04.

Neuroprotective Effect Evaluation
H 2 O 2 -induced HT22 cells are often used as a classic model of nerve damage in vitro.Therefore, we selected this model to evaluate the neuroprotective activities.Trolox was used as a positive control.According to the findings presented in Fig. 5 and Table S3, the EtOAc and n-BuOH fractions exhibited strong neuroprotective effects, with cell survival rates ranging from 84.30 1.63 to 84.31 5.20%.Compounds 1, 3 and 8 showed potent neuroprotective activities with cell survival rates of 82.76 ± 3.05, 81.19 ± 5.43 and 84.17 ± 7.65%, respectively, much stronger than that of trolox (78.06 ± 0.37%).All the findings indicated that the The findings of the present study show that the EtOAc and n-BuOH fractions, and compounds 1, 3, and 8 exhibit three kinds of antioxidant capacities.In comparison with other fractions of the seeds of C. bursa-pastoris, the EtOAc and n-BuOH fractions has more potent in vitro antioxidant activities.Compared with the positive controls trolox, compounds 1, 3, and 8 showed more strong scavenging abilities to free radical and ferric reducing antioxidant powers, and could protect HT-22 cells from oxidative damage.In further study in the future, we will explore the effects and mechanisms of these compounds at the animal level, and even whether they can penetrate the bloodbrain barrier.Since good blood-brain barrier (BBB) permeability is the key to exerting drug efficacy in the treatment of central nervous system diseases [31].whereas, this study indicated that these compounds would be instructive for further study.

Conclusion
In this study, the chemical constituents of shepherd's purse seeds were systematically separated to afford one new compound and fourteen known compounds, and their antioxidant activities were evaluated in vitro.The EtOAc and n-BuOH fractions, and compounds 1, 3, and 8 from shepherd's purse seeds exhibited strong scavenging abilities to free radical and ferric reducing antioxidant powers, and could protect HT-22 cells from oxidative damage.Taken as a whole, this study offers fresh perspectives on the utilization of this edible medicinal plant.