Antiliver Fibrosis Screening of Active Ingredients from Apium graveolens L. Seeds via GC-TOF-MS and UHPLC-MS/MS
Authors: Yi Zhu
Yao Zhao
Junping Hu
Xiaomei Wang
Ming Qiao
Xinling Wang
Jianhua Yang
Journal: Evidence-based Complementary and Alternative Medicine (2020)
Discipline: CLINICAL MEDICINE
Keywords: Apium graveolens L.; celery seeds; antiliver fibrosis effects; HSC-LX2 cells; aqueous ethanol
Affiliations: College of Pharmacy, Xinjiang Medical University, Urumqi 830011, China, Department of Pharmacy, The First Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China

Abstract:  Although several studies have been performed on Apium graveolens L.(celery) seeds, their antiliver fibrosis effects remain to be unexplored. Firstly, we detected the effects of celery seeds extracted with different concentrations of aqueous ethanol on the proliferation of HSC-LX2 cells. Then, we detected the effects of fractions of the optimal effect extract on the proliferation and apoptosis of HSC-LX2 cells. Finally, the compounds of petroleum ether (PP), ethyl acetate (PE), n-butyl alcohol (PB), and water fractions (PW) of the optimal effect extract were determined by GC-TOF-MS and UHPLC-MS/MS, to confirm the potentially antifibrotic compounds combined with pharmacodynamic experiment of monomer compounds in vitro. The results revealed that 60% ethanol extract of celery seeds (60-extract) exhibited remarkable inhibition effect on the proliferation of HSC-LX2 cells compared with 95% ethanol and aqueous extract. Besides, it validated that the inhibition rates of PP, PE, PB, and PW on the proliferation of HSC-LX2 cells were 75.14%, 73.52%, 54.09%, and 43.36%, and their percentage of apoptotic cells were 37.5%, 4.3%, 0.7%, and 0.1% at high doses, respectively. Additionally, it was manifested that apigenin, aesculetin, and butylphthalide have major contribution to the overall compounds of celery seeds, and the inhibition effects on the cell proliferation clearly elevated with increase in their contents. In essence, apigenin, aesculetin, and butylphthalide may hopefully become the natural products of antiliver fibrosis, which laid a foundation for the subsequent development of celery seeds as antiliver fibrosis drugs. 

Retracted: No

PubPeer comments:
 Fig. 3  a Western blots showing the expression of Jag1 in miR-NC and miR-143 mimics transfected CAMA-1 cells. b Western blots showing the efect of miR-143 overexpression on the Rho- signalling pathway in CAMA-1 cells.  For comparison: *[left]* Figure 4 from [Wang et al (2019)] .  Effect of Acetylshikonin on the autophagy-related protein expression as revealed by western blot analysis. The Figure shows that the expression of LC3-I, LC30II and Beclin-1 increases with increasing drug doses, while the expression of p62 decreased and the expression of Vps34 remained almost unchanged. *[right]* Figure 6 from [Zhu, Li Chen (2020)] .  Effect of nootkatone on the cell cycle related protein cyclin-B1 in HXO-Rb44 human retinoblastoma cells. The Figure depicts that nootkatone suppresses cyclin B1 expression dose-dependently.  , 
 *[left]* Fig 3 a,b, again. *[right]* Figure 8 from [Wang et al (2019)] .  Effect of Acetylshikonin on the mTOR PI3K AKT signalling pathway as depicted by western blot analysis. Acetylshikonin caused dose-dependent decrease in the expression of p-mTOR, p-PI3K and p-AKT, while no visible effect was found on the expression of mTOR, PI3K and AKT.  , 
 *[left]* Fig 2f from [Du et al (2020)] .  Western blot analysis showing the expression of Bax and Bcl-2 in miR-NC and miR-143 mimics transfected CAMA-1 cells. *[right]* Fig 2C from [Liu Guo (2020)] .  Effect of miR-NC or miR-9 mimics on the expression of Bax and Bcl-2.  , 
 *[right]* Fig 2C,D from [Du et al (2020)] .  DAPI staining of miR-NC and miR-143 mimics transfected CAMA-1 cells. ...  AO EB staining of miR-NC and miR-143 mimics transfected CAMA-1 cells . *[left]* Fig 1(E) from [Chen et al (2019)] .  AO EB staining for apoptosis . *[middle]* Figure 4 from [Li et al (2018)] .  AO EB staining showing induction of apoptosis in AMC-HN-8 cells upon treatment with Mukonal .  , 
 *[right]* Fig 3c,d from [Du et al (2020)] .  c Transwell assay showing cell migration in miR-NC and miR-143 mimics transfected CAMA-1 cells. d Transwell assay showing cell invasion in miR-NC and miR-143 mimics transfected CAMA-1 cells. *[left]* Figure 7 from [Du et al (2019)] .  Inhibition of (A) cell migration and (B) invasion by oridonin at 30 u00b5M concentrations as depicted by transwell assay . Two pairs of panels overlap.  , 
 *[upper]* Fig 2c from [Du et al (2020)] .  DAPI staining of miR-NC and miR-143 mimics transfected CAMA-1 cells . *[lower]* Figure 3A from [Chen et al (2020)] .  DAPI staining showing miR-151 overexpression induces apoptosis in the miR-151 overexpressing PC-3 cells .  UPDATE: Another sighting! Figure 2 from [Yu, Zhou Li (2018)] .  Detection of apoptosis in untreated AGS, cisplatin-treated AGS, and cisplatin-treated AGS cells transfected with miR-136 by AO EB and DAPI staining.  , 
 Among the References we find  Hua F, Li CH, Chen XG, Liu XP (2018) Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf MEK ERK cascade. Int J of Mol Med 41:3485u20133492 . There is no accompanying citation in the text. This is relevant because of the surprisingly far-reaching influence of [Hua et al (2018)] . Google Scholar informs us that their in-vitro study of Daidzein has been cited **71** times. Many of these are review papers, but we also find a surprising number of papers using it in  Apoptosis Assay paragraphs to illustrate the use of flow-cytometry and Annexin V PI staining to quantify cell apoptosis. Indeed, Hua et al did apply the method, but they were not pioneers, nor is their explanation superior to many others. Alternatively, the paper is cited as a precedent for a phytochemical (daidzein) inhibiting cancer proliferation by interrupting the cell cycle. Given all the earlier precedents, the choice again invites some explanation. It is almost as if all these papers were prepared by a third-party service (a papermill ), following a template from one paper to the next, without checking its contents. Consistent with this notion, the papers came to my attention by recycling the same figures (despite a lack of authors in common). - [Jia et al (2019)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR3 cells was performed as described previously [17]. - [Luo et al (2019)] .  Many plant-derived molecules have been reported to interfere with the **ROS** level in the cancer cells [13]. ... The slides were then covered with a covers lip and examined with a fluorescent microscope. Then 4u2019,6-diamidino-2-phenylindole (DAPI) and annexin V propidium iodide (PI) staining was performed as described previously [13]. - [Zeng et al (2019)] .  The slides were then covered with a covers lip and examined with a fluorescent microscope (BDBiosciences, San Jose, CA, USA). The annexin V PI staining was performed as described previously [8]. - [Wu et al (2019)] *[retracted]*,  The slides were covered with a coverslip and examined with a fluorescent microscope (BD Biosciences, San Jose, CA, USA). Annexin V PI staining was performed as described previously [13]. - [Yang, Chen Wang (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as described previously 16 . - [Yang et al (2019)] .  The slides were covered with cover slips and examined with a fuorescent microscope. DAPI and annexin V propidium iodide (PI) staining were performed as described previously [14] *[14 is a misprint for 13]*. - [Huang et al (2019)] .  The slides were covered with cover slip and examined under fluorescence microscope. Annexin V propidium iodide (PI) staining of the Icariin-treated HeLa cells was performed as described previously [18]. - [Liu et al (2019)] .  The distribution of the breast cancer SK-BR-3 cells in different cycle phases was performed by flow cytometry after PI staining, following the method reported by Hua et al [11]. ...After overnight incubation at 4u00b0C, the cells were subjected to PI staining and flow cytometry. - [Liu et al (2019)] .  After covering the glass slides with cover slips, the cells were analyzed under a fluorescent microscope (Nikon Instruments, Inc., NY, USA). For determining percentage of apoptotic cells, Annexin V PI staining assay using a FACSCalibur flow cytometry (BD Biosciences, NY, USA) was carried out using a previously reported method [12] - [Yin et al (2019)] .  The slides were covered with covers slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as previously described [13] . - [Zhang et al (2019)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining of the SW48 cells was performed as described previously [11] . - [Ma et al (2019)] .  The slides were cover-slipped and examined under a fluorescent microscope. Annexin V PI staining was performed as described previously [15]. - [Li et al (2019)] .  The slides were cover-slipped and assessed by fluorescent microscopy. ROS estimation was performed as described previously [13]. - [Zhang et al (2019)] .  The slides were covered with a cover slip and examined with a fluorescent microscope. Annexin V PI staining was performed as previously described [12]. - [Wu et al (2020)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the AGS cells was performed as described previously [15]. - [Yu et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [9]. - [Yin et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [10]. - [Wei et al (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR-3 cells was performed as described previously [11]. - [Wen et al (2020)] .  The glass slides laden with cell cultures were then covered with cover slips and then using a fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA,) the cells were analysed. Quantification of apoptosis was done with annexin V PI staining assay which was performed as reported already (Hua et al., 2018). -------------------------------------------------------------- Cell-cycle arrest: - [Li Han (2019)] .  Cell cycle arrest is another important mechanism by which anticancer molecules exert their effects [16] . - [Mao et al (2019)] .  The arrest of the cancer cells at G2 M phase prevents the cancer cells from entering mitosis and as such the molecules that trigger the cell cycle arrest prevent the proliferation and tumourigenesis of cancer cells (Hua et al., 2018). - [Si et al (2019)] .  Previous studies have shown that depletion of cyclin A and B1 and upregulation of p27 and p21 results in the G2 M arrest of the cells during cell division [21]. - [Zhou et al (2019)] .  The arrest of cancer cells at G2 M phase prevents the cancer cells from entering mitosis, so the molecules that trigger cell cycle arrest prevent the proliferation and tumorigenesis of cancer cells [20]. - [Tan et al (2019)] .  Cell cycle arrest caused by several anticancer agents is an essential mechanism in the growth inhibition of cancer cells [19] . - [Wei et al (2019)] .  Furthermore, arrest of the cells at different phases of the cell cycle is an important route by which anticancer agents halt the growth of cancer cells [18]. - [Zeng et al (2019)] .  Besides autophagy, cell cycle arrest is another mechanism by which anticancer agents exert their anticancer effects [19] . -------------------------------------------------- Another list of papers cite Hua et al (2018) as precedents for methods which those authors did not in fact apply. - [Zhang et al (2019)] .  For determination of the ROS and mitochondrial membrane potential levels, the A549 cells were treated with 0, 9, 18, and 36 u00b5M concentrations of murrayanine for 24 hours and then the ROS and mitochondrial membrane potential levels in the A549 cells were determined, as described previously [11] *(in fact Hua et al did not measure or even mention ROS or mitochondrial membrane potentials)*. - [Guo et al (2019)] .  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15] . - [Xu et al (2019)] . Hua et al appear in the References but not in the text at all. - [Du et al (2019)] .  **Osteosarcoma** causes significant mortality world over and the treatments available are not efficient and are associated with a number of side-effects [11]. - [Yu et al (2019)] .  Several of the natural products have been reported to induce apoptosis and autophagy through production of ROS [18]. - [Liu et al (2019)] .  The colony formation assay was carried out as described previously [15] *[Hua et al did not perform colony-formation assays]* - [Zhu et al (2020)] .  Estimation of ROS was measured according to a method previously described [8]. - [Du et al (2020)] . Hua et al appear in the References but not in the text at all. , 
 #6 Fig 2c again *[upper]*:  Another sighting! The two left-hand panels of Fig 5 from [Wu Li (2017)] .  Effect of indicated doses of GA on induction of MCF-7 cell apoptosis as detected by DAPI staining, determined by flow cytometry.  , 
 #2 *[left]* Fig 3 a,b, again.  Original source for some bands appears to be Fig 3D of [Qiu et al (2018)] .  ,