CXCR4; Inflammation/Immunology
The targets of Baohuoside I are CXC chemokine receptor 4 (CXCR4) and nuclear factor κB (NF-κB)[1]
The targets of Baohuoside I are c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK)[2]

At 12–25 μM, baohuoside I, an inhibitor of CXCR4, suppresses the expression of CXCR4. In a dose-dependent manner, baohuoside I (0-25 μM) inhibits NF-κB activation and the invasion of cervical cancer cells provoked by CXCL12. Baohuoside I also prevents breast cancer cells from invasively growing[1]. At 24 hours, 11.5 hours, and 48 and 72 hours, respectively, baohuoside I reduces the viability of A549 cells with IC50s of 25.1 μM, 11.5 μM, and 9.6 μM. In A549 cells, baohuoside I (25 μM) stimulates the p38MAPK and JNK signaling cascades, increases ROS levels, and suppresses the caspase cascade[2]. The proliferation of esophageal squamous cell carcinoma Eca109 cells is considerably and dose-dependently inhibited by baohuoside I (3.125, 6.25, 12.5, 25.0, and 50.0 µg/mL), with an IC50 of 4.8 µg/mL at 48 hours[3].

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1. In human cervical cancer HeLa cells, Baohuoside I downregulates CXCR4 expression in a dose- and time-dependent manner. The inhibitory effect can be observed as early as 12 hours after incubation, with the minimum effective concentration of 12.5 μM, and 25 μM Baohuoside I significantly inhibits CXCR4 expression after 24 hours of treatment[1]
2. In breast cancer MDA-MB-231 cells, liver cancer HepG2 cells, and pancreatic cancer HPAC cells, 25 μM Baohuoside I downregulates CXCR4 expression after 24 hours of treatment, with the most significant inhibitory effect on MDA-MB-231 cells, and it suppresses CXCR4 expression in a dose-dependent manner in these cells[1]
3. The downregulation of CXCR4 by Baohuoside I is not mediated through proteasomal or lysosomal degradation pathways but occurs at the transcriptional level, reducing CXCR4 mRNA levels in a dose-dependent manner[1]
4. In HeLa cells, Baohuoside I inhibits the constitutive activation of NF-κB in a dose-dependent manner[1]
5. Pretreatment with 25 μM Baohuoside I can effectively abrogate CXCL12-induced invasion of HeLa cells and MDA-MB-231 cells[1]
1. It exhibits cytotoxicity against human non-small cell lung cancer A549 cells, inhibiting cell viability in a time- and concentration-dependent manner. The half-maximal inhibitory concentration (IC50) values are approximately 25.1 μM at 24 h, 11.5 μM at 48 h, and 9.6 μM at 72 h[2]
2. It induces apoptosis in A549 cells. After treatment with 25 μM Baohuoside I for 24 h, the proportion of apoptotic cells increases from 8.9±1.1% in the control group to 58.5±2.5%, the proportion of cells in the sub-G1 phase rises from 0.13±0.02% to 18.31±0.9%, and TUNEL staining shows a significant increase in positive cells[2]
3. It reduces the mitochondrial membrane potential (DWM) of A549 cells, increasing the green/red fluorescence intensity ratio to 4-fold that of the control group, and promotes the release of cytochrome c (Cytc) from mitochondria to the cytoplasm[2]
4. It upregulates the BAX/Bcl-2 ratio in A549 cells, activates caspase 3 and caspase 9, and leads to the degradation of poly (ADP-ribose) polymerase (PARP)[2]
5. It induces excessive production of reactive oxygen species (ROS) in A549 cells. After treatment with 25 μM Baohuoside I for 12 h, the proportion of DCF-positive cells increases from 4.1±0.9% to 64.3±2.9%, and ROS generation starts to rise at 6 h and persists until 24 h[2]
6. It activates the JNK and p38MAPK pathways in A549 cells in a concentration- and time-dependent manner. Phosphorylation activation of JNK and p38MAPK can be detected at 9 h and persists until 24 h, with no significant change in ERK phosphorylation level[2]
7. Pretreatment with the ROS scavenger NAC (1 mM) can significantly reverse Baohuoside I-induced ROS accumulation (reduced to 23.4±2.7%), mitochondrial membrane potential reduction, cell apoptosis (reduced to 25.9±3.1%), and cell death (reduced from 59.6±2.6% to 27.2±3.3%), as well as inhibit the activation of JNK and p38MAPK pathways[2]
8. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK (50 μM) can only partially inhibit Baohuoside I-induced apoptosis in A549 cells[2]
9. Co-incubation of p38MAPK inhibitor SB203580 (25 μM) or JNK inhibitor SP600125 (25 μM) with 25 μM Baohuoside I for 24 h can effectively attenuate apoptosis in A549 cells[2]
1. It exhibits proliferative inhibitory activity against human esophageal squamous cell carcinoma Eca109 cells in a dose- and time-dependent manner, with a half-maximal inhibitory concentration (IC50) of 24.8 μg/ml at 48 h, and cell growth is almost arrested at 50 μg/ml[3]
2. It induces apoptosis of Eca109 cells in a dose-dependent manner. After treatment with 50 μg/ml for 48 h, the proportion of apoptotic cells in the sub-G1 phase increases from 3.26% in the control group to 49.21%[3]
3. It downregulates the mRNA expression levels of β-catenin, survivin, and cyclin D1 in Eca109 cells in a dose-dependent manner[3]
4. It downregulates the protein expression levels of β-catenin, survivin, and cyclin D1 in Eca109 cells in a dose-dependent manner[3]

In nude mice, baohuoside I (25 mg/kg) reduces the expression of survivin, cyclin D1, and β-catenin protein levels[3].

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Baohuoside-I inhibits in vivo tumor growth in a xenograft tumor model of human esophageal cancer cells. We investigated the in vivo anticancer activity of Baohuoside I using a xenograft model of human esophageal squamous cell carcinoma cells. Briefly, exponentially growing firefly luciferase-tagged Eca109 cells were injected into the flanks of Babl/c nude mice. One week after cancer cell injection, Baohuoside-I was intralesionally administered (25 mg/kg body weight, once a day). Mice were subjected to Xenogen bioluminescence imaging on a weekly basis for an additional three weeks. As shown in Fig. 5, the Baohuoside-I treatment group exhibited a significantly decreased Xenogen imaging signal when compared with the control group. In fact, quantitative analysis revealed that Baohuoside-I-mediated inhibition of xenograft tumor growth was statistically significant (p<0.01) at three weeks after treatment, even though the tumors were not completely eliminated (Fig. 5A and B). Histologic analysis (H&E staining) indicated that the Baohuoside-I treatment group exhibited a decreased cellularity in the tumor mass.[3]

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1. In the human esophageal cancer Eca109-luc nude mouse xenograft model, intralesional injection of Baohuoside I at a dose of 25 mg/kg once daily for three weeks significantly inhibits tumor growth, and the bioluminescent imaging signal intensity is significantly reduced (p<0.01)[3]
2. Histological analysis shows that the cellular density of tumor tissue in the treatment group is decreased[3]
3. The protein expression levels of β-catenin, survivin, and cyclin D1 in tumor tissue are reduced by 40%, 41%, and 45%, respectively (all p<0.01)[3]

In this work, researchers investigate baohuoside I, a component of Epimedium koreanum, as a regulator of CXCR4 expression as well as function in cervical cancer and breast cancer cells. We observed that baohuoside I downregulated CXCR4 expression in a dose- and time-dependent manner in HeLa cells. Treatment with a pharmacological proteasome and lysosomal inhibitors did not have a substantial effect on baohuoside I’s ability to suppress CXCR4 expression. When we investigated the molecular mechanism of action, it was observed that the suppression of CXCR4 expression occurred at the level of mRNA. The decrease in the level of CXCR4 expression caused by baohuoside I was correlated with inhibition of the CXCL12-induced invasion of both cervical and breast cancer cells. Overall, our results show that baohuoside I exerts its antimetastatic effect through the downregulation of CXCR4 expression and, thus, has the potential to play a role in the suppression of cancer metastasis[1].

Western Blotting[1]
For detection of CXCR4, baohuoside I-treated whole cell extracts were lysed with RIPA buffer [150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% deoxycholate, and 5 mM ethylenediaminetetraacetic acid (EDTA)] enriched with a complete protease inhibitor cocktail tablet and then incubated on ice for 30 min with regular vortexing before being centrifuged at 14000 rpm and 4 °C for 15 min. The protein concentration was determined by using the bicinchoninic acid (BCA) protein assay kit. The protein samples were boiled in SDS sample buffer for 5 min and were resolved on a 10% SDS–polyacrylamide gel. After electrophoresis, proteins were transferred onto a polyvinyl difluoride (PVDF) membrane, which was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with the primary antibody at the appropriate final concentration followed by hybridization with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. For each step, the membrane was washed with TBST three times for 10 min and the transferred proteins were incubated with supersignal pico-chemiluminescent substrate or dura-luminol substrate for 2 min according to the manufacturer’s instructions and visualized with imagequant LAS 4000.

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Invasion Assay[1]
The in vitro invasion assay was conducted using the Bio-Coat Matrigel invasion assay system according to the manufacturer’s instructions. Cancer cells (5 × 104 per milliliter) were suspended in medium and seeded into the Matrigel-precoated transwell chambers with polycarbonate membranes with a pore size of 8 μm. After preincubation with or without baohuoside I (25 μM), transwell chambers were then placed into 24-well plates, to which was added the basal medium only or basal medium containing 100 ng/mL CXCL12. After incubation (24 h for HeLa and MDA-MB-231), the upper surface of transwell chambers was wiped off with a cotton swab and invading cells were fixed and stained with a Diff-Quick stain. The invading cell numbers were counted in five randomly selected microscope fields (100×).

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1. Cell Culture: Immortalized human cervical cancer HeLa cells are cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Breast cancer MDA-MB-231 cells, pancreatic cancer HPAC cells, and liver cancer HepG2 cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotics. All cells are maintained at 37℃ in an atmosphere of 5% CO₂ and 95% air, and passaged with 0.25% trypsin-EDTA for 3-5 minutes when the cell confluence reaches 80%[1]
2. Western Blotting: Collect cells treated with Baohuoside I and lyse them with RIPA buffer (150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% deoxycholate, and 5 mM ethylenediaminetetraacetic acid (EDTA)) enriched with a complete protease inhibitor cocktail. Incubate on ice for 30 minutes with regular vortexing, then centrifuge at 14000 rpm and 4℃ for 15 minutes. Determine the protein concentration using a bicinchoninic acid (BCA) protein assay kit. Boil the protein samples in SDS sample buffer for 5 minutes and separate them by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, transfer the proteins onto a polyvinylidene fluoride (PVDF) membrane, block the membrane with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST), incubate with the primary antibody at the appropriate final concentration, followed by hybridization with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. Wash the membrane with TBST three times for 10 minutes at each step, incubate the transferred proteins with chemiluminescent substrate for 2 minutes according to the manufacturer's instructions, and visualize the protein bands with an image analysis system[1]
3. RT-PCR: Extract total cellular RNA using Trizol reagent and synthesize cDNA using a reverse transcription premix according to the manufacturer's protocols. Analyze the relative expression of CXCR4 by quantitative RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primer sequences are as follows: CXCR4 (sense: 5′-CCG TGG CAA ACT GGT ACT TT-3′; antisense: 5′-TTT CAG CCA ACA GCT TCC TT-3′) and GAPDH (sense: 5′-CAG CCT CAA GAT CAT CAG CA-3′; antisense: 5′-GTC TTC TGG GTG GCA GTG AT-3′). The RT-PCR mixture contains 2.5 μL of 10× Taq reaction buffer, 0.5 μL of each 10 mM dNTP, 1 μL of each forward and reverse primer, and 2 μL of template DNA in a final volume of 25 μL. Resolve the amplification products by 1.5% agarose gel electrophoresis stained with safe dye and photograph with an image analysis system[1]
4. Real-Time Quantitative PCR: Perform real-time PCR on the cDNA using specific primers for CXCR4 and GAPDH with a SYBR Green kit on a real-time PCR instrument. The PCR thermal profile is 95℃ for 10 minutes, followed by 40 cycles of 95℃ for 10 seconds and 55℃ for 30 seconds, and a final cooling step at 40℃ for 30 seconds. Normalize the crossing point (Cp) value of CXCR4 to the Cp value of GAPDH for relative quantification[1]
5. Invasion Assay: Conduct in vitro invasion assay using Matrigel-precoated transwell chambers with polycarbonate membranes (pore size 8 μm). Suspend cancer cells (5×10⁴ cells per milliliter) in medium and seed them into the upper chamber. After pretreatment with or without 25 μM Baohuoside I, place the transwell chambers into 24-well plates containing basal medium or basal medium supplemented with 100 ng/mL CXCL12 in the lower chamber. After 24 hours of incubation for HeLa and MDA-MB-231 cells, wipe the upper surface of the transwell chambers with a cotton swab, fix and stain the invading cells, and count the number of invading cells in five randomly selected microscope fields (100×)[1]
6. Electrophoretic Mobility Shift Assay (EMSA): Use an oligonucleotide probe containing the NF-κB binding motif, end-labeled with DIG-ddUTP. Incubate 10 μg of sample protein with the DIG-labeled probe at room temperature for 30 minutes for the binding reaction. Separate the DNA-protein complexes by electrophoresis on a 6% nondenaturing polyacrylamide gel using 0.5× TBE as the running buffer. After electrophoresis, transfer the gel to a nylon membrane and detect chemiluminescently, then quantify the signal intensity with an image analyzer[1]
1. Cell Culture: Human non-small cell lung cancer A549 cells are cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37℃ in a 5% CO₂ humidified atmosphere. Cells are periodically tested for mycoplasma infection and found to be negative[2]
2. Cell Viability Assay (MTT Assay): Cells (1×10⁴ cells/well) are seeded in a 96-well plate and treated with 6.25, 12.5, 25 μM Baohuoside I or 1 mM NAC for 24, 48, or 72 h. After removing the medium containing MTT, dimethyl sulfoxide (DMSO) is added to each well to dissolve the formed crystals. After mixing, the absorbance of the cells is measured at 540 nm using a microplate reader[2]
3. Cell Cycle Analysis: Cells (1×10⁶ cells/ml) are treated with 6.25, 12.5, 25 μM Baohuoside I for 24 h, washed with PBS, harvested with trypsin-EDTA, counted, fixed with 75% ethanol, and stored at 4℃ prior to DNA content analysis. Samples are resuspended in hypotonic propidium iodide (PI) solution (50 μg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100, 0.1 mg/ml ribonuclease A) and incubated in the dark for 1 h. PI-stained samples are analyzed with a flow cytometer[2]
4. Apoptosis Analysis (Annexin V/PI Double Staining): A549 cells (1×10⁶ cells/ml) are preincubated with 50 μM Z-VAD-FMK or 1 mM NAC for 1 h, then treated with 6.25, 12.5, 25 μM Baohuoside I for 24 h; alternatively, cells are co-incubated with 25 μM Baohuoside I and 25 μM SB203580 or 25 μM SP600125 for 24 h. Washed cells are resuspended in Annexin-V binding buffer, stained with FITC-conjugated Annexin-V and PI at room temperature for 15 min in the dark, followed by the addition of binding buffer. Apoptotic cells are detected using a flow cytometer[2]
5. TUNEL Analysis: A549 cells (2×10⁵ cells/ml) treated with 6.25, 12.5, 25 μM Baohuoside I are fixed with paraformaldehyde and permeabilized with Triton-X100 at 4℃ for 5 min. After washing, cells are labeled with TUNEL reaction mixture at 37℃ for 60 min, with fragmented DNA labeled as green spots; a second labeling with DAPI (1:20000 in PBS) is performed to visualize all nuclei. After washing, cells are photographed using a fluorescence microscope[2]
6. Measurement of Mitochondrial Membrane Potential (DWM): A549 cells are seeded to 70-80% confluency and treated with 6.25, 12.5, 25 μM Baohuoside I for 24 h, or preincubated with 1 mM NAC or 5 μM FCCP for 1 h. After washing twice with PBS, JC-1 is added to the cells for 30 min. After removing JC-1, cells are harvested by trypsinization and resuspended in PBS. The fluorescence intensity of JC-1 retained by 10,000 cells per sample is measured at 530 nm (green fluorescence, FL-1) and 590 nm (red fluorescence, FL-2) with a flow cytometer, and the change in DWM is evaluated by the green/red fluorescence ratio[2]
7. Intracellular ROS Generation Detection: Cells (1×10⁶ cells/ml) are treated with 6.25, 12.5, 25 μM Baohuoside I for 12 h, or preincubated with 1 mM NAC for 1 h, then incubated with 10 μM DCF-DA at 37℃ for 15 min. Intracellular ROS oxidizes DCF-DA to the fluorescent compound 2',7'-dichlorofluorescein (DCF), which is detected by a flow cytometer at an excitation wavelength of 480 nm and an emission wavelength of 525 nm[2]
8. Preparation of Cytosolic and Mitochondrial Fractions: Cells (2×10⁶ cells/ml) are washed with PBS and resuspended in isotonic homogenization buffer. After 80 strokes in a homogenizer, unbroken cells are spun down at 30g for 5 min. Mitochondrial fractions are separated by centrifugation at 800g for 10 min and 14000g for 30 min, respectively[2]
9. Western Blot Analysis: A549 cells (2×10⁶ cells/ml) treated with 6.25, 12.5, 25 μM Baohuoside I for 24 h are washed twice with ice-cold PBS, and proteins are extracted with lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 0.02% sodium azide, and 1% NP-40. Equal amounts of supernatant protein (50 μg) are denatured by boiling in SDS sample buffer, separated by 10% SDS–PAGE, and transferred to polyvinylidene fluoride membranes by semi-dry blotting. Membranes are blocked with 5% skim milk containing Tween 20, probed with primary antibodies against PARP, cleaved PARP, Bcl-2, BAX, caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, and β-actin, washed three times with TBS, and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Membranes are visualized with chemiluminescence reagents, and protein expression is quantified using image analysis software[2]
1. Cell Culture: Human esophageal squamous cell carcinoma Eca109 cells are cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml phytomycin at 37℃ in a humidified atmosphere of 5% CO₂. When the confluency reaches 80%, the cells are digested with 0.25% trypsin for subculture[3]
2. Cell Viability Assay (MTT Assay): Cells (1×10⁴ cells/well) are seeded in 96-well plates, and Baohuoside I at concentrations of 3.125, 6.25, 12.5, 25.0, and 50.0 μg/ml is added, with 10 replicate wells for each concentration. The cells are incubated for 24, 48, and 72 h respectively. MTT solution (5 mg/ml in PBS) is added to each well and incubated for 4 h. After centrifugation at 1500×g for 5 min, the medium is carefully removed, and 0.1 ml of dimethyl sulfoxide (DMSO) is added to each well to dissolve the crystals. After shaking, the absorbance is measured at 570 nm using a microplate reader, and the inhibition rate of cell viability by the drug is calculated with the cell viability of the blank control group as 100%[3]
3. Apoptosis Analysis (PI Staining): Eca109 cells are treated with Baohuoside I at concentrations of 0-50 μg/ml for 48 h, then collected and resuspended in PBS containing 50 μg/ml PI, 0.1% Triton X-100, 0.1 mmol/L EDTA(Na)₂, and 50 μg/ml RNase. After incubation in the dark for 30 min, analysis is performed with a flow cytometer (Ex=488 nm, Em=530 nm), and cells in the sub-G0 peak are regarded as apoptotic cells[3]
4. Western Blot Analysis: Total cellular proteins of Eca109 cells are extracted with cell lysis buffer. After sonication for 20 sec, the supernatant is collected by centrifugation at 1500×g for 10 min. 20-100 μg of protein samples are separated by 10% SDS-PAGE gel (electrophoresis at 120 V for 2 h) and transferred to a nitrocellulose membrane (transfer at 135 mA for 2 h). The membrane is blocked overnight with TBST buffer containing 5% milk, washed three times, incubated with primary antibodies against β-catenin, survivin, and cyclin D1 at room temperature for 2 h, washed three times with TBST, incubated with fluorochrome-labeled secondary antibody for 1 h, and imaged with an infrared imager after four washes[3]
5. RT-PCR Analysis: Total cellular RNA is extracted with TRI reagent. 3-5 μg of RNA is used to synthesize cDNA by adding oligo(dT)18, dNTP, and reverse transcriptase. Using cDNA as a template, PCR amplification is performed with specific primers (cyclin D1: annealing temperature 55℃, 35 cycles; survivin: annealing temperature 55℃, 38 cycles; β-actin: annealing temperature 55℃, 35 cycles). The amplified products are separated by 1.5% agarose gel electrophoresis and analyzed with an image analysis system[3]
6. Establishment of Stably-Tagged Eca109-luc Cell Line: The recombinant plasmid containing the firefly luciferase gene is transfected into Eca109 cells by lipofection. Stable cell lines are obtained by selection with 1.0 μg/ml puromycin for 7 days, and luciferase activity is detected with an in vivo imaging system after adding 150 μg/ml D-luciferin[3]

Xenograft tumor model of human esophageal squamous cell carcinoma. [3]
The use and care of animals were carried out by following the guidelines approved by the Institutional Animal Care and Use Committee. Female Balb/c nude mice (4- to 6-weeks-old) were used. Subconfluent Eca109-Luc cells were harvested and resuspended in PBS to a final density of 2x107 cells/ml. Prior to injection, cells were resuspended in PBS and analyzed by 0.4% trypan blue exclusion assay (viable cells >90%). For subcutaneous injection, ~1x106 Eca109-Luc cells in 200 µl PBS were injected into the left flank of each mouse using 27G needles. At 1 week after tumor cell injection, Baohuoside-I (25 mg/kg per mouse) was injected intralesionally once a day, whereas the 10 mice intended for vehicle treatment were administered an equal volume of PBS.

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Xenogen bioluminescence imaging.[3]
Small animal whole body optical imaging was carried out as described. Briefly, mice were anesthetized with isoflurane attached to a nosecone mask equipped with the Xenogen IVIS imaging system and subjected to imaging weekly after subcutaneous injection. For imaging, mice were injected (i.p.) with D-Luciferin sodium salt at 100 mg/kg body weight in 0.1 ml sterile PBS. Acquired pseudo-images were obtained by superimposing the emitted light over the grayscale photographs of the animal. Quantitative analysis was carried out with Xenogen's Living Image V2.50.1 software as described. Animals were sacrificed after 3 weeks, and tumor samples were retrieved for histological evaluation and Western blot analysis.

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1. Establishment of Human Esophageal Cancer Nude Mouse Xenograft Model: Female Balb/c nude mice aged 4-6 weeks are selected. Eca109-luc cells are resuspended in PBS to a density of 2×10⁷ cells/ml (trypan blue staining shows viable cell rate>90%), and 200 μl of cell suspension (containing 1×10⁶ cells) is subcutaneously injected into the left flank of each nude mouse[3]
2. Administration Protocol: One week after tumor cell injection, nude mice in the treatment group receive intralesional injection of 25 mg/kg Baohuoside I once daily, while the control group receives an equal volume of PBS, with continuous administration for three weeks[3]
3. In Vivo Imaging Detection: Nude mice are anesthetized with isoflurane weekly, and D-luciferin sodium salt aqueous solution at 100 mg/kg body weight is intraperitoneally injected. Images are collected by an in vivo optical imaging system, and the fluorescence signal intensity is quantified with imaging analysis software[3]
4. Tissue Processing: After three weeks of administration, nude mice are sacrificed, and tumor tissues are dissected. Part of the tissues are fixed with 10% formalin, embedded in paraffin for hematoxylin-eosin (H&E) staining, and part are used for Western blot analysis[3]

1. Due to the large amount of efflux of the apical efflux transporter, the bioavailability of sucralose I is low [2]. 2. The sucralose I-phospholipid complex can improve its absorption in the Caco-2 cell monolayer model [2]. 3. Vitamin E tocopherol polyethylene glycol succinate 1000 can enhance the intestinal absorption of sucralose I in a four-site rat intestinal perfusion model [2].

[1]. Baohuoside I suppresses invasion of cervical and breast cancer cells through the downregulation of CXCR4 chemokine receptor expression. Biochemistry. 2014 Dec 9;53(48):7562-9.

[2]. Reactive oxygen species-mediated mitochondrial pathway is involved in Baohuoside I-induced apoptosis in human non-small cell lung cancer. Chem Biol Interact. 2012 Jul 30;199(1):9-17.

[3]. The flavonoid Baohuoside-I inhibits cell growth and downregulates survivin and cyclin D1 expression in esophageal carcinoma via β-catenin-dependent signaling. Oncol Rep. 2011 Nov;26(5):1149-56.

Icariside II is a glycosyloxyflavonoid, chemically named 3,5,7-trihydroxy-4'-methoxy-8-isopentenylflavonoid, in which the hydroxyl group at the 3-position has been converted to α-L-rhamnoside. It has multiple activities such as plant metabolite, anti-inflammatory agent, antitumor agent and apoptosis inducer. It is functionally related to α-L-rhamnosylpyranose.
According to reports, piracetam I has been found in Epimedium pubescens, Epimedium acuminatum and other organisms with relevant data.

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1. Piracetam I, also known as Icariside II, is extracted from the stems and leaves of Epimedium koreanum and is traditionally used in traditional Chinese medicine to treat osteoporosis, menstrual disorders and joint pain[1].
2. Previous studies have shown that bacitracin I has anticancer activity against a variety of cancer cells, including prostate cancer, myeloma, osteosarcoma and skin cancer. Its mechanism of action includes reducing the level of hypoxia-inducible factor (HIF) 1α protein in human osteosarcoma cells, inhibiting the cyclooxygenase (COX) 2/prostaglandin E2 pathway in prostate cancer PC3 cells, blocking the signal transduction and transcription activator (STAT) 3 signaling pathway in U266 multiple myeloma cells, and inducing apoptosis in breast cancer MCF7 cells through intrinsic and extrinsic signaling pathways [1]
3. Bacitracin I exerts its anti-metastatic effect by downregulating CXCR4 expression, and the CXCR4/CXCL12 axis is a key mediator of tumor metastasis. Tumor cells that highly express CXCR4 tend to metastasize to organs that highly secrete CXCL12[1]
1. Acorin I, also known as icariin II, is a flavonoid compound isolated from Epimedium koreanum Nakai, which has anti-inflammatory and anticancer activities[2]
2. Acorin I is the main bioactive metabolite of icariin in vivo. In the perfusion rat intestinal model, icariin can be rapidly hydrolyzed into acorin I[2]
3. The mechanism by which acorin I induces apoptosis in A549 cells involves the ROS-mediated mitochondrial pathway and the MAPK pathway. ROS, as an upstream signal, can activate the JNK and p38MAPK pathways, thereby triggering mitochondrial dysfunction and activation of apoptosis-related molecules [2]. 4. Apoptosis induced by cyproheptadine I involves caspase-dependent and non-caspase-dependent mechanisms [2]. 1. Cyproheptadine I is a flavonoid compound extracted from the bark of Acorus calamus, with the molecular formula C27H30O10, a molecular weight of 514, and a purity of >96% [3]. 2. Cyproheptadine I inhibits the proliferation of esophageal cancer cells and induces their apoptosis by inhibiting the β-catenin-dependent signaling pathway and downregulating the expression of downstream target genes survivin and cyclin D1 [3]. 3. Cyproheptadine I has potential therapeutic value for esophageal squamous cell carcinoma and provides experimental evidence for new treatment options for esophageal cancer [3].

C27H30O10

514.5211

514.183

113558-15-9

5488822

Light yellow to yellow solid powder

1.5±0.1 g/cm3

759.4±60.0 °C at 760 mmHg

253.9±26.4 °C

0.0±2.7 mmHg at 25°C

1.666

4.65

5

10

6

37

874

5

O1[C@]([H])([C@@]([H])([C@@]([H])([C@]([H])([C@]1([H])C([H])([H])[H])O[H])O[H])O[H])OC1C(C2=C(C([H])=C(C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])=C2OC=1C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])O[H])O[H])=O

NGMYNFJANBHLKA-LVKFHIPRSA-N

InChI=1S/C27H30O10/c1-12(2)5-10-16-17(28)11-18(29)19-21(31)26(37-27-23(33)22(32)20(30)13(3)35-27)24(36-25(16)19)14-6-8-15(34-4)9-7-14/h5-9,11,13,20,22-23,27-30,32-33H,10H2,1-4H3/t13-,20-,22+,23+,27-/m0/s1

5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one

Baohuoside I; 113558-15-9; Icariside II; BAOHUOSIDEI; CHEBI:82619; 5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one; CHEMBL560116; 4H-1-Benzopyran-4-one, 3-[(6-deoxy-alpha-L-mannopyranosyl)oxy]-5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methyl-2-buten-1-yl)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 32 mg/mL (~62.19 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.04 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 20 mg/mL (38.87 mM) in 0.5% CMC/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)