Alistatin (multiple concentrations; 24 hours) effectively inhibited the proliferation of primary human umbilical vein endothelial cells, with an average IC50 of 3.14 μM[1]. Alistatin (multiple concentrations; 24 hours) effectively inhibited the proliferation of primary human dermal lymphatic endothelial cells, with an average IC50 of 2.54 μM, and completely inhibited cell proliferation at a concentration of 5 μM[1]. Alistatin (multiple concentrations; 24 hours) effectively inhibited the proliferation of primary human pulmonary lymphatic endothelial cells, with an average IC50 of 2.63 μM[1]. Alistatin (10-50 μM; 24 hours) dose-dependently induced apoptosis in primary human dermal lymphatic endothelial cells, with significant effects observed at concentrations of 10 μM and above, and its pro-apoptotic efficacy was stronger than that of hypericin[1]. Alistatin (5-30 μM; 48 hours) can induce G1 phase cell cycle arrest in primary human dermal lymphatic endothelial cells at a concentration of 5 μM, and can induce apoptosis (manifested as an increase in the number of cells in sub-G1 phase) at a concentration of 30 μM [1]. Alistatin (30 μM; 48 hours) can activate the intrinsic apoptotic pathway of primary human dermal lymphatic endothelial cells at a concentration of 30 μM, manifested as the activation of caspase-3 and caspase-9, while caspase-8 was not activated [1]. Alistatin (20 μM; incubation at 37°C for 20 minutes) can cause rapid loss of mitochondrial membrane potential in primary human dermal lymphatic endothelial cells after treatment at a concentration of 20 μM for 20 minutes [1]. Alistatin (1–5 μM; 16 h) altered the levels of MRP1, MRP2, BCRP, and P-gp proteins in human colon adenocarcinoma HT-29 cells in a time- and light-dependent manner, including decreased MRP2 levels at T0– and T6+, increased MRP1 levels at T0–, and increased P-gp levels at T6+ and T6–[2]. Alistatin (1–5 μM; 16 h) altered the levels of CYP3A4 protein in cultured human colon adenocarcinoma HT-29 cells in a concentration-, time-, and light-dependent manner, including increased levels at T0–, T6–, and T6+ (1 μM), and decreased levels at T0+ (5 μM)[2]. In human colon adenocarcinoma HT-29 cells, alisttarin (1–5 μM; 16 h) induced BCRP mRNA expression at T0–, while hypericin/alisttarin combined treatment reduced MRP1, MRP2, and P-gp mRNA expression at T0–[2]. In vitro, alisttarin (1–5 μM; 16 h) regulated CYP3A4 mRNA expression in human colon adenocarcinoma HT-29 cells in a concentration-, time-, and light-dependent manner, including induction at T0+ (1 μM) and T6+ (5 μM) and reduction at T6–[2]. Alisttarin (5–10 μM; 30 min) significantly reduced BCRP activity in human colon adenocarcinoma HT-29 cells, but had no significant effect on MRP1 or MRP2 activity[2]. Alistatin (10 μM; 16 h) significantly reduced CYP3A4 activity in human colon adenocarcinoma HT-29 cells and oxaliplatin-resistant HT-29-OxR cells, and this effect was independent of photoactivation [2]. Alistatin (0.01–10 mM; 25 min) exhibited concentration-dependent DPPH radical scavenging activity, with scavenging rates of 5.76%, 12.03%, 17.89%, and 28.02% at concentrations of 0.01, 0.1, 1, and 10 mM, respectively [3]. Alistatin (0.01–10 mM; 20 min at 50 °C) had very low reducing power, with absorbance values of 0.002, 0.002, 0.068, and 0.085 at concentrations of 0.01, 0.1, 1, and 10 mM, respectively [3]. Alistatin (0.01–10 mM; reaction time 10 min) cannot chelate ferrous ions in the concentration range of 0.01–10 mM[3]. Alistatin (0.01–1 mM; incubation at 25°C for 90 min) exhibits concentration-dependent hydroxyl radical scavenging activity, with scavenging rates of 18.18 ± 6.54%, 33.0 ± 2.75%, and 55.84 ± 8.21% at concentrations of 0.01, 0.1, and 1 mM, respectively[3]. Alistatin (1–5 μM; pre-incubation for 16 h, followed by PDT incubation for 24–48 h) at a concentration of 5 μM, after 16 h of pre-incubation followed by 50 nM hypericin-mediated PDT, significantly enhanced apoptosis in HT-29 colon adenocarcinoma cells[4]. When 16 hours of pre-incubation with 5 μM aristolin (1-5 μM; 16 hours of pre-incubation followed by 24 hours of PDT) was performed, followed by 50 nM hypericin-mediated PDT, the cell cycle distribution of HT-29 colon adenocarcinoma cells was restored to normal 24 hours after PDT, offsetting the accumulation of S-phase cells induced by PDT alone [4]. When 16 hours of pre-incubation with 5 μM aristolin (1-5 μM; 16 hours of pre-incubation followed by 6-24 hours of PDT) was performed, followed by 50 nM hypericin-mediated PDT, the expression of MMP2 and MMP9 in HT-29 colon adenocarcinoma cells was significantly inhibited 6 hours after PDT [4]. When HT-29 colon adenocarcinoma cells were pre-incubated for 16 hours with 5 μM Aristofolin (1-5 μM; pre-incubation for 16 hours, PDT followed by 24 hours, and then seeded for 3 hours), followed by 50 nM hypericin-mediated PDT, cell adhesion of HT-29 colon adenocarcinoma cells was significantly reduced [4]. When HT-29 colon adenocarcinoma cells were pre-incubated for 16 hours with 5 μM Aristofolin (1-5 μM; pre-incubation for 16 hours, PDT followed by 6 hours, and then seeded for 1 week), clonogenic ability of HT-29 colon adenocarcinoma cells was effectively inhibited [4].

In female Wistar rats with MT-450 breast tumors, Aristoforin (2 mM; sc (peritumoral); daily; 14 days) significantly inhibited tumor-induced lymphangiogenesis, reduced peritumoral lymphatic density compared with DMSO control, and did not change tumor volume at the study endpoint [1].

Western Blot Analysis [2]
Cell Types: Human colon adenocarcinoma HT-29 cells
Tested Concentrations: 1 μM, 5 μM (incubated for 16 hours)
Incubation Duration: 16 hours (incubation); analysis was performed at T0-, T0+, T6-, and T6+ time points
Experimental Results: At T0- (dark conditions, immediately after treatment), MRP2 and BCRP protein levels decreased, while MRP1 protein levels increased. At T0+ (after photodynamic therapy, immediately after treatment), MRP2 protein levels increased, while BCRP and MRP1 protein levels decreased. At T6- (dark conditions, 6 hours after treatment), MRP2, BCRP, and P-gp protein levels increased. At T6+ (6 hours post-photodynamic therapy), MRP2 and BCRP protein levels decreased, while P-gp protein levels increased. At T0- (immediately post-treatment in darkness), CYP3A4 protein levels increased by 2.15-fold (1 μM) and 2.55-fold (5 μM) compared to the control group, respectively. At T0+ (immediately post-treatment in darkness), CYP3A4 protein levels at a concentration of 5 μM decreased by 0.74-fold compared to the control group. At T6- (6 hours post-treatment in darkness), CYP3A4 protein levels at a concentration of 1 μM increased by 1.11-fold (1 μM) and 1.21-fold (5 μM) compared to the control group, respectively. At T6+ (6 hours post-treatment in darkness), CYP3A4 protein levels at a concentration of 1 μM increased by 1.98-fold compared to the control group.

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Cell cycle analysis [4]
Cell Types: HT-29 colon adenocarcinoma cells
Tested Concentrations: 1-5 μM (pre-incubation for 16 hours, followed by PDT treatment, and incubation for 24 hours after PDT treatment)
Incubation Duration: 16 hours (pre-incubation); 24 hours (incubation after PDT treatment)
Experimental Results: When used in combination with 50 nM hypericin-mediated PDT, it partially reversed the S phase accumulation caused by PDT alone, restoring the cell cycle distribution to a level similar to that of untreated control cells.

Animal/Disease Models:Wistar rats (female) [1]
Doses: 2 mM (100 μL per injection)
Route of Administration: Subcutaneous injection (peritumor); once daily; 14 days
Experimental Results: Compared with the DMSO control group, the density of podocyte-positive lymphatic vessels around the tumor was significantly reduced. At sacrifice, the remaining tumor volume was comparable to that of the control group.

[1]. Hyperforin and aristoforin inhibit lymphatic endothelial cell proliferation in vitro and suppress tumor-induced lymphangiogenesis in vivo. Int J Cancer. 2009;125(1):34-42.

[2]. Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells. Biomed Pharmacother. 2016;81:38-47.

[3]. DNA-protective activities of hyperforin and aristoforin. Toxicol In Vitro. 2015;29(3):631-637.

[4]. The pro-apoptotic and anti-invasive effects of hypericin-mediated photodynamic therapy are enhanced by hyperforin or aristoforin in HT-29 colon adenocarcinoma cells. J Photochem Photobiol B. 2012;117:115-125.

C37H54O6

594.82

849215-53-8

Typically exists as solids at room temperature

CC(C)C([C@@]12[C@@](C)([C@H](C[C@@](C1=O)(C(OCC(O)=O)=C(C2=O)C/C=C(C)\C)C/C=C(C)\C)C/C=C(C)\C)CC/C=C(C)/C)=O

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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