Na+/K+-ATPase

Convolvulin is non-toxic to normal cells and has an effective growth-inhibiting effect on cancer cells (MCF-7, A549, and HepG2 cells; 0~10 μM; 24 hours) [1]. There was no discernible cytotoxicity in PBMC when volvoluvulin (0.5 to 500 µM) was present. The cell cycle can be stopped in the G2/M phase using volvulin (2 µM) on MCF-7 cells [1]. Convolvulin, which is nontoxic to normal cells, can effectively inhibit the growth of cancer cells in MCF-7, A549, and HepG2 cells. In three cancer cells—MCF-7, A549, and HepG2—Strophanthidin inhibited the expression of checkpoint and cyclin-dependent kinases in comparison to the untreated controls. Proteins can be localized from the nucleus to the cytoplasm and cell membrane by using volvulin. With an aglycone moiety and no sugar units, convolvulin is a monosaccharide cardiac glycoside. By inhibiting several biochemical signaling pathways and stopping the cell cycle in the G2/M phase via both p53-dependent and p53-independent mechanisms, volvulin causes apoptosis [1].

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Lung cancer is the most prevalent in cancer-related deaths, while breast carcinoma is the second most dominant cancer in women, accounting for the most number of deaths worldwide. Cancers are heterogeneous diseases that consist of several subtypes based on the presence or absence of hormone receptors and human epidermal growth factor receptor 2. Several drugs have been developed targeting cancer biomarkers; nonetheless, their efficiency are not adequate due to the high reemergence rate of cancers and fundamental or acquired resistance toward such drugs, which leads to partial therapeutic possibilities. Recent studies on cardiac glycosides (CGs) positioned them as potent cytotoxic agents that target multiple pathways to initiate apoptosis and autophagic cell death in many cancers. In the present study, our aim is to identify the anticancer activity of a naturally available CG (Strophanthidin) in human breast (MCF-7), lung (A549), and liver cancer (HepG2) cells. Our results demonstrate a dose-dependent cytotoxic effect of strophanthidin in MCF-7, A549, and HepG2 cells, which was further supported by DNA damage on drug treatment. Strophanthidin arrested the cell cycle at the G2/M phase; this effect was further validated by checking the inhibited expressions of checkpoint and cyclin-dependent kinases in strophanthidin-induced cells. Moreover, strophanthidin inhibited the expression of several key proteins such as MEK1, PI3K, AKT, mTOR, Gsk3α, and β-catenin from MAPK, PI3K/AKT/mTOR, and Wnt/β-catenin signaling. The current study adequately exhibits the role of strophanthidin in modulating the expression of various key proteins involved in cell cycle arrest, apoptosis, and autophagic cell death. Our in silico studies revealed that strophanthidin can interact with several key proteins from various pathways. Taken together, this study demonstrates the viability of strophanthidin as a promising anticancer agent, which may serve as a new anticancer drug. [1]

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Str/Strophanthidin 0.1 nmol/L stimulated the Na+, K+-ATPase activities (P<0.05), but had no effect on HR, LVP, and +/-dp/dt(max). Str 1 nmol/L increased +dp/dt(max) (P<0.05) and Na+, K+-ATPase activities (P<0.01). Str 10 and 100 nmol/L significantly increased both LVP (P<0.05) and +dp/dt(max) (P<0.05 or P<0.01), and had no significant effects on Na+, K+-ATPase activities. However, Str 1-100 micromol/L at first enhanced the LVP and +dp/dtmax (P<0.01), then reduced them resulting from irregular contraction, and effects of Str on Na+, K+-ATPase activities revealed a concentration-dependent inhibition (P<0.01). Conclusion: The positive inotropic effects and irregular contraction produced by Strophanthidin/Str at higher concentrations result from the inhibition of Na+, K+-ATPase activities, and the positive inotropic effects of Str at lower concentrations are not related to the inhibition of the Na+, K+-ATPase activities. [2]

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We have investigated the effects of inhibiting the Na-K pump with Strophanthidin on the intracellular Ca2+ concentration ([Ca2+]i), sarcoplasmic reticulum (s.r.) Ca2+ content and membrane currents. s. r. Ca2+ content was measured by integrating the Na-Ca exchange current resulting from application of 10 mM caffeine. The application of strophanthidin increased both diastolic and systolic [Ca2+]i. This was accompanied by an increase of s.r. Ca2+ content from a resting value of 17.9+/-1.5 micromol/l to 36.9+/-3.3 micromol/l (n=16) after 5 min. Systolic fluxes of Ca2+ into and out of the cell before and during strophanthidin application were also measured. Ca2+ efflux (measured as the integral of the Na-Ca exchange tail current) rose steadily in the presence of strophanthidin, while Ca2+ influx (the integral of the L-type Ca2+ current) was reduced. In spite of this, s.r. Ca2+ content rose substantially. In the presence of Cd2+ (100 microM), which inhibits the L-type Ca2+ current, strophanthidin had negligible effects on current suggesting that Ca2+ influx via Na-Ca exchange during depolarization does not account for the increase of s.r. Ca2+ content. This suggests that changes of Ca2+ flux during systole are not responsible for the strophanthidin-induced increase of s.r. Ca2+. We conclude that the primary mechanism by which the cardiac cell gains Ca2+ when the Na-K pump is inhibited is by a net influx during diastole [3].

Determination of the cardiac sarcolemmal Na+ , K+ -ATPase activities [2]
The cardiac sarcolemmal Na+ , K+ -ATPase activities were determined by colorimetry. The suspension of cardiac sarcolemma (50-70 µg proteins per mL) was divided into 8 groups (8 samples each group) and incubated with saline or different concentrations (0.1, 1, 10, 100 nmol/L and 1, 10, 100 µmol/L) of Str at 37 ºC for 10 min according to the method provided by manufacturer. Enzyme active unit was expressed as mmol inorganic phosphate per gram sarcolemmal protein per hour (mmol·h-1·g-1 protein).

Cell viability assay [1]
Cell Types: MCF-7, A549 and HepG2 Cell
Tested Concentrations: 0~10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited the proliferation of three different cancer cells.

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Cell Viability Assay [1]
The antiproliferative effect of Strophanthidin on MCF-7, A549, HepG2, and normal cells such as L132, WRL68, and PBMCs was determined by performing an MTT assay. About 4,000 cells per well were plated in a 96-well cell culture microplate and incubated overnight in complete media (DMEM containing 10% FBS with antibiotic) for cells to adhere to the plate. Cells were then treated with various concentrations ranging from 15 to 0.1 μM of strophanthidin for 24 h in serum-free media. An MTT assay was performed to identify cell viability. The absorbance of solubilized formazan was read at 570 nm and at the reference wavelength (non-specific readings) of 650 nm using a multimode plate reader.

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Cell Cycle Analysis [1]
Approximately 1 × 105-106 cells were seeded and incubated for overnight growth before treating with Strophanthidin for 24 h. An appropriate number of cells were added to a conical tube and centrifuged at 1,000 rpm for 3 min. Then the cells were washed with chilled phosphate-buffered saline (PBS) and vortexed for 10 s to attain single-cell suspension. Then the cells were fixed with 4.5 ml of chilled ethanol (100%) for 30 min at 4°C. The cells were then rinsed with cold PBS to remove ethanol and then incubated with 10 μg/ml of RNase for 1–2 h in the dark at 37°C. After incubation, the fixed cells were stained with 0.25 μg/ml of propidium iodide for 30 min, and cell cycle distribution was measured by a flow cytometer.

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Gene Expression Studies Through Real-Time PCR [1]
Total cellular RNA was isolated using the TRIzol® reagent as per manual instructions. The cells were treated with Strophanthidin for 24 h, and the total RNA was isolated in the TRIzol reagent. Purity and concentrations were estimated using NanoDrop. A Verso complementary DNA (cDNA) synthesis kit was used for the synthesis of cDNA by following manufacturer instructions. A total of 2 μg of pretreated RNA was used for the synthesis of cDNA. Real-time quantitative PCR was performed using the Origin SYBR Green Master Mix in the Roche LightCycler 480 system. RNA expression levels were normalized to that of GAPDH and calculated using the ΔΔCt method, and the log2 values were plotted in the graphs. All the primers used in this study are listed in Supplementary Table 1.

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Enzyme-Linked Immunosorbent Assay [1]
Briefly, the cells were treated with lethal doses of Strophanthidin for 24 h, and the total cell lysate was extracted with a RIPA lysis buffer. Enzyme-linked immunosorbent assay (ELISA) antibodies for caspases 3, 7, 8, and 9 were used. The experimentation was processed by following the manufacturer guidelines. Likewise, we performed ELISA to understand the role of Strophanthidin in pathway activation, cellular growth, and apoptosis. We have used a PathScan® MAP Kinase Multi-Target Sandwich ELISA Kit, to understand the phosphorylation of Phospho-MEK1/2 (Ser217/Ser221), Phospho p38 MAPK (Thr180/Tyr182), Phospho p44/42 MAPK (Thr202/Tyr204), and Phospho-SAPK/JNK (Thr183/Tyr185). The absorbance was measured at 450 nm. The experiment was repeated thrice, and the obtained results were plotted in bar charts.

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Western Blot Analysis [1]
Cells were initially treated with Strophanthidin for 24 h. Treated cells were then harvested and lysed in lysis buffer containing 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A, along with a protease inhibitor cocktail, on ice for 30 min. After centrifugation at 14,000 g for 15 min at 4°C, the supernatant was collected as total cellular protein content. The concentration of total proteins was estimated by the Bradford protein estimation assay. An equal concentration (30 μg) of total protein was resolved on 12–15% of SDS-polyacrylamide gel electrophoresis (PAGE) for different-sized proteins and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was then blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4°C overnight. After being washed three times with TBST containing 150 mM NaCl, 10 mM Tris, and 0.1% Tween 20 with pH 7.4, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h and washed with TBST for five to six times in 5-min intervals. Immunoreactive proteins were detected with a chemiluminescent ECL substrate and quantified using the C-DiGit chemiluminescent western blot imaging system (LI-COR). Mean densitometry data from independent experiments were normalized to those of control experiments. Antibodies were procured as follows: caspases 3, 7, and 9 and PARP-1 were obtained from Cell Signaling Technology and Chk1, Chk2, cyclin D1, p53, AKT, p38, MEK1, mTOR, CDK6, SAPK/JNK, C-Myc, BAX, JAK, STAT3, GSK3α, β-catenin, Beclin 1, p62, LC3, PI3K, and GAPDH were used.

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Immunofluorescence [1]
Immunofluorescence was done to understand the protein migration after treating with Strophanthidin. Approximately 0.3 × 106 cells were seeded on top of the coverslips in a 6-well plate. After 16 h, the cells were treated with Strophanthidin and incubated for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde for 20 min and again incubated with 0.1% Triton X for 20 min. After the coverslip was washed with 1X TBS four to five times, it was blocked with 5% BSA for 1 h. Then the coverslip was incubated with a primary antibody overnight at 4°C. The coverslip was then washed with 1X TBS and incubated in a secondary antibody (Alexa Fluor 488) for 2 h. Then the coverslip was washed and incubated with 0.1 μg/ml concentration of DAPI for 20 min in the dark and washed five to six times with 1X TBS. Then the coverslip was transferred to clean glass slides coated with ProLong Gold Antifade Mountant. Excess amount of Antifade was removed, and the slides were sealed with wax and observed under a fluorescent microscope with 40X magnification.

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Quantification of s.r. Ca2+ content and sarcolemmal Ca2+ fluxes [3]
The inward Na-Ca exchange currents produced by applying 10 mM caffeine to a voltage-clamped cell were integrated and converted to total Ca2+ fluxes as described previously. Net movements of Ca2+ during and following voltage-clamp pulses were calculated by integrating membrane currents. The integration of the Ca2+ current during depolarization was done with respect to the level of current before depolarization. Integration began, following the capacity current, when the membrane current crossed this zero level and continued until the end of the pulse. Integration of the tail current began at the point of inflection between the end of the capacity current and the start of the (slower) tail current. One complication found in the present experiments in guineapig cells was that, following the initial transient inward current produced by caffeine, current relaxed to a value which was often more inward than the initial level (see Fig. 2). The origin of this current is not known. We used the final steady-state level as the baseline for integration. Any error introduced by this may affect the basal measurement of s.r. Ca2+ content. It will not, however, affect the estimate of changes of s.r. Ca2+ as the level of this steady-state current was unaffected by Strophanthidin. The Na-Ca exchange currents were first corrected for the activity of non-electrogenic Ca2+ extrusion pathways. Briefly, one needs to correct for the fact that, during a caffeine response, some of the Ca2+ is removed from the cytoplasm by mechanisms other than Na-Ca exchange and does not, therefore, generate current. The magnitude of this current was estimated as follows. The rate constant of decay of the caffeine response was measured (1) under control conditions (kcont) and (2) with the Na-Ca exchange inhibited by 10 mM Ni2+ (kNi). Multiplying a measured Na-Ca exchange flux by kcont/(kcont–kNi) gives the corrected total Ca2+ flux. This correction was used for the Na-Ca exchange fluxes activated by either caffeine or on repolarization (tail currents). The multiplying factor used (obtained from guinea-pig myocytes) was 1.19 (Choi and D. Eisner, unpublished observations). Cell volume was calculated from the membrane surface area obtained from the membrane capacitance:volume ratio of 5 pF/pl for the guinea-pig. All changes of Ca2+ content and sarcolemmal fluxes are expressed in relation to this calculated cell volume

Preparation of the isolated hearts Guinea pigs of either sex, weighing 250±20 g, were used. The hearts were rapidly excised, mounted through aorta, and perfused on a modified Langendorff apparatus at a constant perfusion pressure (10 kPa). A polyethylene cannula containing saline was introduced into the left ventricle cavity and was connected with a pressure transducer, by which heart rate (HR), left ventricular pressure (LVP), and its first derivatives (±dp/dtmax) were recorded on a polygraph system. The hearts were perfused with K-H buffer solution (37 ºC, pH 7.4, saturated with 95 % O2 and 5 % CO2). The K-H buffer solution contained the following (in mmol/L): NaCl 118, NaHCO3 25.0, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, and glucose 11.0. All the hearts were first perfused with K-H solution for 20-30 min for stabilization in a Langendorff apparatus and then were randomly divided into 8 groups (6-8 hearts each group). The hearts were perfused with K-H buffer solution in control group, and in seven Strophanthidin/Str groups with K-H solution containing different concentrations of Strophanthidin/Str (0.1, 1, 10, 100 nmol/L or 1, 10, 100 µmol/L). HR, LVP and ±dp/dtmax were continuously recorded for 20 min. [2]
Isolated guinea-pig hearts were perfused through aorta in a Langendorff mode. Heart rate (HR), left ventricular pressure (LVP), and first derivatives (+/-dp/dt(max)) of LVP were recorded by eight-channel physiological instrument. Cardiac sarcolemmal Na+, K+-ATPase activities were determined with colorimetry. [2]

6185 mouse LD50 intravenous 330 ug/kg CRC Handbook of Antibiotic Compounds, Vols.1- , Berdy, J., Boca Raton, FL, CRC Press, 1980, 8(2)(224), 1982
6185 mouse LD50 intracrebral 63 ug/kg BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Arzneimittel-Forschung. Drug Research., 11(908), 1961
6185 cat LD50 intravenous 224 ug/kg Journal of Pharmacology and Experimental Therapeutics., 103(420), 1951 [PMID:14908859]
6185 cat LDLo unreported 260 ug/kg Archives Internationales de Pharmacodynamie et de Therapie., 148(471), 1964 [PMID:14181242]
6185 rabbit LDLo intravenous 110 ug/kg Naunyn-Schmiedeberg's Archiv fuer Experimentelle Pathologie und Pharmakologie., 185(329), 1937

[1]. Strophanthidin Attenuates MAPK, PI3K/AKT/mTOR, and Wnt/β-Catenin Signaling Pathways in Human Cancers. Front Oncol. 2020;9:1469. Published 2020 Jan 17.

[2]. Relationship between cardiotonic effects and inhibition on cardiac sarcolemmal Na+,K+-ATPase of strophan-thidin at low concentrations. Acta Pharmacol Sin. 2003;24(11):1103-1107.

[3]. Strophanthidin-induced gain of Ca2+ occurs during diastole and not systole in guinea-pig ventricular myocytes. Pflugers Arch. 1999;437(5):731-736.

Strophanthidin is a 3beta-hydroxy steroid, a 14beta-hydroxy steroid, a 5beta-hydroxy steroid, a 19-oxo steroid, a member of cardenolides and a steroid aldehyde. It is functionally related to a 5beta-cardanolide.
Strophanthidin has been reported in Adonis aestivalis, Erysimum pulchellum, and other organisms with data available.
3 beta,5,14-Trihydroxy-19-oxo-5 beta-card-20(22)-enolide. The aglycone cardioactive agent isolated from Strophanthus Kombe, S. gratus and other species; it is a very toxic material formerly used as digitalis. Synonyms: Apocymarin; Corchorin; Cynotoxin; Corchorgenin.

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In conclusion, our in vitro and in silico experiments demonstrate that a natural compound Strophanthidin has a potent anticancer effect on breast, lung, and liver cancer cells. We have demonstrated the possible hypothetical mechanism of apoptosis in these three cancer cells (Figure 10). Strophanthidin hinders the expression of PI3K in MCF-7 cells, which is a key protein for PI3K/AKT/mTOR signaling. This inhibition leads to further activation of AKT, and the inhibition of mTOR and also shows multistep inhibition of p53, which in turn leads to the deregulation of anti-apoptotic protein (Bcl-2) and apoptosis. Suppression of mTOR leads to the downregulation of Beclin 1 and stimulates the inhibition of LC3 and p62 complex to inhibit autophagy. Inhibition of MEK1 was identified in A549 cells due to the downregulation of PI3K and plays a crucial role by inhibiting Gsk3α and β-catenin, which can directly target c-Myc and cyclin D1 to influence apoptosis. Dysregulated MEK1 targets p38MAPK and inhibits the expression of STAT3 and c-Myc to initiate apoptosis. A549 cells exhibit solid autophagic flux compatible with their proliferation rate, and strophanthidin inhibits the expression of LC3 and p62 complex through MEK1 inhibition; hence, it seems that this compound can inhibit autophagy. The inhibition of autophagy exacerbates apoptosis, which is a critical mechanism for a cancer cell to maintain the cellular energy and nutritional homeostasis. MEK1 also activated the expression of BAX by multistep stimulation, which could be an important factor for the activation of initiator caspase 7 and further leads to the expression of caspase 3 and causes apoptosis through death receptor signaling and caspase-mediated apoptosis as witnessed in HepG2 cells. Moreover, strophanthidin exhibited a differential expression that shows the p53-dependent and p53-independent apoptosis in cancer cells. To the best of our knowledge, this is the first study to identify the changes in various biochemical apoptotic signal transduction pathways like MAPK signaling, PI3K/AKT/mTOR, and Wnt/β-catenin pathways to initiate apoptosis. Conclusively, this study revealed for the first time that strophanthidin induced apoptosis by the attenuation of multiple biochemical signaling pathways and by arresting cell cycle at the G2/M phase through p53-dependent and p53-independent mechanisms. [1]

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In conclusion, Our results suggest that Strophanthidin at lower concentrations still has the positive inotropic effects, which involve a mechanism other than Na+ , K+ -ATPase inhibition. [2]

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In conclusion, we have shown that a measured gain of s.r. Ca2+ content is accompanied by a calculated loss of Ca2+ during systole suggesting, therefore, that there is an increased influx of Ca2+ between each pulse during diastole that overcomes the increased net efflux during systole and accounts for the increase in s.r. Ca2+ content. [3]

C23H32O6

404.49658

404.22

C, 68.29; H, 7.97; O, 23.73

66-28-4

6185

White to off-white solid powder

1.432 g/cm3

620.7ºC at 760 mmHg

169ºC

214.9ºC

1.674

1.898

3

6

2

29

777

8

O=C1OCC([C@H]2CC[C@@]3([C@@H]4CC[C@]5(O)C[C@H](CC[C@]5(C=O)[C@H]4CC[C@]23C)O)O)=C1

ODJLBQGVINUMMR-HZXDTFASSA-N

InChI=1S/C23H32O6/c1-20-6-3-17-18(4-8-22(27)11-15(25)2-7-21(17,22)13-24)23(20,28)9-5-16(20)14-10-19(26)29-12-14/h10,13,15-18,25,27-28H,2-9,11-12H2,1H3/t15-,16+,17-,18+,20+,21-,22-,23-/m0/s1

(3S,5S,8R,9S,10S,13R,14S,17R)-3,5,14-trihydroxy-13-methyl-17-(5-oxo-2H-furan-3-yl)-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-10-carbaldehyde

k-Strophanthidin; Strophanthidin; strophanthidin; Convallatoxigenin; Strophanthidine; Corchsularin; k-Strophanthidin; Corchorgenin; Erysimupicrone; ...; 66-28-4; k Strophanthidin; NSC 86078; NSC86078; NSC-86078; Apocymarin

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)

Ethanol : ~25 mg/mL (~61.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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