Natural product; anticancer; anthelmintic; Notch

After 48 hours of treatment, brucine D had dose-dependent effects on H460 and A549 cell viability, with IC50 values of 0.5 and 0.6 μM, respectively [1]. In H460 and A549 cells, brucine D (0.125-1.0 μM) dose-regulatedly enhances chromatin condensation, annexin V-induced cell swarming, and caspase cell death [1].

---

Bruceine D (BD) Showed Potent Antifeedant Activity against DBM [1]
Bruceine D was identified, and its capacity for protecting flowering Chinese cabbage from the feeding of larvae of DBM was evaluated by root and leaf application. Azadirachtin was reported as a preeminent feeding deterrent with systemic action; therefore, azadirachtin was employed for comparison.

---

No-Choice Feeding Assays via Root Application [1]
After incubation with 100 μg/mL bruceine D or azadirachtin at 24 and 48 h, the antifeedant activity against third instar larvae of DBM was evaluated (Figure 2A). With root application of bruceine D, flowering Chinese cabbage leaves showed excellent antifeeding activity, reducing consumption by 93.80% and 96.83%. In contrast to bruceine D, azadirachtin was ineffective through this application, which showed no feeding deterrent at 24 h of treatment and at a 22.60% antifeedant rate for 48 h of treatment. Then, the contents of bruceine D and azadirachtin in leaves were quantified by HPLC-HRESIMS (Figure 2C and D). Both bruceine D and azadirachtin possessed substantial systemic behavior and were continuously translocated to leaves as a function of time (Figure 2B). After root application for 24 and 48 h, the concentrations of bruceine D were 38.69 μg/g (fresh weight, FW) and 108.45 μg/g (FW) which were significantly (p < 0.05) greater than that of azadirachtin with concentrations of 21.43 μg/g (FW) and 36.88 μg/g (FW). Obviously, bruceine D presented better systemic properties than azadirachtin within 48 h. The dosage of bruceine D in leaves was sufficient to prevent the feeding behavior of DBM, while azadirachtin only weakly affected the intake.

---

No-Choice Feeding Assays via Leaf Application [1]
Bruceine D (BD) showed a strong antifeedant effect. To evaluate the antifeedant activity against third instar larvae of DBM, a series of concentrations from 2.5 to 160 μg/mL were used to coat flowering Chinese cabbage leaf discs, and azadirachtin was employed as a positive control with different concentrations viz., 20, 40, 80, 160, 320, 640 μg/mL. After feeding for 24 h, the two compounds showed a dose-dependent antifeedant activity behavior. The values of AFC50 of bruceine D and azadirachtin were 0.11 and 0.68 μg/cm2, respectively, which indicated that the antifeedant activity of bruceine D was approximately 6.2-fold stronger than azadirachtin under our experimental conditions.

---

Bruceine D (BD) inhibited HCC cell viability and induced cellular apoptosis in vitro [2]
To identify therapeutic agents for HCC, we sought to discover a Notch inhibitor in HEK293T cells containing a luciferase-based RBP-Jκ-Luc reporter with ICN (the Intracellular Domain of Notch) -driven luciferase in response to Notch signaling activity (Fig. S1A). In a screen of 1600 natural products, BD showed the strongest inhibition to RBP-Jκ-Luc activity (Fig. S1B). To determine whether BD is an HCC inhibitor, Huh7 and Hep3B cells were treated for 48 h with various concentrations of BD. CCK8 assay revealed strong dose- and time-dependent inhibition of the proliferation of both cell types, and higher sensitivity was observed in HCC cells than in human hepatic cells L-02 (Fig. S2A, B). Similar to the effects on cell survival, BD also inhibited cancer cell colony formation of Huh7 and Hep3B in a concentration-dependent manner (Fig. S2C). Moreover, BD induced a dose-dependent increase of apoptosis, especially early apoptosis, in Huh7 and Hep3B cells, compared with control cells (Fig. S2D). These results confirmed that the BD inhibition of Notch signaling was accomplished through arrest of cell proliferation and induction of apoptosis on HCC cells.

---

Bruceine D (BD) suppressed protein synthesis of Jagged1 in HCC cells [2]
To understand the underlying mechanism of BD inhibition of Notch signaling, we next determined the effect of BD on Notch signaling–related proteins in Huh7 and Hep3B cell lines. Notably, while we observed no change in Notch 1, 2 or 3, Notch ligand Jagged1, cleaved Notch receptor NICD, and Notch-specific downstream protein Hes1 were all clearly downregulated by BD (Fig. 4A).

---

Jagged1 is a β-catenin target gene and required for Bruceine D (BD)-mediated proliferation inhibition of HCC Inhibition of Jagged1 is connected to a reduced tumor burden in malignant HCC. To illustrate the relationship of Jagged1 with the effects of BD, we performed gain- and loss-of-function assays of Jagged1 in HCC cells (Fig. 5A). Trypan blue exclusion test showed that Huh7 cells containing abundant Jagged1 display the ability to proliferate despite the cytotoxic effect of BD (Fig. 5B). In keeping with the effect of BD inhibition, knockdown of Jagged1 led to reduced cell viability and increased apoptosis compared with controls (Fig. 5B). In addition, the group that received BD combined with shJagged1 group showed no difference to the group that received shJagged1 alone. These results demonstrate that Jagged1 is required for the effects of BD.

---

Bruceine D (BD) induced proteasome-dependent degradation of β-catenin [2]
We further speculated whether BD downregulated Jagged1 expression through the Wnt pathway. To investigate this hypothesis, we tested the effect of BD on the β-catenin/Tcf4 transcriptional complex in HCC cells. Huh7 cells were co-transfected with a luciferase reporter gene containing 3 tandem Tcf consensus binding sites (TOP) or a mutated Tcf binding site (FOP) and the Renilla-luciferase reporter as a normalizing transfection control. In accordance with our hypothesis, BD effectively suppressed TOP-luciferase, but not FOP-luciferase, activity in a dose-dependent manner (Fig. 6A).

Bruceine D, a quassinoid, was identified in Brucea javanica (L.) Merr. and displayed outstanding systemic properties and excellent antifeedant activity against the diamondback moth (DBM, Plutella xylostella L.), beet armyworm ( Spodoptera exigua Hübner), and cotton leafworm ( Spodoptera litura Fabricius). Its antifeedant effect on third instar larvae of DBM was approximately 6.2-fold stronger than that of azadirachtin. When Bruceine D (BD) was applied to roots at a concentration of 100 μg/mL for 24 and 48 h, its concentration in flowering Chinese cabbage ( Brassica campestris L. ssp. chinensis var. utiliz Tsen et Lee) leaves was 38.69 μg/g (fresh weight, FW) and 108.45 μg/g (FW), respectively. These concentrations could achieve 93.80% and 96.83% antifeedant effects, which were significantly greater than those of azadirachtin. Similar to azadirachtin, bruceine D also posed a potent growth inhibition effect on insect larvae. After feeding with 20 μg/g bruceine D, no pupae were observed. The results demonstrated that bruceine D is an effective botanical insect antifeedant with outstanding systemic properties, causing potent pest growth inhibitory activity[1].

---

The present study was designated to ascertain the anthelmintic activity of the dried fruits of Brucea javanica and to isolate and characterise the active constituents. The methanol extract from the fruits of B. javanica showed significant anthelmintic activity against Dactylogyrus intermedius (EC(50) (median effective concentration) value=49.96 mg l(-1)). Based on this finding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known quassinoids showing potent activity, bruceine A and Bruceine D (BD). Both bruceine A and D exhibited significant activity against D. intermedius with EC(50) values of 0.49 mg l(-1) and 0.57 mg l(-1), respectively, which were more effective than the positive control, mebendazole (EC(50) value=1.25 mg l(-1)). In addition, the 48-h median lethal concentration (LC(50)) for bruceine A and D against the host (Carassius auratus) was 10.6-fold and 9.7-fold higher than the EC(50) for D. intermedius. These results provide evidence that the isolated compounds might be potential sources of new anti-parasitic drugs for the control of Dactylogyrus. This is the first report on an in vivo anthelmintic investigation for B. javanica against D. intermedius.[3]

Hepatocellular carcinoma (HCC) is known for high mortality and limited available treatments. Aberrant activation of the Wnt and Notch signaling pathways is critical to liver carcinogenesis and progression. Here, we identified a small molecule, bruceine D (BD), as a Notch inhibitor, using an RBP-Jκ-dependent luciferase-reporter system. BD significantly inhibited liver tumor growth and enhanced the therapeutic effects of sorafenib in various murine HCC models. Mechanistically, BD promotes proteasomal degradation of β-catenin and the depletion of its nuclear accumulation, which in turn disrupts the Wnt/β-catenin-dependent transcription of the Notch ligand Jagged1 in HCC. Our findings provide important information about a novel Wnt/Notch crosstalk inhibitor that is synergistic with sorafenib for treatment of HCC, and therefore have high clinical impact[2].

Cell viability assay [2]
Cells were cultured overnight in 96-well plates (3000 cells/well) and were then treated with Bruceine D (BD) or sorafenib at the indicated concentration for 24, 48, or 72 h. Then, cells were treated with 10 μl Cell Counting Kit-8 (CCK8) reagent for 1 h on each well. Optical density was measured at 450 nm and normalized to background absorbance of the medium in the absence of cells. All samples were assayed in triplicate.

---

Soft agar colony formation assay and clonogenic assay [2]
Cells (1000/well) in 6-well plates were incubated with or without Bruceine D (BD) (0–1 μM) in 1 ml 0.33% basal medium eagle agar containing 10% FBS or in 3.5 ml 0.5% basal medium eagle agar containing 10% FBS. The cultures were maintained at 37 °C in a 5% CO2 incubator for 10–14 days. Cell colonies were counted under a microscope using the Image-Pro Plus software program.

---

Apoptosis analysis by flow cytometry Cell apoptosis was examined with an Annexin V-FITC Apoptosis Detection Kit. Cells were treated for 24 h with Bruceine D (BD) or sorafenib alone or combined before staining with Annexin V-FITC and propidium iodide (PI). After incubation at room temperature for 5 min in the dark, the apoptosis was analyzed by flow cytometry.

---

Trypan blue exclusion assay [2]
Huh7 cells were cultured overnight in 6 well-plates, incubated with or without Bruceine D (BD) for 24 h. The cells were stained with trypan blue dye according to the recommended protocol.

---

Immunocytochemistry (ICC) and immunohistochemistry (IHC) [2]
Cells were plated on a 35-mm glass plate and grown overnight before treatment with Bruceine D (BD) (0.5–1 μM) for 24 h. DMSO was used as the untreated control. The primary β-catenin antibody was diluted 1:100 and added for overnight incubation at 4 °C. Afterwards, a 1:200 diluted Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody was added to the fixed Huh7 cells. The coverslips with cells were then mounted onto slides with Vectashield mounting medium containing DAPI stain (CST). Fluorescent imaging was captured by LAS AF Lite software and Leica TCS SP8 X microscope

---

Cell transfections [2]
T-REx-HeLa cells were transiently transfected with luciferase reporter plasmid and pcDNA4/TO-N100-GV using X-tremeGENE HP DNA Transfection Reagent in Opti-MEM medium. After 24 h of transfection, cells were treated with 1 μg/ml tetracycline in the presence of the tested compounds. After 24 h of incubation, cells were lysed for 5 min in Glo lysis buffer, and the luciferase activity was measured by mixing lysate and Bright-Glo luciferase assay reagent in 96-well Nunc MaxiSorp plates. After 5 min of incubation at room temperature, the luminescence in individual wells was monitored in a microplate reader.
HEK293 cells were transfected with Notch1 NEXT plasmid or pcDNA3 plasmid (control) together with 2 μl of X-tremeGENE HP DNA Transfection Reagent in Opti-MEM medium. After 24 h of transfection, HEK293 cells were incubated with DAPT (GSI-IX) or Bruceine D (BD) for another 24 h, and whole cells were extracted for western blot analysis.
Huh7 cells were transiently transfected with shCTNNB1 and shJAG1 with lipo2000 transfection, according to the manufacturer's recommendations. After 24 h of transfection, the cells were treated with either DMSO or Bruceine D (BD) for another 24 h, and whole cells were extracted for western blotting.

---

Luciferase reporter assay [2]
Briefly, cells were seeded into 96-well plates and co-transfected with pTOPflash or pFOPflash vector together with renilla luciferase reporter vector pRL-TK as the control for transfection efficiency. After 12 h of transfection, cells were incubated with Bruceine D (BD) for another 24 h. Luciferase and renilla activities were measured and normalized according to the recommended protoco. For drug screening, HEK293 cell lines containing RBP-Jκ Reporter were seeded into 96-well plates. After overnight incubation, a library of 1600 natural products at 10 μM were added to each well for 24 h, using the Promega E1500 kit detect the luciferase activity.

Insect Growth Regulatory Assay[1]
Bruceine D was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5000 μg/mL and then added to the DBM larvae diet at final concentrations of 2.5, 5, 10, and 20 μg/g. Freshly molted third instar larvae (n = 100 per group) were placed in insect rearing containers. Sufficient food was supplied after starvation for 3 h. Larvae fed diets with DMSO were used as controls. The weight of larval body and diet consumption were measured after treatment with 10, 20 μg/g bruceine D diets at 6, 12, 24, 48 h. The number of individuals who underwent pupation and eclosion was counted at 12 h intervals for the treatment and control groups. Simultaneously, the development processes and phenotypes of the larvae were observed.

---

In vivo anthelmintic efficacy assay [3]
The anthelmintic activity of fractions and the pure compounds such as Bruceine D (BD) was determined against D. intermedius. Tests were conducted in glass tank of 5-l capacity, filled with 2 l aerated groundwater, each containing the test samples and five previously infected fish. The water pH ranged from 7.0 to 7.5, dissolved oxygen between 6.2 and 7.8 mg l−1 (72–85% saturation) and the water temperature was constant at 24 ± 1 °C. Initial tests were conducted to get moderate concentration boundaries to avoid the mortality of fish at high concentrations. The derived fractions and pure compounds were dissolved in 1 ml dimethysulphoxide (DMSO) and made up to 100 ml with distilled water, which were used for the preparation of the different concentrations of the test solutions. The methanol extract and isolated fractions were conducted at a different series of concentrations based on the initial tests, respectively. All the experiments were repeated independently in duplicate, except for the pure compounds 1 and 2, which were conducted in triplicate, following the same protocol for the B. javanica fractions, using five concentrations, 0.5, 1.0, 1.5, 2.0 and 2.5 mg l−1. To discard the possible effects of DMSO on the parasites, a negative control was included using distilled water containing the corresponding percentage of DMSO as the test sample; Mebendazole was used as a positive control with a different series of concentrations of 0.6, 1.0, 1.5, 2.0 and 2.5 mg l−1. After 48 h, all the surviving goldfish in all the treatment and control groups were euthanised by a spinal severance for necropsy. The lamella branchialis were placed on glass slides, and the numbers of parasites on the gills were counted under a light microscope at 4 × 10 magnification to determine the mean number of parasites per infected goldfish.

---

Acute toxicity assay [3]
Acute toxicity of the pure compounds (1 and 2)/Bruceine D (BD) was assayed using the healthy goldfish. The tests were conducted in duplicate using 10 individuals in a tank of 5 l capacity, containing 2-l test solutions at 24 ± 1 °C during the experiment. The pure compounds were conducted at a different series of concentrations based on the initial tests. Control groups were set under the same test conditions without chemicals. The death of fish was recorded when there was no opercula movement and the goldfish no longer responded to mechanical stimulus (touch by glass rod). Any observed dead fish was removed from the medium in time to avoid deterioration of the water quality. After a 48-h exposure, fish mortalities in the treatment and control groups were recorded.

---

Subcutaneous HCC xenograft model study [2]
Four-week-old male Balb/c nude mice were housed and maintained under specific pathogen-free conditions. For in vivo studies, 5 × 106 Huh7 cells in 100 μl PBS were injected subcutaneously into the right flanks of the mice. Based on the data from a pilot study, the mice were randomly divided into 3 groups (n = 6 per group) and were treated with saline with 0.9% sodium chloride (vehicle) or Bruceine D (BD) at doses of 0.75 mg/kg and 1.5 mg/kg body weight by tail vein injection once daily for 10 consecutive days. Tumor volume was calculated according the formula: tumor volume (mm3) = 1/2 × (tumor length) × (tumor width)

[1]. Bruceine D Isolated from Brucea Javanica (L.) Merr. as a Systemic Feeding Deterrent for Three Major Lepidopteran Pests. J Agric Food Chem. 2019 Apr 17;67(15):4232-4239.
[2]. Bruceine D inhibits hepatocellular carcinoma growth by targeting β-catenin/jagged1 pathways. Cancer Lett. 2017 Sep 10;403:195-205.
[3]. In vivo anthelmintic activity of bruceine A and bruceine D from Brucea javanica against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Vet Parasitol. 2011 Apr 19;177(1-2):127-33.

Bruceine D is a quassinoid that is 13,20-epoxypicras-3-ene substituted by hydroxy groups at positions 1, 11, 12, 14 and 15 and oxo groups at positions 2 and 16. Isolated from the ethanol extract of the stem of Brucea mollis, it exhibits cytotoxic activity. It has a role as a metabolite, an antineoplastic agent and a plant metabolite. It is a delta-lactone, a pentol, a quassinoid, an organic heteropentacyclic compound and a secondary alpha-hydroxy ketone. It derives from a hydride of a picrasane.
Bruceine D has been reported in Brucea mollis, Samadera indica, and Brucea javanica with data available.

---

Systemicity is a desirable property for insecticides. Many phytochemicals show good systemic properties and thus are natural sources of novel systemic insecticidal ingredients. Bruceine D, a quassinoid, was identified in Brucea javanica (L.) Merr. and displayed outstanding systemic properties and excellent antifeedant activity against the diamondback moth (DBM, Plutella xylostella L.), beet armyworm ( Spodoptera exigua Hübner), and cotton leafworm ( Spodoptera litura Fabricius). Its antifeedant effect on third instar larvae of DBM was approximately 6.2-fold stronger than that of azadirachtin. When bruceine D was applied to roots at a concentration of 100 μg/mL for 24 and 48 h, its concentration in flowering Chinese cabbage ( Brassica campestris L. ssp. chinensis var. utiliz Tsen et Lee) leaves was 38.69 μg/g (fresh weight, FW) and 108.45 μg/g (FW), respectively. These concentrations could achieve 93.80% and 96.83% antifeedant effects, which were significantly greater than those of azadirachtin. Similar to azadirachtin, bruceine D also posed a potent growth inhibition effect on insect larvae. After feeding with 20 μg/g bruceine D, no pupae were observed. The results demonstrated that bruceine D is an effective botanical insect antifeedant with outstanding systemic properties, causing potent pest growth inhibitory activity. [1]

---

Hepatocellular carcinoma (HCC) is known for high mortality and limited available treatments. Aberrant activation of the Wnt and Notch signaling pathways is critical to liver carcinogenesis and progression. Here, we identified a small molecule, bruceine D (BD), as a Notch inhibitor, using an RBP-Jκ-dependent luciferase-reporter system. BD significantly inhibited liver tumor growth and enhanced the therapeutic effects of sorafenib in various murine HCC models. Mechanistically, BD promotes proteasomal degradation of β-catenin and the depletion of its nuclear accumulation, which in turn disrupts the Wnt/β-catenin-dependent transcription of the Notch ligand Jagged1 in HCC. Our findings provide important information about a novel Wnt/Notch crosstalk inhibitor that is synergistic with sorafenib for treatment of HCC, and therefore have high clinical impact. [2]

---

The present study was designated to ascertain the anthelmintic activity of the dried fruits of Brucea javanica and to isolate and characterise the active constituents. The methanol extract from the fruits of B. javanica showed significant anthelmintic activity against Dactylogyrus intermedius (EC(50) (median effective concentration) value=49.96 mg l(-1)). Based on this finding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known quassinoids showing potent activity, bruceine A and bruceine D. Both bruceine A and D exhibited significant activity against D. intermedius with EC(50) values of 0.49 mg l(-1) and 0.57 mg l(-1), respectively, which were more effective than the positive control, mebendazole (EC(50) value=1.25 mg l(-1)). In addition, the 48-h median lethal concentration (LC(50)) for bruceine A and D against the host (Carassius auratus) was 10.6-fold and 9.7-fold higher than the EC(50) for D. intermedius. These results provide evidence that the isolated compounds might be potential sources of new anti-parasitic drugs for the control of Dactylogyrus. This is the first report on an in vivo anthelmintic investigation for B. javanica against D. intermedius. [3]

C20H26O9

410.4150

410.157

C, 58.53; H, 6.39; O, 35.08

21499-66-1

21499-66-1

122784

White to off-white solid powder

1.6±0.1 g/cm3

661.3±55.0 °C at 760 mmHg

235.8±25.0 °C

0.0±4.5 mmHg at 25°C

1.670

-0.5

5

9

0

29

855

11

O1C([H])([H])[C@@]23C4([H])C([H])([H])C5([H])C(C([H])([H])[H])=C([H])C(C([H])([C@]5(C([H])([H])[H])C2([H])C([H])(C([H])([C@]1(C([H])([H])[H])[C@@]3(C([H])(C(=O)O4)O[H])O[H])O[H])O[H])O[H])=O

JBDMZGKDLMGOFR-KQSRGDCESA-N

InChI=1S/C20H26O9/c1-7-4-9(21)13(23)17(2)8(7)5-10-19-6-28-18(3,14(24)11(22)12(17)19)20(19,27)15(25)16(26)29-10/h4,8,10-15,22-25,27H,5-6H2,1-3H3/t8-,10+,11+,12+,13+,14-,15-,17-,18+,19+,20+/m0/s1

(1R,2R,3R,6R,8S,12S,13S,14R,15R,16S,17R)-2,3,12,15,16-pentahydroxy-9,13,17-trimethyl-5,18-dioxapentacyclo[12.5.0.01,6.02,17.08,13]nonadec-9-ene-4,11-dione

Bruceine-D; Bruceine D; 21499-66-1; Brucein D; CHEBI:68931; C08752; (1R,2R,3R,6R,8S,12S,13S,14R,15R,16S,17R)-2,3,12,15,16-Pentahydroxy-9,13,17-trimethyl-5,18-dioxapentacyclo[12.5.0.01,6.02,17.08,13]nonadec-9-ene-4,11-dione; (1R,2S,3R,3aR,3a1R,4R,6aR,7aS,11S,11aS,11bR)-1,2,3a,4,11-pentahydroxy-3,8,11a-trimethyl-1,2,3,3a,4,7,7a,11,11a,11b-decahydro-5H-3,3a1-(epoxymethano)dibenzo[de,g]chromene-5,10(6aH)-dione; AC1L9BNP; Bruceine D

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: 82~100 mg/mL (199.8~243.7 mM)
H2O: ~5 mg/mL (~12.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.09 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.09 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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