Natural product

Antibacterial minimum inhibitory concentration assay and synergistic effects [1]
IVS/Isovalerylshikonin was isolated from A. euchroma with 98.7% purity (Supplementary Fig. S1) and showed marginal antibacterial activity against the drug-resistant test strain S. aureus RN4220 with an MIC of 16 mg/L (Table 1; Supplementary Fig. S2). As a positive control, the antibiotic vancomycin inhibited the growth of strain RN4220 with an MIC of 2 mg/L. Growth kinetics, ethidium bromide efflux assay and effect of isovalerylshikonin on induction of msrA at the transcriptional level [1]
As shown in Supplementary Fig. S3, the growth curves of bacteria treated with the vehicle or with 0.25 × MIC of Isovalerylshikonin/IVS almost overlapped. The growth of strain RN4220 treated with 0.25 × MIC of STM was almost completely halted within 12 h and grew quickly thereafter. Compared with STM alone, strain RN4220 treated with STM (0.25 × MIC) combined with IVS (0.25 × MIC) grew at a slower rate. Thus, a synergistic effect between STM and IVS against strain RN4220 was observed.
Ethidium bromide was quickly depleted to <50% by vehicle-treated bacteria from 1–60 min, followed by a slower reduction, and the final level of fluorescence was ca. 30% (Fig. 1). IVS inhibited bacterial efflux against strain RN4220 (P < 0.05), although this inhibition was weaker than in the positive control carbonyl cyanide m-chlorophenylhydrazone.
Expression of msrA mRNA in bacteria treated with vehicle was set as the reference level of induction (1-fold). Strain RN4220 was incubated with STM (32 mg/L, 0.0125 × MIC) resulting in substantial expression of msrA mRNA, which was decreased significantly when the bacteria were incubated with STM and Isovalerylshikonin/IVS (P < 0.01). However, the level of mRNA expression in bacteria treated with STM combined with IVS was higher than in those treated with the vehicle (Supplementary Fig. S4).

In vivo infection and acute toxicity studies [1]
The 7-day survival curve of mice infected with strain RN4220 was recorded. Vehicle group mice died within 24 h of infection, as did those treated with Isovalerylshikonin/IVS (40 mg/kg) alone or with STM (10 mg/kg) alone (Fig. 2). Most mice administered a combination of STM (10 mg/kg) and IVS (10 mg/kg) died within 24 h of infection, with a survival percentage of 20%, and the rest died within the following 24 h. Eight of the combination group mice treated with the combination of STM (10 mg/kg) and IVS (20 mg/kg) died within 2 days (48 h) of being infected by strain RN4220, such that the final 7-day survival percentage of this group was 10%. Combination group mice administered STM (10 mg/kg) and a high dose of IVS (40 mg/kg) died slowly within 24 h of infection, and six died within 3 days of infection, with a final 7-day survival percentage of 40%. Mice in the positive control group injected with vancomycin (110 mg/kg) had a 60% survival rate at 7 days of infection with strain RN4220. Isovalerylshikonin/IVS significantly suppressed bacterial levels in infected mice (Supplementary Fig. S6), increasing the in vivo antibacterial activity of STM. The acute toxicity of IVS in mice was measured and it was found to have a 50% lethal dose (LD50) of 2.584 g/kg, indicating that IVS is a low-toxicity compound (Supplementary Fig. S8).

Growth kinetics, ethidium bromide efflux assay, total RNA extraction and real-time PCR [1]
Growth kinetics of strain RN4220 were determined as previously described with some modifications. Isovalerylshikonin/IVS was evaluated in a bacterial efflux assay as described previously. Total RNA was isolated from bacteria using TRIzol reagent according to the manufacturer's instructions. The reverse transcription step was carried out using a RevertAidTM First Strand cDNA Synthesis Kit to synthesise cDNA. PCR analysis was performed using an ABI 7300 real-time fluorescent quantitative PCR system. Detailed methods are provided in the Supplementary material.

In vivo infection and acute toxicity studies [1]
In vivo infection and acute toxicity studies were performed according to previously published methods, with some modifications.

In drug development, animal model efficacy and safety studies are crucial. This study demonstrated the synergistic effect of isovalerylshikonin/IVS and STM on drug-resistant Staphylococcus aureus infection in a mouse model. In addition, this study also determined the acute toxicity of a single exposure to IVS in mice, with an LD50 of 2.584 g/kg (see Supplementary Materials). [1]

[1].Isovalerylshikonin, a new resistance-modifying agent from Arnebia euchroma, supresses antimicrobial resistance of drug-resistant Staphylococcus aureus. Int J Antimicrob Agents. 2019 Jan;53(1):70-73.

[2].Use of matrix‐assisted laser desorption/ionization mass spectrometry for the rapid detection of low‐mass components in the Alkanna tinctoria pigments fraction. Journal of mass spectrometry, 1998, 33(1): 89-91.

(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydro-2-naphthyl)-4-methyl-3-pentenyl isovalerate has been reported in Lithospermum erythrorhizon, and relevant data are available. Isovalerylshikonin has been reported in Onosma heterophylla, and relevant data are available. Antimicrobial resistance is the greatest threat to the treatment of bacterial infectious diseases. Developing resistance modifiers (RMAs) is a promising strategy to mitigate the spread of antimicrobial resistance in bacteria. In this study, isovalerylshikonin (IVS), a natural product isolated from the traditional Chinese medicinal herb Arnebia euchroma, showed certain antimicrobial activity against drug-resistant Staphylococcus aureus RN4220, with a minimum inhibitory concentration (MIC) of 16 mg/L. Furthermore, a synergistic effect between IVS and streptomycin (STM) was detected by microdilution antimicrobial checkerboard assay, which reduced the minimum inhibitory concentration (MIC) of STM against RN4220 strain by up to 16-fold. The synergistic mechanism was investigated using ethidium bromide efflux assay and reverse transcription PCR. In vitro experiments showed that IVS significantly inhibited bacterial efflux and msrA mRNA expression. The in vivo synergistic activity of IVS and STM was detected using a mouse peritonitis/sepsis model. The results showed that IVS and STM synergistically reduced bacterial counts in peritoneum, spleen, and liver tissues and improved mouse survival rate within 7 days. Acute toxicity tests of IVS showed that the median lethal dose (LD50) of a single exposure to IVS in mice was 2.584 g/kg. Overall, the low-toxicity RMA compound IVS exhibited synergistic antimicrobial activity against methicillin-resistant Staphylococcus aureus both in vitro and in vivo. Its mechanism of action is achieved by inhibiting msrA mRNA expression and reducing bacterial efflux. Furthermore, these data support the view that IVS is a potential RMA compound against MsrA efflux pump resistance. [1]

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This study aimed to discover a novel RMA compound against MsrA efflux pumps. IVS, isolated from A. euchroma, was confirmed to be an effective RMA compound against MsrA efflux pump resistance, exhibiting synergistic antibacterial activity against Staphylococcus aureus RN4220 both in vitro and in vivo. Its mechanism of action is through inhibition of msrA mRNA expression and reduction of bacterial efflux. In addition, IVS is a low-toxicity drug with an LD50 of 2.584 g/kg in mice, and can be considered a potential treatment for infections. [1]

C21H24O6

372.41200

372.157

52387-14-1

76549-35-4

479497

Typically exists as solid at room temperature

1.246g/cm3

570.1ºC at 760 mmHg

107ºC

198.6ºC

1.578

3.717

2

6

7

27

656

1

CC(C)CC(=O)OC(CC=C(C)C)C1=CC(=O)C2=C(C=CC(=C2C1=O)O)O

UTOUNDHZJFIVPK-QGZVFWFLSA-N

InChI=1S/C21H24O6/c1-11(2)5-8-17(27-18(25)9-12(3)4)13-10-16(24)19-14(22)6-7-15(23)20(19)21(13)26/h5-7,10,12,17,22-23H,8-9H2,1-4H3/t17-/m1/s1

[(1R)-1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] 3-methylbutanoate

Isovalerylshikonin; 76549-35-4; [1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] 3-methylbutanoate; NSC344556; 52387-14-1; Butanoic acid, 3-methyl-, 1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxo-2-naphthalenyl)-4-methyl-3-pentenyl ester; Alkannin isovalerate; SCHEMBL13389448;

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.)