Natural product

A simple, precise, and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin (1) together with its derivatives acetylshikonin (2), and beta-acetoxyisovaleryl-shikonin (3) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP-18 F(254S )TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100-600 ng for shikonin and acetylshikonin, and 100-1800 ng for beta-acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1-3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively. [1]

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Shikonin and its derivatives are important red colored naphthoquinone pigments found in a large number of Arnebia species, including A. euchroma, that are responsible for the various pharmacological activities exhibited by the plant. The precise separation of each naphthoquinone is essential for total quality evaluation and bioactivity analysis of herbal formulations of A. euchroma. Furthermore, the overexploitation of this useful plant has resulted in species becoming endangered. With this in mind, a simple and rapid preparative scale HPLC method with single compound recovery for the isolation and purification of two shikonin derivatives (i. e. acetylshikonin, beta-acetoxyisovaleryl-shikonin) from cell suspension cultures of A. euchroma is presented. The compounds were separated on a C(18) column within 10 min using acetonitrile/methanol (95:5) as mobile phase in isocratic mode. The isolated compounds were found to be more than 98% pure. The LOD for acetylshikonin and beta-acetoxyisovalerylshikonin was estimated at 0.063 and 0.146 mug/mL, respectively, while the LOQ was found to be 0.209 and 0.487 mug/mL, respectively. The recoveries accomplished for both the shikonin derivatives were in the range of 94.7-96.8%. The repeatability, expressed as %RSD, of acetylshikonin and beta-acetoxyisovalerylshikonin was found to be 1.74 and 1.27, respectively[2].

[1]. Simultaneous densitometric determination of shikonin, acetylshikonin, and beta-acetoxyisovaleryl-shikonin in ultrasonic-assisted extracts of four Arnebia species using reversed-phase thin layer chromatography. J Sep Sci. 2009 Sep;32(18):3239-45.

[2]. Isolation and purification of acetylshikonin and beta-acetoxyisovalerylshikonin from cell suspension cultures of Arnebia euchroma (Royle) Johnston using rapid preparative HPLC. J Sep Sci. 2008 Mar;31(4):629-35.

Butanoic acid, 3-(acetyloxy)-3-methyl-, 1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxo-2-naphthalenyl)-4-methyl-3-pentenyl ester, (S)- has been reported in Alkanna tinctoria with data available.

C23H26O8

430.4477

430.162

69091-17-4

155245

Brown to black solid powder

1.3±0.1 g/cm3

583.6±50.0 °C at 760 mmHg

196.5±23.6 °C

0.0±1.7 mmHg at 25°C

1.572

5.54

2

8

9

31

802

1

CC(=CC[C@@H](C1=CC(=O)C2=C(C=CC(=C2C1=O)O)O)OC(=O)CC(C)(C)OC(=O)C)C

BQSAGDWOHVQNFB-SFHVURJKSA-N

InChI=1S/C23H26O8/c1-12(2)6-9-18(30-19(28)11-23(4,5)31-13(3)24)14-10-17(27)20-15(25)7-8-16(26)21(20)22(14)29/h6-8,10,18,25-26H,9,11H2,1-5H3/t18-/m0/s1

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

69091-17-4; [(1S)-1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] 3-acetyloxy-3-methylbutanoate; Butanoic acid, 3-(acetyloxy)-3-methyl-, 1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxo-2-naphthalenyl)-4-methyl-3-pentenyl ester, (S)-; DTXSID60219131; ((1S)-1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl) 3-acetyloxy-3-methylbutanoate; DTXCID80141622; beta-acetoxyisovalerylshikonin; (S)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 3-acetoxy-3-methylbutanoate;

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 : ~50 mg/mL (~116.16 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 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.

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Solubility in Formulation 2: 2.5 mg/mL (5.81 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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