Liquid liquid fractionation with acid base precipitation along with sephadex isolation using column

Chaitarya Mvnl...could you kindly suggest some solvents for the liquid-liquid fractionation as well as the protocol for the whole process from fractionation to precipitation to sephadex Isolation? Thanks

Here is one reference for extractions for these compounds:

http://web.nchu.edu.tw/pweb/users/taiwanfir/research/140.pdf

Here is another- expraction procedure visible: https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-2006-957763

Dear Mitchel,

Find in attached a paper on:

HPLC/UV Analysis of Polyacetylenes, Phenylpropanoid and Pyrrolidine Alkaloids

in Medicinally Used Codonopsis Species

Jing-Yu He, Shu Zhu and Katsuko Komatsu

Phytochem. Anal. 2014, 25, 213–219

You'll find in:

Isolation and identification of standard compounds

Dried and powdered Codonopsis Radix (1.0 kg) (TMPW No. 26991) was

extracted by steeping in methanol for three times (10 L, 48 h) at room temperature. The combined methanol extracts were evaporated under

reduced pressure and then lyophilised to yield a brown solid residue

(303.0 g). The residue redissolved in distilled water was applied to a

Diaion HP 21 column (9 cm × 55 cm) and successively eluted with H2O,

20, 40, 60 and 80% MeOH (v/v) and MeOH (5.0 L of each solvent). After

detecting each eluent by TLC, the eluents with similar chemical compositions

were combined and dried by reduced evaporation and

lyophilisation to give fractions A (289.0 g), B (5.1 g), C (3.1 g), D (2.7 g), E

(2.6 g) and F (2.1 g), respectively. In the experimental procedure, fractions

were monitored by the TLC and HPLC/UV methods to guide further

isolation and purification. Fraction A contained saccharides rather than

target compounds. Fraction B was subjected to an ODS-A column

(4.5 cm × 50 cm) and eluted with H2O/MeOH (8:2–4:6, v/v; 1.5 L of each

ratio) to obtained four fractions. Fraction B-2 (0.7 g) was subjected to a

Sephadex LH-20 column (2.5 cm × 70 cm) and eluted with H2O/MeOH

(6:4, v/v; 5.0 L) to provide 13 fractions. Fraction B-2-7 was further purified

by semi-preparative HPLC (solvent: MeOH/0.1% trifluoroacetic acid

(TFA) water solution, 15:85 v/v; flow rate: 6 mL/min) to yield

codonopyrrolidium B (1). Fraction C was subjected to an ODS-A column

(4.5 cm× 50 cm) and eluted with H2O/MeOH (7:3–3:7, v/v; 1.0 L of each

ratio) to give five fractions, and fraction C-3 (0.5 g) was purified by semipreparative

HPLC (solvent: MeOH/0.1% TFA water solution, 30:70–45:55 v/v,

linear gradient in 45min; flow rate: 6 mL/min) to yield codonopyrrolidium

A (2). To exclude TFA from the compounds obtained, codonopyrrolidium

B (1) and codonopyrrolidium A (2) were each dissolved in 0.5%

hydrochloric acid and then subjected to an ODS-A column (1.5 cm× 20 cm). After elution with MeOH/0.5% HCl water solution (0:100, 50:50, v/v; 300mL

of each ratio), the chlorides of codonopyrrolidium B (22.3mg) and

codonopyrrolidium A (15.2mg) were obtained from the 50% MeOH

fraction. Fraction D was chromatographed on an ODS-A column

(4.5cm×50cm) and eluted with H2O/MeOH (6:4–2:8, v/v; 1.0 L of each

ratio) to produce three fractions. Fraction D-2 (0.6 g) was purified by

semi-preparative HPLC (solvent: MeOH/0.1% TFA water solution,

30:70–55:45 v/v, linear gradient in 50 min; flow rate: 6 mL/min) to afford

tangshenoside I (3) (9.8mg) and cordifolioidyne B (4) (3.5mg). Fraction

E was subjected to a silica gel column (100–200 mesh; 4.5 cm× 50 cm)

and eluted with CHCl3/MeOH (9:1–1:1, v/v; 1.5 L of each ratio) to obtain

nine fractions. Fraction E-2 (0.2 g) was chromatographed on a silica gel

column (100–200 mesh; 1.5 cm× 45 cm) and eluted with CHCl3/MeOH

(8:2, v/v; 1.0 L) to produce eight fractions, and fraction E-2-8 was subsequently

chromatographed on a Sephadex LH-20 column (1.5cm× 50 cm)

and eluted with H2O/MeOH (1:1, v/v; 3.0 L) to obtain lobetyol (7) (3.8mg).

Fraction E-4 (0.3 g) was repeatedly applied to a Sephadex LH-20 column

(2.5 cm× 70 cm) and eluted with H2O/MeOH (1:1, v/v; 5.0 L) to provide

lobetyolin (6) (14.1mg). Fraction E-6 (0.2 g) was subjected to an ODS-A

column (2.5 cm× 50 cm) and eluted with H2O/MeOH (6:4, v/v; 1.0 L) to

produce eight fractions. Fraction E-6-5 was subjected to a Sephadex

LH-20 column (2.5 cm× 70cm) and eluted with H2O/MeOH (1:1, v/v; 4.0 L)

to obtain lobetyolinin (5) (7.0mg). All of the isolated compounds were

identified by comparison of their spectral data (MS, 1H NMR and 13C NMR

spectra are shown as online Supporting information) with data reported

in the literature (Mizutani et al., 1988; Ishimaru et al., 1991, 1992; Cuendet

et al., 2001; Mei et al., 2008; Tsai and Lin, 2008). The purity of each

compound was confirmed by HPLC/DAD to be higher than 97% at six

wavelengths (205, 215, 230, 254, 267 and 284 nm).

Best wishes

Dear Mitchel,

Find in attached a paper on:

HPLC/UV Analysis of Polyacetylenes, Phenylpropanoid and Pyrrolidine Alkaloids

in Medicinally Used Codonopsis Species

Jing-Yu He, Shu Zhu and Katsuko Komatsu

Phytochem. Anal. 2014, 25, 213–219

You'll find in:

Isolation and identification of standard compounds

Dried and powdered Codonopsis Radix (1.0 kg) (TMPW No. 26991) was

extracted by steeping in methanol for three times (10 L, 48 h) at room temperature. The combined methanol extracts were evaporated under

reduced pressure and then lyophilised to yield a brown solid residue

(303.0 g). The residue redissolved in distilled water was applied to a

Diaion HP 21 column (9 cm × 55 cm) and successively eluted with H2O,

20, 40, 60 and 80% MeOH (v/v) and MeOH (5.0 L of each solvent). After

detecting each eluent by TLC, the eluents with similar chemical compositions

were combined and dried by reduced evaporation and

lyophilisation to give fractions A (289.0 g), B (5.1 g), C (3.1 g), D (2.7 g), E

(2.6 g) and F (2.1 g), respectively. In the experimental procedure, fractions

were monitored by the TLC and HPLC/UV methods to guide further

isolation and purification. Fraction A contained saccharides rather than

target compounds. Fraction B was subjected to an ODS-A column

(4.5 cm × 50 cm) and eluted with H2O/MeOH (8:2–4:6, v/v; 1.5 L of each

ratio) to obtained four fractions. Fraction B-2 (0.7 g) was subjected to a

Sephadex LH-20 column (2.5 cm × 70 cm) and eluted with H2O/MeOH

(6:4, v/v; 5.0 L) to provide 13 fractions. Fraction B-2-7 was further purified

by semi-preparative HPLC (solvent: MeOH/0.1% trifluoroacetic acid

(TFA) water solution, 15:85 v/v; flow rate: 6 mL/min) to yield

codonopyrrolidium B (1). Fraction C was subjected to an ODS-A column

(4.5 cm× 50 cm) and eluted with H2O/MeOH (7:3–3:7, v/v; 1.0 L of each

ratio) to give five fractions, and fraction C-3 (0.5 g) was purified by semipreparative

HPLC (solvent: MeOH/0.1% TFA water solution, 30:70–45:55 v/v,

linear gradient in 45min; flow rate: 6 mL/min) to yield codonopyrrolidium

A (2). To exclude TFA from the compounds obtained, codonopyrrolidium

B (1) and codonopyrrolidium A (2) were each dissolved in 0.5%

hydrochloric acid and then subjected to an ODS-A column (1.5 cm× 20 cm). After elution with MeOH/0.5% HCl water solution (0:100, 50:50, v/v; 300mL

of each ratio), the chlorides of codonopyrrolidium B (22.3mg) and

codonopyrrolidium A (15.2mg) were obtained from the 50% MeOH

fraction. Fraction D was chromatographed on an ODS-A column

(4.5cm×50cm) and eluted with H2O/MeOH (6:4–2:8, v/v; 1.0 L of each

ratio) to produce three fractions. Fraction D-2 (0.6 g) was purified by

semi-preparative HPLC (solvent: MeOH/0.1% TFA water solution,

30:70–55:45 v/v, linear gradient in 50 min; flow rate: 6 mL/min) to afford

tangshenoside I (3) (9.8mg) and cordifolioidyne B (4) (3.5mg). Fraction

E was subjected to a silica gel column (100–200 mesh; 4.5 cm× 50 cm)

and eluted with CHCl3/MeOH (9:1–1:1, v/v; 1.5 L of each ratio) to obtain

nine fractions. Fraction E-2 (0.2 g) was chromatographed on a silica gel

column (100–200 mesh; 1.5 cm× 45 cm) and eluted with CHCl3/MeOH

(8:2, v/v; 1.0 L) to produce eight fractions, and fraction E-2-8 was subsequently

chromatographed on a Sephadex LH-20 column (1.5cm× 50 cm)

and eluted with H2O/MeOH (1:1, v/v; 3.0 L) to obtain lobetyol (7) (3.8mg).

Fraction E-4 (0.3 g) was repeatedly applied to a Sephadex LH-20 column

(2.5 cm× 70 cm) and eluted with H2O/MeOH (1:1, v/v; 5.0 L) to provide

lobetyolin (6) (14.1mg). Fraction E-6 (0.2 g) was subjected to an ODS-A

column (2.5 cm× 50 cm) and eluted with H2O/MeOH (6:4, v/v; 1.0 L) to

produce eight fractions. Fraction E-6-5 was subjected to a Sephadex

LH-20 column (2.5 cm× 70cm) and eluted with H2O/MeOH (1:1, v/v; 4.0 L)

to obtain lobetyolinin (5) (7.0mg). All of the isolated compounds were

identified by comparison of their spectral data (MS, 1H NMR and 13C NMR

spectra are shown as online Supporting information) with data reported

in the literature (Mizutani et al., 1988; Ishimaru et al., 1991, 1992; Cuendet

et al., 2001; Mei et al., 2008; Tsai and Lin, 2008). The purity of each

compound was confirmed by HPLC/DAD to be higher than 97% at six

wavelengths (205, 215, 230, 254, 267 and 284 nm).

Best wishes

Dear Mitchel,

Find in attached a paper on:

A polyacetylene compound from herbal medicine regulates genes associated with thrombosis in endothelial cells

Akira Kawamura, Maria Iacovidou, Anna Takaoka, Clifford E. Soll, and Michael Blumenstein

Bioorg Med Chem Lett. 2007 December 15; 17(24): 6879–6882.

Hoping to be helpful

Thankyou Jack silver and Talaat.I El-Emary... The information you have provided is very resourceful.My work has now been made easier. I offer my sincere gratitude to you.

Thank you too Mitclel

 The best  method for isolating polyacetylenes in herbal plants is use of  high performance liquid chromatography with diode array detector and mass spectrometry.

Kindly read this paper

 J Pharm Biomed Anal. 2006 Jun 7;41(3):683-93. Epub 2006 Mar 7.

Bioactive polyacetylenes in food plants of the Apiaceae family: occurrence, bioactivity and analysis.

Christensen LP1, Brandt K.