Isolation of Bioactive lipids

BIOACTIVE LIPIDS

from Leeuwenhoekiella parthenopeia

Paulina Corral 2023

INDEX

Introduction

extraction protocol

results

INTRODUCTION

Leewenhoekiella parthenopeia is a marine bacterium belonging to the rare biosphere isolated from the Gulf of Naples employing seasonal and diel sampling strategies.

What does the extract do??

Its membrane lipidic extract inhibits the tumor cell viability of a panel of tumor cells (prostate, glyoblastome and breast).

lipid Extraction protocol (Bligh and Dyer, 1959)

• Prepare the monophasic mixture creating a ratio of 0.8:2:1 (v/v) of cell suspention in 3.5% of NaCl solution:methanol:chloroform.

• Gently mix by inversion for 1 hour, centrifuge and collect the supernant.
• Disrupt the monophase by adding 150 μL of KCl 0.2 M until see a biphasic layer.
• Centrifuge for 3 min at 6000 rpm and collect the lower phase of chloroform in a weighted empty glass vial or resistant solvents tube.

• Dry the extract with nitrogen gas/speed vaccum or under fume hood. Weight and store at -20ºC.

*Check here to see the detailed protocol and go to material and methods

dereplication of crude lipid extract

According to the Dictionary of natural products peaks 7,8,13',14,15,19 are known molecules

+ info

tlc of crude lipid extract

254 nm

After sprayH2SO4 5%

UV

After oven 160 ºC 5'

Solvent system: Chloroform:AcetiC aciD 90%:Methanol 80:15:5 StaiN: Sulphuric acid H2SO4 5%

Figure. TLC of total lipid extract in analytic TLC glass back, exhibiting 4 different visualization modes. A. Under UV light, B. 254 nm, C. after spraying with sulphuric acid solution 5%, D. H2SO4 5% after oven at 160ºC for 5 minutes.Line1. Crude extract as reference Line 2. Crude extract whitout pigment *This solvent system is used to determine polar lipids in bacterial and archaeal membranes as chemotaxonomic marker. Commonly used in ratio 65:30:4 of CHCl3:Acetic acid 90%: MeOH.

scaling up to fractioning by column of silica

Solvent systeM: Cloroform, AcetiC aciD, Methanol 80:15:5

Aim: obtaining > 0.5 mg of each fractions for initial antiproliferative tests. Volume of the column: 500 ml Loaded ammount: 200 mg of total lipidic extract. TLC running collection: 134 tubes, speed with dropping collection time of 1 minute per tube.

Each 3 tubes were checked by TLC using the total lipid extract as reference. Fractions with similar migration were collected in the same round flask, then dried in a rotavapor and weighed.

comparison of TLC in solvent systems

hexane:ethyl acetate 1:1

CHCL3: Acetic Acid: MeOH 80:15:5

H2SO4 and oven

UV

TLC no stained

H2SO4 and oven

1-34

35-54

55-118

119-134

Ref.

1-34

1-34

35-54

35-54

55-118

119-134

55-118

Ref.

119-134

Ref.

1-34

35-54

55-118

119-134

Ref.

While with the first solvent system CHCL3:acetic acid: MeOH 80:15:5 all lipids are visualized except the fractions 1-34 (that contain the yellow pigment), with Hexane:Ethyl acetate 1:1 the bands are visible since these mobile phase allows the retention of pigments and their proper separation.

Test of fractions from column

1-34 (contained the yellow carotenoid pigment)

35-54 (The major lipid in the TLC)

55-118 (lipids in minor count)

119-134 (The color is pink, red)

Antiproliferative tests of all range of fractions (4 in total) were performed in triplicate, confirming that the most promising is 1-34 and 119-134 eluted in CHCl3

preparative TLC of fraction 1-34

Solvent system: Hexane: Ethyl acetate 1:1

Preparative TLC of 20 mg of the fraction 1-34 from the column was performed to retain the pigment and observe the bands.

0.8 mg

Active Fractions: 3 and 6

The work continue only with the lower band 6 since it was discussed that it is more simple to isolate and identificate. Then a colum was proposed to recover 3 main fractions: Top, medium (pigment) and bottom zone.

3 mg

column in Hexane: EtoAC 1:1

To obtain more amount of the fractions, 200 mg of crude extract was loaded in a column (26/07/2023). In the column the fractions corresponds to the oposite side respect to the TLC. Those from down in the colum of the corresponds to the fractions in the top in TLC.

Volume of the column: 500 ml Amount of extract loaded: 200 mg of total lipidic extract. 1st Solvent: 360 ml of Hexane:EtoAC 1:1 2nd Solvent: 100 ml of EtoAC: MeOH;H2O 75:15:10

TLC fraction collection: 4 fractions in total colour. - 1 no color 10 mg - 2 light pigmented fraction 18 mg - 3 pigment 25 mg - 4 colour less and washing (2nd solvent) 12 mg

SUmmary TLC in Hexane: EtoAC 1:1

sprayed H2SO4

TLC running collection: 4 fractions As observed, the fraction 1 and 2 are better observed after sprayed with H2SO4 (white spots), but not visualized after oven. The same happened with the 4.

UV

Oven 160 ºC

Summary TLC of Fraction 4 CHCL3: Acetic Acid: MeOH 80:15:5

IIodine

Oven 160 ºC

TLC fraction 4 from the column. Right side sprayed with H2SO4 followed by oven, and left side revealed with iodine vapors. Line 1: Reference Line 2; Fraction 4 eluted with CHCl3 Line 3: Fraction 4 eluted with MeOH As observed after the oven and iodine, the fraction evidenced at least 4 fractions, from wich has 7 other spots, but only two are the major lipids.

CHCL3: Acetic Acid: MeOH 80:15:5

Preparative TLC of fraction 4

CHCL3: Acetic Acid: MeOH 80:15:5

12 mg of fraction 4 was dissolved in CHCL3 and loaded for TLC. 6 bands were scrapped and eluted with: CHCl3: ISO 9:1 CHCl3: ISO 7:3 EtoAC:MeOH;H2O 85:10:5

Weights:1: 0.7 mg 2: 0.6 mg 3: 0.8 mg * 4: 2 mg * (testing on a panel of tumor cells) 5: 3 mg * (testing on a panel of tumor cells) 6: not easy to separate from the closer band (5). * For NMR

NMR results

CHCL3: Acetic Acid: MeOH 80:15:5

Preparative TLC of fraction 4 from the column. Sprayed with H2SO4 followed by oven.

* NMR candidate 3 ?? 4 WB-3559B C37H68N2O7 Mass 652.94

5 Sulfobacin A C34H69NO6S Mass 619.986 Sulfobacin SL3 C33H65NO6S Mass 603.93 and/or Flavocristamide A C34H67NO6S 617.96

PUtative components of lipid extract

MASS expected of the isolated compounds

ongoing

chemical structure of active compounDS

In vitro and in vivo essays

THANKS!