The Art and Science of Natural Products Purification:

Basic Preparative Phytochemistry for Plant Scientists

Participants’ handbook

Novara, September 27, 2016

Summary of the activities – Aim of the workshop (presentation) – The extraction of plant biomass (presentation)

– Non-chromatographic purification methods of plant

exctracts (laboratory experience) • Partition • Crystallization • Steam distillation

– Flash chromatography (laboratory experience) – Conclusion

Aim of the workshop

a) Critically evaluate the pros and cons of various preparative phytochemical techniques (extraction, partition, crystallization, chromatography)

b) Exemplify the methods discussed with laboratory experiences based on plants and compounds of interest for the TriForC project

The extraction of plant biomasses

– The polarity range of plant secondary metabolites (PSM) spans a large range of polarity, and an ideal solvent capable to capture the whole plant metabolome does not exist

– Methanol, ethanol, acetone or mixtures of them are a good approximation to the «ideal solvent», but

• Methanol is a poor solvent for highly apolar compounds

• Methanol is a reactive solvent (formation of methyl acetals)

• Acetone is a poor solvent for highly glycosidated or charged PSM (polydesmosidic saponins, anthocyanins)

– Prior knowledge on the polarity of the compounds of interest is necessary for their complete extraction

Issues to consider for the selection of an extraction solvent

– Facility of removal (boiling point, foaming)

– Density, viscosity, and penetration in plant tissues

– Cost and safety

Examples:

Ether and THF can form explosive peroxides

Butanol foams during evaporation and is high-boiling

Acetonitrile is toxic and expensive

Chloroform is toxic

Laboratory Experience #1: Divide et impera: the fractionation of a plant extracts

(mate, Ilex paraguariensis)

– Liquid/liquid partition: the Kupchan partition scheme

– Liquid/solid partition: the TriForC protocol

– Comparison of the results by TLC analysis of the partitioned extracts

Mate (Ilex paraguariensis A. St. Hil.)

Caffeine-rich infusion, particularly popular in Argentina, where it enjoys a

"national infusion” status.

Based on the leaves of Yerba mate (Ilex paraguariensis A. St. Hil, a bush from

the IIlicaceace family).

Traditionally prepared in a gourd recipient from which it is drunk. The gourd is

nearly filled with yerba, hot water is added, and the hot infusion is drunk from a

straw. Also available in tea bags, and prepared like a normal infusion.

Domesticated by the Jesuits in Paraguay in the XVII century. Production is

now partitioned between Brazil (53%), Argentina (37%), and Paraguay (10%).

Mate: A herb for the metabolic syndrome?

Mate: Mechanisms of activity and constituents

Cardioprotection: decrease of LDL cholesterol and increase of HDL

cholesterol

Weight control: decreased lipid absorption by inhibition and modulation of the

expresion of lipases

Glycemic control: Activation of AMPK

Major classes of constituents:

methylxanthines (mainly

caffeine)

saponins

polyphenolics

caffeoylquinic acids

PARTITION

The Kupchan liquid-liquid partition

Based on the sequential fractionation of an extract between two phases of different polarity Pros: simple and efficient total recovery of the compounds Cons: Time- and solvent consuming Formation of emulsions Formation of precipitates at the interface of the

solvents

A little history

Salomon Morris Kupchan (1922-1976) was an American natural products chemist who pioneered the study of natural anticancer drugs from plants.

Active at the universities of Wisconsins and then Virginia

Discoverer of maytansinoids. Important studies in the field of triterpenoid alkaloids

Father of Charles A. Kupchan (1958), a popular political author and expert in EU issues

The Europeans are making a choice that will inevitably give Europe more geopolitical clout—at the expense of American primacy. … And the impetus will come not just from Europe's new ambition, which will gather slowly, but also from America's domestic politics and its schizophrenic reaction to the rise of Europe.

The original Kupchan protocol(s)

In his work, Kupchan used various partition schemes and there is not a «single» reference partition protocol.

All schemes are based on the partition of a crude methanol extracts with solvents of increasing lipophilicity.

Kupchan original protocol-1

Kupchan, S., M. Recent Advances in the Chemistry of Terpenoid Tumor Inhibitors. University of Wisconsin, 1969

Kupchan original protocol-2

Kupchan, S., M. Recent Advances in the Chemistry of Terpenoid Tumor Inhibitors. University of Wisconsin, 1969

Kupchan original protocol-3

Kupchan, S., M. Recent Advances in the Chemistry of Terpenoid Tumor Inhibitors. University of Wisconsin, 1969

The modified Kupchan partition

The Kupchan-UPO partition

The Kupchan-UPO partition in practice

1. 1 g of a mate extract are dissolved in 40 mL of methanol and transferred into a separator funnel. 10 mL Water are then added

2. The suspension is extracted twice with 50 mL of petroleum ether (upper phase) 3. The water-methanol phase is taken back to the separatory funnel and diluted with

25 mL water 4. The suspension is extracted with EtOAc (2 x 30 mL) (upper phase) 5. The wate phase is taken back to the separatory funnel, and 1 g NaCl is added 6. The suspension is extractec with THF (2 x 30 mL) 7. The petroleum ether, EtOAc and THF phases are analyzed by TLC

The UPO solid-liquid partition

Based on absorption on a solid matrix (silica gel) and de-absorption with solvents of growing affinity for the solid phase (petroleum ether/ethyl acetate/THF)

Quicker than the Kupchan partion method

Avoids the formation of emulsions and precipitates

Irreversible absorption possibile with highly polar compounds

The liquid-solid UPO partition

The UPO liquid/solid partition in practice

1. 3 g of a mate extract are dissolved in acetone (or acetone-methanol), 9 g of silica gel are added, and the slurry is evaporated under reduced pressure

2. The extract adsorbed on silica gel is deposited on top of a layer of 9 g celite packed into a suction filter and protected with a paper filter

3. The three elution volumes (90 mL) are sequentially passed through the adsorbed extract, that is stirred with caustion before suction.

4. The three fractions are analyzed by TLC

Markers for the TLC analysis

Laboratory Experience #2:

– Crystallization of ursolic acid from a crude mate extract

– Purification of zerumbone by steam-distillation of shampoo ginger

Crystallization

Ursolic acid is the major triterpenoid acid from mate

Steam Distillation

The Clevenger apparatus

Shampoo ginger (Curcuma zerumbet)

Zerumbone is the major constituent of the essential oil from shampoo ginger

Laboratory Experience #3:

Purification of celastrol from a thunder god vine (Tripterygium wilfordii Hook f.) extract by flash chromatography

Flash chromatography vs gravity chromatography

Column Cartrigdge

Flash chromatography in a nutshell

Invented by W. Clark Still (1946) in 1978 at Columbia University Uses smaller size particles (250-400 mesh vs 70-230 mesh) silica gel compared to gravity column chromatography Uses a pressurized gas to drive the solvent through the solid phase Currently done with pre-packed plastic cartriges and not with glass Columns and automatized

Clark Still is famous for being an eccentric, and, after giving up research, has spent the past 8 years building an airplane in his garage

Conclusion

– Q&A section

– Take home message