extraction and isolation of natural products Pharmacognosy •"Pharmacognosy" derives from two Greek words, "pharmakon" or drug, and "gnosis" or knowledge. ... •Natural products

Pharmacognosy

• "Pharmacognosy" derives from two Greek words, "pharmakon" or drug, and "gnosis" or knowledge. While representing a classical field of science,

• Its scope includes the study of the physical, chemical, biochemical and biological properties of drugs, drug substances, or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources.

Herbal medicine

1 : the art or practice of using herbs and herbal preparations to maintain health and to prevent, alleviate, or cure disease.

2 : a plant or plant part or an extract or mixture of these used in herbal medicine.

• Natural products are important sources for drug

development. The amounts of bioactive natural

products in natural medicines are always fairly

low. Today, it is very crucial to develop effective

and selective methods for the extraction and

isolation of those bioactive natural products.

• In the USA, approximately 49% of the population has tried natural medicines for the prevention and treatment of diseases

• Natural products have provided the primary sources for new drug development. From the 1940s to the end of 2014, nearly half of the FDA approved chemical drugs for the treatment of human diseases were derived from or inspired by natural products

• Extraction is the first step to separate the desired natural products from the raw materials. The

• extraction of natural products stages

– (1) the solvent penetrates into the solid matrix;

– (2) the solute dissolves in the solvents;

– (3) the solute is diffused out of the solid matrix;

– (4) the extracted solutes are collected.

Any factor enhancing the diffusivity and solubility in the above steps will facilitate the extraction.

Factors affection Extraction

• Selection of the solvent: Selectivity, solubility, cost and safety should be considered.

• Polarity of the solvent and the solute: Based on the law like dissolves like, solvents with a polarity value near to the polarity of the solute are likely to perform better and vice versa. Alcohols (EtOH and MeOH) are universal solvents in solvent extraction for phytochemical investigation.

• Particle size: the finer the particle size is, the

better result the extraction achieves. The

extraction efficiency will be enhanced by the

small particle size due to the enhanced

penetration of solvents and diffusion of

solutes.

• Temperature: High temperatures increase the

solubility and diffusion. Temperatures that too

high, however, may cause solvents to be lost,

leading to extracts of undesirable impurities

and the decomposition of thermolabile

components.

• Duration: The extraction efficiency increases with the increase in extraction duration in a certain time range. Increasing time will not affect the extraction after the equilibrium of the solute is reached inside and outside the solid material.

• Amount of the solvent: The greater the solvent-to-solid ratio is, the higher the extraction yield is;

Extraction methods

• Traditional methods: maceration, percolation and reflux extraction, usually use organic solvents and require a large volume of solvents and long extraction time.

• Modern extraction methods: critical fluid extraction (SFC), pressurized liquid extraction (PLE) and microwave assisted extraction (MAE), have also been applied in natural products extraction – Advantages: lower organic solvent consumption,

shorter extraction time and higher selectivity.

Pre-extraction preparation of plant

samples • Pre-preparation of plant materials such as grinding and drying also

influences the preservation of phytochemicals in the final extracts.

• Fresh vs. dried samples

• Grinded vs. powdered samples

• Drying of plants samples

• Fresh vs. dried samples: Both fresh and dried sample is used in medicinal plants studies. In most cases, dried sample is preferred considering the time needed for experimental design.

• Grinded vs. powdered samples: Lowering particle size

increases surface contact between samples and

extraction solvents. Grinding resulted in coarse smaller

samples; meanwhile, powdered samples have a more

homogenized and smaller particle, leading to better

surface contact with extraction solvents. This particular

pre-preparation is important, as for efficient extraction

to occur, the solvent must make contact with the target

analytes and particle size smaller than 0.5 mm is ideal

for efficient extraction.

• Air-drying

usually takes from 3-7 days to months and up to a year

depending on the types of samples dried (e.g. leaves or seed).

Plant samples, usually plants leaves with stem were tied

together and hang to expose the plant to air at ambient

temperature. This drying method does not force dried plant

materials using high temperature; hence, heat-labile

compounds is preserved.

Drying of plants samples:

Microwave-drying

• Microwave-drying uses electromagnetic

radiation that possesses both electric and

magnetic fields. This method can shorten

drying time but sometimes causes

degradation of phytochemicals.

Oven-drying

• Oven-drying uses thermal energy to remove

moisture from the samples. This sample

preparation is considered as one of the easiest

and rapid thermal processing that can

preserved phytochemicals.

Freeze-drying

• Freeze-drying is a method base on the principle of sublimation.

• Sublimation is a process when a solid is changed into gas phase without entering the liquid phase.

• Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase.

• Sample is frozen at -80°C to -20°C prior to lyophilisation to solidify any liquid (eg. solvent, moisture) in the samples. After an overnight (12 h) freezing, sample is immediately lyophilized to avoid the frozen liquid in the sample from melting.

• However, freeze-drying is a complex and expensive methods of drying compared to regular air drying and microwave-drying. Thus, the use is restricted to delicate, heat-sensitive materials of high value.

Maceration

• Maceration This is a very simple extraction method with the disadvantage of long extraction time and low extraction efficiency. It could be used for the extraction of thermo-labile components.

• Maceration involved soaking plant materials (coarse or

powdered) in a stoppered container with a solvent and allowed to stand at room temperature for a period of minimum 3 days with frequent agitation. The processed intended to soften and break the plant’s cell wall to release the soluble phytochemicals. After 3 days, the mixture is pressed or strained by filtration.

• Digestion

This is a form of maceration in which gentle heat is used during the process of extraction.

Percolation

• Percolation is more efficient than maceration because it is a continuous process in which the saturated solvent is constantly being replaced by fresh solvent.

• This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is generally used.

Decoction

• In this process, the crude drug is boiled in a

specified volume of water for a defined time;

it is then cooled and strained or filtered. This

procedure is suitable for extracting water-

soluble, heat-stable constituents.

Reflux extraction

• Reflux extraction is more

efficient than percolation,

maceration and Decoction.

• It requires less extraction

time and solvent. It cannot

be used for the extraction of

thermo-labile natural

products.

Soxhlet extraction

• The Soxhlet extraction method integrates the advantages

of the reflux extraction and percolation, which utilizes the

principle of reflux and siphoning to continuously extract the

herb with fresh solvent. The Soxhlet extraction is an

automatic continuous extraction method with high

extraction efficiency that requires less time and solvent

consumption than maceration or percolation.

Hot Continuous Extraction (Soxhlet)

• In this method, the finely ground crude drug is

placed in a porous bag or “thimble” made of

strong filter paper, which is placed in a

chamber of the Soxhlet apparatus

Pressurized liquid extraction (PLE)

• It applies high pressure in extraction. High pressure

keeps solvents in a liquid state above their boiling point

resulting in a high solubility and high diffusion rate of

lipid solutes in the solvent, and a high penetration of

the solvent in the matrix.

• PLE dramatically decreased the consumption of

extraction time and solvent amount.

Supercritical fluid extraction (SFE)

• It uses supercritical fluid (SF) as the extraction solvent. SF has similar solubility to liquid and similar diffusivity to gas, and can dissolve a wide variety of natural products. Their solvating properties dramatically changed near their critical points due to small pressure and temperature changes.

• Supercritical carbon dioxide (S-CO2) was widely used in SFE because of its attractive merits such as low critical temperature (31 °C), selectivity, inertness, low cost, non-toxicity, and capability to extract thermally labile compounds.

Ultrasound assisted extraction

• also called ultrasonic extraction or sonication, uses ultrasonic wave energy in the extraction. Ultrasound in the solvent producing cavitation accelerates the dissolution and diffusion of the solute as well as the heat transfer, which improves the extraction efficiency.

• It is applicable for the extraction of thermo-labile and unstable compounds.

Microwave assisted extraction

• Microwaves generate heat by interacting with polar compounds such as water and some organic components in the plant matrix following the ionic conduction and dipole rotation mechanisms.

• Advantages: such as increasing the extract yield, decreasing the thermal degradation and selective heating of vegetal material.

Enzyme assisted extraction

• The structure of the cell membrane and cell wall,

micelles formed by macromolecules such

polysaccharides and protein, and the coagulation and

denaturation of proteins at high temperatures during

extraction are the main barriers to the extraction of

natural products. Cellulose, α-amylase and pectinase

are generally employed in EAE.

distillation

• Hydro and steam distillation Hydro distillation and

steam distillation are commonly used methods for the

extraction of volatile oil.

Separation methods

• The components in the extract from Previous methods are complex and contain a variety of natural products that require further separation and purification to obtain the active fraction or pure natural products. The separation depends on the physical or chemical difference of the individual natural product.

• Chromatography, especially column chromatography, is the main method used to obtain pure natural products from a complex mixture.