434 phg 434 phg recent approaches in medicinal plants analyses prof. mohammed abdulaziz al-yahya 1

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434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

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To carry phytochemical study the following points must be fulfilled: 1- Selection of promising plant materials. 2- Proper collection of selected plants. 3- Authentication of plant material. 4- Drying of plant materials. 5- Garbling of the dried plants 6- Packing, storage and preservation 7- Grinding of the dried plants. 8- Extraction and fractionation of constituents. 9- Methods of separation and purification. 10- Methods of identification of isolated compounds (structure elucidation e.g.: UV, IR, MS, 1 H-NMR and 13 C-NMR). 3

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Page 1: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

434 PHG

Recent Approaches in Medicinal Plants Analyses

Prof. Mohammed Abdulaziz Al-Yahya

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Page 2: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

Phytochemical Study

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Page 3: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

To carry phytochemical study the following points must be fulfilled:

1- Selection of promising plant materials. 2- Proper collection of selected plants. 3- Authentication of plant material. 4- Drying of plant materials. 5- Garbling of the dried plants 6- Packing, storage and preservation 7- Grinding of the dried plants. 8- Extraction and fractionation of constituents. 9- Methods of separation and purification. 10- Methods of identification of isolated compounds (structure elucidation e.g.: UV, IR, MS, 1H-NMR and 13C-NMR).

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1.SELECTION OF PROMISING PLANT MATERIAL

The choice of promising plant depends upon the following:

1- A plant which has a biological activity. 2- A plant used in folk medicine. 3- A plant which shows a particular toxicity.

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2. Proper collection of selected plants

Drug may be collected from: 1- Wild plants. 2- Cultivated plants.

Wild plantWild plant Cultivated plantCultivated plantDisadvantageDisadvantage AdvantageAdvantage1- 1- Scattered in large or Scattered in large or unlimited areaunlimited area

Present in limited area.Present in limited area.

2-2- Difficult to reach Difficult to reach Easy to reachEasy to reach3-3- The collector must be The collector must be highly skilled botanistshighly skilled botanists

The collector must not be The collector must not be skillful person skillful person

4-4- Deficiency may occur due Deficiency may occur due toto continuous collection continuous collection

Continuous supplyContinuous supply5

Page 6: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

Rules for collectionRules for collection The material is best collected when the The material is best collected when the organ in question organ in question

has reached its optimal state of development:has reached its optimal state of development:

1- 1- Roots and rhizomesRoots and rhizomes are collected at the end of the are collected at the end of the vegetation period, i.e. usually in the autumn. vegetation period, i.e. usually in the autumn.

2- 2- BarkBark is collected in the spring.is collected in the spring.

3- 3- LeavesLeaves and and herbsherbs are collected at the flowering stage.are collected at the flowering stage.

4- 4- FlowersFlowers are usually gathered when fully developed.are usually gathered when fully developed.

5- 5- FruitsFruits and and seedsseeds are collected when fully ripe. are collected when fully ripe.

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The following precautions should be considered during stage of collection:

a- The proper time of the day, time of the year and maturity stage of collection is particularly important because the nature and quantity of constituents may vary greatly in some species according to the season and time of collection.

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Page 8: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

●The most advantages time of collection is when the plant containing the active principals is highest in its content, example:

1- Time of year e.g. Hyoscyamous contain less amount of alkaloids in winter than in summer.

2- Time of the day e.g.1 Cardiac glycoside in Digitalis leaves are in higher amount at afternoon than in the morning. e.g.2 Solanaceous plants have higher quantities of alkaloids in the morning than in the afternoon.

3- Stage of maturity e.g. Solanaceous plants have higher quantities of alkaloids when collected in the flowering stage.

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b- The collected plant should be free from any contamination. The main causes of contamination are:

i- Collection of mixtures of plants by error. ii- Collection of closely similar species growing side by side and incorrectly assumed to be the same. iii- Collection of plants which have a parasite within it.

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Page 10: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

c- Collecting plants which are free from diseases (i.e. which are not affected by viral, bacterial, fungal infection). This may cause:

i- Infection may seriously alter plant metabolism and unexpected products could be formed possibly in large amounts (causing confusion). ii- Infection may cause the presence of products of microbial synthesis (causing confusion).

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3. Authentication of plant material

This may be confirmed by:

1- Establishing the identity by a taxonomy experts.

2- Collection of a common species in their expected habitat by a field botanist.

3- By comparing the collecting plant with a voucher specimen (herbarium sheet)

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4. Drying of plant materials

DryingDrying The most common method for preserving plant

material is drying.

- Enzymatic processes take place in aqueous solution. Rapid removal of the water from the cell will, therefore, largely prevent degradation of the cell constituents.

- Drying also decreases the risk of external attack, e.g. by moulds.

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Note: Drying should be carried out as quickly as possible without using high temperatures to prevent chemical changes of thermo-labile constituents e.g. volatile oils.

Living plant material has a high water content: * leaves may contain 60-90% water. * Roots and rhizomes 70-85%. * Wood 40-50%. * The lowest percentage is found in seeds, often no more than 5-10%.

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Drying is done in:

-Shade and in sunlight (Natural drying). - Hot air drying or by freeze-drying (Artificial drying).

Aim of drying:

1- Ease of transport. 2- Ease of grinding 3- Inhibit the growth of microorganisms. 4- Preservative of active constituents.

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Changes may occur during the drying:

1- Size and weight: Drug when drying will be smaller in size and lose 80-90 % of their original weight.

2- Shape and appearance: Black pepper on drying show polygonal reticulation (due to presence of stone cell in the hypodermis)

3- Color: Tea leaves change from green to dark brown, almost black.

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Freeze-drying

• Freeze-drying (Lyophilization) is a very mild method.

• Frozen material is placed in an evacuated apparatus which has a cold surface maintained at -60 to -80 °C. Water vapor from the frozen material then passes rapidly to the cold surface.

• The method requires a relatively complicated The method requires a relatively complicated apparatus and is much more expensive than hot-air apparatus and is much more expensive than hot-air drying. For this reason, it is not used as a routine drying. For this reason, it is not used as a routine method, but it is method, but it is very important for drying heat-very important for drying heat-sensitive substances, e.g. sensitive substances, e.g. antibioticsantibiotics and and proteins proteins.

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5- Active constituent: Slow drying of vanilla pods lead to obtain vanillin from glucovanillic alcohol.

4- Odor: Vanilla pods odorless when fresh and on drying acquire a fragrant, pleasant aromatic odor due to liberation of vanillin which has a charr. or nice odor.

17vanillin Glucovanillin

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How drying can change the yieldFresh Willow (Salix) Leaves

Vaccuum Dried Air Dried

Good yield of Condensed Tannins (Polyphenols)

Good yield of Simple Phenolic GlycosidesBad yield of Simple

Phenolic Glycosides

Bad yield of Condensed Tanins (Polyphenols) 18

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5. Garbling of the dried plants

Garbling is the final step in the preparation of a crude drug.

Garbling consists of the removal of extraneous matter, such as other parts of the plant, dirt and added adulterants.

Excessive dust can clog percolators and result in a turbid

extract which is hard to clarify.

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Drugs with essential oils deteriorate quickly through evaporation, oxidation and polymerization of the substances constituting the essential oil.

Tannins on the other hand, have an almost unlimited durability.

6. Packing, storage and preservation

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7. Grinding of the dried plants

The plant is ground into small particle size to facilitate extraction.

Large particles take a longer time for complete extraction than small ones.

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In order to keep crude drugs as long as possible:

1. It is essential to store them in a dry condition in carefully

closed containers.

2. It is also advisable to store them in light resistant containers such as, tin cans, amber glass container. because - even if light does not affect the active constituents it almost causes changes in the appearance of the drug, especially loss of color.

3. It is also necessary to protect the drug against insect attack.

4. Drugs must always be stored at as low temperature as possible because high temperature accelerate all chemical reactions.

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8- EFFECTIVE EXTRACTION

There is no general (universal) method for the extraction of plant materials.

The precise mode of extraction depends on: 1- The texture of the plant material. 2- The water content of the plant material. 3- The type of substances to be extracted or nature of active constituents.

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Page 24: 434 PHG 434 PHG Recent Approaches in Medicinal Plants Analyses Prof. Mohammed Abdulaziz Al-Yahya 1

Extraction: is the separation of medicinally active portion of plants or animal tissues through the use of selective solvent and suitable methods of extraction.

The principal methods of extraction are: A- Maceration B- Infusion C- Percolation D- Decoction E- Digestion F- Continuous hot extraction technique (Soxhlet extraction process). G- Liquid-liquid extraction H- Solvent-solvent ppt. I- Distillation 24

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A-Maceration: - This method is used for cold water soluble active constituents. - It consists of macerating the plant material in cold water (15-20 °C) for several hours. e.g. liquorice.

B- Infusion - Infusion is used for water-soluble and easily extracts principles. - The plant material is placed in a pot and wetted with cold water. Immediately afterwards, boiling water is poured over it, then left to stand, covered with a lid for about 15 min after which the tea is poured off. e.g. herbal tea.

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C- Percolation: The plant material is placed in percolator and macerated with the solvent for several hours, continuous feeding of solvent until complete extraction is occur.

Principle of action: - The instrument used to hold the powder is called a percolator. - The liquid coming from the percolator impregnated with the soluble constituents is called the percolate. - The residual drug remaining in the percolator after the extraction of the soluble constituents is called the marc.

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D- Decoction

- It was used for water soluble and heat stable constituents. - The method involves boiling the drug with water for 10 min, then allowing it to cool to about 40°C.

E- Digestion - This method is suitable for hard barks or woods which are difficult for water to penetrates.

- Digestion is also considered as macerated but at relatively elevated temperature 35-40C but not exceed 50C. e.g. cinnamon.

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F-Continuous hot extraction technique (Soxhlet extraction process)

-This procedure is the classical chemical and commonest method of extraction of organic constituents.

-The powdered material is continuous extracted successively in a Soxhlet apparatus with a range of solvents of increasing polarity.

(Starting with least polar solvent and ending with the most polar one: Petroleum ether then chloroform then ethyl acetate then, methanol and finally water).

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Extraction with each solvent is continued until side tube of Soxhlet is colorless. 29

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G- Liquid-liquid extraction:• In this technique, the solute molecules are partitioned between two immiscible solvents. • The amount of solute in each phase will depend upon the relative solubility in each solvent which in turn is related to their polarity. • It is measured by the partition coefficient (K) which is constant.

Partition coefficient (K) = mole fraction of solute in phase 1 (upper phase) mole fraction of solute in phase 2 (lower phase)

• The success of this method depends upon the selectivity of the solvents for the required compound.

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H-Solvent-solvent precipitation:

The extract dissolved in a suitable solvent, is mixed with a less polar miscible solvent causing the selective precipitation of the less soluble plant constituents.

e.g. Precipitation of triterpenoid saponins from methanol extract of Phytolacea dodecandra by the addition of acetone.

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I- Distillation Methods:

• There are two types of traps:

One for oils lighter than water and the other for oils heavier than water.

• These two types differ only in the mechanism of the return of the aqueous layer to the distillation flask, keeping the volatile oil layer in its position.

Types of distillation are used:

1-Water and steam distillation 2-Direct steam distillation

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Points for consideration in the distillation method:1- It is often necessary to subject the plant material to special treatment prior to steam distillation e.g. cut or crushed. Crushing or cutting facilitates penetration of water into oil- containing structures in the plant. e.g. Oil cells, glandular hairs.

2- For removal of water or moisture which might be present in the prepared volatile oil, anhydrous sodium sulfate is usually used.

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Choice of solventsChoice of solvents

1.1. Be highly selective for the compound to be extracted.Be highly selective for the compound to be extracted.2.2. Have a high capacity for extraction.Have a high capacity for extraction.3.3. Not react with the extracted compound. Not react with the extracted compound. 4.4. Have a low price.Have a low price.5.5. Be harmless to man and to the environment.Be harmless to man and to the environment.6.6. Be completely volatile.Be completely volatile.

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According to the pharmacopoeias, According to the pharmacopoeias, ethanolethanol is the solvent is the solvent of choice for obtaining classic extracts. of choice for obtaining classic extracts.

The ethanol is usually mixed with water The ethanol is usually mixed with water to induce to induce swelling of the plant particlesswelling of the plant particles and and to increase the porosity to increase the porosity of the cell wallsof the cell walls which facilitates the diffusion of which facilitates the diffusion of extracted substances from inside the cells to the extracted substances from inside the cells to the surrounding solvent. surrounding solvent.

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As a general empirical rule: Non polar solvents (petroleum ether and hexane) will dissolve non-polar compounds (fats and waxes).

While polar solvents (methanol, ethanol and water) dissolve polar compound (alkaloid salts and sugars). (Like dissolve like)

The affinity of the solute for the organic phase may

be greatly increased by using mixture of solvents

instead of single ones (used mixtures of solvent to

increase the solubility). 36

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Example: solublization of an aliphatic carboxylic acid in ethanol, acetone and a mixture of both.

Hydrogen bond

Hydrogen bond

In a mixture of acetone and ethanolIncrease solubility of carboxylic acid by addition of ethanol and acetone. And the solubility increased due to the formation of hydrogen bonds.

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Fresh Leaves or FlowersHomogenize for 5 min in MeOH-H2O (4:1) (10 x Vol or Wt.), Filter

Residue Filterate

Extract with EtOAc (x5), Filter

Residue

Neutral Extract(Fats & Waxes)

Evaporate

Filterate Fibre (mainly Polysaccharides)

CHCl3 Extract

Evaporate to 1/10 vol (<40 ºC)Acidify to 2M H2SO4

Extract with CHCl3 (x3)

Dry, Evaporate

Moderately Polar Extracts(Terpenoids & Phenolics)

Aqueous acid layer

Basify to pH 10 with NH4OH, Extract with CHCl3-MeOH (3:1, twice)

CHCl3-MeOH Extract Dry, evaporate

Basic Extract (Most Alkaloids)

Aqueous basic layer Evaporate , extract with MeOH

Methanol Extract is Polar(Quaternary alkaloids & N-oxides) General Procedure for extracting fresh plant tissues

and fractionating into different classes according to Polarity

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