434 PHG

Recent Approaches in

Medicinal Plants Analyses

Introduction

The use of plant-derived medicinals dates back many

centuries although it is still under estimation in modern

medicine.

Plants remain the most important source of natural

drugs.

- More than 30% of prescription drugs are natural

products.

- More than 60% of anticancer and anti-infective drugs

are natural products.

The main sources of drugs are as follows:

1- Natural substances:

From plants, microorganisms, marine, animals,- etc.

(totally obtained from nature).

* e.g. of plant origin: Alkaloid and glycoside, volatile or

fixed oil.

2- Semisynthetic substances:

These are drugs that are manufactured by partial synthesis.

A natural starting material can be modified by chemical or

biochemical means to produce a substance having specific

pharmacological activities. e.g. steroidal hormones and

corticosteroids.

3- Synthetic substances:

These are drugs which are manufactured by total synthesis

(i.e. complete synthetic process or processes).

Medicinal plants information

sources

1- Herbals:

These are documents (books, manuscripts,….etc) that provide

information about the uses of many plants in folk medicine.

2- Medical botany مسح نباتات في منطقه معينه

- These are published books and periodicals ascribing the native

flora of various regions of the world and the medicinal uses

for each plant.

- It also may include the synonyms of plants and the constituents.

- Many countries, nowadays are represented by books or review

publications on medicinal botany.

e.g. Flora of Saudi Arabia (Dr. Mijahid)

3- Ethnobotany عالقة االنسان بالنباتات المحيطه به

This means the study of the plants in their relationship to human.

Many report describing the habitual use and relationship of man and the

surrounding flora are available.

4- Herbaria (herbarium)تجفيف النبات

Herbarium is a representative whole plant or organ

of plant which is preserved to provide a reference

specimen when required.

Information about:

1-The name (including synonymous names).

2-Date and place of collection.

3-Any field notes that the collector could

gather the information including their:

use and toxicity (if any) should be recorded

on the so called “Herbarium sheet”.

5- Phytopharmacological surveys:

These are surveys which are concerned with the biological activities of plant

extracts or constituents and they are available in specialized periodicals or

books.

When we need to know the biological activities and

or chemical constituents of plants?

To achieve one or more of the following goals:

1- The discovery of new therapeutic agents.

2- The disclosing of new sources of economic materials for the synthesis

of complex chemical substances.

3- Isolation of a novel chemical structure often prompts the chemist to a

successful synthesis of a series of synthetic compounds which may have

some medicinal value.

5- The knowledge of the chemical constituents of plants and their chemical

structure will help studying their biosynthetic mechanisms and hence may

facilitate their synthesis in laboratories.

6- Knowing the chemical constitutes of plants would help

expanding the area of chemotaxonomy.

4-The knowledge of the chemical constituents of toxic plants will certainly help

treating their poisonous effect to humans and animals.

Phytochemical Study

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- Grinding of the dried plants.

6- Garbling of the dried plants

7- Packing, storage and preservation

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).

1. Selection of promising plant materials:

The choice of promising plant depends upon the following:

1- A plant which have a biological activity.

2- A plant used in folk medicine.

3- A plant which show a particular toxicities.

2. Proper collection of selected plants

Drug may be collected from:

1- Wild plants. 2- Cultivated plants.

Wild plant Cultivated plant

Disadvantage Advantage

1- Scattered in large or

unlimited area

Present in limited area.

2- Difficult to reach Easy to reach

3- The collector must be

highly skilled botanists

The collector must not be

skillful person

4- Deficiency may occur due

to continuous collection

Continuous supply

Rules for collection

The material is best collected when the organ in question

has reached its optimal state of development:

1- Roots and rhizomes are collected at the end of the

vegetation period, i.e. usually in the autumn.

2- Bark is collected in the spring.

3- Leaves and herbs are collected at the flowering stage.

4- Flowers are usually gathered when fully developed.

5- Fruits and seeds are collected when fully ripe.

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.

●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.

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 it has a parasite within it.

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).

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. مكانها األصلي

3- By comparing the collecting plant with a voucher specimen

(herbarium sheet)

4- Drying of plant materials

Drying

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.

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%.

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.

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.

5- Active constituent:

Slow drying of vanilla pods lead to obtain vanillin from glucovanillic alcohol.

CH3O

Glu-O

CH2OH CH2OHCH3O

HO

CHOCH3O

HO

Enz

H2OEnz

[O]

VanillinGlucovanillic alcohol (glucoside)

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.

5- Grinding of the dried plants

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

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

6- 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.

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.

7- Packing, storage and preservation

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 a temperature as

possible. B/c high temperature accelerate all chemical

reactions.

8- Extraction and fractionation of constituents

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.

Extraction: is the separation of medicinally active portion of

plants or animal tissues through the use of selective

solvent and suitable methods extraction.

The principal methods of extraction are: 1- Maceration

2- Infusion

3- Percolation

4- Decoction

5- Digestion

6- Continuous hot extraction technique (Soxhlet extraction

process).

7- Liquid-liquid extraction

8- Solvent-solvent ppt.

9- Distillation

1-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.

2- Infusion - Infusion is used for water-soluble and easily extracts principles.

-A popular method of preparing teas. This tea / "herbal tea"

preparation method typically involves pouring hot water over plant

matter, waiting for a period of time and then removing the plant

matter before consumption.

3- 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.

4- Decoction

- It was used for water soluble and heat stable constituents.

- The method involves boiling the drug with water for 10 min,

5- 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.

6-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 (Fig.) 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).

Extraction with each solvent is continued until side

tube of soxhlet is colorless.

7- 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.

8-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.

9- 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

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.

eg. 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.

Choice of solvent

1. Be highly selective for the compound to be extracted.

2. Have a high capacity for extraction.

3. Not react with the extracted compound.

4. Have a low price.

5. Be harmless to man and to the environment.

6. Be completely volatile.

According to the pharmacopoeias, ethanol is the solvent of choice for

obtaining classic extracts.

The ethanol is usually mixed with water to induce swelling of the plant

particles and to increase the porosity of the cell walls which facilitates

the diffusion of extracted substances from inside the cells to the

surrounding solvent.

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).

Example: solublization of an aliphatic carboxylic acid in

ethanol, acetone and a mixture of both.

-R-C

O------------H-O-CH2-CH3

O-H

In ethanol

In acetone R-C

O-H---------O=C

O

CH3

CH3

Hydrogen bond

Hydrogen bond

R-C

OH

O HO-C2H5

O=C

CH3

CH3

(ethanol)

(acetone)

In a mixture of acetone and ethanol

Increase solubility of carboxylic acid by

addition of ethanol and acetone.

And the solubility increased due to the

formation of hydrogen bonds.

Phytopharmacological screening

● After a new substance (expected to be a new drug)

has been developed (extracted, isolated).

-The next step is to subject it to a specified set of

procedures in experimental animals.

● The aim is to determine whether or not the compound

possesses the desired action.

Pharmacological screening:

Requirements:

1- Animal

have a large problem such as:

a- Cost b- Feeding

c- Selection: e.g. : dogs (heart/ vascular)

Rats (analgesia/ inflammation)

rabbits ( CNS)

Monkey ( tumor, virus)

2- Tissues:

All types of muscle, heart, nerve structure.

3- Time:

It take from 2-3 months and also need duplication for experiments.

4- Solubility: drug action depend on solubility so most probably in

animal used: oral, i.p.

Phytopharmacological screening:

● Antimicrobial activity

Cinnamomum verum (Eugenol)

Thymus vulgaris (Thymol)

Lavendula officinalis (Linalool)

● Antineoplastic activity

Catharanthus roseus (Vincrestine, vinblastin)

Taxus brevifolia (Taxol)

● Antimalarial:

Cinchona succirubra (Quinine)

Artemisia annua (Artemisinin)

● Hypoglycemic:

Garlic (Allium sativum) (Allicin)

● Cardiotonic

Digitalis purpurea (Digoxin and Digitoxin)

Strophanthus kombe (K-strophanthoside)

● Antiarrythemic

Cinchona succirubra (Quinidine)

Type of pharmacological screening:

1-Blind screening

The aim is to see if a new compound has any useful

pharmacological activity.

2- Simple screening

The aim is to find a substance having a particular property.

e.g. A single test for the conc. of sugar in blood may be

used to screen compounds for hypoglycemic activity.

3- Programmed screening

In this type of screening the most important pharmacological

action of the new compound is fully investigated.

Phytopharmacological screening

The organization of blind screening Screening for pharmacological activities proceeds from general

observation on intact animals to the more specific techniques

using isolated organs.

It might include the following tests:

A- Preliminary observations on the behaviour of conscious

animals (neuropharmacological tests)

B- The cardiovascular test.

C- Isolated organ preparations especially the isolated

guinea pig ileum.

a) Neuropharmacological tests:

These tests were developed to determine the central

actions of drugs.

● It can detect: sedatives, hypnotics, tranquillizers,

pscychomotor stimulants, muscle relaxant,

analgesics, atropine-like drugs, antipyretics,

peripheral vasodilators.

● The animal used are: mice.

● The route of administration is important:

- The compound is given parenterally (I.P) in order to avoid

the possibility of poor absorption from the intestinal tract.

● Different doses are used.

- For the performance of the test 3 animals are used per dose.

The signs observed in the primary test for pharmacological activity are:

1- Consciousness or awarness.

2- Stereotype: is the frequental mechanical repetition of a movement.

e.g. Morphine: cause the mice to have circular movement.

Amphetamine: cause them to have searching movement.

3- Motor activity and spontaneous activity, this measures stimulation and

depression of the autonomic nervous system.

4-The pain response: this measures analgesia and sedation.

5- The touch response, may indicate the presence of anesthetic activity.

6- Central excitation, is indicated by:

- Tremors.

- Convulsions.

- Restlessness and irritability.

7- The autonomic responses includes:

- Pupil size. - Hypothermia

- Secretion - Skin color

- Heart rate - Respiratory rate

b) The cardiovascular tests:

These tests describe the action of the new substance

on blood pressure of the intact anesthetized animals.

The following conclusions can be made:

1- If the test substance cause brief fall in B.p. (↓in B.p.)

therefore it may have:

- Muscarinic activity like ACH

- Direct vasodilator activity

- Cardiodepressant activity.

2- If the test substance causes a brief rise in blood pressure

(↑ in B.p.) therefore it may have:

- Sympathomimetic activity or

- Direct vasopressor activity.

3- If the test substance cause a prolonged fall in B.p. (↓↓B.p.)

(more than 5 min), it may have: Ganglian blocking

4- If the test substance cause a prolonged rise in B.p. (↑↑B.p.)

(5minutes or more) it may have:

Monoamine oxidase inhibiting activity.

5- If the test substance cause a fall in B.p. after a delay of 45 sec.

it may have: Histamine-releasing activity like morphine.

7- If after administration of the test substance,

the responses to epinephrine injection is diminished

- Sympatholytic activity.

6- If after administration of the test substance,

the response to ACH is diminished.

- Parasympatholytic activity like atropine.

9- If the test substance cause the response to epinephrine is to be augmented

above those obtained before the administration of the substance i.e. these may

be monoamine oxidase inhibition.

This will confirm the observation of prolonged rise in B.p. produced by the test

substance itself.

8- If the test substance cause the response to ACH to be augmented above the

control, the test substance may have:

- Anticholinesterase activity like neostigmine.

c) Isolated organ preparations especially the isolated

guinea pig ileum.

Isolated preparation have the following advantages:

1- Free from interference by central action (e.g. circulating

hormones).

2- Relatively small amounts of the material is required.

3- Drug effect is tested directly without the factor of

absorption, metabolism, etc.

4- Several preparation can be tested on a single animal.

Screening methods:

Ideal method such as:

1- Antimicrobial

2- Anticancer

3- Antiviral

4- Hepatoprotective

5- Antioxidant

Antimicrobial Screening of Natural Products Using

Agar Dilution Method

- The Screening methods for the detection of antimicrobial

activity of natural products fall into three groups, including:

- Bioautographic

- Dilution methods (turbidimetric assay).

- Agar diffusion (plate assay)

The bioautographic is known as:

Qualitative technique since this method will only give

an idea of the presence or absence of substances with

antimicrobial activity.

Procedure:

1- The desired concentration of natural products is dissolved in

very small amount of DMSO.

2- The solution is added to 10 ml of the melted agar medium and

mixed will.

3- The agar is poured into the Petri dish often desired dimensions

and cooled.

4- The desired test organism are then applied a lines from the edges

to the center of the plate.

5- The plate is then incubated and growth of organisms is recorded.

Results:

1- In the above figure five organisms were used to screen natural

products A, B and C at 1mg/10 ml agar.

2- The used organisms are:

E: Escherichia coli S: Staphylococcus aurous C: Candida albicans

B: Bacillus subtillus Ps: Pseudomonas aurogenosa

3- The results revealed that:

a. Natural products A and B are active against:

B: Bacillus. Subtillus S: Staphylococcus aurous C: Candida albicans

b. Natural products C is active against:

B: Bacillus. Subtillus S: Staphylococcus aurous

c. The fourth plate is the control showing growth of all organisms

The Dilution methods (turbidimetric assay) and agar diffusion

(plate assay) are considered as:

Quantitative assays once they determine the minimal

inhibitory concentration (MIC).

MIC is the lowest concentration of an antimicrobial that will

inhibit the visible growth of a microorganism after overnight

incubation.

MIC are important in diagnostic laboratories to confirm

resistance of microorganisms to an antimicrobial agent and

also to monitor the activity of new antimicrobial agents.

Turbidimetric assay:

The test organisms growing in test tubes that contain

different concentrations of the antibiotic.

There is a direct relationship of the concentration of

antibiotic to the growth of the test organism, and by

measuring the growth of the organism which is indicated

by the turbidity of the content of the test tube, the

antibiotic titer can be determine.

Clear tubes indicate a higher antibiotic conc. than turbid

tubes, and the lowest concentration of antibiotic hat

completely prevents the appearance of turbidity is known

as MIC.

Plate assay:

filter paper discs are impregnated with solutions of

antibiotic of varying concentrations , allowed to dry,

placed on agar media seeded with an appropriate test

organism, and incubated.

As the concentration of the antibiotic increase, its

diffusion through the agar medium increases; therefore,

the size of the clear zone of growth inhibition around

the filter paper disc is related to the concentration of

antibiotic.

Antioxidant Screening of Natural Products Using TLC

Oxidation of a wide range of chemical compounds and the production

of free radicals (contains one or more unpaired electrons) at the

cellular level are believed to be involved in the cause of many cancers.

Plant derived natural products such as flavonoids, terpenoids and

steroids have received considerable attention in recent years due to

their diverse pharmacological properties including antioxidant and

antitumor activity.

Antioxidants are compounds that help to inhibit many oxidation

reactions caused by free radicals there by preventing or delaying

damage to the cells and tissues.

Antioxidants: classification

Non-Enzymatic antioxidant:

Alpha tocopherol (vitamin E)

Beta Carotene

Ascorbic acid (vitamin C)

Antioxidant enzymes:

Superoxide dismutase (SOD)

Glutathione peroxidase enzyme

The catalase enzyme

Free radicals and cancer

Many forms of cancer are thought to be the result of reactions between

free radicals and DNA, resulting in mutations that can adversely affect

the cell cycle.

Natural antioxidants are presents

in fruits and vegetable

WHO recomandation:

eat 5 fruit or vegetable per a day

EXPEREMENT

Conditions:

* Plant extract rich in antioxidant are:

- Orange or lemon peel (dry and coarsely powder).

- Green Tea leaves (coarsely powder).

* Standard antioxidant (References):

Gallic acid, Ellagic acid, Arbutin, Hydroquinone & Rutin

(at concentration = 5-10 mg/5 ml MeOH).

* Solvent System:

-For orange peel, Lemon peel and green tea leaves is (for flavonoids):

Ethylacetate : Formic acid : H2O (8 : 1 : 1)

Or CHCl3 : Ethylacetate (6 : 4)

* Detection :

1. DPPH solution (20 mg/100 ml in MeOH), must be freshly

prepared. (DPPH= 1,1 Diphenyl-2-picrylhydrazyl)

2. Heat in Oven at 60 οC.

Procedure:

1. Spotting of the plate.

2. Develop the plate.

3. Dry plates at open air for 15-20 seconds.

4. Dip the plate in DPPH which in Petri dish.

5. Dry plate in dark for 90 seconds at room temperature then for 30

seconds at 60 οC.

6. Free radical scavenging zone were identified immediately as

yellow area against violet purple background plate

Cancer is a class of diseases in which a group of cells display

uncontrolled growth, invasion (intrusion on and destruction of

adjacent tissues) and sometimes metastasis (spread to other

locations in the body via lymph or blood).

Causes:

- Tobacco smoke

- Infectious agents

- Radiation

- Chemical (Benzene)

- Diet (coloring agent, preservative)

- Pesticide

- Drug

Anticancer Screening of Natural Products :

There are currently four structural classes of plant derived anticancer

agents on the market. These are:

1. The vinca alkaloids (vinblastine, vincristine): were isolated from

Catharanthus roseus and are used for the treatment of a wide

variety of cancers, including leukemia, bladder cancer and testicular

cancer.

2. The taxanes (paclitaxel and taxol): were isolated from Taxus brevifolia

and are used for the treatment of a wide variety of cancer including

Ovarian, Lung, Gastric, Cervical, Prostate & Colon cancer

3. The camptothecin derivatives (topotecan and irinotecan).

Uses: Ovarian cancers, Colorectal cancer.

Cytotoxicity assays

1- Brine shrimp lethality assay:

Artemia salina is commonly known as the brine shrimp.

Larvae were hatched at 26 ºC in filtered seawater.

A lamp was placed above the tank. After 24 hrs, hatched nauplii

were used for the assay.

Plant extract with different concentration was added to brine solution.

The experiment was run in five replicates of each concentration.

The number of dead and survivor brine shrimps in each tube was

counted after 24 h.

To ensure that mortality observed in the bioassay is related to

bioactive compounds, dead larvae in each treatment were compared

to the dead larvae in the control.

The percentage of mortality was calculated as: percentage of survival

in the control-percentage of survival in the treatment

The brine shrimp assay is one of the best and rapid biological and

toxicological assays for all the labs.

Cirrhosis is the formation of fibrous tissue in the liver from replacing dead

liver cells. The death of the liver cells can be caused by viral hepatitis,

alcoholism or contact with other liver-toxic chemicals.

Diagnosis is done by checking levels of Serum glutamic oxaloacetic

transaminase and Serum glutamic pyruvic transaminase

Method:

1- Animals (rabbits/ rats)

- Control (saline) - Standard (Silymarin)

- Extracts or isolated compounds

2- Induction cirrhosis in animals by using:

- CCl4 or CHCl3 (reversible)

- Galactosamine or α- amanitin (irreversible)

3- Administer extract and standard 24 hours measure enzymes

Hepatoprotective Screening of Natural Products

Although random compound screening in animal models

is still a useful approach to discover new drugs.

Disadvantages: - It requires a large amount of compound.

- Its sensitivity is low.

- It is extremely laborious.

- Since the amount of active constituents present in natural

products is usually very small, it is impractical, in most cases,

to supply sufficient quantities of pure natural compounds for

animal experimentation.

High-throughput technologies:

• High-throughput screening (HTS) is an approach to drug discovery

that has gained widespread popularity over the last three or four

years.

• HTS is the process of assaying a large number of potential effectors

of biological activity against targets.

• The methods of HTS are applied to the screening of combinatorial

chemistry, genomics, protein, and peptide libraries.

• The goal of HTS is to accelerate drug discovery by screening large

libraries often composed of hundreds of thousands of compounds.

Advantage:

• HTS decreases the amount of testing compound required such that only

microgram quantities are needed. This is advantageous for certain natural

products that are difficult to isolate and purify, and permits compounds that are

difficult to synthesize to be assayed.

• The number of compounds assayed has increased from 100 000 per year to

100 000 per day.

• HTS need:

Highly specialized and expensive screening lab to run an HTS operation .

High-throughput screening (HTS)

Using robotics, data processing and control software, liquid handling devices,

and sensitive detectors.

HTS allows a researcher to quickly conduct millions of biochemical, genetic

or pharmacological tests.

The results of these experiments provide starting points for drug design and

for understanding the interaction or role of a particular biochemical process

in biology.

Methodology and equipment

HTS uses automation to run an assay, or screen, of a library

of candidate compounds against a target.

Note:

Automation: Typically, an integrated robot system consisting of

one or more robots transports assay-microplates

from station to station for sample and reagent

addition, mixing, incubation, and finally readout

or detection.

Assay plate preparation

The key labware or testing vessel of HTS is the microtiter plate:

a small container, usually disposable and made of plastic, that

features a grid of small, open divots called wells.

Modern microplates for HTS generally have either 384, 1536,

or 3456 wells. These are all multiples of 96, reflecting the original

96 well microplate with 8 x 129mm spaced wells.

To prepare for an assay, the researcher fills each well of the plate

with some biological matter such as protein or some cells, or an

animal embryo.

After some incubation time has passed to allow the biological

matter to absorb, bind to, or to react with the compounds in the

wells, measurements are taken across all the plate's wells, either

manually or by a machine.

Manual measurements are often necessary when the researcher is

using microscopy to seek changes or defects in embryonic

development caused by the wells' compounds, looking for effects

that a computer could not easily determine by itself. Otherwise, a

specialized automated analysis machine can run a number of

experiments on the wells. In this case, the machine outputs the

result of each experiment as a grid of numeric values, with each

number mapping to the value obtained from a single well.

A high-capacity analysis machine can measure dozens of plates in

the space of a few minutes like this, generating thousands of

experimental data points very quickly.

High-Throughput Screening

Up to 100,000 samples/24 hours