434 PHG
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 معينه منطقه في نباتات مسح
- 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 به المحيطه بالنباتات االنسان عالقة
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) النبات تجفيف
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 plantWild plant Cultivated plantCultivated plantDisadvantageDisadvantage AdvantageAdvantage
1- 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 reach
3-3- 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 supply
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.
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. األصلي مكانها
3- By comparing the collecting plant with a voucher specimen (herbarium sheet)
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.
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
EnzH2O
Enz
[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 sidetube 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 waterand 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 solventChoice of solvent
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.
According to the pharmacopoeias, According to the pharmacopoeias, ethanolethanol is the solvent of choice for is the solvent of choice for
obtaining classic extracts. obtaining classic extracts.
The ethanol is usually mixed with water The ethanol is usually mixed with water to induce swelling of the plant to induce swelling of the plant
particlesparticles and and to increase the porosity of the cell wallsto increase the porosity of the cell walls which facilitates which facilitates
the diffusion of extracted substances from inside the cells to the the diffusion of extracted substances from inside the cells to the
surrounding solvent. 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.
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.
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- Feedingc- 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: classificationAntioxidants: classification Non-Enzymatic antioxidant: Non-Enzymatic antioxidant: Alpha tocopherol (vitamin E)Alpha tocopherol (vitamin E) Beta CaroteneBeta Carotene Ascorbic acid (vitamin C)Ascorbic acid (vitamin C)
Antioxidant enzymes:Antioxidant enzymes: Superoxide dismutase (SOD)Superoxide dismutase (SOD) Glutathione peroxidase enzymeGlutathione peroxidase enzyme The catalase enzymeThe catalase enzyme
Free radicals and cancerFree radicals and cancer
Many forms of cancer are thought to be the result of reactions betweenMany forms of cancer are thought to be the result of reactions between
free radicals and DNA, resulting in mutations that can adversely affectfree radicals and DNA, resulting in mutations that can adversely affect
the cell cycle. the cell cycle.
Natural antioxidants are presents in fruits and vegetable
WHO recomandation:
eat 5 fruit or vegetable per a day
EXPEREMENTConditions: * 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 modelsis 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
