Prinsip Uji Bioaktifitas Natural

7/29/2019 Prinsip Uji Bioaktifitas Natural

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UJI BIOAKTIFITAS TANAMAN OBATNUR PERMATASARI


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WHY THE SUDDEN INTEREST INNATURAL PRODUCTS ?

Low/absent toxicity ?

Complete biodegradability

Renewable sources Low cost

Tropical forest responsible for production ofmajor pharmaceutical drugs.

New advances in the analysis and assaying ofplant materials


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Complex formulations vs single component

Synergy (pharmacodynamics and

pharmacokinetics) chemical complexity

TRADITIONAL MEDICINE:how do herbs differ from conventional drugs?


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HERBS THERAPEUTIC USES

DRUG DISCOVERY ACTIVE INGREDIENTSIN HERBS HAVE THERAPEUTIC POTENTIAL HERBAL MEDICINES CANNOT BEDISMISSED SOME ARE EFFICACIOUS USE CAUTIOUSLY
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IMPORTANCE OF PLANTS IN MEDICINE

ATROPINE

CAFFEINE

THEOPHYLLINE

COLCHICINE

DIGOXIN

ERGOTAMINE

MORPHINE

PENICILLIN

PHYSOSTIGMINE

QUINIDINE

QUININE

COCAINE

TAXOL

SALICIN


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HERBAL MEDICINE

ANECDOTAL, TRADITIONAL, FOLKLORE UNSUBSTANTIATED THERAPEUTIC CLAIMS

LITTLE TESTINGNO STANDARDIZATION SOME REMEDIES ARE POISONOUS


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HERBAL MEDICINE - CONS

ADULTERATION OTHER DRUGS,TOXINS BLOOD CONCENTRATION VARIES SIDE EFFECTS, DRUG INTERACTIONS


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HERBAL MEDICINES

EASY ACCESS NO Rx INEXPENSIVE SOME THERAPEUTIC EFFICACY PLACEBO EFFECT


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Procedure for obtaining the active principlesfrom plants

Medicinal plants extracts fraction

structure


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Interdisciplinary approach

Chemist

Biologist

Pharmacologist

Botanist Agronomist

etc


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Classification of bioassay (Bioassays are typically conducted to measure the

effects of a substance on a living organism)

General screening bioassay

Preliminary information onpharmacological potential of material

Specialized screening bioassay

Prediction of mechanism of action andtherapeutic indications
http://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Organism


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Levels of testing

DRUG + receptor

BINDING+ transduction

system (second

messenger; enzyme)

BIOCHEMICAL TESTING

functional

whole or

part organs

ISOLATED TISSUE EXPERIMENTS

Anaesthetised or

conscious animals

WHOLE ANIMAL EXPERIMENTS


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Classification of bioassay `general screening

bioassay`

Broad screening bioassay (animal model) in vivo

Primary screening bioassay (lethality and cellgrowth inhibition)


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ANIMAL MODEL in vivo

HIPOESTROGEN OVARIECTOMY

Tikus dengan diet induksi atherogenicTikus dengan induksi rokok

Tikus dengan inflamasi (model rhematoid arthritis)Tikus dengan tukak lambung (induksi indometacin)


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Mempelajari arteriogenesis dan angiogenesis dengan melakukan

ligasi arteria femoralis

Tikus diabet yang diinduksi streptozotocin


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Classification of bioassay `specialized

screening bioassay`

Lower organism (bacteria, yeast, fungi, viruses,insects, protozoa)

Isolated subcellular system (enzymes ,

receptor) Isolated cellular system (cell culture)

Isolated organs of vertebrates (Isolatedorgan)

Whole animals (to detect of activity of the organsystem)


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Cell culture

Advantages Cost + time effective information

Reliable+ reproducible

Human cells

increase relevance

Permit studies not possible in animals


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Cell culture

Disadvantages Lack multisystemic integration

Heterotypic interaction are lost

Lack of the several systemic compound

Lack effects of other systems Limited models

Dedifferentiation

loss of the differentiated properties


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Cell culture Primary culture (directly from human,animal or

plant tissue)

Extended culture (multipassage culture) cell

strain/ cell line Established (transformed) cell/ continous cell

line/ immortalized cell line


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Primary tissue culture A culture derived directly from a tissue Best resembling natural tissue

Limited growth potential

Limited life span

May give rise to a cell strain (20-100 generations) or be

immortalized (an infinite life span)


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To imitate ischemia, 2 105 cells on beads were pipettedinto a conical centrifuge tube and allowed to settle, and the

excess media were removed to a separate flask (hypoxia-

volume restriction). The headspace of the centrifuge tube

was flushed with N2 containing 5% carbon dioxide andincubated at 37C undisturbed for the specified period of

time

In vitro model: Cell culture(related to hypoxia-ishemia)


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MACAM ORGAN

Pembuluh darah terpisah : aorta strip ( tikus, kelinci, marmot) dipotong spiral sehingga membentuk lembaran

aorta ring ( tikus, kelinci, marmot)

pembuluh darah ekor tikus terpisah

Usus terpisah (dengan atau tanpa saraf) , digunakan ileum

Jantung terpisah (atrium, jantung terpisah/lanedorf)Trakea terpisah

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Toxicity of chemicals is determined in thelaboratory

The normal procedure is to expose test animals By ingestion, application to the skin, by inhalation, gavage,

or some other method which introduces the material intothe body, or

By placing the test material in the water or air of the testanimals environment

Measure of toxicity


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Toxicity is measured as clinical endpoints whichinclude Mortality (death)

Reproductive tox (teratogenesis,reproductionperformance,perinatal and postnatal tox) Carcinogenicity (ability to cause cancer), and, Mutagenicity (ability to cause heritible change

in the DNA)

Measure of toxicity


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Three terms are commonly used to describe theduration of dose(s)

AcuteSubchronic

Chronic

Duration of toxicity


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Application of a single or short-term (generally

less than a day) dosing by a chemical

Animal: mouse, rat, female,maleExamination: death animal in a 14 day period(weight, behavioral, lethargy, food consumption etc)Information: LD50,target organ, reversibility,

dose-response

Duration of Exposure:Acute Exposure

FDA PRECLINICAL PHARMACOLOGY & TOXICOLOGY


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FDA PRECLINICAL PHARMACOLOGY & TOXICOLOGY

REQUIREMENTS

DRUGS Two Species - Rodent & Non-rodent

Clinical Route & Schedule

Pharmacokinetics - Optional

BIOLOGICALS

Most Relevant Species

Clinical Route & Schedule

Biodistribution

Ref: DeGeorge, Cancer Chemother. Pharmacol., 41, 173-185, 1998.


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REPRESENTATIVE SURFACE AREA TO WEIGHT RATIOS (km) FOR VARIOUSSPECIES(Freireich, et al, Cancer Chem otherapy R eports, 1966, 50: 219-244)

Species Body Weight

( kg )

Surface Area

( m2 )

Km

Factor

Mouse 0.02 0.0066 3.0

Rat 0.15 0.025 5.9

Monkey 3.0 0.24 12

Dog 8.0 0.40 20

Human

Child

Adult

20

60

0.80

1.6

25

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Measures of Toxicity:


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LD50

The amount (dose) of a chemical which produces death in 50%of a population of test animals to which it is administered by

any of a variety of methods

mg/kg

Normally expressed as milligrams of substance per kilogram ofanimal body weight

Measures of Toxicity:The Median Lethal Dose

f


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LC50

The concentration of a chemical in an environment (generallyair or water) which produces death in 50% of an exposed

population of test animals in a specified time frame

mg/L

Normally expressed as milligrams of substance per liter of airor water (or as ppm)

Measures of Toxicity:The Median Lethal Concentration

Toxicity


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LD50 measured in mg/kg of body weight

LD50 ExamplesSupertoxic < 0.01mg dioxin, botulism, mushroom

Extreme. Toxic 15g water, table sugar

Toxicity

D ti f E


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Toxic symptoms are expressed after repeatedapplications for a timeframe less than half the lifeexpectancy of the organism (90 days)

Examination: body weight, food consumtion,respiratory and cardiovascular distress, motor andbehavioral abnormalities etc

At the end of the 90-day blood and organ collectedfor analysis

Duration of Exposure:Subchronic Exposure

D ti f E


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Expression of toxic symptoms only after

repeated exposure to a chemical in dosesregularly applied to the organism for a time

greater than half of its life-expectancyMice : 18 m 24 mRats : 2-2.5 y

Duration of Exposure:Subchronic Exposure


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Response (symptoms) could be on the molecular,cellular, organ, or organism level(interference w/receptor,membrane function,cellular energyproduction, binding ti biomolc, pertubation in calsiumhomeostasis etc)

Local vs. Systemic Reversible vs. Irreversible Immediate vs. Delayed Graded vs. Quantal

degrees of the same damage vs. all or none

What is a Response?


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There are three primary routes by which organisms areexposed to poison

Oral

Dermal

Inhalation

Primary Routes of Exposure


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Primary Routes of Exposure:Oral ExposureAny exposure which occurs when the chemical is taken inthrough the mouth and passes through the gastrointestinaltract

ADME (target organ adverse effect is dependentupon the concentration of active compound at the

target site for enough time, Not all organs are affectedequally, greater susceptibility of the target organ, higher

concentration of active compound Liver, Kidney Lung, Neurons, Myocardium, *Bonemarrow


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Primary Routes of Exposure:Dermal ExposureExposure of the skin

Animal : back (0.5 of liquid and 0.5 g of solid, 1-inch square, oneintact and two abraded skin sites, 4 h)

Examination: erithema,edema, corrosive action

RESEARCH: STATEGY TO DECIDE WHICH PLANTS TO


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STUDY:

Random selection. Noting which plants are eaten by animals.

Focus on botanical relatives of known plants ofinterest.

Pay attention to the knowledge of indigenouspeople


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Choosing the right bioassay or test system is crucial to the success of adrug research program.

The test should be simple, quick and relevant as there are usually a larg

number of compounds to be analyzed.

Human testing is not possible at such early stage, so the test has to bedone in vitro first. Because in vitro tests are cheaper, easier to carry out

less controversial and can be automated than in vivo one.

In vivo tests needed to check the drugs interaction with specific target

and to monitor their pharmacokinetics properties.

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RESEARCH: STATEGY TO CHOICE BIOASSAY

III-Identifying a bioassay


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y g y

2-In vitro tests

They do not involve live animals. Instead, specific tissues, cells, or

enzymes are isolated and used.

Enzyme inhibitors can be tested on pure enzyme in solution.

Receptor agonist and antagonists can be tested on isolated tissues or cell

Antibacterial drugs are tested in vitro by measuring how effectively they

inhibit or kill bacterial cells in culture

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y g y

3-In vivo tests

In vivo tests on animals often involve inducing a clinical condition in the

animal to produce observable symptoms.

The animal is then treated to see whether the drug alleviates the problem

by eliminating the observable symptoms. For example, the development

of non-steroidal inflammatory drugs was carried out by inducing

inflammation on test animals.

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3-In vivo tests

The animals used may be transgenic i.e,some mouse genes are replaced

by human genes so the mouse produces the human receptor or enzyme.

Or the mouses gene may be altered to be susceptible for some disease

such as breast cancer.

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3-In vivo tests

There are several problems associated with in vivo testing. It is slow and

also causes animal suffering. There are also many problems of

pharmacokinetics and the result obtained may be misleading. For

example, penicillin methyl ester is hydrolyzed in mice into active penicillin

while it is not hydrolyzed in humans or rabbits. Also, thalidomide is

teratogenic in rabbits and humans while it is not in mice.

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y g y

4-Test validity

Sometimes the validity of testing procedure is easy and clear. For examplthe antibacterial drug can be tested by its effect on killing bacteria. Local

anaesthetics are tested by their effect on blocking action potential in

isolated nerve.

In other cases, the testing procedure is more difficult. For example, there

is no animal model for antipsychotic drug.

Thus, validity of the test should be carried out.

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