UJI BIOAKTIFITAS TANAMAN OBAT

NUR PERMATASARI

WHY THE SUDDEN INTEREST IN NATURAL PRODUCTS ?

Low/absent toxicity ? Complete biodegradability Renewable sources Low cost Tropical forest responsible for production of major pharmaceutical drugs. New advances in the analysis and assaying of plant materials

TRADITIONAL MEDICINE: how do herbs differ from conventional drugs?

Complex formulations vs single component Synergy (pharmacodynamics and pharmacokinetics) chemical complexity

HERBS THERAPEUTIC USES

DRUG DISCOVERY ACTIVE INGREDIENTS IN HERBS HAVE THERAPEUTIC POTENTIAL HERBAL MEDICINES CANNOT BE DISMISSED SOME ARE EFFICACIOUS USE CAUTIOUSLY

IMPORTANCE OF PLANTS IN MEDICINE ATROPINE CAFFEINE THEOPHYLLINE COLCHICINE DIGOXIN ERGOTAMINE MORPHINE PENICILLIN PHYSOSTIGMINE QUINIDINE QUININE COCAINE TAXOL SALICIN

HERBAL MEDICINE
ANECDOTAL, TRADITIONAL, FOLKLORE UNSUBSTANTIATED THERAPEUTIC CLAIMS LITTLE TESTING NO STANDARDIZATION SOME REMEDIES ARE POISONOUS

HERBAL MEDICINE - CONS

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

HERBAL MEDICINES
EASY ACCESS NO Rx INEXPENSIVE SOME THERAPEUTIC EFFICACY PLACEBO EFFECT

Procedure for obtaining the active principles from plants

Chemical diversity

bioassay

Medicinal plants extracts fraction structure <-- pure constituents structure elucidation modification toxicology

synthesis

Interdisciplinary approach
Chemist Biologist Pharmacologist Botanist Agronomist etc

Classification of bioassay

(Bioassays are typically conducted to measure the effects of a substance on a living organism)

General screening bioassay Preliminary information on pharmacological potential of material Specialized screening bioassay Prediction of mechanism of action and therapeutic indications

Levels of testing
DRUG + receptor + transduction system (second BINDING functional messenger; enzyme) whole or BIOCHEMICAL TESTING part organs

ISOLATED TISSUE EXPERIMENTS

Anaesthetised or conscious animals

WHOLE ANIMAL EXPERIMENTS

Classification of bioassay `general screening bioassay`

Broad screening bioassay (animal model) in vivo Primary screening bioassay (lethality and cell growth inhibition)

ANIMAL MODEL in vivo

HIPOESTROGEN OVARIECTOMY

Tikus Tikus Tikus Tikus

dengan dengan dengan dengan

diet induksi atherogenic induksi rokok inflamasi (model rhematoid arthritis) tukak lambung (induksi indometacin)

Mempelajari arteriogenesis dan angiogenesis dengan melakukan ligasi arteria femoralis

Tikus diabet yang diinduksi streptozotocin

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 (Isolated

organ)

Whole animals (to detect of activity of the organ

system)

Cell culture
Advantages Cost + time effective information Reliable+ reproducible Human cells increase relevance Permit studies not possible in animals

Disadvantages Lack multisystemic integration

Heterotypic interaction are lost Lack of the several systemic compound

Cell culture

Lack effects of other systems Limited models Dedifferentiation

loss of the differentiated properties

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

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)

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

To imitate ischemia, 2 105 cells on beads were pipetted into a conical centrifuge tube and allowed to settle, and the excess media were removed to a separate flask (hypoxiavolume restriction). The headspace of the centrifuge tube was flushed with N2 containing 5% carbon dioxide and incubated at 37C undisturbed for the specified period of time

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 dll

Measure of toxicity
Toxicity of chemicals is determined in the laboratory 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 into the body, or By placing the test material in the water or air of the test animals environment

Measure of toxicity
Toxicity is measured as clinical endpoints which include Mortality (death) Reproductive tox (teratogenesis,reproduction performance,perinatal and postnatal tox) Carcinogenicity (ability to cause cancer), and, Mutagenicity (ability to cause heritible change in the DNA)

Duration of toxicity
Three terms are commonly used to describe the duration of dose(s) Acute Subchronic Chronic

Duration of Exposure:Acute Exposure

Application of a single or short-term (generally less than a day) dosing by a chemical Animal: mouse, rat, female,male Examination: death animal in a 14 day period (weight, behavioral, lethargy, food consumption etc) Information: LD50,target organ, reversibility, dose-response

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

FDA PRECLINICAL PHARMACOLOGY & TOXICOLOGY REQUIREMENTS DRUGS Two Species - Rodent & Non-rodent Clinical Route & Schedule Pharmacokinetics - Optional

BIOLOGICALS

Most Relevant Species Clinical Route & Schedule Biodistribution

REPRESENTATIVE SURFACE AREA TO WEIGHT RATIOS (km) FOR VARIOUS SPECIES (Freireich, et al, Cancer Chemotherapy Reports, 1966, 50: 219-244)

Species Mouse Rat Monkey Dog Human Child Adult

Body Weight Surface Area Km ( kg ) ( m2 ) Factor 0.02 0.15 3.0 8.0 20 60 0.0066 0.025 0.24 0.40 0.80 1.6 3.0 5.9 12 20 25 37

Measures of Toxicity: The Median Lethal Dose

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 of animal body weight

Measures of Toxicity: The Median Lethal Concentration

LC50 The concentration of a chemical in an environment (generally air 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 air or water (or as ppm)

Toxicity
LD50 measured in mg/kg of body weight LD50 Examples

Supertoxic
Extreme. Toxic Very Toxic Toxic Mod. Toxic Slight. Toxic Non-Toxic

< 0.01mg
<5mg 5-50mg 50-500mg 500mg-5g 5g-15g >15g

dioxin, botulism, mushrooms

heroin, nicotine morphine, codeine DDT, H2SO4, Caffeine aspirin, wood alcohol ethyl alcohol, soaps water, table sugar

Duration of Exposure: Subchronic Exposure

Toxic symptoms are expressed after repeated applications for a timeframe less than half the life expectancy of the organism (90 days) Examination: body weight, food consumtion, respiratory and cardiovascular distress, motor and behavioral abnormalities etc At the end of the 90-day blood and organ collected for analysis

Duration of Exposure: Subchronic Exposure

Expression of toxic symptoms only after repeated exposure to a chemical in doses regularly applied to the organism for a time greater than half of its life-expectancy Mice : 18 m 24 m Rats : 2-2.5 y

What is a Response?
Response (symptoms) could be on the molecular, cellular, organ, or organism level

(interference w/receptor,membrane function,cellular energy production, binding ti biomolc, pertubation in calsium homeostasis etc)

Local vs. Systemic Reversible vs. Irreversible Immediate vs. Delayed Graded vs. Quantal degrees of the same damage vs. all or none

Primary Routes of Exposure

There are three primary routes by which organisms are exposed to poison Oral Dermal Inhalation

Primary Routes of Exposure: Oral Exposure

Any exposure which occurs when the chemical is taken in through the mouth and passes through the gastrointestinal tract ADME (target organ adverse effect is dependent

upon the concentration of active compound at the target site for enough time, Not all organs are affected

equally, greater susceptibility of the target organ, higher concentration of active compound Liver, Kidney Lung, Neurons, Myocardium, *Bone marrow

Primary Routes of Exposure: Dermal Exposure

Exposure of the skin Animal : back (0.5 of liquid and 0.5 g of solid, 1-inch square, one intact and two abraded skin sites, 4 h) Examination: erithema,edema, corrosive action

RESEARCH: STATEGY TO DECIDE WHICH PLANTS TO STUDY:

Random selection. Noting which plants are eaten by animals. Focus on botanical relatives of known plants of interest. Pay attention to the knowledge of indigenous people

RESEARCH: STATEGY TO CHOICE BIOASSAY

Choosing the right bioassay or test system is crucial to the success of a drug research program. The test should be simple, quick and relevant as there are usually a large number of compounds to be analyzed.

Human testing is not possible at such early stage, so the test has to be done 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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III-Identifying a bioassay 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 cells. Antibacterial drugs are tested in vitro by measuring how effectively they inhibit or kill bacterial cells in culture

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III-Identifying a bioassay

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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III-Identifying a bioassay

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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III-Identifying a bioassay

3-In vivo tests

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

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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III-Identifying a bioassay 4-Test validity

Sometimes the validity of testing procedure is easy and clear. For example,

the 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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