penjelasan mengenai apa itu bioassay

1. Urip Harahap
PROGRAM MAGISTER ILMU FARMASI
FAKULTAS FARMASI USU
2024
TTM-1 dan TM 2

2. Setelah mengikuti kuliah ini mahasiswa mampu:
Menjelaskan esai dan prinsip dasar esei biologis
Mendiskusikan keuntungan relatif esei biologis dibandingkan dengan metoda
fisika dan kimia
Menggambarkan problema utama jika dikaitkan dengan penggunaan hewan
atau jaringan hewan dalam esei biologi
Menjelaskan standar yang digunakan dalam esei biologi dalam term yang
umum
Menggambarkan makna aktivitas biologi
Menjelaskan efek yang diperoleh dalam esei biologi pada hewan atau jaringan
Mendiskusikan aspek disain eksperimen esei biologi
Memahami teknik-teknik esei biologis baik dalam menguji bahan alam yang
belum pernah diketahui kahsiatnya secara farmakologis (kualitatif) dan bahan
atau obat yang telah ditehui aktivitas biologinya (kuantitatif).

3. ▪ Approach to the experimental pharmacology has been
changed in past few years. It is due to the
advancement in learning through computer based
experimentations.
▪ Although it is effective, less expensive and save lives
of thousands of experimental animals, understanding
and implementation of such kind of knowledge for
further study or in industries had certain limitations.
Hence, there is a modify in the usual approach of
gaining of knowledge, practical skills and developing
attitudes in students.
▪ A need has, therefore, been felt for a manual that
documents why and how a practical should be
conducted.

4. ▪ Farmakologi, studi tentang obat-
obatan, terdiri dari banyak bidang.
Farmakologi eksperimental adalah
salah satu fokus utama dalam
penemuan obat.
▪ Kegiatan praklinis dan klinis bersama-
sama adalah untuk penemuan
kandidat obat.
▪ Tahap praklinis: in vitro,
menggunakan jaringan dan cell lines
dan in vivo, menggunakan hewan
sebagai sistem eksperimental
▪ The preclinical phase validates the pharmacokinetic and
pharmacodynamic properties of the drug in order to support the
upcoming clinical phase.
▪ So drug discovery involves many steps including active compound
identification, pharmaceutical profiling, safety or toxicology and
efficacy evaluation, and analysis using in vitro cell lines and in vivo
animal models
▪ Even though the in vitro techniques are well established, the drug’s
activity or effect in a whole organism needs to be determined
through in vivo studies in laboratory animals due to some species’
genetic similarity with humans and also in order to be cost effective
and help reduce tragedies in human trials of a novel chemical entity
▪ Thus, these animal models provide fundamental information
regarding toxicity and other relevant disease information prior
to the initiation of a clinical study of that particular candidate
compound. Also, the in vitro studies with relevant cells from the
human system play a major role in the analysis of a particular drug

5. ▪ Pengaruh Teknik atau perlkauan,
variasi pada hewan, variasi waktu
ke waktu, variasi pada bahan uji
→ seagai respons untuk
memperkirakan potensi hasil uji
yang diperoleh
▪ Agar hasil uji memuaskan maka
perlu ada standar untuk
mereduksi pengaruh tersebut
▪ Kelompok hewan yang sama akan
memberi respons yang berbeda
dari waktu ke waktu dan hewan
yang berbeda juga memberi level
resposn yang berbeda dari waktu
ke waktu
▪ Bioassay procedures to describe,
evaluate, and predict the potential
hazard of toxic materials to
organisms and ecosys tems, and
the health-related aspects of
polluted waters continue to receive
widespread attention.
▪ Various chemical, physical, and
biological phenomena may alter
chemical structure and therefore
the fate and toxicity of certain
compounds in natural and
laboratory toxicity test sys tems.
Journal (Water Pollution Control Federation,
55:6 1983
Cell Culture Assays
▪ Cell culture assays play an important role in
drug research along with animal tests with
the principle of ‘3Rs’—Reduction,
Refinement and Replacement—due to its
quick and easy way of drug response
analysis, as well as high throughput in
assays, simplicity and cost-effectiveness
Tujuan: Untuk mengevaluasi
sitotoksisitas bahan kimia dan skrining
obat melalui viabilitas sel in vitro dan uji
sitotoksisitas menggunakan kultur sel
Bioassays in Experimental and Preclinical
Pharmacology

6. ▪ It is relatively easy to screen extracts
and other materials for cytotoxic
effects through automated screening
procedures used in industry as well as
by the research organizations, like the
National Cancer institute, USA running
60 different cancer cell lines
▪ It is based on one or more mammalian
cell lines in actively growing condition
undergoing mitotic division.
▪ The cells are cultured in a microliter-
well plate; the rate of multiplication
and growth is measured by the
intensity of a colour directly
proportional to the number of cells
present
CYTOTOXICITY TESTING
▪ The cells are cultured in a microliter-well plate; the rate of multiplication and
growth is measured by the intensity of a colour directly proportional to the
number of cells present
▪ Different types of experiments are employed, the most basic one being a
comparison of the rate of proliferation of a cancer cell line after a specified
period of time in the presence and in the absence of the test material.
▪ For determination of the cytotoxic effect whether cytostatic or cytocidal, two
sets of identical cells are exposed to the test agent under identical conditions
for the same period of time. At the end of the exposure period one set of cells
is assayed, whilst in the other set the medium containing test material is
replaced by the fresh medium
▪ The cells a re then incubated for a fixed period of time before the assay for
the cell growth. If the test material has only cytostatic effect, the cells will
grow and undergo mitosis in the fresh medium. On the other hand, if it has
cytocidal effect, no such growth of cells will be observed
▪ Two majo r techniques are used to assess the cell growth. The first one uses
either 3-(4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide (MTI) or
2,3- bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5 carboxanilide
sodium (X-IT) salt.

7. ▪ The MTT method was first introduced in 1986 by Cole
(Cole, 1986) followed by the XTI method in the year] 988
(Scudiero et al., 1988). Both the reagents Mn and Xn are
me tabolica11y reduced by the mitochondria present in
the viable cells to a coloured formnzan produce the inte
nsity of which is measured spectrophotometricatly in a
plate reader.
▪ The use of XTI is preferred since theformazan produced
is soluble in water, whereas the solubilisation step is
required if [vlTT is used (Skehan et al., 1996). However,
many cell lines arc not so efficient in reducing XTT
compa red to MTT; but with the addition of phenazine
methosulphate (P.MS) the reduction becomes much
better (Scudiero et al.,1988).

8. ▪ Th e second major technique preferred in testing cytotoxicity is
the sulpho-rhodamine B (SRB) assay. This is based on the uptake of
the negatively charged pink aminozanthine dye (SRB) by the basic
amino acids present in the cells. The greater the number of cells,
the greater the amount of dye being taken up. After fixing, the cel1
s are lysed releasing the dye that gives more intense colour and
greater absorbance (Skehan et al., 1996).
▪ Th e SRB assay is simple, sensitive, reproducible and faster than
the formazan assay. lt gives better linearity, good signal-to-noise
ratio, and has a stable end-point that does not require a time-
sensitive measurement compared to the MTI' or XTT assays
(Fricker and Buckley, 1996, Keepers et al., 1991 ). However, both the
formazan based and SRB assays are being followed in different
laboratories.

9. Advantages and
Disadvantages of
Cell Line Studies
Advantages
▪ A large number of extracts and other materials can be screened for cytotoxic effects
using throughput a utomated screening procedures .
▪ Compared to in vivo and in vitro studies very small concentratwn of the material is
required
▪ The results are obtained quickly
Disadvantages
▪ Sophisticated and expen sive equipments, like fluoresce micrscope, confocal
microscope, flow-cytometer etc. under aseptic condition are required.
▪ Results are inconsistent with wate r insoluble maten als exceptmg when they are
soluble in less than 1 % DMSO solution.
▪ Results obtained with the cell lines may n ot corroborate with those of the in vivo
studies
The cell line studies provide a quick indication regarding the efficacy of the test material as
well as its probable mechanism of action. A number of test materials can be screened at a
time. However, in order to confirm the results obtained by the cell line studies, both in vitro
and in vivo studies have to be carried out

10. ASSAY
https://www.slideshare.net/FarazaJaved/biological-assay
▪ Assay adalah prosedur investigasi
(analisis) dalam laboratorium (medicine),
farmakologi, biologi lingkungan, dan biologi
molekuler untuk menilai secara kualitatif
atau mengukur secara kuantitatif
keberadaan atau jumlah atau aktivitas
fungsional suatu entitas target (analit)
bisa berupa obat atau senyawa biokimia
atau sampel organik
Type of assay

11. ▪ Zat pembanding harus memiliki tindakan serupa dengan bahan uji, yaitu
menujukkan kurva dosis-respons yang paralel dan menolak pengujian yang
tidak menunjukkan paralelisme
▪ The assay must be balanced in time, all responses being obtained
simultaneously or so balanced that secular variation may be discounted in
analysis
▪ The living material is allotted to dosage groups in such a way that variation in
response will not bias the result or the estimate of error
▪ An estimate of error must be available from the internal evidence of the assay
itself →These are restrictions which must be placed on any assay of whatever
form, in addition there are other highly desirable points which should be
adhered to if at all possible
▪ The assay should be completely balanced, with equal numbers per group and
equal numbers and spacing of groups on whatever scale is used (often a log
scale)
▪ A standard preparation should be employed as a routine, either a substance
carefully calibrated against an international standard, or a local standard if no
international standard exists

12. CHEMICAL ASSAY
▪ It is the study of the separation, identification, and quantification of
the chemical components of natural and artificial materials
▪ Spectrophomotetry, Spectrofluomtery, Chromatohraphy
IMMUNOASSAY
▪ Suatu teknik yang memanfaatkan pengikatan antara antigen
dan homologh antibodi untuk mengidentifikasi dan mengukur
antigen atau antibodi spesifik dalam sampel
https://www.slideshare.net/FarazaJaved/biological-assay
TYPES OF
ASSAY
BIOLOGICAL ASSAY
▪ Prosedur pengujian biologi untuk memperkirakan konsentrasi bahan obat farmakologis
dalam produk yang diformulasikan atau material bulk
▪ Potensi spesifik obat diberikan pada hewan atau relawan manusia, respon obattersebut
dibandingkan dengan standar

13. MICROBIAL ASSAYS
▪ Microbiological assay: qualitative or quantitative dtermination of chemical compound from a simple or
even complex material with use of microorgnisms
▪ Used to determine the potency of a drug in animal or man and monitoring and controlling antimicrobial
chemotherapy
▪ Many antimicrobial agents , wich inhibit the growth of microorganisms (antibiotics) or are essential for
their growth
Keuntungan:
▪ Simpel, spesifik, ruang/tempat, material dan waktu yang dibutuhkan minimal
▪ Relatif murah
▪ Convenionent method
Keterbatasan
▪ Memerlukan teknis yang trampil
▪ Perlu kalibrasi yang tepat
▪ Less reproducible
▪ Chances of greater error
▪ Tidak digunakan jika tersedia uji kimia atau fisika yang baik

14. BIOESAI
▪ Estimation of concentration or potency of a substance by
measurement o biological respons it produces
• i.e Observation of pharmacological effects on
❖ Living tissues, or cells
❖ Microorganisms
❖ Animals
• Also known as BioStandardiztion

15. ▪ Bioassay: singkatan untuk BIOLOGICAL ASSAYS (pengujian biologis) dan
merupakan jenis eksperimen ilmiah.
▪ Bioassay lazimnya dilakukan untuk mengukur efek suatu zat pada organisme
hidup.
▪ Bioassay mungkin bersifat kualitatif atau kuantitatif.
▪ Bioassay kualitatif digunakan untuk menilai efek fisik suatu zat yang mungkin
tidak dikuantifikasi, seperti perkembangan atau deformitas yang tidak normal.
▪ Bioassay kuantitatif melibatkan estimasi konsentrasi atau potensi suatu zat
dengan mengukur respons biologis yang dihasilkannya.
▪ Bioassay kuantitatif biasanya dianalisis menggunakan metode biostatistik
What is Bioassay?

16. • Bioassay adalah penggunaan organisme hidup untuk menguji keberadaan suatu
senyawa atau untuk menentukan jumlah senyawa yang ada dalam sampel.
Organisme yang digunakan sensitif terhadap senyawa yang akan dilakukan
pengujian.
• Dengan demikian, efek yang diamati biasanya adalah kematian atau kesehatan
organisme uji yang memburuk. Bergantung pada organisme uji, sampel tanah,
udara, atau cairan dapat diuji.

17. • Contoh: sejarah klasik dari bioassay adalah penggunaan burung
kenari oleh para penambang di abad yang lalu, karena kenari
lebih sensitif ketimbang manusia terhadap gas berbahaya
seperti metan. Burung kenari bereaksi dengan cepat bahkan
sejumlah kecil gas saja. Ini memberi kesempatan penambang
untuk melarikan diri.
• Beberapa spesies tanaman juga dapat digunakan untuk
melakukan bioassay di dalam air. Namun, yang lebih umum,
organisme uji adalah organisme bersel tunggal seperti alga,
kutu air (khususnya spesies yang disebut Daphnia magna.
Water flea, any member of
the crustacean order Anomopoda (class
Branchiopoda),

18. Principles of Bioassay
Estimasi atau
Determinasi
Agen:
Fisik
Kimia
Biologis
Mengukur dan membandingkan
besar respons bahan uji dengan standar
pada sistem biologis sesuai dengan kondisi
standar
Bioassay: instrumen yang dapat digunakan untuk
mengestimasi dan penemuan zat aktif biologis dan
aplikasinya penting karena secara farmakologi memiliki
sensitivitas dan memeiliki spesifisifitas
Metode kimia: metode yang sangat kompleks,
memerlukan dosis kimia tinggi dalam ujinya dan
komposisi kimia tersebut yang menunjukkan
tindakan farmakologis
▪ Bioassay didefinisikan sebagai estimasi atau penentuan konsentrasi atau potensi zat fisik,
kimia atau biologi dengan cara mengukur dan membandingkan magnitud respon bahan uji
dengan standar (pada sistem biologis yang sesuai pada kondisi standar)
▪ Bioassay: perangkat (tool) yang dapat mengestimasi dan menemukan senyawa aktif dan
merupakan cara yang penting sebab sensitive dan spesifik dan diaplikasi dalam farmakologi.
▪ Metode kimia adalah metode yang sangat kompleks, karena membutuhkan dosis dan
komposisi kimia yang tinggi menunjukkan tindakan farmakologis obat (bahan uji) (expl. Uji
kimia in vitro analite penghambat -glucosidase)

20. Bioassays are essential in the development of new drugs, and monitoring pollutants in
the environment. Environmental bioassays are generally a broad-range survey of
toxicity, and a toxicity identification evaluation is conducted to determine what the
relevant toxicants are.
The use of bioassays include:
1. measurement of the pharmacological activity of new or chemically
undefined substances
2. investigation of the function of endogenous mediators
3. determination of the side-effect profile, including the degree of drug
toxicity
4. measurement of the concentration of known substances (alternatives
to the use of whole animals have made this use obsolete)
5. Assessing the amount of pollutants being released by a particular
source, such as wastewater or urban runoff.

21. Metode untuk mengukur efek obat sangat diperlukan agar
kita dapat membandingkan sifat-sifat zat yang berbeda, atau
substans yang sama pada circumstance yang berbeda,
persyaratan ini dipenuhi oleh teknik bioassay, yang
didefinisikan sebagai estimasi konsentrasi atau potensi suatu
bahan dengan cara megukur respons biologisnya pada living
systems

22. Is the main method
for finding new drugs
in pharmaceutical
companies
Two different concepts
Bioactive compound
screening
The Functional
elucidation (mechanism
study of an herbal
medicine)
In vitro or in vivo, in
situ (occasionally)
Biological and pharmacological methods to explain
why an herb has a known therapeutic effect
Bioassays are performed for both purposes
▪ In practice, functional elucidation is more
complicated and challenging. With a well set up
bioassay, screening of an extract or a fraction is
usually terminated if the result is negative
▪ However, for functional evaluation of an herbal
medicine with a known function, the study
should not be stopped if a negative result is
obtained from one assay. Other bioassays with
different mechanisms should continue to be
tried out for the test sample

23. For example, red clover (Trifolium pratense) and black cohosh
(Actaea racemose) have been used by women with menopause
to relieve their hot flash symptoms, so their methanol extracts
were both studied on estrogenic activity beginning with the
estrogenic receptor (ER) binding assay
Red clover →aktivitas
esterogenik beriktan
langsung dengan ER
Black cocosh, tidak berikatan dengan ER
→tidak ada aktivitas esterogenik, tetapi
berikatan dengan R 5-HT1A dan 5-HT7
→
efek via regulasi sistem saraf
▪ Enzyme or receptor binding studies are usually the first step of a
series of bioassays for new drug development or mechanism
study of herbal medicines
Traditional Herbal Medicine Research Methods Identifi cation, Analysis, Bioassay, and
Pharmaceutical and Clinical Studies. Edited by Willow J.H. Liu, 2011

24. • Bioesai: prosedur untuk menjelaskan (mendeskripsikan),
mengevaluasi, dan memprediksi potensi bahaya bahan
beracun bagi organisme dan ekosistem, serta aspek
yang berhubungan dengan kesehatan.
• Berbagai fenomena kimiawi, fisik, dan biologi mungkin
akan mengubah struktur kimiawinya, oleh karena itu
nasib (fate), respons, dan toksisitasnya bisa uji di
laboratorium dan/atau di alam (natural)
http://www.jstor.org/stable/25041968
BIOESAI DALAM
PERSPEKTIF
TOKSIKOLOGI
LINGKUNGAN

25. • Determination of drugs potency
• Determine the therapeutic advantage of
one drug over another treatments
• Screening of new agents
• Determination of the pharmacological
activities of a new drug
• Establishment of SAR
Application of
bioassay in
pharmacology

26. • Bioassay is less precise
• In bioassay, active constituent and
its structure do not have to be
known
• Bioassay is more sensitive

27. Biologicals Substances which
cannot be completely
characterised by physicochemical
techniques alone, and which
therefore require the use of some
form of bioassay.
NOTE!!!:

28. ▪ Membandingkan analit (senyawa/bahan uji) dengan
sediaan standar internasional yang sama selain untuk
mengetahui berapa banyak bahan uji yang diperlukan
untuk menghasilkan efek biologis yang sama seperti
yang dihasilkan oleh standar
▪ In a biological assay, animals are used as if they were test
tubes, as a convenient way of observing a reaction usually
called the response.
▪ This response may be the death of the animal, or changes
in its growth rate, blood constituents, or other tissues.
Sometimes each animal can only be used once, sometimes
it can be used in repeated assays
→

29. ▪ Prinsip: Potensi sampel uji dibandingkan dengan
sediaan standar dengan menentukan kerjanya
pada otot jantung
▪ Sediaan Standar dan Satuan (Unit): Sediaan
standar adalah campuran daun digitalis kering
dan serbuk (1 unit = 76 mg)
▪ Persiapan Ekstrak: sejumlah tertentu serbuk
diekstraksi dengan alkohol (dehydrated) dalam
ektraktor selama 6 jam sampai Ekstrak akhir 10
ml (5 ml alkohol + 5 ml air) per 10 g serbuk
digitalis, lalu disimpan di antara 50C dan -50C
→

30. ▪ Prinsip: Potensi oksitosin ditentukan dengan
membandingkan aktivitasnya dengan
Sediaan Standar Oksitosin pada kondisi
metode pengujian yang sesuai.
▪ Persiapan Standar: Sediaan standar adalah
standar internasional ke-4 untuk Oksitosin,
didirikan pada tahun 1978, terdiri dari
peptida oksitosin sintetis beku-kering
dengan albumin manusia dan asam sitrat
(disedakan dalam ampoul yang mengandung
12,5 Unit)
→

31. ◼ Whole animals
◼ Isolated organs of vertebrates
◼ Lower organisms e.g. fungi, bacteria,
insects, molluscs, lower plants, etc.
◼ Cultured cells such as cancer cells and
tissues of human or animal organs
◼ Isolated sub-cellular systems, such as
enzymes, receptors, etc

32. • Bacteria can be used in bioassays. For
example, the use of bacteria to detect
and determine the amount
of antibiotics or compounds that might be
carcinogens in a sample has been
practiced for decades.

33. • Bioassay bakteri lain sangat berguna yaitu
penggunaan bakteri yang dirancang untuk
berfluoresensi (memancarkan cahaya). Jika bakteri
dirusak oleh senyawa beracun yang ada dalam
sampel uji, bakteri gagal berfluoresensi.
• Penurunan fluoresensi populasi bakteri diukur
dengan spektrofotometer. Tingkat penurunan
dibandingkan dengan standar untuk menentukan
konsentrasi senyawa beracun tersebut

34. ◼ Stimulus (Test sample, drug candidate,
potential agrochemical, etc)
◼ Subject (Animal, Tissues, Cells, Sub-cellular
orgenlles, Biochemicals, etc.)
◼ Response (Response of the subject to
various doses of stimulus)

35. ◼ Untuk memprediksi beberapa jenis potensi
terapi, baik secara langsung atau dengan
analogi dari senyawa uji.
◼ Bioassay: istilah yang umum digunakan
untuk uji biologis dan biasanya jenis
percobaan in vitro
◼ Bioassay biasanya dilakukan untuk
mengukur efek senyawa (zat) pada
organisme hidup atau sampel hidup lainnya.

36. ➢ In Silico Screenings
➢ Non- physiological Assays
➢ Biochemical or Mechanisms-Based Assays
➢ In Vitro Assays
◼ Assays on Sub-cellular Organelles
◼ Cell based Bioassays
◼ Ex-Vivo Assays
▪ Tissue based Bioassays
▪ NMR Based Drug Discovery
▪ In Vivo Bioassays
▪ Animal-based Assays/Preclinical Studies
▪ Human trial/Clinical Trials

37. • The results of the assay/bioassay need to
validated by monitoring the effect of an
available known compound (Standard).
• Without judicious choice of standard and its
reproducible results in an assay system, no
screening can be claimed credible.

38. ◼ In Vitro: In experimental situation
outside the organisms. Biological or
chemical work done in the test tube( in
vitro is Latin for “in glass”) rather than
in living systems
◼ Examples include antifungal,
antibacterial, organ-based assays,
cellular assays, etc

39. ◼ Activity Assays
DPPH assay
Xanthine oxidase inhibition assays
Superoxide scavenging assay
Antiglycation assay
◼ Bioassays (cell-based)
DNA Level
Protein Level
RNA Level
Immunology assay
◼ Toxicity Assays
MTT assay
Cancer cell line assays

40. ◼ InVivo:Test performed in a living
system such as antidiabetic
assays, CNS assays,
antihypertensive assays, etc.

41. ◼ AnimalToxicity
Acute toxicity
Chronic toxicity
◼ Animals Study
Animal model with induced disease
Animal model with induced injury
◼ Pre-ClinicalTrials
◼ ClinicalTrials

42. • Active principle of drug is unknown
• Active pple cannot be isolated, e.g. insulin, posterior
pituitary extract etc.
• Chemical method is either
– not available
– if available, too complex,
– insensitive to low doses e.g. Histamine can be
bioassayed in microgram conc.
• Unknown Chemical composition, e.g. long acting thyroid
stimulator.
• Chemical composition of drug variable but has same
pharmacological action e.g. cardiac glycosides isolated
from diff sources, catecholamines etc.

43. • The uses of bioassay are:
▪ To measure the pharmacological
activity of new or chemically
undefined substances to investigate
the function of endogenous mediators
▪ To measure drug toxicity and
unwanted effects.
Bioassays are critical for:
▪ Product development and manufacturing
▪ Ensuring continued quality, safety and efficacy of
biopharmaceutical products
▪ Bioassay plays a key role in the development of new drugs

44. REFERENCE
1. https://www.bioassay.de/biological-assays/
2. https://www.sciencedirect.com/topics/agricultural-and-
biological-sciences/bioassays
3. https://www.omicsonline.org/bioassay-peer-reviewed-open-
access-journals.php
4. RRJOB | Volume 3 | Issue 2 | April - June, 2015