This document provides details on bioassay methods for several compounds including vasopressin, digitalis, d-tubocurarine, histamine, and 5-hydroxytryptamine (5-HT).
It describes two common bioassay methods for vasopressin - the first uses rats to measure changes in blood pressure, the second uses rats and measures anti-diuretic activity. For digitalis, it outlines guinea pig and pigeon bioassays measuring the lethal dose. The d-tubocurarine bioassay uses rabbits to measure head drop or isolated frog muscle to measure contraction reduction. Histamine is assayed using guinea pig ileum or other tissues and measuring contraction. Finally, 5
Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunumVISHALJADHAV100
Overview of Discussion
Objective
Principle
Requirements
Experimental specifications (conditions)
Drugs and solutions used in rabbit intestine experiment
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Result and interpretation
Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunumVISHALJADHAV100
Overview of Discussion
Objective
Principle
Requirements
Experimental specifications (conditions)
Drugs and solutions used in rabbit intestine experiment
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Result and interpretation
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Isolation, Identification and Analysis of PhytoconstituentsDr. Siddhi Upadhyay
Isolation, Identification and Analysis of Phytoconstituents
a) Terpenoids: Menthol, Citral, Artemisin
b) Glycosides: Glycyrhetinic acid & Rutin
c) Alkaloids: Atropine,Quinine,Reserpine,Caffeine
d) Resins: Podophyllotoxin, Curcumin
Expt. 5 Bioassay of oxytocin using rat uterine horn by interpolation methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of oxytocin standard solution
Preparation of De Jalon solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Graphical presentation of DRC
Calculation
Result and interpretation
Liquid oral topic in Industrial Pharmacy contains many topics like solution, elixirs, syrups, emulsion, and suspension. This topic includes general introduction, types, formulation, components, uses, and Quality control tests. These are also beneficial in other subjects like Pharmaceutics.
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Isolation, Identification and Analysis of PhytoconstituentsDr. Siddhi Upadhyay
Isolation, Identification and Analysis of Phytoconstituents
a) Terpenoids: Menthol, Citral, Artemisin
b) Glycosides: Glycyrhetinic acid & Rutin
c) Alkaloids: Atropine,Quinine,Reserpine,Caffeine
d) Resins: Podophyllotoxin, Curcumin
Expt. 5 Bioassay of oxytocin using rat uterine horn by interpolation methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of oxytocin standard solution
Preparation of De Jalon solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Graphical presentation of DRC
Calculation
Result and interpretation
Liquid oral topic in Industrial Pharmacy contains many topics like solution, elixirs, syrups, emulsion, and suspension. This topic includes general introduction, types, formulation, components, uses, and Quality control tests. These are also beneficial in other subjects like Pharmaceutics.
Insulin bioassay is the measurement of insulin potency by biological activity measurement using a standard biological system. An often used technique is to inject insulin samples into animals, usually rats or rabbits, and then track their blood sugar levels to see how well insulin lowers blood sugar. An alternate strategy is to measure glucose absorption or metabolic reactions using cultured cells sensitive to insulin, including adipocytes or muscle cells. By giving important information on insulin potency, purity, and stability, these tests guarantee the quality of insulin products intended for therapeutic use. Bioassays continue to be necessary for insulin preparation quality control and regulatory clearance even with the development of analytical methods.
Screening models for immunomodulatory agents:- Introduction for immunostimulants and immunosuppressant, Models for immunomodulatory agents, Screening for immunostimulants, screening for immunosuppressant
Expt. 6 Study of effect of drugs on gastrointestinal motilityVISHALJADHAV100
Objective
Principle
Requirements
Preparation of Tyrode solution
Procedure
Kymograph recording of contractions
Observation table
Result and Interpretation
Similar to Bioassay vasopressin digitalis ACTH (20)
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
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Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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2. BIOASSAY OF VASOPRESSIN
The vasopressor activity is estimated by comparing the activity of preparation being
examined with that of the Standard Preparation of arginine vasopressin (AVP) under the
conditions of a suitable method of assay.
STD
Synthetic AVP peptide acetate with human albumin and citric acid (supplied in ampoules
containing 8.20 Units),
METHOD AIP 1966
Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a
suitable alpha-blocker. For example 10 ml/ kg of body weight of a solution prepared by
dissolving 5 mg of Phenoxybenzamine HCl in 0.1 ml of ethanol (95%) + 0.05 ml of 1M
HCl+ diluting to 5 ml 0.9% NaCl.
3. After 18 hrs, anaesthetise the rat with ananaesthetic that will maintain a prolonged &
uniform Blood Pressure. After 45 to 60 mins, tie the rat on its back to the operating table
by its hind legs.
Trachea is cannulated to maintain respiration & Carotid artery is cannulated to record
the BP.
Then cannulate the femoral vein inj. of STD & Test vasopressin
Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision &
cannula.
4. At this stage inject through the venous cannula 200 Units of heparin to prevent clotting
Responses to 2 doses of STD & 2 doses of Test preparation of vasopressin are taken with
help of BP apparatus. Usually 3-5 mU of vasopressin may give appropriate rise in BP.
Based on the responses obtained (4 pointed graphical method bioassay)conc. of
vasopressin can be obtained.
5. METHOD B
Based on the Anti diuretic activity of vasopressin
Sixteen Male albino rats 2 groups each group contain 8 rats. STD gp 0.006 unit &
Test gp Equivalent dose
After injection animals are allowed to metabolic cage collect the urine by using
graduated measuring cylinder.
After first collected urine of each animal volume is recorded at intervals of 15min for
3-4hrs
6. After 4 hrs urine flow stops & observations are discontinued. Time (in min) for
excretion of the half the volume of urine is calculated for each group.
Crossover test is carried out after 24hrs
Mean values obtained from the results of 2 gps.
Mean values should be between 96-135 min.
If, both mean time values are same both should contain the same units
If the times are different then relative potency can be calculated by comparing the STD.
8. BIOASSAY OF DIGITALIS
PRINCIPLE Potency of the test sample is compared with that of the standard
preparation by determining the action on the cardiac muscle.
STD The standard preparation is a mixture of dried & powdered digitalis leaves
(1 unit = 76 mg.)
Preparation of Extracts
Exact amount of the powder is extracted with dehydrated alcohol in a continuous
extraction apparatus for 6hrs. The final extract should contain 10 ml (5 ml. alcohol +5 ml.
water) per 10 g. of digitalis powder. It should be stored in between 5oC & – 5oC.
9. Guinea-pig Method (Endpoint method)
STD & Test sample extracts are diluted with normal saline in such a way that 1g of digitalis
powder is diluted to 80 ml.
A guinea pig is anaesthetized with a suitable anaesthetic. It is dissected on the operation
table. The jugular vein is traced out by removing adhering tissues & cannulated by means
of venous cannula.
A pin is inserted in the heart such that it gets inserted in the apex of the heart. In this
way, we can observe the heart beats by up and down movements of the pin.
10. The injection is continued through venous cannula untill the heart is arrested in systole.
The amount of extract required to produce this effect is taken as the lethal dose of the
extract.
Another set of 19 animals of the same species are used for this experiment & the
average lethal dose is determined. Lethal dose should be occasionally checked.
The lethal dose of the test sample is determined in a similar way using minimum 6
guinea-pigs of the same strain. The potency of the test sample is calculated in relation to
that of the std. preparation by dividing the average lethal dose of the sample to the test
& expressed as units per gram.
11. 2. Pigeon Method
Minimum 6 pigeons are used for testing each sample. The weight of the heaviest pigeon
should not exceed twice the weight of the lightest pigeon. Food is withdraw 16- 28 hrs
before the experiment.
Pigeons are divided on the basis of their sex, weight and breed into 2 gps. They are
anaesthetized with anesthetic ether. One side of the wing is dissected & the alar vein is
cannulated by means of a venous cannula. Dilutions are made with normal saline.
Test sample & STD sample is infused through cannula. In pigeons, stoppage of heart is
associated with a characteristic vomiting response called ‘emesis’.
The milk from the crop sac of pigeons is being ejected out. This may be taken as the end
point response of digitalis.
12. The lethal dose per kg of body weight is determined for each pigeon.
Potency of the test sample = Mean lethal dose of STD / Mean lethal dose of the test.
14. BIOASSAY OF d-TUBOCURARINE
1.Rabbit Head-drop Method :
Principle:
d-Tubocararine HCl is injected into the marginal vein of a rabbit’s ear till the
rabbit’s neck muscles are relaxed such that the animal cannot hold its head
up. The total amount of test sample required to produce the endpoint is
compared with the total amount of the STD sample required to produce
similar endpoint.
Selection of Rabbits:
Rabbits weighing 2 kg are used. Animals should be free from disease,
obtained from a healthy colony and should be accustomed with the
experimental procedure.
15. BIOASSAY OF d-TUBOCURARINE
Experimental Procedure:
Rabbit is placed in a holder with its head protruding outside. The head
should be freely movable.
Minimum 8 rabbits are used. They are divided into 2 groups each
containing 4 rabbits.
First group will receive STD sample and the second group will receive the
sample under test d-Tubocurarine solution is injected at a constant speed
by infusion apparatus through the marginal vein.
Injection should be given at a rate of 0.4ml/min and should take about 10
min. Dose 0.012% w/v in saline.
16. BIOASSAY OF d-TUBOCURARINE
Infusion is continued till the rabbit will not be in a position to hold its
head erect or there will be no response by focusing light on the eyes.
Rabbits recover immediately from the effect of curarization.
During the experiment there is a possibility of respiratory embarrassment
which is treated by injecting Neostigmine methyl sulphate (0.05 mg.) and
atropine sulphate immediately through the marginal ear vein.
Cross-over test is carried out to minimize biological error due to animal
variation.
Those rabbits which received the STD sample on the first day will be
given test sample on the second day of experiment and vice versa.
Mean dose which produces head drop of the test sample is compared
with the mean dose of standard preparation.
17. BIOASSAY OF d-TUBOCURARINE
2.Frogs Rectus Abdominus muscle Preparation:
A frog is pithed and laid on its back on a cork covered board to which it is
pinned. The skin covering the abdomen is cut away and the rectus
abdominus muscle of one side is dissected.
The muscle is then pinned to the cork by four pins to keep its normal
length while a thread is sewn through each end.
It is then mounted in the organ bath containing frog's Ringer solution
which contains: NaCl, 6.5gm.; KCl, 0.29 gm.; CaCl2, 0.24 gm.; NaHCO3,
0.4 gm.; glucose, 1.5 gm. and distilled water 2000 ml.
Oxygenation is carried out to keep the tissue alive. The muscle is
stabilized for 30-45 min. in order to get critical quantitative response.
18. BIOASSAY OF d-TUBOCURARINE
Frogs Rectus Abdominis muscle Preparation:
The responses are recorded using isotonic frontal writing lever with 1g
tension.
Two similar contractions with the same concentration of Ach are
obtained.
Three doses of the STD sample and one intermediate dose of the test
sample are selected & the reduction in height of contraction induced by
Ach is noted down.
Ach contraction is recorded on slow moving drum for 90 sec. d-
Tubocurarine is allowed to act for 30 sec.
The percentage reduction at each dose levels is calculated and log dose
response curve of the STD drug is plotted. A linear response will be
obtained. The potency of test sample is calculated from the standard
curve.
20. BIOASSAY OF HISTAMINE
Histamine is present in almost all the animal tissues mostly within mast cell &
basophil granules.
Tissues rich in histamine are skin, gastric and intestinal mucosa, lungs, liver &
placenta.
Non-mast cell histamine is present in brain, epidermis, gastric mucosa &
growing regions.
Histamine also exerts a various other immune regulatory functions by
modulating the functions of monocytes, T cells, macrophages, neutrophils,
eosinophils, B cells & dendritic cells.
Histamine is also present in blood, most body secretions, venoms & pathological
fluids. It is now known to play important physiological roles. Histamine receptors
(H1, H2, H3 & H4) present on target cells.
Bioassay of histamine on isolated guinea pig ileum can be determined by graded
bioassay procedure i.e. i) Matching bioassay, ii) Interpolation bioassay, iii)
Bracketing assay, iv) Multiple point assays.
21. Bioassay of histamine in biological samples can be studied by using different
bioassay methods. Depending upon pharmacological action, histamine can be
assayed by:
Contractile effect of isolated ileum of guinea pig,
Contractile effect of uterus of guinea pig, and
Fall in blood pressure of anaesthetized and atropinized cat
Bioassay of histamine using guinea pig ileum:
Principle
Guinea pig ileum is very useful preparation for the bioassay methods. It is more
sensitive to histamine. Contractile response of histamine to ileum is due to
presence of H1 receptor.
22. Preparation of Standard and other solution:
Prepare the stock of Tyrode solution. Also prepare the standard stock
solution of histamine (1 mg/ml) & then different concentrations of
histamine by serial dilution method.
Procedure:
Sacrifice the 24 hr fasted guinea pig by stunning on the head.
Fix the animal on the dissecting board by tying its legs.
Open the abdominal cavity by a small horizontal cut followed by vertical
midline incision and expose the abdominal organs.
23. Trace the ileocaecal junction by lifting the caecum, then go upwards up to 8 cm
from the junction and cut the 2-3 cm long segment of ileum muscle (excluding
the terminal 5-8 cm, contains excess of excitatory α-adrenergic receptor, which
may interfere in the study).
Immediately place it in a petri dish containing aerated warm Tyrode solution.
Slowly remove the mesentery attached to the muscle & then gently clean the
lumen of ileum by passing luke warm Tyrode solution through it with a pipette
or syringe.
Mount the preparation in the inner organ bath containing Tyrode solution (20
ml) maintained at 37ºC. Tie the bottom end of the muscle to the hook of
aeration tube and the upper end to the isotonic frontal lever by a thread
without closing the lumen.
24. Adjust the magnification of response to 7-10 fold. Aerate the tissue with O2
or carbogen slowly (40-60 bubbles per minutes).
Stabilize the tissue for 30 min by applying a tension of 0.5 g weight attached
to the lever, during which wash the tissue with fresh Tyrode solution once in
every 10 min.
Use 5 min time cycle with contact time of 30 sec for recording the contraction
dependent responses of tissue due to histamine on the smoked drums.
Record the responses of STD & test compounds.
Record the responses of test compound i.e. unknown concentration of
histamine with gradually increasing volume, till obtaining a response (T) which
would lie on the linear portion (30-70%) of the CRC. Fix the response obtained
due to volume of T.
25. Record the graded responses of STD solution & test solution of histamine.
The potency of the test sample is calculated in relation to that of the std.
preparation by dividing the average lethal dose of the sample to the test and
expressed as units per gram.
Potency = Lethal dose of STD /Lethal dose of test= unit/g.
27. BIOASSAY OF 5-HT
5-Hydroxytryptamine (5HT) is the biologically active local hormone, low
molecular weight & also an important neurotransmitter in the brain &
periphery.
It was first detected in serum (serotonin) & enterochromaffin cells of gut
mucosa (enteramine) & later both were identified to be 5HT. Today the terms
5HT & serotonin are used interchangeably.
About 90% of body's content of 5-HT is localized in the intestines; most of the
rest is in platelets & brain.
It is also found in wasp and scorpion sting, and is widely distributed in
invertebrates and plants (banana, pear, pineapple, tomato etc.).
The concentration of 5-HT in biological samples can be assayed by:
1. Isolated fundus strip of rat stomach,
2. Isolated terminal colon of rat,
3. Isolated rat uterus & 4. Perfused rabbit ear.
28. Isolated fundus strip of rat stomach
Principle:
Rat fundus preparation is a very sensitive tissue for the bioassay methods &
is useful to study the action of several substances like 5-HT, ACh, PGE2 &
bradykinin.
Fundus strip preparation is slow contracting muscle.
Fundus part of stomach can be easily identified by its grey colour & situated
above the pink coloured thick pyloric part.
A zig-zag preparation of the fundal strip is prepared so as to expose
maximum portion of the tissue to drug.
Preparation of Standard and other solution:
Prepare the stock of Krebs solution. Prepare the STD stock solution of
serotonin (1 mg/ml) & then different concentrations of serotonin by serial
dilution method.
29. British Journal of Pharmacology, Volume: 120, Issue: S1, Pages: 142-147, First
published: 08 September 2011, DOI: (10.1111/j.1476-5381.1997.tb06791.x)
30. Procedure:
Sacrifice the 24 hr fasted rat by stunning on the head. Fix the animal on the
dissecting board by tying its legs. Open the abdominal cavity by a small
horizontal cut followed by vertical midline incision and expose the abdominal
organs.
Identify the stomach and separate it from the abdomen by gently cutting its
cardiac and pyloric end. Place it in a petri dish containing aerated warm Krebs
solution.
Incise the fundus of the stomach (upper grey part) from pyloric part (pink
and thick part). Cut the fundus from the lesser curvature and open it
longitudinally. Then cut the fundus at the midline into two equal parts. Give
alternate transverse cuts (zig-zag cut) on opposite sides of the muscle to
make a fundal strip preparation.
31.
32. Mount the preparation in the inner organ bath containing Krebs solution (20
ml) maintained at 37ºC. Tie the bottom end of the muscle to the hook of
aeration tube & the upper end to the isotonic frontal lever by thread. Adjust
the magnification of response to 10-15 fold. Aerate the tissue with O2 or
carbogen slowly (40-60 bubbles per minutes).
Apply 1 g load & stabilize the tissue for 30 min during which wash the tissue
with fresh Krebs solution once in every 10 min.
Use a 5 min time cycle with contact time of 90s (since the fundus strip muscle
contracts slowly and relaxes slowly) for recording the contraction due to
serotonin.
Record the graded responses of STD solution & test solution of serotonin.
The potency of the test sample is calculated in relation to that of the std.
preparation by dividing the average lethal dose of the sample to the test and
expressed as units per gram.
34. ACTH (Adrenocorticotropic hormone, Corticotropin) is polypeptide tropic
hormone (39 amino acids) secreted by the Anterior Pituitary Gland.
ACTH stimulates the production of Cortisol a steroid hormone important
for regulating Glucose, Protein & Lipid Metabolism, suppressing the immune
system response & helping to maintain Blood Pressure.
Official Preparations:
Corticotropin injection: Is a sterile solution, in a suitable diluent, of the
polypeptide from the pituitary glands of mammals. Potency range should be
80.0 – 120.0 % of USP cortiotropin units.
Corticotropin for injection, antimicrobial agent. Repository corticotropin
injection is corticotropin in a sterile solution of partially hydrolyzed gelatin & is
intended for S.C. & I.M route use. This solution has been adopted as the
reference STD for the bioassay.
35. Packing: Preserve in single-dose or multiple-dose containers of Type-1 glass.
Storage: Store in cold place.
Labeling: Injection recommends intravenous administration.
Purpose and rationale:
This is a historical assay method. Administration of pituitary ACTH decrease
the ascorbic acid present in the adrenals. The depletion of adrenal ascorbic
acid is a function of the dose of ACTH administered. This relationship
has been used for a quantitative assay of ACTH.
36. Solutions:
Solution A: Five units of test or standard dissolved in 0.25 ml of 0.5% phenol
solution and diluted with 8.1 ml of 15% gelatin solution (Now 0.5ml contain
300 mU ACTH).
Solution B: Three ml of solution A diluted with 6 ml
gelatin solution. Now concentration reduced to 100 mU ACTH/ 0.5 ml.
Solution C: Again 3 ml of solution B diluted with 6 ml of gelatin solution, the
resulting solution contains 33 mU ACTH/ 0.5 ml.
37. Procedure
Male Wistar rat (100-200 g) are hypophysectomized (pituitary gland
removed by surgery) one day prior to the test.
For one test with 3 dose of Test preparation and STD solution used for the
study.
Number of hypophysectomized rats required: at least 36 (preferably
60).
The hypophysectomized rats are randomly distributed in to six groups. Each
rat receives subcutaneous 0.5 ml of the various concentrations of test or
standard.
38. Procedure
Three hours after injection, the animals are anesthetized and both adrenals
removed, freed from extraneous tissue and weighed.
The rats are sacrificed and the scull opened to verify completeness of
hypophysectomy.
The adrenals are homogenized in glass tubes contains 200 mg pure sand &
8.0 ml of 4% trichloroacetic acid and the ascorbic acid determined (Roe and
Kuether , 1943).
The potency ratio including confidence limits is calculated with the 3 + 3
point assay.
39. Ascorbic acid determination:
Reagents
0.02% ascorbic acid solution
85 % sulfuric acid (9N H2SO4)
0.02 g/ml of dinitrophenol hydrazine in 9N H2SO4
0.06 g/ml of thiourea are dissolved in distilled water
Charcoal
Preparation of 0.02% ascorbic acid solution
100 mg L-ascorbic acid are dissolved in 100 ml of 4% trichloroacetic acid
(1mg/ml solution) (Solution A= 1 % solution)
2 ml of Solution A diluted in 10 ml of 4% trichloroacetic acid to achieve a
0.2% ascorbic acid solution (solution B)
1 ml of solution B diluted in 10 ml of 4% trichloroacetic acid to achieve a
0.02% ascorbic acid solution (solution C)
40. Preparation of other solutions
Sulfuric acid (85%) is obtained by adding 900 ml concentrated sulfuric
acid to 100 ml distilled water.
Two gm dinitrophenol hydrazine are dissolved in 100 ml 9N H2SO4 (75 ml
distilled water & 25 ml concentrated sulfuric acid).
Six gm thiourea are dissolved in 100 ml distilled water.
Calibration
Trichloroacetic acid (4%) is added to 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 ml
of the 0.02% ascorbic acid solution (solution C) and 1.0, 1,5 and 2.0 ml of
the 0.2% ascorbic acid solution to reach a final volume of 8.0 ml (Solution
B).
100 mg charcoal is added to each sample and thoroughly mixed by
shaking for 1 min.
After 5 min the solutions are filtered
41. Ascorbic acid determination:
An aliquot of 0.1 ml of the 6% thiourea solution is added to 2.0 ml of the
filtrate followed by 0.5 ml dinitrophenyl hydrazine solution.
The mixture is shaken and heated for 45 min at 57°C in a water bath.
The solutions are placed in an ice-cold water bath and with further cooling
2.5 ml of the 85% sulfuric acid are added.
The calibration curve is established at a wave length of 540 nm using the
solutions without ascorbic acid as blank.