Biological Screening of Herbal Drugs

Chapter 12

Biological Screening of Herbal Drugs

INTRODUCTION

It is well-known that drugs when administered to the body never produce ‘new’ effects but get by modifying existing physiological systems. Observation of visible effects of plant extracts on intact animals can give information of their pharmacological activity and possible use as therapeutic agents. The species commonly used for this purpose are mouse and rat. Availability of suitable worksheets is essential to enable systematic observation of valuable symptoms. Elaborate procedures, using mice and cats, have been described by Irwin (1964). Reinhard (1982) has published a simplified scheme for mice. His article also contains descriptions of a number of tests for specific activities, which can be performed in mice. Malone (1977) has devised screening protocols for rats, which are suitable for working with natural products, partly purified fractions or pure compounds. Rats are injected intraperitoneally with the samples and observed at defined time intervals for one day. Observations are then performed once a day for one week, after which the animals are killed and examined. The test protocol contains observation of 58 parameters and has been named by Hippocrates, the ‘father of medicine’. Sandberg (1967) made minor modifications to the original protocol (55 parameters). Malone (1977) has published a modified worksheet with 63 parameters and has also discussed computerization of the procedure, allowing comparison of the pharmacological profile of an unknown sample with similar profiles of known drugs.

Modern chemical methods have led to a dramatic increase in the number of natural or synthetic molecules available for pharmacological research. At the same time, recent developments in cellular and molecular pharmacology provide an increasing number of selective tests able to identify the activity and the mechanisms of action of biologically active molecules. Paradoxically, however, the availability of numerous sophisticated techniques does not necessarily make pharmacological research much easier. Molecular graphics have not yet proven very serviceable in the investigation of novel molecules. On the other hand, the probability of assessing the biological activity of new drugs by stochastic screening with modern reliable methods remains limited, unless viable working hypotheses can first be made as to their overall effect.

This difficulty could be partly overcome by intermediate screening methods, on the basis of global or functional tests. Such methods have been developed in various domains of biology, such as cardiovascular research or in the identification of antimitotic or immunosuppressive drugs. However, only very few methods are available as yet for application to the cases of neurotropic substances. Most global tests for screening neurotropic drugs are outdated; adequate behavioral tests are only capable of detecting a few transmitters like activities, and such a situation represents three limiting factors in an area characterized by particularly rapid developments. The number of identified transmitter substances has increased from less than 10 to over 50 in a few years. In addition, several are neuropeptides, an important, but still relatively unexplored class of biologically active molecules. Peptides of marine origin represent an important source of natural substances still awaiting systematic screening.

NEED FOR PHYTOPHARMACOLOGICAL EVALUATION

To demonstrate a pharmacological effect, nothing can replace observation of animal models; but as they are expensive and often difficult to interpret, simpler tests are used. These tests require less effort and also make possible a better understanding of the mechanisms of action of substances being tested. Nonanimal models are becoming smaller and smaller while still remaining representative of a living organism.

By means of finely adjusted multidisciplinary efforts and of a choice of tests that accurately represent future therapeutic applications, research centres, such as the National Cancer Institute in the United States, have been able to select active substances with some success. Thus, the combination of several selective, sensitive, and specific tests (such as the model of P-338 leukaemia in vivo versus astrocytoma in vitro) has made it possible to detect directly up to 90% of clinically active antitumor compounds. These methods have also helped to eliminate substances that give false positive results, such as cardenolides, saponosides, flavonoids, and terpenic lactones. At the cost of a huge effort applied to more than 100,000 plant extracts, only about 10 particularly promising antileukaemic substances were selected, among these were: indicine N-oxide, maytansine, Hom harringtonine, taxol and its derivatives, and 4-beta-hydroxywithanolide E (whose 17-alpha side chain removes all its cardioactivity).

Of course, most pharmaco-chemical research are performed with more limited means, but the scientific literature flows with interesting results. These may be categorized into two groups according to the possible methods of approach. One approach is to demonstrate new pharmacological activities, or even future clinical applications, from raw materials or natural substances already known. For example; hypericin inhibits monoamine oxidases A and B from rat brain, which may explain its antidepressive properties; 5 to 6 g of pectin ingested daily significantly decrease cholesterol levels by inhibiting the reabsorption of bile; trigoneiline, from fenugreek, displays a hypoglycaemic effect in animals with experimental alloxan-induced diabetes; sulphur compounds from garlic and onions, and phenylpropane derivatives from the essential oil of nutmeg, have displayed good properties against platelet aggregation; gossypol, obtained from raw cottonseed oil, is well-known as a male contraceptive agent, acting after 4 to 5 weeks of treatment, without affecting the testosterone level—its molecular mechanism of action towards lipid membranes has been elucidated.

The second type of approach is the discovery of new natural substances displaying pharmacological or even new therapeutic effects. This is the royal road par excellence that most often leads to patents being taken out. Publications in this field are numerous, which can be further explained by the following examples.

Withanolide F has an anti-inflammatory action, demonstrated by the classical plantar oedema test in rats, which is five times that of phenylbutazone and comparable to that of hydrocortisone (a substance with no effect on the central nervous system).
Certain tetracyclic sesquiterpenes isolated from sponges of the genus phyllospongia have comparable antiinflammatory effects in vivo.
New triterpenic saponosides, such as dianosides A and B isolated from Dianthus superbus L. var longicalycinus (Carycphyilaceae) have analgesic properties at subcutaneous doses of 10–30 mg/kg as measured by the acetic acid test in mice.

These few results, taken as examples, demonstrate—if such a demonstration is necessary—that this approach to research leads along an extremely interesting trail. It is a technique permitting innovation of the type currently much sought. The accumulation of scientific knowledge also leads to the development of a rigorous pharmacological vigilance, particularly with regard to natural substances that are considered a priority to be of secondary therapeutic value. Examples that come to mind are: glycyrrhizin, whose not inconsiderable mineralocorticoid activity induces iatrogenic hypertension with hypokalaemic and metabolic alkalosis; the pyrrolizidine alkaloids present in the Boraginaceae and Asteraceae (particularly the genera Senecic and Eupatorium), which induce fatty degeneration of liver cells and eventually necrosis and fibrosis, caused by certain bifunctional alkylating pyurrole metabolites that bind to DNA; diterpene esters of the phorbol and ingenol types, present in the Euphorbiaceous and Thymeliaceae, which are in fact cocarcinogenic substances.

The terpenes, as with the flavonoids, certain molecules in the environment, though considered inactive in their normal state, may nevertheless show some activity when combined with an appropriate vector. Such is the case of epoxylathyrol, a diterpene present in the latex and seeds of Euphorbia lathysis L. This plant contains natural esters that have no activity on cultures of hepatic tumors cells. However, Schroeder et al. (1979) have synthesized a series of aliphatic esters with chain lengths ranging from 2 to 20 carbon atoms and have performed tests in vitro. The cytotoxicity curves demonstrate that the dibutyrate ester represents the optimal chain length, revealing an activity that the nonesterified epoxylathyrol does not possess. All this shows how greatly the interaction between the human body and molecules in our environment may be modifiable, and how research on substances thought to be devoid of interest may lead to surprises.

NEW STRATEGIES FOR EVALUATING NATURAL PRODUCTS

The fundamental problem for chemists working on natural products used to be that of choosing which pharmacological principles and methods to use to understand the possible biological use of any given substance.

The RICB (Reseau d’interaction chimie—Bioiogique or Chemical Biological Interaction Network) has come up with lots of objectives for helping the chemists as described below

The RICB helps chemists by providing assistance from biologists, who use their knowledge and sophisticated methods of observation for the pharmacological activity of any given substance. Rather than the current classical procedure of observing an overall pharmacological e.g., measurement of femoral artery blood flow in dogs), it is now possible to observe the effect of the substance on any one of the underlying components of the overall peripheral pharmacological effect (e.g. binding to α- or β-adrenergic receptors, or to angiotensin or vasopressin receptors, or the effect of posterior pituitary vasopressin). The RICB aids biologists by providing new tools for examining receptors, neuromodulators, ion channels, and membrane-coupling mechanisms. It can thus be said that, thanks to derivatives of yohimbine (indole alkaloids from the Rubiaceae and Apocynaceae), the alpha-1 and alpha-2 subtypes of adrenergic receptors were distinguished. Likewise, forskolin, a labdane diterpene, enabled progress to be made in understanding the adenylate cyclase system. Morphine, ouabain and tetrodotoxin are other examples of biological agents derived from the chemistry of natural materials. Biologists can also make use of the chemists’ extra skills in resolving certain problems at the frontiers of biology, such as extraction, labelling, and molecular modelling.

In general, the RICB has also provided a discussion forum for a collaboration between two disciplines that are relatively unfamiliar with each other and for developing new strategies in the evaluation of natural or synthetic substances. By using different animal models, the phyto-pharmacological potentials of different plant species, including Nelumbo nucifera and Leucas lavendulaefolia have been reported.

ERRORS IN SCREENING PROCEDURES

Any screening procedure has a characteristic error rate. This is inevitable because in high throughput screening it is necessary to compromise with some accuracy or precision to achieve the requisite speed. Thus, when a large number of compounds are carried through a particular screen, some of the compounds are classified incorrectly. A screen may be used in an absolute sense, so that compounds that pass a certain criterion are termed positives, whereas those that fail to meet the criterion are termed negatives. Compounds that pass, but should have failed, are false positives. In general, false positives are tolerable, if they are not too numerous, because they will be rectified later. Compounds that fail, but should have passed, are false negatives. False negatives are lost forever if the failure eliminates them from further testing.

All screening procedures are based on assumptions of analogy. They have different degrees of relevance or predictability. Studies in phase II clinical trials predict the results with high probability in large clinical trials. But even here there is the possibility of false-positive or falsenegative results. The relevance of a test is much less in early pharmacological tests, such as used in high-throughput screening. Generally, the relevance is inversely proportional to the simplicity of the test.

In any case, one is confronted with the problem of falsepositive results (type I errors) and false-negative results (type II errors).

In each step, two sources of error for false-positive results have to be taken into account:

a = error of the first type due to the model
α = error of the first type due to statistics

In the error of the first type, a compound is considered to be active, but is actually ineffective. This type of error is clarified during further development, after negative clinical trials at the latest.

However, there are two sources of error for false-negative results:

b = error of the second type due to the model
β = error of the second type due to statistics

In the error of the second type, a compound is considered to be ineffective, but is actually effective.

This type of error will never be clarified; an effective drug has just been missed. Perhaps another investigator will test this compound under different aspects.

The statistical errors derive from the fact that a pharmacological test is performed only several times or in a limited number of animals. One can specify the probability that a decision made is incorrect, that is, a drug candidate is erroneously identified as effective when it is actually ineffective. Usually, this risk is set to 5% (P < 0.05) and is called the statistical error of the first type or type I error. The error of the second type or type II error is connected to the type I error by statistical rules.

Usually, screening is performed sequentially. Tests in high-throughput screening are followed by tests in isolated organs, then in small animals, and special tests in higher animals, until the compound is recommended for further development and for studies in human beings. From each step, not only errors of type I, but also from type II, arise. As a consequence, many effective compounds are lost

There are two ways to circumvent this obstacle:

to increase the number of compounds entering the screening procedure dramatically, hope for a reasonable number of true positives, and accept a high rate of false-negative results (White 2000) as followed in the ultra-high-throughput screening.
To performs tests with high relevance, meaning tests with high predictive value in whole animals at an early stage (Vogel and Vanderbeeke 1990).

The literature on high-throughput screening includes some publications dealing with false-negative results (Jones and King 2003; Colland and Daviet 2004; Heller-Uszynska and Kilian 2004).

Zhang et al. (1999, 2000) studied the role of falsenegative results in high-throughput screening procedures. They presented a statistical model system that predicts the reliability of hits from a primary test as affected by the error in the assay and the choice of the hit threshold. Thehit confirmation rate, as well as false-positive (representing substances that initially fall above the hit limit but whose true activity is below the hit limit) and false-negative (representing substances that initially fall below the hit limit but whose true activity is in fact greater than the hit limit) rates have been analysed by computational simulation. The Z-factor and the Zi-factor were introduced to characterize the reliability of high-throughput assays.

The problem of type II errors, that is, false-negative results, also exists in many other physiological and pharmacological studies (Martorana et al. 1982; Bar- ros et al. 1991; Sand Kuhler et al. 1991; Waldeck 1996; Williams et al. 1997). For example, Pollard and Howard (1986) reinvestigated the staircase test, a well-accepted primary screening method for anxiolytics, and found several false-negative results for clinically active anxiolytics.

SCREENING METHODS FOR ANALGESIC AGENTS

Centrally Acting Analgesics

Hot plate method

The paws of mice and rats are very sensitive to heat even at temperatures which do not damage the skin. They respond by jumping, withdrawal of paws, and licking of paws. The time until these responses occur can be prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetyl salicylic acid or phenyl acetic acid type do not generally affect these responses.

The hot plate consists of an electrically heated surface. The temperature is controlled for 55°–56°C. Adult albino rats are used for the test. The animals are placed on the hot plate, and the time until either licking or jumping occurs is recorded by a stopwatch. The delay in response is recorded after administration of the standard or the test compound.

Haffner’s tail clip method

In this method, the raised tail phenomena in mice are observed. Six mice per group are used. A clip is applied to the base of the tail of mice, and the reaction time is noted. The test compounds are administered orally to fasted animals. The animal quickly responds to the stimuli by biting the clip or the tail near the location of the clip. The time between stimulation onset and response is measured by a stopwatch.

Tail immersion test

This method is based on the observation that morphinelike drugs are selectively capable of prolonging the reaction time of the typical tail-withdrawal reflex in rats induced by immersing the end of the tail in warm water of 55°C.
Adult albino rats are used for the test. They are placed in individual cages leaving the tail hanging out freely. The animals are allowed to adapt to the cages for 30 min. before testing. The lower 5 cm portion of the tail is marked. This part of the tail is immersed in a cup of freshly filled water of exactly 55°C. Within a few seconds, the rat reacts by withdrawing the tail. A stopwatch records the reaction time. The reaction time is determined before and periodically after either oral or subcutaneous administration of the test substance. A withdrawal time of more than 6 s is regarded as a positive response.

Radiant heat method

This test is useful for quantitative measurements of pain threshold against thermal radiation in man and for evaluation of analgesic activity. It is very useful for discriminating between centrally acting morphine-like analgesics and nonopiate analgesics.
The animal is put into a small cage with an opening for the tail at the rear wall. The investigator holds the tail gently. By the opening of a shutter, a light beam exerting radiant heat is directed at the end of the tail. For about 6 s, the reaction of the animal is observed. The mouse tries to pull the tail away and turns the head. The shutter is closed with a switch as soon as the investigator notices this reaction. Mice with a reaction time of more than 6 s are not used in the test. The escape reaction is the end point of this test. Before administration of the test compound or the standard, the normal reaction time is determined. The test compounds and the standard are administered either orally or subcutaneously. The analgesiometer can also be used to measure analgesic activities.

Formalin test in rats

Rats weighing 180–300 g are administered 0.05 ml of 10% formalin into the lower surface of the front paw. The test drug is administered simultaneously either subcutaneously or orally. Each individual rat is placed in a clear plastic cage for observation. Readings are taken and scored according to a pain scale. Pain responses are indicated by elevation of the paw or excessive licking and biting of the paw. Analgesic response or protection is indicated if both paws are resting on the floor with no elevation of the injected paw.

Tooth pulp stimulation

This test is based on the fact that stimulation of the tooth pulp induces characteristic reactions, such as licking, biting, chewing, and head flick which can be observed easily.

Adult healthy rabbits are used for the test. Rabbits are anaesthetized and their pulp chambers exposed with a high-speed dental drill. On the day of the experiment, clamping electrodes are placed into the drilled holes. After an accommodation period of 30 min, stimulation is started to determine the threshold value. The stimulus is applied with a frequency of 50 Hz and duration of 1 s. The electrical current is started at 0.2 mA and increased until the phenomenon of licking occurs. The test substance is eitherinjected intravenously or given orally. The animals serve as their own controls.

Grid shock test

This test measures the analgesic properties by the ‘flinchjump’ procedure in rats. The floor of the box used is wired with stainless steel wire, spaced about 1 mm apart. The stimulus is given in the form of an electric current, 30 cycles per second with duration of 2 ms per pulse. With increasing shock intensities, the mice flinch, exhibit a startling reaction, increase locomotion, or attempt to jump. The behaviour is accurately reflected on the oscilloscope by marked fluctuations of the pulse and defined as the pain threshold response. The current as measured in milliamperes is recorded for each animal before and after administration of the drug.

Electrical stimulation of the tail

This method is based on the fact that since the tail of mice is known to be sensitive to any stimulus, the stimulus can be varied either by the duration of the electric shock or by an increase in the electric current to check the efficacy of the analgesic agent.

Male mice weighing 20 g are placed in special cages. A pair of clips is attached to the tail, and the positive electrode is placed at the end of the tail. Electric current at an intensity of 40–50 V is applied. The frequency of the stimulation is 1 shock/second, and the pulse duration is 2.5 ms. The normal response time range of the stimuli is 3–4 sec. Following administration of the drug, the response time is registered at 15 min intervals until the reaction time returns to control levels.

Peripherally Acting Analgesics

Pain in inflamed tissue (Randall-Selitto test)

This method is based on the principle that inflammation increases the peripheral analgesic sensitivity to pain. Inflammation decreases the pain reaction threshold, but the threshold is readily elevated by nonnarcotic analgesics of the salicylate-amidopyrine type as well as by the narcotic analgesics.

Groups of healthy albino rats (130–175 g) are used. The animals are starved 18 to 24 h before administration. To induce inflammation, 0.1 ml of a 20% suspension of Brewer’s yeast in distilled water is injected subcutaneously into the plantar surface of the left hind paw of the rat. After three hours, pressure is applied through a tip to the plantar surface of the rat’s foot at a constant rate to the point when the animal struggles, squeals, or attempts to bite. Each animal is tested for its control pain threshold. Any animal with a control pain threshold greater than 80 g is eliminated and replaced. The mean applied force is determined for each time interval.

Writhing test

Pain is induced by injection of irritants into the peritoneal cavity of mice. The animals react with a characteristic stretching behaviour which is called writhing. The test is suitable to detect analgesic activity. An irritating agent such as phenyl quinone or acetic acid is injected intraperitoneally to mice, and the stretching reaction is evaluated.

Mice of either sex of weight 20–25 g are used. Phenyl quinone in a concentration of 0.02% is suspended in a 1% suspension of carboxy methyl cellulose. About 0.25 ml of this suspension is injected intraperitoneally. The mice are placed individually into glass beakers and are observed for a period often one minute. The number of writhes is recorded for each animal. A writhe is indicated by a stretching of the abdomen with simultaneous stretching of at least one hind limb.

SCREENING METHODS FOR ANTIDIABETIC AGENTS

Diabetes mellitus is a metabolic disorder characterized by increased blood glucose level associated with discharge of glucose in urine. There are two major types of diabetes mellitus, that is, insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM). Insulin-dependent diabetes mellitus, also called type 1 diabetes, occurs due to complete loss of pancreatic β-islet cells and hence there is insulin deficiency. Noninsulin dependent diabetes mellitus, also called as type 2 diabetes, is due to insulin resistance. Insulin resistance is developed due to defects at the receptor level or insulin signaling at the postreceptor level. This defect may be in the effector cells such as the skeletal muscle, the adipose tissue etc., or in the β-islet cells. A large number of drugs including herbs and minerals with suspected antidiabetic activity have been successfully tested in the laboratory. The various animal models to screen antidiabetic activity are listed in this section.

Models for Insulin-Dependent Diabetes Mellitus

Alloxan-induced diabetes

Alloxan is a cyclic urea compound which induces permanent diabetes. It is a highly reactive molecule which produces free radical damage to β-islet cells and causes cell death. Alloxan at a dose level of 100 mg/kg in rats produces diabetes. In rabbits, a dose level of 150 mg/kg infused through a marginal ear vein produces diabetes in 70% of the animals.
Albino rats of either sex weighing 150–200 g are injected with a single dose of alloxan monohydrate (100 mg/kg body weight) dissolved in normal saline (0.9%) by intraperitoneal route. The animals are kept for 48 h during which Foodland water is allowed ad libitum. The blood glucose level shows the triphasic response with hyperglycaemia for 1 h followed by hypoglycaemia that lasts for 6 h and stable hyperglycaemia after 48 h. The animals showing fasting blood glucose level above 140 mg/dl after 48 h of alloxan administration are considered diabetic. Drug samples to be screened are administered orally for a period of six weeks. After six weeks of treatment, blood samples are collected from 8 h fasting animals through a caudal vein. Serum is separated by cooling centrifuge (2–4°C) at 3000 r.p.m. for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase method (GOD-POD kit) using an autoanalyser.

Streptozotocin-induced diabetes

Streptozotocin is a broad-spectrum antibiotic which causes β-islet cells damage by free radical generation. Streptozotocin induces diabetes in almost all species of animals excluding rabbits and guinea pigs. The diabetogenic dose of Streptozotocin varies with species. In mice, the dose level is 200 mg/kg through i.p. and in beagle dogs 15 mg/ kg through i.v. for three days. Adult albino rats of either sex weighing 150–200 g is injected with Streptozotocin (60 mg/kg body weight) prepared in citrated buffer (pH 4.5) solution by i.p. route. The citrated buffer is prepared by mixing 53.9 parts of 0.1 M citric acid and 46.1 parts of 0.2 M disodium hydrogen orthophosphate and finally adjusted to a pH of 4.5. The blood glucose level shows the same triphasic response as seen in alloxan-treated animals. Animals showing fasting blood glucose level above 140 mg/ dl after 48 h of Streptozotocin administration are considered diabetic. Drug samples to be screened are administered orally for a period of six weeks. After six weeks of treatment, blood samples are collected from 8 h fasted animals through a caudal vein. Serum is separated by cooling centrifuge (2–4°C) at 3000 r.p.m. for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase method (GOD-POD kit) using an autoanalyser

Virus-induced diabetes

Viruses are one of the etiological agents for IDDM. They produce diabetes mellitus by infecting and destroying β-islet cells of pancreas. Various human viruses used for inducing diabetes include RNA picornovirus, coxsackie B4 (CB-4), and encephalomyocarditis (EMC-D).

Six- to eight-week-old mice are inoculated by 0.1 ml of 1:50 dilution of D-variant encephalomyocarditis (EMC) through i.p. route. The 0.1 ml of the above dilution contains 50 PFU (plaque-forming units) of EMC virus. Mortality due to this concentration of virus is approximately 10–20%. A less-infecting variant produces a comparable damage by eliciting autoimmune reactivity to the β-islet cells. Infected animals are considered hyperglycemic if their nonfasting levels exceed by 250 mg/dl the levels of uninfected animals of the same strain. Drug samples to be screened are administered orally for a period of six weeks. After six weeks of drug treatment, blood glucose estimation is done to determine the antidiabetic activity

Insulin antibodies-induced diabetes

A transient diabetic syndrome can be induced by injecting guinea pigs with antiinsulin serum. It neutralises the endogenous insulin with insulin antibodies. Diabetes persists as long as the antibodies are capable of reacting with the insulin remaining in circulation.

Preparation of Antibody:

Bovine insulin, dissolved in acidified water (pH 3.0) at a dose of 1 mg is injected to guinea pigs weighing 300–400 g. Antiinsulin sera is collected after two weeks of antigenic challenge.

Adult albino rats are injected with 0.25–1.0 ml of guinea pig antiinsulin serum. Insulin antibodies induce a dosedependent increase of blood glucose level up to 300 mg/ dl. Slow rate intravenous infusion or an intra-peritoneal injection prolongs the effect for more than a few hours. However, large doses and prolonged administration are accompanied by ketonemia. The drug sample to be screened is administered by a suitable route, and blood glucose level is analysed to determine the activity.

Hormone-induced diabetes

Dexamethasone:

Dexamethasone is a steroid possessing immunosupression action which causes an autoimmune reaction in the islets and produces type 1 diabetes.

Adult rats weighing 150–200 g is injected with dexamethasone at a dose level of 2 5 mg/kg body weight by i.p. twice a day. The repeated injection of the same dose level is carried out for a period of 20–30 days resulting in IDDM. The sample to be screened is administered through a suitable route, and blood glucose level is analysed to determine the activity.

Genetic Models

Nonobese diabetic mouse

Nonobese diabetic mouse (NOD) is a model of IDDM. Hypoinsulinemia is developed which is caused by autoimmune destruction of pancreatic β-islet cells in association with autoantibody production.
Mice are breed at the laboratory by sib-mating over 20 generations. After 20 generations of sib-mating, spontaneous development of IDDM in mice is obtained. Diabetes develops abruptly between 100 and 200 days of age. Weight loss, polyurea, and severe glucosuria are common. The animals are treated with the drug sample to be screened. Blood sample is analysed for glucose level to determine activity.

Bio-breeding rat

Diabetes is inherited as an autosomal recessive trait and develops with equal frequency and severity among males and females. Insulin deficiency and insulitis are due to autoimmune destruction of pancreatic β-islet cells. Spontaneous diabetes is diagnosed in a noninbreed but closed out-breed colony of rats at bio-breeding laboratories.

Rats are breed at the laboratory by sib-mating over 20 generations. After 20 generations of sib-mating, spontaneous development of IDDM in rats is obtained. The onset of clinical diabetes is sudden and occurs at about 60–20 days of age. The clinical presentation of diabetes in the bio-breeding (BB) rat is similar to that of its human counterpart. Marked hyperglycaemia, glycosuria, and weight loss occur within a day of onset and are associated with decreased plasma insulin, that if untreated will result in ketoacidosis. The animals are treated with the drug sample to be screened for a required period of time. The blood sample is analysed for glucose level to determine activity.

Models for NIDDM

Streptozotocin-induced neonatal model for NIDDM

Streptozotocin causes severe pancreatic β-cells destruction, accompanied by a decrease in pancreatic insulin stores and a rise in plasma glucose level. In contrast to adult rats, the treated neonates partially regenerate and become normoglycaemic by three weeks of age. In the next few weeks, the β-cell number increase, mainly from the proliferation of cells derived from ducts, leads to hyperinsulinemia, and shows symptoms similar to insulin resistance.

Neonatal rats are treated with Streptozotocin (90 mg/kg body weight) prepared in citrated buffer (pH 4.5) by i.p. at birth or within the first 5 days following birth. After six weeks, the rats develop symptoms similar to NIDDM. Rats showing fasting blood glucose level above 140 mg/dl are considered diabetic. Further steps are similar to that of the alloxan-induced model. The drug sample to be screened is administered by a suitable route, and blood glucose level is analysed to determine the activity

Other Chemically Induced NIDDM Models

Adrenaline-induced acute hyperglycaemia

Adrenaline is a counter-regulatory hormone to insulin. It increases the rate of glycogenolysis and glucose level in blood causing acute hyperglycaemia.

Adult albino rats are injected at a dose level of 0.1 mg/ kg through s.c. route. The dose produces peak hyperglycemic effect at 1 h and lasts up to 4 h. The drug sample to be analysed is administered through a suitable route, and blood glucose level is determined. Oral hypoglycaemic agents can be screened by this method.

Chelating Agents

Dithizone-induced diabetes

Organic agents react with zinc in the islets of Langerhans causing the destruction of β-islet cells, producing diabetes. Severe necrosis and disintegration of β-cells (insulin producing cells) were observed, while α-cells (cells which produce glucoagon which maintains the glucose level in the blood) remain unaltered. Compounds such as dithizone, EDTA, 8-hydroxy quinoline are used to induce spontaneous type 2 diabetes in experimental animals. Dithizone at a dose level of 40–100 mg/kg (i.e.) produces type 2 diabetes in mice, cats, rabbits, and golden hamsters.

Adult rabbits weighing 1.8–2 kg is divided into two groups of six animals each. An exactly weighed amount of dithizone is dissolved in dilute ammoniacal solution (0.2 to 0.5%). The solution is warmed to 60 -70°C for 10 min to aid solubility of dithizone. Dithizone injection at a dose level of 50–200 mg/kg produces triphasic glycaemic reaction. Initial hyperglycaemia is observed after 2 h and normoglycemia after 8 h, which persists for up to 24 h. Permanent hyperglycaemia is observed after 24–72 h. The drug sample to be analysed is administered through a suitable route, and the blood glucose level is determined.

Models for Insulin Sensitivity and Insulinlike Activity

Euglycaemic clamp technique

This method has proved to be a useful technique of quantifying in vivo insulin sensitivity. A variable glucose infusion is delivered to maintain euglycemia during insulin infusion. The net glucose uptake is quantified, and the sensitivity of the body tissue to insulin is determined.

Adult albino rats weighing 150–200 g are fasted overnight and anaesthetized with pentobarbital (40 mg/kg i.p.). Catheters are inserted into a jugular vein and a femoral vein for blood collection and insulin and glucose infusion, respectively. To evaluate the insulin action under physiological hyperinsulinemia (steady-state plasma insulin concentration during the clamp test is around 100 μU/dl) and maximal hyperinsulinemia, two insulin infusion rates, that is, 6 and 30 mU/kg/min are used. The blood glucose concentrations are determined from samples collected at 5 min intervals during the 90-min clamp test. The glucose infusion rate is adjusted so as to maintain basal level. The glucose metabolic clearance rate is calculated by dividing the glucose infusion rate by the steady-state blood glucose concentration. The drug sample to be analysed is administered through a suitable route, and the blood glucose level is determined.

Assay for insulin and insulin-like activity

This assay involves comparing two standard solutions of insulin with the test drug for its insulin-like activity.

Four groups of six rabbits weighing at least 1.8 kg are used. Two standard solutions of insulin containing one unit and two units respectively and two dilutions of sample whose potency is being examined are prepared. As diluent, a solution of 0.1 to 0.25% w/v of either m-cresol or phenol and 1.4 to 1.8 w/v of glycerol acidified with hydrochloric acid to a pH between 2.5 and 3.5 is used. Each of the prepared solution (0.5 ml) is injected subcutaneously. After 1 h and 2.5 h of each injection, a suitable blood sample is taken from the ear vein of each rabbit, and the blood sugar level is determined preferably by glucose oxidase method.

EVALUATION OF ANTIDIARRHOEAL AGENTS

Castor Oil-Induced Diarrhoea in Rats

The method followed here is the method of Awouters et al. (1978) with modification. The original method included only male wistar rats (220–250 g), where they were starved overnight before treatment with the selected drug in the next morning. In the present study, rats of either sex (180–200 g) are fasted for 18 hours. Animals are housed in six in each. Varying doses of the test drugs are administered orally by gavage as suspension to different groups of animals. The next group received diphenoxylate (5 mg/ kg) orally as suspension as standard drug for comparison. Other group that served as control is treated with the control vehicle only.

One hour after treatment, each animal receives 1 ml of castor oil orally by gavage and then observed for defecation. Up to 4th hour after the castor oil challenge, the presence of characteristic diarrhoeal droppings is noted in the transparent plastic dishes placed beneath the individual rat cages.

The effects of the test drug like the standard antidiarrhoeal agent, diphenoxylate, are calculated based on the frequency of defecation when compared to untreated rats. Both substances also should reduce greatly the wetness of faecal droppings.

Gastrointestinal Motility Tests

Rats are fasted for 18 h and placed in different cages containing six in each. Each animal is administered orally with 1 ml of charcoal meal (3% deactivated charcoal in 10% aqueous tragacanth). Immediately after that the first few groups of animals are administered orally with the test drug at varying doses. Next group receives atropine (0.1 mg/kg, i.p), the standard drug for comparison. The last group is treated with aqueous tragacanth solution as control. Thirty minutes later, each animal is killed, and the intestinal distance moved by the charcoal meal from thepylorus is cut and measured and expressed as a percentage of the distance, the charcoal meal has moved from the pylorus to the caecum.

The antidiarrhoeal test drugs decrease propulsion of the charcoal meal through the gastrointestinal tract when compared with the control group by this model which is comparable to that of atropine (standard drug) which reduces the motility of the intestine significantly.

PGE2 -Induced Enteropooling

In this method, rats of the same stock as above are deprived of food and water for 18 h and are placed in six perforated cages with six animals per cage. The first few groups of rats are treated with varying doses of the test drug. The last two groups are treated with 1 ml of 5% v/v ethanol in normal saline (i.p.). The last group of this is then treated with the control vehicle, which served as control. Immediately afterwards, PGE2 is administered orally to each rat (100 μg/kg) in 5% v/v ethanol in normal saline. After 30 minutes, each rat is killed and the whole length of the intestine from the pylorus to the caecum dissected out and its contents are collected in a test tube and the volume is measured.

PGE2 induces significant increase in the fluid volume of rat intestine when compared with control animals receiving only ethanol in normal saline and control vehicle. The antidiarrhoeal test drugs inhibit this PGE2 -induced enteropooiing. Statistical analysis is performed by student’s ‘t’ test, and in all the cases results are expressed as mean ± SE.

SCREENING METHODS FOR ANTIFERTILITY AGENTS

Antifertility agents are substances which prevent reproduction by interfering with various normal reproductive mechanisms in both males and females. An ideal contraceptive agent is one which possess 100% efficacy, reversibility of action, which is free from side effects and is easy to use.

Ancient literature has mentioned the use of a number of plants/preparations for regulation of fertility in the form of emmenagogues, ecbolic, abortifacients, and local contraceptives. For centuries, virtually every indigenous culture has been using plants and/or their various parts in one or the other form to restrict its population. Women have used herbs since time immemorial to control their fertility. The information was passed on from mother to daughter; midwives and wise women all possessed this knowledge, but most of these plant’s activities and their mechanism of action were not scientifically studied.

There are approximately 2,50,000 species growing on earth. It stands to reason that not all of them can be used to regulate fertility; therefore, some criteria have to be laid down for selecting plants to evaluate their antifertility potential. Three options are available:

Investigation of plants that have folkloric/traditional reputation as contraceptives.
Evaluation of plants that are known to contain constituents which theoretically affect the female cycle and thus produce antifertility effects, for example, oestrogenic sterols, isoflavones, and coumestans or those, which have a potential to contract the uterus; and
Random collection of plants for mass screening.

During the last six decades, sporadic attempts have been made by Indian investigators to evaluate antifertility plants. But there is variation in the reports given by various investigators on the same plant part (from inactivity to 100% activity). This appears to be due to inadequate attention given to proper botanical identification, authentication, and testing procedure. In spite of the detailed description of plants found in ancient ayurvedic and unani literature, documented experimental or clinical data on them are lacking. Furthermore, the efficacies of these plants have not yet been confirmed through repeated investigations.

Screening Methods for Antifertility Activity in Females

Antifertility action of drugs acting in females may be due to:

Inhibition of ovulation
Prevention of fertilization
Interference with transport of ova from oviduct to endometrium of the uterus
Implantation of fertilized ovum
Distraction of early implanted embryo

Screening Methods for Antiovulatory Activity

Cupric acetate-induced ovulation in rabbits

Rabbits are reflex ovulators. They ovulate within a few hours after mating or after mechanical stimulation of vagina or sometimes even the mere presence of a male or administration of certain chemicals like cupric acetate.

In this screening method, cupric acetate is used for the induction of ovulation. The rabbit ovulates within a few hours after an i.v. injection of cupric acetate (0.3 mg/ kg using 1% cupric acetate in 0.9% saline). Injection of antiovulatory drugs, 24 h before the induction procedure prevents ovulation.

Sexually mature female albino rabbits, weighing 3–4 kg, are used for the study. Animals are kept in isolation for at least 21 days to ensure that they are not pregnant and to prevent the induction of ovulation by mating. They are then treated with the test drug, and 24 h later an i.v. injection of cupric acetate is given. The rabbits are sacrificed, and the ovaries are examined 18–24 h later. The total number of ovulation points on both the ovaries is recorded for each animal. Then the ovaries and uterus are excised and preserved in 10% buffered formalin and subjected to histopathological evaluation.

HCG-induced ovulation in rats

Immature female albino rats do not ovulate spontaneously and do not show cyclic changes of the vaginal epithelium. Priming with human chorionic gonadotropin (HCG) induces follicular maturation, followed by spontaneous ovulation two days later. Injection of an antiovulatory drug before the induction procedure will prevent ovulation. This principle is used for screening potential antiovulatory agents.

Immature female albino rats (24–26 days old) are used for the experiment. The animals are treated with various test drugs in different dose levels. After the administration of the test drug, exogenous HCG is given to induce ovulation. After two days, the animals are sacrificed. Their ovaries are preserved in a 10% buffered formalin and subjected to histopathological evaluation. The results are compared with the control group.

Screening Methods for Oestrogenic Compounds: In Vivo Methods

A primary therapeutic use of oestrogen (both in vivo and in vitro) is in contraception. The rationale for these preparations is that excess exogenous oestrogen inhibits FSH and LH and thus prevents ovulation.

Assay for water uptake

The principle of the assay is based on the observation that the uterus responds to oestrogens by increased uptake and retention of water. A peak in the uptake is observed six hours after administration.

Ovariectomized adult animals may be used for this experiment. It is simpler to use immature 18-day-old mice, or 22-day-old rats obtained two days before the beginning of the experiment. The animals are randomly grouped. The control group is given 0.1 ml of cottonseed oil (vehicle for estradiol) subcutaneously. The oestrogen control group is given doses ranging from 0.01 to 0.1 μg to establish a doseresponse curve. In the initial test, the test compound is given to groups at a high and low dose. In subsequent tests, it is given over a range of doses to provide the dose-response curve. All doses are given in 0.1 ml of cottonseed oil.

Five hours after treatment, the animals are killed by cervical fracture and the uteri are quickly excised. The operation is begun by a longitudinal slit through the skin of the abdomen and through the body wall. The uterus is picked up with the forceps and severed from the vagina. The uterine horns are separated from the connective tissues and are then cut at their constriction point near the ovary. The uteri are kept moist by placing them on dump (not wet) filter paper and by covering them with damp filter paper. They are then rapidly weighed in a sensitive balance. The uteri are dried in an oven at 60°C, for 24 h and are reweighed. The percentage increase in water over control can be calculated and compared with the values of other groups.

Procedure for ovariectomy: The animals are anaesthetized with ether. A single transverse incision is made in the skin of the back. This incision can be shifted readily from one side to the other, so as to lie over each ovary in turn. A small puncture is then made over the site of the ovary, which can be seen through the abdominal wall, embedded in a pad of fat. The top of a pair of fine forceps is introduced, and the fat around the ovary is grasped, care being taken not to rupture the capsule around the ovary itself. The tip of the uterine horn is then crushed with a pair of artery forceps and the ovary together with the fallopian tube removed with a single cut using a pair of fine scissors. Usually no bleeding is observed. The muscular wound is closed by absorbable sutures, and the outer skin wound is closed by nylon suture.

Four-day uterine weight assay

This assay is based on the observation that oestrogens cause an increase in protein synthesis and thus bring about an increase in uterine weight. A peak is observed after about 40 h.

Immature or Ovariectomized albino mice or rats can be given the test drug intramuscularly in cottonseed oil for three consecutive days. On the fourth day, animals are killed by cervical fracture, the uteri rapidly excised, and the uterine contents gently squeezed out (results are unreliable if the uterine contents are not removed). The uteri are weighed immediately in the wet state. They are then dehydrated in an oven at 100°C for 24 h and reweighed to obtain the dry weight increase. The log dose is plotted against the wet weight to produce a sigoid curve, and the ED50 can be determined for comparison of the test compound with estradiol

Vaginal cornification

This assay is based on the fact that rats and mice exhibit a cyclical ovulation with associated changes in the secretion of hormones. This leads to changes in the vaginal epithelial cells. The estrus cycle is classified into the proestrus, estrus, metestrus, and diestrus stage. Drugs with oestrogenic activity change the animals from whatever stage they were into the estrus stage.

Adult female albino rats having a regular estrus cycle are used for the study. Animals are treated with various test and standard drugs. Change in the vagina can be observed by taking vaginal smears and examining these for cornified cells, leucocytes, and epithelial cells in the normal animals and treated animals twice daily over a period of four days. Any drug which changes the animals into the estrus stage skipping other stages is considered to have oestrogenic activity.

The experimental procedure for taking vaginal smears

Holding the animal on the ventral side up, a drop of normal saline is inserted into the vagina with a Pasteur pipette. Care must be taken to avoid damage or injury to the vagina so as to prevent pseudopregnancy. The drop of normal saline should be aspirated and replaced several times. It is then transferred to a microscope slide and allowed to dry. The smears are fixed by placing the slide in absolute alcohol for 5 sec, allowing it to dry, and staining it with a 5% aqueous methylene blue solution for 10 min. The excess stain is washed off with tap water, and the slide is dried and observed using a low power microscope.

Chick oviduct method

The weight of the oviduct of young chicken increases depending on the dose of natural and synthetic oestrogen. This principle is used for the screening of oestrogenic compounds.

Seven-day-old pullet chicks are injected subcutaneously twice daily with solutions of the test compound in various doses for six days. Doses (0.02–0.5 μg) of 17β-estradiol per animal serve as standard. Six to ten chicks are used for each dosage group. On the day after the last injection, the animals are sacrificed, and the weight of the body and oviduct is determined

SCREENING METHODS FOR ANTIINFLAMMATORY AGENTS

WHO has identified 2000–2010 as the decade for musculoskeletal disorders. Herbal drugs like holy basil (tulsi; Ocimum sanctum), turmeric (Curcuma longa), Indian olibanum tree (Boswellia serrata), ginger (Zingiber officnale), etc. are widely used for the treatment of various inflammatory disorders. They are not only found to be safer and have fewer side effects, but they also cover a large domain of mechanisms involved in inflammation thus proving to be more beneficial than synthetic drugs. Inflammation expresses the response to damage of cells and vascularissues. The five basic symptoms of inflammation—redness, swelling, heat, pain, and deranged function, have been known since the ancient Greek and Roman era.

The major events occurring during this response are an increased blood supply to the affected tissue by vasodilation, increased capillary permeability caused by retraction of the endothelial cells which allows the soluble mediators of immunity to reach the site of inflammation and leukocytes migration out of the capillaries into the surrounding tissues. Neutrophils, monocytes, and lymphocytes also migrate towards the site of infection. The development of inflammatory reactions is controlled by the following systems: cytokines, complement, kinin and fibrinocytic pathways; by lipid mediators (prostagiandins and leukotrienes) released from different cells; and by vasoactive mediators released from mast cells, basophils, and platelets.

The response is accompanied by the clinical signs of erythema, oedema, hyperalgesia, and pain. Inflammatory responses occur in three distinct phases, each apparently mediated by different mechanisms:

Acute transient phase: Characterized by local vasodilatation and increased capillary permeability

Sub-acute phase: Characterized by infiltration of leukocytes and phagocytic cells.

Chronic proliferative phase: Tissue degeneration and fibrosis occur.

Drugs preventing acute and sub-acute inflammation can be tested using the following models: paw oedema in rats, croton oil ear oedema, pleurisy tests, UV-erythema in guinea pigs, oxazolone-induced ear oedema in mice, granuloma pouch technique, and vascular permeability. The effectiveness of drugs which work at the proliferative phase can be measured by methods for testing granuloma formation, such as the cotton pellet granuloma, adjuvant induced arthritis, glass rod granuloma, and PVC sponge granuloma.

Testing of Drugs Preventing Acute and Sub-Acute Inflammation

Paw oedema

This technique is based upon the ability of antiinflammatory agents to inhibit the oedema produced in the hind paw of the rat after injection of a phlogistic agent (irritant). Rats with a body weight between 100 and 150 g are required. Many irritants have been used, such as brewer’s yeast, formaldehyde, dextran, egg albumin, kaolin, Aerosil®, and sulphated polysaccharides like carrageenan. The animals are fasted overnight. The control rats receive distilled water while the test animals receive drug suspension orally. Thirty minutes later, the rats are subcutaneously injected with 0.1 ml of 1% solution of carrageenan in the foot pad of the left hind paw. The paw is marked with ink and immersed in the water cell of a plethysmometer up to this mark. Thepaw volume is measured plethysmograph Cally immediately after injection, 3 and 6 h after injection, and eventually 24 h after injection. The paw volumes for the control group are then compared with those of the test group

Croton oil ear oedema in rats and mice

This method mainly evaluates the antiphlogistic activity of topically applied steroids.

For this method, mice (22 g) or rats (70 g) are required. For tests in mice, the irritant is composed of (v/v): 1 part croton oil, 10 parts ethanol, 20 parts pyridine, and 69 parts ethyl ether; for rats the irritant is composed of (v/v): 4 parts croton oil, 10 parts ethanol, 20 parts pyridine, and 66 parts ethyl ether. The standard and the test compound are dissolved in this solution. Irritants are applied on both sides of the right ear (0.01 ml in mice or 0.02 ml in rats under ether anaesthesia). Controls receive only the irritant solvent. The left ear remains untreated. Four hours after application, the animals are sacrificed under anaesthesia. Both ears are removed, and discs of 8 mm diameter are cut. The discs are weighed immediately and the weight difference between the treated and untreated ear is recorded indicating the degree of inflammatory oedema.

Pleurisy test

Pleurisy is the phenomenon of exudative inflammation in man. In experimental animals, pleurisy can be induced by several irritants, such as carrageenan, histamine, bradykinin prostaglandins, mast cell degranulators, and dextran. Leukocyte migration and various biochemical parameters involved in the inflammatory response can be measured easily in the exudate.

Male rats weighing 220–260 g are required. The animal is lightly anaesthetized with ether and placed on its back. The hair from the skin over the ribs on the right side is removed and the region cleaned with alcohol. A small incision is made into the skin under the right arm. The wound is opened and 0.1 ml of 2% carrageenan solution is injected into the pleural cavity through this incision. The wound is closed with a clip. One hour before this injection and 24 and 48 h thereafter, rats are treated (subcutaneously or orally) with the standard or the test compound. A control group receives only the vehicle. The animals are sacrificed 72 h after carrageenan injection and pinned on a dissection board with the forelimbs fully extended. About 1 ml of heparinized Hank’s solution is injected into the pleural cavity through an incision. The cavity is gently massaged to mix its contents. The fluid is aspirated out of the cavity using a pipette. The aspirated exudates are collected in a graduated plastic tube. About 1 ml (the added Hank’s solution) is subtracted from the measured volume. The values of each experimental group are averaged and compared with the control group. The white blood cell number in the exudate is measured using a Coulter counter or a haematocytometer.

Ultraviolet erythema in guinea pigs

Antiinflammatory agents delay the development of ultraviolet erythema on albino guinea pigs. They are shaved on the back 18 h before testing. The test compound is suspended in the vehicle and half the dose of the test compound is administered orally 30 min before ultraviolet exposure. Control animals are treated with the vehicle alone. The guinea pigs are placed in a leather cuff with a hole of 1.5–2.5 cm size punched in it, allowing the ultraviolet radiation to reach only this area. An ultraviolet burner is warmed up for about 30 min before use and placed at a constant distance (20 cm) above the animal. Following a 2 min ultraviolet exposure, the remaining half of the test compound is administered. The erythema is scored 2 h and 4 h after exposure

Oxazolone-induced ear oedema in mice

The oxazolone-induced ear oedema in mice is a model of delayed contact hypersensitivity that permits the quantitative evaluation of the topical and systemic antiinflammatory activity of a compound following topical administration.

Mice of either sex (25 g) are required. A fresh 2% solution of oxazolone in acetone is prepared. This solution (0.01 ml) is injected on the inside of both ears under anaesthesia. The mice are injected 8 days later, again under anaesthesia, with 0.01 ml of 2% oxazolone solution (control) or 0.01 ml of oxazolone solution in which the test compound or the standard is dissolved, on the inside of the right ear. The left ear remains untreated. The maximum of inflammation occurs 24 h later. At this time the animals are sacrificed under anaesthesia and a disc of 8 mm diameter is punched from both ears. The discs are immediately balance. The weight difference is an indicator of the inflammatory oedema.

Granuloma pouch technique

Irritants such as croton oil or carrageenan produce aseptic inflammation resulting in large volumes of exudate, which resembles the sub-acute type of inflammation. Rats (150–200 g) are selected for the study; the back of the animals is shaved and disinfected. With a very thin needle, an air pouch is made by injection of 20 ml of air under ether anaesthesia. Into the resulting air pouch 0.5 ml of a 1% solution of croton oil in sesame oil is injected. After 48 h, the air is withdrawn from the pouch and 72 h later any resulting adhesions are broken. Instead of croton oil, 1 ml of a 20% suspension of carrageenan in sesame oil can be used as irritant. Starting with the formation of the pouch, the animals are treated every day either orally or subcutaneously with the test compound or the standard. On the fifth day, the animals are sacrificed under anaesthesia. The pouch is opened, and the exudate collected in glass cylinders. The average value of the exudate of the controls and the test groups is calculated.

Vascular permeability

This test is used to evaluate the inhibitory activity of drugs against increased vascular permeability, which is induced by a phlogistic substance. Mediators of inflammation, such as histamine, prostaglandins, and leukotrienes are released following stimulation of mast cells. This leads to a dilation of arterioles and venules and to an increased vascular permeability. As a consequence, fluid and plasma proteins are released and edemas are formed. Vascular permeability is increased by subcutaneous injection of the mast celldegranulating compound 48/80. The increase of permeability can be recognised by the infiltration of the injected sites of the skin with the dye Evan’s blue.

Male rats (160 and 200 g) are used. About 5 ml/kg of 1% solution of Evan’s blue is injected intravenously. One hour later, the animals are dosed with the test compound orally or intraperitoneally. After 30 min, the animals are lightly anaesthetized with ether and 0.05 ml of 0.01% solution of compound 48/80 is injected subcutaneously at three sites. About 90 min after the injection of compound 48/80, the animals are sacrificed by ether anaesthesia. The abdominal skin is removed, and the dye-infiltrated areas of the skin measured. The percent inhibition in the treated animals as compared to the control group is calculated.

Testing of Drugs Preventing the Proliferative Phase (Granuloma Formation) of Inflammation

Cotton pellet granuloma

Foreign body granulomas are induced in rats by the subcutaneous implantation of pellets of compressed cotton. After several days, histologically giant cells and undifferentiated connective tissue can be observed besides fluid infiltration. The amount of newly formed connective tissue can be measured by weighing the dried pellets after removal. More intensive granuloma formation has been observed if the cotton pellets are impregnated with carrageenan.

Male and female rats with an average weight of 200 g are used. The back skin is shaved and disinfected with 70% ethanol. An incision is made in the lumbar or neck region. Subcutaneous tunnels are formed, and a sterilized cotton pellet is placed with the help of a blunted forceps. The animals are treated for seven days subcutaneously or orally. They are then sacrificed, the pellets taken out and dried. The net dry weight, that is, after subtracting the weight of the cotton pellet is determined. The average weight of the pellets of the control group as well as that of the test group is calculated. The percent change of granuloma weight relative to the vehicle control group is determined.

Adjuvant arthritis in rats

Adjuvant-induced arthritis in rats exhibit many similarities to human rheumatoid arthritis. An injection of complete Freund’s adjuvant into the rat’s paw induces inflammation as a primary lesion with a maximum inflammation after three to five days. Secondary lesions occur after a delay of approximately 11 to 12 days and are characterized by inflammation of noninjected sites (hind legs, forepaws, ears, nose, and tail), a decrease in weight and immune responses.

Male rats with an initial body weight of 130 to 200 g are used. On day 1, rats are injected in the sub-plantar region of the left hind paw with 0.1 ml of complete Freund’s adjuvant. The adjuvant consists of 6 mg mycobacterium butyricum thoroughly ground with a mortar and pestle and suspended in heavy paraffin oil (Merck) to give a concentration of 6 mg/ml. Dosing with the test compounds or the standard is started on the same day and continued for 12 days. Both paw volumes and body weight are recorded on the day of injection. The paw volume is measured plethysmographically with equipment as described in the paw oedema tests. On day 5, the volume of the injected paw is measured again, indicating the primary lesion and the influence of therapeutic agents on this phase. The severity of the induced adjuvant disease is determined by measuring the noninjected paw (secondary lesions) with a plethysmometer. The animals are not dosed with the test compound or the standard from day 12 to 21. On day 21, the body weight is determined again and the severity of the secondary lesions evaluated visually and graded according to the following scheme:

Sponge implantation technique

Foreign body granulomas are induced in rats by subcutaneous implantation of a sponge. Sponges used for implantation are prepared from polyvinyl foam sheets (thickness: 5 mm). Discs are punched out to a standard size and weight (10.0 ± 0.02 mg). The sponges are then soaked in 70% v/v ethanol for 30 min., rinsed four times with distilled water, and healed at 80°C for 2 h. Before implantation in the animal, the sponges are soaked in sterile 0.9% saline in which either drugs, antigens, or irritants have been suspended. Typical examples include 1% carrageenan, 1% yeast,1% zymosan A, 6% dextran, heat killed. Bordctelhi pertussis, or 0.5% heat-killed Mycobacterium tuberculosis.

Sponges are implanted in rats weighing 150–200 g under ether anaesthesia. An incision is made, and separate cavities are formed into which sponges are inserted. Up to 8 sponges may be implanted per rat. The incision is closed with Michel clips and the animals maintained at a constant temperature of 24°C. For short-term experiments, the animals are treated with the test drug or standard once before implantation orally or subcutaneously. For long-term experiments, the rats are treated daily up to 3 weeks.

Glass rod granuloma

Glass rod-induced granulomas reflect the chronic proliferate phase of inflammation. Of the newly formed connective tissue, not only can the wet and dry weight be measured, but also the chemical composition and mechanical properties. Glass rods with a diameter of 6 mm are cut to a length of 40 mm and the ends rounded off. They are sterilized before implantation. Rats are anaesthetized with ether, the back skin shaved and disinfected. From an incision in the back region, a subcutaneous tunnel is formed with a blunted forceps. A glass rod is introduced into this tunnel. The incision wound is closed by sutures. The animals are kept in separate cages. The rods remain in situ for 20 or 40 days. Animals are treated orally. At the end of 20 days the animals are sacrificed. The glass rods are removed together with the surrounding connective tissue, which forms a tube around the glass rod. By incision at one end, the glass rod is extracted, and the granuloma sac inverted forming a plain piece of pure connective tissue. Wet weight of the granuloma tissue is recorded. The specimens are kept in a humid chamber until further analysis. Biochemical analyses, such as determination of collagen and glycosaminoglycans, can also be performed.

SCREENING METHODS FOR ANTIPYRETIC AGENTS

Treatment with antipyretics has been very important in the preantibiotic era. Nevertheless, for treatment of acute viral diseases and for treatment of protozoal infections like malaria, reduction of elevated body temperature by antipyretics is still necessary. For antiinflammatory compounds, an antipyretic activity is regarded as a positive side effect. To evaluate these properties, fever is induced in rabbits or rats by injection of lipopolysaccharides or Brewer’s yeast.

Antipyretic Testing in Rats

The subcutaneous injection of Brewer’s yeast suspension is known to produce fever in rats. A decrease in temperature can be achieved by administration of compounds with antipyretic activity.

Procedure

A 15% suspension of Brewer’s yeast in 0.9% saline is prepared. Groups of six male or female wistar rats with a body weight of 150 g are used. By insertion of a thermocouple to a depth of 2 cm into the rectum the initial rectal temperatures are recorded. The animals are fevered by injection of 10 ml/kg of Brewer’s yeast suspension subcutaneously in the back below the nape of the neck. The site of injection is massaged in order to spread the suspension beneath the skin. The room temperature is kept at 22–24°C. Immediately after yeast administration, food is withdrawn. 18 h post challenge, the rise in rectal temperature is recorded. The measurement is repeated after 30 min. Only animals with a body temperature of at least 38°C are taken into the test. The animals receive the test compound or the standard drug by oral administration. Rectal temperatures are recorded again 30, 60, 120, and 180 min postdosing.

Evaluation

The differences between the actual values and the starting values are registered for each time interval. The maximum reduction in rectal temperature in comparison to the control group is calculated. The results are compared with the effect of standard drugs, for example, aminophenazone 100 mg/ kg p.o. or phenacetin 100 mg/kg p.o.

Modifications of the method

Stitt and Shimada (1991) and Shimada et al. (1994) induced fever in rats by microinjecting 20 ng PGE1 directly into one of the brain’s circumventricular organs of the rat known as the organum vasculum laminae terminalis.

Luheshi et al. (1996) induced fever by intraperitoneal injection of 100 μg/kg lipopolysaccharide into rats and measured the inhibition of fever by interleukin-1 receptor antagonist.

Telemetry has been used to record body temperature in animals (Riley et al. 1978; Gallaher et al. 1985; Clement et al. 1989; Guillet et al. 1990; Kluger et al. 1990; Bejanian 1991; Watkinson et al. 1996; Miller et al. 1997).

Antipyretic Testing in Rabbits

Lipopolysaccharides from Gram-negative bacteria, for example, E. coli, induce fever in rabbits after intravenous injection. Only lipopolysaccharide fractions are suitable, which cause an increase of body temperature of 1°C or more at a dose between 0.1 and 0.2 μg/kg after 60 min. In the rabbit, two maxima of temperature increases are observed. The first maximum occurs after 70 min and the second after 3 h.

Procedure

Rabbits of both sexes and of various strains with a body weight between 3 and 5 kg can be used. The animals are placed into suitable cages and thermocouples connected with an automatic recorder are introduced into the rectum. The animals are allowed to adapt to the cages for 60 min. Then 0.2 ml/kg containing 0.2 μg lipopolysaccharide are injected intravenously into the rabbit ear. After 60 min, the test compound is administered either subcutaneously or orally. Body temperature is monitored for at least 3 h.

Evaluation

A decrease of body temperature for at least 0.5°C for more than 30 min as compared with the temperature value before administration of the test compound is regarded as positive effect. This result has been found after 45 mg/kg phenylbutazone s.c. or 2.5 mg/kg indomethacin s.c

Modifications of the method

Cashin and Heading (1968) described a simple and reliable assay for antipyretic drugs in mice, using intracerebral injection of pyrogens. Davidson et al. (1991) tested the effect of human recombinant lipocortin on the pyrogenic action of the synthetic polyribonucleotide polyinosini:polycytidylic acid in rabbits. Yeast-induced pyrexia in rats has been used for antipyretic efficacy testing by Loux et al. (1982) and Cashin et al. (1977). van Miert et al. (1977) studied the effects of antipyretic agents on fever and ruminal stasis induced by endotoxins in conscious goats. Petrova et al. (1978) used turpentine-induced fever in rabbits to study antipyretic effects of dipyrone and acetylsalicylic acid. Lee et al. (1985) studied the antipyretic effect of dipyrone on endotoxin fever of macaque monkeys. Loza Garcia et al. (1993) studied the potentiation of chlorpromazine-induced hypothermia by the antipyretic drug dipyrone in anesthetized rats. Shimada et al. (1994) studied the mechanism of action of the mild analgesic dipyrone preventing fever induced by injection of prostaglandin E1 or interleukin-1β into the organum vasculosum terminalis of rat brain.

SCREENING METHODS FOR ANTIULCER AGENTS

An ulcer is a local defect, or excavation of the surface of an organ, or tissue, which is produced by the sloughing of inflammatory necrotic tissue. The term ‘peptic ulcer’ refers to a group of ulcerative disorders of the upper GIT, which appears to have in common, the participation of acid pepsin in their pathogenesis. A peptic ulcer probably results due to an imbalance between aggressive (acid, pepsin, and H. pylori) and defensive (gastric mucous, bicarbonate secretion, prostaglandins, innate resistance of the mucosal ceils) factors. In gastric ulcers, acid secretion is normal or low.

Pylorus Ligation in Rats

This principle is based on ulceration induced by accumulation of acidic gastric juice in the stomach (Shay et al. 1945).

The requirements include: stereo microscope, adult albino rats (150–170 g), anaesthetic ether, plastic cylinder, 0.1 N sodium hydroxide.

Adult albino rats weighing 150–170 g is starved for 48 h although they have access to drinking water. Normally ten animals are used per dose and as control. After they are ether anaesthetized, a midline abdominal incision is made, and the pylorus ligated. The abdominal wall is closed by sutures and test compounds are given either orally by gavage or injected subcutaneously. The animals are placed for 19 h in plastic cylinders with an inner diameter of 45 mm being closed on both ends by a wire mesh. The animals are then sacrificed using carbon dioxide anaesthesia. The abdomen is opened, and a ligature is placed around the aesophagus close to the diaphragm. The stomach is removed, and the contents drained into a centrifuge rube. Along the greater curvature, the stomach is opened and pinned onto a cork plate. The mucosa is then examined with a stereo microscope.
The evaluation is done by counting the numbers of ulcers and the severity graded according to the following scores:

0 = no ulcers; 1 = superficial ulcers; 2 = deep ulcers;

3 = perforations

The volume of gastric content is measured after centrifugation. Acidity is determined by titration with 0.1 N NaOH. Ulcer index U is calculated using the following formula:

Ulcers Through Immobilization Stress

The principle behind this method involves psychogenic factors, such as stress, which play a major role in the pathogenesis of gastric ulcers in man.

The requirements include: adult albino rats, anaesthetic ether, CO2 anaesthesia, and a stereo microscope. A group of 10 adult albino rats (150–170 g) per dose of the test drug and for controls are used. Food and water are withdrawn 24 h before the experiment. After oral and subcutaneous administration of the test compound or a placebo solution, the animals are slightly anaesthetized with ether. Both the upper and lower extremities are fixed together and the animals wrapped in wire gauge. They are horizontally suspended in the dark at 20°C for 24 h and finally sacrificed using carbon dioxide anaesthesia. The stomach is removed, fixed on a cork plate, and the number and severity of the ulcers registered with a stereo microscope.

Stress Ulcer by Cold Water Immersions

The principle behind this assay is that cooling the rats in water when they are restrained according to the previous model accelerates the occurrence of gastric ulcers and shortens the time of necessary immobilization. In thismodel, the gastric ulcer formation is mainly due to gastric hypermotility, which could lead to mucosal over-friction.

The requirements include: wistar rats, cages, Evans blue dye, 2% formol saline, CO2 anaesthesia, and a magnifier. Groups of 8–10 wistar rats weighing 150–200 g are used. After oral administration of the test compounds, the rats are placed vertically in individual restraint cages in water at 22°C for 1 h. They are then removed, dried, and injected intravenously through a tail vein with 30 mg/ kg Evans blue. After 10 min, they are sacrificed using CO2 anaesthesia and their stomachs removed. Formol saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greatest curvature, washed in warm water, and examined under a threefold magnifier.

The evaluation is done by measuring the lengths of the longest diameter of the lesions. This is summated to give a total lesion score (in mm) for each animal; the mean count in control rats should be about 25 (range 20–28). Inhibition of the lesion production is expressed as a percentage value.

SCREENING METHODS FOR HEPATOPROTECTIVE AGENTS

A toxic or repeated dose of a known hepatotoxin is administered to induce liver damage in experimental animals. The test substance is administered before or after the toxin treatment. If the hepatotoxicity is prevented or reduced, the test substance is effective. There are various models of inducing hepatotoxicity in rodents (rats and mice)

Hepatitis in Long-Evans Cinnamon Rats

The Long-Evans Cinnamon strain of rats has been recommended as a useful model to study genetically transmitted hepatitis and chronic liver disease. It has been speculated that this strain of rats is prone to liver diseases due to excessive copper accumulation in the liver.

Long-Evans Cinnamon rats are housed in temperature and humidity-controlled rooms at a 12:12 light/dark cycle. Groups of 6–10 rats are given different diets based on a 15% purified egg protein diet and supplemented with vitamins or drugs. Drugs are applied via mini pumps intraperitoneally implanted under ether anaesthesia. The occurrence of jaundice is easily observable as the time when the ears and tail turn yellow and the urine becomes bright orange, staining the fur in the lower abdominal region. Usually the jaundice progressively worsens, ending in death of the animal within about a week. Incidence of jaundice and mortality versus time are used as parameters to measure the extent of hepatoprotective activity

Allyl Alcohol-induced Liver Necrosis in Rats

In this method allyl alcohol is used as a liver necrosisinducing agent in rats. Albino rats weighing 120–150 g are used. On the first day, food but not water is withdrawn. After 6 h, the compounds to be tested for protective activity are administered i.p or orally. After 1 h, the animals are dosed orally with 0.4 ml/kg of a 1.25% solution of allyl alcohol in water. Next morning, the treatment with the potentially protective drugs is repeated. Food but not water is withheld until the third day. Next morning, the animals are sacrificed and the liver is removed. The parietal sides of the liver are checked using a stereomicroscope with 25 times magnification. Focal necrosis is observed as white-green or yellowish hemorrhagic areas clearly separated from unaffected tissue. The diameter of the necrotic areas is determined using an ocularmicrometer. These values are added for each animal to obtain an index for necrosis.

Carbon Tetrachloride-induced Liver Fibrosis in Rats

Chronic administration of carbon tetrachloride to rats induces severe disturbances of hepatic function together with histologically observable liver fibrosis. This model is used for the screening of hepatoprotective agents.

Albino rats are treated orally twice a week with 1 mg/ kg carbon tetrachloride, dissolved in olive oil 1:1, over a period of 8 weeks. The animals are kept under standard conditions (day/night rhythm: 8:00 AM to 8:00 PM; 22°C room temperature; standard diet; water ad libitum). Twenty animals serve as controls receiving only olive oil; 40–60 animals receive only the carbon tetrachloride. Groups of 20 rats receive in addition to carbon tetrachloride, the compound under investigation in various doses by gavage twice daily (with the exception of the weekends, when only one dose is given) on the basis of the actual body weight. The animals are weighed weekly. At the end of the experiment (8 weeks), the animals are anaesthetized and exsanguinated through the caval vein.

The serum is analysed for parameters like total bilirubin, total bile acids, 7 S fragment of type IV collagen, procollagen III N-peptide. The liver, kidney, aortic wall, and tail tendons are prepared for determination of hydroxyproline. They are weighed and completely hydrolysed in 6 N HCl. Hydroxyproline is measured by HPLC and expressed as mg/mg wet weight of the organs.

For histological analysis, three to five pieces of the liver weighing about 1 g are fixed in formalin and Carnoy solution. Three to five sections of each liver are embedded, cut, and stained with azocarmine aniline blue (AZAN) and evaluated for the development of fibrosis using a score of 0–IV

The values of all the parameters of the test group are compared with the control group using suitable statistical methods.

Bile Duct Ligation-induced Liver Fibrosis in Rats

Ligation of the bile duct in rats induces liver fibrosis, which can be evaluated histologically and by determination of serum collagen parameters. This model is used for the screening of hepatoprotective agents.

Albino rats are anaesthetised and laparotomy is performed under aseptic conditions. A midline incision in the abdomen is made from the xiphosternum to the pubis, exposing the muscle layers and the linea alba, which is then incised over a length corresponding to the skin incision. The edge of the liver is then raised, and the duodenum pulled down to expose the common bile duct, which pursues an almost straight course of about 3 cm from the hilum of the liver to its opening into the duodenum. There is no gall bladder, and the duct is embedded for the greater part of its length in the pancreas, which opens into it by numerous small ducts. A blunt aneurysm needle is passed under the part of the duct selected, the pancreas is stripped away with care, and the duct is double ligatured with cotton thread.

The peritoneum and the muscle layers as well as the skin wound are closed with cotton stitches. The animals receive normal diet and water ad libitum throughout the experiment. Groups of 5–10 animals receive the test compound in various doses or the vehicle twice daily for 6 weeks. They are then sacrificed, and the blood harvested for determination of bile acids. 7 S fragment of type IV collagen, and procoilagen III N-peptide. The liver is used for histological studies and for hydroxyproline determinations. Control animals show excessive bile duct proliferation as well as formation of fibrous septa. This is consistent with complete biliary cirrhosis. The value of the test is compared with the control using suitable statistical analysis.

Galactosamine-induced Liver Necrosis

A single dose or a few repeated doses of D-galactosamine causes acute hepatic necrosis in rats. Prolonged administration leads to cirrhosis. This model is used for the screening of hepatoprotective agents.

Adult albino rats weighing 110–180 g is injected intraperitoneally three times weekly with 500 mg/kg D-galactosamine over a period of one to three months. The test substances are administered orally with food or by gavage. The control group receives only vehicle or food without drugs. The rats are sacrificed at various time intervals and the livers excised and evaluated by light microscopy and immunohistology using antibodies against macrophages, lymphocytes, and the extracellular matrix component.

Country-made Liquor Model

Country-made liquor (CML, containing 28.5% alcohol) is used to produce hepatotoxicity in this model. CML is administered orally at a dose of 3 ml/100 g/day for 30 days, which results in severe fatty changes in liver.

Rats are divided into groups of eight each. The control group receives 1% gum acacia as vehicle, corn oil (1 ml/100 g/day), and glucose isocaloric to the amount of alcohol. The positive control group receives CML (3 ml/100 mg/day) in two divided doses and corn oil (1 ml/100 g/day) in a single dose. Other test groups receive drugs in respective doses along with CML (3 ml/100 g/day) and corn oil.

After 21 days, the blood is withdrawn for analysis of SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The rats are sacrificed, and the livers dissected out for histopathological analysis. The value of the test is compared with the control using suitable statistical analysis.

Paracetamol Model

This model is used to produce experimental liver damage only in mice, since rats are resistant to paracetamol-induced hepatotoxicity. Paracetamol administered orally as a single dose of 500 mg/kg in mice produces hepatotoxicity.

Adult albino mice are used for the study. Paracetamol is administered as a single dose of 500 mg/kg. After 48 h, they are treated with the test drugs for 5 days. At the end of the experiment, blood is withdrawn for biochemical analysis of SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The liver is subjected to histopathological studies. The value of the test is compared with the control using suitable statistical analysis.

Partial Hepatectomy Model

In this method, partial hepatectomy (removal of 70% of liver mass) is done and the action of drugs on the regenerationof liver cells studied. Hepatoprotective agents improve the regeneration capability of liver.

Rats are used for this study as they can withstand surgical infections better than mice. They are anaesthetized using light ether anaesthesia. A median line incision reaching 3–4 mm posteriorly from the xiphoid process of the sternum is done and the large median lobe of the liver with the left lateral lobe taken out. These lobes are ligated by coarse linen and excised. Around 68 ± 2% of the total hepatic parenchyma is also removed. The peritoneum is closed using absorbable suture and the integument closed using nonabsorbable surgical suture. Various hepatoprotective drugs can be screened for their activity using these hepatectomized rats.

At the end of the screening experiments, the blood is collected for analysis of the serum. The following parameters are determined: SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The animals are sacrificed. The liver is excised out, weighed, and subjected to histopathological evaluation. The value of the test is compared with the control using suitable statistical analysis.

SCREENING METHODS FOR WOUND-HEALING AGENTS

The extracts obtained from plants are usually made into different formulations, either as ointment or as lotion and applied to the skin wound. Sometimes it is used internally or even injected if required depending on the nature of the constituents. The models usually used for the evaluation of the wound-healing activity can be described as follows:

Excision Wound Model

Four groups of animals containing ten in each group are to be anaesthetized by open mask method with anaesthetic ether. The rats are depilated on the back. One excision, wound is inflicted by cutting away 500 mm2 full thickness of skin of a predetermined area. Rats are left undressed to the open environment. Then the drug, that is, the reference standard (0.2% w/w nitrofurazone ointment), simple ointment BP (control), and test drug ointment or different other forms are administered till the wound is completely healed. This model is used to monitor wound contraction and epithelialization time. Epithelialization time is noted as the number of days after wounding required for the scar to fall off leaving no raw wound behind. Wound contraction is calculated as percent reduction in wound area. The progressive changes in wound area are monitored planimetrically by tracing the wound margin on a graph paper every alternate day. To determine the changes in healing of wound measurement of wound, area on graph paper is expressed as unit (mm2). For histopathological examination, tissues are collected from the completely healewound when the scar is removed. A transverse section of tissue is prepared from each group of rat and stained with haematoxilin and eosin to reveal the tissue section clearly. Then the tissues are observed under microscope to study different histopathological phenomenon.

Incision Wound Model

Four groups of animals containing ten in each group are anaesthetized, and two paravertebral long incisions of 6 cm length are made through the skin and cutaneous muscles at a distance of about 1.5 cm from midline on each side of the depilated back of rat. Full aseptic measures are not taken and no local or systemic antimicrobials are used throughout the experiment. All the groups are treated in the same manner as mentioned in case of excision wound model. No ligature is to be used for stitching. After the incision is made, the parted skin is kept together and stitched with black silk by 0.5 cm apart. Surgical thread (No. 000) and curved needle (No. 11) are used for stitching. The continuous threads on both wound edges are tightened for good adoption of wound. The wound was left undressed. The ointment of extract, standard drug (nitrofurazone ointment), and simple ointment BP is applied to the wound twice daily or feeded daily until complete recovery, to the respective groups of animals.

Tensiometer

It consists of a 6 × 12-inch wooden board with one arm of 4-inch long, fixed on each side of the possible longest distance of the board. The board is placed at the edge of a table. A pulley with bearing is mounted on the top of one arm. An alligator clamp with 1-cm width is tied on the tip of another arm by a fishing line (20-lb test monofilament) in such a way that the clamp could reach the middle of the board. Another alligator clamp is tied on a longer fishing line with 1 litre polyethylene bottle on the other end.

Tensile strength of wound represents the promotion of wound healing. Usually wound-healing agents promote the gaining of tensile strength. Tensile strength (the force required to open the healing skin) was used to measure the amount of healing. The instrument used for this purpose is called as tensiometer, which is explained as above. This was designated on the same principle as the thread tested in textile industry. One day before performing the experiment (measurement of tensile strength) the sutures are removed from the stitched wounds of rats after recovery and tensile strength is measured as follows.

Determination of Tensile Strength

The sutures are removed on ninth day of wounding and the tensile strength is measured on 10th day. Extract ointments along with simple ointment (control) and nitrofurazone ointment (standard) are administered throughout theperiod, twice daily for 9 days. On 10th day again the rats are anaesthetized, and each rat is placed on a stack of paper towels on the middle of the board. The amount of the towels could be adjusted in such a way so that the wound is on the same level of the tips of the arms. The clamps are then carefully clamped on the skin of the opposite sides of the wound at a distance of 0.5 cm away from the wound. The longer pieces of the fishing line are placed on the pulley and finally to polyethylene bottle, and the position of the board is adjusted so that the bottle receive a rapid and constant rate of water from a large reservoir, until the wound began to open. The amount of water in the polyethylene bag is weighed and considered as tensile strength of the wound. The mean determinations are made on both sides of the animals and are taken as the measures of the tensile strength of the wound. The tensile strengths of the extract and nitrofurazone ointment treated wounds are compared with control. Tensile strength increment indicates better wound-healing promotions of the applied drug

Dead Space Wound

Three groups of animals containing ten in each group are anaesthetized by open mask method with anaesthetic ether. Dead space wounds are created by subcutaneous implantation of sterilized polypropylene tubes (2.5 × 5 cm). The test drug is administered at different doses based on the design of the experiment for a period of ten days. The granuloma tissues formed on the tubes are harvested on the 10th postwounding day. The buffer extract of the wet granuloma tissue is used for the determination of lysyl oxidase activity, protein content, and tensile strength. Part of granuloma tissue is dried, and the acid hydrolysate is used for the determination of hydroxyproline, hexosamine, and hexuronic acid.

The progresses of wound healing in excision and incision wound method have to be studied. The measurement of the tensile strength, that is, the effect of the extract and standard drug on the wound-healing process by incision wound method have to be studied. Results are expressed as mean ± SE and compared with the corresponding control (simple ointment) values; p-values are calculatedby student’s t-test by comparing with control. Percentages of wound contractions are calculated with respect to the corresponding 0 day’s wound area (mm2 ).

The contractions of wound with all the drugs comparing with simple ointment (control) are measured. The epithelialization period of the wound area of the extract treated group are compared with standard drug treated group. Tensile strength of wounds of rats treated with standard drug (nitrofurazone ointment), in case of incision wound model is measured increment in tensile strength indicates better wound healing.

The histopathological examination of the tissues of the wound area treated with extract, standard drug is performed. In these studies, test drugs (herbals) with good activity showed rapid increase in tissue regeneration in skin wounds, more relative fibrosis. The skin adrenal structures like pilocebaceous glands, sweat glands, etc. are better presented in wounds treated with extract compared to standard drug treated animal wounds.

The changes in the biochemical parameters affecting wound healing in dead space wound model like, granuloma weight, lysyl oxidase activity, as well as protein content are to be measured which is usually increased with effective test drugs. The hydroxyproline, hexuronic acid, hexosamine level are measured which are increased considerably. The observed increase in tensile strength could be attributed to the increase in lysyl oxidase activity which is responsible for cross-linking and maturation of collagen. The reduction in granuloma weight is also due to better maturation of collagen, which invariably leads to shrinkage of granulation tissue. However, in this case, the observed increase in tensile strength is not only due to increased cross linking via lysyl oxidase but also due possibly to interactions (noncovalent, electrostatic) with the ground substance as evidenced by a highly significant increase in the hexosamine content.

Wound healing involves different phases such as contraction, epithelialization, granulation, collagenation, etc. The glycosidal mixture of extract of Centella asiatica has been reported to be responsible to enhance incised wounds healing (Rosen et al., 1967) and in stimulating collagen in human skin fibroblast cell (Vogel and DeSouza, 1980).

Biological Screening of Herbal Drugs

Biological Screening of Herbal Drugs

INTRODUCTION

NEED FOR PHYTOPHARMACOLOGICAL EVALUATION

NEW STRATEGIES FOR EVALUATING NATURAL PRODUCTS

ERRORS IN SCREENING PROCEDURES

SCREENING METHODS FOR ANALGESIC AGENTS

Peripherally Acting Analgesics

SCREENING METHODS FOR ANTIDIABETIC AGENTS

Models for Insulin-Dependent Diabetes Mellitus

Hormone-induced diabetes

Genetic Models

Models for NIDDM

Other Chemically Induced NIDDM Models

Chelating Agents

Models for Insulin Sensitivity and Insulinlike Activity

EVALUATION OF ANTIDIARRHOEAL AGENTS

Gastrointestinal Motility Tests

PGE2 -Induced Enteropooling

SCREENING METHODS FOR ANTIFERTILITY AGENTS

Screening Methods for Antifertility Activity in Females

Screening Methods for Antiovulatory Activity

Screening Methods for Oestrogenic Compounds: In Vivo Methods

Vaginal cornification

The experimental procedure for taking vaginal smears

Chick oviduct method

SCREENING METHODS FOR ANTIINFLAMMATORY AGENTS

Testing of Drugs Preventing Acute and Sub-Acute Inflammation

Croton oil ear oedema in rats and mice

Testing of Drugs Preventing the Proliferative Phase (Granuloma Formation) of Inflammation

SCREENING METHODS FOR ANTIPYRETIC AGENTS

Antipyretic Testing in Rats

Antipyretic Testing in Rabbits

Procedure

SCREENING METHODS FOR ANTIULCER AGENTS

Pylorus Ligation in Rats

Ulcers Through Immobilization Stress

Stress Ulcer by Cold Water Immersions

SCREENING METHODS FOR HEPATOPROTECTIVE AGENTS

Hepatitis in Long-Evans Cinnamon Rats

Allyl Alcohol-induced Liver Necrosis in Rats

Carbon Tetrachloride-induced Liver Fibrosis in Rats

Bile Duct Ligation-induced Liver Fibrosis in Rats

Country-made Liquor Model

SCREENING METHODS FOR WOUND-HEALING AGENTS

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