Men, Medicine and Nature

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Men, Medicine and Nature

1. INTRODUCTION

Sound health and sound mind is the primary concern of a man. Again good health helps to maintain a happy mind. That’s why it is called that “health is wealth”. But very usually man is facing obstacles in keeping his good health right from the very beginning of his journey in this world. Naturally for the survival, man fights against any disastrous happening with him and thus wants to overcome any obstacle. Suffering from various ailments and injuries was the commonest occurrence for the ancient man. And whenever man used to become helpless with this type of incidents he used to apply his instinct and sixths senses to use something that were available in his surroundings to allay his sufferings. In most cases it was the plant or plant parts that were used as remedies. It is not certain when and how man learned to use the plant appropriately but it is thought that animal was the first guide to show him the use of plants. Because animals naturally know or feel how to get relief from the unusual internal conditions. We still observe such incidents incase of our pets-it’s an example. On the other hand, Muslim faith informs us that Adam was the first guide to make us understand about the virtue of plants by self-taking of ‘Gandam’ fruit. But previous one has much more logical and scientific basis. By and by, whether it was the guidance of animals or basic instinct of man or sudden practices on assumptions, man came to know about the therapeutic value of many plants or plant parts and thus their use are coming off from generation to generation. On the starting there was no particular writing method to preserve these experiences and verbally those were supplied to various persons and continuous using on similar conditions made man to keep the experience in their mind. Later these were written down and still these are being used as those were or in somewhat modified form. The most important thing today is that those are now being tried to prove scientifically and in this way synthetically development are being advanced to meet the greater necessity or to conform the purity and specificity.

As for example, we can set some of our modern drugs like digitoxin, atropine and the narcotic analgesics like morphine that have been developed from plants. Again thousands of plant metabolites are being used in the treatment of variety of diseases. A few interesting examples of plant metabolites include taxol from Taxus brevifolia, vincristine and vinblastine from Vinca rosea Linn., all of which are important anticancer agents being used clinically. In the current popular field of chemotherapy, cepheranthine isolated from Stephania cepharantha and Stephania sasaki is being used as a prophylactic in the management of tuberculosis. So it is clear that the primitive use of plants or herbs played a great role in the development of modern medicine. Though the plant or plant parts was the main concern of treatment, at that time whenever man became unsuccessful to overcome any injuries or diseased conditions, he used to apply religious beliefs and thus offered prayers for the relief.

Thus often happening of these types of situations created some verses or phrases or words or prayers or tortures or other physical acts to reduce his sufferings. There was another reason for the development of those rhythmic verses or words or prayers or phrases-that was the intention of the religious chiefs to take or increase their power over the general people, because they claimed that these religious facts were given to them by the ancestral Gods. As a result this became confined in the hands of the religious chiefs for many countries and it became a sort of secret magic and once it took the secondary importance. However all those practices are collectively known as traditional medicine as these were being practiced from generation to generation with the traditional belief?

The basic concept of this system of medicine has been very comprehensively described by the World Health Organization in this way: “Traditional medicine is the sum total of all knowledge and practice, whether explicable or not, used in the diagnosis, prevention and elimination of physical, mental, or social imbalance, relying exclusively on practical experience and observations handed down from generation to generation verbally or in writing”

1.2 Medicinal Plants-The Best Friend to Human Being in Nature

The universe is the greatest store that includes everything for the suitable survival of animals and plants. For this reason, the greatest creation of God, man can enjoy a comfortable and smooth life by using the plants and other animal as sources of food, clothing, shelter and medicine.

The contributions of the plants are numerous in every sector of human life. It helps to growing up of the human body and also protects human being from sickness by being used as medicine. A large number of plants are used as medicinal agents in this world. Specifically in Bangladesh about two hundred fifty species are used as medicinal plants.

In nature plants of several varieties are available which are responsible for various pharmacological actions. They are termed as medicinal plants. On the other hand, some of them produce harmful effects on animal systems. They are termed as toxic or poisonous plants. It has now been established that the plants which naturally synthesize and accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils and contain minerals and vitamins possess medicinal properties. A medicinal plant may thus be defined as a plant which, in one or more of its organs contains substances that can be used for therapeutic uses or which are precursors for the synthesis of useful drugs.

However, ideally a definition of medicinal plants should include the following:

a) Plants or plant parts used medicinally in galenical preparation (e.g. decoctions, infusions, etc).

b) Plants used for extraction of pure substances either for direct medicinal use or for the synthesis of medicinal compounds (e.g. synthesis of sex hormones).

c) Food, spice and perfumery plants used medicinally

d) Microscopic plants, e.g. fungi, actinomycetes, used for isolation of drugs, especially antibiotics.

Fibre plants, e.g. cotton, flax, jute, used for the preparation of surgical dressings.

1.3 Historical Background of Medicinal Plants

Since disease, decay and death have always co-existed with life, the study of diseases and their treatment must also have been contemporaneous with the dawn of human intellect. The primitive man must have used as therapeutical agents and remedial measures those things which he was able to procure most easily. There is no authentic record of medicines used by the primitive man.

Illness, physical discomfort, injuries, wounds and fear of death had forced early man to use any natural substance that he could lay his hand on, without any resistance, for relieving the pains and sufferings caused by these abnormal conditions and for preserving his health against disease and death.

Primitive peoples in all ages have had some knowledge of medicinal plants, derived as the result of trial and error. These primitive attempts at medicine were based on intuition, guesswork, superstition, or trial and error. Most savage people have believed that disease was due to the presence of evil spirits in the body and could be driven out by the use of poisonous or disagreeable substances calculated to make the body an unpleasant place in which to remain. The knowledge regarding the source and use of the various products suitable for this purpose was usually restricted to the medicine men of the tribe. As civilization progressed the early physicians were guided in great part by these observations.

Rig Veda (4500-1600 BC), which is the oldest book in the library of man supplies various information on the medical use of plants in the Indian subcontinent. It was noted that Indo-Aryans used the soma plant (Amanita muscaria, a narcotic and hallucinogenic mushroom) as a medicinal agent. It is unfortunate that the Ayur Veda is no more available in its original form but the most authentic and original texts considered as the renowned representatives of the original Ayur Veda, are the encyclopedic Agnivesha or Charaka Samhita and Sushruta samhita. The Indo-Aryans used the plant for sacrificial purposes and its juice is described in the ancient Aryan literature as stimulating beverage. The word oshadhi literally means heat -producer. When the Indo-Aryans came to use the soma plant for therapeutic purposes, they came to possess knowledge of the medicinal properties and uses of herbs and plants. Hence, Oshadhi applied to all herbs and medicinal plants. The Vedas made many references to healing plants including Sarpagondha (Rauwolfia serpentina), while a comprehensive Indian herbal, the Charaka Samhita, cites more than 500 medicinal plants.

As far as records go, it appears that Babylonians (about 3000years BC) were aware of a large number of medicinal plants and their properties. As evident from the Papyrus Ebers (about 1500 BC), the ancient Egyptians possessed a good knowledge of the medicinal properties of hundreds of plants. Many of the present day important plant drugs like henbane (Hyoscyamus spp.), mandrake (Mandragora officinarum), Opium (latex of Papaver somniferum fruit), pomegranate (Punica granatum), castor oil (oil of Ricinus communis seeds), aloe (juice of Aloe spp.), onion (Allium cepa) and many other were in common use in Egypt about 4500 years ago.

The Pen Tsao, the earliest known Chinese pharmacopoeia, appeared around 1122 BC attributed to the legendary emperor Shen Nung, this authoritative work described the use of Chaulmoogra oil (from the seed of Hydrocarpus kurzii ) to treat leprosy.

The practice of herbal medicine flourished most during the Greek civilization. When historical personalities like Hippo crates (born in 460 BC) and Theophrastus (born in 370 BC) practiced herbal medicine as he was distinguished physician, practicing and researching into herbal medicine. His materia medica consists of some 300 to 400medicinal plants. The far ranging scientific work of Aristotle (384-322 BC), a Greek philosopher, included an effort to catalogue the properties of the various medicinal herbs at that time. The Greek writer–physician Dioscorides (60 AD) who wrote the famous treatise De Materia Medica, (published in78AD) which contained the description of 600 medicinal plants. Two of the 37 volumes of books written by Pliny De Elder (23-70 AD) which included a large number of medicinal plants. The great Greek pharmacist-physician Galen (133-200 AD), who wrote about 500 volumes of books describing hundreds of recipes and formulation of medicinal preparations containing both plant and animal ingredients. Allopathic and Homeopathic systems of medicine today are based on the doctrine expatiated by Galen.

After the dark ages were over, there came the period of the herbalists and encyclopedists, and the monasteries of Northern Europe produced vast compendiums of true and false information regarding plants, stressing in particular the medicinal value and folklore. It was about this time that the curious “Doctrine of Signatures” came into being. It was developed by Paracelsus (1490-1541 AD), a Swiss alchemist and physician. According to this superstitious doctrine all plants possessed some sign, given by the Creator, which indicated the use for which they were intended. Thus a plant with heart-shaped leaves should be used for heart ailments, the liver leaf with its three lobed leaves for liver troubles, and so on. Many of the common names of our plants of today owe their origin to this curious belief.Such names as heartsease, Solomon’s-seal, dogtooth violet, and liverwort carryon the old superstition.

From this crude beginning the study of drugs and drug plants has progressed until now Pharmacognosy and Pharmacology are essential branches of medicine⁷.

1.4 Medicinal Plants Used In Traditional Medicine

Hakim Mohammed Said, a noted traditional medicine expert, advocates that Arab, Chinese and Indian systems of medicines are not static but progressing from generation to generation. Recent trend is to integrate the traditional medicine with modern medicines. Best therapeutic results are said to be obtained often with the traditional Chinese system.

We have seen that some manufacturers of traditional medicines use wrong parts of medicinal plants in preparing their medicines. Medicinal plants used in traditional medicine are often collected in the wrong season, at the wrong time of the day and at the wrong stage of their growth due to ignorance on the part of the collectors. These problems greatly affect the quality of traditional medicine, prepared from plant origin.

1.5 Contribution of Medicinal Plants to Modern Medicine

Plants and man are inseparable. The human race started using plants as a means of treatment of diseases and injuries from the early days of civilization on earth and in its long journey from ancient time to modern age the human race has successfully used plants and plant products as effective therapeutic tools for fighting against diseases and various other health hazards.

Scientists identified and isolated different chemical constituents from plants, which have been used to prepare modern medicines. In course of time their synthetic analogues have also been prepared. In this way, ancient uses of Datura plants have led to the isolation of hyoscine, hyoscyamine, atropine and tigloidine; Cinchona bark to quinine and quinidine, Rawolfia serpentina to reserpine and rescinnamine, Digitalis purpurea to digitoxin and digoxin, Opium to morphine and codeine, Ergot to ergotamine and ergometrine, Senna to sennosides, Catharanthus roseus to vinblastine and vincristine⁴.

A recent survey by the United Nations Commission for Trade and Development (UNCTAD) indicated that about 33% of drugs, produced in the developed countries, are derived from plants (UNCTAD/GATT 1974: Annual Report, Geneva, Switzerland) and that if microbes are added, 60% of medicinal products are of natural origin⁵. According to some sources almost 80% of present-day medicines are directly or indirectly derived from plants¹⁰. Surprisingly, this large quantity of modern drugs comes from less than 15% of the plants, which are known to have been investigated pharmacologically, out of an estimated 250000 to 50000 species of higher plants growing on earth¹¹. More than 47% of all drugs, used in Russia, are obtained from botanical sources¹².

At present, thousands of plant metabolites are being successfully used in the treatment of variety of diseases¹¹. A few striking examples of plant metabolites include taxol from Taxus brevifolia¹³, vincristine and vinblastine from Vinca roseus¹⁴. All of which are important anticancer agents being used clinically. In the current popular field of chemotherapy, cepharanthine, isolated from Stephania cepharantha and Stephania sasaki¹⁵ is being used as a prophylactic in the management of tuberculosis.

Even today 80% of the rural population of most developing countries of the world, depends on herbal medicine for maintaining its health and well being¹⁶. The consumption of medicinal plants is increasing in many developed countries, where 35% of drugs contain active principles from natural origin¹⁷.

In China, about 15000 factories are involved in producing herbal drugs, herbal medicines have been developed to a remarkable standard by applying modern scientific technology in many countries, such as, China, India, Bangladesh, Srilanka, Thailand and United Kingdom. In these countries, the dependence on allopathic drugs has been decreased to greater extent¹⁸.

One hundred and seventy drugs from different plants, which are or once official in the USP or NF, were used by the North American Indians. In 1960, 47% of drugs, prescribed by physicians in the United States of America, were from natural sources¹⁹. In 1967, 25% of the products, which appeared in 1.05 billion prescriptions filled in the United States, contained one or more ingredients derived from higher plants²⁰.

In the United States, in 1980, the consumers paid 8 billion dollars for prescription drugs in which the active ingredients are still derived from plants⁵. 47% of some 300 million new prescriptions written by physicians in America in 1961, contained as one or more active ingredients, a drug of natural origin²¹. “Modern medicine still has much to learn from the collector of herbs”, said Dr. Halfdan Mahler, Director General of the World Health Organization. Many of the plants, familiar to the witch doctor, really do have the healing power that tradition attaches to them. The age-old art of the herbalist must be tapped¹⁰.

Thus it is apparent that whatever progress, science might have made in the field of medicine over the years, plants still remain as the primary source of supply of many important drugs, used in modern medicine. Indeed, the potential of obtaining new drugs from plant sources is so great that thousands of substances of plant origin are being studied for activity against such formidable foes as heart diseases, cancer, and aids⁶. In this way, modern medicine will continue to be enriched by the introduction of newer and more potent drugs from plant sources.

1.6 Chemical Constituents of Medicinal Plants

Plants have been serving the animal kingdom as its source of energy (food, fuel) as well as its means of shelter and sustenance since the very beginning of its existence on earth’s surface habitable for the animals.

In addition to providing the animal kingdom its food, fuel and shelter, each of these plants has been synthesizing a large variety of chemical substances since their first day of life on earth. These substances include, in addition to the basic metabolites, phenolic compounds, terpenes, steroids, alkaloids, glycosides, tannins, volatile oils, contain minerals, vitamins and a host of other chemical substances referred to as secondary metabolites which are of no apparent importance to the plants own life. But many of these compounds have prominent effects on the animal system and some possess important therapeutic properties which can be and have been utilized in the treatment and cure of human and other animal diseases since time immemorial. These secondary metabolites differ from plant to plant. Thus, the plant kingdom provides a tremendous reservoir of various chemical substances with potential therapeutic properties.

The chemical constituents, which are capable of influencing the physiological systems of the animal body by exerting some pharmalogical actions, are designated as the active chemical constituents or simply active constituents. In short, it may be said that the chemical constituents present in the medicinal plants constitute the most important aspect of all medicinal plants.

Green leaves are the sites of a great deal of such chemical activity. The chemical substances with medicinal properties found in some plants are the products of such chemical processes. The occurrence of the active chemical substances in all parts of the plant body or they may be accumulated in higher concentrations in some specific part.

The types of chemical constituents are as follows:

A. Alkaloids and amides

· Pyridine group

· Tropane group

· Isoquinoline group

· Quinoline group

· Quinolizidine group

· Indole group

· Steroidal group

· Imidazole group

· Phenylthylamine group

· Alkaloidal amines

B. Antibiotic and Anti-inflammatory principles

C. Bitter and Pungent principles

D. Volatile oils and Fixed oils

E. Glycosides

· Anthraquinone glycosides

· Cardiac glycosides

· Saponin glycosides

· Thiocyanate glycosides

· Other glycosides

F. Gum-resins and Mucilage

G. Vitamins and Minerals.

1.9 Inflammation – An Overview

Inflammation is the characteristic response of mammalian tissue to injury. Whenever the tissue is injured there follows at the site of injury a series of events that tend to destroy or limit the spread of the injurious agent.

Inflammation is fundamentally a protective response. It is closely intertwined with the process of repair. Inflammation serves to destroy, dilute, or wall off the injurious agent, but it in turn, sets into motion a series of events that, as far as possible, heal and reconstitute the damage tissue. Without inflammation, infections would go unchecked, wounds would never heal, and injured organs might remain permanent festering sores. However, inflammation may be potentially harmful, causing life-threatening hypersensitivity reactions, progressive organ damage, and scarring.

Etiology of inflammations:

The agents that injure tissue and therefore evoke the inflammatory response include:

1. Physical agents: e.g. Physical agents such as excessive heating or cooling, ultraviolet or ionizing radiation or mechanical trauma.

2. Chemical agents: e.g. Chemical substances including toxins from various bacteria.

3. Hypersensitivity: e.g. reaction of antibody or of sensitized lymphocytes with bacterial or other antigens.
4. Infection: Microbial infection is a very important cause of inflammation. Microorganism may injure tissue in several ways by release of exo- or endo-toxins, by intracellular multiplication followed by cell death as seen in many viral infections.
5. Necrosis: From almost any cause leads to release of substances, which induce inflammation in adjacent living tissue.

Cardinal signs of inflammation:

1. Redness

2. Swelling

3. Heat

4. Pain

5. Loss of function

Types of inflammations:

According to its duration and predominant inflammation cell type, inflammation is divided into acute or chronic pattern. The vascular and cellular responses of both acute and chronic inflammation are mediated by chemical factors derived from plasma or cells and triggered by inflammatory response.

Acute Inflammation:

Acute inflammation is the immediate and early response to an injurious agent. This is relatively non-specific, its main role, being to clear away dead tissues, protect against local infection, and allow the immune system access to the damaged area. The major components of acute inflammation are:-

1. Alteration in vascular caliber that lead to an increase in blood flow.
2. Structural changes in the microvasculature that permit the plasma proteins and leukocytes to leave the circulation.
3. Emigration of the leukocytes from the microcirculation and their accumulation in the focus of injury.

i. Vascular changes

Inflammation causes change in vascular process in the affected areas. Such changes are described in the following steps:

ii. Change in vascular flow and caliber

Change in vascular flow and caliber being very early after injury and develop at varying rates, depending on the severity of the injury. The changes occur in the following order:

Injurious agents cause an inconstant and transient vasoconstriction of arterioles then follow the vasodilation. The first involves the arterioles and then results in opening of new capillary bed in the area. Slowing of the circulation is brought about by increased permeability of the microvasculature, with the outpouring of protein-rich fluid into the extravascular tissues then leads to stasis. The increased permeability is the cause of edema.

iii. Increased vascular permeability (vascular Leakage)

Increased vascular permeability is the hallmark of acute inflammation. The loss of protein from the plasma reduces the intravascular osmotic pressure and increases the osmotic pressure of the interstitial fluid. Together with the increased hydrostatic pressure owing to vasodilation, this leads to a marked outflow of fluid and its accumulation in the interstitial tissue. The mechanisms purposed for endothelial permeability they are:-

i. Formation of endothelial gaps in venules (elicited by histamine, bradykinin, leukotrienes, substance-P, and many other chemical mediators).

ii. Cytoskeletal reorganization. The endothelial cells undergo a structural reorganization of the cytoskeleton, such that endothelial cells retract from one another.

iii. Increased transcytosis across the endothelial cytoplasm: Transcytosis occurs across channels consisting of clusters of interconnected, uncoated vesicles and vacuoles called the vesiculovacuolar organelle, many of which are located close to intercellular junctions.

iv. Direct endothelial injury, resulting in endothelial cell necrosis and detachment: This effect is usually encountered in necrotizing injuries and is due to direct damage to the endothelium by the injurious stimulus, as, for example, by severe burns or lytic bacterial infections.

v. Delayed prolonged leakage is a curious but relatively common type of increased permeability that begins after a delay of 2 to 12 hours, lasts for several hours or even days, and involves venules as well as capillaries.

vi. Leukocytes-mediated endothelial injury: leukocytes adhere to endothelium relatively early in inflammation. As seen subsequently, such leukocytes may be activated in the process, releasing toxic oxygen species and proteolytic enzymes, which then causes endothelial injury or detachment- resulting in increased permeability.

vii. Leakage from new blood vessels: New vessels sprouts remain leaky until endothelial cells differentiate and form intercellular junctions. In addition, certain factors that cause angiogenesis also increase vascular permeability.

iv.Cellular events: Leukocyte Extravasation and Phagocytosis

A critical function of inflammation is the delivery of leukocytes to the site of injury. The sequence of events in this journey called extravasatyion, includes: —–

1) Margination, rolling and adhesion of leukocytes in the lumen

2) Transmigration across the endothelium (also called diapedesis)

3) Migration in interstitial tissues toward a chemotactic stimulus

v. Adhesion and transmigration

These occur largely due to interactions between complementary adhesions molecules on the leukocytes and on the endothelium. Chemical mediator’s adhesion pairs include:

1) The selectins (E, P and L) which bind through their lectin (sugar binding) domains to oligosaccharides (e.g. sialylated Lewis X), which themselves are covalently bound to cell surface glycoproteins.

2) The immunoglobulin family, which includes the4 endothelial ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1).

3) The integrins, which function as receptors some of the members of the immunoglubin family and the extracellular matrix. The principal integrin reveptors for ICAM-1 are the b-integrins LFA-1 and MAC-1 (CD11a/CD18 and CD11b/CD18), and those for VCAM-1 are the integrins a₄b₁ (VLA4) and a₄b₇.

It is now thought that neutrophils adhesion and transmigration in acute inflammation occur by a series of overlapping steps: –

1) Endothelial activation: Mediators present at the inflammatory sites increase the expression of E-selectin and P-selectin be endothelial cells.

2) Leukocyte rolling: There is an initial rapid and relatively loose adhesion, resulting from interactions between the selectins and their carbohydrate ligands.

3) Firm adhesion: The leukocytes are then activated by chemokines or other agents to increase the avidity of their integrins.

4) Transmigration: This is mediated by interaction between platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) on leukocytes and endothelial cells.

vi. Chemotaxis and leukocyte Activation

Adherent leukocytes emigrate through interendothelial junctions, traverse the basement membrane, and move towards the site of injury along a gradient of chemotactic agents. Neutrophils emigrate first and monocytes follow. Chemotactic agents for neutrophils include bacterial products, complement fragments, arachidonic acid metabolites (e.g. Leukotriene B-4) and certain cytokines.

Chemotaxis involves binding of chemotactic agents to receptors on leukocytes, phospholipase C activation, incteased intracellular calcium, activation of protein kinase-C, protein phosphorylation leading to activation of intracellular contractile proteins. Locomotion is controlled by the effects of calcium ions and phosphoinositols on actin regulatory proteins such as gelsolin and filamin.

vii. Phagocytosis

Phagocytosis involves attachment of opsonized particles to Fe and C3b receptors on the surface of leukocytes; Engulfment by pseudopods encircling the phagocytosed paricles, creating a phagosome; Fusion of lysosomal granules with the phagosome, leading to de-granulation; Killing and /degradation of bacteria.

viii. Extracellular Release

During phagocytosis, leukocytes release; Lysosomal enzymes by regurgitation during feeding, reverse endocytosis and cytotoxic release; Oxygen derived active metabolites; Products of arachidonic acid metabolism.

ix. Defects in Leukocyte function

These interfere with inflammation and inctease susceptibility to infection. They include both genetic and acquired defects.

Deficiency in the number of circulating cells (neutropenia)

1) Defects in adherence (e.g. leukocyte adhesion deficiency) type I and II

2) Defects in migration and chemotaxis

3) Defects in phagocytosis (e.g. diabetes mellitus)

4) Defects in mivrobicidal activity. In chronic grnulomatous disease there are ingerited defects in NADPH oxidase leading to a defect in the respiratory burst, hydrogen peroxide production and the MPO-hydrogen peroxide halide bactericidal mechanism.

x. Major events in inflammation

F Hyperemia

F Exudation of fluid

F Cellular Exudation

F Emigration of leukocytes

xi. Outcome of acute inflammation

Acute inflammation may results in:

1. Complete resolution, with regeneration of native cells and restoration of the site acute inflammation to normal.

2. Healing by connective tissue replacement and scarring, which occurs after substantial tissue destruction, when the inflammation occurs in tissues that do not regenerate or when there is abundant fibrin exudation.

3. Abscess formation.

4. Progression to chronic inflammation.

Chronic Inflammation:

Chronic inflammation is of longer duration and is associated histologically with the presence of lymphocytes and macrophages, the proliferation of blood vessels, fibrosis, and tissue necrosis.

Chronic inflammation arises under the following settings:

1.Persistent infections by certain microorganism, such as tubercle bacilli having low toxicity and evoke an immune reaction called delayed hypersensitivity.

i. Prolonged exposure to potentially toxic agents, either exogenous or endogenous.

ii. Autoimmunity: under certain conditions, immune reactions are set up against the individual’ own tissues, leading to autoimmune diseases.

1.10. CENTRAL NERVOUS SYSTEM DEPRESSANT

Central nervous system depression or CNS depression refers to physiological depression of the central nervous system that can result in decreased rate of breathing, decreased heart rate, and loss of consciousness possibly leading to coma or death. CNS depression often results from the use of depressant drugs such as alcohol, opioids, barbiturates, benzodiazepines,general-anesthetics, and anticonvulsants such as valproate semisodium used to treat epilepsy. Drug overdose is often caused by combining two or more depressant drugs, although overdose is certainly possible by consuming a large dose of one depressant drug. CNS depression can also be caused by the accidental or intentional inhaling of certain volatile chemicals such as Butanone (contained in Plastic Cement).

Other common causes of CNS depression are metabolic disturbances, such as hypoglycaemia.

CNS depression is treated within a hospital setting by maintaining breathing and circulation. Individuals with reduced breathing may be given supplemental oxygen, while individuals who are not breathing can be ventilated with bag valve mask ventilation or by mechanical ventilation with a respirator. Sympathomimetic drugs may be used to attempt to stimulate cardiac output in order to maintain circulation. CNS Depression caused by certain drugs may respond to treatment with an antidote.

2. OBEJECTIVE AND RATIONALITY OF THE PRESENT STUDY:

The use of plant products- crude extracts and metabolites in the treatment of various diseases has not been yet abolished from the modern world. Rather in many parts of the world, herbal medicine is being popular day by day and the usage rate is increasing. Besides in underdeveloped and developed countries, traditional herb based therapy has always got a significant contribution to the total health care system. Since Bangladesh is enriched with medicinal plants, the present study may direct a significant way of making best use of these resources. Majorities of our population, who are impoverished, have to rely on the indigenous system of medication due to their inability to meet the cost of modern medicine. Many of the indigenous plants of our country don’t have any scientific bases behind their folklore use. Again plants do have varied types of molecular entities within and it is not unlikely that a particular plant that has got traditional indication for a particular disease may exhibit beneficial role in some other ailments. That is why the present study- bioactivity guided phytochemical investigation of Piper betle Linn. (Piperaceae) is primarily aimed at —

1. Rationalization of the traditional use of the selected plant

2. Exploration of possible newer medicinal activities of the same plant and

3. Isolation of the bioactive principles.

2.1. Present Study Protocol

1. Extraction of the powdered plant parts of leaf of Mimosa pudica.

2. Preliminary biological screening of the crude extract for analgesic,anti-inflammatory activity and CNS depressant for which the plant has popular folklore usage.

3. Establishment of dose – response relationship.

4. Analysis of statistical significance of all the experiments conducted.

3. PLANT DESCRIPTION:

Mimosa pudica:

Mimosa pudica (from Latin: pudica “shy, bashful or shrinking”; also called sensitive plant and the touch-me-not), is a creeping annual or perennial herb often grown for its curiosity value: the compound leaves fold inward and droop when touched or shaken, re-opening minutes later. The species is native to South America and Central America, but is now a pantropical weed.

3.2. Description:

Mimosa is a deciduous, small to medium-sized tree that can grow 20 to 40 feet tall. It is a member of the legume (Fabaceae) plant family and is capable of fixing nitrogen. The bark is light brown and smooth while young stems are lime green in color, turning light brown and covered with lenticels. Leaves are alternately arranged and bipinnately compound (6 to 20 inches long), having 20 to 60 leaflets per branch. The leaf arrangement gives mimosa a fern-like or feathery appearance. The flowers are fragrant and pink in color, about 1½ inches long. Fruits are flat and in pods, a characteristic of many legumes. Pods are straw-colored and 6 inches long containing 5 to 10 light brown oval-shaped seeds about ½ inch in length. Pods typically persist on the plant through the winter months.

Mimosa reproduces both vegetatively and by seed. Seeds require scarification in order to germinate. This characteristic allows the seed to remain dormant for many years. Normally seeds are dispersed in close proximity of the parent plant; however, seeds can also be dispersed by water. Wildlife may also contribute to the spread of mimosa through the ingestion and excretion of the seeds. Vegetative reproduction occurs when trees are cut back, causing quick resprouting and regrowth.

Extracts of the plant have been shown in scientific trials to be a moderate diuretic, depress duodenal contractions similar to atropine sulphone, promote regeneration of nerves, and reduce menorrhagia (Modern-natural 2001). Anitdepressant activity has been demonstrated in humans (Martínez and others 1996). Root extracts are reported to be a strong emetic (Guzmán 1975).”

The leaves are bipinnate, meaning that they are compound leaves consisting of many leaflets arranged on side-branches off the main axis. These leaflets are called pinnae, and these are sub-divided into many little leaflets called pinnules, thus giving the plant a fern-like appearance. The pinnae are 2.5 to 5cm long and are made up of elliptical, 0.5cm long pinnules arranged in rows of opposite pairs.

3.4. Taxonomy and nomenclature

Scientific classification
Kingdom:	Plantae
(unranked):		Angiosprms
(unranked):			Eudicots
(unranked):			Rosids
Order:			Fabales
Subfamily:			Mimosoideae
Genus:			Mimosa
Species:			M. pudica

Biological:

There are no known biological control agents for the control of mimosa. The plants like a warm sunny position and a relatively humid atmosphere.

Chemical:

Mimosa seedlings and small trees can be controlled by applying a 2% solution of glyphosate or triclopyr plus a 0.25% non-ionic surfactant to thoroughly wet all leaves. Systemic herbicides such as glyphosate and triclopyr can kill entire plants because the chemicals travel through a plant from the leaves and stems to the actively growing roots. Triclopyr is a selective herbicide for many broad-leaved plant species and should be considered for sites where native or other desirable grasses are meant to be conserved.

The cut-stump and basal bark herbicidal methods should be considered when treating individual trees or where the presence of desirable species preclude foliar application. Stump treatments can be used as long as the ground is not frozen. Horizontally cut stems at or near ground level. Immediately apply a 25% solution of glyphosate or triclopyr and water to the cut stump making sure to cover the outer 20% of the stump. Basal bark applications are effective throughout the year as long as water is not standing at time of application. Apply a mixture of 25% triclopyr and 75% basal oil to the base of the tree trunk to a height of 12-15 inches from the ground. Thorough wetting is necessary for good control; spray until run-off is noticeable at the ground line. Applications should be made to cut stumps within one minute of cutting.

For larger trees stem injections of imazapyr or triclopyr can be used. For trees already chopped down apply these herbicides to the stem and stump to prevent resprouting. For saplings, apply triclopyr as a 20% solution in commercially available basal oil, diesel fuel, or kerosene (2.5 quarts per 3-gallon mix) with a penetrant (check with herbicide distributor) to young bark as a basal spray. For resprouts and seedlings thoroughly wet all leaves with a surfactant in water with and triclopyr or glyphosate herbicide as a 2% solution (8 ounces per 3-gallon mix between July to October) or clopyralid as a 0.2- to 0.4% solution (1 to 2 ounces per 3-gallon mix between July to September).

PLANT DESCRIPTION

Documented Properties

& Actions:Antibiotic, antimicrobial, anti-neurasthenic, antispasmodic, diuretic, nervine, poison, sedativePlant

Chemicals

Include:Ascorbic-acid, crocetin, crocetin-dimethyl-ether, D-glucuronic-acid, D-xylose, linoleic-acid, linolenic-acid, mimosine, mucilage, norepinephrine, oleic-acid, palmitic-acid, sitosterol, stearic-acid

3.8. Chemical constituents

Mimosa pudica contains the toxic alkaloid mimosine, which has been found to also have antiproliferative and apoptotic effects. The extracts of Mimosa pudica immobilize the filariform larvae of Strongyloides stercoralis in less than one hour. Aqueous extracts of the leaf of the plant have shown significant neutralizing effects in the lethality of the venom of the monocled cobra (Naja Kaouthia). It appears to inhibit the myotoxicity and enzyme activity of cobra venom.

4. MATERIALS AND METHODS

4.1 Preparation of the Plant Extract

Sample

The sample was the leaf of the plant, Mimosa pudica to study its analgesic,anti-inflammatory activity and CNS depressant..

Collection of sample

The samples were collected from the Botanical garden of the Jahangirnagar University and verified with Taxonomy Division of the Botany Department of the university.

Method of drying and pulverizing

The fresh leaf were washed with water immediately after collection. Then they were dried in oven under 40-50⁰C for 48 hours. When the roots were properly dried they were ground by using a mechanical grinder to a coarse powder. Before grinding the machine was cleaned to avoid contamination. The coarse powder was then taken in a clean airtight container and prepared for extraction.

Method of extraction

The powdered leaves were weight (116gm) and filled in a clean extraction bag. The bag was placed in a soxhlet extractor. Then petroleum ether (500mL) was used for extraction. The extraction process was repeated for 10 cycles and in the same way methanol was used subsequently.

The extracts were collected and dried separately on water bath. The dried extracts were kept in desiccators.

Application of the extracts

Definite amount of the dried extract that was under study was weighed and dispersed in suitable amount of alcohol to get suitable means to administer to the experimental animals.

The experiments were carried out on albino mice (Swiss Strain). They were obtained from animal house of the Department of Pharmacy, Jahangirnagar University, Savar, Dhaka. Rats of 2-3 months old, weighing 200-250g, were used. The rates were housed in plastic cages. They were maintained at room temperature under conditions of natural light and dark schedule. The rats were fed with ‘rat chow’- prepared according to the formula developed by the Bangladesh Council for Scientific and Industrial Research (BCSIR), Dhaka. They were allowed to drink water ad libitum. The standard drug and extracts were administered to the stomach with the help of feeding needle fitted to syringe.

Standard Drug and its Solutions

Acetyl Salicylic Acid (Aspirin) (Purity 99.99%) was obtained from the store of the Pharmacy Department of Jahangirnagar University and used as standard. Solutions of the drug were prepared according to various dosages regimens in 0.2 ml of ethanol/100 g. B.W. Which were adjusted to a volume of 1 ml/100 g. B.W. with distilled water during feeding. Doses of the drug were selected as reported in different literature and pilot experiments were carried out in the study. All drugs were administered orally.

Inflammatory Agent

Among the many methods used for screening and evaluation of anti-inflammatory drugs, one of the most commonly employed techniques is based upon the ability of such agents to inhibit the acute inflammatory edema produced in the hind paw of the rat following injection of a phlogistic agent. In this experiment, the phlogistic agent of choice was carrageenin; a mucopolysaccharide derived from Iris sea moss, Chondrus carrageenin (Sigma, Japan) was prepared as 1% solution in water for injection.

Experimental Design

The following experimental study was designed to demonstrate the anti-inflammatory effect of Mimosa pudicaextract and to compare the effect with a standard anti-inflammatory drug (viz. Aspirin) on induced acute inflammation.

4.2.2 Carrageenin Induced Inflammation

Acute inflammation was induced as described by Winter et all (1962). A volume of 0.05 ml, 1% carrageenin was injected through a 26-gauge needle into the planter aponeurosis of the right hind paw of the rats. The rats were pretreated with test drugs before an hour of carrageenin injection. The maximum linear cross section of the joint (between the central region of the planter aponeurosis and origin of the extensor hallucis dorsis) was measured before carrageenin administration and similar measurement were made 3 and 4-hours after the administration of carrageenin to assess the progress of local inflammation edema. The mean increase in anterior posterior diameter of paw of each group at 1^st, 2^nd and 3^rd hour after carrageenin injection was calculated to observe the dependent activity of drugs.

For standard drug testing, increase in paw-diameter 3 hours after carrageenin administration was considered as a measure of effect. The percentage of edema with test drugs at different doses compared to control group was calculated.

4.2.3 Formalin test

The antinociceptive activity of the drugs was determined using the formalin test described by Dubuission and Dennis (1977). Control group received 5% formalin. 20 µl of 5% formalin was injected into the dorsal surface of the right hind paw 30 min after administration of Mimosa pudica extract (100 and 200 mg/kg, p.o.) and 30 min after administration of Diclofenac Na (10 mg/kg, i.p.). The mice were observed for 30 min after the injection of formalin, and the amount of time spent licking the injected hind paw was recorded. The first 5 min post formalin injection is referred to as the early phase and the period between 15 and 30 min as the late phase. The total time spent licking or biting the injured paw (pain behavior) was measured with a stop watch.

Grouping of Animals and Their Treatment

The animals in which inflammation was induced by formalin and carrageenin injection were grouped as follows. The animals were pretreated with the vehicle or drugs one hour prior to formalin and carrageenin injection.

Group I: This group consists of 3 mice. They were received vehicle, i.e., ethanol in a volume of 0.2 mo/100 g B.W. in dilute solution orally and served as control.

Group II: This group consisted of 3 mice, which Received indomethacin 10 mg/Kg. B.W. orally.

Group III (Methanolic extrac): This group included 3 mice that received Mimosa pudica 100 mg/mice orally.

Group IV (Methanol extract): This group included 3 mice that received Mimosa pudica 200 mg/mice orally.

4.2.4 Analgesic Activity

Experimental Animal

Young Swiss-albino mice of either sex aged 7-8 weeks, average weight 20-35 gm were used for the experiment. The mice were purchased from the animal Research Branch of the International Centre for Diarrheal Disease and Research, Bangladesh (icddr, b). They were kept in standard environmental condition (at 24.0±0°C temperature & 55-65% relative humidity and 12 hour light/12 hour dark cycle) for one week for acclimation after their purchase and fed (icddr, b) formulated rodent food and water ad libitum. The set of rules followed for animal experiment were approved by the institutional animal ethical committee.

Experimental Design

Twelve experimental animals were randomly selected and divided into four groups denoted as group-I, group-II, group-III and group-IV, consisting of 3 mice in each group. Each group received a particular treatment i.e. control, standard and the dose of the extracts of the plant respectively. Prior to any treatment, each mouse was weighed properly and the dose of the test sample and control materials was adjusted accordingly.

Method of Identification of Animals

Each group consisted of three mice. As it was difficult to observe the biologic response of three mice at a time receiving same treatment, it was quite necessary to identify individual animal of a group during the treatment. The animals were marked as M-1=Mice 1, M-2=Mice 2 and M-3=Mice 3,

Preparation of Test Materials

In order to administer the crude extract at dose of 100 mg/kg and 200mg/kg body weight of mice, required amount of extract was measured and was triturated unidirectional way by the addition of small amount of suspending agents (Tween-80). After proper mixing of extracts and suspending agent, normal saline was slowly added. The final volume of the suspension was made 5 ml. To stabilize the suspension, it was stirred well by vortex mixture. For the preparation of Indomethacin at the dose of 10 mg/kg-body weight, required amount of Indomethacin was taken and a suspension of 5 ml was made.

Grouping of Animals and Their Treatment

The animals in which pain was induced by acetic acid injection for writhing test were grouped as follows. The animals were pretreated with the vehicle or drugs half an hour prior to acetic acid injection.

Group I: This group consists of 3 mice. They were received vehicle, i.e., ethanol in a volume of 0.2 mo/100 g B.W. in dilute solution orally and served as control.

Group II: This group consisted of 3 mice, which Received diclofenac 10 mg/Kg. B.W. orally.

Group III (Methanolic extrac): This group included 3 mice that received Mimosa pudica root 100 mg/mice orally.

Group IV (Methanol extract): This group included 3 mice that received Mimosa pudica root 200 mg/mice orally.

Grouping of Animals and Their Treatment

The animals in which pain was induced by formalin injection for licking test were grouped as follows. The animals were pretreated with the vehicle or drugs half an hour prior to formalin injection.

Group I: This group consists of 3 mice. They were received vehicle, i.e., ethanol in a volume of 0.2 mo/100 g B.W. in dilute solution orally and served as control.

Group II: This group consisted of 3 mice, which Received diclofenac 10 mg/Kg. B.W. orally.

Group III (Methanolic extrac): This group included 3 mice that received Mimosa pudica root 100 mg/mice orally.

Group IV (Methanol extract): This group included 3 mice that received Mimosa pudica root 200 mg/mice orally.

Procedure:

At zero hour test samples, and Diclofenac-Na were administered orally by means of a long needle with a ball-shaped end. For control group acetic acid was administered by means of a syringe at that time.

After 30 minutes acetic acid (0.7%) was administered intraperitoneally to each of the animals of all the groups.

30 minutes interval between the oral administration of test materials and intra-peritoneal administration of acetic acid was given to assure proper absorption of the administered samples.

Five minutes after the administration of acetic acid, number of squirms or writhing were counted for each mouse for fifteen minutes.

Figure 8: Schematic representation of procedure for screening of analgesic property on mice by acetic acid induced method for Mimosa pudica root extracts.

Counting of Writhing

Each mouse of all groups were observed individually for counting the number of writhing they made in 15 minutes commencing just 5 minutes after the intraperitoneal administration of acetic acid solution. Full writhing was not always accomplished by the animal, because sometimes the animals started to give writhing but they did not complete it. This incomplete writhing was considered as half-writhing. Accordingly two half writhing were taken as one full writhing.

5. CNS depressant activity

5.1. Hole Cross Test

The method used was done as described by Takagi et al ^[19]. A steel partition was fixed in the middle of a cage (30 cm×20 cm×14 cm h). A hole (diameter 3 cm) was made in the steel partition at a height of 7.5