Biological Investigations of Catharanthus roseus (Apocynaceae)

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Biological Investigations of Catharanthus roseus (Apocynaceae)

Chapter-1
Introduction

1.1. Rationale and Objective of the Present Study:

Herbal medicine or botanical medicine or phytomedicine refers to the use of any plant’s seeds, berries, roots, leaves, bark, or flowers for medicinal purposes, (Barrett et al., 1999). It has long (about 3000 yrs) been considered as “the people’s medicine” for its accessibility, safety & the ease with which can be made, (Rabin, 2001). In this regard, the value of natural products can be assessed using 3 criteria: (1) The rate of introduction of new chemical entities of wide structural diversity, including serving as templates for semi synthetic & total synthetic modification. (2) The number of diseases treated or prevented by these substances & (3) Their frequency of use in the treatment of disease, (Chin et al 2006).

According to WHO, from 119 plant-derived pharmaceutical medicines, about 74% are used in modern medicine in ways that correlate directly with their traditional uses. Major pharmaceutical companies are currently conducting extensive research on plant materials gathered from the rain forests and other places for their potential medicinal value, (Jin, 2003). WHO also estimated that 4 billion people, 80% of the world’s population, presently use herbal medicine for some aspect of primary health care. Herbal medicine is a common element in Ayurvedic, homeopathic, naturopathic, traditional, oriental, Native American& Indian medicine, (Jin, 2003). Even among prescription drugs, at least 25% contain at least one compound derived from higher plants. The percentage might be higher if we include over-the-counter (OTC) drugs, Duke, 1990).

In developing countries, about 75% of the world populations rely on traditional medicine for their primary health care (Matu and Staden, 2003). Bangladesh, having a large variety of plant kingdom provided the ancient culture for the practice of herbal medicines.

The high cost of imported conventional drugs and/ or inaccessibility to western health care facility, imply that traditional mode of health care is the only form of health care that is affordable and available to our rural people. On the other hand, even when western health facilities are available, traditional medicine is viewed as an efficient and an acceptable system from a cultural perspective (Munguti, 1997). As a result, traditional medicines usually exist side by side with western forms of health, healing, weight loss or gain or maintenance, to survival and more.

New drugs derived from natural sources have been launched on the market during 2000-2005. These new drugs have been approved for the treatment of cancer, neurological diseases, infectious diseases, cardiovascular and metabolic diseases, immunological, inflammatory and related diseases, and genetic disorders, which encompass many of the common human diseases. Besides new drugs launched on the market from 2000 to the present, there are a variety of new chemical entities from natural sources undergoing clinical trials, (Newman et al., 2003).

The potential benefits of herbal medicines could lie in their high acceptance by patients, efficacy, relative safety, and relatively of low costs, (Thomas et al., 2001).Thus documentation of indigenous knowledge on the use of plants and providing an inventory of useful plants from local flora can be a great help for accurate use of traditional medicines. Identification and isolation of the active constituents from traditionally used ohyto-therapy can ensure the health care. In addition, herbal drugs can also be scientifically modified for better pharmacological activity and to establish safe and effective drugs. Moreover, there’s an added healing benefit to self-made remedies, (Cech, 2000).

Herbs are nutritional foundation nutrients and good alternative medicines to nourish the body’s deepest and most basic elements. From Aloe to St. John’s Wort, herbal medicines are mainstream in modern civilization. As we enter the 21st century and a new millennium, it may be urged that interest in herbal medicines &natural products is at an all time high. This phenomenon has been mirrored by an increasing attention to phytomedicine by the health professions inclusive of pharmacy and medicine. Thus to develop a phytomedicine is definitely a way of benefit of many more years for the humanity with a look to the future with a great deal of anticipation.

Thus the rationality of the ongoing study lies in meeting the challenge of developing herbal medicines for our own survival, which needs a systematic research on indigenous medicinal plants with scientific approach. And also to strengthen the existing health care system, chemical and biological analyses of an indigenous plant, Catharanthus roseus (Family: Apocynaceae) is the primary objective of the present study.

1.2. The Plant Family – Apocynaceae:

Catharanthus roseus is a renowned medicinal plant, belonging to the family Apocynaceae; and is a rich source of alkaloids, which are distributed in all parts of the plant. The alkaloid content of C. roseus varies considerably in various parts; the maximum being in the root bark which ranges from 0.15 to 1.34 % and even up to 1.79 in some strains.1 The plant contains about 130 alkaloids of the indole group out of which 25 are dimeric in nature. Two of the dimeric alkaloids vinblastine and vincristine mainly present in the aerial parts, have found extensive application in the treatment of human neoplasma. Among the monomeric alkaloids ajmalicine (raubacine) found in the roots has been confirmed to have a broad application in the treatment of circulatory diseases, especially in the relief of obstruction of normal cerebral blood flow.

1.2.1 . Apocynaceae Species Available in Traditional Use:

Table- Traditional Uses of Catharanthus roseus in various developed and developing countries.
Country Traditional Used
Australia Hot water extract of dried leaves is taken orally for menorrhagia, diabetes and extract of root bark is taken orally as febrifuge.
Bangladesh The hot water extract of dried entire plant is taken orally by human for diabetes mellitus.
China Hot water extract of the aerial parts is taken orally as a menstrual regulator.
Cook Island Decoction of dried leaves used orally to treat diabetes, hypertension and Cancer.
Dominica Hot water extract of leaves is taken orally by pregnant woman to combat primary inertia in childbirth and the boiled leaves are drink to treat diabetes.
England Hot water extract of dried entire plant is taken orally for the curing of diabetes.
Europe Decoction of dried leaves is taken orally for diabetes mellitus.
France Hot water extract of entire plant is taken as an antigalactagogue.
French Guina Hot water extract of entire plant is taken orally as a cholagogue.
India The hot water extract of dried entire plant is taken orally by human for cancer. Hot water extract of dried leaves is taken orally to Hodgkin’s disease. The root extract is taken orally for menorrhagia.
Jamaica Hot water extract of dried leaves is taken orally for diabetes.
Kenya Hot water extract of dried leaves is taken orally for diabetes.
Mexico Infusion of whole plant is taken orally for stomach problem.
Mozambique Hot water extract of leaves is taken orally for diabetes and rheumatism and the root extract is taken orally as hypotensive and febrifuge.
North Vietnam Hot water extract of the aerial parts is taken orally as a menstrual regulator.
Pakistan Hot water extract of dried ovules is taken orally for diabetes.
Peru Hot water extract of dried entire plant is taken orally by human adults for cancers, heart disease and leishmaniasis.
Philippines Hot water extract of root is taken orally by pregnant women to produce abortion.
South Africa Hot water extract of dried leaves is taken orally for menorrhagia and diabetes.
South Vietnam Hot water extract of the entire plant is taken orally by human adults as an antigalactagogue.
Taiwan Decoction of dried entire plant is used orally by human adults to treat diabetes mellitus and liver disease.
Thailand Hot water extract of dried entire plant is taken orally for diabetes.
USA Hot water extract of leaves are smoked as a euphoriant.
Venda Water extract of dried root is taken orally for venereal disease.
Vietnam Hot water extract of dried aerial parts is taken orally as drug in Vietnamese traditional medicine, listed in Vitnamese pharmacopoeia (1974 Edition).
West Indies Hot water extract of leafy stems is taken orally for diabetes.

1.2.2 . Chemistry of Vinblastine:

1.2.2.1 . Vinblastine:

Vinblastine is an antimicrotubule drug used to treat certain kinds of cancer, including Hodgkin’s lymphoma, non-small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. It is also used to treat Langerhans cell histiocytosis. Vinblastine was traditionally obtained from Catharanthus roseus, also known as Vinca rosea, a Madagascar Periwinkle. It is generated in the plant by the joining of two alkaloids catharanthine and vindoline.

Vinblastine

1.2.2.2 .Biosynthesis of Vinblastine:

Vinblastine may be isolated from the Madagascar Periwinkle (Catharanthus roseus), along with several of its precursors- catharanthine and vindoline. Extraction is costly and yields of vinblastine and its precursors are low. Enantioselective synthesis has been of considerable interest in recent years, as the natural mixture of isomers is not an economical source for the required C16’S, C14’R stereochemistry of biologically active vinblastine. Initially, the approach depends upon an enantioselective Sharpless epoxidation, which sets the stereochemistry at C20. The desired configuration around C16 and C14 can then be fixed during the ensuing steps. In this pathway, vinblastine is constructed by a series of cyclization and coupling reactions which create the required stereochemistry.
The overall yield may be as great as 22%, which makes this synthetic approach more attractive than extraction from natural sources, whose overall yield is about 10%. Stereochemistry is controlled through a mixture of chiral agents (Sharpless catalysts), and reaction conditions (temperature, and selected enantiopure starting materials).

Figure 1.1: Biosynthesis of Vinblastine

1.2.3 . Chemistry of Vincristine:

1.2.3.1. Vincristine:

Vincristine is an anti-neoplastic drug found in the Madagascar periwinkle (Catharanthus roseus). It is clinically used to treat a range of cancers including various lymphomas and sarcomas, advanced testicular cancer, breast cancer and acute leukemia.12 Vincristine belongs to a group of bisindole alkaloids derived from tryptophan. Vincristine was approved by the Food and Drug Administration (FDA) in 1984. It is available in the trade under the names Oncovin, Vincasar, and Vincrex. This drug was previously known as leurocristine (LCR).

Vincristine

1.2.3.2. Biosynthesis of Vincristine:
Vincristine belongs in a group of alkaloids that derive from tryptophan. The structure of vincristine is derived by coupling of two alkaloids, catharanthine and vindoline. The biosynthesis of vincristine is summarized in First,
(1) Catharanthine is oxidised by a peroxidise catalyst
(2) Which forms a peroxide which acts as a leaving group. When the peroxide leaves, the carbon-carbon bond is broken and the intermediate electrophilic ion
(3) Is attacked by the nucleophilic vindoline
(4) The molecule is then reduced in the dihydropyridinium ring by NADH-dependent 1,4-addition, giving the substrate for hydroxylation.
(5) Finally, reduction by NADH yields vincristine. charged oxygen to form catharanthine N-oxide
(6) When the carbon-carbon bond is broken by the trifluoroacetic
anhydride, the intermediate electrophilic ion
(7) is attacked by the nucleophilic vindoline
(8). The substrate is then reduced in the dihydropyridinium ring and oxidized by ferric chloride and oxygen
(9). Finally, vincristine is formed by reduction by sodium borohydride.
There are other methods for the synthesis of vincristine.The synthesis of (+)-vincristine has been accomplishedthrough a stereoselective coupling of demethylvindoline and the eleven-membered carbomethoxyverbanamine precursor.4 The oxidation of 17-hydroxy-11-methoxytabersonine, followed by regioselective acetylation with mixed anhydride method yielded demethylvindoline.4 Fukuyama’s first de novo syntheses of these alkaloids was published in 2002 and 2004, but researchers continue to find new and efficient methods that allow for the assembly of key substructures of vincristine.

Figure 1.2: Biosynthesis of Vincristine

1.3. Introduction to Catharanthus roseus:

1.3.1. Taxonomic hierarchy of the investigated plant (Wikipedia, 2006):

Kingdom Plantae – Plants

Subkingdom Tracheobionta

Superdivision Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Asteridae

Order Gentianales

Family Apocynaceae

Genus Catharanthus G. Don

Species Catharanthus roseus (L.) G. Don

1.3.2. Plant Description:

Family: Apocynaceae
Pronunciation: cath-ar-AN-thus ROW-zee-us
Common names:
This species was formerly known as Vinca rosea

Names
Periwinkle
Nityakalyani
Billaganneru
Ainskati
Nayantra
Rattanjot
Sada bahar
Sadaphul
Ushamanjairi

Botanical Trait:

Nature: Perennial herb.
Appearance: An erect bushy perennial herb and evergreen shrub.
Height: Grows to a height of 90 centi metre with a spread of one m.
Stem Erect
Leaves Simple,opposite,exstipulate,petiolate.The inflorescence is racemose.
Flowers: Soft pink, tinged with red.
Petals Five petals appearing in spring and autumn.
Varieties There are three varieties as given below
Rose purple flowers White flower White flowers with a rose purple spot in the centre. First type is being cultivated because of its higher alkaloid content. Recently, two white flowered varieties named “Nirmal” and “Dhawal” have been released by the CIMAP,Lucknow.

Vinblastine sulphate (sold as Velban) is used particularly to treat Hodgkin’s disease besides lymphocarcoma, choriocarcinoma, neuroblastoma, carcinoma of breast, lungs and other organs in acute and chronic leukemia. Vincristine sulphate (sold as Oncovin) arrest mitosis in metaphase and is very effective for treating acute leukaemia in children and lymphocytic leukemia. It is also used against Hodgkin’s disease, Wilkins’s tumor, neuroblastoma and reticulum cell sarcoma. Today India is the third largest manufacture of Vinblastine and Vincristine in the world and is exporting these alkaloids to European countries. High demand and low yield of these alkaloids in the plant has led to research for alternative means for their production. Vinblastine is also modified structurally to yield deacetyl vinblastine amide (Vindesine) introduced recently as Eldisine for use in the treatment of acute lymphoid leukemia in children. Biochemical coupling of alkaloids Catharanthine and Vindoline to get dimeric compounds is also achieved.

Beside these, tissue culture technique is developed for the development of these dimeric alkaloids. In the present communication a detailed application of C. roseus including traditional uses in various developed and developing countries, pharmacological activities and the application of various biotechnological tools viz.

Optimization of Media Composition, Phytohormones, pH, Temperature, Light, Aeration, Elicitors, Mutagenesis, High Cell Density Culture, Selection of Superior cell lines, Bioreactors and Immobilization Methods, Hairy root culture, In Vitro Somatic embryogenesis, Biosynthesis of alkaloids in Catharanthus, Metabolic and Genetic Engineering in alkaloids biosynthesis, Coupling method for Alkaloids biosynthesis, Cellular Compartmentation has been applied for the enhancement of important secondary metabolites present in different parts of Catharanthus.

(a) Whole Plant

(b) Flowres

(c)Seeds

(d) Leaves

(e) Stems

Figure 1.3: (a) Whole Plant, (b) Flowers, (c) Seeds, (d) Leaves (e) Stems of Catharanthus roseus

1.3.3. Cultivation Practices:
Cultivated in Bangladesh
Type Annual

Climate No specific climatic requirements but mostly Tropical and Sub-tropical area The plant tolerates heat and drought and can be relied on to flower in the hottest weather.

Soil
Light sandy soils rich in humus are preferred for large scale cultivation of the plant Soil must be moist but well-drained, as too much moisture could lead to bacterial fungus or stem rot.

Rain

Rainfall of 100 cm or more is considered ideal for raising it as a commercial crop under rainfed conditions
Light requirements It can be grown in sun or shade
Flowers Pink, red, white Takes a long time to flower when started from seed.
Stem The brittle stems break easily.

Propagation
Propagation is by seed or cuttings. Softwood cuttings can be taken and rooted during summer. Should be kept in the dark until the seed germinates
and do not over-water.
Propagation Through Seeds
Seeds required
per hectare 2 to 3 kg

Seeding period March to April
Seed
germination
period About 10 days
time

Planting material Within two months
Seed spacing
45cm x 30cm or 45cm x 45 cm

Height 15 cm to 30 cm

Harvesting
Ready for harvest of root after one year.The crop is cut about 7.5 cm above the ground and dried for stems, leaves and seeds.Then the whole field is copiously irrigated and ploughed and the roots are collected.

1.3.4. Medicinal Use:
Catharanthus roseus is a tropical plant used in traditional herbal medicine in regions of the world where it historically grows. Madagascar periwinkle, the common name of this medicinal and ornamental plant, indicates where the species originated. The plant has a long history of use in Ayurvedic medicine, traditional Chinese medicine and other healing systems. Western medical science began researching Catharanthus roseus and its extracts during the 20th century, finding several compounds useful in cancer treatment. Older texts may refer to the plant by its earlier Latin name, Vinca rosea.
Records indicate that Catharanthus roseus has been used as a medicinal herb for centuries. Although native to Madagascar, the plant has naturalized throughout subtropical Asia, Africa and the Americas and has been used both ornamentally and medicinally. All parts of the plant have been used in regional herbal medicine, including the dried root, leaves, flowers and stalks. Alkaloids used in modern medicine are extracted from the whole dried plant. To help preserve the plant in the wild, it is cultivated for medicinal use in many areas of the world.
Ayurvedic medicine and other traditional herbal systems use Catharanthus roseus for the treatment of diabetes. Insect stings are relieved using a juice from the leaves. Herbal use in the Caribbean includes using extracts from the flowers as an eyewash for infants. The flowers are also used for treating asthma and excess gas. Other traditional herbal treatments include using the plant for painful menstruation, tuberculosis and rheumatism.
Catharanthus roseus in the mid-20th century after learning of its use as a diabetes treatment in the Caribbean and Asia. The plant contains dozens of alkaloids, including vinblastine, which was found to have anti-tumor properties. Medication made from this alkaloid is used to treat Hodgkin’s lymphoma, an immune system cancer. A second alkaloid, vincristine, is utilized for treating leukemia in children. It has been credited with significantly improving the survival rate of victims of childhood leukemia.
Numerous research studies have been conducted on the plant and its extracts. Laboratory studies suggest that it does have potential for treating diabetes. Moreover, antibacterial properties have been found in the extracts of the leaves. The flower petals, seeds and other parts of the plant exhibit antioxidant properties.
In addition to its medicinal uses, Catharanthus roseus is a popular ornamental plant. This subtropical flowering plant can be grown year round in warm regions. It is widely used in temperate zones as an annual bedding plant and continues blooming throughout the season. The five petaled flowers range from white to various shades of pinks, purples and deeper reddish colors. The plant’s ability to thrive in poor soil and full sunshine or partial shade makes it a popular garden addition. Which are used for making medicines to treat a number of diseases:
• Brain Health:
Daily supplements made with the active ingredients found in Vinca rosea help to improve blood supply to the brain and increase the level of oxygen and glucose that the brain can effectively utilize. These supplements are also highly effective in preventing the abnormal coagulation of blood and in raising the levels of serotonin, the blood neurotransmitter, in the brain. Serotonins are bunches of neurons in the central nervous system that play a critical role in memory, sleep, appetite, heart function and muscle control. Deficiencies of serotonin are likely to cause schizophrenia, phobias, migraine and bulimia.
• Prevention of Dementia:
The main alkaloid in Vinca rosea is known as vincamine. Vincamine has blood thinning and memory-enhancing properties and is effective in the treatment of vascular dementia. The condition is caused when the arteries that supply blood to the brain develop plaques. Vascular dementia is the most common dementia after Alzheimer disease. Vascular dementia is not one disease but a number of syndromes related to numerous vascular mechanisms. This form of dementia is a preventable condition and can be corrected with early detection and diagnosis.
• Anti-Cancer Properties:
Derivatives of Vinca rosea have shown efficacy in the treatment of leukemia and Hodgkin’s Disease. Extracts of the plant have demonstrated significant anticancer properties against a number of different cell types. The highest level of efficacy is seen in the multi-drug resistant tumors. The Vinca rosea alkaloids that are used primarily for the treatment of cancer include vinblastin and vincristine.

1.3.5. Chemistry of Catharanthus roseus:

Some reported compound isolated from Catharanthus roseus are mentioned below:

Chapter 2
Experimental

2.1. Plant Being Experiment:

Catharanthus roseus included in Apocynaceae was investigated in this study.

Plant Name Family Plant part used

Catharanthus roseus Apocynaceae Leaves

2.2 Investigation of Catharanthus roseus:

2.2.1. Collection and preparation of Plant Material:
The plant under investigation was collected from Dhaka, Savar in November 2012 and the genus as well as family was determined in Laboratory. The leaves were separated from plant and sun dried for several days. Then the leaves were powdered handly in the phytochemical laboratory room, Department of Pharmacy, Daffodil International University.

2.2.2. Extraction of Plant Material:
About 80 grams of the powdered material was taken two different round bottom flasks and 1.5 liters methanol was added to soak. The container with its content was sealed by aluminum foil and kept for 15 days performing occasional shaking and stirring. Then the whole moisture was filtered through Whatman no.1 filter paper and filtrate concentrated at room temperature. The concentrated was then air dried and to solid residue. Then the solid residue was weighed by an analytical balance that was 5.07 grams.

2.2.3. Solvent-Solvent Partition of Crude Extract:
The solvent-solvent partition was performed using the protocol desined by Kupchan and modified by Wegnen (in 1993). The total crude extract was dissolved in percent 50 ml methanol. Then it was extracted respectively with n-hexane, carbon tetrachloride and dichloromethan. The four fractions (aqueous, n-hexane, carbon tetrachloride and dichloromethane) were dried by evaporation and stored for further analysis.

2.2.3.1. Modified Kupchan Partition (Beckett and Stenlake, 1986):
2.2.3.2. Preparation of Mother Solution:
5 grams of crude extract was triturated with 45 ml of 10% methanol and was dissollved completely. The mother solution was partitioned with n-hexane, carbon tetrachloride and dichloromethane respectively (three solvent of different polarity). In subsequent stages of each of fraction was analyzed in separately for detection of antimicrobial effect, DPPH free radical activity, and antioxidant property.

2.2.3.3. Partitioning with n-Hexane:
The mother solution was taken in separating funnel, and 100ml of n-Hexane was added to funnel and it was shaked and kept for several time in non-agited condition. Then the organic portionb was collected. The portion were collected three times according to the same way, was evaporated in atmosphere.

2.2.3.4. Partitioning with Carbon Tetrachloride:
The mother solution was taken in separating funnel extracted with CCl4 (100*3). The fraction were collected together and evaporated.

2.2.3.5. Partitioning with Dichloromethane:
The mother solution after partitioning with n-Hexane and carbon tetrachloride 5ml water was added and shaked for proper mixing. Then the mother solution was taken in separatin funnel and was extracted with CH2Cl2 (100*3). The portion was collected and evaporated. The aqueous methalonic fraction was preserved as aqueous fraction.

After Evaporation the Weight of Different Fraction Obtained are as Follows:
Plant Fraction Weight

Catharanthus roseus n-Hexane soluble fraction 1.20gm
Carbontetrachloride soluble fraction 1.00gm
Dichloromethane soluble fraction 0.95gm
Aqueous soluble fraction 1.30gm

90 ml methanol+10ml water

Extraction with n-Hexane(1000 x 3)

Extraction with CCl4 (1000 x 3)
+12.5 ml Water

Extraction with CH2Cl2 (100 x 3)
+ 16ml Water

Figure 2.1: Schematic representation of the modified Kupchan partitioning of methalonic crude extract of experiment plant.
Chapter 3
Design of Biological Investigation

3.1. General Approaches to Drug Discovery from Natural Sources:

Natural product compounds are the source of numerous therapeutic agents. Recent progress to discover drugs from natural product sources has resulted in compounds that are being developed to treat cancer, resistant bacteria and viruses and immunosuppressive disorders. Many of these compounds were discovered by applying recent advances in understanding the genetics of secondary metabolism in actinomycetes, exploring the marine environment and applying new screening technology. In many instances, the discovery of a novel natural product serves as a tool to better understand targets and pathway in the disease process (Gullo et al, 2004)

New medicines have been discovered with traditional, empirical and molecular approaches. The traditional approach makes use of drug that has been found by trial and error over many years in different cultures and system of medicine. Examples include drugs like morphine, quinine and ephedrine that have been in widespread use for a long time, and more recently compounds such as the ant malarial artemisinin. The empirical approach builds on an understanding of a relevant physiological process and often develops a therapeutic agent from a naturally occurring lead molecule. Example includes tubocurarine and other muscle relaxants, propranolol and other ß-adrenoceptor antagonists, and cimetidine and other H2 receptor blockers. The molecular approach is based on the availability or understanding of a molecular target for the medicinal agent. With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach.

The major advantage of natural products for random screening is the structural diversity. Bioactive natural products often occur as a part of a family of related molecules so that it is possible to isolate a number of homologues and obtain structure-activity relationship. Of course, lead compounds found from screening of natural products can be optimized by traditional medicine chemistry or by application of combinatorial approaches. Overall, when faced with molecular targets in screening assays for which there is no information about low molecular weight leads, use of a natural products library seems more likely to provide the chemical diversity to yield a hit than a library of similar numbers of compounds made by combination synthesis. Since only a small fraction of the world’s biodiversity has been tested for biological activity, it can be assumed that natural products will continue to offer novel leads for novel therapeutic agents. Molecules discovered from natural source provide the source of inspiration for the major source of inspiration for drug discovery. In particular, these compounds are important in the treatment of life-threatening conditions (Wikipedia, 2007)

3.2. Design of Biological Investigation:

In early times, all drugs and medicine agents were derived from natural substances and most of these remedies were obtain from higher plants. Today, many new chemotherapeutic agents are synthetically derived, based on “rational” drug design. The study of natural products has advantage over synthetic drug design in that leads optimally to materials having new structural features with novel biological activity. Not only do plants continue to serve as important sources of new drugs, but phytochemical derived from them are also extremely useful as lead structures for synthetic modification and optimization of bioactivity. The starting materials for about one-half of the medicines we use today come from natural sources. Virtually every pharmacological class of drugs includes a natural products prototype. The future of plants as sources of medicinal agents for use in investigation, prevention, and treatment of diseases is very promising (Setzer, W.N. 1999).

Natural products are naturally derived metabolites and/or by products from microorganisms, plant, or animals. The major advantage of natural products for random screening is the structural diversity. Bioactive natural products often occur as a part of a family of related molecules so that it is possible to isolate a number of homologues and obtain structure-activity relationship. Of course, lead compounds found from screening of natural products can be optimized by traditional medicinal chemistry or by application of combinatorial approaches.

Overall, when faced with molecular targets in screening assays for which there is no information about low molecular weight leads, use of a natural products library seems more likely to provide the chemical diversity to yield a hit than a library of similar numbers of compounds made by combinatorial synthesis. Since only a small fraction of the world’s biodiversity has been tested for biological activity, it can be assumed that natural products will continue to offer novel leads for novel therapeutic agents.

3.3. Experimental Design:

3.3.1. Evaluation of Antioxidant Activity:

Antioxidant activity of different extracts was evaluated depending on their free radical scavenging activity, Antioxidants that scavenge free radicals are known to possess an important role in preventing these free radical induced-diseases. There is an increasing interest in the antioxidants effects of compounds derived from plants, which could be relevant in relations to their nutritional incidence and their role in health and diseases (Steinmetz and Potter, 1996; Aruoma, 1998; Bandoniene et al., 2000; Pieroni et al., 2002; Couladis et al., 2003).

3.3.2. Microbiological Investigations:

The in vitro antimicrobial study was designed to investigate the antibacterial as well as antifungal spectrum of the crude extracts by observing the growth response. The rationale for these experiments is based on the fact that bacteria and fungi are responsible for many infectious diseases, and if the test materials inhibit bacterial or fungal growth then they may be used in those particular diseases. However, a number of factors viz. the extraction method inoculate volume, culture medium composition, pH and incubation temperature can influence the results.

3.3.3. Brine Shrimp Lethality Test: A Rapid Bioassay:

Brine shrimp lethality bioassay (Mclaughlin et al., 1976; Meyer et al., 1986) is a rapid and comprehensive bioassay for the bioactive compounds of natural and synthetic origin and is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. The method utilizes in vivo lethality in a simple zoological organism (Brine shrimp nauplii) as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products.
The brine shrimp assay has advantages of being rapid (24 hours), inexpensive, and simple (e.g., no aseptic techniques are required). It easily utilizes a large number of organisms for statistical validation and requires no special equipment and a relatively small amount of sample (2-20 mg or less). Furthermore, it does not require animal serum as is needed for cytotoxicities.

Chapter 4
Evaluation of Antioxidant Activity

4.1. Relation and Objective:

There is considerable recent evidence that free radical induced oxidation damage to biomolecules. This damage causes cancer, aging, neurodegenerate diseases, atherosclerosis, malaria and several other pathological events in living organisms (Halliwell et al, 1992).

Antioxidants which scavenge free radicals are known to process an important role in preventing these free radical include diseases. There is an increasing interest in the antioxidants effects of compound derived from plants, which could be revelent in relations to their neutritional incidence and their role in health and diseases (Steinmetz and potter, 1996; Aruoma, 1998; Bandonience et al, 2000; Pieroni et al, 2002; Couladis et al, 2003)

A number of reports on the isolation and testing of plant derived antioxidants constitude a broad range of substances including phenolic or nitrogen containing compounds and carotenodis (Shahidi et al, 1992; Velioglu et al, 1998; Pieta et al, 1998)

Lipid peroxidation is one of the main reasons for deterioration of food products during processing and storage. Synthetic antioxidant such as tert-butyl-1-hydroxytoluene (BHT), Butylated hydroxyanisole (BHA), Propyl gallate (PG), and tert-butylhydroquinone (TBHQ) are widely used a food additives to increase shelf life, especially lipid peroxidation. However, BHT and BHA are known to have not toxic and carcinogenic effects on humans (Ito et al, 1986; Wichi, 1988), but abnormal effects on enzyme systems (Inatani et al, 1983). Therefore, the interest in natural antioxidant, especially of plant origin, has greatly increased in recent years (Jayaprakasha & Jaganmohan Rao, 2000)

4.2. Antioxidant Evaluation by DPPH Method:

4.2.1. Principle:

The present study was aimed at evaluating the In vitro free radical scavenging activity of Catharanthus roseus using 1,1-diphenyl-2-picrylhydrazyl (DPPH) by the method of Brand-Williams et al., 1995. 2.0 ml of a methanol solution of the extract at different concentration were mixed with 3.0 ml of a DPPH methanol solution (20 ?g/ml). The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the plant extract as compared to that of tert-butyl-1-hydroxytoluene (BHT) by UV spectrophotometer.

The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color. The color turns from purple to yellow as the molar absorptive of the DPPH radical at 517 nm reduces when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H. DPPH radical scavenging activity is described as IC50 which is the concentration of samples to produce 50% reduction of the DPPH.

4.2.2. Materials and Methods:

DPPH was used to evaluate the free radical scavenging activity of various compounds and medicinal plants (Choi et al., 2000; Desmarchelier et al., 1997).

4.2.2.1. Materials:

1,1-diphenyl-2-picrylhydrazyl UV-spectrophotometer
tert-butyl-1-hydroxytoluene (BHT) Beaker (100 & 200 ml)
Distilled water Test tube
Methanol Light-proof box
Dichloromethane Pipette (5 ml)
Carbon tetra chloride Micropipette (50-200 µl)
n-hexane Amber reagent bottle

4.2.2.2. Methods:

• Inhibition free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 – Asample/Ablank) X 100
Where Ablank is the absorbance of the control reaction (containing all reagents except the test material).

• Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract concentration.

• BHT was used as positive control.

• Tests carried out in triplicate and average value was taken.

Decolorization of purple-colour of DPPH

Figure4.2.1. Schematic representation of the method of assaying free radical scavenging activity.

4.2.3. Results and Discussion:

Different partitionates of methanolic extract of experimental plants were subjected to free radical scavenging activity developed by the method of Brand-Williams et al., 1995.

4.2.3.1. Results and discussion of the test samples of Catharanthus roseus (leaves):

In this investigation, crude extract and aqueous soluble fraction of methanolic extract showed the highest free radical scavenging activity with IC50 value15.95 and 19.45 ?g/ml respectively. At the same time, n-hexane, carbon tetrachloride, dichloromethane soluble fraction of methanolic extract showed free radical scavenging activity with the IC50 value of 58.25, 52.85.0 and 146.09 ?g/ml respectively

Table4.2.1: IC50 Values of Standard and Different Partitionates Catharanthus roseus (Leaves):
Code Sample IC50 (µg/ml)
HSF n-Hexane soluble fractions 58.25
CTSF Carbon tetrachloride soluble fractions 52.85
DMSF Dichloromethane soluble fractions 146.09
CME Crude methanolic extract 15.95
AQF Aqueous soluble fractions 19.45
AA Ascorbic Acid 3.25
BHT Tert-butyl-1-hydroxy toluene 24.50
HSF: n-Hexane soluble fraction; CTSF: Carbon tetrachloride soluble fractions
DMSF: Dichloromethane soluble fraction; CME: Crude methanolic extract;
AQF: Aqueous soluble fractions; AA: Ascorbic Acid; BHT: Tert-butyl-1-hydroxy toluene

Figure 4.2.2: IC50 Values of Standard and Different Partitionates of Catharanthus roseus (Leaves)

Table 4.2.2: IC50 value of Ascorbic Acid:

SL. Absorbance
of blank Concentration
(?g/ml) Absorbance
of extract % Inhibition IC50
(?g/ml)
1 0.325 500 0.005 98.461 3.25
2 250 0.006 98.153
3 125 0.011 96.615
4 62.5 0.012 96.307
5 31.25 0.015 95.384
6 15.625 0.038 88.307
7 7.813 0.098 69.846
8 3.906 0.139 57.230
9 1.953 0.175 46.153
10 0.977 0.186 42.769

Figure 4.2.3: Free radical scavenging activity of ascorbic acid.

Table 4.2.3: IC50 value of tert-butyl-1-hydroxytoluene (BHT):

SL. Absorbance
of blank Concentration
(?g/ml) Absorbance
of extract % Inhibition IC50
(?g/ml)
1 0.325 500 0.016 95.076 24.5
2 250 0.068 79.076
3 125 0.097 70.153
4 62.5 0.135 58.461
5 31.25 0.159 51.076
6 15.625 0.175 46.153
7 7.813 0.206 36.615
8 3.906 0.225 30.769
9 1.953 0.238 26.769
10 0.977 0.258 20.615

Figure 4.2.4: IC50 value of tert-butyl-1-hydroxytoluene (BHT).

Table 4.2.4: IC50 Value of n-Hexane Soluble Fraction of Catharanthus roseus (leaves):