Biological investigation of Caryota urens
1.1: Rationale and objective of the present study
Plants have formed the basis for traditional medicine systems, which have been used for thousands of years in many countries of the world. These plant-based systems continue to play an essential role in health care, and it has been estimated by the World Health Organization that approximately 80% of the world’s inhabitants rely mainly on traditional medicines for their primary health care (Schultes et al.,1972 ).
Plants have provided a source of inspiration for novel drug compounds, as plant-derived medicines have made large contributions to human health and well-being (Nelson 1982). Approximately 119 pure chemical substances extracted from higher plants are used in medicine throughout the world (Farnsworth et al., 1985). More than 30% of the pharmaceutical preparations are based on plants (Shinvari and Khan, 2003). The plant is a biosynthetic laboratory and the remedial phyto-elements produced inside a plant through a cascade of biochemical reactions significantly contribute to the traditional and modern medicines. Chemical diversity of secondary plant metabolites that results from plant evolution may be equal or superior to that found in synthetic combinatorial chemical libraries (Vagelos, 1991).
The number of higher plant species ( angiosperms and gymnosperms) on this planet is estimated at 250,000 (atensu E.S.et al,1978), with a lower level at 215,000 (Cronquist,1981; Cronquist,1988) and an upper level as high as 500,000 (Tippo et al.,1977; Schultes,1972). Of these only about 6% have been screened for biologic activity, and a reported 15% have been evaluated phytochemically (Verpoorte, 2000) About 2.5 million species of higher plants and the majority of these have not been investigated in detail for their pharmacological activities (Ram et al., 2003)
The goals of using plants as sources of therapeutic agents are (Daniel et al., 2001)
a) to isolate bioactive compounds for direct use as drugs, e.g. digoxin, digitoxin, morphine, reserpine, taxol, vinblastine, vincristine;
b) to produce bioactive compounds of novel or known structures as lead compounds for semisynthesis to produce patentable entities of higher activity and/or lower toxicity, e.g., metformin, nabilone, oxycodon (and other narcotic analgesics), taxotere, teniposide, verapamil, and miodarone, which are based, respectively, on galegine, ?9- tetrahydrocannabinol, morphine, taxol, podophyllotoxin, khellin, and khellin;
c) to use agents as pharmacologic tools, e.g., lysergic acid diethylamide, mescaline, yohimbine; and
d) to use the whole plant or part of it as an herbal remedy, e.g., cranberry, echinacea, feverfew, garlic, etc.
There are several familiar approaches for lead searching from the plants and the isolated bioactive compounds are utilized in three basic ways (Cox, P.A., 1994):
- Unmodified natural plant products where ethno medical uses suggested clinical efficacy, e.g.,digoxin, digitoxin (1), morphine (2) .
- Unmodified natural products of which the therapeutic efficacy was only remotely suggested by indigenous plant use, e.g., vincristine
- Modified natural or synthetic substances based on a natural product used in folk medicine, e.g., aspirin
With high throughput screening methods becoming more advanced and available, these numbers will change, but the primary discriminator in evaluating one plant species versus another is the matter of approach to finding leads. There are some broad starting points to selecting and obtaining plant material of potential therapeutic interest. However, the goals of such an endeavour are straightforward.
Chemical diversity of secondary plant metabolites that results from plant evolution may be equal or superior to that found in synthetic combinatorial chemical libraries. It was estimated that in 1991 in the United States, for every 10,000 pure compounds (most likely those based on synthesis) that are biologically evaluated (primarily in vitro), 20 would be tested in animal models, and 10 of these would be clinically evaluated, and only one would reach U.S. Food and Drug Administration approval for marketing. The time required for this process was estimated as 10 years at a cost of $231 million (U.S.) (Vagelos, 1991). Most large pharmaceutical manufacturers and some small biotechnology firms have the ability to screen 1,000 or more substances per week using high throughput in vitro assays. In addition to synthetic compounds from their own programs, some of these companies screen plant, microbial, and marine organisms.
The work described in this dissertation is an attempt to isolate and characterize the chemical constituents of indigenous medicinal plants,Caryota urens (Fam. Arecaceae)to evaluate the possible pharmacological, toxicological and microbiological profiles of the crude extracts as well as purified compounds.
2.1: The plant family:
The plant under investigation Caryota urens belongs to the family Arecaceae or Palmae.Arecaceae are a family of flowering plants, the only family in the monocot order Arecales. Over 230 genera with around 3000 species(real palm trees.com2012)are currently known, most of which are restricted to tropical, subtropical, and warm temperate climates. Most palms are distinguished by their large, compound, evergreen leaves arranged at the top of an unbranched stem. However, many palms are exceptions, as palms in fact exhibit an enormous diversity in physical characteristics. As well as being morphologically diverse, palms also inhabit nearly every type of habitat within their range, from rainforests to deserts.
Palms are among the best-known and most extensively cultivated plant families. They have been important to humans throughout much of history. Many common products and foods are derived from palms, and palms are widely used in landscaping for their exotic appearance, making them one of the most economically important plants
Kingdom : Plantae
Clade : Angiosperms
Clade : Monocots
Clade : Commelinids
Order : Arecales
Palms range from tiny understory plants to towering trees, and are found throughout the tropics and subtropics. Some commercially important palms include coconut (Cocos nucifera), date (Phoenix dactylifera) and oil palm (Elaeis guineensis)
Palms, considered in a dietetical and medicinal point of view, are of the highest importance to the inhabitants of tropical regions. Their stems yield starch (sago) sugar, and wax; their terminal leaf buds are boiled and eaten as a kind of cabbage; their fruits yield oil, sugar, and resins; and their seeds form articles of food, and yield, by pressure, fixed oil.
In the abundance of sugar and starch which the palms yield, this family resembles the grasses. However, they are distinguished from the latter in containing, in some cases, a large quantity of fixed oil. To these three principles are chiefly due the nutritive qualities of palms. However, these substances being non-nitrogenized, are merely fat making and heat yielding, and without the addition of proteine compounds (found in the seeds, and probably in other edible parts of palms), would be insufficient to support life.
Wax, astringent matter (tannin), and resinous principles, are useful products obtained from palms, the ashes obtained by the combustion of palm leaves yield potash.
The only resinous substance used in medicine and the arts, and which is obtained from the palms, is Dragon’s blood, the produce of Calamus Draco.(henriettasherbal.com)
2.3 Habitate and Distribution :
Most palms grow in the tropics. They are abundant throughout the tropics, and thrive in almost every habitat therein. Their diversity is highest in wet, lowland tropical forests, especially in ecological “hotspots”
Only an estimated 130 palm species grow naturally beyond the tropics, mostly in the subtropics. Palms inhabit a variety of ecosystems. More than two-thirds of palm species live in tropical forests, where some species grow tall enough to form part of the canopy and shorter ones form part of the understory. Some species form pure stands in areas with poor drainage or regular flooding, including Raphia hookeri which is common in coastal freshwater swamps in West Africa. Other palms live in tropical mountain habitats above 1000 meters,. Palms may also live in grasslands and scrublands, usually associated with a water source, and in desert oases such as the date palm. A few palms are adapted to extremely basic lime soils, while others are similarly adapted to very acidic serpentine soils.
|Figure 2.1: Distribution of plants of Arecaceae family|
Palms are a monophyletic group of plants, meaning the group consists of a common ancestor and all its descendants. Extensive taxonomic research on palms began with botanist H.E. Moore, who organized palms into 15 major groups based mostly on general morphological characteristics. The following classification, proposed by N.W. Uhl and J. Dransfield in 1987, is a revision of Moore’s classification that organizes palms into six subfamilies. A few general traits of each subfamily are listed.
Coryphoideae are the most diverse subfamily, and are a paraphyletic group, meaning all members of the group share a common ancestor, but the group does not include all the ancestor’s descendants. Most palms in this subfamily have palmately lobed leaves and solitary flowers with three, or sometimes four carpels. The fruit normally develops from only one carpel.
Subfamily Calamoideae includes the climbing palms, such as rattans. The leaves are usually pinnate; derived characters (synapomorphies) include spines on various organs, organs specialized for climbing, an extension of the main stem of the leaf-bearing reflexed spines, and overlapping scales covering the fruit and ovary.
Subfamily Nypoideae contains only one one species, Nypa fruticans<href=”#cite_note-11″>], which has large, pinnate leaves. The fruit is unusual in that it floats, and the stem is dichotomously branched, also unusual in palms.
Subfamily Ceroxyloideae has small to medium-sized flowers, spirally arranged, with a gynoecium of three joined carpels.
The Arecoideae are the largest subfamily, with six diverse tribes containing over 100 genera. All tribes have pinnate or bipinnate leaves and flowers arranged in groups of three, with a central pistillate and two staminate flowers.
The Phytelephantoideae are a monoecious subfamily. Members of this group have distinct monopodial flower clusters. Other distinct features include a gynoecium with five to ten joined carpels, and flowers with more than three parts per whorl. Fruits are multiseeded and have multiple parts. Currently, few extensive phylogenetic studies of Arecaceae exist.
In 1997, Baker et al. explored subfamily and tribe relationships using chloroplast DNA from 60 genera from all subfamilies and tribes. The results strongly showed the Calamoideae are monophyletic, and Ceroxyloideae and Coryphoideae are paraphyletic. The relationships of Arecoideae are uncertain, but they are possibly related to Ceroxyloideae and Phytelephantoideae. Studies have suggested that the lack of a fully resolved hypothesis for the relationships within the family is due to a variety of factors including difficulties in selecting appropriate outgroups, homoplasy in morphological character states, slow rates of molecular evolution important for the use of standard DNA markers, and character polarization. However, hybridization has been observed among Orbignya and Phoenix species, and using chloroplast DNA in cladistic studies may produce inaccurate results due to maternal inheritance of the chloroplast DNA. Chemical and molecular data from non-organelle DNA, for example, could be more effective for studying palm phylogeny.
Table2.1:Subfamily and Genus of Arecaceae family
|Coryphoideae||Corypheae||Thrinacinae||Thrinax, Chelyocarpus, Crysophila, Itaya, Schippia, Thrinax, Coccothrinax, Zombia, Trachycarpus, Guihaia, Rhapis, Rhapidophyllum, Chamaerops, Maxburrietia|
|Livistoninae||Livistona, Pholidocarpus, Brahea, Johannesteijsmannia, Licuala, Pritchardia, Pritchardiopsis, Serenoa, Copernicia, Colpothrinax, Acoelorraphe, Washingtonia|
|Coryphinae||Corypha, Nannorrhops, Chuniophoenix, Kerriodoxa|
|Borasseae||Lataniinae||Borassodendron, Latania, Borassus, Lodoicea|
|Hyphaeninae||Hyphaene, Medemia, Bismarckia|
|Calaminae||Eleiodoxa, Salacca, Daemonorops, Calamus, Calospatha, Pogonotium, Ceratolobus, Retispatha|
|Plectocomiinae||Myrialepsis, Plectocomiopsis, Plectocomia|
|Lepidocaryeae||Mauritia, Mauritiella, Lepidocarym|
|Ceroxyleae||Ceroxylon, Oraniopsis, Juania, Louvelia, Ravenea|
|Hyophorbeae||Gaussia, Hyophorbe, Synecanthus, Chamaedorea, Wendlandiella|
|Arecoideae||Caryoteae||Arenga, Caryota, Wallichia|
|Iriarteae||Iriarteinae||Dictyocaryum, Iriartella, Iriartea, Socratea|
|Dypsidinae||Vonitra, [Chrysalidocarpus: now Dypsis], Neophloga, [Neodypsis: now Dypsis], Phloga, Dypsis|
|Euterpeinae||Euterpe, Prestoea, Neonicholsonia, Oecocarpus, Jessenia, Hyospathe|
|Archontophoenicinae||Archontophoenix, Chambeyronia, Hedyscepe, Rhopalostylis, Kentiopsis, Mackeea, Actinokentia|
|Linospadicinae||Calyptrocalyx, Linospadix, Howea, Laccospadix|
|Ptychospermatinae||Drymophloeus, Carpentaria, Veitchia, Normanbya, Wodyetia, Ptychosperma, Ptychococcus, Brassiophoenix, Balaka|
|Areninae||Loxococcus, Gronophyllum, Areca, Siphokentia, Hydriastele, Gulubia, Nenga, Pinanga|
|Oncospermatinae||Deckenia, Acanthophoenix, Rocheria, Oncosperma, Tectiphiala, Verscheffeltia,Phoenicophorium, Nephrosperma|
|Butiinae||Butia, Jubaea, Jubaeopsis, Cocos, Syagrus, Lytocaryum, Parajubaea, Allagoptera, Polyandrococos|
|Attaleinae||Attalea [Scheelea, Orbignya, Maximiliana: these three genera are now included in Attalea]|
|Bactridinae||Acrocomia, Gastrococos, Aiphanes, Bactris, Desmoncus, Astrocaryum|
|Geonomeae||Pholidostachys, Welfia, Calyptronoma, Calyptrogyne, Asterogyne, Geonoma|
|Phytelephantoideae||Palandra, Phytelephas, Ammandra|
Arecaceae is the first modern family of monocots that is clearly represented in the fossil record. Palms first appear in the fossil record around 80 million years ago, during the late Cretaceous Period. The first modern species, such as Nypa fruticans and Acrocomia aculeata, appeared 69-70 million years ago, confirmed by fossil Nypa pollen dated to 70 million years ago. Palms appear to have undergone an early period of adaptive radiation.
Figure 1: Fossil of Permineralized Nypa prop
By 60 million years ago, many of the modern, specialized genera of palms appeared and became widespread and common, much more widespread than their range today. Because palms separated from the monocots earlier than other families, they developed more interfamilial specialization and diversity. By tracing back these diverse characteristics of palms to the basic structures of monocots, palms may be valuable in studying monocot evolution. Several species of palms have been identified from flowers preserved in amber including Palaeoraphe dominicana and Roystonea palaea. Evidence can also be found in samples of petrified palmwood.(Wikipedia 2012)
2.5: Characteristics of palmae family
Leaves clustered, terminal, very large, pinnate, or flabelliform, plaited in vernation. Spadix terminal, often branched, or enclosed in a one or many-valved spathe.
Flowers small, greenish ,with bractlets. Perianth six-parted, in two series, persistent; the three outer segments often smaller, the inner sometimes deeply connate. Ovary one, three-celled, or deeply three-lobed; the lobes or cells one-seeded, with an erect ovule, rarely one-seeded.
Fruit occasionally very large. (R. Brown, 1810.)Fruit baccate or drupaceous, with fibrous flesh. Albumen cartilaginous, and either ruminate or furnished with a central or ventral cavity; embryo lodged in a particular cavity of the albumen, usually at a distance from the hilum, dorsal, and indicated by a little nipple, taper or pulley-shaped; Trunk arborescent, simple, occasionally shrubby and branched, rough, with the dilated half-sheathing bases of the leaves or their scars.
Figure 1.2: Patterns of the Arecaceae or Palmae Family
Palms represent the third most important plant family with respect to human use (Johnson, 1998). Numerous edible products are obtained from palms, including the familiar date palm fruits, coconut palm nuts, and various palm oils. Some less well-known edible palm products include palm “cabbage” or “heart-of-palm”, immature inflorescences, and sap from mature inflorescences.
The members of the Palm Family with human uses are numerous.
· Coir is a coarse water-resistant fiber extracted from the outer shell of coconuts, used in doormats, brushes, mattresses, and ropes.
· Some indigenous groups living in palm-rich areas use palms to make many of their necessary items and food. Sago, for example, a starch made from the sago palm is a major staple food for lowland peoples of New Guinea and the Moluccas.
· Recently the fruit of the açaí palm Euterpe has been used for its reputed healthful benefits.
· Saw palmetto (Serenoa repens) is under investigation as a drug for treating enlarged prostates.
· Palm leaves are also valuable to some peoples as a material for thatching, basketry, clothing, and in religious ceremonies .
- Ornamental Uses. Today, palms are valuable as ornamental plants
- Few palms tolerate severe cold, however, and the majority of the species are tropical or subtropical. The three most cold-tolerant species are Trachycarpus fortunei, Rhapidophyllum hystrix and Sabal minor,
2.7: Arecaceae species available in Bangladesh
Table2.1 Arecaceae species available in Bangladesh (Bangladesh National Herbarium, 2005)
| Scientific name
of the plant
|1.Borasses xlabelliser||Tal||Dhaka,Mymensingh, Kishoreganj, Jamalpur,|
|2.Cocos nucifera||Narical||Barisal,Noakhali, Mymensingh
Kishoreganj. Sherpur, Sylhet,
Jamalpur, Dhaka, Chittagong, Coxbazar,Rajshai, Tangail.
|3.Areca catechu||Supari||Noakhali,Chittagong, Gajni,
Kishoreganj, Rajshai, Faridpur, Sylhet,Netrokona, Mymensingh
|4.Calamus tenuis||Bet||Sylhet, Habigang|
|6.Caryota urens||Chaur(Fishtail palm)||Cultivated in the garden|
|7.Oreodoya redia||Royal palm||Cultivated in the garden|
2.8: Description of Caryota urens:
2.8.1:Taxonomic hierarchy (Zipcodezoo.com)
Kingdom: Plantae – Plants
Genus: Caryota urens
Species: Caryota urens(L.)
Botanical name: – Caryota urens L.
(English) : fishtail palm, Indian sago palm, kitul palm, toddy palm, wine palm
(Sanskrit): dirgha, mada
(Tamil) : konda panna, koondalpanai, kundal panai, thippali, tippili
2.9.2:General botanical data:
Caryota urens is an unarmed, hapaxanthic, solitary or clustered, medium-sized palm up to 20 or 30 m tall; bole straight, unbranched palm tree. Leaves 5-6 m long, bipinnate, drooping; leaflets are fish tail shaped. Flowers seen in peculiar pendular spadix, inflorescence is long, resembling a women’s hair. Flowers in a group of three, central female guarded by two male flowers. Fruits globose, reddish when ripe.of three, central female guarded by two male flowers.
2.9.3:Photographs of Caryota urens:
(a) Whole Plants with fruits (b) Leaves (c) fruits
Figure2.9.3: a) Whole Plants with fruites (b) Leaves c) fruites
2.8.4 Ecology and distribution
Native : India, Myanmar, Nepal, Sri Lanka,Bangladesh
Exotic : Papua New Guinea, Thailand, Vietnam
Light: Toddy palm thrives in full sun to part shade.
Moisture: This palm prefers a rich, moist, but well drained soil.
Hardiness: USDA Zone 9 to 10. A mature toddy palm can handle temperatures as low as 26°F (-3°C) without damage, but young palms must be protected from frost. Seeds obtained from populations living at higher altitudes are colder hardy and more frost resistant.
C. urens is monoecious, flowering and leaf flushing continues throughout the year. Since the plants have a determinate growth habit, no new leaves originate after emergence of the 1st terminal inflorescence, which signals the start of the plant’s reproductive phase. Flowering begins at the top of the trunk and often continues downwards for several years. Individual staminate flowers remain open for 16-20 days, while a single inflorescence has flowers opening for about 6 weeks. The pistillate flowers open for 2-3 weeks after all the staminate flowers have bloomed and remain receptive for 3-13 days. C. urens is an obligate out breeder. Fruit development takes 32-38 weeks.
2.8.5:Propagation and management
C. urens can be propagated by seed with direct sowing being a viable method. Exposure of seeds to direct sunlight for 6 hours prior to sowing inhibits germination. Therefore, satisfactory germination could be obtained by placing seeds in a moist, dark environment. Seeds germinate in 18-30 days.
At room temperature the seeds remain viable for 30-90 days, depending on storage conditions.
Local physicians to treat gastric ulcers, migraine headaches, snakebite poisoning and rheumatic swellings, prescribe a porridge prepared from C. urens flour. Plant pacifies vitiated pita, hyperpiesia, burning sensation, and general weakness.
2.8.7:Chemistry of Caryota urens:
It contains three types of Catechin(flavan-3-ol, a type of natural phenol and antioxidantnamely 1-epicatechin, l-gallocatechin and l-epicatechingaliate.(Center of Excellence for Medicinal Plants in Sri Lanka:2006).Catechin is a plant secondary metabolite. Catechin exists in the form of a glycoside.
1-epicatechin(also known under the names L-epicatechin, epicatechol, (-)-epicatechol, l-acacatechin, l-epicatechol, 2,3-cis-epicatechin or (2R,3R)-(-)-epicatechin)is the most common epicatechin isomer.
Caryota urens contain calciumoxalate(skin irritant toxic substance)and possibly irritant proteins.
3.1 GENERAL APPROACHES TO DRUG DISCOVERY FROM NATURAL SOURCES
Despite the progress of science during the past four centuries, Shakespeare’s words “There are more things between heaven and earth…” did not lose their actuality. For many life-threatening illnesses, no effective treatment exists and knowledge about the etiology of diseases is still limited
Traditional, empirical and molecular approaches have been utilized to discover new medicines (Harvey, 1999). The traditional approach provides drugs that have been found by trial and error over many years in different cultures and systems of medicine (Cotton, 1996). Examples include drugs like morphine, quinine and ephedrine that have been in widespread use for a long time. The empirical approach develops a therapeutic agent from a naturally occurring lead molecule and builds on an understanding of a relevant physiological process (Verpoorte, 1989, 2000). Examples include tubocurarine and other muscle relaxants, propranolol and other b-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 (Harvey, 1999). With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach.
Natural products have advantages over synthetic drug design in that it provides lead compounds having new structural features with novel biological activity. One-half of the medicines we use today, has been derived from natural sources. Virtually every pharmacological class of drugs includes a natural product prototype. The future of plants as sources of medicinal agents for use in investigation, prevention, and treatment of diseases is very promising. . Nature has been a valuable source of drugs and will always continue to deliver lead compounds (Martin and Lars, 2004)
3.2 GENERAL APPROACHES TO DRUG DISCOVERY FROM NATURAL SOURCES
Natural products have been the most successful source of drugs ever (Newman et al, 2003). Research progressed along two major lines: ethno pharmacology (medicinal herbs, substances of abuse, ordeal poisons) and toxicology (poisonous plants, venomous animals, arrow and fish poisons) (Heinrich, M., and Gibbons, S., 2001). These strategies have already produced many valuable drugs and are likely to continue to produce lead compounds (Tulp and Bohlin, 2002). Approximately 60% of the world’s population relies entirely on plants for medication (Farnsworth, 1994). Of the 520 new drugs approved between 1983 and 1994, 39% were natural products or derived from natural products and 60–80% of antibacterial and anticancer drugs were derived from natural products (Cragg et al, 1997). Thirteen natural product related drugs were approved from 2005 to 2007 (Butler, 2008), and five of these represented the first members of new classes of drugs: the peptides exenatide and ziconotide, and the small molecules ixabepilone, retapamulin. Current commercial evidence also supports the case for natural products. Of the 20 bestselling non-protein drugs in 1999, nine were either derived from or developed as the result of leads generated by natural products — simvastatin, lovastatin, enalapril, pravastatin, atorvastatin, augmentin, ciprofloxacin, clarithromycin and cyclosporin — with combined annual sales of >US$16 billion. Newer developments based on natural products include the antimalarial drug artemisinin and the anticancer agents taxol, docetaxel and camptothecin (Harvey and Waterman, 1998), (Verpoorte, 1998), (Grabley and Thiericke, 1999).
Today, many new chemotherapeutic agents are synthetically derived, based on “rational” drug design. The study of natural products has advantages over synthetic drug design in that it 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 phytochemicals derived from them are also extremely useful as lead structures for synthetic modification and optimization of bioactivity.
Natural products are naturally derived metabolites and/or by products from microorganisms, plants, or animals (Baker et al., 2000). 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:
The medicinal properties of plants have been investigated in the recent scientific developments throughout the world due to their-
Ø potent antioxidant activities
Ø no side effects and
Ø economic viability.
The antioxidant activity in terms of
Ø DPPH free radical scavenging capability
Ø Antioxidant activity assay by the Phosphomolybdenum method
Ø Determination of total phenolic content
Ø Determination of reducing power by potassium ferricyanide and trichloro acetate
was evaluated to explore the potent antioxidant components from the investigated plants.
3.3.2 Brine shrimp lethality test: A rapid bioassay:
Brine shrimp lethality bioassay (Mclaughlin et al., 1976; Meyer et al., 1986) has been suggested for screening pharmacological activities in plant extracts. It is considered as a useful tool for preliminary assessment of toxicity and is a rapid and comprehensive bioassay for the bioactive compounds of natural and synthetic origin . A simple zoological organism (Brine shrimp nauplii) is utilized in this method to conveniently monitor in vivo lethality for screening and fractionation in the discovery of new bioactive natural products.
The brine shrimp assay has several advantages of being-
Ø rapid (24 hours),
Ø simple (e.g., no aseptic techniques are required).
Ø It easily utilizes a large number of organisms for statistical validation
Ø requires no special equipment and a relatively small amount of sample (2-20 mg or less).
Ø Furthermore, it does not require animal serum as is needed for cytotoxicities.
Brine shrimp toxicity is closely correlated with 9KB (human nasopharyngeal carcinoma) cytotoxicity (p=0.036 and kappa = 0.56). For cytotoxicities ED50 values are generally about one-tenth the LC50 values found in the brine shrimp test. Thus, it is possible to detect and then monitor the fractionation of cytotoxic, as well as 3PS (P388) (in vivo murine leukaemia) active extracts using the brine shrimp lethality bioassay.
3.3.3 Microbiological investigations:
The antibacterial as well as antifungal spectrum of the crude extracts can be ascertained by observing the growth response with the help of in vitro antimicrobial study. These experiments are rationalized on the fact that many infectious diseases are caused by bacteria and fungi and if the test materials inhibit bacterial or fungal growth then they may be used in those particular diseases.
However, a number of factors can influence the results like-
Ø the extraction method
Ø inocula volume,
Ø culture medium composition,
Ø pH and
Ø incubation temperature.
3.3.4 Thrombolytic activity investigation
Cerebral venous sinus thrombosis (CVST) is a common disorder that is often accompanied by significant morbidity and mortality. In anticoagulation therapy the intravenous heparin is the first line of treatment for CVST, because of its efficacy, safety and feasibility. However, thrombolytic therapy with its ability to produce rapid clot lysis has long been considered as an attractive alternative.Thrombolytic drugs like tissue plasminogen activator(t-PA) ,urokinase, streptokinase etc. play a crucial role in the management of patients with CVST.
3.3.5 Membrane stabilizing activity investigation:
Inflammatory cells produce a complex mixture of growth differentiation cytokines as well as physiologically active arachidonate metabolites .In addition they possess the ability to generate reactive oxygen species(ROS) that can damage cellular biomolecules which in return augement the stae of inflammation.(Cochrane, 1991)The erythrocyte membrane resembles to lysosomal membrane and such as the effect of drugs on the stabilization of erythrocytes can be extrapolated to the stabilization of lysosomal membrane(Omale 2008).Therefore when membrane stabilizes they interfare in the release and in the action of mediators like histamine, serotonin, prostaglandine, leukotrines etc. (Shinde et al., 1999)
4.1 Rationale and objective
Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many of which are based on their uses in traditional medicine. Plants produce a diverse array of bioactive molecules that are particularly important in the treatment of life-threatening conditions. Oxidation reactions initiated by excess free radicals have been shown to lead to the formation of tumors, damage of DNA ,mRNA , proteins, enzymes; cause cancer, cardiovascular diseases, nervous disorders, premature ageing, Parkinson’s and Alzheimer’s diseases, rheumatic and pulmonary disorders. Therefore, the need for systematic screening of medicinal plants for antioxidant activity cannot be overemphasized.
Free radicals are atoms or group of atoms that have at least one unpaired electron, making them highly reactive. The potentially reactive derivatives of oxygen are known as reactive oxygen species (ROS) (e.g. superoxide anions, hydrogen peroxide and hydroxyl, nitric oxide radicals), and play an important role in oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA, related to the pathogenesis of various important diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, and neurodegenerative diseases and also in the ageing process.
Antioxidants are the substance that when present in low concentrations compared to those of an oxidisable substrate significantly delays or prevents oxidation of that substance. Antioxidants prevent the oxidative damage by directly reacting with ROS, quenching them and/or chelating catalytic metal ions and also by scavenging free oxygen. Since ancient times, many herbs have been potentially used as an alternative remedies for treatment of many infections, diseases and as food preservatives suggesting the presence of antimicrobial and antioxidant constituents (Tatjana et al., 2005). 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. Plants are the potential source of natural antioxidants. Natural antioxidants or phytochemical antioxidants are the secondary metabolites of plants (Walton and Brown, 1999). Carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, tocotrienols etc., are some of the antioxidants produced by the plant for their sustenance. Beta-carotene, ascorbic acid and alpha tocopherol are the widely used antioxidants (Mccall and Frei, 1999).
Different synthetic antioxidant such as tert-butyl-1-hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) used as food additives to increase shelf life are known to have not only toxic and carcinogenic effects in 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). Plant polyphenols have been studied largely becauseof the possibility that they might underlie the protective effectsafforded by fruit and vegetable intake against cancer and otherchronic diseases (Elena et al., 2006).
Because of the complex nature of phytochemicals, the antioxidant activities of plant extracts must be evaluated by combining two or more different in vitro assays. A number of reports on the isolation and testing of plant derived antioxidants have been described during the past decade.
The purpose of this study was to evaluate different extractives of Albizia chinensis stem bark and Picrasma javanica leaves as new potential sources of natural antioxidants and phenolic compounds.
4.2: Assays for total phenolics
The antioxidative effect is mainly due to phenolic components, such as flavonoids (Pietta, 1998), phenolic acids, and phenolic diterpenes (Shahidi, Janitha, & Wanasundara, 1992). The phenolic compounds exert their antioxidant properties by redox reaction, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Osawa, 1994). Many phytochemicals possess significant antioxidant capacities that may be associated with lower incidence and lower mortality rates of cancer in several human populations (Velioglu et al., 1998). Phenolic compounds are secondary metabolites of plants and can act as antioxidants by many potential pathways such as free radical-scavenging, oxygen radical absorbance, and chelating of metal ions
In the alkaline condition phenols ionize completely. When Folin-Ciocalteu reagent is used in this ionized phenolic solution the reagent will readily oxidize the phenols. Usual color of Folin-Ciocalteu reagent is yellow and after the oxidation process the solution becomes blue. The intensity of the color change is measured in a spectrophotometer at 760 nm. The absorbance value will reflect the total phenolic content of the compound (Harbertson and Spayd, 2006).
|Phenols + Na2CO3||Complete ionization||Ionized phenols|
|Ionized phenols + Folin-Ciocalteu
4.2.2 Materials & Methods:
Total phenolic content of A. chinensis and P. javanica extractives was measured employing the method as described by Skerget et al.,(2005) involving Folin-Ciocalteu reagent as oxidizing agent and gallic acid as standard (Majhenic et al., 2007).
|Ø Folin-Ciocalteu reagent (10 fold diluted)||Ø UV-spectrophotometer|
|Ø Na2CO3 solution (7.5 %)||Ø Vial|
|Ø tert-butyl-1-hydroxytoluene (BHT)||Ø Beaker (100 & 200ml)|
|Ø Ascorbic acid||Ø Test tube|
|Ø Methanol||Ø Pipette (1ml)|
|Ø Chloroform||Ø Pipette (5ml)|
|Ø Carbon tetra chloride||Ø Micropipette (50-200 µl)|
|Ø Distilled water|
220.127.116.11 Composition of Folin-Ciocalteu reagent:
|3||Sodium Tungstate Dihydrate||10.0|
|5||Phosphoric Acid 85 % solution in water||5.0|
|6||Molybdic Acid Sodium Dihydrate||2.5|
4.2.3 Standard curve preparation:
Gallic acid was used here as standard. Different gallic acid solution were prepared having a concentration ranging from 100 µg / ml to 0 µg / ml. 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na2CO3 (7.5 % w/v) solution was added to 0.5 ml of gallic acid solution. The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 760 nm. After plotting the absorbance in ordinate against the concentration in abscissa a linear relationship was obtained which was used as a standard curve for the determination of the total phenolic content of the test samples.
4.2.4 Sample preparation:
2 mg of the extractives was taken and dissolved in the distilled water to get a sample concentration of 2 mg / ml in every case. The samples along with their concentration for the total phenolic content measurement are given in the Table 6.
Table 4.1: Test samples for total phenolic content determination
|Plant part||Sample code||Test Sample||Calculated amount (mg/ml)|
|Fruit of C.urens||MESAC||Methanolic extract of fruit of C.urens||2.0|
|HXSF||Hexane soluble partitionate||2.0|
|CTCSF||Carbon tetrachloride soluble partitionate||2.0|
|AQSF||Aqueous soluble partitionate||2.0|
4.2.5 Total phenolic compound analysis
To 0.5 ml of extract solution (conc. 2 mg/ml), 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na2CO3 (7.5 % w/v) solution was added. The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 760 nm by UV-spectrophotometer and using the standard curve prepared from gallic acid solution with different concentration, the total phenols content of the sample was measured. The phenolic contents of the sample were expressed as mg of GAE (gallic acid equivalent) / gm of the extract.
|2.5 ml Folin-Ciocalteu reagent (10 times diluted)|
|2.0 ml Na2CO3 (7.5 % w/v) solution|
|0.5 ml of diluted extract
(Conc. 2.0 mg/ml)
|Incubated for 20 minutes at room temperature|
|Absorbance measured at 760 nm|
Figure 4.1: Schematic representation of the total phenolic content determination
4.3 Antioxidant activity assay by the Phosphomolybdenum method
The Phosphomolybdenum method was based on the reduction of Molybdenum, Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate-Mo (V) complex with a maximal absorption at 695 nm. The assay is successfully used to quantify vitamin E in Whole plant, roots and trunks. As it being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts (Prieto et al., 1999). Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid. The study reveals that the antioxidant activity of the extract exhibits increasing trend with the increasing concentration of the plant extract.
4.3.1 Materials and Method:
The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure described by Prieto et al. (1999). The assay is based on the reduction of Mo (VI)–Mo (V) by the extract and subsequent formation of a green phosphate-Mo (V) complex at acid pH. A 0.4 ml extract was combined with 4 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybda