Evaluation Of Phytochemicals And Antioxidant Potentials Of Crotalaria Juncea Seeds

Crotalaria juncea (Fabaceae) commonly known as Sunn-Hemp, is used for summer cover crop it returns nitrogen to the soil, suppresses weeds and nematodes, improves soil tilth. It is traditionally used as antidiabetic, aphrodisiac, antineoplastic, antimicrobial, learning and memory enhancing property with anti-venom activities. The plant contains substantial amounts of alkaloids which is responsible for its pharmacological activity.
Oxidative stress and impaired antioxidant system have been implicated in the pathophysiology of diverse disease states. The phytochemical screening and antioxidant property of seeds of Crotalaria juncea, used in the management and treatment of various diseases, were studied. The extracts of Crotalaria juncea seeds with various solvents were screened for the presence of phytochemicals, their total antioxidant activity and their inhibition of 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) radical were evaluated. The studies found that Crotalaria juncea seeds have potent antioxidant properties and types of phytochemicals; alkaloids, glycosides, saponins and tannins however, steroids, flavonoids, reducing sugars showed not their presence. In conclusion study revealed that C. juncea seeds have significant pharmacological properties therefore further study warranted to identify the active principles.

INTRODUCTION
Medicinal plants:
Medicinal plants have been accounted for therapeutically purpose since historic times and their contribution in maintaining human health is invaluable. Indian has great diversity of plant species and their role in traditional system of medicine is well versed. Indian Ayurvedic system of medicine is used worldwide for management of various human ailments and has taken extensive growth.
Medicinal plants have been used as traditional treatments for numerous human diseases for thousands of years. Many diseases like diabetes, cancer etc. continues to be a major cause of morbidity and mortality throughout the world and there is renewed interest in the discovery of novel compounds that can be used to fight these diseases. Numerous studies have validated the traditional use of medicinal plants by investigating the biological activity of extracts of such plants, which have good therapeutics potential (Palombo EA, 2006).
Large numbers of promising molecules have come out of Unani and Ayurvedic experimental base including Rauwolfia alkaloids for hypertension, Psoralens in Vitiligo, Holarrhena alkaloids in Ameobiasis, Guggulsterons as hypolipidemic agents, Mucuna pruriens for Parkinson's disease, and many other steroidal lactones and glycosides as immunomodulators (Patwardhan, 2000, Mahdi et al., 2013).
Medicinal plants have always been considered as a source for healthy life for people. Remedial properties of medical plants are very useful in healing various diseases and the advantage of these medicinal plants are natural (Kalemba and Kunicka, 2003). In many parts of the world, medicinal plants have been used for its antibacterial, antifungal and antiviral activities for hundreds of years (Ali et al., 1998; Barbour et al., 2004; Yasunaka et al., 2005).
Herbal medicines are in great demand in both developed and developing countries as a source of primary health care owing to their attributes having wide biological and medicinal activities, high safety margins and lesser costs. Herbal molecules are safe and would overcome the resistance produced by the pathogens as they exist in a combined form or in a pooled form of more than one molecule in the protoplasm of the plant cell (Lai and Roy, 2004; Tapsell et al., 2006). Even with the advent of modern or allopathic or synthetic medicine, Balick and Cox (1996) have noted that a number of important modern drugs have been derived from plants used by indigenous people.
Traditional use of medicine is recognized as a way to learn about potential future medicines. Researchers have identified number of compounds used in mainstream medicine which were derived from "ethnomedical" plant sources (Fabricant and Farnsworth, 2001). Plants are used medicinally in different countries and are a source of many potent and powerful drugs (Srivastava, et al., 1996; Mahesh and Sathish, 2008).
Natural products are the chemical compounds found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design. The medicinal use of natural products'compounds that are derived from natural sources such as plants, animals or micro-organisms have immense importance in drug discovery and have also been the sole means to treat diseases and injuries. The structural analysis of natural compounds and the ability to synthesize them allowed chemists to modify them in order to suppress or enhance certain characteristics such as solubility, efficiency or stability in the human body. Newman (2008), estimates that about 60% of the drugs that are now available'including household names such as artemisinin, camptothecin, lovastatin, maytansine, paclitaxel, penicillin, reserpine and silibinin'were either directly or indirectly derived from natural products.

Phytochemicals

Phytochemicals are chemical compounds that occur naturally in plants (Phyto means "plant" in Greek). For many decades, the use of synthetic chemicals as drugs has been effective in the treatment of most diseases but their adverse effects is considerable as evident by many complexities arises at long term as well as short term exposure. Moreover, from ancient to modern history, many traditional plant based medicines are playing an important role in health care. Phytochemicals are natural bioactive compounds found in vegetables, fruits, medicinal plants, leaves, flowers and roots which act as a defense system to combat against diseases. The phytochemicals from natural products cover a diverse range of chemical entities such as polyphenols, flavonoids, steroidal saponins, organosulphur compounds and vitamins. A number of bioactive compounds generally obtained from terrestrial plants such as isoflavones, diosgenin, resveratrol, quercetin, catechin, sulforaphane, tocotrienols and carotenoids are proven to reduce the risk of cardiovascular diseases and aid in cardioprotection which is the leading cause of death globally. The cardioprotective effects of the various phytochemicals are perhaps due to their antioxidative, antihypercholesteroemic, antiangiogenic, anti-ischemic, inhibition of platelet aggregation and anti inflammatory activities that reduce the risk of cardiovascular disorders. The multi-faceted role of the phytochemicals is mediated by its structure-function relationship and can be considered as leads for cardiovascular drug design in future (Vasanthi et al., 2012).

Based on their chemical structure, phytochemicals can be classified into the following groups:

Many phytochemicals like galactomannan, Taxol groups used in treatments of diabetes and cancer respectively. Phytochemicals are a large group of plant-derived compounds hypothesized to be responsible for much of the disease protection conferred from diets high in fruits, vegetables, beans, cereals, and plant-based beverages such as tea and wine (Arts, I.C. and P.C. Hollman, 2005). Of the numerous phytochemicals (such as alkaloids, tannins, flavonoids and terpenes) present in active extracts, tannins and flavonoids are thought to be responsible for antidiarrhoeal activity by increasing colonic water and electrolyte reabsorption. Others act by inhibiting intestinal motility. As some of the active ingredients are potentially toxic, there is a need to evaluate the safety of plant preparations. A few clinical trials have evaluated the safety and tolerability of traditional and herbal medicine preparations used to treat diarrhoea and generally indicate that minimal side effects are observed. However, with the increased popularity of plant-derived and herbal medicines, the benefits and potential dangers of these medicines must be considered (Palombo EA, 2006).

Alkaloids
Alkaloids are a structurally diverse group of over 12,000 cyclic nitrogen-containing compounds that are found in over 20% of plant species (Ashihara et al., 2006). Although no single classification exists, alkaloids are often distinguished on the basis of a structural similarity (e.g. indole alkaloids) or a common precursor (e.g. benzylisoquinoline, tropane, pyrrolizidine, or purine alkaloids).
Examples of some alkaloid secondary metabolites that are in common usage as psychotropic medicines, social drugs, or hallucinogens and have been used in insect studies either as simple tools for the modulation of specific neurotransmitter targets or, alternatively, in insect models of drug abuse and addiction.

Terpenes
Terpenes are a diverse group of more than 30,000 lipid-soluble compounds. Their structure includes 1 or more 5-carbon isoprene units. As a broad group, terpenes exhibit a range of toxicity from deadly to entirely edible and this is in keeping with their broad range of ecological roles, which include antimicrobial properties and a range of properties that attract symbiotic for the purposes of pollination, seed dispersal, and secondary protective roles. Monoterpenes can also function as antigerminative, phytotoxic allelopaths (Almeida et.al, 2010; Martino et.al, 2010). One other key property of terpenoids is that they are generally present in complex mixtures that play multiple, differing, or additive ecological roles for the plant (Wink M, 2003). Many terpenoids containing herbal extracts have thus resisted the identification of a single active component, while adequate standardization of herbal extracts has also proved to be elusive.

Structures of selected terpenes, including the monoterpenes 1, 8-cineole and geraniol, the sesquiterpene, valerenic acid, the diterpene, ginkgolide A, and the triterpene, ginsenoside.

Phenolics
Polyphenols (also known as phenolics) are compounds contain phenol rings. Phenolics are ubiquitously found across the plant kingdom, with ~10,000 structures identified to date. Structurally, they share at least one aromatic hydrocarbon ring with one or more hydroxyl groups attached. Phenolics range from simple low-molecular weight compounds, such as the simple phenylpropanoids, coumarins, and benzoic acid derivatives, to more complex structures such as flavanoids, stilbenes, and tannins phenolics, and flavonoids in particular, are ubiquitous in plants and therefore represent an important component of a normal human diet. Epidemiological studies have suggested associations between consumption of phenolic-rich foods or beverages and various diseases, such as stroke, cardiovascular disease, and cancer (Steffen LM,2006) and neurologic disorders such as dementia. (Vingtdeux et.al, 2008; Commenges et.al, 2000). Cognitive performance in elderly populations has also been shown to be associated with tea, but not coffee, consumption (Feng L et.al, 2008) and the consumption of polyphenol-rich foods such as chocolate, red wine, and tea. The anthocyanins that give grapes their purple color, the isoflavones, the phytoestrogens from soy and the tannins that give tea its astringency are phenolics.
It has been suggested that flavonoid-rich foods may limit neurodegeneration and prevent or reverse normal or abnormal deteriations in cognitive performance (Spencer J, 2010). However, the majority of the research in this area is concentrated on the effects of single molecules and the following includes a review of evidence surrounding the 3 most promising single molecule candidates. The chemical structure of curcumin, EGCG and resveratrol are shown in figure:

Glycosides
Glycosides are the molecules in which a sugar is bound to a non-carbohydrate moiety, usually a small organic molecule. Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. These can be activated by enzyme hydrolysis, which causes the sugar part to be broken off, making the chemical available for use. In animals and humans, poisons are often bound to sugar molecules as part of their elimination from the body. An example is the cyanoglycosides in cherry pits that release toxins only when bitten by herbivore. Glycosides are compounds containing a carbohydrate and a noncarbohydrate residue in the same molecule. Glycyrrhizin inhibits liver cell injury caused by many chemicals and is used in the treatment of chronic hepatitis and cirrhosis in Japan. It also inhibits the growth of several DNA and RNA viruses, inactivating herpes simplex virus particles irreversibly. Cardiac glycosides are drugs used in the treatment of congestive heart failure and cardiac arrhythmia. These glycosides are found as 7 secondary metabolites in several plants and in some animals. Some of these compounds are used as arrowhead poisons in hunting (Filippos et al., 2007).

Flavonoids
Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids belong to the polyphenol family. Flavanoids can be visualized as two benzene rings which are joined together with a short three carbon chain. One of the carbons of the short chain is always connected to a carbon of one of the benzene rings, either directly or through an oxygen bridge, thereby forming a third middle ring, which can be five or six-membered. The flavonoids consist of 6 major subgroups: chalcone, flavone, flavonol, flavanone, anthocyanins and isoflavonoids.Together with carotenes, flavanoids are also responsible for the coloring of fruits, vegetables and herbs.
Flavonoids are derived from 2-phenylchromen-4-one (2-phenyl-1-4-benzopyrone) and are commonly known for their antioxidant activities. Flavonoids, which are widely distributed in plants, fulfill many functions including producing yellow, red or blue pigmentation in flowers and protection from attacks by microbes and insects. Compared to other active plant compounds, they are low in toxicity. Flavonoids are referred to as nature's biological response modifiers because of their inherent ability to modify the body's reaction to allergens, viruses and carcinogens. They show anti-allergic, anti-inflammatory, antimicrobial and anticancer activity (Rauha et al., 2000; Cushnie and Lamb, 2005; Filippos et al., 2007; Spencer and Jeremy, 2008).

Saponins
Saponins are the glycosides of 27 carbon atom steroids, or 30 carbon atom triterpenes in plants. They are found in various plant parts; leaves, 6 stems, roots, bulbs, flowers and fruits. They are characterized by their bitter taste and their ability to haemolyze red blood cells. They are used medically as expectorant, emetic and for the treatment of excessive salivation, epilepsy, chlorosis and migraines. They are used in Ayurvedic medicine as a treatment for eczema, psoriasis and for removing freckles. Saponins are believed to be useful in the human diet for controlling cholesterol. Digitalis-type saponins strengthen the heart muscle causing the heart to pump more efficiently (Oakenfull and Sidhu, 1990). Saponins also inhibit cancer tumor growth in animals, particularly, lung and blood cancers, without killing normal cells. Saponins are the plant's immune system acting as an antibiotic to protect the plant against microbes and fungus (Shideler, 1980; Chatterrjee and Chakravorty, 1993).

Steroids
Steroid is a terpenoids lipid drug, used to improve the performance of a person and gives him more physical strength. They may cause side effects for human body on over doses which may lead to hormonal imbalance. The practice of steroid abuse is seen commonly in sports which is treated as illegal. Steroids (short for corticosteroids) are synthetic drugs that closely resemble cortisol, a hormone that your body produces naturally. Steroids work by decreasing inflammation and reducing the activity of the immune system. They are used to treat a variety of inflammatory diseases and conditions. Corticosteroids are different from anabolic steroids, which some athletes use to build bigger muscles. Examples of corticosteroid medications include triamcinolone, cortisone, prednisone, and methylprednisolone. Steroids decrease inflammation and reduce the activity of the immune system. Inflammation is a process by which the body's white blood cells and chemicals protect the body against infection and foreign organisms such as bacteria and viruses.
In certain diseases, however, the body's defense system (immune system) doesn't function properly and is overactive. This may cause inflammation to work against the body's own tissues and cause tissue damage. Inflammation is characterized by redness, warmth, swelling and pain. Steroids reduce the production of inflammatory chemicals in order to minimize tissue damage. Steroids also reduce the activity of the immune system by affecting the function of white blood cells.

Corticosteroids can be taken to treat:
' Arthritis
' Asthma
' Autoimmune diseases such as lupus and multiple sclerosis
' Skin conditions such as eczema and rashes
' Some kinds of cancer
Steroids are strong medicines, and they can have side effects, including weakened bones and cataracts. Because of this, you usually take them for as short a time as possible.Steroidogenesis is the biological process by which steroids are generated from cholesterol and transformed into other steroids. A number of drugs target the mevalonate pathway:
' Statins (used to reduce elevated cholesterol levels in patients)
' Bisphosphonates (used in treatment of various bone-degenerative diseases)

Progesterone

Tannins
Tannins are polyphenols that are obtained from various parts of different plants belonging to multiple species. Deriving it name from the technical word 'tanning' that meant converting animal hides to leather through chemical processes; tannin is basically used for this function. It is found in abundance in the tree bark, wood, fruit, fruitpod, leaves, and roots and also in plant gall.
Tannins are found as shapeless yellowish or light brown masses like powder, flakes or sponge. Apart from tanning, tannins are also used in dyeing, photography, refining beer and wine as well as an astringent in medicines. Significantly, tannins form a vital element of tea, for example, tannins are found in tea and coffee and consuming too much of these beverages without milk may lead to calcium and iron deficiency in the body and often lead to osteoporosis (a diseases where bones become fragile) and anemia. Consuming food that is rich in vitamin C also helps in neutralizing tannin's effects on iron absorption.
Tannins can also be effective in curbing hemorrhages as well as restrict bare swellings. While tannins are proved haemostatics, they are also beneficial when applied on mucosal coating in mouth. Hence, herbs possessing tannins are widely used as mouthwashes, eyewashes, snuff and even as vaginal douches and also treat rectal disorders. The good thing is that tannins' anti-inflammatory effect helps to control or curb all indications of gastritis, enteritis, oesophagitis and irritating bowel disorders. This action is possible by involving lymph stasis and neutralizing the autolytic enzymes. Conventionally, tannins have also been used to cure diarrhea. In most rural areas diarrhea is caused due to the irritation of the enteritis or the small intestine and is the reason for many deaths worldwide.

Ellagic acid

Reducing sugars
A reducing sugar is a carbohydrate that is oxidized by a weak oxidizing agent (an oxidizing agent capable of oxidizing aldehydes but not alcohols, such as the Tollen's reagent) in basic aqueous solution. The characteristic property of reducing sugars is that, in aqueous medium, they generate one or more compounds containing an aldehyde group. A reducing sugar contains aldehyde or ketone in its molecular structure.Glucose is the most common carbohydrate. This monosaccharide serves as the main source of energy for living things. It can be absorbed directly into the blood from the intestines due to its simple chemical structure. The presence of aldehyde makes glucose a reducing sugar. Glucose can be stored as starch in plants and glycogen in animals to provide an energy source later.

Antioxidants
An antioxidant is a molecule that inhibits the oxidation of free radicals generated due to different chemical reactions in our bodies. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell (Russel J et.al, 2009). Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Oxidative stress is damage to cell structure and cell function by overly reactive oxygen-containing molecules and chronic excessive inflammation. Oxidative stress seems to play a significant role in many human diseases, including cancers (Dabelstein & Werner, 2007). Antioxidants are the compounds that act as radical scavengers, prevent the radical chain reactions of oxidation and are recognized good potential in promoting health and lowering the risk of various diseases.

The three major antioxidant vitamins are beta-carotene, vitamin C, and vitamin E.
Beta-carotene and other carotenoids: Among the 600 or more carotenoids in foods, beta-carotene, lycopene and lutein are well-known leaders in the fight to reduce the damage from free radicals. Foods high in carotenoids may be effective allies against prostate cancer (beta-carotene); cancers of the mouth, pharynx, esophagus, stomach, colon and rectum (lycopene); and may help decrease your risk of macular degeneration (lutein). Foods high in carotenoids include red, orange, deep-yellow and some dark-green leafy vegetables, like tomatoes, carrots, spinach, Brussels sprouts, sweet potatoes, winter squash and broccoli.

Vitamin C: Perhaps the best-known antioxidant, vitamin C offers a wide-variety of health benefits, including protecting from infection and damage to body cells, helping produce collagen (the connective tissue that holds bones and muscles together); protecting your body from bruising by keeping capillary walls and blood vessels firm; and helping in the absorption of iron and folate. To take advantage of these benefits, eat foods rich in vitamin C like citrus fruits (oranges, grapefruits and tangerines), strawberries, sweet peppers, tomatoes, broccoli and potatoes.

Review Literature
Crotalaria juncea
Kingdom : Plantae
Family : Fabaceae
Genus : Crotalaria
Species : C. juncea
Part used : Seeds
Common name : Sunn-Hemp Seeds of C. juncea

Crotalaria juncea, known as Sunn or Sunn hemp, is a tropical Asian plant of the legume family (Fabaceae). Grown as a source of green manure, fodder and the lignified fiber obtained from its stem, it bears yellow flowers and elongate, alternate leaves. Sunn hemp is an excellent choice for a summer cover crop for growers because it returns nitrogen to the soil, suppresses weeds and nematodes, improves soil tilth and water holding capacity, and reduces erosion in fields otherwise left without plant cover. Sunn hemp forms a symbiotic relationship with soil bacteria that remove nitrogen (N) gas from the atmosphere and transforms nitrogen to plant-available forms.
The generic name Crotalaria means "rattle" and refers to the noise made by the seed pods when they have matured. Research has been conducted in the US on sunn hemp since the 1930s when it was reported to improve soil quality (Cook and White, 1996). Sunn hemp is also grown to provide biologically fixed nitrogen (N2) to subsequent crops, suppress weeds and nematodes, add organic matter to soils, and in some parts of the world, it is used as a forage crop.
Sunn hemp can grow to a height of six feet under favorable growing conditions with stem diameters that can reach 2 inches. The root system consists of a long tap root, with many well developed lateral roots (Figure 1). The inflorescence is a terminal raceme, with yellow flowers typical of the sub-order Faboideae (Papilionaceae) or pea group (Figures 2 and 3). There are several flowering types depending on the location of origin and growing conditions. Most commonly they are day-neutral, but some types are short-day. Days to flower ranges from 30-35 days in New Delhi, India (Chaudhury et al., 1978) to 60-70 days in Homestead, Fla. (Abdul-Baki et al., 2001). Mature pods bear numerous seeds that are dark grey and loose in the pod at maturity.

Fig. (a) Sunn hemp 'Tropic Sun' shoot and root development (b) Sunn hemp 'Tropic Sun' in flower

Fig. (a) Sunn hemp 'Tropic Sun' seed. (b) Maturing seed pods of 85-day-old
In order for growers to get the best return on their investment, Sunn hemp as a summer crop must be terminated prior to the main stem getting too woody for the equipment to manage. Since each operation has its own set of tools and production preferences, the limits of "too woody" will vary among operations. Sunn hemp is a promising summer annual cover crop for the southeast due to the many benefits it provides to the farming system. Seed availability and consequently the higher cost of seed may limit adoption at least in the short term.
Medicinal importance of C. juncea

Antibacterial Activity of C. juncea
C.juncea Linn. (leguminoceae), is used as medicine in the traditional system of Indian medicine for the treatment of anaemia, impetigo, menorrhagia, psoriasis etc. The ethanol extract of flowers part (CJFEE) and seeds part (CJSEE) were evaluated for the antibacterial activity by the agar disc diffusion method. The presence of steroids, triterpenes, flavonoids, phenolics and glycosides were reported in the extracts by the preliminary chemical tests. Results revealed that CJSEE possess significant antibacterial activity against the E. coli, K. pneumonia, P. aeruginosa, S. aureus and V. chlorae. Hence, study concluded that seed part possess significant antibacterial activity which may be linked to its phenolic content. (Chouhan and Singh, 2010).

Anti-inflammatory and Anti-ulcerogenic properties
C. juncea Extract (CJE) significantly inhibited the development of chronic joint swelling induced by Complete Freund's Adjuvant (CFA) in rats for 12 days whereas the standard indomethacin produced anti-arthritic effect till day 21. The effect of (CJE) was dose-dependent and linear, but for a short period compared to the standard. Treatment with (CJE) for 12 days significantly reduced the body weight of the animals and the body weight returned to normal after withdrawal of CJE. This decrease in body weight was not observed in any other animals in normal and standard groups. This may be due to CJE alone and not due to any biological factor or disease progression. This indicates the role of CJE in appetite suppressant activity. CJE was found to be anti-ulcerogenic compared to the normal control and indomethacin group of animals, a definite advantage in chronic therapy in RA. The report clearly showed CJE significantly inhibited adjuvant induced arthritis in rats. It also possesses anti-ulcerogenic property which may be due to its appetite suppressant effect. This indicates that the significant decrease in the paw volume on day 12 was due to the CJE alone and not due to any other bio-logical reasons. However, the present investigation re-quires the clinical trials to substantiate the report.(Purnima Ashok et al., 2006, G.P. Rajani, S. Arulmozhi, Basavaraj Hulkoti, B.G. Desai and R. Rajendran,2006)
Aims & Objectives
Aim of the study 'To evaluate the Phytochemicals and antioxidant potential of Crotalaria juncea seeds'

With achieving following objectives:

' Investigation for the presence of different types of phytochemicals in seeds of Crotalaria juncea.

' Antioxidant activity of Crotalaria juncea seeds by performing different assays.

Materials and Methods
Plant Material:
The Crotalaria juncea seeds were collected from authorized dealer at local market of Lucknow and identified by National Botanical Research Institute, Lucknow.

Extraction and fractionation
Extraction:
Seeds of C. juncea (800g) were shade dried and percolated with ethanol (600ml) and stand for overnight at room temperature and filter, this process was repeated three to four times for complete extraction. Combined extract concentrated under reduced pressure at temp 40-50??C on a rotary evaporator. Residue obtained 58.54g.

Fractionation:
About 250 g of seeds of C. juncea was successively extracted (to 16-18 hrs each solvent) by Petroleum ether, Chloroform, Methanol and Aqueous methanol by Soxhlet apparatus. The chemical compounds of different nature present in seeds of C. juncea were extracted according to their polarity by soxhlet extraction.

Solvents Polarity Residue Obtained
Petroleum ether Non-Polar 6.1336g
Chloroform Semi-Polar 7.84g
Methanol Polar 16.3917g
Aq. Methanol Highly Polar 7.3825g

Fig: 1 diagram showing fractionation of seeds of C. juncea

Phytochemical screening
1.0g of dry residue was dissolved in 10 ml of ethanol and following test for the analysis of different types of phytochemicals was performed, according to method described by Trease and Evans (1989) with a little modifications.

1. Test for alkaloids:
i. Mayer's test: 1ml of alcoholic extract taken in a test tube, addition of 0.2 ml dil. HCl & 1ml of Mayer's reagent- Yellowish buff precipitate.
ii. Dragendroff's test: 1ml extract + 0.2 ml dil.HCl + 1ml Dragendroff's reagent- Orange brown precipitate.
2. Test for glycosides: 0.5ml of alcoholic extract, addition of 1 ml water+ few drops of aq. NaOH- Yellow coloration
3. Test for steroids: 1ml extract + 1ml sulphuric acid- formation of Red color.
4. Test for Flavonoids: 0.5ml of extract + few drops conc. HCl. immediate development of red color.
5. Test for reducing sugars: 2ml of extract + 1ml mixture of equal vol. of Fehling's solution A & B and boiled for 5 minutes. , formation of brick red color precipitate.
6. Test for Tannins: 5ml extract + 2ml of 5% FeCl3- a Greenish black precipitate.
7. Test for Saponins: 1ml extract + 10ml distilled water shaken in graduated cylinder for 15 minutes- formation of 1cm layer of foam.
Antioxidant Activity
The study of the antioxidant activity of C. juncea seeds was concluded by performing the two different assays:

1) Total antioxidant activity (TAA)
2) DPPH assay

Total Antioxidant Activity (TAA):
Total antioxidant activity of aq. seed extract was determined according to the method of Prieto et al., 1999. An aliquot of C.juncea seed extract of 1.0 ml (1.0 mg /ml) was combined with 1.0 ml of reagent solution (0.6M sulphuric acid +28M sodium phosphate+4mMammonium molybdate). The tubes were incubated in a boiling water bath at 95??C for 90 min. Then the samples were cooled to room temperature and the absorbance was measured at 695 nm against blank prepared in the same condition by replacing sample with 1.0 ml of distilled water. All the analyses were performed in triplicate and the result were averaged. Antioxidant capacity was expressed as Ascorbic acid equivalent (m mol /g).Further on antioxidant activity was determined using absorbance recorded by double beam spectrometer.

DPPH Assay:
DPPH is a common abbreviation for an organic chemical compound 2,2-diphenyl-1-picrylhydrazyl. The percentage of antioxidant activity (AA %) of each substance was assessed by DPPH free radical assay. The samples were reacted with DPPH radical in methanol solution. The reaction mixture consisted of adding 1.0 mL of sample, 1.0 of mL of DPPH radical solution. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep violet to light yellow) were read absorbance at certain wavelength after 100 min of reaction using a double beam spectrophotometer. The mixture of methanol 1.0 mL and DPPH solution (1.0 ml) serve as blank. Ascorbic acid used as standard. The scavenging activity percentage (AA %) was then determined.
Antioxidant Activity %= [Abs control - Abs (sample)/Abs (control.)]*100

Instrumentation
Following are the instruments and apparatus used in the study
' Soxhlet Extraction apparatus
' Rotary Evaporator
' Distillation Units
' Column Chromatography/TLC
' Ph meter
' Centrifuge
' Hot-Air oven
' Weighing machine
' Double Beam Spectrophotometer

Soxhlet Extraction apparatus: This apparatus is used for extraction of mixture of compounds from seeds, flowers or leaves of medicinal plants. It is a hot continuous process of extraction, generally at 50-70 degree Celsius.
In this method, the finely ground crude drug is placed in a porous bag or 'thimble' made of strong filter paper, which is placed in chamber of the Soxhlet apparatus. The extracting solvent in flask is heated, and its vapors condense in condenser. The condensed extractant drips into the thimble containing the crude drug, and extracts it by contact. When the level of liquid in chamber rises to the top of siphon tube, the liquid contents of chamber siphon into flask. This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated. The advantage of this method, compared to previously described methods, is that large amounts of drug can be extracted with a much smaller quantity of solvent. This effects economy in terms of time, energy and consequently financial inputs. At small scale, it is employed as a batch process only, but it becomes much more economical and viable when converted into a continuous extraction procedure on medium or large scale.

Rotary Evaporator & Distillation Units: These apparatus are used for concentrating the extract thereby reduces its volume. They are also used for recovery of various compounds or solvents and helps in its distillation.
A rotary evaporator (or rotovap) is a device used in chemical laboratories for the efficient and gentle removal of solvents from samples by evaporation. When referenced in the chemistry research literature, description of the use of this technique and equipment may include the phrase "rotary evaporator", though use is often rather signaled by other language (e.g., "the sample was evaporated under reduced pressure").Rotary evaporators are also used in molecular cooking for the preparation of distillates and extracts.
A simple rotary evaporator system was invented by Lyman C. Craig. (Craig, et.al, 1950) It was first commercialized by the Swiss company B??chi in 1957. Rotary evaporation is most often and conveniently applied to separate "low boiling" solvents such an n-hexane or ethyl acetate from compounds which are solid at room temperature and pressure. However, careful application also allows removal of a solvent from a sample containing a liquid compound if there is minimal co-evaporation (azeotropic behavior), and a sufficient difference in boiling points at the chosen temperature and reduced pressure.

Column & Thin Layer (TLC) Chromatography: The procedure follows the general principle of separating the various mixtures of compounds obtained after extraction. Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.
Column chromatography: Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.
Thin Layer chromatography: It involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used.
pH meter: A pH meter is an electronic device used for measuring the pH (acidity or alkalinity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.
Weighing machine: Weighing scales is a measuring instrument for determining the weight or mass of an object.
Centrifuge: A centrifuge is a piece of equipment, generally driven by an electric motor (or, in some older models, by hand), that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. A centrifuge is also used to separate the components of blood in blood banks. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances to separate out along the radial direction (the bottom of the tube). By the same token lighter objects will tend to move to the top (of the tube; in the rotating picture, move to the centre).
Hot-Air oven: Hot air ovens are electrical devices used in sterilization. They were originally developed by Pasteur. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 ??C (122 to 572 ??F). There is a thermostat controlling the temperature. These are digitally controlled to maintain the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminum trays and may have an on/off rocker switch, as well as indicators and controls for temperature and holding time. The capacities of these ovens vary. Power supply needs vary from country to country, depending on the voltage and frequency (hertz) used. Temperature sensitive tapes or other devices like those using bacterial spores can be used to work as controls, to test for the efficacy of the device in every cycle.
Double beam spectrophotometer:
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.
If you pass white light through a colored substance, some of the light gets absorbed. A solution containing hydrated copper (II) ions, for example, looks pale blue because the solution absorbs light from the red end of the spectrum. The remaining wavelengths in the light combine in the eye and brain to give the appearance of cyan (pale blue).Some colorless substances also absorb light - but in the ultra-violet region. Since we can't see UV light, we don't notice this absorption. Different substances absorb different wavelengths of light, and this can be used to help to identify the substance - the presence of particular metal ions, for example, or of particular functional groups in organic compounds. The amount of absorption is also dependent on the concentration of the substance if it is in solution. Measurement of the amount of absorption can be used to find concentrations of very dilute solutions. An absorption spectrometer measures the way that the light absorbed by a compound varies across the UV and visible spectrum.

Results
Phytochemicals analysis
The phytochemical screening of seeds of C. juncea revealed the presence different types of phytochemicals. The result is tabulated below in Table I.
Table I: Phytochemicals present in C. juncea seeds
PHYTOCHEMICALS RESULT
Alkaloids Present
Glycosides Present
Steroids Absent
Flavonoids Absent
Reducing Sugars Absent
Saponins Present
Tannins Present

Total Antioxidant Activity
Total antioxidant activity is a quantitative assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and subsequently the formation of a green phosphate/Mo (V) complex at acid pH was recorded with the maximal absorption at 695 nm through spectrophotometer. The antioxidant activity is expressed in number of equivalents to Ascorbic acid. Different extracts of C. juncea seeds; petroleum ether, chloroform, methanolic, aq. alcoholic fractions were obtained by soxhlet apparatus and methanolic extract obtained at room temperature showed number of equivalents to ascorbic acid standards as recorded by calibration graph. The result is given in Table II.

Table II Total Antioxidant Activity of C. juncea seeds different extracts equivalent to Ascorbic acid standard.
Extracts 1000 ??g equivalent to ascorbic acid in ??g
Petroleum ether fraction 1.1
Chloroform fraction 1.5
Methanol fraction 1.6
Aq. alcoholic fraction 1.5
Methanolic (RT) 1.2

Free radical-scavenging activity: DPPH assay
DPPH free radical scavenging method is a widely used method to evaluate the free radical scavenging ability of plant materials. In this assay, an antioxidants reduces the DPPH (2,2-diphenyl-1-picrylhydrazyl) radial (purple colour) to a yellow coloured compound, 2,2-diphenyl-1-picryl-hydrazyl-hydrate. The extent of colour change depends on hydrogen donating ability of the antioxidants. It has been documented that ascorbic acid, tocopherol, cysteine, glutathione, gallic acid and few other compounds can reduce and decolorize DPPH radical. DPPH free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in methanol. This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to colorless ethanol solution. The use of the DPPH assay provides an easy and rapid way to evaluate antioxidants by spectrophotometry, so it can be useful to assess various products at a time. The DPPH assay result obtained of different fractions and extracts of C. juncea seeds is depicted below in table III.

Table III: DPPH scavenging activity of different extracts of C.juncea at different concentrations
Extracts Concentration ??g/mL % Inhibition
Petroleum ether fraction 400 14.46
800 14.18
1000 14.18
Chloroform fraction 400 16.13
800 27.53
1000 23.64
Methanol fraction 400 30.45
800 56.05
1000 69.68
Aq. Alcoholic fraction 400 46.59
800 72.04
1000 85.53
Methanol extract 400 34.63
800 59.38
1000 72.46

Fig. % inhibition of DPPH radical by different fractions and extract of C. juncea seeds a concentration of 1mg/mL.

Discussion
Our result demonstrates that seeds of C. juncea showed the presence of alkaloids, glycosides, tannins and saponins, however, flavonoids, reducing sugars and steroids were absent. Over all result is depicted in table I. Secondary metabolites (phytochemicals) and other chemical constituents of medicinal plants account for their medicinal value. For example, saponins are glycosides of both triterpenes and steroids having hypotensive and cardio depressant properties (Varadarajan et al. 2008; Olaleye, 2007). As revealed in our studies that C. juncea seeds have types of phytochemicals and due to presence of different types of phytochemicals, C. juncea documented to have several medicinal properties (Reddy V.R. et al. 1999).
The antioxidant potential of C. juncea seeds were performed by different in vitro methods. The antioxidant activity of different extracts was evaluated by two different assays; Total Antioxidant Activity and DPPH assay. The result is represented in Table II and Table III respectively. Total Antioxidant Activity of different extract of seeds of M. pruriens showed 1000 ??g equivalent to ascorbic acid from range 0.3 to 1.9 ??g; hexane fractions showed 1.1 ??g, chloroform fraction represent 1.5 ??g, methanol fraction accounted 1.6 ??g and aq. alcoholic fraction, methanol extract showed 1.5 and 1.2 ??g respectively.
Percentage inhibition of DPPH by petroleum ether fraction showed 14.46% for 400 ??g/mL, 14.18% for 800 ??g/mL and 14.18% for 1000 ??g/mL. Similarly, chloroform fraction showed 16.13% for 400 ??g/mL, 27.53% for 800 ??g/mL and 23.64% for 1000 ??g/mL. Moreover, methanol fraction showed 30.45% for 400 ??g/mL, 56.05% for 800 ??g/mL and 69.68% for 1000 ??g/mL. Aq. Alcoholic fraction, methanol extract inhibit 46.59% for 400 ??g/mL, 72.04% for 800??g/mL, and 85.53% for 1000 ??g/mL and 34.63% for 400 ??g/mL, 59.38% for 800 ??g/mL, 72.46% for 1000 ??g/mL of DPPH respectively.
All the extracts inhibited DPPH, indicating their antioxidant activity. Hexane fraction showed least potential to inhibit DPPH radical; however, methanol fraction, aq. alcoholic fraction and methanol extract occupied highest potential to inhibit DPPH radical with a little difference between them, while chloroform fractions showed moderate activity to inhibit DPPH radical.
The DPPH test provides information on the reactivity of compounds with a stable free radical DPPH that gives a strong absorption band at 517nm in visible region. When the odd electron becomes paired off in the presence of a free radical scavenger the absorption reduces and the DPPH solution is decolorized as the colour changes from deep violet to light yellow. The degree of reduction in absorbance is reflective of the radical scavenging (antioxidant) power of the compounds (Brand-Williams et al., 1995).

Conclusion
The present study evaluates presence of different types of chemical compounds and antioxidant efficiency of Crotalaria juncea seeds. On the basis of above findings it can be concluded that C. juncea have different types of chemical compounds. Aq. alcoholic fraction and methanol extract showed highest radical scavenging activity against DPPH radical. The antioxidant activity and different types of compounds present may be responsible for C. juncea medicinal account. Therefore, further study warranted to explore and identify the active principles of C. juncea seeds that could be used in management of various diseases.

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