The present study was designed to compare phytochemicals, antimicrobial and antioxidant activity of different extracts of leaves of
Analysis of Phytochemicals in various extracts of
Comparative evaluation of antibacterial and antioxidant activity of various extracts of
The disc diffusion method was used to detect antibacterial activity of different extracts of
The findings suggested that phytochemicals present in
Azadirachtaindica (Neem) is a fast growing evergreen popular tree of meliaceae family found in most tropical countries like India, Africa, America [1]. It has been used in Ayurvedic medicine for more than 4000 years due to its medicinal properties. Neem is called “arista” in Sanskrit,a word that means ‘perfect’, complete and imperishable [2]. The tree is regarded as “village dispensary” in India. The importance of the neem tree has been recognized by the US National Academy of Science, which publish a report in 1992 entitled “Neem- a tree for solving global problems” [3]. It possesses maximum useful non-wood products (leaves, fruits, bark, flowers, seed, gum, oil, and neem cake) than any otherspecies. It is known to have Antiallergenic, antidermatic, antifeedant, antifungal, antiinflammatory, antipyorrhoeic, antiscabic, cardiac, diuretic, insecticidal, larvicidal, nematicidal, spermicidal and other biological activities. Quercetin and ß-sitosterolwerefirstpolyphenolic flavonoids purified from fresh leaves of neem and were known to have antifungal and antibacterial activities. Numerous biological and pharmacological activities have been reported including antibacterial [4], antifungal [Kher and Chaurasia 1997] and anti-inflammatory. Earlier investigators have confirmedtheir role as anti-inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, antifungal, antibacterial, andante tumour activities [5].
Azadirachtaindica is considered harmless to human, animal, birds, beneficial insects and earthworms and has been approved by the US Environment Protection Agency for use on food crops [6]. Biological active compound isolated from different parts of the plant include: azadirachtin, meliacin, gedunin, salanin, nimbin,valassinand many other derivatives of these principles. Each part of the neem tree has some medicinal property and is thus commercially exploitable. Several pharmacological activities and medicinal applications of various parts of neem are well known. Cyclic trisulphide and tetrasulphide isolated from the steam distillate of fresh, matured neem leaves have antifungal activity against trichophytonmentagrophytes.
It has been accepted that drugs based on allopathy are expensive and also exhibit toxic effect on normal tissues and on various biological activities. It is largely accepted fact that numerous pharmacologically active drugs are derived from natural resources including medicinal plants [7].
A study confirmed that ethanolic fraction of neem leaf (EFNL) treatment effectively up regulated the pro-apoptoticgenes and proteins including p53, Bcl-2-associated Xprotein (Bax), Bcl-2-associated death promoter protein (Bad) caspases, phosphatase and tensin homolog gene (pTEN),and c-Jun N-terminal kinase (JNK) [11].
A methanol extract of the leaves exerts antipyretic effect in male rabbits [12]. The plant also possesses analgesic activity mediated through opioid receptors in laboratory animals.
The aqueous extract of leaf also possesses potent immune stimulant activity as evidenced by bothhumoral and cell-mediated responses [13]. Leaf extract at100 mg/kg after three weeks of oral administration causes higher IgM and IgG levels along with increased titer of anti albumin antibody [14].
Aqueous extract of neem leaves significantly decreases blood sugar level and prevents adrenaline as well as glucose-induced hyperglycaemia [15]. The aqueous leaf extract when orally fed, also produceshypoglycaemia in normal rats and decreased blood glucose levels in experimentally-induced diabetes in rats [16]. Neem leaf aqueous extract produces antiulcer effect in rats exposed to restraint – cold stressor ethanol orally by preventing mucus depletion and mast cell degranulation [17].
Oral administration of aqueous extract of Neem leaf shows anti-fertility effect in mice [18]. Neemseed and leaf extracts are effective against malarial parasites [19]
In the present work, we have tried to analyses various extracts from
Leaves were washed thoroughly with distilled water and surface sterilized with 0.1% HgCl2and then air dried for 4-5 days.
The aqueous extract was prepared according to Sripanidkulchai
Two bacterial strains were used in this study-one gram positive (
Bacterial suspensions were prepared from over night cultures by the direct colony method. Colonies were taken directlyfrom the plate and suspended into 1 ml of sterile 0.85% saline. The turbidity of initial suspension was adjusted comparing with 0.5 McFarland Standard which is equivalent to 108colony forming units (CFU)/ml. Bacterial lawn was spread on nutrient agar plates using a sterile cotton swab.
The qualitative antimicrobial activity of different extracts of
Ferric chloride is acidic in nature; it produces H3O+ions when dissolved in water. Compounds with a phenol group will form a blue, violet, purple, green, or red-brown colour upon addition of aqueous ferric chloride. Extracts were treated with 3-4 drops of 5% ferric chloride solution. The appearance of bluish or greenish black coloration indicates the presence of pyrogallol or catechol tannins. Formation of bluish black colour indicates the presence of phenols.
The conjugation in flavonoid compounds produces a yellow colour, while the extended conjugation in the resultant anthocyanidin shifts the colour further out to the red region of the visible spectrum. The dramatic change in colour makes this a simple visual test for the presence of flavones. Extracts were treated with few drops of 10% lead acetate solution. Formation of yellow colour precipitate indicated the presence of flavonoids
Fehling’s solution contains blue alkaline cupric hydroxide solution, which upon heating with plant extract gets reduced to yellow or red cuprous oxide. Extracts were treated with 2-3drops of Fehling reagent and heated to 10 minutes. Appearance of red colour precipitate indicated the presence of reducing sugars.
Mercury salt reacts with the lone pairs of nitrogen that are present, then the typical off-white precipitate falls out. An uncharged mercury salt is, thus obtained. Extracts were dissolved individually in dilute hydrochloric acid and filtered. Filtrates were treated with 2-3 drops of Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicated the presence of alkaloids.
The reaction is due to the presence of the hydroxyphenyl group, C6H5OH in the amino acid molecule; and any phenolic compound which is unsubstituted in the 3,5 positions such as tyrosine, phenol and thymol will give the reaction. Solutions of nitric acid containing mercuric nitrate reacts with phenols, producing red colours or yellow precipitates which react with nitric acid to form red solution. The reaction probably depends on the formation of a nitro compound; which then reacts with phenol.20 µl of millon reagent was added to the test tube containing 1ml of extract and then heated in water bath for 10 minutes. The samples were cooled and 10µl of 1% sodium nitrite solution. Red colour indicated the presence of proteins.
Xanthoprotein test gives positive results with proteins, peptones, peptides, amino acids and other primary amines, including ammonia. Proline and hydroxyproline give yellow colour with ninhydrin, while other acids give blue to purple colour.4-5 drops of 0.1% ninhydrin solution were added to 1ml of extract. Purple colour indicated the presence of amino acids.
Salkowski’stest detect presence of cholestrol. Presence of double bond in one cholestrol ring is responsible for its ability to form coloured product in the presence of concentrated inorganic acid and it should yield a layering of colour. Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of concentrated Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicated the presence of triterpenes.
The DPPH radical scavenging assay of various extracts of
DPPH radical scavenging activity (%) =1-As/Ac×100
Where Ac-Absorbance of control, As-Absorbance of test sample.
The yield obtained of the different extracts was shown in.
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| 1. | Aqueous | 95mg | 1.9 |
| 2. | Methanol | 200mg | 2 |
| 3. | Chloroform | 60mg | 0.6 |
| 4. | Ethyl acetate | 530mg | 5.3 |
The result showed that the yield of ethyl acetate extract was higher than other extracts. Fig. 1
It may be due to the different polarity of the solvents used.
The screening of phytochemicals was done in all the four extracts by performing various tests as listed in materials and method section.
The ferric chloride test formation of greenish black colour revealed that phenolics were present inthe methanol, ethyl acetate and aqueous extracts.The chloroform extract did not reveal presence of phenolics.
Inthe lead acetate test formation of yellow precipitate revealed that flavonoids are present in all the extracts (fig. 4.3). Control contains lead acetate. The yellow precipitate was detected in all the extracts.
The Fehling’s test for carbohydrates revealed the presence of reducing sugars in aqueous, methanol and ethyl acetate extracts, whereas chloroform extract showed absence of carbohydrate. Formation of green or red colour indicated the presence of reducing sugar (Fig. 4.3).
The Mayer’s test for alkaloids revealed that aqueous, methanol, chloroform, ethyl acetateand crude methanolshow presence of alkaloids (Fig. 4.4).In this (A) is control which contain only reagent. In all extracts, yellow colour precipitate or turbidity was observed. Formation of yellow colourindicatedpresence of alkaloids.
Formation of yellow colour in the ethyl acetate extract revealed the presence of amino acids. The control in the contains the conc. HNO3. The yellow colour was absent in the aqueous, methanoland chloroform extract, showing the absence of amino acids.
In salkowski’s test, formation of reddish brown colour revealed that triterpenoids were present in all the extracts. Reddish brown colour was formed in the aqueous, methanol, chloroform and ethyl acetate extract of
The result showed that various phytochemicals were present in the aqueous, methanol, chloroform and ethyl acetate extract. Alkaloids, flavonoids, terpenoids were present in all the four extracts. Tannins were absent in the ethyl acetate extract and phenols were only present in the ethyl acetate extract. Sterols and phlobatannins were absent in all the four extracts. Saponins were only present in the aqueous extract and amino acids were only present in the ethyl acetate extract. “++” indicates the more intense colour as compared to other extracts.
The antimicrobial activity of different extracts of
In the present study, we found that methanolic extract of
In DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity the results showed that percentage inhibition increased with increasing concentration from 2.5µg/ml to 10 µg/ml. DPPH antioxidant assay is based on the ability of a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron at 517 nm and is visible with deep purple color. When DPPH accepts an electron donated by an antioxidant compound, it is decolorized and can be quantitatively measured from the changes in the absorbance. Free radical scavenging effect of the corresponding different
DPPH radical scavenging activity (%) =1-As/Ac×100
Where Ac-Absorbance of control, As-Absorbance of test sample.
IC50 value is a measure of the effectiveness of substance in inhibiting a specific biological or biochemical function. The IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half. The IC50 is the concentration of an inhibitor where the response or binding) is reduced by half. In the present study, the different extracts of
Methanolic extract of
Many of the existing synthetic drugs cause various side effects. Hence, natural plant based drug compounds could be useful in meeting this demand for newer drugs with minimal side effects.
The antimicrobial activity of the aqueous, methanol, chloroform and ethyl acetate extracts of
In the present project entitled
The DPPH free radical scavenging activity was performed to study the antioxidant potentials of
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