Qualitative Phytochemical Investigation of Azadirachta Indica Leaves Using Standard Extraction Techniques

Azadirachta indica, another name for neem, is a naturally occurring herb that comes from the neem tree. The extract is made from the seeds of the tree and has numerous traditional uses. Neem is well recognized for its insecticidal and pesticidal properties, though it is also utilized in hair and dental treatments. In the past, fever, infections, inflammation, skin disorders, and dental problems have all been treated with the leaves, blossoms, seeds, fruits, roots, and bark of the neem tree. The medicinal use of neem leaf, in particular, have been documented. Neem leaf and its constituents have been demonstrated to possess immunomodulatory, anti-inflammatory, antihyperglycemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic, and anticarcinogenic properties [1]. Neem is a fast-growing perennial tree that ranges in size from 10 to 15 meters. It needs minimal yearly rainfall (400–800 mm) and grows well in regions with temperatures as high as 48–50 °C. The plant also grows well in poor, degraded, or mined soils. However, growth is impacted by frosts and low temperatures (poor growth below 14 °C). Since neem trees are the storage or repository of a wide variety of abundant bioactive secondary metabolite (BASM), they remain an ideal research topic. Since the bulk of BASM are present in the leaves and seeds, isolating or extracting the bioactive components does not need destroying the entire plant. Furthermore, because it is perennial, the annual replenishment of leaves and seeds makes whole-plant harvesting impossible. The high proportion of water-soluble chemicals in neem BASM promotes DIY extraction and usage in conventional medicine. Additionally, most of these metabolites are biodegradable, GRAS-compliant, and environmentally benign bioactive compounds that are safe for both people and the environment [2].

Azadirachta indica tree belongs to the family Meliaceae which is found in abundance in tropical and semitropical regions like India, Bangladesh, Pakistan, and Nepal. The flowers are used as element tonic, and for treatments of fever and nasal polyposis. The stem bark is used for the treatment of diarrhoea and ameobic dysentery. The leaves are also used as element tonic, to stimulate gastric secretion, for treatment of fever and as insecticide. Furthermore, in Thai land, the young leaves and young flowers of Siamese neem tree are commonly consumed as a cooked vegetable. The objective of this research was to investigate the antioxidant activity of the aqueous extracts of leaves of Siamese neem tree from several extracting and drying methods including percolation, decoction, maceration, soxhlet extraction, spray drying and freeze drying [3].

Taxonomical Classification

· Kingdom:               Plantae

· Subkingdom:         Tracheobionta

· Division:                Magnoliophyta

· Class:                     Eudicot

· Subclass:                Rosidae

· Order:                    Sapindales

· Family:                  Meliaceae

· Genus:                   Azadirachta

· Species:                 indica

MATERIALS AND METHODS

2.1. Plant Material

Fresh leaves of Azadirachta Indica (Neem) were collected during the appropriate season from a specified geographical location Near Boys’ Hostel–2, Paramedical Campus, Uttar Pradesh University of Medical Sciences (UPUMS), Saifai, Etawah, Uttar Pradesh, India. The collected plant material was taxonomically authenticated by a qualified botanist from K.K.P.G. College, Etawah.

2.2. Leaf Drying & Powdering

The collected leaves were thoroughly washed water to remove extraneous matter, shade-dried at room temperature for a period of 10–15 days, and subsequently pulverized into a coarse powder using a suitable mechanical grinder.  

2.3. Preparation of Soxhlet Extraction

2.3.1. Ethanolic Extraction:

The powdered drug was separately extracted using vapora apparatus with 80% ethanol (60–80 °C; 1: 5, w/v) for 7-8 hrs. and then filtered. Ethanol was removed using a vaporate to yield dried leaf 80% ethanolic Soxhlet extracts [4].

2.3.2. Aqueous Extraction:

The powdered drug was separately extracted using soxhlet apparatus with 100% Distilled Water (100 °C; 1: 5, w/v) for 5-6 hrs. and then filtered.  Distilled water was removed using a evaporat to yield dried leaf Soxhlet extracts.

2.4. Phytochemical Screening of Azadirachta indica Extracts:

Each of the Azadirachta indica Extracts to phytochemical analysis for the active constituents found in the Extracts and findings were recorded as present (+) or absent (-) according to the color change.

2.4.1. Tests for alkaloids: 1 gm of  plant extract was dissolved in 10 ml of  Distilled water and filtered. The filtrate was used to test the presence of alkaloids.

Mayer’s Test: Filtrates was treated with Mayer’s reagent. Formation of yellow cream-colored precipitate indicates the presence of alkaloids.

Dragendorff’s Test: Filtrate were treated with Dragendorff’s reagent. Formation of reddish-brown colored precipitate indicates the presence of alkaloids.

Wagner’s test: Filtrate was treated with Wagner’s reagent. Formation of reddish-brown colored precipitate indicates the presence of alkaloids.

Hager’s Test: Filtrates was treated with Hager’s reagent. Formation of yellow colored precipitate indicates the presence of alkaloids.

2.4.2. Tests for carbohydrates:

Molisch’s test: 1 ml of extract solution was treated with few drops of Molisch’s reagent in a test tube and 2ml of concentrated H₂SO₄ was added carefully along the sides of the test tube, formation of purple to violet color ring at the junction indicates the presence of carbohydrates.

Fehling’s test: 1 ml of plant extract solution was mixed with 5 ml of fehling’s solution [Equal volume of Fehling’s A (Copper sulphate in distilled water) and Fehling’s B (Potassium tartarate and Sodium hydroxide in distilled water) reagents are mixed] and boiled. Formation of brick red precipitate indicates the presence of reducing suger.

Benedict’s test: Take  the 2ml  test solution with few drops of Benedict’s reagent, (alkaline solution containing cupric citrate complex) and boil on water bath upon boiling, reddish brown precipitate forms if reducing sugars are present.

2.4.3. Tests for Glycoside:

Keller killiani’s test: 2ml plant extracts was shaken with 1 ml glacial acetic acid containing a trace amount of ferric chloride. 1 ml of conc. sulphuric acids was added carefully by the side of the test tube, blue color appears in the acetic acid layer and red colour at the junction of the two liquids indicates the presence of glycosides.

2.4.4. Tests for steroids:

Salkowaski test:  2ml Plant extract in chloroform with few drops of concentrated Sulphuric acid, shake well and allow to stand for some time, red color appears in the lower layer indicates the presence of sterols and formation of yellow colored lower layer indicates the presence of triterpenoids.

2.4.5. Tests for Protein and Amino acids: 100 mg of plant extract was taken in 10 ml of water and filtered. The filtrate was used to test the presence of protein and amino acids.

Millon’s test: 2 ml of filtrate was treated with 2 ml of Millon’s reagent in a test tube and heated in a water bath for 5 minutes, cooled and added few dropped of NaNo₂ solution. Formation of white precipitate, which turns to red upon heating, indicating the presence of proteins and amino acids.

Ninhydrine test: Take 2 ml of filtrate extract, 0.25% Ninhydrine reagent was added in a test tube and boiled for 2 minutes. Formation of blue colour indicates the presence of amino acids.

Biuret test: 2 ml of filtrate was treated with 2 ml of 10% sodium hydroxide solution in a test tube and heated for 10 minutes. A drop of 7% copper sulphate was added in the above mixture. Formation of purplish violet colour indicates the presence of proteins.   

2.4.6. Tests for phenolic:

Ferric chloride test: 2 ml of plant extract solution were allowed to react with 2 ml of 1% ferric chloride solution in test tube. Formation of greenish- black colour indicates the presence of phenolic nucleus [5].

2.4.7. Tests for flavonoids:

Shinoda test (Magnesium hydrochloride reduction test): 2 ml Plant extract, few fragments of magnesium metal were added in a test tube, followed by drop wise addition of concentrated hydrochloric acid. Formation of magenta colour indicates the presence of Flavanoids.

Alkaline reagent test: To the test solution add few drops of sodium hydroxide solution, formation of an intense yellow color which turns to colorless on addition of few drops of dilute acetic acid indicates the presence of flavonoids.

RESULTS AND DISCUSSION

Table 1: Phytochemical analysis of Azadirachta indica (Neem) extracts