Evaluation of Phytochemicals, Total Flavonoids Content and Antioxidant Activity of Leucaena Leucocephala (Lam.) De Wit.

Leucaena leucocephala (Lam.) De Wit. originates from Central America and Mexico and belongs to Fabaceae family. It is also known as Acacia leucocephala (Lam.), Mimosa leucocephala (Lam.) and Leucaena glabrata (Rose). It is widely distributed and cultivated in more than 150 countries especially in tropical and Sub-tropical zones. Leucaena leucocephala is a perennial evergreen, flowering, non-climbing, thornless, fast-growing, small to moderate size tree with a long life. In the genus Leucaena, about 23 well-defined species of small trees are present (KHALID et al ., 2024). Leucaena is derived from Greek words in which “leuc” means “white” and “cean” means “new” mentioned to the whitish flowers. The flowers are also mentioned by species name: leucocephala in which “leu” means “White” and “cephala” means “Head” (Zayed et al ., 2018). Leucaena leucocephala is a medium-wood plant that can be recognized by its physical characteristics, such as its thornless growth ang height of 7 to 18 meters. This plant typically has bipinnate leaves with 6-8 pairs of pinnae bearing 11-23 pairs of leaflets that its usual size is between 8-16 mm long. Flowers are round, light yellow that quickly develops into a fruit with flat brown pods that are 13-18 mm long and contain 15-30 flat oval seeds (Zarina et al ., 2017).   Leucaena leucocephala are medium sized fast – growing plant. It is also derived as “Miracle Tree” for its excellent multipurpose uses. The trees has many uses like controls soil erosion, fuel wood, timber, fodder, green manure etc. It also has medicinal properties like Antimicrobial, Anthelmintic, Antibacterial, Anti- proliferative, Anti- diabetic, Anticancer, Cancer preventive, Diuretic, Anti-inflammatory, Antioxidant, Antitumor, Antihistaminic, Nematicide, Pesticide, Anti- androgenic, Hypo-cholesterole (Deivasigamani, 2018). Leucaena leucocephala is a multipurpose tree, valued for its wood, which is used to create paper pulp, tiny furniture, and high-quality charcoal. Its seeds, young leaves, and young shoots can be eaten as vegetables. Additionally, seeds can be used as jewellery or as an alternative to coffee. It is planted to promote soil fertility, control erosion, prepare sites for reforestation, and provides shades for commercial crops like coffee and cacao. Cattles, water buffalo and goats are frequently fed the protein-rich leaves and legumes (Verma, 2016).  Leucaena leucocephala are medicinal plants which is used to control stomach ache, like contraception and abortifacient (Zayed et al ., 2016). It is used to cure intestinal worms, measles, scurf, diabetes. The roasted seeds are used to increase the flow of menstruation (Chaurasia et al ., 2015). Leucaena leucocephala (Chinese petai) leaves and seeds are used as medicinally for Indonesians. It leaves contain active flavonoids that reduce pain. It seeds have anthelmintic properties, insomnia, swelling, kidney inflammation, and diabetes. The plant used by people as wound medicine. Because of it has no side effects towards the skin irritation it uses as traditionally (Septina et al ., 2020). It’s bark and roots are used as household remedies. The roots contains emmenagogic and abortive qualities (De Angelis et al ., 2021).

MATERIALS AND METHODS

2.1 Plant Collection

The leaves and stem of the plant were collected from Navrangpura area of Ahmedabad district of Gujarat state. The plant parts were washed with water and dried at a room temperature for 2-3 days. After drying, the plant parts were made into a coarse powder using a grinder, and stored in air-tight plastic box at room temperature.

2.2 Plant Extracts Preparation

The extracts were prepared by using cold extraction method. The dried powder of the plant was dissolved in the solvent (methanol and chloroform) using the ratio of 1:10 g/ml to their respective conical flasks and which was then covered it with parafilm and aluminium foil and then incubate for 48 hours at a room temperature. All the samples were separately filtered by using the Whatman filter paper 1. The filtrate are transferred into petri-plates for solvent evaporation for 2-3 days at a room temperature. The petri-plates were stored in laboratory refrigerator for the further process. The percentage yield was calculated by the standard formula-

Yield (%) = (Weight of Dry Extract ÷ Weight of Plant Powder Taken) × 100

2.3 Qualitative Analysis (Phytochemicals Screening)

30 mg extract was dissolved in 30 ml methanol and chloroform solvents separately to prepare 1mg/ml concentration of the stock solution. Both the methanolic and chloroform extracts were further subjected for phytochemical analysis to determine the presence of various secondary metabolites like alkaloids, phenols, quinones, steroids, flavonoids, saponins, carbohydrates, proteins and glycosides. Preliminary phytochemical screening was done by following methods (Monisha et al ., 2017) (Sahrawat et al ., 2018) (Shah et al ., 2021).

• Test for Alkaloids

Mayer’s Test: Take 1ml extract and add 2ml Mayer’s reagent, the formation of white creamy ppt. or yellow colour indicates the presence of alkaloids.

Wagner’s Test: Take 1ml extract and add 1ml Wagner’s reagent, the formation of red brown ppt. indicates the presence of alkaloids.

• Test for Carbohydrates

Fehling’s Test: Take 1ml extract and add 1ml Fehling’s A solution then add 1ml Fehling’s B solution and incubate in water bath for 2-4 minutes, the formation of red colour settled in bottom of test tube indicates the presence of carbohydrates.

• Test for Glycosides

Ferric Chloride Test: Take 1ml extract and add 0.5ml Glacial acetic acid then add few drops of 1% Ferric chloride solution then add conc. H₂SO₄, the formation of brownish or red-green colour indicates the presence of glycosides.

Ammonia Test: Take 1ml extract and add 3ml Chloroform then add 1ml 10% Ammonia solution, the formation of pink colour indicates the presence of glycosides.

• Test for Terpenoids

Salkowaski’s Test: Take 1ml extract and add 1ml Conc. H₂SO₄ then incubate in water bath for 2-4 minutes, the formation of greyish colour indicates the presence of terpenoids.

• Test for Proteins

Nitric acid Test: Take 1ml extract and add Conc. Nitric acid, the formation of yellow colour indicates the presence of proteins.

• Test for Phenols

Ferric chloride Test: Take 1ml filtrate and add 5% Ferric chloride solution, the formation of dark green or blue or black colour indicates the presence of phenols.   

Folin Ciocalteu Test:  Take 1ml filtrate and add 1ml Folin Ciocalteu reagent, the formation of blue-green colour indicates the presence of phenols.

Lead acetate Test: Take 1ml extract and add 0.5ml Lead acetate solution, the formation of white precipitates indicates the presence of phenols.

• Test for Tannins

Ferric chloride Test: Take 1ml filtrate and add 5% Ferric chloride solution, the formation of dark green or blue or black colour indicates the presence of tannins.

• Test for Quinones

Sodium hydroxide Test: Take 1ml extract and add 1ml of 2% NaOH, the formation of blue green or red colour indicates the presence of quinones.

• Test for Flavonoids

Shinoda’s Test: Take 1ml extract and add few drops of Conc. HCl, the formation of pink scarlet colour indicates the presence of flavonoids.

Lead acetate Test: Take 1ml extract and add few drops of 10% lead acetate solution, the formation of yellow precipitates indicates the presence of flavonoids.

Sodium Hydroxide Test: Take 1ml extract and add 3ml of 2% NaOH, the formation of yellow colour seen then add dil. H2SO4 the yellow colour will disappears which indicates the presence of flavonoids.

• Test for Saponins

Foam Test: Take 1ml extract and add 3ml distilled water and shake it vigorously, the formation of foam layers indicates the presence of saponins.

• Test for Steroids:

Salkowaski’s Test: Take 2ml extract and shake with chloroform then add Conc. H₂SO₄ side by side, the formation of red colour at the lower chloroform layer indicates the presence of steroids.

2.4 Quantitative Analysis

The stock solution was prepared with the concentration of 1mg/ml in the methanol solvent. In case of sample, 10 mg of dry extract was dissolved in 10 ml of solvent (methanol) to make stock solution. Similarly, the standard solutions (Quercetin) were prepared with a concentration of 1mg/ml.

2.4.1 Quantification of Total Flavonoids Content

The total flavonoids content was determined by using colorimetric method with slight modifications. The standard solution used in the concentration gradient series of 10mg/ml to 100mg/ml. To the series, 0.3ml of 5% Sodium nitrite, 0.3ml of 10% Aluminium chloride, 2ml 1M Sodium hydroxide are added. Similarly for plant extract, 1ml plant extract, 0.3ml of 5% Sodium nitrite, 0.3ml of 10% Aluminium chloride, 2ml 1M Sodium hydroxide are added. Final volume made up to 5ml in both standard and extract and incubate for 5 minutes. The absorbance was determined at 510nm. The flavonoid content was calculated from standard curve of Quercetin and expressed as mg QEg^-1 (Quercetin equivalent) using the formula,

TFC = (C ×V) / M

Where, C = concentration of quercetin obtained from the standard calibration curve in mg/ml

V = volume of the extract in ml

M = weight of the extract in g

2.5 Antioxidant Activity

2.5.1 DPPH Radical Scavenging Assay

Antioxidant activity was done by using the DPPH radical scavenging assay. The DPPH is a 2,2- diphenyl-1-picrylhydrazyl reagent. The DPPH was freshly prepared by dissolving 4mg DPPH in 100ml of methanol to make 0.004% DPPH solution. Ascorbic acid was used as standard on the concentration of 0.2mg/ml. The concentration gradient series ranging from 40μg/ml to 200μg/ml, and it is replaced with 0.2mg/ml sample extract when the sample is under process. 2ml of prepared DPPH was added in standard and sample extract and incubate in complete darkness for 15 minutes. The absorbance was measured by using a spectrophotometer at λ = 517nm, a gradual decrease was determined. The test was repeated in triplicates to avoid any errors. The scavenging activity is calculated based on the percentage of DPPH radical scavenged (I%) with the help of the following formula,

I% = [(A_{control –} A_sample)/ A_control] × 100

Where, A_control is the absorbance of control (containing all reagents except the sample), and A_sample is the absorbance of sample.      

RESULT AND DISCUSSION

3.1 Yield (%) value

The yield (%) of plant extracts is calculated for the purpose of quantification of the phytochemical constituents with respect to the dry plant extract. The methanolic leaf extract 10.49% yield and in stem 5.26% yield observed. While in chloroform leaf extract 3.33% and in stem 1.37% yield was observed. The standard formula used was,

Yield (%) = (Weight of Dry Extract ÷ Weight of Plant Powder Taken) × 100

Calculations:

1. Yield (%) for Methanolic Leaf Extract = (1.049 ÷ 10) × 100 = 10.49%
2. Yield (%) for Methanolic Stem Extract = (0.526 ÷ 10) × 100 = 5.26%
3. Yield (%) for Chloroform Leaf Extract = (0.333 ÷ 10) × 100 = 3.33%
4. Yield (%) for Chloroform Stem Extract = (0.137 ÷ 10) × 100 = 1.37%

3.2 Qualitative Analysis

Methanolic extract of leaf and stem of Leucaena leucocephala (Lam.) De Wit. evinced the presence of alkaloids, glycosides, proteins, flavonoids, saponins, and steroids. Whereas, the chloroform extract of leaf revealed only the occurrence of alkaloids and glycosides and in stem alkaloids, terpenoids, proteins and steroids are occurred. This result proves that the polar methanol solvent tends to extract more phytochemicals from the plant sample as compared to the non-polar chloroform solvent.

Table 1: Preliminary Phytochemical Tests