Exploring the Phytochemical Composition and Antioxidant Properties of Acacia paradoxa, Acacia nilotica, and Acacia auriculiformis Barks

Genus Acacia was considered to contain some 1352 species leading to 1986.That year, Lesile Pedley questioned the monophyletic nature of the genus, and proposed a split into three genera: Acacia sensustricto (161 species). Senegal (231 species) and Racosperma (960 species) in 2013, Pedley published a paper 834 new combinations in Racosperma for most species most of which were formerly placed in Acacia. All but 10 of these species are native to Australia, where it constitutes the largest plant genus. At the 2011 International Botanical conference held in in Melbourne, Australia, the decision to use the name Acacia rather than the proposed Racosperma for this genus was upheld. Other Acacia taxa continue to be called acacia by those who choose to consider the entire group as one genus. Seed pod fossils of Acacia parschugiana and Acacia cyclosperma are known form their Tertiary deposits. A funicles of Acacia auriculiformis have an antifungal efficacy against the Aspergillus ochraceous and Curvularia lunata. Acacia auriculiformis may also include neutralizing radical generated by fungal Laccase as evidence by it enhance DPPH Radical scavenging activity and Laccase inhibition (Mihara et al., 2005) Rajbir S. et al. obtained the antioxidant kaempferol from the methanol extract of Acacia nilotica (Kalaivani T et al., 2010) While the antioxidant Umbelliferon was also recovered from the bark of Acacia nilotica. Plant parts of Acacia nilotica possess antioxidant potential. Bark and leaves methanol and Ethanol extract of Acacia nilotica Respectively were showed the antioxidant activity (Singh et al., 2010) (Osman et al., 2014). Since ancient times, Acacia nilotica has been used in traditional medicine to cure a variety of illness. Its antibacterial, Antifungal, Antioxidant, antidiabetic, antihypertensive, antispasmodic, antiviral properties. Acacia paradoxa is a plant in the family Fabaceae. Its common names include kangaroo acacia, kangaroo thorn, prickly wattle, hedge wattle and paradox acacia. The large shrub or tree up to 2 to 4 metres (7 to 13 ft) tall and has a similar width, it has ribbed branchlets that are often arched downward.

MATERIALS AND METHODS

Plant collection

A Bark of Acacia nilotica, Acacia paradoxa, Acacia Auriculiformis respectively collected in February month of 2025 from Ahmedabad district, Gujarat, India. After collection bark was dried in shade for 1 week followed by grind it and make a fine powder for further phytochemical studies.

Extract Preparation

The cold extraction methodology, the weighed about 10gm of plant powder using a Digital Balance and transferred into Separate Flasks. Then added 100ml of Methanol and Acetone to their Respective Flasks Containing the powder. The Flask then sealed and incubate for 24 Hours at room Temperature. After the 24 hour of Incubation period, the prepared extract was filtered using Whatman Filter paper no. 1 in petri dishes. Prior to filtration the empty petri dishes were weighed on weighing balance to measure the initial weight. The Filtered extracted were carefully Transferred to the filter paper in petri dishes. After filtration the petri dishes Containing the filtered extract were left aside for approximately for half an hour to allow for the evaporation of the solvent. The petri dishes were then weighed again to measure the weight of filtered extract. The difference between the initial and final weights of petri dishes provided the Yield Extract value. The yield of each extract prepared in different solvents was calculated by using the formula (El Mannoubi, I., 2023). 

% Yield = The mass of extract after solvent Evaporation Total Mass of Plant Material ×100

Qualitative Phytochemical Analysis

Test for Alkaloid

A little portion of the crude extract was diluted in diluted hydrochloric acid and filtered.

Mayer’s Test: Take 2ml of extract add 1ml of Mayer’s reagent side by side; white creamy precipitate indicates the presence of alkaloids.

Wager’s Test: Take 2ml of extract and add 2ml of wager’s reagent side by side. Reddish Brown precipitate indicates the presence of Alkaloids.

Test for Phenols

Ferric Chloride Test: Mix 2ml extract with 1-2 Drops of 5% Ferric chloride, the bluish black colour indicates the presence of Phenols.

Lead Acetate Test: Mix 2ml extract with 0.5ml lead acetate, white precipitates indicate the presence of phenols.

Test for Flavonoids

Alkaline Test: 1ml extract with 10% sodium hydroxide; yellow colour was seen then add dilute Hydrochloric acid; yellow colour disappears that indicates presence of flavonoids.

Zinc-HCL Test: Take 1ml extract mix with Zinc dust and add Conc. HCL, Magenta colour Show the presence of Flavonoids.

Test for Tannins

Lead acetate Test: 1ml extract was treated with 10%lead acetate solution, resulting in white precipitates indicating tannins.

Ferric Chloride: 1ml extract was treated with 1ml 5% ferric chloride solution. The presence of tannin is indicated by the green hue.

Test for Lignin

Labat Test: 1ml extract and 1ml Gallic acid was taken, olive green precipitates show the presence of lignin.

Test for Steroid

Liberman Burchard’s Test: In 1ml extract add 1ml chloroform with 2ml Acetic Anhydride and 1-2 drops H2SO4, Array of colour change Blue- green- Red (ring at junction) is seen.

Test for Terpenoids

Salkoski Test: Take 1ml extract treated with 1ml chloroform, filter it and add 1ml conc. H2SO4, yellow precipitates show that terpenoid is present. (Banu, K. S. and Cathrine, L. 2015).

Total Phenolic Content

The Total Phenolic content were determined by Folin-ciocalteu reagent method. 1ml extract of three selected Bark Acacia nilotica, Acacia paradoxa, Acacia Auriculiformis respectively and standard Gallic acid (100-1000 µg/ml) were taken and 1.5ml 1N Folin ciocalteu reagent added. 10ml Distilled water and 4ml 20% sodium carbonate respectively added to mixture. Make final volume 25ml with Distilled water. After 30minutes of incubation, the absorbance at 765nm was measured using UV visible spectrophotometer (Shimadzu UV-1800, Shimadzu corporation, Kyoto, Japan) A result of total phenolic content was represented as a milligram Gallic acid equivalent per gram (mg GAE/g). (Sembiring et al., 2018).

Total Flavonoid content

Total flavonoid content was determined by aluminium chloride method. 1ml extract of three selected Bark Acacia nilotica, Acacia paradoxa, Acacia Auriculiformis respectively and standard Quercetin (100-1000 µg/ml) Were taken and 0.1ml 10% Aluminium chloride added. Followed by 0.1 ml 1M Sodium Acetate added. Make final volume upto 10ml with distilled water. Absorbance at 415nm was measured using UV visible spectrophotometer (Shimadzu corporation Kyoto, Japan). A result of total flavonoid content was reported as a milligram Quercetin equivalent per gram (mg QE /g). (Quettier-Deleu et al., 2000).

Total Tannin Content

Total Tannin content was determined by Folin Denis Method with some modifications. 1ml extract of three selected bark and Standard Tannic acid (100-1000 µg/ml) were taken and 0.1ml Folin Denis reagent added. Then added 1ml of 7.5%Na2CO3. Make final volume 10ml with Distilled water. Shake the constituents’ particles and mix it properly.  After 30 Minutes of Incubation of the absorbance of 700nm was measured using UV visible spectrophotometer (Shimadzu UV-1800, Shimadzu corporation, Kyoto, Japan) A result of Total Tannin Content was represented as a milligram Tannin acid equivalent per gram (mg TAE/g). (Vala, M., & Maitreya, B., 2022).

Antioxidant Activity  

The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to determined antioxidant potential of three selected Acacia species. Take 0-1000 ml extract and 100-1000 mg/ml Standard Ascorbic acid and make final volume 1ml using Methanol and Acetone solvents respectively and 3ml DPPH Solutions was added followed by, incubate for 30 minute in Dark Condition and the absorbance at 517nm was measured using UV-Visible spectrophotometer (Shimadzu UV- 1800, Shimadzu corporation Kyoto, Japan) and check the inhibition using following formula.

Inhibition (%) =    Control – Test      × 100

                    Control

Where, Control is the absorbance of the control (DPPH solution without the addition of Bark extract) and Test is the absorbance of reaction mixture samples (in the presence of Bark extract). IC50 Value obtained from the results of DPPH method, indicates the sample quantity was derived from a co-relating the discoloration of the sample with its concentration. (Stankovic, M. S. 2011).

RESULT

Yield Extract Value:

Graph:1 Yield Extract of three Selected Acacia Species

In methanolic solvent, the Acacia nilotica Bark extract yield is 4.18 %, the Acacia auriculiformis bark extract is 3.11% and Acacia paradoxa Bark extract is 7.53%. In Acetone solvent, the Acacia nilotica Bark extract yield is 8.9 %, the Acacia auriculiformis bark extract is 2.99 %, the Acacia paradoxa Bark extract yield is 8.98 %. Acacia paradoxa shows the maximum extract in the Acetone extract.

Qualitative phytochemical Analysis:

Table: 1 Qualitative phytochemical screening of three Acacia species

Phytochemical constituents include Alkaloids, Flavonoids, Tannin, Phenols, Lignin. According to table, the preliminary phytochemical screening of selected bark reveals the presence of phytochemicals in methanol and acetone extracts. Certain test for flavonoids reveals the existence of phytochemical in selected bark in both Methanol and Acetone extract. Additionally, testing for phenols have shown that these phytochemicals are present in Acacia nilotica, Acacia paradoxa, Acacia auriculiformis in Methanolic extract. Similar to this, testing of phenols, alkaloids have shown that Acacia nilotica, Acacia paradoxa, Acacia auriculiformis contain these phytochemicals in acetone extract. Lignin indicating their presence in Acacia paradoxa bark.

Quantitative Analysis

Table:2 Total Phenol, Flavonoid and Tannin Content of three Selected Acacia genus bark.

Results shows as Mean ± Standard Deviation. In this result TPC means Total Phenol Content, TFC means Total Flavonoid Content, TTC means Total Tannin Content.

Total Phenolic content

Table 2 shows the total phenolic content of three selected acacia species bark’s extract. Acacia Nilotica shows 1709.2 ±0.04 mg GAE/g phenol in methanol extract and 2947.2±0.04 mg GAE/g in acetone extract. Acacia auriculiformis methanol extract shows 3844.2±0.10 mg GAE/g phenolic content and acetone extract shows the Higher phenol content 5270.2±0.01 mg GAE/g. Acacia paradoxa shows 2770.2±0.0 mg GAE/g phenolic content in methanol extract and 1690.2±0.00 mg GAE/g phenolic content in acetone extract.

Total Flavonoid Content

Table 2, shows the total flavonoid content of three selected acacia species bark’s extract. Acacia nilotica shows 514.66 ±0.04 mg QE/g Flavonoid in methanol extract and 834.75±0.06 mg QE/g Flavonoid content in Acetone extract. Acacia auriculiformis shows 1376.33±0.07 mg QE/g Flavonoid in methanol extract and 2473.25±0.07 mg QE/g Flavonoid content in Acetone extract. Acacia paradoxa shows mg 176.33±0.00 mg QE/g Flavonoid in methanol extract and 82.25±0.00mg QE/g in acetone extract.

Total Tannin Content

Table 2 shows the total Tannin content of three selected acacia species bark’s extract. Acacia nilotica shows 231.57±0.04 mg TA/g Tannin in methanol extract and 190.14±0.00 mg TA/g Tannin content in Acetone extract. Acacia auriculiformis shows 310.14±0.02 mg TA/g Tannin in methanol extract and 245.14±0.02mg TA/g Tannin in acetone extract. Acacia paradoxa shows mg 160.14±0.05 TA/g Tannin in methanol extract and 80.14±0.07mg TA/g Tannin in acetone extract.

Antioxidant Activity

DPPH Radicle scavenging Activity

Graph 2: DPPH Scavenging activity of selected three Bark in Methanol extract.

Graph 2 shows the DPPH scavenging activity of bark of three Acacia species in methanol extract. Acacia nilotica bark shows 60 percentage inhibition at 1000 µg/ml, 45.23 percentage inhibition at 800µg/ml concentration, 37 percentage inhibition at 600 µg/ml, 50 percentage inhibition at 400 µg/ml, 55 percentage inhibition at 200 µg/ml concentration respectively. Acacia auriculiformis bark shows the 52.26 percentage inhibition at 1000 µg/ml, 32.08 percentage inhibition at 800µg/ml concentration, 27.9 percentage inhibition at 600 µg/ml concentration, 23.47 percentage inhibition at 400 µg/ml concentration, 15.6 percentage inhibition at 200 µg/ml concentration. Acacia paradoxa bark shows 70 percentage inhibitions at 1000 µg/ml, 56 percentage inhibition at 800µg/ml concentration, 59 percentage inhibition at 600 µg/ml concentration, 55 percentage inhibition at 400 µg/ml concentration, 36 percentage inhibition at 200 µg/ml concentration.

Graph 3: DPPH Scavenging activity of selected three bark in Acetone extract.

Graph 3 shows the DPPH scavenging activity of bark of three Acacia species in methanol extract. Acacia nilotica bark shows 71.82 percentage inhibition at 1000 µg/ml, 45.23 percentage inhibition at 800µg/ml concentration, 60.5 percentage inhibition at 600 µg/ml concentration, 54.95 percentage inhibition at 400 µg/ml concentration, 37.75 percentage inhibition at 200 µg/ml concentration. Bark of Acacia auriculiformis in Acetone extract shows 20.1 percentage inhibition at 1000 µg/ml, 17.08 percentage inhibition at 800µg/ml concentration, 16.82 percentage inhibition at 600 µg/ml concentration, 6.812 percentage inhibition at 400 µg/ml concentration, 3.49 percentage inhibition at 200 µg/ml concentration. Bark of Acacia paradoxa in Acetone extract shows 70 percentage inhibition at 1000 µg/ml, 56 percentage inhibition at 800µg/ml concentration, 59 percentage inhibition at 600 µg/ml concentration 55 percentage inhibition at 400 µg/ml concentration, 36 percentage inhibition at 200 µg/ml concentration.

Graph 4: IC₅₀ Values of antioxidant activity (DPPH) of three selected Bark extract.