Green Exploration of Persea americana Seed: A Potential Source of Natural Antioxidants and Antimicrobial Compounds

Medicinal plants have long been used as an important source of therapeutic agents for the treatment of various human diseases. Plant-derived compounds have gained increasing attention due to their effectiveness, low toxicity, and wide availability. According to global health estimates, nearly 80% of the world’s population relies on traditional plant-based medicines for primary healthcare ^[12].

Plants synthesize numerous secondary metabolites such as alkaloids, flavonoids, tannins, phenolics, and terpenoids, which contribute to their biological activities including antimicrobial, antioxidant, and anti-inflammatory properties. These compounds play an important role in plant defense mechanisms against pathogens and environmental stress ^{[2] [1]}. The emergence of antibiotic-resistant microorganisms has created an urgent need for the development of alternative antimicrobial agents. Natural products derived from medicinal plants are considered promising candidates because they often act through mechanisms different from conventional antibiotics ^[11]. Persea americana (avocado), belonging to the family Lauraceae, is widely cultivated in tropical and subtropical regions. While the pulp of avocado fruit is commonly consumed for its nutritional value, the seed is often discarded as waste despite containing several bioactive compounds such as phenolics, flavonoids, tannins, and glycosides ^{[3] [6]}.

Previous studies have reported that avocado seed extracts exhibit significant antioxidant and antimicrobial activities. The presence of high levels of phenolic compounds and flavonoids contributes to their biological activity against various microbial pathogens ^{[7] [3]}. Therefore, the present study aimed to evaluate the phytochemical constituents, antioxidant activity, and antimicrobial potential of avocado seed extracts.

MATERIALS AND METHODS

Collection of Plant Material

Fresh seeds of Persea americana were collected from local markets in Tiruchirappalli, Tamil Nadu, India. The collected samples were authenticated based on morphological characteristics. The seeds were washed thoroughly with distilled water to remove adhering impurities and air-dried under sterile conditions.

Preparation of Seed Powder

The cleaned seeds were cut into small pieces and shade dried at room temperature (25–30°C) for two weeks until a constant weight was obtained. The dried material was pulverized into fine powder using a mechanical grinder and stored in sterile, airtight containers at room temperature until further use.

Extraction of Plant Material

Approximately 10 g of powdered material was extracted with 100 mL of solvent (distilled water and ethanol separately) using a rotary shaker at 150 rpm for 24 hours at room temperature. The extracts were filtered through Whatman No.1 filter paper and concentrated if necessary. The filtrates were stored at 4°C for subsequent analysis.

Phytochemical Screening

Preliminary phytochemical analysis was carried out to detect the presence of alkaloids, flavonoids, tannins, phenols, saponins, steroids, terpenoids, and glycosides using standard qualitative methods. Each test was performed in triplicate to ensure reproducibility.

Thin Layer Chromatography (TLC)

TLC analysis was performed using silica gel-coated plates as the stationary phase. The mobile phase consisted of ethanol:chloroform:water in the ratio of 5:4:1. The samples were spotted on the plates and developed in a TLC chamber. The spots were visualized under UV light (254 nm) and further confirmed using anisaldehyde–sulphuric acid reagent.

The retention factor (Rf) was calculated using the formula:

Distance travelled by compound

Rf =

Distance travelled by solvent

UV–Visible Spectroscopy

UV–Visible spectroscopic analysis of the extracts was performed in the wavelength range of 300–1100 nm using a UV–Vis spectrophotometer. The absorbance values were recorded to identify characteristic peaks corresponding to bioactive compounds.

FTIR Analysis

Fourier Transform Infrared (FTIR) spectroscopy was carried out to identify functional groups present in the extracts. The dried extract was mixed with potassium bromide (KBr) and pressed into pellets. The samples were scanned in the range of 4000–400 cm?¹ and the spectral data were recorded.

Antioxidant Activity

DPPH Radical Scavenging Assay

The antioxidant activity was determined using the DPPH radical scavenging method. Various concentrations of the extract were prepared and mixed with DPPH solution. The reaction mixture was incubated in the dark for 30 minutes and absorbance was measured at 517 nm using a spectrophotometer. The percentage inhibition was calculated.

Hydrogen Peroxide Scavenging Assay

Hydrogen peroxide scavenging activity was evaluated by mixing the extract with hydrogen peroxide solution. After incubation, absorbance was measured at 230 nm. The percentage inhibition was calculated based on control values.

Antimicrobial Activity

Antibacterial activity was assessed using the agar well diffusion method. Test organisms included Escherichia coli, Klebsiella pneumoniae, and Pseudomonas species. Sterile Mueller-Hinton agar plates were inoculated with bacterial cultures (adjusted to 0.5 McFarland standard). Wells were created using a sterile cork borer, and different concentrations of the extract were added. Plates were incubated at 37°C for 24 hours. The zone of inhibition was measured in millimeters. All experiments were performed in triplicate.

Statistical Analysis

All experiments were conducted in triplicate and results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using standard methods.

RESULTS AND DISCUSSION

PHYTOCHEMICAL SCREENING

The preliminary phytochemical analysis of aqueous and ethanolic extracts of Persea americana seed revealed the presence of several bioactive compounds (Table 1). The aqueous extract showed the presence of alkaloids, tannins, phenols, saponins, steroids, coumarins, and glycosides, whereas the ethanolic extract exhibited comparatively fewer constituents.

Table 1. Phytochemical screening of aqueous and ethanolic extracts of Persea americana seed

The higher number of phytochemicals detected in the aqueous extract suggests that polar solvents are more effective in extracting bioactive compounds from plant materials. Secondary metabolites such as phenols, tannins, and alkaloids are well known for their antimicrobial and antioxidant properties. The presence of these compounds indicates the potential therapeutic value of the seed extract ^[2].

Figure 1. Phytochemical screening results of aqueous extract

Figure 2. Phytochemical screening results of ethanolic extract

These findings are consistent with earlier reports indicating that Persea americana seeds are rich in phenolic compounds and other secondary metabolites responsible for biological activity ^[3].

THIN LAYER CHROMATOGRAPHY (TLC)

Table 2. TLC analysis of avocado seed extract showing Rf values

Figure 3. TLC chromatogram of avocado seed extract

TLC analysis of the extracts demonstrated distinct spots corresponding to different phytochemical constituents (Table 2). The aqueous extract exhibited a spot with an Rf value of 0.52, while the ethanolic extract showed a spot at 0.81. The variation in Rf values indicates the presence of compounds with different polarities. The observed chromatographic separation confirms the complexity of phytochemical composition in the extracts. TLC serves as a rapid and effective preliminary technique for identifying plant metabolites ^[4]. Similar chromatographic profiles have been reported in earlier studies, supporting the presence of phenolic and flavonoid compounds in avocado seed extracts ^[10].

UV–VISIBLE SPECTROSCOPY

Table 3. UV–Visible absorption peaks of avocado seed extract

Figure 4. UV–Visible spectrum of avocado seed extract

The UV–Visible spectroscopic analysis revealed an absorption peak at 675.7 nm (Table 3), indicating the presence of chromophoric compounds. Absorption in this region is typically associated with conjugated systems and aromatic compounds, including phenolics and flavonoids. These compounds contribute significantly to antioxidant activity due to their ability to donate electrons and neutralize free radicals ^[4]. The observed spectral pattern is consistent with previous studies on plant extracts rich in antioxidant phytochemicals ^[5].

FTIR ANALYSIS

Table 4. FTIR functional group identification of avocado seed extract

Figure 5. FTIR spectrum of avocado seed extract

FTIR analysis of the extract revealed several characteristic absorption bands corresponding to different functional groups (Table 4). The peaks observed at 3367 cm?¹ (N–H stretching), 2129 cm?¹ (C≡N stretching), 1640 cm?¹ (C=C stretching), 1289 cm?¹ (N–O stretching), and 682 cm?¹ (C–Br stretching) indicate the presence of amines, nitriles, alkenes, nitro compounds, and alkyl halides, respectively. The identification of these functional groups confirms the presence of diverse bioactive compounds in the extract. Functional groups such as amines and alkenes are commonly associated with biologically active molecules, which may contribute to the observed antioxidant and antimicrobial properties. FTIR spectroscopy is widely used to identify functional groups and confirm the presence of phytochemicals in plant extracts ^[5].

ANTIOXIDANT ACTIVITY (DPPH ASSAY)

Table 5. DPPH radical scavenging activity of avocado seed extract

Figure 6. DPPH antioxidant activity of avocado seed extract

The DPPH assay demonstrated a concentration-dependent increase in antioxidant activity. The percentage inhibition increased from 40.25% at 500 µg/mL to 73.67% at 1000 µg/mL. The increasing radical scavenging activity with increasing concentration indicates the strong antioxidant potential of the avocado seed extract. This activity may be attributed to the presence of phenolic compounds, flavonoids, and tannins detected in the phytochemical screening. These compounds can donate hydrogen atoms to neutralize free radicals, thereby preventing oxidative damage. Previous studies have also reported high antioxidant activity in avocado seed extracts due to their rich phenolic content ^{[3] [10]}.

HYDROGEN PEROXIDE SCAVENGING ACTIVITY

Table 6. Hydrogen peroxide scavenging activity of avocado seed extract

Figure 7. H?O? scavenging activity of avocado seed extract

The DPPH radical scavenging assay demonstrated a concentration-dependent increase in antioxidant activity of the extract (Table 5). The percentage inhibition increased from 40.25% at 500 µg/mL to 73.67% at 1000 µg/mL. The increase in radical scavenging activity with concentration indicates the strong antioxidant potential of the extract. This activity may be attributed to the presence of phenolic compounds, tannins, and other phytochemicals capable of donating hydrogen atoms to stabilize free radicals ^[9]. Similar findings have been reported in avocado seed extracts, where high phenolic content contributed to significant antioxidant activity ^[3].

ANTIMICROBIAL ACTIVITY

Table 7. Antibacterial activity of avocado seed extract against pathogenic bacteria

E.coli	k. pnemoniae
Pseudomonas sp

Figure 8. Antibacterial activity (zone of inhibition) of avocado seed extract