Evalution Of Phytochemicals, Antioxidant Potential, And Antibacterial Properties of Plant: Pennisetum Setaceum (Forssk.) Chiov

Pennisetum setaceum (Forssk.) Chiov., commonly referred to as purple fountain grass, is a type of purple-pigmented ornamental grass from the Poaceae family. Pennisetum setaceum (Forssk.) Chiov., which originated in Africa, is one of the most commonly used ornamental grasses worldwide. It has several landscaping applications, including border, erosion control, rock garden, and urban garden. It requires a warm temperature to produce vibrant leaf color. Purple fountain grass was selected because it is evergreen, grows in hot regions, and is drought tolerant (Gonzalez-Rodriguez et al., 2010). It belongs to the C4 grasses, which have a unique photosynthetic pathway. The shrub blooms widely throughout the summer and winter. The plant has wonderful qualities, including spikes of purple flowers that gracefully shoot from its numerous long colorful leaves (Willams & Black et al., 1996)

Fig:1: Pennisetum setaceum (Forssk.) Chiov.

Antimicrobial activity describes a substance’s ability to kill or decrease microorganisms such as bacteria, viruses, fungi, and parasites. Medicinal plants have an important function in preventing infections and diseases caused by bacteria. Various plant parts, such as leaves, stems, and roots, were used for treatment, which was caused by bacteria, viruses, and fungus (Huang et al., 2019).

MATERIALS AND METHODS:

2.1 Plant sample collection:

Healthy and mature plant parts leaves and stem of Pennisetum setaceum (Forssk.) Chiov. was collected from campus area of Gujarat University on 9th January, 2025.

2.2 Prepration of plant extract:

Plant parts were sun-dried and crushed into powder form. Cold extraction method was used to form plant extracts. For this, took 10 g of plant powder in 100 ml of methanol, D.W., and hexane. Then put them in a rotary shaker for 24 hours. After 24 hours, the solutions were filtered with Whatman filter paper number 1. After filtering, the solutions are poured into petri plates and air-dried. To make the stock solution, 30 mg of dried plant extract was dissolved in 30 ml of methanol, D.W., and hexane. The stock solutions can later be used to perform various activities.

2.3 The qualitative analysis:

Secondary metabolites, including alkaloids, carbohydrataes, glycosides, saponin, phytosterols, phenols, tannins, flavonoids, protiens, and amino acids are studied using standerd techniques (Harbone, J. B. 1984).

Table: 1: Qulitative analysis of phytochemical constituenys (Shaikh and patil, 2020)

2.4 Qulitative analysis:

Several quantitative methods are used to quantify the estimation of secondary metabolites. Secondary metabolites help in adaptation and defence against several stress- releated diseases.

2.4.1 Total phenol content (TPC):

The Folin-Ciocalteu method has used for total phenolic content determination. Gallic acid is used as standard. Various concentratuins of plant extract are taken like (0.2, 0.4, 0.6, 0.8, 1.0 mg/ml). Then add 0.5 ml of the folin-Ciocalteau reagent then add 2 ml of 20% Na2CO3 to the test tube. Total concentration of test tube will be 3 ml. The test tubes were boiled for one minite in water bath. After the test tubes were cooled down at room temprature, the abosrbance was measured at 650 nm (Somnwathi et al., 2015). The total phenolic content of extracts was calculated using the gallic acid equivalent for each gram of dry weight (mg GAE/g). The quantity of phenolic content in the plant sample was calculated by using the formula below:

Total phenolic content: GAE × V

                                          m

          GAE:  Gallic acid equivalent (mg/ml)

               V: Volume of plant extract

2.4.2 Total Tannin content (TTC):                           

For determination of total tannin concentration, Folin-Ciocalteu method was used with slide modifications. For TTC, tannic acid is used as a standard. Took different rant of plant extract concentration like 0.2, 0.4, 0.6, 0.8, 1 mg/ml. Then add 7.5 ml of distilled water. Then add 0.5 ml folin phenol reagent to the mixture. Add 1 ml of 35% Na2CO3 and diluted with 10 ml of distilled water. The mixture was thoroughly mixed and kept at room temperature for 30 minutes. Take absorbance at 725 nm. (Lahare et al., 2021). Tannin content was calculated in mg of TAE/g of extracts. A calibration curve is created with tannic acid as the standard. To determine the tannic concentration of a plant sample, use the following formula:

            Total Tannin content: TAE ×V

                                                  m

TAE: Tannic acid equivalent (mg/ml)

                 V: Volume of plant extract

2.4.3 Total flavonoids content (TFC):

The colorimetric test was used to determine the total flavonoid content. Quercetin is used as standard. Different concentrations of plant extract are taken like (0.2, 0.4, 0.6, 0.8, 1.0 mg/Ml). Take100 μl of extract with 4 ml of distilled water. Then, 0.3 ml of 5% sodium nitrite was added. After 5 minutes, 0.3 ml of 10 % aluminium chloride was added. Then add 2 ml of 1 M sodium hydroxide for 6 minutes. The mixture was diluted with 3.3 ml of distilled water and well mixed. The absorbance was measured at 510 nm (Zhishen et al., 1999). Using Quercetin as the standard, a calibration curve is created. To determine the flavonoid content of a plant sample, use the following formula:

Total Flavonoids content: QE × V

                                              m

QE: Quercetin equivalent (mg/ml)

                  V: Volume of plant extract

2.5 Antioxidant activity:                                          

Antioxidants are chemical or natural molecules that can prevent cell damage. Antioxidants are stable compounds that transfer electrons to neutralize free radicals, reducing their harmful effects. Antioxidants have several functions, including hydrogen donation, radical scavenging, peroxide decomposition, singlet oxygen quenching, synergy, enzyme inhibition, and metal chelation. The present study examined the antioxidant activity of Pennisetum setaceum (Forssk.) Chiov. plants using several antioxidant methods (Gameti et al., 2023).  

2.5.1 DPPH ASSAY:

1 ml of methanol and 1 mg of leaf extract. The standard and plant extract series were prepared in triplicate, with 1 ml of extract in each test tube and concentration ranging from 20 to 100 mg/ml. The standard is ascorbic acid. DPPH is light sensitive, so a fresh solution was prepared by dissolving 4 mg powdered DPPH in 100 ml of methanol and storing it in a dark area. After making series of extracts at various concentrations, 3 ml of DPPH solution was added and incubated for 20 to 30 minutes in dark. After some incubation, the purple solution created by the addition of DPPH wills turn pale yellow. The absorbance was measured at 517 nm (Pattani et al., 2023).  The radical scavenging activity was calculated using the following equation:

%inhibition= Control - Sample   × 100

                            Control

2.5.2 PMA ASSAY:

The method given by Prieto et al., (1999) was used with some modification. In this experiment, 0,2 ml of plant extract (1mg/ml stock) and standard ascorbic acid (0.2-1 ml) were mixed with Phosphomolybladate reagent. The reaction mixture was then incubated in a water bath for 90 minutes. After cooling to room temperature, the absorbance was measured at 695 nm. The antioxidant ability was reported as mg of ascorbic acid equivalents AAE/g of extract (Mankad et al., 2021).

          Antioxidant capacity: AAE × V

                                                   m

AAE: Ascorbic acid equivalents (mg/ml)

               V: Volume of plant extract

2.6: Antibacterial activity:

Antimicrobial susceptibility has been examined using the agar well-diffusion technique. Escherichia coli and Staphylococcus aureus have been used to test the extract of Pennisetum setaceum (Forssk.) Chiov. for its capacity to inhibit the growth of the infection. To create colonies for sensitivity testing, each bacterial strain was cultivated into nutrient agar plates and incubated for 18 to 24 hours at 37°C. After an overnight incubation period, colonies were selected using a sterile disposable inoculating loop and then transferred to a glass tube containing sterile. Before the plates were streaked with bacteria, wells 5 mm in diameter were punched into the medium using sterile bores. All of the plates were filled with the bacterial sample. A sterile cotton swab was put into the solution. It was moved and gently pushed against the fluid level to remove any extra inoculum. After the bacterial inoculation of the plate, 50 μl aliquots of each sample extract after the bacterial inoculation of the plates. To select the bacteria, three plates were used for each extract, and the same extract was poured in each plate. The plates are put in an incubator that is regulated at 37°C. After a 24-hor incubation period, each plate was examined for an inhibited zone. Using a measuring instrument, the inhibited zones were measured in mm.

RESULT AND DISCUSSION

3.1 Qualitative analysis:

Table: 1: Phytochemical screening or qualitative analysis

(‘+’ Presence of phytochemicals, ‘_’ Absence of phytochemicals)

3.2 Quantitative analysis:

3.2.1 Total phenol content:

The maximum value of phenol is obtained in methanol extract of leaves with value of 6.301 ± 0.00 GAE/g while the lowest value of phenol is obtained in D.W. extract is 0.253±0.005 TAE/g in plant leaves.

Table: 2: Total phenolic content of plant sample

3.2.2 Total tannin content:

The maximum concentration of total tannin content is obtained in methanol extract are 3.358±0.071 TAE/g in plant leaves. The lowest concentration of tannin is obtained in D.W. extract is 0.262±0.009 TAE/g in plant leaves.

Table: 3: Total Tannin content of plant sample

3.2.3 Total flavonoids content:

The maximum concentration of total flavonoid content is obtained in methanolic extract of leaves with value of 6.919±0.055QE/g. The lowest concentration of tannin in D.W. extract of leaves with value of 1.566±0.006 QE/g.

Table: 4: Total flavonoid content of plant sample

3.3 Antioxidant activity:

3.3.1: DPPH ASSAY:

Graph 1: standard graph for DPPH assay

Graph 2: IC50 value of leaves and stem

The highest IC50 value is 20.728, present in the hexane extract of leaves. Lowest IC50 value is 5.204, present in methanolic leaves extract. The highest IC50 value present in hexane stem extract is 15.421 while the lowest IC50 is present in methanol stem extract is 8.129.

3.3.2 PMA ASSAY:

Graph 3: Total PMA of plant sample

3.4: Antibacterial activity:

Table: 5: Result of antimicrobial activity of Pennisetum setaceum (Forssk.) Chiov. plant

The methanol and acetone extracts of stems show the zone of inhibition is 16 mm and 20 mm against E. coli bacteria. And the methanol extract of leaves shows a 14 mm of zone inhibition. And the acetone extract of stem shows 10 mm of zone inhibition.

Fig: 2: Antibacterial activity in stem Methanolic and acetone extract

Fig: 3: Antibacterial activity in leaf methanol and acetone

Fig: 4: Antibacterial activity in stem acetone