Journal of Plant Biochemistry & Physiology
Open Access

Total Phenolic Profile and Antiviral Activity of Senna Alexandrina

Sumayya Khadim^{* *}Correspondence: Sumayya Khadim, Department of Chemistry,Government College Women University Faisalabad, Faisalabad, Pakistan, Tel: 923030824237, Email:

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Introduction

Humans and animals are also at risk from viral illnesses. Because viral diseases have become drug-resistant and have the potential to spread quickly, they are responsible for a large number of deaths worldwide. Efforts are now being done to manage viral infections utilizing currently available antiviral medicines. Many people in the world's poorest countries, such as Africa, depend on medicinal plants for their health [1,2]. The South region of the world, has a wealth of plants used as an alternative medicine throughout history [3,4]. Plants that are used for medicinal purposes are getting increasingly popular. Many plant species have anti-inflammatory, antibacterial, antiviral, anticancer, and antioxidant properties. [5]. The Prophet's Medicine mentions Senna alexandrina. However, in Saudi Arabia, S. Sana makki, C. Senna angustifolia Vahl, and C. Senna are some of the other names for this species. In each of these nations, the plant can be found. In the year 2016 this plant is cultivated for its therapeutic benefits. The FDA has approved this herbal laxative as an over-the-counter herbal medication for adults and children [6]. Pods have been shown to be vanquished by leaves in several investigations. Elansary and his colleagues are also treated with typhoid, spleen, and anthelmintic infusions. Natural phytochemicals such as flavonoids and phenolic compounds are found in plants, including leaves, fruits, barks, stems, and roots, and can be used to treat a variety of maladies, including viral disorders [7,8]. Antioxidant effects of flavonoids, particularly phenolic compounds, have been studied and proven to be efficient [9]. The antioxidant activities of these bioactive compounds may be attributable to the activation of antioxidant enzymes, chelating metal catalysts, and scavenging free radicals to combat oxidative stress [10]. The goal of this study was to see if the medicinal plant Senna alexandrina might be used as a supplemental and alternative antiviral treatment by analyzing their phenolic profiles. We also wanted to see if the water and NADES extracts had any antioxidant, antibacterial, or antiviral properties. Toxic organic solvents can be replaced by deep eutectic natural solvents (NADES). They are non-toxic, biocompatible, and environmentally friendly. The extraction of components, particularly phytochemicals, from the selected medicinal plants was done with NADES [11]. The extraction efficiency of the water and NADES extracts is assessed by measuring the extracted total phenolic and total flavonoids.

Materials and Methods

Chemical

Various chemicals were employed in this project. Sodium carbonate (Daejum), Gallic acid (AppliChem), Methanol (Merck), Aluminum Chloride (Merck), Distilled water, Sodium nitrate (RDH), Glucose (RDH), and Citric acid (AppliChem). Folin-Ciocalteu reagent (Sigma Aldridge), Quercetin (AppliChem), DPPH (Alpha Chemical), Sodium carbonate (RDH).

Sample collection and preparation

The leaves of Senna alexandrina were collected in July 2019, collected from fresh fields.

Spectrophotometric analysis

The phenolic compounds were extracted using a method described previously, modified slightly [12,13]. Briefly, the plant materials were dried in the shade before being pulverized. Each sample and distilled water was placed into a blender in a 1:5 ratio and blends it. Then sample was poured in the beaker covered with Aluminum foil and left for 3 days. Similar approach was utilized for NADES solvent. The sample was filtered with filter paper the next day [14].

Preparation of NADES

NADES were prepared by mixing glucose, Citric Acid and distilled water in 1:1:1 ratio and placing it on the Hot Plate at 40-45°C temperature with constant stirring until the solution became clear at room temperature.

Analysis of total phenolic content

The quantity of phenols in the extract was obtained by using an assay Folin-Ciocalteu, [15], with little modification. Briefly, as a standard, a 1% solution of Gallic acid was utilized. We dissolved the Gallic acid (0.2 g) in 20 mL of distilled water for this experiment. In methanol, the dilutions of 1 µl/ml, 5 µl/ml, 10 µl/ml, 15 µl/ml, 20 µl/ml, 25 µl/ml, 50 µl/ml, 80 µl/ml, 100 µl/ml, and 125 µl/ml were produced. 100 µl of dilution were measured one at a time, after that we measured 500 µl of H2O and 100 µl of reagent (Folin.c) and added into the test tube. After 6 minutes 1 mL of 7% Na2CO3 (3.85 g of Na2CO3 dissolve in 50 mL H2O) and 500 µl H2O were added. After 90 minutes, at 760 nm readings were taken, a straight curve was plotted, and the same approach was used to determine the phenolic content of samples. The solutions were made in triplicate.

Analysis of total flavonoid content

Flavonoid in plant extracts has been calculated by complex forming assay of Aluminium chloride. Modified by some researchers. Quercetin (0.2 g) was dissolved in 20 mL of water. Then, in methanol, dilutions of 1 µl/ml, 5 µl/ml, 10 µl/ml, 15 µl/ml, 20 µl/ml, 25 µl/ml, 50 µl/ml, 80 µl/ml, 100 µl/ml, and 125 µl/ml were produced. Using a micropipette, measure 100 µl of dilution and added into the test tube, add 500 µl H2O and 100 µl of 5% NaNO3 (0.5 g of NaNO3 in 10 ml H2O) after 6 min, then add 10% AlCl3 (1 g of AlCl3 dissolved in 10 ml H2O) 150 µl . After 6 minutes, 1 M NaOH (200 µl) was added (dissolve 0.4 g in 10 ml water). A UV–Vis Spectrophotometer set to 510 nm was used to measure the absorbance of the blank solution and the mixture. The standard solution was Quercetin. The solutions were made in triplicate. The results were presented as mg quercetin equivalent QE g-1 of dry weight DW.

Scavenging Activity

The water and NADES extract's ability to scavenge the 2,2-diphenyl-1-picrylhydrazyl stable free radicals was assessed using the DPPH test [16-20]. The antioxidant is added into the methanol and freshly prepared DPPH solution (200 μM), and the antioxidant's ability to scavenge the free radical of stable DPPH is measured at 517 nm. Extracts at various amounts were added to a methanol DPPH solution. After 30 minutes, absorbance was measured at 517 nm at room temperature in the dark. The IC50 value is the sample concentration necessary to scavenge 50% of DPPH free radicals.

FTIR analysis

The characterization of plant materials was done using this method. The sample contained the active functional groups were discovered in the 400-4000 nm wavelength range using this approach.

HPLC analysis

It is used for assessment of the amount of Phenols, Flavonoids, and antioxidants present in the extracts. The phenolic components were isolated, described, and measured using this amazing chromatography. Each plant extract test sample was dissolved in 24 mL methanol (and homogenized) were used to perform HPLC. After 16 mL of distilled water, 10 mL of 6 M HCl was added. The combination was then thermostated at 95°C for 2 hours. Prior to HPLC analysis, the final solution was filtered using a 0.45 µm nylon membrane filter. The Shim-pack CLC-ODS (C18), 25 cm × 4.5 mm, 5 µm column was used to separate plant samples using gradient HPLC. The chromatographic separation was carried out with the following mobile phase gradients: A (H2O:Acetic Acid-94:6, pH=2.27), B (Acetonitrile 100%) 0-15 min =15% B, 15-30 min =45% B, 30-45 min =100% B With 1 mL/min flow rate and UV-VIS detector at 280 nm wavelength at room temperature. Each compound's identity was determined by comparing the retention period, UV-VIS spectra, and peaks with those previously obtained by injecting standards. External calibration of the standards was used to perform the quantification.

Antiviral assay

Viral inoculum preparation: Poured the 10 ml secondary culture media in flacon tubes and placed in centrifuge at 14000 rpm for 10 min at room temperature. Supernatant discarded and added remaining 10 ml culture repeat it 2 times. Added activation media (20 ml MgCl2 and 1.5 µl acetosyringone) in pellet and gently mix it. After that placed the tube in dark for 3 h and then ready to inoculate in host plant.

Administration to the local host plant: Subsequently the Cotton curl Multan virus was injected on Nicotiana benthamiana plants that had been cultivated. Water and NADES extract of Senna alexandrina were infiltrated to the 10 host plants of local lesion for one day in a row. Distilled water was used as a control. Plants were selected for homogeneity and cultivated in a growth room at 28°C, 16:8 h light/dark cycle with all treatments repeated three times.

Statistical Analysis

Three tests were conducted, with the findings displayed as a ± standard derivation. Pearson's correlation coefficient and other statistics were calculated using Excel 2007. An ANOVA test was run on Windows using the SPSS 16.0 program to determine the statistical significance of the various treatments. The statistical significance level was set at 0.05, the lowest achievable value.

Results

The water and NADES extracts of Senna alexandrina were collected and submitted to various analyses in the current investigation. Their reported characteristics and findings are detailed and discussed here;

Analysis of total phenolic content

Phytochemicals like phenols mostly present in plant extracts contain hydroxyl groups that provide antioxidant activity [21]. By using the standard method of the Folin-Ciocalteu reagent total phenolic content was calculated. For this purpose, as a standard 1% solution of Gallic acid was used as shown in Table 1 and Figure 1. Senna alexandrina the water extract showed more phenolic content of 318.2 mg GAE/g as compared to NADES extract of 296.8 mg GAE/g as shown in Figure 2.

Table 1: Regression equation and R2 values of Gallic acid (GA) and Quercetin (Q) standards.

Figure 1: Final standard graph of TPC.

Figure 2: TPC mg GAE g-1 of water extract of Senna alexandrina (SW) and NADES extract of Senna alexandrina (SN).

Analysis of total flavonoid content

The flavonoids in extracts were calculated using a spectrophotometric method. 1% solution of Quercetin was used as a standard solution, its standard curve is shown in Figure 3 value is given in Table 1. Water extracts of Senna alexandrina showed more TFC as compared to the NADES extracts. The value of TPC of all samples is shown in graphs Figure 4.

Figure 3: Final standard graph of TFC.

Figure 4: TFC mg GAE g– 1 of water extract of Senna alexandrina (SW) and NADES extract of Senna alexandrina (SN).

Antioxidant assay

Antioxidant-rich plant species have been used to treat various ailments. The conventional scavenging method of free radicals was employed to calculate the antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl. When it comes to antioxidant activity, UV radiations were employed to analyze water and NADES extracts of Senna alexandrina. UV radiation was absorbed differently by each sample. SW and SN samples absorb 1.052 and 1.325 cm-1, respectively. Samples SW and SN, on the other hand, show percent inhibition of 47.6 and 34, respectively. The graph below shows the comparison of the antioxidant activity of all samples (Figure 5).

Figure 5: DPPH (% radical scavenging activity) of SW and SN.

HPLC results

The phenolic components were isolated, described, and quantified using this chromatography. In the Water extract of Senna alexandrina nine phenolic acids and one flavonoid (Quercetin) were extracted as shown in Table 2. In NADES extract of Senna alexandrina six phenolic acids including Gallic Acid and one flavonoid (Quercetin) were extracted as shown in Table 3.

Table 2: Water extract of Senna alexandrina.

Table 3: NADES extract Senna alexandrinea.

FT-IR results

Infrared spectroscopy, often known as FTIR, which uses infrared light to shake bonds between the molecules inside the sample that absorbs it. Because the majority of the samples have different subatomic bonds or different subatomic bond configurations, FTIR can be used to provide compound data on particles inside the specimen. Based on the maximum value in the region of IR, the FTIR spectroscopy was utilized so that the functional group could be determined. The presence of numerous compounds in SW and SN has been revealed by FTIR spectroscopy analyses, with various peaks values according to stretching frequency in the water and NADES extracts of Senna alexandrina.

Water extracts of Senna alexandrina: The existence of numerous compounds in the Aqueous extracts of Senna alexandrina is confirmed by FT-IR spectroscopy as shown in Table 4 and Figure 6.

Table 4: FT-IR results of aqueous extracts of Senna alexandrina.

Figure 6: FTIR results of SW.

NADES extracts of Senna alexandrina: FTIR spectra of SN extract confirmed that many other functional groups also detected as shown in Table 5 and Figure 7.

Table 5: Results of FT-IR spectroscopy of Senna alexandrina NADES extract.

Figure 7: FTIR Spectrum analysis of NADES extract of Senna alexandrina.

Antiviral activity

Simultaneous infiltration of inhibitor (Water extract of Senna alexandrina, NADES extract of Senna alexandrina, and cotton leaf virus was done on Nicotiana bathaminia that was grown in a standard condition required for best growth mentioned above.

Nades extract of Senna Alexandrina as an Inhibitor

Plants that were infiltrated with NADES extract of Senna alexandrina inhibitor and cotton leaf Multan virus could not survive showed in (Figure 8) of 2 weeks analysis under optimum condition.

Figure 8: SN as an inhibitor

Water extract of Senna Alexandrina

Plants that were infiltrated with Water extract of Senna alexandrina inhibitor and cotton leaf Multan virus survived as shown in the following Figure 9 of 2 weeks analysis under optimum condition.

Figure 9: SW as an inhibitor.

Discussion

Senna alexandrina the water extract showed more phenolic content of 318.2 mg GAE/g as compared to NADES extract of 296.8 mg GAE/g as shown in Figure 2. Plant species exhibit a wide range of phenolic compounds, which are diverse secondary metabolites [22,23]. In this study, the phenol content was different from what was reported previously. Senna alexandrina showed TPC of 318.2 mg GAE g−1 DW, which was lower than that reported (3761 mg GAE g−1 DW) [24]. It is possible that these medicinal plants could differ in their phenolic compounds and phytochemicals depending on the phytogeography area and season during which they were harvested caused variation [25,26]. Different extraction techniques and solvent polarities could have caused the variations as well. Among other factors, plant maturity and genotype may have contributed to the variation in phenolic compound concentrations.

In plants there are many functions of flavonoids, they have been suggested to be secondary antioxidant defenses in plants in response to various stresses [27]. Water extracts of Senna alexandrina showed more TFC as compared to the NADES extracts. The value of TPC of all samples is shown in graphs (Figure 4). This study's results differ from those reported in the literature [28]. There may be similar factors responsible for the variations in TPC as well.

Antioxidant activity results of Senna alexandrina are similar to those reported in the literature. SW and SN samples absorb 1.052 and 1.325 cm-1, respectively. Samples SW and SN, on the other hand, show percent inhibition of 47.6 and 34, respectively. The sample SW absorbed the least amount of UV light but shows a maximum of % inhibition its mean that the anti-oxidant activity of sample SW is at its peak. Basically, a lower IC50 value was connected with a stronger DPPH radical-scavenging activity. It has been postulated that samples with an IC50 of less than and equal to 50-100 g/mL are strong antioxidants, samples with an IC50 of 101-150 µg/mL are moderate antioxidants, and samples with an IC50 of greater than 150 µg/mL are weak antioxidants. The DPPH radical is a preferred substrate for measuring antioxidant activity fast due to its stability in the radical state and simplicity of assay. According to recent research, phenolic chemicals extracted from plants may be responsible for the antioxidant action, particularly flavonoids, due to -OH groups, which possess redox properties [29].

The phenolic components were isolated, described, and quantified using HPLC. In the Water extract of Senna alexandrina nine phenolic acids and one flavonoid (Quercetin) were extracted as shown in Table 2. In NADES extract of Senna alexandrina six phenolic acids including Gallic Acid and one flavonoid (Quercetin) were extracted as shown in table 3. There are differences in chemistry between them. The presence of these compounds is also reported in the literature [30].

The existence of numerous compounds in the water extracts of Senna alexandrina is confirmed by FT-IR spectroscopy as shown in (Figure 6). The existence of six functional groups in Senna alexandrina aqueous extracts is demonstrated. The strong absorption at 3492.45 cm-1 indicates the O-H, N-H, and O-H of –CO2H are present. This indicates that carbonyl compounds were present in the aqueous extract. Other groups are absorbed at a slower rate as shown in Table 4. In FTIR of SN the prominent band at 1720.19 cm-1 stretching frequency indicate carbonyl (C=O) group which is associated with conjugated aromatic compounds. The band at 1222.65 cm-1, 1108.87 cm-1, 1074.16 cm-1 and 1025.94 cm-1 was due to the presence of C-O and C-N (normally weak and difficult to find) stretch groups, respectively. The intense bands at 1222.65 cm-1 and 1108.87 cm-1 stretching suggest that the esters and secondary alcohols are present in the extract. These results are according to those reported in the literature [31,32].

Conclusion

Plants that were infiltrated with water extract of Senna alexandrina inhibitor and cotton leaf Multan virus survived in 2 weeks analysis under optimum condition. While plants that were infiltrated with NADES extract of Senna alexandrina could not survive. SW extract contains a large amount of phenolic acid along with Quercetin that inhibits the virus to attack and as result plants survive. Quercetin and p-coumaroyl show antiviral activity by inducing autophagy and inhibiting viral mRNA synthesis. So, the results are following the literature. [33,34]. The Senna alexandrina presented antiviral and higher levels of antioxidant activity due to their phenolic compounds. This means that these herbs can be used in pharmaceuticals to boost the body's endogenous antioxidant system and reduce free radical levels. To validate their use in the management of viral diseases, further experiments and clinical studies are recommended.

Acknowledgement

The authors are thankful to the Higher Education Commission (HEC) of Pakistan for financial assistance. Furthermore, Government College Women University Faisalabad and University of Agriculture Faisalabad are graciously acknowledged for supporting this work.

Conflict of Interest

The authors declare that there is no conflict of interest

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