Introduction

Since time immemorial, plants have been utilized by mankind to serve numerous livelihood requirements (Kanjikar, 2019; Poddar et al., 2020). Besides oxygen demand, plants have aided us human beings in different aspects of life including shelter, food, clothing, medicine, etc (Dar et al., 2017; Georgievna et al., 2020; Prasathkumar et al., 2021). Plants are a source of rich chemical compositions like carbohydrates, glycosides, flavonoids, terpenoids, carotenoids, polyphenols, vitamins, proteins, steroids, tannins, etc. (Saxena et al., 2013). The presence of these chemical species makes plants a magnificent part of different traditional medicine systems. Famous medicinal systems like Ayurveda, Traditional Chinese Medicine (TCM), Unani medicine, incorporate several medicinal plants, which prove beneficial in treating several human ailments (Patwardhan et al., 2005). Globally, a high population depends upon plants to fulfill basic needs of life including medicinal needs (Jamshidi-Kia et al., 2018). Medicinal plants have been proving beneficial in drug discovery and design (Velmurugan et al., 2020). India is among the vast countries in terms of area and possesses huge floral diversity. India is home to more than 45,000 plant species out of which maximum have been reported of medicinal properties (Mehraj et al., 2018). Kashmir Valley, a division of Jammu and Kashmir Union Territory in India, falls in Westernmost Himalayan ranges. It is known for its extraordinary beauty and high mountain ranges covered with green belt. Due to soothing climatic conditions and nutritious soil quality, Kashmir Valley bears huge floral diversity including several varieties of medicinal plants (Nengroo & Rauf, 2019). Portulaca oleracea L. (common purslane) is an agricultural weed growing across the globe and is ranked among top most eight common plants growing on earth (Srivastava et al., 2021). It belongs to the family Portulacaceae, which consists of more than 120 succulent shrub and herb species (Zhou et al., 2015). Portulaca oleracea L. is locally known as “Nunnar” in Kashmir and is usually found in apple orchids and vegetable crops including carrots, cabbage, cauliflower, spinach, etc. Although purslane is a weed, its stems and leaves are entirely edible bearing a salty taste. In Kashmir, people cook leaves and stems of the weed and eat it with rice. Portulaca oleracea L. is of high medicinal value and has been listed among the most used medicinal plants globally by the World Health Organization (Sedaghati et al., 2019). The above ground portions of the plant are a part of folk medicine used in several countries due to its high anti-asthmatic, antipyretic, anti-ascorbic, febrifuge, diuretic, antiseptic, antitussive, vermifuge, and antispasmodic effects (Catap et al., 2018; Alu’datt et al., 2019; Khazdair et al., 2019). Different extracts of the plant have been shown with hypocholesterolemic, bronchodilatory, neuropharmacological, wound healing, analgesic, hypolipidemic, anti-inflammatory, anti-aging, antibacterial, hypoglycemic, antioxidant, and skeletal muscle relaxant (Kaveh et al., 2017; Sallam & Anwar, 2017; Ahmadi et al., 2021). Other studies have reported that Portulaca oleracea L. shows protective effects against several chronic disorders including heart diseases and cancer (Zhao et al., 2013). The plant is considered an immune booster and suppressor of heart attack due to the high values of omega-3 fatty acids (Simopoulos, 2004). Keeping in the high medicinal value of P. oleracea, the present research was designed to assess the phytochemicals, antioxidant, antibacterial, and antifungal activity of different extracts (aqueous, ethanolic, methanolic, acetone, and hexane) of the plant. We performed DPPH, NBT, and FRAP assays to estimate the antioxidant potential of P. oleracea. The antibacterial activity of the extracts was evaluated against S. aureus, E. coli, and M. luteus. Antifungal activity was estimated against Fusarium oxysporum and Aspergillus flavus.

Materials and Methods

Collection of Plant Material

P. oleracea was collected from apple orchids of Central Kashmir’s Chadoora area (33.9451° N, 74.7966° E, elevation of 1,577 m), a tehsil in district Budgam, Jammu, and Kashmir. The plant was authenticated by the Department of Botany, Madhyanchal Professional University, Bhopal, Madhya Pradesh. The aerial plant parts including flowers, stem, and leaves were collected in September and cleaned with tap water.

Sample Preparation

The aerial parts of the plant were shade dried for 3 weeks at a temperature of about 29-30^oC. These parts were cut into small pieces manually and stored in airtight plastic bags to avoid exposure till further use.

Extracts Preparation

The dried aerial parts of P. oleracea were incubated at 50^oC for 1 h, in order to remove all the remaining moisture. Then, the plant material was ground thoroughly till a fine powder is obtained. The powder was suspended in the appropriate solvents used in the study (20% w/v): water, methanol (95%), ethanol (95%), acetone (70%), and hexane (70%). For methanolic, ethanolic, acetone, and hexane extractions, suspensions were placed under refluxing fitted with a magnetic stirrer at 20^oC for 48 h. The aqueous extract was prepared by boiling the water and plant powder mixture for 3 h. All the suspensions were allowed to settle down and the liquid phase was passed through filtration with Whatman filter paper no. 1. The filtrate in each case was allowed to dehydrate at 70^oC with a rotatory evaporator. The final crude extracts were prepared by mixing each crude extract with dimethyl sulfoxide (DMSO) to obtain a final concentration of 10 mg/mL. These extracts were then stored at 4^oC until further use.

Phytochemical Screening

Qualitative Analysis

Phenolics

Ferric chloride test was performed to investigate the presence of phenolics in different solvent extracts of P. oleracea. 50 mg of extract was dissolved in 5 mL of distilled water followed by the addition of few drops of 5% ferric chloride. This mixture was then examined for the presence of phenolics, which is confirmed by the appearance of Bluish-black color.

Tannins

The ferric chloride test was performed to monitor the presence of tannins in the extracts. Briefly, 50 mg of the plant extract was taken and then dissolved in 10 mL distilled water. This mixture was shaken thoroughly prior to the addition of few drops of ferric chloride (5%). The presence of tannins is confirmed by the formation of a green precipitate.

Steroids

We performed the steroid test to check its presence in the extracts. Briefly, 50 mg of each extracted sample was added to 2 mL of chloroform followed by the addition of 2 mL sulfuric acid. The mixture was allowed to settle down for a few minutes. The presentation of red color by the mixture confirms the presence of steroids.

 Alkaloids

The presence of alkaloids in the extracts was monitored by the execution of Wagner’s test. In brief, 50 mg from each extract was added with a small quantity of hydrochloric acid followed by filtration. The filtrate was placed in a test tube and added with Wagner’s reagent. The Wagner’s reagent produces a reddish-brown precipitate in reaction with alkaloids.

Flavonoids

The presence of flavonoids was confirmed by the execution of the alkaline reagent test. About 50 mg of different solvent extracts were added with a dilute ammonia solution (5 mL) and followed by filtration. Then, the filtrate was added with few drops of concentrated sulfuric acid and let to settle down. The yellow color formation reveals the presence of flavonoids in the sample.

Saponins

The presence of saponins in the experimental extracts was confirmed by the Forth test. Briefly, about 50 mg of different P. oleracea extracts were dissolved in distilled water to a total volume of 20 mL. This mixture was then thoroughly mixed by continuous shaking for 20 min. The formation of foam to a height of 2 cm from the surface of the mixture confirmed presence of saponins.

Terpenoids

The presence of terpenoids in different plant extracts were confirmed by the Salkowski test. To 50 mg plant material, 2mL chloroform and 3mL concentrated sulfuric acid were carefully added. A reddish-brown coloration signified the presence of terpenoids.

Anthraquinones

30 mg of the extracts and few drops of 10% ammonia solution were added. The appearance of a pink color precipitate indicates the presence of anthraquinones.

Quantitative Analysis

Total Flavonoids

We estimated the total phenol content of different P. oleracea extracts via the aluminum chloride spectrophotometric analysis. In brief, 200 µL of each crude extract and/or catechin solution (standard) was added with 1mL distilled water and 70µL of 5% sodium nitrite. Thereafter, this mixture was supplied with 150 µL of aluminum trichloride (10%) and placed over incubation for 10 min. After the incubation period, distilled water and 0.5mL of 1M NaOH was added to this solution to a final volume of 2.5mL. Finally, the absorbance studies were performed at 510 nm against blank. The total flavonoid content in the extracts was expressed in the equivalents of mg (+)-catechin g^-1 of the dry matter (DM) of the extract.      

Total Phenols

We performed the Folin-Ciocalteu method (slight modification) in the determination of total phenols within different extracts of P. oleracea. In brief, 100 µL from each extract was mixed with Folin-ciocalteu reagent and deionized water. Then, 2mL of 75% sodium carbonate solution was added to the mixture and left untouched at room temperature for 2.5 h. Finally, the absorbance was recorded at 765nm and total phenols were expressed in terms of mg hydroxytyrosol equivalents g^-1 of the DM of the extract.

Antioxidant Activity

DPPH Assay

The antioxidant potency of different P. oleracea was estimated through 2,2-diphenyl-1picrylhydazyl (DPPH) scavenging efficacy. We prepared the stock solution of DPPH by dissolving 24 mg of DPPH in absolute methanol (100mL). Then, the extract solutions were prepared by dissolving 1 mg/mL of crude extracts in the absolute methanol followed by dilution for the forth analysis. Afterward, we added DPPH (1 mL) to 100 µL of different P. oleracea extracts prepared in altering concentrations of 100, 200, 300, 400, and 500 µg/ml. These mixtures were well shaken prior to transfer into a dark room for 15 min at 25 ^oC. Finally, the absorbance was taken at 517 nm using methanol as blank. BHT was used as positive control and DPPH activity was determined by the following equation:

NBT Assay

The determination of superoxide anion radical scavenging efficacy of different solvent extracts of P. oleracea was analyzed by nitroblue tetrazolium (NBT) reduction. In brief, the reaction mixture comprises of a final volume 3mL Tris-HCL buffer (16mM concentration and pH=8), maintaining 0.3 mM NBT (0.5mL), 0.93 mM NADH (0.5mL) and 1 mL of different solvent extracts with different concentrations viz 100, 200, 300, 400, and 500 µg/ml followed by the addition of 0.5mL of 0.12mM phenazine methosulphate (PMS). The complete reaction mixture was let to settle down followed by 6min incubation at room temperature. Finally, absorbance was measured at 560nm against blank and ascorbic acid was taken as the positive control.     

Ferric Reducing/Antioxidant Power (FRAP) Assay

The FRAP method was used to determine the antioxidant activity of different extracts of P. oleracea. In brief, 1 ml of each solvent extract with altering concentrations of 100, 200, 300, 400, and 500 µg/ml, were mixed with 2.5 ml of 1% potassium ferricyanide and 2.5 ml of phosphate buffer (pH 6.6). This mixture was subjected to incubation for half an hour at 50^oC followed by the addition of 2.5ml of 10% trichloroacetic acid. Afterward, this mixture was placed for 10 min of centrifugation at 3,000 rpm. The supernatant then evolved was diluted in 2.5 ml of distilled water. This mixture was supplied with 0.5 ml of 0.1% fresh ferric chloride and shaken vigorously. Finally, the absorbance of the samples was taken at 700 nm. Absorbance is directly proportional to the ferric reducing potential of the extracts.

Antimicrobial Activity

Microbial Culture

Five different extracts of P. oleracea were investigated for antimicrobial activity against five microbes including E. coli, S. aureus, M. luteus, Fusarium oxysporum, and Aspergillus flavus. These microbes were collected from the Laboratory of Pharmacy Department, MPU, Bhopal, Madhya Pradesh. Nutrient broth was used to culture bacteria while fungi in PDB (potato dextrose broth) media. All the microbes were cultured for 24 h at 37^oC (bacteria) and 30^oC (fungi) for 24 h.

Antibacterial Assay

Different solvent extracts of the aerial parts of P. oleracea were examined for their antibacterial potential via the disc diffusion method. In brief, 10 µl of each solvent extract was prepared in 10% DMSO. These solutions were stored in a chiller till further use. 10⁵ CFU/mL (hemocytometer) of the bacterial (E. coli, S. aureus and M. luteus) suspensions were spread gently by using sterile cotton bud over Mueller-Hinton agar. Afterward, discs of 6 mm diameter were sterilized for 20 min at 115^oC followed by the loading of extract concentrations. Streptomycin (30 mg) was used as the positive control. The plates bearing the bacterial cultures were placed with the contact of dried and sterilized discs. The areas were labeled followed by subjecting each disc to incubation for 24 h. Each individual experiment was repeated in triplicates and results were revealed as the diameter (mm) of inhibition zones. The minimum concentration of the extracts that suppressed visual growth of the bacteria was taken as MIC (minimum inhibitory concentration) value.  

Antifungal Assay

A similar disc diffusion method was used to examine the antifungal potential of the extracts. Briefly, 10 µl of each solvent extract was prepared in 10% dimethyl sulfoxide (DMSO). These solutions were stored in a chiller till further use. Fungal (Fusarium oxysporum and Aspergillus flavus) suspensions at a concentration of 10⁵ CFU/mL were spread gently by using sterile cotton bud over potato dextrose agar. Then discs of 6mm diameter were sterilized for 25 min at 120^oC followed by the loading of various extract concentrations. Amphotericin-B (25 mg) was used as a positive control. The plates bearing the bacterial cultures were placed with the contact of dried and sterilized discs. The areas were labeled followed by subjecting each disc to incubation for 72 h. Each experiment was repeated in triplicates and results were revealed as % inhibition of diameter (mm). The minimum concentration of the extracts that suppressed any visual growth of the fungi was taken as MIC value.        

Statistical Analysis

All the experimental data are expressed as mean±SD of 3 independent replicates. Data analysis was carried out with the help of SPSS 18.0 software. Data comparison was done through one-way ANOVA and significant differences among means were determined by using Tukey’s test. The p-value of <0.05 was considered statistically significant.

Results and Discussion

Phytochemical Content Within Different Solvent Extracts of P. Oleracea

The plant P. oleracea L. (purslane) has been reported to have a rich chemical composition mainly flavonoid, terpenoids, alkaloid, and organic acid (Dabbou et al., 2020). Additionally, fatty acids, terpenoids, vitamins, polysaccharides, proteins, sterols, and vitamins have also been found in purslane (Petropoulos et al., 2016). The phytochemical composition of purslane shows significant variations depending upon a number of factors including harvesting time, environment, culture conditions, and solvent nature used for extraction (Uddin et al., 2014). In this study, we aimed at finding the phytochemical composition of purslane growing in Kashmir Valley. The qualitative phytochemical analysis of different solvent extracts of P. oleracea was performed by carrying out different tests. The results (Table 1) indicated the presence of phenols, tannins, steroids, alkaloids, flavonoids, saponins, terpenoids, and anthraquinones. The aqueous extract of P. oleracea showed positive results for phenols, steroids, alkaloids, tannins, flavonoids, terpenoids, and anthraquinones, and negative for saponins. Methanolic extract showed positive tests for the presence of phenols, steroids, alkaloids, flavonoids, terpenoids, and anthraquinones while negative for tannins and saponins. Ethanolic extract showed positive tests for the presence of phenols, steroids, alkaloids, flavonoids, terpenoids, and anthraquinones while negative for tannins and saponins. Acetone and hexane extracts confirmed the presence of phenols, steroids, alkaloids, terpenoids, tannins, and saponins while turned negative for anthraquinones. Hexane extract turned negative for flavonoids as well. Moreover, we determined the total phenolic and flavonoid content of each extract. The results indicated that purslane is very rich in phenolic as well as flavonoid content (Table 2). Out of the five extracts, methanolic and ethanolic extracts showed higher phenolic content, and acetone and methanolic extracts showed higher flavonoid content, respectively. 

Table 1. Phytochemicals present in aqueous, methanolic, ethanolic, acetone, and hexane extracts of P. oleracea (aerial parts).      

The “+” sign denotes “present” and “- ” sign denotes “absent”

Table 2. Total phenols and total flavonoids (mg g^-1 DM) content within different solvent extracts of P. oleracea

Antioxidant Activity of Different Solvent Extracts of P. Oleracea

Several research investigations have revealed that the rich pharmacological and medicinal properties of P. oleracea are attributed to its strong antioxidant potential. Phenolic compounds are diversely distributed among plant species and have attracted much attention because of their strong free radical scavenging and antioxidant potency (Hassan et al., 2017). The antioxidant species have revealed tremendous health benefits via their physiological behavior including antioxidant, anticancer, and antimutagenic (Hassan et al., 2017; Akhtar et al., 2019). Medicinal and dietary plants comprise of high phenolic content and show remarkable inhibition of oxidative stress (Rice-Evans et al., 1996; Tchicaillat-Landou et al., 2018). Total flavonoid and phenol content within plants have been shown to highly determine their antioxidant potential (Ng et al., 2020). P. oleracea bears high concentrations of both phenols, as well as flavonoids as revealed above. This indicated that P. oleracea could possess very high antioxidant activity. Additionally, P. oleracea has been reported to possess higher content of ascorbic acid, omega-3-fatty acids, gallotannins, apigenin, quercetin, kaempferol, and α-tocopherols, which enhances its antioxidant potency (Rahimi et al., 2019). A study performed by Behravan et al. evaluated the effects of ethanolic and extract P. oleracea on H₂O₂-induced DNA damage in human lymphocytes (Behravan et al., 2011). They performed the comet assay and reported that the aqueous extract remarkably suppressed the DNA damage while no such effect was recorded for ethanolic extract. They concluded that the aqueous extract of P. oleracea bears antioxidant components, which could inhibit the induction of oxidative DNA damage against these lymphocytes. Therefore, we investigated the antioxidant activity of different solvent extracts of P. oleracea using DPPH scavenging assay, NBT assay, and FRAP assay.

DPPH Scavenging activity

For DPPH scavenging activity, we evaluated different concentrations of aqueous, methanolic, ethanolic, acetone, and hexane extracts of P. oleracea viz 100, 200, 300, 400, and 500 µg/ml. equal concentrations of BHT were used as the positive control. The results indicated significant scavenging activity of these extracts (Table 3). The DPPH scavenging activity was observed to increase with the increase in extract concentrations. Acetone extract showed higher scavenging potential with 82.3 ± 1.24% at a concentration of 500 µg/ml. The percentage inhibition of aqueous, methanolic, ethanolic, ethanolic, acetone, and hexane extracts ranged from 13.2 ± 0.22% to 49.6 ± 0.72%, 27.31 ± 0.08% to 78.22 ± 2.18%, 26.44 ± 0.14% to 63.7 ± 1.16%, 34.5 ± 0.16% to 82.3 ± 1.24%, 9.7 ± 0.13% to 45.34 ± 0.19%, respectively. The least activity was shown by hexane extract.

Table 3. DPPH scavenging activity of different solvent extracts of P. oleracea.

Nitro-Blue Tetrazolium (NBT) Reducing Activity

Same extract concentrations were evaluated for the NBT assay. Results showed that all the extracts possess strong efficacy to reduce NBT to formazan crystals. The NBT reducing power of each extract was found to be concentration-dependent. Ascorbic acid was used as positive control and showed a percentage inhibition ranging from 63.9 ± 0.02% to 89.367 ± 0.16% at concentrations of 100-500 µg/ml. Acetone and methanolic extracts revealed a strong NBT reducing potential. Aqueous, methanolic, ethanolic, acetone, and hexane extracts revealed a high percentage inhibition at 500 µg/ml viz 51.4 ± 0.17%, 75.25 ± 0.14%, 64.7 ± 0.16%, 80.55 ± 0.24%, 47.34± 0.17%, and 89.367± 0.16%, respectively (Table 4).

Table 4. The NBT reducing potential of different solvent extracts of P. oleracea.   

FRAP

The FRAP assay was performed to monitor the antioxidant potency of these extracts. Results showed that each extract showed remarkable potency towards FRAP. The highest FRAP was recorded for ethanolic extract of the plant ranging from 0.5 ± 0.04 to 1.52 ± 0.005 (100-500 µg/ml). Chlorogenic acid was used as a positive control, which showed higher absorbance of 3.58 ± 0.003148 at 500 µg/ml. The results of FRAP analysis of each extract are given in Table 5.

Table 5. The FRAP of different solvent extracts of P. oleracea.

Antimicrobial Activity of Different Solvent Extracts of P. Oleracea

Portulaca oleracea L. weed is medicinally as well as pharmacologically important. It has been previously shown with antibacterial, antiviral, and antifungal activity against different microbes (Liu et al., 2021). P. oleracea has been reported to induce antifungal effects against dermatophytes (Genera Trichophyton) (Oh et al., 2000). Similarly, a study has reported that the methanolic extract of P. oleracea inhibited bacterial growth against different gram-negative and gram-positive strains of Neisseria gonorrhea, Pseudomonas aeruginosa, and Escherichia coli. The inhibition zones (mm) for the gram-negative bacterial strains were found to be 15, 15, and 14 mm, while for gram-positive 15, 14, and 13 mm, respectively. The same study indicated antifungal activity of methanolic extract of P. oleracea against Candid albicans demonstrating a zone of inhibition of 12 mm (Elkhayat et al., 2008). Key molecular isolations from different parts of the plant have proven beneficial to inhibit virus penetration. Antiviral activity of one such pectic polysaccharide has been reported against herpes virus simplex type 2 via suppression of virus penetration (Dong et al., 2010).

In the current study, we investigated the antifungal and antibacterial activity of different solvent extracts of P. oleracea against M. luteus, S. aureus, E. coli, Fusarium oxysporum, and Aspergillus flavus. The antibacterial activity was examined against one gram-negative strain i.e. E. coli, and two gram-negative strains i.e. S. aureus and M. luteus. The results indicated that all extracts, except hexane, showed potential antibacterial effects against both gram-negative and -positive bacteria (Table 6). Acetone yielded inhibition zones of 13.1 mm, 16.4 mm, and 19.7 with MIC of 0.60 mg/ml, 0.12 mg/ml, and 0.16 mg/ml against gram-negative E. coli and gram-positive S. aureus and M. luteus, respectively. Similarly, Methanolic and aqueous extracts showed inhibition zones of 15.4, 17.4, and 14.3 mm and 12.4, 15.6, and 21.3 mm with MIC values of 0.85, 0.10, and 0.13 mg/ml and 0.70, 0.11, and 0.14 mg/ml, respectively. Ethanolic extract showed the highest antibacterial activity with inhibition zones of 16.7, 21.5, and 23.5 mm against the gram-negative and -positive strains, respectively. Additionally, the MIC values for ethanolic extracts were 0.14, 0.05, and 0.07 mg/ml. Streptomycin used as positive control showed inhibition zones of 28.9, 38.50, 35.40, and MIC values of 0.05, 0.02, and 0.02 mg/ml, respectively. Therefore, ethanolic extract showed comparable antibacterial activity to that of streptomycin.

 Table 6. Antibacterial activity of different solvent extracts of P. oleracea L. (MIC value expressed in mg/ml).

Each value is the mean± SD of three biological replicas, NA=No activity, Streptomycin was used as the positive control

The five different solvent extracts of P. oleracea were evaluated against Fusarium oxysporum and Aspergillus flavus. Each of the extracts except water and hexane revealed significant antifungal activity against positive control of Amphotericin B. Acetone extract of P. oleracea showed a percentage inhibition of 47.35% and 52.15% with MIC values of 1.80 mg/ml and 1.77 mg/ml against Fusarium oxysporum and Aspergillus flavus, respectively. Similarly, methanolic extract yielded a percentage inhibition of 49.55% and 55.81% with MIC values of 1.42 mg/ml against Fusarium oxysporum and Aspergillus flavus, respectively. The positive control showed percentage inhibitions of 8315% and 78% with MIC values of 0.003 mg/ml and 0.0001 mg/ml, respectively. In comparison to the positive control, ethanolic extract showed the highest antifungal activity against Fusarium oxysporum and Aspergillus flavus. The percentage inhibition was found to be 61.23% and 59.10% with MIC values of 0.62 mg/ml and 0.73 mg/ml, respectively (Table 7). 

Table 7. Antifungal activity of different solvent extracts of P. oleracea (MIC value expressed in mg/ml).

Each value is the mean± SD of three biological replicas, NA= No activity. Amphotericin B was used as a positive control.