Introduction

Humans rely heavily on plants as a source of food, feed, traditional medicine, and medication. According to current statistics, at least 35 - 40 thousand different plant species have been utilized for medical purposes [1]. As there are safety concerns with the adverse effects of synthetic chemicals and their medications, the demand for herbal therapies is steadily expanding. According to the World Health Organization (WHO) traditional herbal plants, have a significant role in effectively achieving health goals. India’s ancient system of plant-based treatment and executive food preparation has a long history. As reported by Samhita and Samhita [2] ayurvedic science explained the use of over 700 herbal plants and elaborated it for further medicinal use. Many herbal plants possess antioxidants, antibacterial, antifungal, and other therapeutic uses [3-5]. Recent studies have further elucidated the role of plants in managing newer healthcare usages [6].

Herbal plant medications are a prominent therapy in the traditional system, and these can be employed in modern therapeutics owing to their biological properties. There is a potential threat due to the harmful effects of chemical drugs on the human body. Therefore, various plants and their parts have been utilized for the management of a variety of diseases in Ayurvedic and folk medicine. Plants synthesize primary metabolites for both necessary and specialized tasks, such as growth and development [7-9].

Phenolic compounds are primarily used as a cellular support material and are a key component made up of polymeric phenolics. The bioactivities of phenolic compounds (flavonoids and phenolic acids) may be connected to their ability to chelate metals, inhibit lipoxygenase, and scavenge free radicals [10]. Bioactive polyphenols have gained a lot of interest as they protect the human body from cellular damage resulting from enhanced free radicals, and cause oxidative stress [11,12]. The term “oxidative stress” refers to a disruption in the balance between the production of free radicals, and the status of in vivo antioxidant defense mechanisms [13].

This biological circumstance has a significant impact on the emergence of several illnesses, like aging, cancer, coronary heart disease, Alzheimer’s disease, neurodegenerative disorders, atherosclerosis, cataract formation, and inflammation [11]. Enzymatic antioxidant systems, such as peroxidases, polyphenol oxidase, superoxide dismutase, catalase, etc. lower the effect of free radicals generated reactive oxygen species (ROS), which are responsible for proteins damage, DNA damage, genomic instability, the function of macromolecules, the addition of mutation and ultimately results in cellular functional alteration (Figure 1) [14-15]. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertbutyl hydroxydroquinone (tBHQ), propyl gallate (PG), and others are limited for use since some of them are reported for their harmful effects [16,17].

Figure 1: Scavenging effect of free radicals and its associated diseases with antioxidants and polyphenols.

Antioxidants that can be found in medicinal plants in abundance are prominent therapeutic agents that scavenge free radicals from the body. Natural antioxidants boost the antioxidant capacity. Daily consumption of bioactive compounds such as polyphenols or flavonoids leads to reducing of various life-threatening diseases including heart disease, stroke, pulmonary disease, and different types of cancer [18-20]. The natural antioxidants found in the leaves, fruits, seeds, roots, and bark of plants show anti-inflammatory, antiviral, antimicrobial, and antioxidant properties [2, 21-23]. Plant-based antioxidant agents such as flavonoids, alkaloids, and polyphenols may downregulate the uncontrolled cell division caused by oxidative stress imposed by free radicals [24]. Many plant components regulate the genes or signaling pathways involved in antioxidant activities that internally regulate the inflammatory response [25]. Antioxidants protect cells from ROS-induced DNA damage which is associated with disease prevention [22]. Given the foregoing; the current study was conducted to investigate the antioxidant potential of various category plants, and their cytotoxic potential was also evaluated.

Material & Methods

Reagents & Chemicals

DPPH 2,2, Diphenyl picryl hydrazyl, Gallic acid, L Ascorbic acid, MTT reagent, DMEM high glucose media, FBS, PenStrep, DMSO, trypsin-EDTA were purchased from sigma. Aluminum chloride, Sodium carbonate, Ferrous sulfate, and 2,4,6-tripyridyl-Striazine (TPTZ) were purchased from Himedia. All the other chemicals used were of analytical grade.

Plant material collection and preparation of extracts

Plant material was collected from their natural habitat or procured. The freshly harvested material was properly cleaned, dried and powdered. Each sample was incubated in absolute ethanol for 72 hours. Following that, each extract was centrifuged and resultant ethanolic was filtered with Whatman’s filter paper and the filtrate was evaporated using a Rotatory evaporator under low pressure. A known weight of the obtained extract was dissolved with a known volume of solvent to have an extract with a known concentration. The obtained extract was stored at 4°C until analysis [26].

Determination of total phenolic content (TPC)

The total phenolic content (TPC) was determined using the Folin-Ciocalteu reagent method with some modifications [12]. A suitable amount of aliquot of extract was added into the reaction mixture. The absorbance of the extract was measured at 760 nm against a blank. The total phenolic content of the plant extracts was expressed as milligram Gallic acid equivalents per gram of dry weight (mg GAE/g DW) using the calibration curve of Gallic acid.

Determination of total flavonoid content (TFC)

The total flavonoid content (TFC) was determined using a modified aluminum chloride colorimetric method [22]. An aliquot of 0.04 ml & 0.1ml (1mg/ml) of extract was added in 2% aluminum chloride (0.1ml). To the mixture, 0.8 ml methanol was added. After spinning the mixture, the reaction was incubated for 60 minutes at room temperature in the dark. The absorbance of the solution was expressed as milligram Quercetin equivalent per gram of dry weight (mg QE/g DW) using the calibration curve of quercetin.

Ferric reducing antioxidant power assay

The reducing power was determined according to the method of [27]. An aliquot of 0.03 ml (1mg/ml) of plant extract was added with FRAP reagent in a 1:30 ratio. The reagent included acetate buffer (300mM, pH 3.6), 2,4,6-tri[2-pyridyl]-s-triazine (10mM in 40mM HCl) solution, FeCl3.6H2O (20mmol/liter) in 10:1:1 ration respectively. The absorbance was measured at 593 nm against blank. The absorbance of the extract was expressed as milligram Ferrous sulfate equivalent per gram of dry weight (mg FSE/g DW) using the calibration curve of Ferrous sulfate. 

DPPH• radical scavenging activity

DPPH radical scavenging activity was determined with some modifications [28]. The hydrogen atom-donating ability of the plants was analyzed by decolorization of the methanol solution of DPPH. DPPH produces a violet/purple color in methanol solution and fades to shades of yellow color in the presence of antioxidants. DPPH stock solution was prepared with a concentration of 0.002% in methanol. 1mg/ml extract was prepared using methanol as solvent. 0.1ml of extract was added with 0.7ml of DPPH and volume was made up to 1ml with methanol. A control was prepared by adding 0.7 ml of DPPH and 0.3 ml of methanol. The reaction mixture was incubated and absorbance was measured against the blank. Free radical inhibition percentage was calculated using the following equation. 

Scavenging, DPPH (%) = (Abs control, DPPH – Abs sample, DPPH)/Abs control, DPPH×100

Cytotoxic activity against Human Embryonic Kidney cells

Human embryonic kidney cells (HEK-293) were procured from the National Centre for Cell Sciences (NCCS), Pune, India, and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of inactivated Fetal Bovine Serum (FBS), 1% antibiotic/antimycin cocktail at 37°C in humidifier containing 5% CO₂. The cells were allowed to develop to 80-90% confluence in 25cm² culture flasks before being harvested with 0.25% trypsin/ EDTA solution. The viability of the cells was determined by the trypan blue dye exclusion technique. The cells were sub-cultured in 96-well plates cells, the cell count was adjusted to 9000 cells/ ml using DMEM containing 10%FBS. After 24 hours, when a partial monolayer was formed, the supernatant was removed and 0.1ml of different concentrations of plant samples were added to 96 well plates. The plate was incubated at 37°C for 24h in a 5% CO2 atmosphere. After 24h, the sample was removed from the wells and 0.5mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) working solution was added in each well for 4hrs. After the incubation period, the medium was withdrawn, and each experimental well was filled with 100μl of DMSO, which was further incubated for 20min at room temperature. Finally, MTT activity was measured using a microplate reader at a wavelength of 570nm [29]. The percentage viability of the cell was determined using the equation:

%Cell viability= [OD (Control cells)- OD (treated cells) /OD (control wells)]100

Statistical Analysis

All tests were carried out in triplicates and results were expressed as mean ± standard deviations. Both, the mean and standard deviation were determined using GraphPad Prism. The biochemical parameters were subjected to statistical analysis following One-way ANOVA. Significance difference was adapted to all the parameters under study to test the level of statistical significance p<0.05.

Results

Determination of Total Phenolic and Flavonoid Content:

The total phenolic content (TPC) of standard gallic acid was estimated using the calibration curve (Y = 0.0484x + 0.019, R² = 0.9968) and represented as mg GAE/g DW. The total flavonoid

content (TFC) of standard quercetin was estimated using the calibration curve (Y= 0.0099x-0.044, R²= 0.9985) and represented as mg QE/g DW. The result of total phenolic and flavonoid contents is illustrated in Figures (2-4).

The result of the study showed that the total phenolic and flavonoid content in the flowering group was reported highest in Alstonia scholaris and Tagetes erecta respectively followed by others as shown in Figure 2.

                    A)

                  B)

Figure 2: A) Total phenolic content (TPC) and a standard curve with gallic acid concentration ranging from 0.005 to 0.025mg/ml in the reaction mixture.

B) Total flavonoid content (TFC) and a standard curve with quercetin concentration ranging from 0.025 to 0.25mg/ml in the reaction mixture.

(P.h; Physalis hybrida; T.e; Tagetus erecta; T.m; Tropaeolum majus; A.s; Alstonia scholaris; C.o; Calendula officinalis; T.d; Tabernaemontana divaricate; C.t; Clitoria ternatea)

Likewise, for medicinal plant clusters, Cinnamon verum showed the highest phenolic content and Glycyrrhiza glabra showed the highest flavonoid content among others as shown in Figure 3.

Figure 3: A) Total phenolic content (TPC) B) Total flavonoid content (TFC)

(G.g; Glycyrrhiza glabra; A.i; Azadirachta indica; O.t; Ocimum tenuiflorum; T.a; Terminalia arjuna; C.a; Crinum asiaticum; A.r; Asparagus racemosus; T.c; Tinospora cordifolia; C.v; Cinnamomum verum).

Whereas in the case of trees, the highest phenolic content was found in Tectona grandis and the highest flavonoidcontent was shown by Saraca asoca. Total phenolics and flavonoid content among tree samples used in this study were reported in Figure 4.

Figure 4: A) Total phenolic content (TPC) B) Total flavonoid content (TFC)

(F.be; Ficus benghalensis; F.b; Ficus benjamin; F.e; Fraxinus excelsior; T.s; Tecoma stans; T.g; Tectona grandis; S.a; Saraca asoca)

Determination of Antioxidant Activity

Determination of Free Radical Scavenging Activity:

Plant extracts were evaluated by DPPH assay, to investigate their radical scavenging activity and the results are shown in Figure (5). DPPH is a stable free radical widely used to evaluate the free radical scavenging activity from many plant extracts. DPPH assay undergoes a reduction in the presence of antioxidants. The disappearance of DPPH in the presence of antioxidants is measured spectrophotometrically at 517nm. In the case of flowering plants, the maximum percentage of scavenging activity was shown by Tabernaemontana divaricata 65 to 70%. Whereas in the case of medicinal plants, maximum inhibition was shown by Glycyrrhiza glabra 70 to 80 %. In the case of trees, maximum inhibition was shown by Fraxinus excelsior 70 to 80%, and the results revealed that all the plants showed promising antioxidant activities.

(A)

(B)

(C) 

               Figure 5: Percent scavenging activity of DPPH free radical in different plants.

Determination of Total Antioxidant Capacity

Plants were evaluated by using FRAP assay, to investigate the reducing antioxidant power. The ferric-reducing antioxidant power was expressed as mg FSE/g DW, and calculated using the calibration curve (Y= 1.36x + 0.000005, R² = 0.999) shown in Figure (6).

In the case of flowering plants, the maximum antioxidant power was shown by Tagetus erecta, in medicinal plants Cinnamomum verum, and trees Ficus benjamin and Ficus excelsior showed considerable activity.

A)

B)

   (C) 

Figure 6: Antioxidant potential of different plant groups expressed as mg Ferrous sulfate equivalent per gram of dry weight.

The findings of the studies agreed with earlier reports; nevertheless, variation in value could be caused by differences in the origin of the raw materials under different geographical variations and genotype-environment interaction [14].

Cytotoxic Activity

In the present study, the MTT assay was used to evaluate the cytotoxic potential of four selected plants exhibiting good antioxidant potential as per the parameter’s studies such as TPC, TFC, FRAP, and DPPH. These plants belongs to different clusters of medicinal, and tree plant groups Alstonia scholaris; Glycyrrhiza glabra; Terminalia arjuna; Ficus benghalensis. The result of the cytotoxicity assay conducted against HEK-293 is shown in Figure 7. The cytotoxicity activity was found to be dose-dependent, as the higher the concentration higher the activity was observed. Glycyrrhiza glabra showed 70 to 80% cell viability at 150μg/ml in the case of HEK-293 cell lines. F. benghalensis, A. scholaris, and T. arjuna show no cytotoxicity up to the dose of 150μg/ml in the case of HEK-293 cells. A one-way ANOVA test was done to evaluate the statistical difference and significance.

Figure 7: Growth inhibition of HEK-293 (Human embryonic kidney cells) by (G.g Glycyrrhiza glabra; F.b Ficus benghalensis; A.s Alstonia scholaris; T.a Terminalia arjuna. Cells were seeded at a density of 10×10³ cells/well and treated in a dose-dependent manner for 24 hours, Here C stands for Control, C1 concentration 25μg/ml, C2 50μg/ml, C3 75μg/ml, C4 100μg/ml, C5 125μg/ml, C6 150μg/ml. Cell viability was measured by MTT assay. The data is shown as the average of three separate studies, with standard errors represented by mean ± standard error (p-value <0.01).