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BY 4.0 license Open Access Published by De Gruyter Open Access November 23, 2022

Evaluation of phytochemical and antioxidant potential of various extracts from traditionally used medicinal plants of Pakistan

  • Syed Anis Ali Jafri , Zafar Mahmood Khalid , Muhammad Zakryya Khan and NaqeebUllah Jogezai EMAIL logo
From the journal Open Chemistry

Abstract

The antioxidant potential of various extracts was evaluated using different antioxidant assays such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, ferric reducing antioxidant power (FRAP) assay, and 2,2-azinobis-ethylbenzothiozoline-6-sulphonic acid (ABTS) using UV spectrophotometer. The highest absorbance was observed in ethanolic extracts (EEs) of Euphrasia stricta 71.92 ± 1.22%, 65.77 ± 1.38%, and 67.88 ± 0.74%, followed by methanolic extracts (MEs) 70.14 ± 0.82%, 64.84 ± 0.74%, and 65.48 ± 1.40% for DPPH assay (517 nm), FRAP assay (700 nm), and ABTS assay (734 nm), respectively. The EEs of Euphorbia platyphyllos L. showed the antioxidant activity of 69.76 ± 1.48%, 64.42 ± 0.88%, and 65.54 ± 1.36% and MEs 68.00 ± 1.50%, 62.92 ± 0.64%, and 63.42 ± 0.94% for DPPH, FRAP, and ABTS assays, respectively. So, this research suggested that these medicinal plants possess a significant antioxidant potential and are important source of natural antioxidants and can be effectively used in treating oxidative stress disorders.

Graphical abstract

Graphical presentation of whole experimental process.

1 Introduction

The reactive oxygen species (ROS) are free radicals like hydroxyl radical, nitric oxide radical, hydrogen peroxide, and superoxide anion radical, hypochlorite radical, lipid peroxides, and various singlet oxygen molecules [1]. During the metabolic activities, our body produces certain compounds called free radicals by its own [2]. The normal physiological processes result in lesser production of free radicals in the body, but abnormal functioning or less availability of antioxidant level in the body results in oxidative stress resulting in the generation of these free radicals in high level [3]. These free radicals are the cause of many degenerative and chronic diseases like Parkinson’s disease, arthritis, cancers, stroke, Alzheimer’s disease, immune suppression, atherosclerosis, ageing, diabetes mellitus, chronic inflammatory diseases, ischemic heart disease, and neurodegenerative diseases [4]. On the other hand, some other factors i.e. alcohol, smoking, ionizing radiations, chronic diseases, and environmental pollution are also responsible for the increased level of these free radicals, in addition to natural causes [5].

These free radicals are bound by very vital chemical compounds, the antioxidants, which reduce and prevent us from the harmful effects on normal cells of the body. Some antioxidants produced artificially, such as butylated hydroxyl-toluene and butylated hydroxyl-anisole, which are available commercially are less stable and are very harmful, whereas natural antioxidants are safe with minimal side effects. Due to safety reasons, antioxidants have been preferably obtained from natural products [6]. Fruits, vegetables, seeds, herbs, sprouts, edible mushrooms, and cereals are the natural sources of food which can be used as an efficient source of these antioxidants to minimize the side effects and damage from free radicals [7]. The chemical substances present in plants, which have defensive effects, are called phytochemicals. The phytoconstituents found in the plants, which are responsible for antioxidant potential, are mainly flavonoids, phenols, anthocyanin, iso-flavones, flavones, lignins, catechins, iso-catechins, and coumarins. These phytochemical constituents are mainly determined by measuring total phenolic contents (TPC) and total flavonoid contents (TFC), and the antioxidant effects are determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), Ferric reducing antioxidant power (FRAP), and 2,2-azinobis-ethylbenzothiozoline-6-sulphonic acid (ABTS) assays [8].

The diseased condition by ROS can be effectively improved by compounds called antioxidants that can hunt and neutralize the free radicals. An extensive diversity of free radical hunting or antioxidant components like phenols, vitamins, terpenoids, and flavonoids have been found in plants which possess high antioxidant potentials [9]. The plant-derived polyphenolic constituents might be more useful in vivo with their positive effects as these are proved to be more efficient antioxidants when compared to vitamin E or C in vitro conditions [10].

Different medicinal plants having antioxidant potential have been efficiently applied to treat ROS and are of great importance because of their alimentary radical scavenging diet supplement. The antioxidant potentials of medicinal plants are mainly because of rich source of phyto-nutrients and ingredients like phenols, flavonoids, and terpenoids present in them. The antioxidant potentials of many medicinal plants have been studied for anti-cancer activity, immunomodulator activity, hepatoprotective effects, and hypolipidemic activity [11].

Hence, the evaluation of free radical scavenging activity (RSA) and comparison of various medicinal plants with different protocols using spectrophotometer are effective ways for the study of plants’ secondary metabolites [12].

Timur or Indian prickly ash, Zanthoxylum armatum DC. (syn. Z. alatum Roxb.), widely found in Kashmir, India and in the Himalayan regions of Bhutan at high altitudes of 2,500 m. It is also found in Nepal, Taiwan, Philippines, China, Japan, Malaysia, and Pakistan at 1,300–1,500 m altitudes [13]. Various medicinal plants are commonly used by folk people of Himalayan regions in Pakistan for medicinal and nutritional purposes.

Before the coming of the modern pharmacological medicines, people of Manipur have been using medicinal plants for the treatment of diabetes mellitus. People not only in the rural areas but those living in the urban areas are also using these traditional medicines preferably for herbal treatments at present all over the world [14].

Previous studies showed that bioactivities of the plant extracts were because of high polyphenol, phenolic, and flavonoid contents found in them. A number of oxidizing molecules like singlet oxygen and numerous other free radicals responsible for different diseases can be effectively scavenged by flavonoids usage. The formation of ROS and chelate trace elements involved in free-radical production can be effectively suppressed by using these flavonoids. These phytochemicals can also be used to protect and upregulate antioxidant defenses and scavenging of ROS. Also, the oxidative stress tolerance of plants can be conferred by these phenols and flavonoids. Various plant extracts from many plant sources, e.g., fruits, herbs, vegetables, and cereals, rich in phenols and flavonoids have been efficiently used in the food industry and other health assistances because of their antioxidant properties [15]. These investigations have showed that the higher TPC was estimated using DPPH assay where the wine samples observed also showed the same trend for catechin and gallic acid phytoconstituents and the abundance of phenolics.

Many secondary metabolites of medicinal plants are responsible for their potential natural source for disease prevention and treating different diseases. Lot of researchers have been attracted toward the plants to obtain these natural compounds from plant extracts important for their medicinal properties. The disease ailment potential of various phytochemicals from plants are flavonoids, tannins, alkaloids, and phenolic substances, which are analyzed by DPPH, FRAP, and ABTS antioxidant assays [16]. Phytol is an active ingredient of plants that has been reported to be applied in formulations to treat the cardiovascular diseases and is reported to lower serum levels of triglycerides and/or cholesterol in type II diabetes and obese patients [17].

Keeping in mind the prime importance of medicinal plants and their usage by common people in Himalayan regions, the present study was conducted with aims and objectives to evaluate the antioxidant activity using different antioxidant assays, phytochemicals estimation, and factors responsible for scavenging potentials of various allelopathic medicinal plants from moist temperate Himalayan regions of Pakistan.

2 Material and methods

2.1 Chemicals and reagents

DPPH, methanol, ethanol, trichloroacetic acid, gallic acid, quercetin, rutin, butylated hydroxy toluene, 2-deoxy-2-ribose, EDTA, hydrogen peroxide (H2O2), ascorbic acid, 2-thiobarbituric acid (TBA), Folin–Ciocalteu reagent, nitro blue-tetrazolium (NBT), riboflavin, potassium chloride, aluminum chloride, sodium carbonate, sodium nitrate, sodium hydroxide (NaOH), hydrochloric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, deionized water, potassium dihydrogen phosphate, ferric chloride, potassium ferricyanide, FeCl3, conc. H2SO4, Molisch’s reagent, Fehling’s solution, ammonium thiocyanate, FeSO4, Mayer’s reagent, and chemical solvents (analytical grade) used during the study were purchased from Sigma Chemicals (USA). Deionized water, distilled water (dH2O), water bath, pipette, centrifuge tubes, centrifuge, incubator, vortex shaker, and UV spectrophotometer were available in the laboratory where experimental work was performed.

2.2 Plants collection and identification

Various medicinal plants were collected from different localities of moist Himalayan regions of Pakistan in their growth season. The collected plants were identified by expert taxonomist in the Department of Environmental sciences, Ecology and Biodiversity Lab, International Islamic University, Islamabad, Pakistan. The complete details of these medicinal plant samples used in this experiment are given in Table 1.

Table 1

Traditionally used medicinal plants collected from Himalayan regions of Pakistan

S. no. Scientific name Local name Family Parts used Traditional medicinal uses
1. Trifolium species White clover, Desi siree, Saag Fabaceae Leaves Stomach problems, anti-intestinal helminthic worms, and anticestodal properties
2. Cassia tora Linn. Kikkar, Cassias Fabaceae Leaves and seeds Phytochemical and pharmacological properties
3. Psoralea corylifolia L. Babchi or Bakuchi Fabaceae Leaves and seeds Antibacterial, antitumor, anti-inflammatory, and immunomodulatory activities
4. Urticae folium Nettles or Stinging nettles Urticaceae Seeds and leaves Antimicrobial, antiulcer, and analgesic activities
5. Teraxaci folium Dandelion Asteraceae Whole plant extract Anti-inflammatory, anticarcinogenic, and antioxidative activities
6. Rubi idaei folium Akhriyar and Jammaro Phragmidiaceae Fruits and leaves Antimicrobial properties
7. R. potaninii Maxim Desi drawa and Tunn Anacardiaceae Fruits and leaves Antihypertension to control high BP and antidiabetic properties
8. Lantana camara Big sage, Wild sage, Red sage, White sage, and Tick berry Verbenaceae Whole plant extracts Antimicrobial, fungicidal, insecticidal, anticancer, skin itches, leprosy, rabies, chicken-pox, measles, asthma, and antiulcer properties
9. Lonicera japonica Thunb. Chanbha, Japanese honeysuckle, and Golden and silver honeysuckle Caprifoliaceae Dried leaves, stem, and flowers Anti-inflammatory, to treat fever, headache, cough, thirst, and sore throat
10. Epilobii herba Willow herbs Onagraceae Whole plant extracts Used for benign prostate hyperplasia (BPH), bladder, and hormone disorders
11. Thymus serpyllum L. Breckl and thyme, Breckl and wild, and creeping thyme Lamiaceae Aerial parts Antimicrobial, to treat fever, and antitumor activity
12. Salvia officinalis Garden sage, Common sage, or Culinary sage Lamiaceae Whole plant extract Treating Alzheimer’s disease as neurotoxic
13. Euphrasia herba Eyebright Orobanchaceae Whole plant extract For eyestrain and to relieve inflammation caused by cold, cough, sinus infection, sore throat, and hay fever
14. Equiseti herba Horsetail, Snake grass, and Puzzle grass Equisetaceae Aerial parts Antimicrobial and genotoxicity
15. Millefolii herba Yarrow or common yarrow Asteraceae Leaves and flowers Antimicrobial activity
16. Mentha piperita folium Wild mint Lamiaceae Whole plant extract Antimicrobial activity
17. Euphorbia platyphyllos L. Dhoodal Euphorbiaceae Aerial parts Cytotoxic and apoptotic activities
18. Marrubium vulgare White horehound Lamiaceae Aerial parts Treating stomach problems
19. Melissae folium Lemon Balm, Balm, Common balm, or Balm mint Lamiaceae Whole plant extract Antimicrobial activities
20. Hederae folium Jal bail Araliaceae Leaves and fruit Antimicrobial, antioxidative, hepatoprotective, and antimutagenic activities
21. Fritillaria thunbergii Lilly Liliaceae Whole plant extract Antimicrobial activities
22. Satureja montana Mountain savory or Winter savory Lamiaceae Whole plant extract Antimicrobial activities
23. Gunnera perpensa River pumpkin, Wild rhubarb, Wild ramenas, and Nalcas. Gunneraceae Stem and leaves Antimicrobial, anti-inflammatory, and antioxidative properties
24. Absinthii herba Absinthe, Absinthim, Absinthe wormwood, Grand wormwood, and Wormwood Asteraceae Stalk and leaves Antidepressant and antioxidant activities
25. Viscum album Mistletoe Santalaceae Leaves, fruit, and stalk Antihyperglycemic activity
26. Asperula herba Woodruff and Goldenrod Rubiaceae Leaves and flowers Antibacterial activities
27. Tephrosia purpurea L. Sarphonk, Sharpunkha, Fish poison, and Wild indigo Fabaceae Leaves Anticarcinogenic and antilipid peroxidative effects
28. Tinospora cordifolia (Willd.) Heart-leaved moonseed, Guduchi, and Giloy Menispermaceae Leaves Antibacterial and antifungal properties
29. Ocimum basilicum Basil and Sweet basil Lamiaceae Leaves Antioxidant and anticancer properties
30. Saussurea lappa Costus or Kuth Asteraceae Whole plant For stomach problems
31. Betulae folium Silver birch, Warty birch, European white birch, or East Asian white birch Betulaceae Bark and leaves Anti-inflammatory, antiviral, and anticancer properties
32. Cantaurii herba Centory, Starthistles, Knapweeds, and Centaureas Asteraceae Whole plant Cytotoxic, antifungal, and antimicrobial properties
33. Prunella vulgaris Common self-heal, Woundwort, Carpenter’s herb, Brown-wort, and Blue curls Lamiaceae Leaves and flower Cytotoxic and immunomodulatory activities
34. Echter Ehrenpreis Speedwell and Common gypsy-weed Veroniceae Whole plant Antimicrobial properties
35. Paris polyphilla Love apple and Satuwa Melanthiaceae Leaves Antimicrobial properties
36. Melissa officinalis L. Lemon balm, Balm, Common balm, or Balm mint Lamiaceae Leaves and stalk Genotoxicity and cytotoxicity
37. Hedyotis diffusa White flower and Snake-tongue grass Rubiaceae Whole plant extract Anti-inflammatory, cytotoxic, and antibacterial activities
38. Polygonum aviculare Common knotgrass, Prostrate knotweed, Bird-weed, Pigweed, and Low-grass Polygonaceae Leaves and stalk Antimicrobial and anti-inflammatory properties
39. Salviae off. Folium Sage, also called Garden sage, Common sage, and Culinary sage Lamiaceae Aerial parts Cytotoxic properties
40. Fagopyrum cymosum Buckwheat and Tartary buckwheat Polygonaceae Aerial parts Anti-inflammatory and also to treat fever and headache

2.3 Preparation of plant extracts

The collected plants were washed thoroughly to remove debris and were shade dried. These samples (parts used from each plant) were then grinded into fine powder after drying in an oven for 7 days, at Ecology and Biodiversity Lab, International Islamic University, Islamabad, Pakistan. Three solvents i.e., ethanol, methanol (analytical grade), and water (dH2O) were used for the preparation of plant extracts according to the procedure adopted earlier [18] with some modification. For this purpose, the dried and powdered plant samples of about 100 g each were taken in separate flasks and were extracted with 100 mL of 70% ethanol, 40% methanol, and dH2O using a stirrer. The filtration was achieved using Muslin cloth, the solutions were then centrifuged, and rotary evaporator was used for sample drying through evaporation. Air-tight plastic vials were used to store the collected dried plant samples for further studies. These extracts were ethanolic extract (EE), methanolic extract (ME), and aqueous extract (AE), respectively, of all the plants under study.

2.4 Phytochemical screening at University of Virginia (UVa)

The powdered samples were then shifted to Department of Biology at UVa, USA, where quantitative phytochemical estimation of TPC and TFC, and antioxidant evaluation of these samples was performed by various antioxidant assays using spectrophotometer. Three antioxidant assays (DPPH, FRAP, and ABTS assays) were used to determine the free radical scavenging potentials of these medicinal plants under study.

2.4.1 Determination of TPC

For estimation of TPC, Folin–Ciocalteu reagent was used to measure the TPC of these samples at 765 nm using UV spectrophotometer [19]. Stock solutions of each extract were prepared. The dilutions of all these EEs and MEs (0.5 mL of 1 mg/mL) and standard phenolic compound (gallic acid) using dH2O (5 mL, 1:10 dilution) were made and then mixed with Folin–Ciocalteu reagent solution, adding 4 mL of 1 M aqueous sodium carbonate. The absorbance was measured at 765 nm after the mixture was kept for 30 min for total phenol estimation using spectrophotometer. A common reference compound, gallic acid equivalent (GAE; mg/g of dry mass) curve was used to express the TPCs. All experiments were conducted in triplicates and the data were analyzed as average of three values.

2.4.2 TFCs

Shinoda test: 4 mL of extract solution, 1.5 mL of 50% methanol solution, and a small magnesium chunk were warmed. On adding 5–6 drops of concentrated HCl, red color was observed for flavonoids [20]. To 10 mL volumetric flasks containing 4 mL of water, different plant extracts (1.0 mg/mL) and various dilutions (10–1,000 µg/mL) from Rutin (Standard) were added. Then, 0.3 mL of 5% NaNO2 was added to the above mixture. 1 M NaOH (2 mL) was added after 6 min and 10 mL total volume was made by using dH2O. The solution was mixed well, and absorbance was taken at 510 nm. The Rutin equivalent (mg/g of dry mass) a common reference compound was used to express the TFCs. All experiments were performed in triplicates and the data were analyzed as average of three values.

2.4.3 Qualitative phytochemical screening

Presence or absence of different phyto-constituents (qualitative tests for phytochemicals) in the prepared extracts of important selected medicinal plants (Euphrasia stricta (ES), Euphorbia platyphyllos L. (EP), Epimedium brevicomum Maxim (EB), Viscum album (VA), Psoralea corylifolia L. (PC), Equisetum arvense (EA), Veronica officinalis (VO), Artemisia herba (AH), Fagopyrum cymosum (FC), and Prunella vulgaris (PV)) that have showed higher TPCs and TFCs were tested. Different chemical reagents were prepared. These tests were performed according to the standard procedures with minor modification [21,22,23].

2.4.3.1 Test for tannin/polyphenol [23]

3-4 drops of 10% FeCl3 were added to the diluted extract, the gallic tannins showed blue color, while the solution turned green for the presence of catechol tannin.

2.4.3.2 Test for terpenoids [22]

The reddish-brown color showed the presence of terpenoids when 0.2 g of each sample was mixed with 3 mL of conc. H2SO4.2 mL chloroform.

2.4.3.3 Test for glycosides [21]

Molisch’s reagent test: 5 mL of Molisch’s reagent and concentrated H2SO4 were added to the extract. The glycosides were indicated by violet color.

2.4.3.4 Test for reducing sugar [22]

1 mL of water and 5–8 drops of Fehling’s solution were added to 0.5 mL of plant extract and was heated. The appearance of brick red precipitation indicated the presence of reducing sugar.

2.4.3.5 Test for quinine [22]

The ammonium thiocyanate and freshly prepared FeSO4 solution (1 mL) were added to the extract, and then conc. H2SO4 was added drop by drop. The presence of quinine was indicated by deep red color.

2.4.3.6 Test for saponins [21]

20 mL of dH2O was boiled with 2 g powdered sample. 5 mL of dH2O and 10 mL of filtrate were quivered vigorously. The presence of saponins was indicated by the appearance of frothing.

2.4.3.7 Dil. NH3 tests

To the extract, 5 mL of dilute NH3 solution was added along with conc. H2SO4. The flavonoids were indicated by the appearance of yellow-color.

2.4.3.8 Test for flavonoids [23]

Shinoda test: 4 mL of extract solution, 1.5 mL of 50% methanol solution, and a small magnesium chunk were warmed. 5–6 drops of con. HCl were added. Red color was observed for flavonoids.

2.4.3.9 Test for volatile oils [23]

A small quantity of dilute HCl and 0.1 mL of NaOH were shaken with 2 mL of extract. The presence of volatile oil was indicated by the white precipitate.

2.4.3.10 Dragendroff’s reagent test [21]

2% H2SO4 and 2 mL of extract were warmed with the addition of few drops of Dragendroff’s reagent. The presence of alkaloids was observed by the orange-red color.

2.4.3.11 Test for alkaloids: Meyer’s test [23]

1 mL of Meyer’s reagent was added to 2 mL of extract. The presence of alkaloids was indicated by the pale-yellow precipitate.

2.4.3.12 Test for steroids [23]

A few drops of acetic acid and a drop of conc. H2SO4 were added in 1 g plant extract. The presence of steroids was indicated by the appearance of green color.

2.4.3.13 Test for cardiac glycosides [23]

2 mL of glacial acetic acid with one drop of FeCl3 solution was used to treat the 5 mL of plant extract. The presence of cardiac glycosides was indicated by the appearance of a violet ring or a greenish ring.

2.4.3.14 Phytochemicals effect in seed germination

To observe the cytotoxic effects of phytoconstituents on living cells, the germinating Pisum seeds were taken to represent the living cells. The AEs, ME, and EE were used to check the phytoconstituents effect on Pisum seeds germination by soaking in the solutions for 5 days [24].

2.5 Antioxidant assays

To determine the antioxidant effects of plant extracts, different antioxidant assays for the selected plant extracts were performed according to standard procedures with minor modifications.

2.5.1 DPPH antioxidant assay

DPPH is a stable free radical chemical with purple color that absorbs at 517 nm. The DPPH purple color is converted to yellow color or even colorless if plant sample possess any potential free radical scavenging property. The antioxidant or radical scavenging properties of various plant samples were investigated by applying DPPH antioxidant assay with some modifications in the procedure adopted by Roberta et al. and Blois protocols [25,30].

About 1 mL of test solution of EE, ME and AE was dissolved in equivalent amount of DPPH solution (0.1 mM). The increase in DPPH absorbance of tested samples was measured after 20 min incubation at room temperature, by taking the absorbance at 517 nm. Standard ascorbic acid (1 mM) showed the maximum absorbance of 90.36 ± 1.05 μg/mL, and was taken as a reference solution in this DPPH antioxidant assay.

The formula used to calculate the percent (%) inhibition was

DPPH percentage ( % ) inhibition = ( ( A B A A ) / A B ) × 100 ,

where, A B denotes the absorbance of DPPH radical + methanol; A A represents the absorbance of DPPH radical + sample extract/standard.

2.5.2 FRAP assay

The FRAP assay was used to determine the free RSA or antioxidant activity of medicinal plant extracts [26]. Phosphate buffer (0.2 M) was prepared by taking 800 mL of water in 1,000 mL graduated flask and added with 8 g NaCl, 1.44 g disodium hydrogen phosphate, 0.2 g potassium chloride, 0.24 g potassium dihydrogen phosphate, and pH was adjusted to 6.6 using HCl and the volume was adjusted using deionized water. 1% potassium cyanide was prepared by mixing the potassium ferricyanide (1 g) in 100 mL of deionized water or dH2O. 10% trichloroacetic acid stock solution was prepared by mixing trichloroacetic acid (10 g) in 100 mL of deionized or dH2O. 0.1% ferric chloride stock was prepared by dissolving the ferric chloride (100 mg) in 100 mL of dH2O. The standard ascorbic acid (0.1%) solution was prepared by mixing ascorbic acid (1 mg) in 1 mL of water. A colored compound was formed in the mixture, resulting from the reaction of antioxidants with ferric chloride, trichloroacetic acid, and potassium ferricyanide. The absorbance value of this solution was then measured at 700 nm by spectrophotometer.

The formula used to calculate the percentage (%) inhibition was

FRAP percentage ( % ) inhibition = ( ( A B A A ) / A B ) × 100 .

2.5.3 ABTS assay

ABTS, a radical cation decolorization assay, was also used to investigate the free radical scavenging potential of plant samples [27]. 7 mM ABTS in water and 2.45 mM potassium persulfate (1:1) were used in this experiment, which when mixed and stored in the dark at room temperature for 12–16 h before use resulted in the formation of ABTS+ cation radical by chemical reaction between them. The dilution of ABTS+ solution was prepared with methanol to attain an absorbance value of 0.700 at 734 nm. Then, 3.995 mL of diluted ABTS+ solution was added with 5 μL of plant extract and after 30 min incubation in dark from initial mixing, the absorbance was measured. Each assay was accompanied by running an appropriate solvent blank.

The medicinal plant extracts prepared in various solvents (ethanol, methanol and water) were mixed well using a stirrer. About 25 µL of these extracts were added to the dilution buffer and was vertexed thoroughly. The working solution was placed in freezer until next use. 10 µL of assay buffer was added to separate tubes as a negative control and 10 µL of samples or Trolox standard was also added to the given individual wells of the assay tubes. The solution was again added with 20 µL of sample or Trolox to all tubes with standards and samples of extracts. About 100 µL of the ABTS solution was added to each tube. The tubes were then placed on plate shaker at room temperature. The reaction was allowed to proceed for about 5 min. 50 µL of stop solution per tube was added to stop the reaction. The standard substance used in this experiment was Trolox. The absorbance of each sample was measured for the potential inhibition at a wavelength of 734 nm using UV spectrophotometer.

The formula used to calculate the percentage (%) inhibition was

ABTS + percentage ( % ) Inhibition = ( ( A B A A ) / A B ) × 100 .

2.6 Statistical analysis

Each experiment was repeated in triplicate and was measured as the mean value of three replicative trials as standard error mean (SEM) (mean value ± SEM). For each plant species in all three methods, the mean value and standard deviation (SD) and the SD variance (SDV) were calculated to check the % inhibition or % antioxidant activity. ANOVA was performed to compare data and the data were analyzed using Microsoft Excel 2016.

3 Results and discussion

3.1 Determination of TPC

The antioxidant activity of medicinal plants is usually because of the presence of phenolic and flavonoid contents in them. The antioxidant potential is primarily because of redox properties possessed by phenolic compounds found in the medicinal plants or other plants and fruits [28].

It was observed that the EEs of ES showed higher TPCs of 58.19 ± 1.74 GAE μg/mg followed by 46.05 ± 1.10 GAE μg/mg, and 51.93 ± 1.72 GAE μg/mg for EEs of EP and EB, respectively.

The MEs of ES EP, and EB showed TPC in range of 45.70 ± 1.48 GAE μg/mg, 42.00 ± 1.54 GAE μg/mg, and 44.06 ± 0.64 GAE μg/mg, respectively, as shown in Table 2. As the hydroxyl groups (OH) is responsible for the free radical scavenging ability in them, so rapid screening of antioxidant activity can be effectively applied by using the TPCs of these medicinal plants.

Table 2

TPCs found in various medicinal plants at 765 nm

S. no. Plant sample TPCs at 765 nm mean value ± SD (GAE μg/mg)
EE ME AE
1 ES 58.19 ± 1.74 45.70 ± 1.48 13.00 ± 1.20
2 EP 46.05 ± 1.10 42.00 ± 1.54 12.84 ± 1.24
3 EB 51.93 ± 1.72 44.06 ± 0.64 13.26 ± 0.44
4 VA 42.84 ± 0.48 42.50 ± 0.56 11.08 ± 0.92
5 PC 37.70 ± 0.62 38.12 ± 0.06 8.54 ± 0.88
6 EA 40.56 ± 0.32 36.90 ± 0.18 11.49 ± 0.70
7 VO 44.28 ± 2.00 39.02 ± 1.24 13.56 ± 1.20
8 AH 42.24 ± 0.36 37.16 ± 0.82 13.04 ± 0.18
9 FC 41.82 ± 1.87 40.30 ± 1.41 10.03 ± 1.24
10 PV 36.77 ± 1.84 34.68 ± 0.54 11.26 ± 0.69
11 Hederae folium 34.62 ± 1.63 32.76 ± 1.06 14.40 ± 0.56
12 Salvia divinorum 39.40 ± 1.76 37.08 ± 0.96 12.00 ± 1.66
13 Thymus serpyllum L. 35.23 ± 1.00 33.28 ± 1.44 10.03 ± 0.74
14 Melissa officinalis 42.00 ± 1.36 38.12 ± 1.28 11.20 ± 1.31
15 Cassia tora L. 40.94 ± 1.82 38.74 ± 0.46 13.02 ± 0.46
16 Saussurea lappa 35.79 ± 1.74 34.33 ± 1.04 12.20 ± 1.58
17 Epilobium parvifolium 40.52 ± 1.90 37.96 ± 0.06 9.00 ± 1.64
18 Satureja montana 44.30 ± 1.34 42.12 ± 1.00 13.44 ± 0.80
19 Asperula odorata 38.26 ± 0.53 36.05 ± 0.44 11.56 ± 0.68
20 Gunnera perpensa 34.15 ± 1.72 33.46 ± 1.16 14.66 ± 1.40
21 Fritillaria thunbergii 43.72 ± 1.84 40.78 ± 1.26 12.14 ± 1.56
22 Melissa flava 41.56 ± 0.88 38.42 ± 0.34 9.26 ± 0.68
23 Ocimum basilicum 34.49 ± 1.44 32.80 ± 1.56 11.33 ± 1.78
24 Achillea millefolium 39.35 ± 0.77 36.76 ± 0.34 8.82 ± 0.33
25 Urticae folium 37.06 ± 1.15 34.76 ± 0.55 14.53 ± 0.52
26 Polygonum aviculare 41.98 ± 1.86 40.00 ± 0.98 12.50 ± 1.80
27 Lonicera japonica Thunb. 36.75 ± 1.59 51.64 ± 1.62 11.00 ± 1.4
28 Tinospora cordifolia 39.54 ± 1.88 37.08 ± 0.76 12.36 ± 1.22
29 Paris polyphilla 35.21 ± 0.88 34.62 ± 1.42 13.44 ± 1.68
30 Mentha piperita folium 42.92 ± 0.46 41.36 ± 1.24 10.31 ± 0.60
31 Tephrosia purpurea L. 33.83 ± 0.57 31.08 ± 0.92 14.48 ± 0.43
32 Marrubium vulgare 40.78 ± 0.44 39.64 ± 0.88 13.81 ± 0.66
33 Lantana camara 34.56 ± 1.65 32.50 ± 1.81 10.04 ± 1.90
34 Betulae folium 39.47 ± 0.46 36.82 ± 0.53 8.45 ± 0.94
35 Teraxaci folium 35.42 ± 1.56 34.16 ± 2.12 8.56 ± 1.48
36 Rubi idaei folium 43.33 ± 1.74 42.02 ± 0.58 14.00 ± 1.90
37 Hedyotis diffusa 40.92 ± 1.78 40.50 ± 1.82 13.57 ± 0.74
38 Smilax glabra Roxb. 36.80 ± 1.76 35.63 ± 1.51 10.88 ± 1.64
39 Trifolium repense 41.45 ± 1.75 38.59 ± 1.76 11.24 ± 1.92
40 Cantaurii herba 38.24 ± 0.24 36.18 ± 0.92 12.44 ± 0.88

3.2 Determination of TFC

Flavonoids are the various plant secondary metabolites like flavanols, flavones, and derivatives of tannins found excessively in plants with antioxidant potentials. The presence of free OH groups, especially 3-OH group is mainly responsible for the antioxidant activity of these flavonoids. Plant flavonoids can be used as potential antioxidant in vitro as well as in vivo [29].

It was observed that the EEs of ES, showed higher TFCs, measured as 42.44 ± 1.26 QE μg/mg followed by 43.39 ± 1.05 QE μg/mg and 39.21 ± 1.76 QE μg/mg for EEs of EP and EB, respectively, as shown in Table 3 below.

Table 3

TFCs found in various medicinal plants at 510 nm

S. no. Plant sample TPCs at 510 nm mean value ± SD (QE μg/mg)
EE ME AE
1 ES 42.44 ± 1.26 39.18 ± 0.74 12.10 ± 1.62
2 EP 43.39 ± 1.05 35.88 ± 1.34 10.23 ± 0.44
3 EB 39.21 ± 1.76 38.62 ± 1.98 11.90 ± 1.72
4 VA 36.92 ± 1.80 38.96 ± 0.16 11.72 ± 0.88
5 PC 28.29 ± 1.34 26.73 ± 1.91 9.62 ± 1.54
6 EA 37.80 ± 1.98 34.26 ± 0.69 10.06 ± 1.88
7 VO 33.71 ± 1.36 31.42 ± 1.50 8.64 ± 0.96
8 AH 25.85 ± 0.43 25.15 ± 0.46 14.46 ± 0.62
9 FC 37.53 ± 1.74 35.76 ± 1.14 10.16 ± 1.92
10 PV 26.12 ± 1.65 25.38 ± 1.74 9.94 ± 1.76
11 Hederae folium 24.36 ± 0.82 22.16 ± 0.40 11.66 ± 0.98
12 Salvia divinorum 28.93 ± 1.24 27.88 ± 1.59 10.33 ± 1.34
13 Thymus serpyllum L. 24.44 ± 0.77 23.26 ± 0.94 10.85 ± 0.48
14 Melissa officinalis 30.21 ± 1.56 28.57 ± 0.25 12.43 ± 0.55
15 Cassia tora L. 33.78 ± 1.63 31.00 ± 0.78 12.58 ± 1.26
16 Saussurea lappa 29.35 ± 1.51 26.94 ± 1.12 9.00 ± 1.31
17 Epilobium parvifolium 25.56 ± 1.28 24.04 ± 0.44 10.76 ± 1.86
18 Satureja montana 32.41 ± 0.83 50.72 ± 1.52 6.64 ± 1.64
19 Asperula odorata 27.82 ± 0.96 25.46 ± 1.64 9.33 ± 0.40
20 Gunnera perpensa 21.60 ± 0.52 20.58 ± 0.32 10.49 ± 0.93
21 Fritillaria thunbergii 35.28 ± 0.46 33.44 ± 0.86 8.61 ± 0.79
22 Melissa flava 32.66 ± 1.35 28.80 ± 1.41 6.44 ± 1.96
23 Ocimum basilicum 26.31 ± 0.93 25.74 ± 0.63 8.85 ± 0.38
24 Achillea millefolium 28.84 ± 1.66 24.36 ± 1.22 10.56 ± 1.86
25 Urticae folium 24.38 ± 1.14 22.04 ± 0.54 13.34 ± 1.00
26 Polygonum aviculare 29.52 ± 1.44 27.56 ± 1.68 12.76 ± 0.58
27 Lonicera japonica Thunb. 26.60 ± 1.96 23.64 ± 1.42 11.58 ± 1.46
28 Tinospora cordifolia 24.85 ± 1.72 22.29 ± 1.96 11.64 ± 1.98
29 Paris polyphilla 28.44 ± 0.84 27.88 ± 0.12 9.51 ± 0.62
30 Mentha piperita folium 32.16 ± 0.56 30.33 ± 1.44 11.00 ± 1.18
31 Tephrosia purpurea L. 24.39 ± 1.88 23.96 ± 0.80 13.52 ± 0.86
32 Marrubium vulgare 31.72 ± 1.64 28.58 ± 1.35 11.84 ± 1.54
33 Lantana camara 26.30 ± 1.46 25.06 ± 0.86 8.40 ± 1.66
34 Betulae folium 25.82 ± 0.70 23.69 ± 0.53 10.36 ± 1.48
35 Teraxaci folium 24.46 ± 1.58 21.27 ± 0.67 12.64 ± 0.76
36 Rubi idaei folium 34.62 ± 1.97 32.65 ± 0.54 10.33 ± 1.64
37 Hedyotis diffusa 30.54 ± 0.69 29.37 ± 0.96 10.74 ± 0.41
38 Smilax glabra Roxb. 28.87 ± 0.78 24.54 ± 0.45 8.22 ± 1.20
39 Trifolium repense 22.40 ± 1.42 20.16 ± 1.94 11.08 ± 0.52
40 Cantaurii herba 26.63 ± 0.86 25.94 ± 0.22 9.70 ± 0.82

The TPC and TFC of the plant extracts of ES, [28] EP [28], and EB [29] were evaluated. The Epimedium brevicomum Maxim plant EEs showed the phenolic and flavonoid contents of 101.89 ± 0.52 GAE μg/mg and 265.28 ± 9.62QE μg/mg, respectively (Table 3). The antioxidant and anti-inflammatory activities of the extracts were correlated with the levels of phenol and flavonoid compounds possessed by these medicinal plants.

So, the tested medicinal plants were observed as a rich source of phenolic and flavonoid compounds. However, a detailed phytochemical investigation is required to recognize the potential phenolic and flavonoid contents of these medicinal plants before their application in treatment of oxidative stress related diseases.

3.3 Phytochemical screening of medicinal plants

Table 4 below shows the results for phytochemical screening tests obtained during the experiment on the selected plants extracts. The phytoconstituents found in plants were flavonoid, terpenoid, phenols, quinine, tannin, cardiac glycoside, steroid, alkaloid, volatile oils, and glycosides. The phytochemicals polyphenols, flavonoids, and terpenoids are highly present in most plants ES, EP, EB, VA, PC, EA, VO, AH, FC, and PV while tannins, glycosides, and quinines are highly present in ES, EP, and EB. The constituents terpenoids, cardiac glycosides, and alkaloids are moderately present in ES, EP, EB, VA, and PC. Previous studies have indicated that cytotoxic effect is shown by phytochemicals like tannin, flavonoid, and alkaloid [30]. The anticancer properties are reported for flavonoids, while antiviral properties and antibacterial effects are reported for the presence of saponin [31]. By inhibitory activity toward growth, tannin also indicated the anticancer properties [33]. The results showed that improved insulin resistance, glucose uptake in effected blood pressure, and reduced inflammation were shown by the curcumin present in turmeric [33]. These flavonoids prevent cardiovascular diseases and cancer [32]. So, the tested plants like ES, EP, and EB could be used as anticancerous, antibacterial, antiseptic agents, and antioxidant [33]. The blood sugar was also maintained by the ES. The cardiac glycoside was found abundantly in these plants which are very beneficial for the heart. ES and EB are rich in phenolic compounds, flavonoids, tannins, and terpenoids and has antihelmintic property so stomach problems could be efficiently treated by using these plants [34]. The antioxidant, antibacterial agent, and anti-inflammatory effects were shown because of the polyphenolic compounds, flavonoid, and terpenoid found in ES and Thymus serpyllum. Similarly, the rise in blood pressure prevention and heart diseases were efficiently reduced by using these plant extracts.

Table 4

Phytochemical screening of different medicinal plants

Plants Phytochemical screening
Tannin Cardiac glycosides Reducing sugars Quinine Glycosides Polyphenols Flavonoids Saponins Alkaloids Terpenoids Steroids Volatile oil
ES +++ + +++ ++ +++ +++ ++ ++ ++ +
EP +++ ++ +++ +++ +++ +++ ++ ++ ++ ++ ++
EB +++ +++ +++ ++ +++ +++ ++ ++ ++ ++ ++
VA ++ ++ +++ +++ +++ ++ ++ ++ +
PC ++ + ++ + ++ +++ ++ + + ++
EA ++ ++ ++ +++ ++ ++ ++ ++ +
VO ++ + + ++ +++ +++ + + ++ + +
AH ++ + + + +++ +++ +++ ++ ++ + +
FC ++ + –_ + + +++ ++ + + ++ ++ ++
PV ++ + + ++ +++ ++ ++ + +

+++ indicates highly present, ++ indicates moderate present, + indicates present whereas the –indicates absent.

3.4 Evaluation of antioxidant potential of medicinal plants

3.4.1 DPPH free radical scavenging assay

The highest antioxidant potential was observed in the EEs of ES [25]. The RSA of EE, ME and AE of various medicinal plant extracts was examined as shown in Table 5. The standard antioxidant compound l-ascorbic acid showed the highest RSA of 90.36 ± 1.05 µg/mL. The EEs showed the highest antioxidant activity of 71.92 ± 1.22% followed by MEs with 70.14 ± 0.82%.

Table 5

The inhibitory effects of various medicinal plant extracts in DPPH assay at 517 nm

Sr. No. Plant sample % Free RSA mean value ± SD (µg/mL)
EE ME AE
1 ES 71.92 ± 1.22 70.14 ± 0.82 12.08 ± 0.96
2 EP 69.76 ± 1.48 65.00 ± 1.50 18.34 ± 1.84
3 EB 67.46 ± 1.26 64.78 ± 0.36 13.34 ± 0.25
4 VA 53.94 ± 1.14 51.18 ± 0.66 15.06 ± 1.40
5 PC 53.81 ± 1.48 50.52 ± 1.66 20.50 ± 1.41
6 EA 52.88 ± 1.28 49.86 ± 0.55 15.90 ± 1.12
7 VO 52.74 ± 1.66 51.04 ± 0.56 12.26 ± 1.78
8 AH 52.48 ± 0.54 50.37 ± 0.28 9.44 ± 0.44
9 FC 51.93 ± 0.85 50.84 ± 0.76 25.06 ± 1.42
10 PV 51.79 ± 1.22 48.66 ± 0.82 10.08 ± 1.16
11 Hederae folium 51.72 ± 1.40 50.86 ± 0.62 17.58 ± 1.6
12 Salvia divinorum 51.54 ± 0.48 49.80 ± 0.56 10.08 ± 0.90
13 Thymus serpyllum L. 51.38 ± 0.36 47.16 ± 0.82 16.04 ± 0.18
14 Melissa officinalis 51.17 ± 1.84 49.68 ± 0.54 19.26 ± 0.69
15 Cassia tora L. 51.12 ± 1.00 48.28 ± 1.44 18.03 ± 0.74
16 Saussurea lappa 51.04 ± 1.82 50.74 ± 0.46 15.02 ± 0.78
17 Epilobium parvifolium 50.86 ± 0.88 48.42 ± 0.34 16.26 ± 0.68
18 Satureja montana 50.74 ± 1.15 47.76 ± 0.55 14.03 ± 0.5
19 Asperula odorata 50.70 ± 1.86 49.00 ± 0.98 16.50 ± 1.80
20 Gunnera perpensa 50.45 ± 1.59 46.64 ± 1.62 14.00 ± 1.4
21 Fritillaria thunbergii 50.21 ± 0.88 49.62 ± 1.42 13.64 ± 1.63
22 Melissa flava 50.18 ± 0.46 47.36 ± 1.24 20.31 ± 0.60
23 Ocimum basilicum 49.80 ± 1.76 45.63 ± 1.51 13.38 ± 1.94
24 Achillea millefolium 49.66 ± 0.86 48.30 ± 1.40 24.00 ± 1.78
25 Urticae folium 49.52 ± 1.47 49.04 ± 0.83 13.00 ± 1.76
26 Polygonum aviculare 48.24 ± 1.76 46.63 ± 0.42 23.8 ± 0.28
27 Lonicera japonica Thunb. 46.18 ± 0.42 44.84 ± 0.58 18.33 ± 0.29
28 Tinospora cordifolia 44.02 ± 0.78 42.96 ± 1.88 20.06 ± 0.64
29 Paris polyphilla 42.92 ± 1.86 41.06 ± 0.24 18.28 ± 1.82
30 Mentha piperita folium 42.80 ± 0.58 39.55 ± 0.36 16.40 ± 1.24
31 Tephrosia purpurea L. 42.36 ± 0.40 40.38 ± 1.76 13.70 ± 0.46
32 Marrubium vulgare 41.54 ± 1.62 38.46 ± 0.16 20.14 ± 0.56
33 Lantana camara 39.63 ± 0.6 37.56 ± 0.54 17.29 ± 0.68
34 Betulae folium 37.78 ± 0.42 36.44 ± 1.8 12.14 ± 0.82
35 Teraxaci folium 37.45 ± 0.73 35.52 ± 0.75 15.45 ± 0.62
36 Rubi idaei folium 36.84 ± 1.55 34.6 ± 1.76 12.80 ± 0.33
37 Hedyotis diffusa 35.68 ± 1.76 32.36 ± 0.94 18.54 ± 1.14
38 Smilax glabra Roxb. 30.82 ± 0.8 27.87 ± 0.63 11.55 ± 0.76
39 Trifolium repense 25.56 ± 1.54 21.72 ± 1.09 12.21 ± 1.86
40 Cantaurii herba 19.33 ± 1.74 19.02 ± 0.58 8.00 ± 1.90
Ascorbic acid (standard) 90.36 ± 1.05

The EP showed the RSA of 69.76 ± 1.48% and 65.00 ± 1.50% for its EEs and MEs, respectively compared to the standard l-ascorbic acid. The antioxidant activity shown by EEs and MEs of EB was 67.46 ± 1.26% and 64.78 ± 0.36%, respectively, as shown in Table 5.

Similarly, the VA. and PC showed the RSA of 53.94 ± 1.14 and 53.81 ± 1.48 for its EEs and 51.18 ± 0.66 and 50.52 ± 1.66 for MEs, respectively, followed by EA, VO, FC, PV, Hederae folium, Salvia divinorum, Thymus serpyllum L., Melissa officinalis, and Cassia tora L. using DPPH protocol.

It was observed that the alcoholic DPPH solution reduction is because of the presence of hydrogen donor antioxidant (AH) which reacts with free radicals and converts it to non-radical DPPHH form. The complete reaction can be expressed by this equation, DPPH˙+AH → DPPH-H+A˙. The DPPH remaining in the reaction mixture measured after some time acts in reverse to the antioxidant RSA. The free RSA of the plant extracts from ES, EP, and EB were evaluated using DPPH antioxidant assay. So, these findings from the present study suggested that relatively higher antioxidant potential of the tested medicinal plant samples were involved in the decolorization of the stable DPPH radical solution.

This study is in accordance with the published scientific data on the chemical composition and biological activity of ten species of Euphrasia stricta L: E. rostkoviana Hayne, E. brevipila Burnat et Gremli, E. parviflora Schag., EP, E. montana Jord., E. pectinata Tenore, E. condensata Jord., EB, E. stricta D. Wolff. ex J. F. Lehm., E. salisburgensis Funk, E. maximowiczii Wettst., and VA, and E. reuteri Wettst. The representatives of Euphrasia are the sources of biologically active substances, namely, glycosides, organic acids, lipophylic substances, macro and microelements etc. Flavonoids, phenoloacids, and iridoic glycosides, however, are the main groups. Hypotensive, anti-inflammatory, antimicrobic, antioxidant, and hepatoprotective action of Euphrasia representatives have been revealed experimentally [35]. Our results are in good agreement with previous studies of Pejin et al. [17], where phytol having effective antiradical activity had lowered the production of hydroxyl radicals (˙OH) by 15%, superoxide anion radicals (˙O2) by 23%, and nitric oxide radicals (˙NO) by 38%. The antiradical activity of phytol was evaluated by electron paramagnetic resonance against hydroxyl radical (˙OH), superoxide anion radical (˙O2), methoxy radical (˙CH2OH), carbon dioxide anion radical (˙CO2), as well as toward nitric oxide radical (˙NO) and ˙DPPH radical. The results showed that it reduced the generation of all tested radicals having promising activity against ˙CO2, ˙CH2OH, and ˙DPPH radicals (56, 50, and 48%, respectively) [17].

Similar findings were observed for increased concerns about the safety of synthetic antioxidants, focusing in the use of natural sources from antioxidants. In South Asia, the R. stricta and EP have been very important medicinal plants. Various antioxidant assays like DPPH, metal ion chelating assay, and FRAP Assay were used to evaluate the antioxidant potential of their MEs. The plants taken for antibacterial activity and superoxide radical activity were R. stricta, PC, and VA. The MEs of R. stricta, PC, and VA sheets showed the antioxidant potential were comparable with strong antioxidants observed previously and were always concentration dependent as shown in the present study [36].

This study is also in accordance with the antioxidant studies of phenolic compounds using response surface methodology from EB. To optimize achievement of high extraction yield of the phenolic compounds, three extraction variables using a Central Composite Design (CCD) was employed including ethanol concentration (X 1), extraction time (X 2), and ratio of aqueous ethanol to raw material (X 3). The experimental yield was 4.29 ± 0.033%, under these conditions. The DPPH and FRAP assays were used to evaluate the antioxidant activity [37].

Similar studies were conducted previously where the alcoholic DPPH solution reduction is because of the presence of hydrogen donor antioxidant (AH) which reacts with free radicals and converts it to non-radical DPPHH form. The complete reaction can be expressed by this equation, DPPH + AH → DPPHH + A. The DPPH remaining in the reaction mixture measured after some time acts in reverse to the antioxidant RSA. So, these findings from present study suggested that the relatively higher antioxidant potential of the tested medicinal plant samples were involved in the decolorization of the stable DPPH radical solution [38].

This study is also in accordance with the previous study where EEs of EB showed the maximum radical scavenging potential of 64.96 ± 0.79% followed by 62.59 ± 0.73% and 11.33 ± 1.46% for its MEs and AEs, respectively. The lipophilic and hydrophilic compounds can also be measured for their antioxidant capacities by using this assay. From the results it was observed that the EEs of all samples showed the highest radical scavenging capacities followed by the DPPH radical scavenging ability of MEs and AEs for all experimental samples. Thus, these results for antioxidant potential are in accordance with this present study [39].

3.4.2 FRAP assay

In this study, the Fe3+ to Fe2+ reduction assay was used to check the reducing ability of MEs of various medicinal plants. Here the antioxidant activity of the samples caused changes in color from yellow to pale/light green or blue color in the solution. All the medicinal plant samples displayed the presence of various antioxidants, such as flavonoids and phenolic acid, in considerable amount. The electron donating capacity of a reducing agent (i.e., antioxidants) can be monitored by FRAP assay, resulting in the Fe3+-ferricyanide complex to reduce to give ferrous (Fe2+) ions, so creating a chromogenic complex [40]. The absorbance of this resultant blue-green colored solution of samples was measured at 700 nm which was related to the Fe2+ amount in the mixture. The diverse abilities of these medicinal plant extracts to reduce the ferric ions (Fe3+) were revealed by the present study [41].

The ferric reducing capacities of various tested medicinal plant for ethanolic, methanolic and AEs observed are shown in Table 6. The standard l-ascorbic acid showed 85.48 ± 1.56% at 500 µg/mL concentration. The highest activity of 65.77 ± 1.38% was shown by EEs of ES, followed by 64.84 ± 0.74% and 12.56 ± 0.32% with MEs and AEs at 500 µg/mL, respectively. The EEs of EP showed the percent reducing power of 63.42 ± 0.88%, followed by 60.92 ± 0.64% and 12.56 ± 0.32% for MEs and AEs, respectively. The EEs of EB showed the percent reducing power of 62.88 ± 0.54, followed by 61.68 ± 1.86 and 11.28 ± 0.58 for MEs and AEs, respectively, as shown in Table 6.

Table 6

The inhibitory effects of various medicinal plant extracts in FRAP assay at 700 nm

S. no. Plant sample % Free RSA at 700 nm mean value ± SD (µg/mL)
EE ME AE
1 ES 65.77 ± 1.38 64.84 ± 0.74 12.56 ± 0.32
2 EP 63.42 ± 0.88 60.92 ± 0.64 12.86 ± 0.44
3 EB 62.88 ± 0.54 61.68 ± 1.86 11.28 ± 0.58
4 VA 52.73 ± 0.82 52.46 ± 1.34 9.58 ± 0.45
5 PC 51.86 ± 0.60 48.08 ± 0.94 15.26 ± 0.19
6 EA 51.39 ± 1.56 50.26 ± 0.89 12.68 ± 1.11
7 VO 50.76 ± 1.65 50.18 ± 1.36 8.22 ± 1.04
8 AH 50.62 ± 1.30 46.64 ± 0.48 10.68 ± 1.46
9 FC 50.07 ± 1.90 50.00 ± 1.34 20.14 ± 1.54
10 PV 48.92 ± 1.74 45.46 ± 0.60 10.00 ± 0.48
11 Hederae folium 48.70 ± 0.66 47.13 ± 0.46 14.84 ± 1.28
12 Salvia divinorum 48.52 ± 1.80 46.96 ± 0.16 11.02 ± 0.38
13 Thymus serpyllum L. 48.35 ± 0.43 44.15 ± 0.46 14.52 ± 0.60
14 Melissa officinalis 48.12 ± 1.65 47.38 ± 1.74 14.94 ± 1.76
15 Cassia tora L. 47.84 ± 0.77 45.26 ± 0.94 18.85 ± 0.48
16 Saussurea lappa 47.78 ± 1.63 47.00 ± 0.78 12.55 ± 1.18
17 Epilobium parvifolium 47.66 ± 1.35 45.80 ± 1.41 14.44 ± 1.96
18 Satureja montana 47.48 ± 1.14 46.04 ± 0.54 13.00 ± 1.00
19 Asperula odorata 47.22 ± 1.44 44.56 ± 1.68 12.92 ± 0.54
20 Gunnera perpensa 46.90 ± 1.96 46.64 ± 1.42 11.28 ± 1.44
21 Fritillaria thunbergii 46.74 ± 0.84 43.88 ± 0.12 14.51 ± 0.62
22 Melissa flava 46.66 ± 0.56 45.33 ± 1.44 15.00 ± 1.18
23 Ocimum basilicum 46.57 ± 0.78 44.54 ± 0.45 14.22 ± 1.20
24 Achillea millefolium 46.39 ± 0.24 42.53 ± 0.86 22.16 ± 1.44
25 Urticae folium 46.16 ± 0.64 43.38 ± 0.80 12.26 ± 0.54
26 Polygonum aviculare 44.55 ± 1.54 41.38 ± 0.69 15.44 ± 1.86
27 Lonicera japonica Thunb. 43.66 ± 0.84 40.53 ± 0.38 15.34 ± 1.44
28 Tinospora cordifolia 42.14 ± 0.40 40.42 ± 1.24 15.60 ± 0.48
29 Paris polyphilla 40.60 ± 0.78 39.91 ± 0.52 15.57 ± 0.78
30 Mentha piperita folium 40.33 ± 0.54 38.42 ± 2.34 14.24 ± 0.52
31 Tephrosia purpurea L. 38.84 ± 0.42 36.28 ± 1.84 12.54 ± 0.66
32 Marrubium vulgare 38.27 ± 1.96 34.88 ± 0.64 18.74 ± 0.72
33 Lantana camara 35.62 ± 1.74 32.66 ± 0.95 18.44 ± 1.36
34 Betulae folium 34.48 ± 0.64 31.85 ± 0.91 11.69 ± 0.35
35 Teraxaci folium 34.14 ± 0.96 32.48 ± 0.52 14.44 ± 0.84
36 Rubi idaei folium 33.94 ± 1.22 32.52 ± 0.28 11.62 ± 1.79
37 Hedyotis diffusa 30.51 ± 1.68 25.82 ± 1.56 14.60 ± 1.52
38 Smilax glabra Roxb. 26.86 ± 0.38 20.94 ± 0.72 9.66 ± 0.56
39 Trifolium repense 20.86 ± 1.24 15.26 ± 1.96 8.74 ± 1.22
40 Cantaurii herba 17.62 ± 1.97 13.65 ± 0.54 5.33 ± 1.64
Ascorbic acid (standard) 85.48 ± 1.56

Similarly, the VA and PC showed the RSA of 52.73 ± 0.82 and 51.86 ± 0.60 for its EEs and 52.46 ± 1.34 and 48.08 ± 0.94 for MEs, respectively, followed by EA, VO, FC, PV, Hederae folium, Salvia divinorum, Thymus serpyllum L., Melissa officinalis, and Cassia tora L. using FRAP protocol.

The antioxidant activity of the plant extracts from ES, EP, and EB were evaluated using DPPH and FRAP antioxidant assays. Thus, the sequence for the reducing power based on these experimental results can be organized as: l-ascorbic acid > EE > ME > AE. It was observed that the reductones present in these medicinal plants may be responsible for the reducing properties of the extracts and the reaction may be dependent for inhibiting the free radical chain by providing a hydrogen atom or may be by reaction with certain compounds of peroxides to avoid the peroxide development [42].

This study is also in accordance with the published scientific data on the chemical composition and biological activity of ten species of Euphrasia L: E. rostkoviana Hayne, E. brevipila Burnat et Gremli, E. parviflora Schag., E. montana Jord., E. pectinata Tenore, E. condensata Jord., E. stricta D. Wolff. ex J. F. Lehm., E. salisburgensis Funk, EP, E. maximowiczii Wettst., and E. reuteri Wettst. The representatives of Euphrasia are the sources of biologically active substances, namely, glycosides, organic acids, lipophylic substances, macro- and microelements etc. Flavonoids, phenoloacids, and iridoic glycosides, however, are the main groups. Hypotensive, anti-inflammatory, antimicrobic, antioxidant, and hepatoprotective action of Euphrasia representatives have been revealed experimentally [43].

The present study follows the previous study, where potential antioxidant activities of different extracts (Ethanolic, methanolic, petroleum ether, diethyl ether, ethyl acetate, and AEs) from EP (Euphorbiaceae) were evaluated for DPPH scavenging assay. Except diethyl ether and petroleum ether extracts all the extracts showed very significant DPPH scavenging activity by EP EEs. These results recommended that for most breast cancer treatments, this plant has higher capacity for source of anticancer agent. These results suggest that a dose-response relationship was shown by all the extract samples. Hence, additional studies are required for the isolation and identification of various phenolic compounds from the extracts for the treatment of cancers and tumors [44].

Similar studies were carried out where response surface methodology was used for the extraction of phenolic compounds from EB. For obtaining the highest extraction yield of the phenolic compounds, to optimize three extraction variables, CCD was employed, including ethanol concentration (X 1), extraction time (X 2), and ratio of aqueous ethanol to raw material (X 3). The DPPH and FRAP assays were used to evaluate the antioxidant activity which indicated that the EB phenolic compounds showed significant antioxidant properties. Earlier studies’ results showed that the main phenolic compounds in the extract product was revealed by HPLC analysis as catechin (Cianidanol), gallic acid, vanillic acid, ferulaic acid, p-hydroxybenzoic acid, benzoic acid, caffeic acid, quercetin, and rutin [37].

3.4.3 ABTS assay

In this research, the conversions for the ABTS+ radical cation inhibition or hunting capacities of each plant samples in various solvent extracts were investigated in comparison with the standard ascorbic acid and Trolox. The trial data for ABTS radical scavenging potential of each plant extract is shown in Table 7. The ABTS free radicals were generated by mixing the ABTS in potassium persulphate. The measurement was taken by spectrophotometer at 734 nm after 30 min incubation under dark conditions [45,46,47]. The higher ABTS radical scavenging potential was shown by the tested plant extracts using different solvents.

Table 7

The inhibitory effects of various medicinal plant extracts in ABTS assay at 734 nm

S. no. Plant sample % Free RSA at 734 nm mean value ± SD (µg/mL)
EE ME AE
1 ES 67.88 ± 0.74 66.48 ± 1.40 14.37 ± 0.26
2 EP 64.89 ± 1.36 63.48 ± 0.94 13.75 ± 1.16
3 EB 63.96 ± 0.79 63.22 ± 0.58 11.33 ± 1.46
4 VA 53.86 ± 1.18 50.44 ± 0.92 13.99 ± 0.84
5 PC 52.94 ± 1.12 52.55 ± 1.72 19.38 ± 1.68
6 EA 52.78 ± 0.41 49.66 ± 0.76 12.54 ± 0.96
7 VO 52.33 ± 0.66 51.88 ± 0.42 11.86 ± 0.29
8 AH 52.24 ± 1.03 50.80 ± 1.38 13.00 ± 1.06
9 FC 51.70 ± 0.47 48.20 ± 0.28 20.85 ± 0.33
10 PV 51.64 ± 0.29 51.60 ± 0.54 11.39 ± 0.66
11 Hederae folium 51.42 ± 1.54 49.38 ± 1.42 15.04 ± 1.90
12 Salvia divinorum 51.16 ± 1.65 50.96 ± 1.56 13.35 ± 0.16
13 Thymus serpyllum L. 50.74 ± 1.22 50.64 ± 0.63 15.42 ± 0.76
14 Melissa officinalis 50.52 ± 1.58 47.46 ± 0.96 16.55 ± 1.24
15 Cassia tora L. 50.25 ± 1.32 49.47 ± 1.62 19.60 ± 1.46
16 Saussurea lappa 48.96 ± 0.56 46.44 ± 0.38 15.26 ± 0.69
17 Epilobium parvifolium 48.71 ± 0.18 45.02 ± 1.82 19.64 ± 1.66
18 Satureja montana 48.58 ± 0.94 47.62 ± 0.68 12.71 ± 0.65
19 Asperula odorata 48.26 ± 1.66 47.40 ± 1.21 15.06 ± 1.84
20 Gunnera perpensa 48.15 ± 0.82 46.44 ± 0.58 17.45 ± 0.22
21 Fritillaria thunbergii 47.93 ± 1.78 47.52 ± 0.84 14.20 ± 1.92
22 Melissa flava 47.80 ± 1.34 45.75 ± 1.28 18.51 ± 0.41
23 Ocimum basilicum 47.75 ± 0.80 47.49 ± 0.66 14.96 ± 1.52
24 Achillea millefolium 47.58 ± 0.68 44.77 ± 0.26 22.94 ± 0.82
25 Urticae folium 47.44 ± 0.86 46.00 ± 0.42 13.81 ± 0.65
26 Polygonum aviculare 47.32 ± 1.50 44.54 ± 0.78 21.36 ± 0.39
27 Lonicera japonica Thunb. 44.20 ± 1.68 41.62 ± 1.69 15.26 ± 1.54
28 Tinospora cordifolia 42.82 ± 0.38 39.54 ± 0.86 17.70 ± 1.96
29 Paris polyphilla 41.62 ± 1.50 39.60 ± 1.38 17.80 ± 1.47
30 Mentha piperita folium 41.74 ± 1.92 38.42 ± 0.46 15.24 ± 1.66
31 Tephrosia purpurea L. 40.22 ± 0.55 38.51 ± 2.42 14.06 ± 1.82
32 Marrubium vulgare 39.16 ± 1.94 36.80 ± 1.59 18.60 ± 1.33
33 Lantana camara L. 37.46 ± 0.88 35.98 ± 0.58 16.20 ± 0.24
34 Betulae folium 35.64 ± 1.72 32.33 ± 0.96 12.94 ± 1.18
35 Teraxaci folium 35.16 ± 0.56 33.66 ± 1.38 14.82 ± 0.55
36 Rubi idaei folium 34.80 ± 1.24 32.24 ± 1.82 12.28 ± 1.76
37 Hedyotis diffusa 33.54 ± 0.48 30.16 ± 1.66 16.04 ± 0.74
38 Smilax glabra Roxb. 27.42 ± 0.68 23.36 ± 0.38 13.26 ± 0.92
39 Trifolium repense 21.16 ± 1.66 17.92 ± 0.46 10.64 ± 1.58
40 Cantaurii herba 18.91 ± 1.74 16.08 ± 1.45 6.76 ± 1.98
Ascorbic acid (standard) 88.64 ± 1.22

The highest ABTS radical scavenging ability was shown by the standard ascorbic acid (88.64 ± 1.22%) followed by the scavenging activity for EEs of ES with 67.88 ± 0.74%. The MEs of ES showed RSA of 66.48 ± 1.40% followed by AEs 14.37 ± 0.26% at 500 µg/mL concentration. The EEs of EP showed the maximum radical scavenging potential of 64.89 ± 1.36% followed by 63.42 ± 0.94% and 13.75 ± 1.16% for its MEs and AEs, respectively, at the same concentration as shown in Table 7.

The EEs of EB showed maximum radical scavenging potential of 63.96 ± 0.79% followed by 63.22 ± 0.58 and 11.33 ± 1.46% for its MEs and AEs, respectively, as shown in Table 7. The antioxidant activity of the plant extracts from ES, EP, and EB were evaluated using various antioxidant assays. The lipophilic and hydrophilic compounds can also be measured for their antioxidant capacities by using this assay [48]. From these findings, it was suggested that the EEs of all samples showed the highest radical scavenging capacities followed by the ABTS radical scavenging ability of MEs and AEs for all experimental samples.

Similarly, the VA and PC showed RSA of 53.86 ± 1.18% and 52.94 ± 1.12% for its EEs and 50.44 ± 0.92% and 52.55 ± 1.72% MEs, respectively, followed by EA, VO, FC, PV, Hederae folium, Salvia divinorum, Thymus serpyllum L., Melissa officinalis, and Cassia tora L. using ABTS protocol.

The present study is in accordance with the study conducted earlier in which ABTS˙ radical cation-based assays are considered the most plentiful antioxidant potential assays, together with the DPPH radical-based assays according to the Scopus citation rates [49]. Some antioxidants can undergo oxidation without coupling, whereas others, at the least of phenolic nature, can form coupling adducts with ABTS+, thus this reaction is a particular response for certain antioxidants. These coupling adducts can undergo further oxidative degradation leading to hydrazindyilidene-like and/or imine-like adducts with 3-ethyl-2-imino-1,3-benzothiazoline-6-sulfonate, 3-ethyl-2-oxo-1, and 3-benzothiazoline-6-sulfonate and as marker compounds, respectively [50,51].

This study is also in accordance with the study conducted on the biological activity investigation of moss Bryum moravicum Podp. (Bryaceae) in Germany using ABTS. The representatives of mosses are the assets of biologically live materials, particularly glycosides, natural acids, lipophylic substances, macro- and microelements, and so forth, but flavonoids, phenoloacids, and iridoic glycosides are the main compounds [52]. Hypotensive, anti-inflammatory, antimicrobic, antioxidant, and hepatoprotective motion of Euphrasia representatives have been discovered experimentally [53].

The present study follows the previous study, where potential antioxidant activities of different extracts (methanolic, petroleum ether, ethanolic, diethyl ether, ethyl acetate, and AEs) from EP (Euphorbiaceae) were evaluated for DPPH scavenging assay [54]. The results recommended that for most breast cancer treatments, this plant has higher capacity for source of anticancer agent. These results also suggested a dose-response relationship for all extract samples. Hence, additional studies are required for the isolation and identification of various phenolic compounds from the extracts for the treatment of cancers and tumors [48,55].

Similar studies were also conducted where four Iranian herbs were investigated to evaluate their antioxidant potentials, TPCs and TFCs. The dragon head and thyme showed higher antioxidant activity as compared to others when compared with the DPPH assay results. The TFCs observed in the dragonhead were higher from 10.12 to 22.2 EQ/g extract. Experimental results suggested that the dragonhead and thyme extracts were high in antioxidant capacity and so can be used as an important dietary source of phenolic compounds [56].

Similar findings were also observed for phytochemical detection of Gunnera perpensa L., where it was found that the extracts are rich in steroids, flavonoids, alkaloids, tannins, saponins, and glycosides. The G. Birpensa MEs showed strong inhibition in DPPH and ABTS assays but a weak inhibition (<50%) was noticed in superoxide, nitric oxide, and hydroxyl radical scavenging assays [56]. Our results are also in accordance with these findings.

Present study is also in accordance with the findings where the total alkaloids, peiminine, peimine, and peimisine from the Fitillaria thunbergii bulb were extracted using the supercritical fluid extraction method. The DPPH-RSA assay, ABTS-RSA, and FRAP assay were used to investigate the antioxidant capacity of F. thunbergii extracts. The maximum yield was 3.8 mg/g of total alkaloids, showed 1.3 mg/g peimine, 1.3 mg/g peiminine, and 0.5 mg/g peimisine. When compared to equivalent ascorbic acid (EAA)/100 g, the antioxidant potential was measured as EC50 value for the extracts and it was 5.5 mg/mL for DPPH, was 0.3 mg/mL for ABTS, and for FRAP, the value was EC50 118.2 mg/mL [54].

Similar studies were also conducted for the antioxidant potential of volatile oils and phenolic compounds of Ailanthus altissima. The antioxidant, antimicrobial, and plant toxicity properties from MEs of leaves and their residues were analyzed. The DPPH and FRAP assays were used to measure the antioxidant potentials of extracts. So, a significant support was provided by this study for high antioxidants and cytotoxic activities of species and so can be efficiently used as a natural herbicides and antioxidants source in the food and pharmaceutical industries [49,57]. The findings of our study are further supported by earlier investigations using the same methodologies and protocols [58]. This is also in accordance to studies performed earlier about antiradical activity of ingredient, phytol, that have also shown its promising antioxidant and antimicrobial activity [17].

Similar findings were also observed for the phenolic compounds and antioxidant activities were evaluated in the celery plant (Apium graveolens L.). It was observed that because of compounds such as caffeine, citric acid, coumaric acid, tannin, apigenin, luteolin, folic acid, saponin, and kaempferol, the celery possessed strong anti-oxidant properties to eliminate free radicals. It was suggested that with different compounds and different concentrations, celery can have different therapeutic values and can be effectively used in future studies focusing on other therapeutic and industrial properties [59,60].

Similar studies were also conducted for the antioxidant properties of Arctium lappa Linn for its active oxygen removal and free radical potential. Water produced the largest amount of extract that showed the strongest antioxidant activity, among all the solvents used in extraction [61].

This study is also in accordance with the previous study where 16 plants selected in Yemen were studied for their potential antioxidant properties and phytotoxic activities. Two different solvents (methanol and hot water) were used to extract the dried plant samples and to produce 34 raw extracts. Different types of compounds such as flavonoids, terpenoids, and others were found in phytochemical examination that may be responsible for the antioxidant and antimicrobial activities [62,63].

The potential antioxidant effects in shoot extracts of Asparagus cochininensis (Lour.) were studied in 80 Ming Kun mice, which were randomly divided into four groups (20/group). The plant extract was characterized by strong antioxidant ability in vivo and in vitro and can be used to reduce the radicals in the body and thus prevent aging [64].

Similar findings were also observed for the antioxidant capacities and phenolic compounds identification of Astragali complanate that was performed by ultrasonic extraction. The antioxidant capacity was measured by using DPPH test and the TPC was estimated through Folin–Ciocalteu assay [65].

So, findings of our study are strongly supported by earlier studies and are correlated to abovementioned studies. The presence of phenolic and flavonoid contents in our studied plant extracts are responsible for antioxidant and scavenging effects to free radicals and effective to treat the diseases caused due to accumulation of ROS and free radicals in the body.

4 Conclusion and recommendations

In this research the composition and antioxidant potentials of about 40 medicinal plants were analyzed by using various assays. The medicinal plants were investigated in this experiment along with their used parts and traditional medicinal uses. The free radical scavenging potential observed in these medicinal plant samples was because of the presence of some natural source such as phenol, flavonoid, or tannin contents. In this experiment, the free RSA of various medicinal plant samples was measured using different extracts depending upon the ability to eliminate the free radicals using synthetic DPPH. The reactivity of different compounds with the stable free radicals was because of the odd number of electrons present in them.

The TPCs, TFCs and potential Antioxidant activities of about 40 traditionally used medicinal plants from Pakistan were analyzed by using various assays in this research. The medicinal plants investigated in this experiment along with their used parts and traditional medicinal uses are given in Table 1. The total antioxidant potential of these medicinal plants was because of high amount of polyphenol and other phytochemical components found in them. The AEs showed almost similar and comparable results for all samples regarding free RSA compared to the ascorbic acid. These findings also indicated that all the tested medicinal plants samples are likely to possess significant levels of free RSA although comparatively less than standard ascorbic acid. So, this research suggested that all medicinal plants and particularly ES, EP, and EB possess a significant antioxidant potential and can be efficiently applied as an important antioxidant source for the treatment and inhibition of widely spreading oxidative stress related degenerative diseases like cancer, cardiovascular and inflammatory joint disorders, atherosclerosis, dementia, diabetes, asthma, and eyes related degenerative diseases.

This study showed that EEs of ES plant possessed the highest radical scavenging potential followed by the EP and EB, resulting in significant antioxidant potential for these traditionally used medicinal plants as compared to the highest antioxidant activity of standard ascorbic acid in all three assays (DPPH, ABTS, and FRAP). Different levels of scavenging activity were focused for all extracts in all used assays like DPPH, ABTS, and FRAP assays. It was also observed that the electron donating and/or free radical scavenging properties were responsible for the possible antioxidant potentials of these plant extracts which has been always concentration dependent. The phenol and flavonoid contents from these plants were observed as a potential source of natural antioxidants which can be efficiently used in the inhibition of oxidative stress associated diseases. This research may also lead to additional investigation of other specific compounds in various medicinal plants and their antioxidant potentials in vivo using various antioxidant assays. Thus, from these findings, it was concluded that the ES, EP, and EB medicinal plants are important source of natural antioxidants like phenols, flavonoids, tannins etc., and can be efficiently used in the treatment of various oxidative stress related diseases, most importantly cardiovascular disorders and cancers.

Acknowledgements

This research work is a part of the PhD thesis of Mr. Syed Anis Ali Jafri. The authors are highly thankful to Allama Iqbal Open University, Islamabad, Pakistan, for providing facilities to conduct this research. Parts of research facilities provided by International Islamic University, Islamabad (IIUI during this research are also acknowledged). The authors also acknowledged Dr Michael P. Timko at Department of Biology, University of Virginia, USA for giving an opportunity to conduct research in his Laboratory for this research, as well as the Higher Education Commission (HEC), Pakistan for their financial support (International Research Support Initiative Program) which gave me a golden opportunity to do this wonderful project at a well-reputed and well-equipped laboratory at UVA, USA.

  1. Author contributions: Study design was contributed by Anis Ali Syed and Dr Zafar Mahmood Khalid. Data analysis/interpretations were performed by Anis Ali, Dr NaqeebUllah Jogezai, Muhammad Zakryya Khan, and Dr Zafar Mahmood Khalid. Write up of manuscript was contributed by Anis Ali and Dr NaqeebUllah Jogezai. Critical revision/review was performed by Dr Zafar Mahmood Khalid, Dr. Muhammad Zakryya Khan, and Anis Ali Syed. The final draft was approved by all the authors.

  2. Conflict of interest: The authors state no conflict of interest.

  3. Ethical approval: Institutional Bioethics and Biosafety Committee (IBBC) approved the study through assigned number IIUI (BI&BT)/FBAS-IBBC-2016-06 (approved on September 16, 2016).

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Received: 2022-09-08
Revised: 2022-10-26
Accepted: 2022-10-30
Published Online: 2022-11-23

© 2022 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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