The DPPH* scavenging capacity of MPPE was evaluated by Brand-Williams et al. (42) with slight modifications. The control of DPPH was prepared by dissolving 24 μg of DPPH in 100 mL of methanol. 1 mL of different concentrations of MPPE (25, 100, 200, 400 μL mL^–1) were mixed with 3 mL of DPPH and kept for fifteen minutes in a dark place; then the absorbance was measured at 517 nm. The inhibition of DPPH* was calculated using the following equation.

The superoxide radical scavenging capacity of MPPE was determined using Nishikimi et al. (43) method with slight modification. 1 mL of MPPE concentrations (50–400 μg mL^–1) was mixed with 4.5 mL of Tris-HCl buffer solution (16 mM, pH 8.0), 0.5 mL of 250 μM nitro blue tetrazolium chloride (NBT) solution, 0.5 mL of 400 μM nicotinamide adenine dinucleotide (NADH) and 0.5 mL of 50 μM phenazine methosulfate (PMS). The mixture was incubated for 5 min at room temperature, and its absorbance was recorded at 560 nm compared to a blank sample. The superoxide radical scavenging capacity was calculated using the following formula.

With some modifications, the OH^⋅ scavenging capacity of MPPE was determined according to the method described by Smirnoff and Cumbes (44). 1 mL of MPPE concentrations (50–400 μg mL^–1) were mixed with 1 mL of FeSO₄, saffron (360 μg mL^–1), 1mL H₂O₂, and 0.5 mL of methanol-salicylic acid solution and given a standing time of 30 min at 37°C. The absorbance of the mixture was read at 520 nm against a blank. The reaction of plant extracts decreased the absorbance of bleached saffron, which specifies the decrease in hydroxyl radical scavenging capacity. % inhibition of OH^⋅ was calculated using the following equation:

NO^⋅ scavenging activity of MPPE was assessed by Griess reaction (45). 1 mL of MPPE concentrations were mixed with 2 mL of sodium nitroprusside, 0.5 mL of phosphate buffer saline, and incubated at 25°C for 3 h. After incubation, 1 mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) was added, and the mix was kept stable for 5 min to complete diazotization. Then 1 mL of naphthyl ethylene diamine dihydrochloride was added, then incubated at 25°C for 30 min. The sample color was read at 540 nm absorbance against a blank sample.

The MPPE inhibition ability of α-Glucosidase was assessed following by Bljajić et al. (46) method with slight modifications. 1 mL of MPPE concentrations mixed with α-glucosidase (0.2 U mL^–1 in 0.1 M phosphate buffer, pH 6.9), then incubated for 10 min at 37°C. After incubation, 50 μL p-nitrophenyl-α-D-glucopyranoside solution (1mM in the same buffer) was added to the previous mix and incubated for 5 min to release p-nitrophenol. Finally, the absorbance was read at 405 nm. % Inhibition of α-glucosidase was calculated using the following equation:

The inhibition ability of α-amylase was assessed by Apostolidis, Kwon (47). 0.5 mL of MPPE concentrations were added with standard (acarbose 1mg mL^–1 in water) and 500 μL of the α-amylase enzyme (0.8 U mL^–1 in 20 nM phosphate buffer, pH 6.8) of the porcine pancreas and incubated for 10 min at 25°C, followed by supplementation of 500 μL of soluble starch (0.5% solution in buffer) and standing for 10 min at 25°C. The solution was then added by 1 mL of 3.5-dinitro salicylic acid (96 mM), placed in boiling water for 5 min, and then cooled to room temperature. The plant mixture was diluted with 10 mL of distilled water, and its absorbance was read at 540 nm. The percentage of α-amylase inhibition was calculated using the following equation:

Plants contain phenolic compounds with antioxidant properties. Flavonoids are essential to these substances, possessing a wide range of chemical and biological activities, including radical scavenging properties (64). Therefore, the content of phenolic and flavonoid compounds of medicinal plant extracts was determined in the present study. The TPC of the dietary plants was found to be significantly different at (p < 0.05), as (Figure 1).

The C. sinensis has the highest TPC of 53.18 ± 3.18 mg GAE g^–1, followed by M. longifolia and C. citratus with 50.507 ± 2.96 and 43.433 ± 2.22 mg GAE g^–1, respectively. The results follow the study of Turkmen et al., who reported that C. sinensis and C. citratus give high polyphenol yields (65). However, our study’s TPC of wild peppermint was markedly lower than the previously reported TPC value of 71.43 mg GAE g^–1 (66). The lowest polyphenolic content concentration was 6.8267 1.02 mg GAE g^–1 in the F. vulgare 6.8267 ± 1.02 mg GAE g^–1, as depicted in Figure 1.

Higher total polyphenolic content in tea and wild peppermint leaves is attributed to a phenolic hydroxyl group bond in the flavan-3-ol structure with a strong reducing ability (67). These phenolic compounds may improve foods’ shelf life and antimicrobial properties besides retention of color, taste, and flavor. They are the defense system to neutralize reactive oxygen species (ROS), preventing molecular damage and harmful effects on microorganisms and insects and thus increasing their survival ability (68).

Flavonoids constitute an exceptional class of phenolic compounds with higher antioxidant activity than other phenolic acids. The carbon skeleton of diphenyl propane, which consists of two phenolic rings united with a heterocyclic carbon, is the reason for the strong functional properties of flavonoids (69).

In this colorimetric method, aluminum chloride reacts with adjacent keto or hydroxyl groups of flavones or flavonols to produce acid-stable complexes that quantify the content of flavonoid content of plants (70). The study’s total flavonoid content (TFC) of eight medicinal plants ranged from 3.29 ± 0.50 to 43.19 ± 3.63 mg QE g^–1, as shown in Figure 1. All plants exhibited significant flavonoid content; however, tea leaves had the highest flavonoid content, 43 mg QE g^–1, while the smallest flavonoid content, 3.29 mg QE g^–1, was obtained in the C. absus seed extract. Our results resembled the study of (44), who also reported a high level of flavonoids in tea leaves. Moreover, slight variations in results than previous studies may be due to the difference in sample collection, extraction method, or environmental conditions. The high flavonoid content in tea extract is due to many caffeoyl derivatives in tea leaves, such as caffeic acid, chlorogenic acid, and caffeoylquinic acid, which are significantly responsible for their antioxidant capacity (71). The total flavonoid contents of plants do not conflict with their total phenolic contents, so they can possess a high amount of both functional compounds simultaneously.

The number of flavonoids in M. longifolia was marginally higher than the number of flavonoids (21.0 ± 2.52 mg CE g^–1) in M. arvensis (17.75 ± 1.03 mg CE g^–1). Hajlaoui et al. reported markedly higher flavonoid contents in their study for M. pulegium and M. longifolia 63.93 and 33.83 mg CE g^–1, respectively; this probably depends on their habitats (72). Flavonoids help plants produce metabolizing enzymes such as quinone reductase, glutathione-S-transferase, and uridine 5-diphospho-glucuronyl transferase that detoxify carcinogens and help in their elimination from the body, which is similar to the mechanism of chemotherapy. Due to their antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic, and enzyme-modulatory functions, flavonoids are essential components in various nutraceutical, pharmaceutical, and cosmetic applications in today’s world (73).

The active groups in the MPPE were characterized by FTIR analysis to distinguish the nature of phytogenic compounds in MPPE (monomer or polymer). Figure 2 depicts eight plants’ FTIR absorption spectra of MPPE, i.e., Mentha longifolia, M. arvensis, Tinospora cordifolia, Cymbopogon citratus, Foeniculum vulgare, Cassia absus, Camellia sinensis, and Trachyspermum ammi. C. sinensis and M. arvensis. Absorbance bands detected in the range 500–4,000 cm^–1 in Figures 2A–H are about thirty-two bands seen in the eight plants; 3,955, 3,930, 3,810, 3,665, 3,625, 3,611, 3,358, 3,100, 3,000, 2,998, 2,921, 2,458, 2,845, 2,356, 2,210, 2,150, 1,985, 1,748, 1,747, 1,648, 1,585, 1,480, 1,398, 1,242, 1,215, 1,250, 999, 859, 712, 560, 511, 504, 475, 458 cm^–1. These bands correlated to the stretching vibrations of –C–O–C–, ether bonds, –C–O– terminal methyl, –C–C– groups, aromatic rings, alkyne bonds, and N–H, primary and secondary amines, and O–H groups, respectively.

These bands represent the vibrational stretching bands responsible for compounds such as terpenoids and flavonoids. The 1,640–1,550 cm^–1 absorption peak corresponding to proteins suggests that proteins interact with polyphenol monomers. Additionally, Absorption peaks observed between 3,950 and 3,200 cm^–1 imply alcohols (O–H bands) presumably originating from proteins and carbohydrates present in the sample, which bind with polyphenols and are responsible for stability and increasing the activity of produced polymers. These active groups prove that MPPE contains polyphenolic polymers conjugated with carbohydrates and proteins that enhance their bio-availability and bio-activity. Based on Figures 2A–C, C. sinensis polyphenolic extract exhibited 11 bands from 3,902 to 475 cm^–1, followed by M. longifolia extract with ten peaks from 3,665 to 487 cm^–1, then C. absus phenolic extract with nine peaks (3,900–458 cm^–1).

Muruganantham et al. (74) studied the FTIR spectrum of medicinal plants’ different parts, such as leaf, stem, and root. The false daisy (Eclipta alba and E. prostrate) extracts revealed the presence of bioactive groups such as COO, NH₂, NH, sulfur derivatives, carbohydrates, NO, chlorates, and carbohydrates. These active groups are responsible for the various medicinal properties. At the same time, the active groups of methanolic and aqueous extracts of Bauhinia racemosa leaf extracts revealed the presence of proteins, oils, lipids, phenolic compounds, flavonoids, saponins, tannins, and carbohydrates in FTIR spectra (75). Furthermore, Ragavendran et al. (76) detected COO, NH, NH-CO, polysaccharides, sulfur derivatives, halogen, and hydrocarbons responsible for the various therapeutic actions of Aerva lanata. Similarity, the same active groups were detected in black creeper (Ichnocarpus frutescens) ethanolic extract (77).

Also, Pednekar and Raman (78) found that the wild grape (Ampelocissus latifolia) methanolic leaf extract had transition metal carbonyl compounds and aliphatic fluoro compounds. The active compounds in Senna auriculata, Solanum torvum, Phyllanthus maderaspatensis, and Phyllanthus amarus leaves methanolic extracts act as natural antibiotics. The detected active groups in these plants indicate the existence of amino acids, amides, lipids, carbohydrates, glycogen, cellulose, starch, carotenoids, calotropin, and calotropogenin phosphates. The common active group in all extracts was OH, which can create hydrogen bonds and has the potential to act as an antibacterial agent at low IC50 (79).

It is well known that polymers such as proteins and carbohydrates can stabilize and enhance the bioavailability of medicinal medicines. Consequently, they can also be employed to improve the stability of polyphenols by polymerization or conjugation with polymers. The synthesized quercetin polymers exhibited antioxidant and antibacterial properties against three prevalent bacteria: B. subtilis, E. coli, and S. aureus. Sahiner re-established antioxidant and antibacterial activities by polymerizing rutin acid with the same approach (80, 81). In addition, Sahiner et al. (81) revealed that tannic polymers were antioxidant and anticancer properties against A549 malignant cells compared to cisplatin. In contrast, rutin and tannic acid polymers exhibited excellent drug-release properties.

Quercetin and kaempferol polymers are catalyzed with enzymes with enhanced antioxidants compared to their respective monomers, while oligomeric catechin from HRP exhibits antibacterial activities (82). The polymerization of tannic acid with laccase resulted in the formation of gallic dimers, gallic, partly gallic-glucose ester, and glucose. 68 Several phenolic acids have also been combined with chitosan enzymatically. Balasubramaniam, Murugan (83) used the polymerization of ferulic with chitosan by laccase to improve its antioxidant and antibacterial activity –comparable to the native chitosan activity. Božič, Štrancar (84) studied the effect of pH on phenolic-chitosan polymer activity. They found that pH 4.5 exhibited the most potent antioxidant activity and increased antibacterial activity against Listeria monocytogenes and Escherichia coli, compared to native chitosan. As we know that MPPE contains active groups related to polyphenols, proteins and carbohydrates detected by FTIR, therefore the UHPLC analysis was conducted to detect the phenolic compounds in MPPE in the following section:-

HPLC detected Twenty-five phenolic compounds in eight medicinal plant extracts (Table 3), categorized under three groups: phenolic acids, flavonoids, and alkaloids. Following FTIR results, four medicinal plants in descending order have the most detected phenolic compounds, i.e., C. sinensis > M. longifolia > C. absus > T. cordifolia. In the HPLC profile of C. sinensis, 22 compounds were found. The main compound was 3-Feroylquinic acid (1,302 mg L^–1), also found in M. longifolia and C. absus. C. sinensis has a higher phenolic content, i.e., vanillic acid, ferulic acid, chlorogenic acid, p-coumaric acid, gallic acid, rosmarinic acid, catechin, luteolin, 7-O-neohesperideside, quercetin 3,7-O-diglucoside, Hesperidin, Rutin, quercetin, and caffeine in the range of (560–780 mg L^–1). At the same time, other compounds are of medium content (99–312 mg L^–1). The phenolics in C. sinensis were 20–116% more than those in M. longifolia and C. absus. On the other hand, C. absus is characterized by sinapic acid (450.69 mg/L). At the same time, Carlinoside is the precious compound in C. citratus (525.98 mg L^–1). Additionally, genkwanin 5-O-glucoside is the main compound in M. longifolia. F. vulgare is rich in quercitron and quercetin dihydrate (803–900 mg L^–1). Also, T. cordifolia is rich in alkaloids (palmatine, caffeine, and berberine). T. ammi had a lower content of detected polyphenols than other medicinal plants.

About two-thirds of the phenolics in plant-based foods are flavonoids, which are very important to human health. For instance, caffeine releases fatty acids from stored body fat and inhibits fat absorption, resulting in weight loss. Quercetin’s antioxidant and anti-inflammatory properties may help decrease edema, eliminate cancer cells, regulate blood sugar, and prevent heart disease.

Nevertheless, phenolic acids represent one-third of polyphenols. Due to their antioxidant qualities and predicted health benefits, the scientific community’s interest in these simple phenolic acids continues to increase (23). Phenolic acids spread in many foods, i.e., fruits, cereals, vegetables, tea/coffee, spices, etc.; humans need about 25 mg daily (24).

Phenolic acids, which have a variety of biological uses, are the primary polyphenols generated by plants and serve as the progenitor of bioactive compounds often employed in the pharmaceutical, cosmetic, and food sectors. Phenolic acids are essential for absorbing nutrients, building parts, enzyme activity, protein structure, photosynthesis, and plant communication. The primary benefit of employing phenolic acids is their capacity to be metabolized by natural bacteria; hence, they provide an essential alternative to artificial chemicals that are equally damaging to the environment. While neochlorogenic acid acts as an antioxidant that scavenges potentially harmful free radicals and has been proven to inhibit bone resorption and promote bone formation (85), it also inhibits bone resorption. While chlorogenic acid decreases food cravings, reduces daily calorie intake, and induces body fat reduction through heat production (86), similar to statins used to treat cholesterol and triglyceride issues.

Abundant evidence confirms that a diet rich in vegetables and fruits improves health since they primarily function as antioxidants and prevent oxidative damage-induced illnesses such as stroke, coronary heart disease, and cancer (87–89). The average daily consumption of phenolic acids is 211 mg/day for hydroxy-cinnamic acid and 11 mg/day for hydroxybenzoic acid. At the same time, the population tested exhibited a range of 6 to 987 mg of phenolic acids (90). Exploring the probable significance of phenolic acids in plant life is only one of several research projects; their involvement in food quality is also a major field of study. They have improved foods’ sensorial, antioxidant, and nutritional properties (91).

Besides their roles as food preservatives, the phenolic acids affect fruit ripening and inhibit enzymatic browning (23, 92). Caffeic and ferulic acids (free/esterified) are the most prevalent phenolic acids in most fruits and cereal grains. Also, alkaloids are valuable therapeutic compounds that may be used to treat various disorders, such as malaria, diabetes, cancer, and cardiac dysfunction; for instance, palmatine and berberine play a crucial role in osteoclast apoptosis via the NOS system in osteoclasts (93). It has been claimed that palmatine possesses sedative and antioxidant properties. Palmatine enhances memory in mice by reducing brain acetylcholinesterase activity via the GABA-benzodiazepine pathway and its antioxidant properties (94). Inhibitors of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE 1), acetyl- and butyrylcholinesterases (AChE and BChE) have also been identified (94).

Herbal remedies are frequently utilized as alternatives to pharmaceuticals. According to traditional Indian literature, Tinospora cordifolia (Willd.) Miers (TC), a member of the Menispermaceae family, is a well-known traditional herb used to cure diabetes mellitus and various other diseases. Herbal anti-diabetic medications containing TC are commercially available. TC has been found as a potent CYP2C9 inhibitor, thereby increasing the likelihood of herb-drug interactions (HDI) with drugs metabolized via the CYP pathway. In vivo pharmacokinetic research (95). examined the pharmaco-kinetic HDI of TC extract with Gliclazide (GL) following oral co-administration in Wistar rats. In addition, Mentha arvensis and Mentha longifolia have substantial pharmacological uses as antioxidants, anti-fertility agents, anti-hepatoprotective agents, and antibacterial, antifungal, antimicrobial, anti-inflammatory, and anti-viral agents, among others. Due to phenolic and flavorful essential components, the wild mint essential oil has demonstrated remarkable biological applications (96).

The leaf of Cymbopogon citratus is also used orally with mate tea in Argentina for sore throat, empacho, and as an emetic (97). Gianeti, Mercurio (98) found that green tea-based cosmetics helped keep the skin moist and positively affected the skin’s microrelief. Traditional medicine also uses Cassia absus to treat bronchitis, asthma, cough, leucoderma, renal and hepatic illnesses, constipation, tumors, venereal ulcers, headaches, hemorrhoids, and wound healing. Preliminary research in vitro and in vivo has shown crucial scientific evidence for its use (30).

The antioxidant activity of MPPE was also examined as a free radical scavenging response using a DPPH assay. Free radicals play a significant role in chronic diseases such as cancer, diabetes, cardiovascular diseases, arthritis, and neural diseases (99). Antioxidants react with the DPPH substance, reducing its oxidative properties, proportional to the amount of hydroxyl (OH-) ions prevailing in the reaction mixture. Currently, the research on medicinal plants is more focused on discovering free radical-scavenging substances that translate into their antioxidative, antiradiation, anti-lipid oxidation, and immune modulation properties.

Eight medicinal plants investigated in the study demonstrated considerable antioxidant abilities (Figure 3A). The scavenging ability of MPPE against DPPH* was found to be 4.8 ± 0.92 to 85.21 ± 3.67% for different concentrations varying between 50 and 400 μg. The highest inhibition activity was represented by M. arvensis (20.97 ± 1.39 to 73.57 ± 2.29%, IC₅₀ value 119.9 ± 2.9 μg mL^–1), and C. sinensis (13.34 ± 1.68 to 71.68 ± 2.89%, IC₅₀ value 130.3 ± 2.7 μg mL^–1) at various concentrations. The lowest percentage of inhibition was found in F. vulgare and C. absus, which fluctuated between 4.8 ± 0.96 and 23.8 ± 1.8% with the IC₅₀ value (194 ± 2.7 μg mL^–1).

An earlier study by Zakaria et al. reported that the antioxidant activity of M. arvensis extract was 66.37% with a 100% methanol concentration (100). This variation in the antioxidant activity of M. arvensis may be due to the difference in the analytical method, which was also described by Safdar et al., that antioxidant activity decreases by increasing methanol purity (52). M. longifolia, C. citratus, T. cordifolia, and T. ammi exhibited significant antioxidant activity, which tallied the findings of earlier researchers with some differences (101). The present investigation also affirmed that the scavenging activity is directly proportional to the concentration of plant extracts. All plant samples showed maximum scavenging activity at a concentration of 400 μg, which progressively declined with decreasing concentration until the slightest scavenging activity was observed at 50 μg. Ashraf et al. documented a similar relationship in their experiment (70).

Phenols and flavonoids are well-known antioxidant compounds. In flavonoids, the presence of an O-dihydroxy structure on the B-ring that contains 2, 3 double bonds conjugated with a 4-oxo function on the C-ring; the 3-hydroxyl ions on the C-ring at positions C3 and C5 bonded with 4-production on the A and C-ring are responsible for a high free radical scavenging potential(102). However, in phenolic compounds acids such as 2,3-hydroxybenzoic acid, caftaric acid, gallic acid, and caffeic acid, a 3-hydroxyl structure intensified the antioxidant properties (1, 102).

Hydrogen radicals are considered among the most active and dangerous free radicals for human health among reactive oxygen species, causing cell injury at the site of inflammation in oxidative stress-induced diseases (103). The antioxidant system in the human body protects cells from free radicals once they are produced. Antioxidants are produced in the body by consuming a diet rich in phytochemicals which give rise to transferase, lipase, and protease enzymes involved in DNA repair. Moreover, catalase, Se-glutathione peroxidase, and superoxide dismutase are also produced, which play a major role in enzymatic defense, hydrolyzing superoxide, lipid peroxide, and hydrogen peroxide, thus preventing toxic radicals. Hydroxyl radical (OH) is the most powerful oxidant from exposure to the Fenton reagent (104).

All plants showed significant dose-dependent hydroxyl radical activity in the present study, as shown in Figure 3B. M. arvensis and C. citratus showed the highest hydroxyl scavenging activity compared to other vegetal extracts with IC₅₀ values of 133.3 ± 3.4 and 174.9 ± 3.1, respectively, at the concentration of 400 μg mL^–1 (Table 4), followed by C. sinensis, T. ammi, T. cordifolia, M. longifolia. C. absus and F. vulgare showed lower hydroxyl radical scavenging activity at all extract concentrations. The results are consistent with the previous literature reporting that the ethanolic extract of M. arvensis had good hydroxyl scavenging activity (70%) with a low IC₅₀ value of 52 μg mL^–1 (105). The variation in the result might be due to the plant sample, testing conditions, and the nature of the solvent used to prepare the extract. Furthermore, it is also documented that C. citratus (106) and C. sinensis (107) have potential hydroxyl radical activity due to the number of hydroxyl groups presents in the B ring of the flavonoid content of plant extracts.

The nitric oxide radical is the most toxic among all free radicals. It induces an inflammatory response, and the severity of damage increases with the presence of oxygen to produce peroxynitrite, which further harms living cells and their constituents. The generation of nitric oxide, followed by the combination of sodium nitroprusside and oxygen, forms nitrite. Plant extracts affect nitrite production by directly bonding with oxygen or reacting with NO. The NO scavenging activity of different plant extracts is shown in Figure 3C, indicating that the NO scavenging activity is directly proportional to the concentration of plant extracts. M. arvensis showed maximum NO scavenging activity (81.71 ± 1.1%) at all concentrations compared to the standard with IC₅₀ values of 132.8 ± 3 and 118.3 ± 3.5 μg mL^–1, respectively (Table 4). C. citratus and C. sinensis showed more than 50% NO scavenging activity. M. longifolia, T. cordifolia, T.ammi, and F. vulgare showed an average NO scavenging activity, while C absus showed the lowest NO scavenging activity with an IC₅₀ value of 177.3 ± 2.8 μg mL^–1.

Earlier literature showed that M. arvensis methanolic extracts have moderate NO scavenging activity at 71.28% at 250 μg mL^–1 concentration due to the chelating ability of phenolic compounds (108). Similarly, previous studies reported bioactive compounds in C. citratus, C. sinensis, M. longifolia, T. cordifolia, F. vulgare, and T.ammi significantly inhibited in vitro NO generation that could mitigate harmful effects of inflammatory conditions (108–112). NO scavenging activity of plants originates from tannins, phenols, flavonoids, and alkaloid compounds in plants, particularly catechins in C. sinensis (113). This study implies that indigenous plants can be influential in preventing diseases triggered by nitric oxide.

The superoxide radical is formed in living organisms during aerobic respiration, which causes the generation of hydrogen peroxide. The high oxidizing activity of H₂O₂ badly affects the cells and damages their lipid, protein, and nucleic acid components. Their Antioxidant activity estimates the therapeutic activity of plant extracts. All plant extracts (Figure 3D) demonstrated considerable superoxide radical scavenging activity, which ranged from 28.83 ± 0.7% to 90.15 ± 1.8% at 400 μg mL^–1 in a dose-dependent curve. M. arvensis and C. sinensis exhibited the highest inhibition of superoxide radicals with the IC₅₀ value 144.1 ± 3.3 and 148 ± 2.7 μg mL^–1, respectively. Extracts of M. longifolia, T. ammi, and C. citratus showed moderate superoxide scavenging activity. While F. vulgare, T. cordifolia, and C. absus showed the lowest percentage of superoxide inhibition with an IC₅₀ value of 188.9 ± 3.3 and 156.1 ± 2.8 μg mL^–1 (Table 4).

Our results follow Kumar et al., who reported that T. cordifolia has an average superoxide radical scavenging inhibition property comparable to the standard (114). The superoxide radical activity of the C. absus extract has not been reported earlier; however, methanolic extracts of C. absus showed lower DPPH inhibition percentage in the research of Ahmad et al. (30). Other research also corresponds to the present study’s findings that M. arvensis (115) and C. sinensis (116) have more vigorous superoxide radical scavenging activity than the standard. Previous investigations have also reported phenols, flavonoids, glycosides, phytochemicals (isoorientin, orientin, and isoscoperin), and sugar moieties at positions C-6, C-7, and C-8 as powerful sources of superoxide radicals in plants (117). Similarly, phytochemicals, including alkaloids, flavonoids, methoxy residue, disulfide bond, and hydroxyl groups in M. arvensis and M. longifolia and quercetin, epicatechin gallate, gallic acid, and epigallocatechin gallate in C. sinensis attributes to its higher superoxide anion scavenging potential (118).

Plant extracts can act as antidiabetic agents by their influence on enzymes crucial in carbohydrate digestion or by restricting the hydrolysis of carbohydrates to glucose and other simple sugar compounds. α-glucosidase enzymes convert carbohydrates to glucose in the small intestine after partial breakdown by an α-amylase enzyme from saliva and pancreatic juice. Plant substances are increasingly used to develop natural-origin medicines for curing diabetes, hyperglycemia, and obesity by inhibiting α-glucosidase enzymes. The α-glucosidase inhibition activity of eight MPPEs is demonstrated in Figure 4A. M. arvensis, C. sinensis, and C. citratus had the strongest inhibitory activity, which was even greater than the control Acarbose at 400 and 200 μg mL ^–1 with the IC₅₀ value 125.7 ± 2.4 and 124 ± 2.3 μg mL^–1 (Table 4). F. vulgare, T.ammi, and M. longifolia were more than 50% inhibitive to α-glucosidase at (400 μg mL ^–1) concentration. At the same time, T. cordifolia and C. absus recorded minor α-glucosidase inhibition activity at all concentrations used in the research.

The current study showed weaker inhibitory activity of methanolic extracts of C. absus than a previous contrasting study which reported that hydroalcoholic extracts of C. absus had potent inhibitory activity against α-glucosidase (119). However, The results of the present investigation are per the majority of earlier research, which reported that C. citratus (115), M. arvensis (120), F. vulgare (121) had strong α-glucosidase inhibition activity due to their high phytochemical contents, such as alkaloids, flavonoids, and saponins that bind other non-competitive enzymes (119).

In vivo, the antidiabetic and antihyperglycemic activity of streptozotocin in diabetic rats was caused by C-glycosyl flavones, iso-orientin, and chlorogenic acid naturally occurring in lemongrass extracts (117). Whereas the strong enzyme inhibition activity of F. vulgare extracts may be attributed to the presence of caffeic acid. An in vitro study showed that caffeic acid derivatives (122) and 3,4-di-O-caffeyolquinic acid (123) could inhibit the α-glucosidase activity and angiotensin-converting enzymes, which can reduce the risk of type-2 diabetes mellitus. The main mechanisms of caffeic acid, which regulates blood glucose levels, are stimulating insulin production, triggering glucose uptake by adipocytes in the liver, and reducing the glucose level in the bloodstream. In general, phenolic compounds can alter carbohydrate metabolism by their binding affinity towards hydrolyzing enzymes (121). Present study confirms the antidiabetic activity of selected plant extracts which indicates their suitability for use in therapeutic medicine.

An effective strategy is required to tackle or manage diabetes mellitus by controlling or altering the activity of α-amylase, which prevents or delays the digestion of carbohydrates after their ingestion, eventually modulating the circulatory glucose level (124). Figure 3B depicts the antidiabetic potential of medicinal plant extracts as measured by their α-amylase inhibitory activity along with IC₅₀ values (Figure 4B and Table 4). The percentage of inhibition of different plant extracts at a concentration of 400 μg mL ^–1 against α-amylase is expressed in the following order M. arvensis > F. vulgare > T.ammi > M. longifolia > C. absus > T. cordifolia > C. citratus > C. sinensis. All vegetal extracts impaired α-amylase activity to appreciable levels. The extracts of M. arvensis, F. vulgare, and T.ammi showed the strongest inhibitory activity of α-amylase (IC₅₀ 109 ± 7.1, 117 ± 6.3, and 128 ± 1.1 μg mL ^–1) as compared to the extracts of other plants, while C. sinensis had the weakest inhibitory rate (27.69 ± 1%) with an IC₅₀ value of 113 ± 1.2 μg mL ^–1.

Similar results were reported by Wang et al., that 75% of ethanolic extracts of C. sinensis showed mild inhibition response against the α-amylase activity (125). Furthermore, the remarkable α-amylase inhibition activity of M. arvensis, F. vulgare, and some other Bangladeshi and Indian medicinal plants was also reported in some other studies (120, 121, 124). Another research highlighted that the α-amylase inhibition activity of medicinal plants could be associated with their phenolic compounds, which largely reduces the risk of developing type-2 diabetes among people (126, 127). Therefore, it can be inferred that the dietary plants utilized in the study can be used as medicine or functional food for treating or preventing diabetes mellitus along with healthy lifestyle changes.

Cell viability tested at maximum vegetal extract concentration (400 μg mL ^–1) showed the least amount of living Caco-2 cells in the reaction mixture of T. ammi, C. sinensis, C. citratus, and M. arvensis 15.7 ± 0.4, 39.54 ± 1, 36.29 ± 0.9 and 41.74 ± 1% respectively. Whereas C. absus, F. vulgare, M. longifolia, and T. cordifolia showed the highest cell viability percentage (61.32 ± 0.5, 58.99 ± 0.8, 50.27 ± 0.9, and 48.7 ± 0.8) at 400 μg mL ^–1 concentration as indicated by the initial values. T. ammi extracts exhibited maximum cytotoxicity at the lowest extract concentrations of 25–50 μg mL ^–1. The results are consistent with the reported cytotoxicity of the T. ammi extract against the colon carcinoma cell line HCT116 (128) and breast cancer cell line MCF-7 (129).

The cytotoxicity of the T. ammi extract is due to the presence of thymol compounds. Previous investigations documented that thymol has potential cytotoxicity against various cell lines such as monocyte leukemia THP-1 (130), carcinoma HEp-2 (131), and colorectal adenocarcinoma Caco-2 cell line (132). The cell viability rates determined the IC₅₀ value of plant extracts at different concentrations. In the Caco-2 cell line, death of more than 50% of the cells occurred from 104 ± 4.6, 111 ± 7.2, and 143 ± 2.0μg mL^–1 strengths of M. longifolia, M. arvensis, and T. ammi extracts (Table 4 and Figure 5). The results of the present investigation confirm that therapeutic plant extracts can play a significant role in cancer therapy due to their distinct cytotoxic abilities and rich phytochemical composition. Alami et al. (133) recorded 103 plant species belonging to 47 botanical families used by Moroccans to treat cancer. Aristolochia fontanesii Boiss. & Reut, Marrubium vulgare L., and Allium sativum L. are the most preferred species in Morocco. Medicinal plants used for cancer treatment were classified into four groups: 48 species were used traditionally as anticancer (group a), 41 species were pharmacologically investigated for their anticancer activities (group b), 32 plants with bioactive compounds tested against cancer (group c), and eight plants were clinically investigated for their anticancer effects (group d). Out of 82 plant extracts pharmacologically tested (from plants of group b), only 24 ones show a significant cytotoxic effect. A total of seventy-seven compounds are isolated from plants of group (c). However, only six were clinically evaluated, and most exhibited a beneficial effect on cancerous patients with few side effects.