LC-QTOF-MS Characterization, Antioxidant Activity, and In Vitro Toxicity of Medicinal Plants from the Tri-Than-Thip Remedy

Background The world population has exhibited increased trust in folk medicine, including Thai folk medicine, for the treatment of various illnesses. However, the comparative antioxidant and cytoprotective activities against oxidative damage of Tri-Than-Thip (Tri-TT), a Thai folk remedy, have not been reported. Objectives The purpose of this study was to evaluate the antioxidant capacities of Tri-TT and its herbal constituents, Cassia fistula, Pithecellobium dulce, and Ficus benjamina. Methods Extracts were obtained from Tri-TT and its herbal constituents. The free radical scavenging activities, cytotoxicity, ferric-reducing antioxidant power (FRAP), metal chelating activities, total phenolic compound (TPC) contents, and total flavonoid (TF) contents of Tri-TT extract were investigated, and qualitative analysis of the chemical composition of Tri-TT extract was performed by LC-QTOF-MS. Results Tri-TT extract exhibited remarkable scavenging activities toward DPPH, ABTS, and superoxide anion radicals, with IC50 values of 0.081 ± 0.00, 0.021 ± 0.00, and 0.205 ± 0.057 mg/mL, respectively. The oxygen radical antioxidant capacity (ORAC) and FRAP values of Tri-TT were 6.499 ± 0.67 μM TE/g extract and 1,919.71 ± 63.14 mM FeSO4/mg sample, respectively. P. dulce had the highest scavenging activities and antioxidant capacity followed by C. fistula and F. benjamina. The TPC and TF content of Tri-TT extract were 287.87 mg equivalence/g extract and 225.62 mg catechin equivalent/g extract, respectively. The highest TPC was obtained from P. dulce, and TF content was observed in C. fistula. Using LC-QTOF-MS, a total of 25 compounds were tentatively identified in Tri-TT, including polyphenols such as luteolin, gallic acid, baicalein, apigenin, epicatechin, and ellagic acid. In addition, Tri-TT extract demonstrated nontoxicity (cell viability >90%) to Vero cells at the highest tested concentration of 80 μg/mL. Conclusion This study demonstrated that the Tri-TT remedy is a promising candidate as a natural source of antioxidant activity, suggesting that the polyphenol content of plants may contribute to antioxidant activities.


Introduction
Free radicals include reactive oxygen species (ROS) as well as reactive nitrogen species (RNS). Within cells, ROS function as secondary messengers in intracellular signaling cascades involved in human diseases such as gastric ulcers, hypertension, preeclampsia, neurological disorders, atherosclerosis, inflammatory conditions, certain cancers, and the process of aging [1]. An antioxidant can delay or relieve and inhibit oxidative damage to a target molecule and its ability is to trap free radicals by acting as a free radical scavenger, chelating, and other mechanisms to prevent lipid oxidation, and carbonyl scavengers as a way to avoid lipid oxidation consequences antioxidants. Antioxidants mitigate the effects of free radicals and protect cells from damage. Plants are good sources of antioxidants, and herbs have been used medicinally since ancient times based on reports of folk medicine. Natural sources of antioxidants have been of interest to researchers, as they are inexpensive and natural [2]. In ailand, many plants used in food and medicine have been reported as sources of natural antioxidants. From the types of remedies described in the ai Pharmaceutical Textbook, herbal formulations are commonly added to ai ancient household remedies [3]. ai remedies have shown antioxidant capacity when studied as an herbal formulation, and subsequent deeper studies on their antioxidant activities revealed good antioxidant activity and a potential for development as natural dietary supplements, including Triphala and Jatu-phala-Tiga (JPT). A previous study reported that JPT has strong antioxidant activities, particularly the water extract of the polyherbal tonic.
ese findings rationalize further investigation of JPT infusions as a promising agent for antiaging and oxidative stress prevention [4]. e overall report generated interest in ai drug formulas against free radicals from natural and Tri-an-ip (Tri-TT) remedies and is another interesting ai drug formula.
e Tri-TT remedy is a group of herbs from ai traditional medicine that have historically been used for nourishing breastfeeding mothers, healing wounds, and relieving diarrhea, and this remedy contains many parts of Cassia fistula, Pithecellobium dulce, and Ficus benjamina, which were reported to have good antioxidant capacity [5][6][7]. Although a previous study confirmed the good antioxidant ability of different parts of the herbal elements, there have been a few reports on the roots of the component herbs. erefore, this study aimed to evaluate the antioxidant capacity and cytotoxicity of the Tri-TT remedy and its three individual botanical constituents.

Preparation of the Tri-TT Remedy and ree Botanical
Extracts. One kilogram of dried root powders of P. dulce, C. fistula, and F. benjamina was mixed together in a 1 : 1:1 ratio to obtain Tri-TT. Briefly, both Tri-TT and individual plant ingredient powders were individually macerated with 1000 mL of ethanol at room temperature for 3 days, filtered through Whatman No. 1 filter paper, and dried using a vacuum rotary evaporator (Heidolph, Germany). All extracts were stored at 20°C until further experiments. e extraction yield of each plant extract was calculated as weight percent (% w/w) [4]: Extraction yield (%) � weight of the dry extract weight of the initial dry material × 100. (1)

Free Radical Scavenging
Activities. e antioxidant activity of the extracts was evaluated using the DPPH and ABTS assays described by Ghasemi Pirbalouti et al. [8]. For DPPH radical scavenging assays, 1 mL of sample extract was diluted in 2-fold increment to various concentrations (1.22 to 2500 μg/mL), and 20 μL of each sample at different concentrations was placed in a 96-well plate containing 80 μM DPPH in ethanol solution (180 μL). e 96-well plate was incubated in the dark for approximately 30 min at room temperature. e absorbance of the solution was read at 520 nm. Trolox was used as a positive control and used to construct a calibration curve, and half maximal inhibitory concentration (IC 50 ) values were calculated.
To generate ABTS + , 2 mM ABTS and 2.45 mM potassium persulfate were mixed together at a volume ratio of 1 : 1, and then the mixture was stored in the dark at room temperature for 16 h. e absorbance of the solution was maintained at 0.70 ± 0.05 at 734 nm. Sample extracts (10 μL) at various concentrations (between 1.22 and 2500 μg/mL) were added to a 96-well plate, followed by the addition of 1 mL of ABTS + solution and incubation for 6 min. e absorbance was read at 734 nm. Trolox was used as a positive control and used to construct a calibration curve. Finally, the scavenging activity is expressed as the concentration that caused 50% inhibition of ABTS + , as in the DPPH assay: scavenging activity (%) � Ab control − Abs sample × 100 Abs control . (2)

Metal Chelating
Activity. e ability of the polyherbal extracts to chelate ferrous ions was measured by a previously described colorimetric metal chelating activity (MCA) method [9]. Briefly, 0.1 mM FeSO 4 (0.2 mL) and 0.25 mM ferrozine (0.4 mL) were added to 0.2 mL of plant extract with a concentration range of 0.03 to 62.50 mg/mL. After incubation at room temperature for 10 min, an increase in the absorbance of the stable ferrous-ferrozine complex was detected at 562 nm. EDTA was used as a positive control. MCA was calculated using 2 Evidence-Based Complementary and Alternative Medicine metal chelating activity (%) � Ab control − Abs sample × 100 Abs control . (3)

Single Electron
Transfer-Based FRAP Assay. e ferricreducing antioxidant power (FRAP) activity of the plant extract was determined according to a previous study with minor modifications [10]. e FRAP working solution was freshly prepared by mixing 10 mL of 300 mM acetate buffer, 1 mL of 10 mM TPTZ solution, and 10 mL of 20 mM ferric chloride. Twenty milliliters of each extract was diluted in ethanol to various concentrations of 0.625 to 1.35 mg/mL, added to each well in a 96-well microtiter plate, and incubated at room temperature for 30 min in the dark. e absorbance of the solution was detected at 562 nm by the colored product from an intense blue color complex formed by the reduction of TPTZ to ferrous-TPTZ in the presence of electron donating antioxidants at low pH, and the reducing capacity is expressed as μM Fe 2 SO 4 /mg extract.

Superoxide Anion Radical Scavenging Activity.
Superoxide anions can be found in the process of energy production in cells in the body, which leads to lipid peroxidation. erefore, the superoxide anion scavenging ability and the capacity to reduce the rate of lipid peroxidation were evaluated. is activity was evaluated based on the reduction of NBT according to a previous report with minor modifications [11]. e riboflavin/methionine/illuminate system was used to generate superoxide anion radicals, which reduced NBT to form purple formazan (NBT 2+ ). e reaction mixture contained 100 μL of NBT (400 μg/mL) and 0.4 mL of a solution consisting of riboflavin (30 μg/mL), methionine (30 μg/mL), EDTA (20 μg/mL), and the plant extract at different concentrations (2-fold dilution; 4.88 to 156.25 μg/mL) diluted in 0.05 M PBS (pH 7.4). Photoinduced superoxide radicals were initiated with illumination by a fluorescent lamp (20 W) at 25°C for 25 min. After incubation, the absorbance was measured at 560 nm. e scavenging activity is expressed as the concentration that caused 50% inhibition of superoxide anion radicals (IC 50 ; mg/mL). Catechin was used as a reference compound.

Hydrogen Atom Transfer-Based Assay and Peroxyl Radical
Scavenging Assay (ORAC Assay). An oxygen radical antioxidant capacity (ORAC) assay with some modifications was used to evaluate the antioxidant activity of the extracts against peroxyl radicals generated from the thermal homolysis of AAPH [12]. e assay was carried out in blackwalled 96-well plates with PBS (pH 7.4). e standard was 25 mL of Trolox solution, and the samples were analyzed as 25 mL solutions at various concentrations of 0.2 to 100 μg/ mL (2-fold dilution). All experimental wells received 150 mL of sodium fluorescein (40 nM). After 30 min of incubation at 37°C, 25 μL of AAPH solution was added to the solution. e plate was placed into a microplate reader and analyzed with an excitation wavelength of 485 nm and emission wavelength of 535 nm, every 5 min for 90 min. e antioxidant capacity is expressed as Trolox equivalents per μg of extract (μM TE/μg E).
where R 1 is the fluorescence reading at the initiation of the reaction and R n is the last measurement.

Determination of TPC Content.
e total phenolic compound (TPC) content in plant extracts was determined according to a previous study with minor modifications [13]. Briefly, 120 μL of extract (2.5 mg/mL) was mixed with 1 mL of Folin-Ciocalteu reagent for 5 min. en, 1 mL of 20% w/v sodium carbonate solution was homogenously mixed and allowed to stand for 90 min in the dark at ambient temperature. en, the absorbance was measured at 725 nm. e TPC content was nitrotetrazolium determined with a calibrated curve of gallic acid and is expressed in terms of milligrams of gallic acid equivalents per gram of extract.

Determination of TF Content.
To determine the total flavonoid (TF) content, the plant extracts were analyzed based on a previous study with minor modifications [14]. Briefly, 50 μL of the plant extract (2.5 mg/mL) was combined with 300 μL of 5% (w/v) sodium nitrite, 300 μL of 10% (w/v) aluminum trichloride, and 4 mL of distilled water, after which the solution was homogenously mixed and incubated for 6 min at ambient temperature. e reaction was stopped with 2 mL of 1 M sodium hydroxide after 5 min. e absorbance was recorded at 510 nm.
e TF content is expressed as catechin equivalents per gram dry matter.

MTT Assay.
e plant extracts were tested for in vitro cytotoxicity using Vero cells by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay [15]. Cells (1 × 10 5 /well) were placed in 96-well plates and incubated at 37°C with 5% CO 2 for 24 h. en, different concentrations of samples at 5 to 80 μg/mL (2-fold dilution) were added and incubated for 24 h. Each sample was analyzed in triplicate. After sample incubation, 100 μL/well 5 mg/mL 0.5% MTT was added to the wells and incubated for 4 h. When purple precipitate was clearly visible under a microscope, 100 μL of DMSO was added, and the plate was shaken for 5 min. e absorbance of each well was measured at 540 nm with a microtiter plate reader using DMSO as a blank, and percentages of cell viability were calculated: percentage of cell viability � absorbance of extract treated wells absorbance of untreated wells × 100.

(6)
Evidence-Based Complementary and Alternative Medicine

Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF MS)
Conditions. e composition of Tri-TT extract was analyzed by UHPLC with a column from Zorbax Eclipse Plus C18 Rapid Resolution HD column (150 mm length * 2.1 mm inner diameter, particle size 1.8 μm), using a liquid chromatograph-quadrupole time-of-flight mass spectrometry (LC-QTOF MS) instrument (1290 Infinity II LC-6545 Quadrupole-TOF, Agilent Technologies, USA). e temperature was maintained at 40°C, and the injection volume was 2 μL. Elution was performed with the following 30 min, and mobile phase program was as follows: A: 0.1% formic acid in water, B: acetonitrile, and flow rate: 0.2 mL/min. LC-MS/MS analysis was performed in negative ion mode with a scanning range from m/z 100 to 1500 using a Dual AJS ESI ion source.

Statistical Analysis.
e data are presented as the mean value ± SD value. One-way ANOVA was conducted. Minitab software was used to calculate the significant differences (p < 0.05) between mean values.

Extract Yield.
Our results demonstrated that the yield of the ethanol extract of Tri-TT was 1.489%. e highest extraction yield was found for C. fistula (2.670%), followed by P. dulce and F. benjamina (Table 1).
is finding was consistent with that of previous studies, showing that alcohol extracts have a good percentage yield [16][17][18].

Free Radical Scavenging
Activities, DPPH Assay, and ABTS Assay. DPPH and ABTS assays are the most commonly used antioxidant assays and are spectrophotometric techniques based on quenching of stable-colored radicals [18]. e DPPH radical scavenging activity and ABTS radical content of the Tri-TT extract were 0.081 ± 0.00 mg/mL and 0.021 ± 0.00 mg/mL, respectively. For the herbal components, P. dulce had remarkable DPPH and ABTS free radical scavenging activities, with IC 50 values of 0.07 ± 0.00 and 0.10 ± 0.00 mg/mL, respectively (  [19]. In addition, the ethanolic F. benjamina leaf extract had an inhibition percentage of 44.87 and was usually nontoxic [20]. e DPPH and ABTS assays showed that the P. dulce extract had the strongest antioxidant activity, followed by C. fistula and F. benjamina (Table 2). e results of this study were similar to those of Selvakumar, who reported that the ethanol extract of flowers from these species had an IC 50 value of 85.20 μg/mL [21]. e 50% ethanol extract of C. fistula flowers exhibited an antioxidant activity of 47% at 4 μg/mL, and the methanol extract of F. benjamina leaves demonstrated higher antioxidant potential with a significant IC 50 value of 37.76 at 100 μg/mL [22,23]. Based on the antioxidant activity results from the above report, Tri-TT and its herbal components show good potential to control oxidative stress by the DPPH and ABTS assays, but both methods simulate free radical formation at the in vitro level and generate free radicals not found in the body. erefore, the NBT assay and ORAC assay are important techniques that will confirm the antioxidant capacity of an extract. e superoxide anion radical scavenging activity was estimated by the NBT method [24]. Superoxide radicals are known to be very harmful to cellular components as precursors of more ROS, and the ability of a plant extract to scavenge oxidation and mitigate biological damage is of interest [25]. e superoxide radical scavenging activity was defined as the concentration that produced 50% inhibition of superoxide anion radicals, as shown in Table 2.
e IC 50 value of Tri-TT extract was 20.05 ± 0.057 mg/mL, similar to that of the Triphala remedy, which is a popular tonic drug, and antioxidants from a traditional Ayurvedic herb remedy showed an IC 50 value of 42.95 ± 2.07 μg/mL [26]. e herbal component C. fistula showed the best antioxidant activity of 0.08 ± 0.02 mg/mL. As previously reported, the IC 50 values of ethanolic and water C. fistula fruit extracts were compared, and the C. fistula ethanol extract showed the best NO radical scavenging activity of 1,232.64 ± 1.73 μg mL [6].
According to the ORAC assay, which has been widely used to investigate the scavenging activities of several natural compounds, hydroxyl radicals are major active oxygen species causing lipid peroxidation and enormous biological damage [20,27]. e tested extracts scavenged peroxyl radicals in a concentration-dependent manner, as indicated by the inhibition of fluorescence decay. In this study, C. fistula had remarkable peroxyl radical scavenging properties with an ORAC value of 6.499 ± 0.67 μM TE/g extract (Figure 1). A previous report demonstrated that the IC 50 value of an ethanolic C. fistula extract from flowers showed a better hydroxyl radical scavenging activity (IC 50 ) of 609.03 ± 0.64 μg/mL than that of the aqueous C. fistula fruit extract, which exhibited a moderate activity of 1748.86 ± 0.65 μg/mL. e above activities of the C. fistula ethanol and water extracts clearly indicate strong concentration-dependent activity [6], and it may be concluded that C. fistula was the main component of Tri-TT involved in preventing and reducing intracellular ROS levels.

In Vitro Metal Chelating and FRAP Radical Scavenging
Activity. e FRAP assay is relatively simple and easy to conduct. e FRAP assay measures the potential of antioxidants to reduce the ferric tripyridyl triazine (Fe3+-TPTZ) complex and produce a blue ferrous complex [27]. e ability of a compound to reduce iron(III) to iron(II) generally depends on the presence of reductants [28], which exhibit antioxidative potential by quenching the free radical chain and donating a hydrogen atom [29,30]. e results (Table 2) indicated that Tri-TT had the highest MCA with an IC 50 value of 0.02 ± 0.00 mg/mL. e ion-chelating effect increased with increasing concentrations and stimulated a remarkable reducing power, with an FRAP value of 1,919.71 ± 63.14 mM FeSO 4 /mg (Table 3). e value in this study was higher than that reported in a previous study on traditional ai remedies. Ya-hom Intajak and Jatu-Phala-Tiga remedies had FRAP values of 0.93 ± 0.12 (mmol FeSO 4 / g) and 23.07 ± 1.84 (mM FeSO 4 /mg), respectively [4,31]. In addition, P. dulce possessed the highest reducing power, with an FRAP value of 3,335.38 ± 439.75 mMFeSO 4 /mg, whereas its MCA IC 50 value was 0.01 ± 0.00 mg/mL. Previous reports showed that the FRAP IC 50 value of a P. dulce methanol extract was 13.70 μg/mL [7] and that the extract possessed antioxidant, antibacterial, and antifungal activities [32].
us, it was found that Tri-TT extract was a good choice for antioxidant use according to the FRAP and MCA assay results.

TPC and TF Contents.
TPC and TF contents are indicators widely used to represent antioxidant activity. e high potential of phenolic and flavonoid compounds to scavenge radicals may be explained by their phenolic hydroxyl groups [33]. is study determined the TPC and TF contents of the Tri-TT remedy and individual botanical extracts. e TPC contents and TF contents were 287.87 ± 15.10 mg equivalence/g extract and 225.62 ± 2.056 mg catechin equivalent/g extract, respectively. In recent years, researchers have been interested in the search for new, natural antioxidants. Tri-TT has good antioxidant properties, similar to Tri-phal, Trichin-a-La-Ma-Ka, Tri-Ke-Son-Mat, Tri-Sa-Mo, Tri-Ti-Pa-Ya-Ros, and Tri-Su-Ra-Pon, and previous studies have described Tri-TT as the best antioxidant source among Triremedy groups from ai folk medicine [3,19]. In addition. Tri-TT has higher TPC and TF contents than Ya-hom Intajak and twenty polyherbal remedies either with rejuvenating effects or that are used as health-promoting tonics [31,34]. is study detected significant differences (p > 0.05) in the TPC contents of the three botanical extracts and showed that P. dulce had the highest TPC content, similar to the methanol extract of leaves and bark, which exhibited a TPC content of 0.084 ± 0.24 0.129 ± 0.11 μg/mL gallic acid equivalents [16]. e C. fistula extract exhibited the highest TF content, consistent with a previous study that reported TF contents of methanolic C. fistula leaf and stem extracts of 45.08 ± 1.37 and 4.17 ± 0.20 (quercetin equivalent) mg/g extracted compound, respectively [35]. In 2002, researchers reported the TPC and TF contents of several parts of C. fistula, including young leaves, old leaves, twigs, bark, flower buds, flowers, and pods [36], but this study showed that the roots have higher TPC and TF contents (Table 3). From all the experimental reports above, the results differed due to differences in the reactions and mechanisms of the methods used, which should be considered when comparing the antioxidant activities of plant extracts and compounds in herbs [37].

Cytotoxicity Analysis by the MTT Assay.
Vero cells, also known as African green monkey kidney cells, are recognized by the World Health Organization (WHO) and Chinese Pharmacopoeia in producing vaccines [38]. In the present Table 2: Metal chelating activity (MCA) and free radical scavenging capacities of different extracts of Tri-an-ip and three botanical extracts.

Extracts
MCA assay (IC 50 ; mg/mL) * Radical scavenging properties (IC 50 ; mg/mL) * * DPPH ABTS NBT Tri-TT 0.02 ± 0.00 0.08 ± 0.00 0.02 ± 0.00 0.205 ± 0.057 P. dulce 0.01 ± 0.00 b 0.07 ± 0.00 a 0.01 ± 0.00 a 0.33 ± 0.23 b C. fistula 0.02 ± 0.00 c 0.10 ± 0.00 a 0.01 ± 0.00 a 0.08 ± 0.02 a F. benjamina 0.01 ± 0.00 a 1.69 ± 1.79 b 0.06 ± 0.01 b 1.06 ± 0.25 c * IC 50 of EDTA (a positive control) was 0.01 ± 0.00 mg/mL. * * e IC 50 values of Trolox obtained from the DPPH, ABTS, and NBT assays were 0.025, 0.020, and 0.025 mg/mL, respectively. a-c Values in the same column with different superscripts are significantly different (p < 0.05). Extraction yield (g/100 g dried plant material).  Evidence-Based Complementary and Alternative Medicine 5 study, the cytotoxic effects of extracts of Tri-TT and its three botanical extracts were determined by the MTT assay. Figure 2 shows the viability of Vero cells after treatment with various concentrations of the extracts of Tri-TT and its three botanical extracts. e IC 50 value indicated the concentration that can inhibit 50% cell proliferation and showed that the extracts had cytotoxic ability. A relatively high IC 50 means that the compound is more nontoxic to the cell. Similar to a previous report about three-component herbal remedies, Vero cells were exposed to quercetin 3-O-rutinoside, kaempferol 3-Orutinoside, and kaempferol 3-O-robinobioside from the ethanol extract of F. Benjamina leaves, and antiviral activity was evaluated by the plaque assay and exhibited low toxicity [39]. In addition to normal cell testing, herbal components have also been found to inhibit cancer cells and have shown varying activities from toxic to safe. P. dulce bark and leaf lipophilic fractions were assessed for their cytotoxic activity using an MTT cell viability assay against two different cancer cell lines, namely, hepatocellular carcinoma and colon carcinoma cells, and the lipophilic extract was reported to possess significant cytotoxic activity in a colon carcinoma cancer line [40], and it was shown that there was cytotoxic potential of P. dulce  leaf extracts on breast cancer cells (MCF-7 cell line) at 400 mg/mL [41]. In addition, a C. fistula methanol extract reduced prostate human cancer cell line viability in a dosedependent manner in the MTT assay. e lowest viability of cancer cells was observed with 30 μg, at 5.06%, and the vehicle control showed 97.77% cell viability [42].

Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS)
Conditions. e qualitative analysis of compounds in the Tri-TT remedy infusion using LC-QTOF-MS in negative mode revealed chemical constituents and the known antioxidant-related constituents, with phenolic acids and flavonoids being the major components. Within the phenolic acid group, catechins such as epicatechin (RT � 6.76) and epigallocatechin (RT � 6.  [44]. e last herb component, P. dulce, contained many chemical groups with antioxidant effects that were similar to those of the two previous herbs, such as glycosylated compounds and flavonoids (quercetin-3-glucoside, luteolin-7-O-glucoside, and kaempferol-3-O-rhamnoside) as well as fatty acids (azelaic acid, etc.) [45]. e chemicals found in the herbal components and Tri-TT remedy belonged to similar chemical groups as the primary and secondary metabolite constituents of plant parts, with an emphasis on phenolic compounds and flavonoids with potent antioxidant activities ( Figure 3). is paper also assessed the antioxidant and free radical abilities of plant parts.

Conclusions
is study showed that the Tri-TT remedy and its three botanical constituents have complicated chemical constituents according to LC-QTOF-MS fingerprint analysis, as well as remarkable antioxidant, superoxide radical scavenging, hydroxyl radical scavenging, cytotoxic and protective activities against induced oxidative stress in the body. e ethanol extract of Tri-TT could be a significant material for the prevention of several diseases and could be Data Availability e data that support the findings of this study are available on request from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.