Phytochemical Profiling and Antioxidant Activities of the Most Favored Ready-to-Use Thai Curries, Pad-Ka-Proa (Spicy Basil Leaves) and Massaman

Food is one of the factors with the highest impact on human health. Today, attention is paid not only to food properties such as energy provision and palatability but also to functional aspects including phytochemical, antioxidant properties, etc. Massaman and spicy basil leaf curries are famous Thai food dishes with a good harmony of flavor and taste, derived from multiple herbs and spices, including galangal rhizomes, chili pods, garlic bulbs, peppers, shallots, and coriander seeds, that provide an array of health benefits. The characterization of phytochemicals detected by LC-ESI-QTOF-MS/MS identified 99 components (Masaman) and 62 components (spicy basil leaf curry) such as quininic acid, hydroxycinnamic acid, luteolin, kaempferol, catechin, eugenol, betulinic acid, and gingerol. The cynaroside and luteolin-7-O-glucoside found in spicy basil leaf curry play a key role in antioxidant activities and were found at a significantly higher concentration than in Massaman curry. Phenolic and flavonoid compounds generally exhibit a bitter and astringent taste, but all the panelists scored both curries higher than 7 out of 9, confirming their acceptable flavor. Results suggest that the Massaman and spicy basil leaves contain various phytochemicals at different levels and may be further used as functional ingredients and nutraceutical products.


Introduction
Recently, not only increasing pollution caused by industrial development but also lifestyle, eating and exercising habits, workload, and less relaxing life conditions have been impacting human health in various ways [1], leading to increased stress and the overproduction of free radicals.Unbalanced free radicles produced in the body as a result of stress, anabolism, and catabolism cause macromolecular changes in proteins, fats, carbohydrates, and DNA, increasing the risk of non-communicable diseases and conditions such as stroke, high blood pressure, diabetes, and cancer [2].Therefore, maintaining balanced food compositions with high levels of antioxidants is beneficial for general wellness.Thai curries and related products, such as fried curry dumplings, steamed buns, and instant stir-fried curries, are an integral part of the historical, cultural, and ethnic background of local Thais and those who have Chinese, Indonesian, and Indian descent.Historically, Thais have used herbs and spices and even curry paste containing galangal rhizomes, chili pods, garlic bulbs, peppers, shallots, and coriander seeds, with health benefits linked to their anti-aging, anti-inflammation, anti-cancer, and antioxidation properties [3][4][5], to treat or relieve common complaints such as stomachache, flu, and acne, following ancestral traditions [6].Scientific data have shown that phenolic compounds, especially ferulic acid and flavonoids, are antioxidant agents [7,8] that inhibit reactive oxygen species (ROS) and related molecules such as nitric oxide, nitric oxide synthase, and xanthine oxidase, as well as toxic agents produced from free radicals [9].Allicin derived from crushed or damaged garlic has anti-cancer properties [10], while eugenol plays a key role in antioxidant and antimicrobial activity [11], and the procyanidin oligomer obtained from cinnamon exhibits anti-diabetes properties [12].
Thailand, known as "The Land of Smiles", is globally renowned for its mouth-watering culinary dishes [6], with Massaman curry regularly recognized as one of the most delicious dishes in the world [13].Massaman curry combines the sweetness, saltiness, and creaminess of coconut milk with the slight sourness of roasted groundnuts and the distinctive aroma of more than 15 types of herbs and spices such as shallot, dried finger chili, galangal, cumin, lemongrass, clove, kefir lime fruit, cinnamon bark, coriander root, cardamom, and the turmeric rhizome.Massaman curry paste is claimed to be the richest combination of raw materials [14].Four large Thai companies (Mae-Ploy, Namjai, Aroy-D, and Maesri) produce and export seasoning and famous recipe blends such as green curry, red curry, Tom-Yum, Pad-Thai, and Massaman.Thailand is the second largest global curry paste exporter, following India [15].
Spicy basil or Pad-ka-proa is consumed throughout the country by both Thais and tourists.This dish can be cooked quickly, within 5 min, and is combined with various hot spices to suit people of all ages, from children to adults.The taste is mildly salty, sweet, and hot, with the aromas of garlic, pepper, and, particularly, holy basil leaves, which are also used for the treatment of various conditions in Ayurveda medicine due to their biological anti-inflammatory, anti-diabetic, and anti-enteritic activities that ward off the symptoms of malaria and ameliorate heart disease [16].Basil is called the "Mother Medicine of Nature" or "The Queen of Herbs" [17].The main phytochemicals contained in basil leaves are phenolic acids, flavonoids, propenyl phenol, and terpenoids, particularly, ursolic acid, which is often used as a biomarker [18].Ursolic acid exhibits anti-inflammatory, antioxidant, anti-apoptotic, and anti-carcinogenic effects [19].
While there are interesting and convincing studies promoting the consumption of plant materials to enhance human health through the bioactive compounds they contain, a systemic database of the bioactive molecules in Massaman and Pad-ka-proa has not been reported.Therefore, this research aimed to identify and characterize the phenolic compounds contained in Massaman and Pad-ka-proa by LC-ESI-QTOF-MS/MS.These results will be useful for the functional food industry, medicinal applications, and healthconscious customers.
Methanol, acetonitrile, and acetic acid were HPLC-grade and purchased from RCI Labscan, Bangkok, Thailand.

Preparation of Curry Powders
Massaman curry and spicy basil leaf curry were made by mixing the ingredients listed in Table 1.All ingredients were purchased from a local market in Hat Yai, Songkhla province, Thailand.After grading and washing with a 100 ppm chlorine solution at a ratio of 1:3 (ingredient: solution) for 15 min, the fresh raw materials were rinsed with tap water 2 times to remove excess chlorine residue to lower than 1 ppm.The cleaned ingredients of each curry recipe were blended to a paste before drying using a drum dryer (DD-D12L16, Chareontut, Samutprakarn, Thailand) at 110-120 • C for 2-3 min to obtain dried curry powder with a moisture content of 4-6%.The spicy basil leaf curry was dried in a rotary hot air oven (HS-169, AT Packing, Nonthaburi, Thailand) at 70 • C for 16-18 h to obtain a moisture content of 4-6%.Each dried sample was ground with a high-speed mixer (WF-20B, Thaigrinder, Thailand) until the powder size was lower than 60 mesh (250 µm) (Laboratory test sieve, Endecotts, UK).Flow charts showing an overview of making the Massaman and spicy basil leaf curry powders are presented in Figures 1 and 2, respectively.

Sample Preparation and Extraction
Each curry powder sample was extracted following the method described by Srisook et al. [20] with some modifications, including using 80% ethanol and 24 h instead of 95% and 5 days.All powders from each curry sample were extracted with 80% ethanol at a ratio of 1:10 (curry powder: 80% ethanol) and stirred in the dark at 25 • C for 24 h.The mixtures were then separated by vacuum suction using a Buchner funnel before centrifugation (CR22GIII, Hitachi, Japan) at 4 • C for 20 min at 7100× g.The ethanol was completely removed using an evaporator (N-1000, EYELA, Rikakikai, Japan) before freeze-drying (KD-330cr, I.T.C., Bangkok, Thailand) at −25 • C until reaching a moisture content of 6-8%.Each curry powder sample was extracted following the method described by Srisook et al. [20] with some modifications, including using 80% ethanol and 24 h instead of 95%   Each curry powder sample was extracted following the method described by Srisook et al. [20] with some modifications, including using 80% ethanol and 24 h instead of 95%

Total Phenolic Content (TPC) Determination
(TPC) was determined using the method described by Singleton and Rossi [21] with some modifications, including using a well plate instead of a test tube.Briefly, 20 µL of the sample extract was added to a 96-well plate, followed by 100 µL of 10% Folin reagent (v/v).After incubation in the dark at 30 • C for 6 min, 7.5% Na 2 CO 3 (anhydrous) (w/v) was added, and the mixture was incubated for another 30 min.The absorbance was measured at 765 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Singapore, Singapore).TPC was measured using gallic acid as the standard agent at concentrations of 0-100 µg/mL with R 2 = 0.999.The standard curve is shown in Figure S1.

Total Flavonoid Content (TFC) Determination
(TFC) was determined using the method described by Chandra et al. [22] with some modifications, including using a well plate instead of a test tube.Briefly, 100 µL of the sample extract was mixed with 100 µL of 2% AlCl 3 •6H 2 O (w/v) and incubated in the dark at 30 • C for 60 min.The absorbance of the mixture was then measured at 420 nm by a microplate reader (Varioskan LUX, Thermo Scientific, Singapore) using rutin as the standard agent at concentrations of 0-80 µg/mL with R 2 = 0.998.The standard curve is shown in Figure S2.

DPPH Radical Scavenging Activity
2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity was determined following the method of Ding et al. [23].First, 100 µL of the sample extract was mixed with 100 µL of 0.2 mM DPPH in 95% ethanol.The mixture was then incubated in the dark for 30 min at 30 • C. Finally, the absorbance was measured at 517 nm by a microplate reader (Varioskan LUX, Thermo Scientific, Singapore, Singapore) using Trolox as the standard agent at concentrations of 0-12 µg/mL with R 2 = 0.998.The standard curve is shown in Figure S3.

ABTS Radical Scavenging Activity
The 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay was determined as described by Arnao et al. [24].The ABTS radical was generated by incubating 7.4 mM ABTS solution in the dark at 30 • C for 12 h.The radical solution was then diluted to obtain an absorbance of 1.1 ± 0.02 at 734 nm.Then, 20 µL of the sample extract was mixed with 280 µL of the radical solution and kept in the dark for 2 h at 30 • C. The absorbance of the mixture was measured at 734 nm by a microplate reader (Varioskan LUX, Thermo Scientific, Singapore) using Trolox as the standard agent at concentrations of 0-110 µg/mL with R 2 = 0.999.The standard curve is shown in Figure S4.

Ferric Reducing Antioxidant Power (FRAP) Assay
The ferric-reducing antioxidant power (FRAP) assay was determined following the method of Benzie and Strain [25].A freshly prepared FRAP solution containing 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mM HCl and 20 mM FeCl 3 •6H 2 O (ratio 10:1:1) was warmed at 37 • C for 30 min.Then, 15 µL of the sample extract was mixed with 285 µL of the FRAP solution and incubated for 30 min at 37 • C. The absorbance of the mixture was measured at 593 nm by a microplate reader (Varioskan LUX, Thermo Scientific, Singapore) using Trolox as the standard agent at concentrations of 0-100 µg/mL with R 2 = 0.999.The standard curve is shown in Figure S5.

Oxygen Radical Absorbance Capacity (ORAC) Determination
The ORAC (oxygen radical absorbance capacity) was determined following the method of Huang et al. [26].A sample solution of 25 µL was mixed with 150 µL of fluorescein solution 81.6 nM.The mixture was incubated at 37 • C for 15 min and then 25 µL of AAPH 153 mM was added.Fluorescence (excitation wavelength at 485 nm and emission wavelength at 530 nm) was read at 2 min time intervals for 90 min by a microplate reader (Varioskan LUX, Thermo Scientific, Singapore) using Trolox as the standard agent at concentrations 0-170 µg/mL with R 2 = 0.998.The standard curve is shown in Figure S6.
The antioxidation activities were previously studied [27], and the details are shown in Table 2.

Characterization of Phenolic Compound Profiles by LC-ESI-QTOF-MS/MS
All extract samples were examined for phenolic and flavonoid compounds following the modified method of Araujo et al. [28] and profiling by LC-ESI-QTOF-MS/MS-positive and -negative electrospray ionization at the University Center Laboratory with ISO accreditation.Each sample (2 µL) was injected into a Zorbax Eclipse Plus C18 Rapid Resolution HD column (150 mm length × 2.1 mm inner diameter) and performed at 25 • C. The mobile phases were solvent A, a mixture of methanol: acetonitrile: water: acetic acid (10:5:85:1, v/v), and solvent B, a mixture of methanol: acetonitrile: acetic acid (60:40:1, v/v), with a flow rate of 0.2 mL/min.Wavelengths (λ) at 230, 257, 280, 325, 368, and 450 nm were used to detect the compounds in the sample.Mass spectrometry was run on a Dual AJS ESI for ion source with an MSQ-TOF (model: G6545A, Agilent, Beijing, China) and mass spectrometer range of 100-1500 m/z.Electrospray ionization (ESI) was performed when the gas temperature reached 325 • C with a flow rate of 13 L/min and a nebulizer pressure set at 35 psig for the introduction source.Data were analyzed by MassHunter WorkStation Software Quantitative Analysis Navigator V8 and WorkStation Software Qualitative Analysis Workflows V8 with database MassHunter METLIN PCD.An overview diagram of the characterization of the phenolic profiles by LC-ESI-QTOF-MS/MS is presented in Figure S7.

Sensory Evaluation
Massaman curry powder and spicy basil leaf curry powder were cooked with the ingredients shown in Table 3 and then presented to 50 panelists for sensory evaluation using a 9-point Hedonic scale following the method of Wichchukit and O'Hahony [29] with ethical approval no.PSU-HREC-2023-008-1-1.Appearance, color, odor, taste, texture, and overall liking attributes were scored.Plain rice was served with the curry dishes, and mouthfeel was rinsed using normal water and fresh cucumber.This study complied with the Declaration of Helsinki and was approved by the human research ethics committee of Prince of Songkla University (PSU-HREC-2023-008-1-1).

Statistical Analysis
The experiment was set up using a completely randomized design (CRD).All quality parameters were performed with eight repetitions.Differences in mean values and variations were tested using ANOVA with Tukey's test (p < 0.05).Statistical analysis of the data was carried out using SPSS statistics software version 22 (IBM, New York, NY, USA).

Total Phenolic and Flavonoid Contents and Antioxidant Activities
Phenolic compounds are important phytochemical constituents showing redox properties responsible for antioxidant activity with diverse benefits in the human diet [30].The results indicated that the TPC of spicy basil leaf curry was significantly 6-fold higher than Massaman curry (p < 0.05) (Table 2), while the TFC value of Massaman curry was similar to spicy basil leaf curry.Lu et al. [31] studied the antioxidant capacity and contained phenolic compounds of 18 spices in curry powder, including star anise, fennel, cumin, angelica dahurica root, green prickleyash, Sichuan pepper, dried tangerine peel, white pepper, nutmeg, galangal, dried ginger, tsaoko amomum fruit, villous amomum fruit, dried chili pepper, bay leaves, cinnamon, and mustard, and found that total flavonoids were higher than total phenolics.Total flavonoids were reported as major constituents found in cardamom, clove, cinnamon, black pepper, cumin seed, fennel seed, red chili, coriander, and ginger [32].Akullo et al. [33] stated that garlic bulbs extracted with ethanol provided higher TPC than TFC, explaining the reason why spicy basil leaf curry contained higher TPC than Massaman curry, even when the spicy basil leaf curry was mainly garlic (45%) and dried basil leaves (35%).Chaudhary et al. [34] reported that using methanol as the solvent for basil leaf extraction provided higher TFC than TPC.Solvent type plays a key role in extraction due to polarity and leads to various phytochemicals.
In this study, antioxidant DPPH and ABTS activities were assessed using Trolox as a standard and determined mainly via hydrogen and electron transfer [35,36].The FRAP assay was determined by the electron transfer ability of antioxidants by reducing the colorless complex ferric ion (Fe 3+ ) to the blue ferrous complex (Fe 2+ ) [36], while the ORAC assay measured the ability to transfer hydrogen atoms to RO•/ROO• radicals generated by AAPH thermolysis in the presence of a probe that quantified antioxidant oxidation [37].The results showed that the spicy basil leaf curry expressed higher antioxidant activity values than Massaman curry in all assays (p < 0.05) (Table 2).Dat-arun et al. [8] reported that fresh Massaman curry paste provided DPPH with 11.81 ± 0.06 mg GAE/100 g crude extract and FRAP with 0.311 ± 0.006 mg TE/100 g crude extract.However, to date, no scientific information on spicy basil leaf curry is available.Juntachote and Berghofer [38] found that basil leaves (Ocimum sanctum Linn) recorded DPPH with IC 50 20.6 µg extract/mL.Pearson's correlations of the TPC, TFC, DPPH, ABTS, FRAP, and ORAC assays were significantly correlated at p < 0.01 with r > 0.974, while TFC was significantly correlated with ORAC at p < 0.05 with r = 0.898, as shown in Table S1.This result suggests that the phytochemicals contained in both curries effectively inhibited the peroxyl radical generated in the human body [39].Schaich et al. [40] found that DPPH, ABTS, and the ORAC assay had a good relationship and reacted with radicals through a similar mechanism with some modifications.For instance, antioxidants react with DPPH by transferring electrons and/or giving a hydrogen atom back to active molecules or radicals [41].ABTS radicals were in an inactive form by taking electrons and hydrogen atoms from the antioxidant, while the ORAC assay evaluated the ability of an antioxidant to quench radicals by hydrogen atom transfers independently of electron transfers.The FRAP mechanism is based on electron transfer rather than hydrogen atom transfer [42].

Characterization of Polyphenols in Spicy Basil Leaf Curry and Massaman Curry Using LC-ESI-QTOF-MS/MS
Qualitative identification of the polyphenols in the spicy basil leaf and Massaman curries was conducted by LC-ESI-QTOF-MS/MS in both the negative and positive ionization modes (Tables 4 and 5), and the contained phytochemicals and their biological activities are listed in Table 6.The major constituents found in spicy basil leaf curry were flavonoids and derivatives, comprising 17 compounds, 14 terpenes, 10 phenolics and derivatives, and 21 other types including quinones, alkaloids, chromones, capsaicinoids, flavonoidal alkaloids, and steroidal saponins (Table 4).The main flavonoid derivative was identified as 6-C-beta-D-Xylopyranosyl-8-C-alpha-L-arabinopyranosylapigenin (with abundance: 43.35 × 10 5 ), which agreed with the finding of various herbs and spices [43].Apigenin is a flavonoid compound that effectively downregulates the expression and secretion of pro-inflammatory cytokines through the IL-23/IL-17/IL-22 axes [44].Apiin was the second-most abundant flavonoid (abundance: 40.55 × 10 5 ), which is mainly found in celery leaves, parsley leaves, and bell peppers.In this experiment, apiin was found in fresh green and red chili, which are in the same genus as bell pepper (Capsicum annuum L.) [45].Adem et al. [46] reported that apiin, hesperidin, rutin, and diosmin were the most effective agents against SARS-CoV-2 Mpro when compared with Nelfinavir (positive control).Cynaroside A (abundance: 31.11× 10 5 ) was found in coriander, basil, eggplant, and ginger rhizome [47].Both cymaroside and luteolin-7-O-glucoside expressed multiactivity including anti-cancer, anti-bacterial, and antioxidant activity [48].Song and Park [49] stated that luteolin and luteolin-7-O-glucoside increased the function of heme oxygenase-1, which exhibited a critical role in maintaining cellular redox homeostasis against oxidative stress.The highest terpene contents in the curry were capsianoside II (abundance: 52.23 × 10 5 ) and capsianoside I (abundance: 52.23 × 10 5 ), which were mainly liberated from chili (Capsicum annuum L.).The main ingredient used in spicy basil leaf curry was chili pods at 11%.Capsianosides, particularly capsianoside F, exhibited tight junction permeability of the human intestine, which mitigated leaky gut syndrome [50,51].Dihydrocapsaicin (abundance: 18.81 × 10 5 ) is commonly found in chili pods and diminishes TNFα-mediated activation of NFkB and its molecular targets in endothelial cells while also inducing upregulation of nitric oxide and exhibiting antioxidant properties [52].Phenolics and derivatives such as N-feruloyltyramine (abundance: 63.50 × 10 5 ) are commonly found in garlic bulbs [53] and mitigate several cardiovascular disorders through cyclooxygenase enzymes I and II [54].These enzymes play a key role in P-selection, which mediates the formation of platelets and leukocytes in activated endothelial cells [54].Quinic acid, a phenolic compound (abundance: 44.58 × 10 5 ), is found in cinchona bark, coffee beans, tobacco leaves, carrot leaves, and apples.The National Center for Advancing Translational Sciences [55] reported multiple functions for quinic acid such as acting as an antioxidant that has shown anti-cancer activity through apoptosis-mediated cytotoxicity in breast cancer cell testing in mice models [56].N-trans-Feruloyloctopamine (abundance: 7.79 × 10 5 ) is mainly found in garlic and shows high potential as a tyrosinase inhibitor [57], relating to melanin production and skin cancer or carcinoma cells [58].Extracted garlic skin containing N-trans-feruloyloctopamine inhibited cell proliferation and invasion in hepatocellular carcinoma cells [59].6 ′ -Hydroxysimvastatin has been used as a cholesterol-lowering and anti-cardiovascular disease drug [60], while hydrocodone, also found in this study, is a morphinane-like compound commonly used in combination with acetaminophen to control moderate to severe pain [61].The results showed that spicy basil leaf curry contained high amounts of phytochemicals with various health-promoting properties.The Massaman curry in this experiment contained 54 flavonoids and derivatives, 23 phenolics and derivatives, 8 terpenes, and 13 other types including quinones, alkaloids, chromones, and ketones, as shown in Table 5. Apigenin 7-O-glucoside is a main flavonoid compound (abundance: 167.45 × 10 5 ) generated from several plants [62].Candida spp., which is the most common cause of yeast infection, is inhibited by Apigenin 7-O-glucoside, which also shows cytotoxic effects on colon cancer cells and cervical cancer HeLa cells as well as alleviating DSS-induced colitis [63].Apigenin 7-O-glucoside significantly exhibited these mentioned activities [64][65][66].Kaempferol 4 ′ -glucoside was also found in Massaman curry at an abundance of 155.85 × 10 5 .It has various properties including anti-cancer, anti-inflammatory, antioxidant, anti-depressant, and anti-epilepsy properties, and it also improves cerebral blood flow [67].Chang et al. [68] reported that kaempferol 4 ′ -glucoside showed anti-inflammatory activity by inhibiting NO generation, iNOS protein, and iNOS mRNA level by retarding NF-κB-mediated iNOS gene transcription.Kaempferol also showed significant inhibition of NSCLC (non-small cell lung cancer) cell proliferation (p < 0.05) and inhibited the mesenchymal-epithelial transition in progressive lung cancer by promoting NSCLC cell autophagy, leading to NSCLC cell death in a rat model [69].Yu et al. [70] stated that kaempferol reduced inflammatory bowel disease (IBD) by inhibiting IL-1β, IL-6, TNF-α, CRP, and NO secretion as well as retarding regenerated blood vessels of high intestinal microvascular density [71].Luteolin (abundance: 91.64 × 10 5 ) is a flavone type that is generally present in plants, with multiple functions such as antioxidant, anti-inflammatory, and antiallergic properties, in particular, against liver disorders, including metabolic-associated fatty liver disease, hepatic fibrosis, and hepatoma [72,73].He et al. [74] reported that luteolin inhibited Aβ-induced oxidative stress, mitochondrial dysfunction, and neuronal apoptosis via a PPARγ-dependent mechanism, one of the pathways for Alzheimer's disease in rat models.Wang et al. [75] stated that luteolin inhibited herpes simplex virus 1 (HSV-1) infection, enhanced antiviral type I interferon production, and activated the cytoplasmic DNA-sensing cGAS-stimulator of the interferon gene (STING) pathway.The main phenolic acid and derivative compound in Massaman curry was glucocaffeic acid (abundance: 52.54 × 10 5 ), which is common in both herbs and spices [76,77].The multifunctions of caffeic acid have been addressed as anti-cancer, antiviral, and anti-inflammatory activities [78].Caffeic acid recovered ischemia-induced synaptic dysfunction in mouse hippocampal slices [79].The results indicated that caffeic acid (1-10 µM) did not directly affect synaptic transmission and plasticity but indirectly affected other cellular targets to correct synaptic dysfunction.Quinic acid (abundance: 40.84 × 10 5 ) and N-Feruloyltyramine (abundance: 30.35 × 10 5 ) were also identified in spicy basil leaf curry.One terpene found in this experiment was cofaryloside (abundance: 16.11 × 10 5 ).Cofaryloside I-II has been reported in Yunnan Arabica coffee beans [80][81][82], but no scientific information is currently available.Betulinic acid (abundance: 13.19 × 10 5 ) was the second-most abundant terpene compound found in this experiment.Melo et al. [83] reported that betulinic acid is present in various plants.Betulinic acid (50 mg/mL in water) elevated the plasma hormone levels of insulin and leptin and decreased levels of ghrelin hormone in high-fat feed rats.Other biological activities of betulinic acid include anti-HIV, anti-inflammatory, and anti-cancer activities [84,85].Maslinic acid (abundance: 9.60 × 10 5 ) was also found in this experiment.Maslinic acid is commonly found in many types of plants and exhibits many health aspects such as hypoglycemic effects, anti-inflammatory effects, neuroprotective effects, antioxidant effects, and anti-tumor effects [86,87].Cao et al. [88] found that maslinic acid administration favored probiotic bacterial growth in PD mice, which helped to increase striatal serotonin, 5-hydroxyindole acetic acid, and γ-aminobutyric acid levels, reduced levels of tumor necrosis factor-alpha and interleukin 1β in the substantia nigra pars compacta, and significantly prevented dopaminergic neuronal-related Parkinson's disease in a rat model.
The LC-ESI-QTOF-MS/MS results indicated that Massaman curry was higher in the number of polyphenolic types and variety of flavonoids and derivatives compared with spicy basil leaf curry activity.However, spicy basil leaf curry provided higher antioxidant activity based on TPC, TFC, DPPH, ABTS, FRAP, and ORAC, possibility because cynaroside A from basil leaves has strong antioxidant effects, as reported by [49, 89,90], where cynaside A exhibited good antioxidant activity with a lower IC 50 than quercitrin, rutoside, and protocatechuic acid [89].
Using the LC-ESI-QTOF-MS/MS technique indicated the possibility of toxins from Massaman curry and spicy basil leaf curry as podophyllotoxin (abundance: 3.12 × 10 5 ) and clitidine (abundance: 4.61 × 10 5 ), respectively.Podophyllotoxin is mostly generated in the rhizome of Podophyllum species, which grow widely across the Himalayan and Western China regions.Physicians have attempted to use this toxin for external genital and perianal warts caused by the human papillomavirus (HPV).HPV can be an opportunistic infection (OI) of HIV [91,92].Clitidine is created by the poisonous mushroom (Clitocybe acromelalga), and some molds contaminate dried herbs and spices as well as nuts [93][94][95].Therefore, safety awareness is needed.Clitidine may not be as harmful as aflatoxins and ochratoxins, but its presence indicates that the drying and storage processes for curry powders must be considered and submitted to the Thai FDA for controlling and warning entrepreneurs, companies, and consumers as well as public sectors.Data on the thermal degradation of podophyllotoxin could be destroyed at 114-118 • C [96], and clitidine has no information.Therefore, preventive systems such as washing with proper detergents, for example, bisodium carbonate, acetic acid, ozone, and calcium hydroxide [97], drying conditions, and storage with high vacuum values as well as irradiation may need to be applied.This is the first report using LC-ESI-QTOF-MS/MS to confirm phytochemicals and non-volatile compounds contained in mixed herbs and spices or curries supporting the body and wellness.This finding generally suggests not only the potential health impact or bioactivity associated with the unique composition of polyphenolics and flavonoids in Massaman or spicy basil leaf curry but also suggests that toxicity due to plant and mold toxin contamination also needs to be considered and managed.In addition, the identified phytochemical profiling found in both curries provided great evidence to extend the intensity determination of specific biological compounds further to obtain more value-added applications including functional ingredients and food, nutraceuticals, and medicinal products.-Anti-inflammatory.
-Inhibits the growth of both DNA and RNA viruses. -Anti-cancer.
-Inhibits the proliferation of HeLa and ME-180 cells.
-Inhibits electron transfers and metal chelating activities.
-Inhibits the activity of prostaglandin F 2α and angiotensin-I-converting enzyme.-Inhibits the activity of the MAPK and NF-kB pathways and SOCS3 in the signal transducer and activator of transcription 3 (STAT3) pathway.-Anti-cancer.
-Decreases G-actin and cytochalasin D and increases F-actin.
-Inhibits homozygous mutations in PTEN and TP53 genes in the human prostate cancer cells line and inhibits mutation in codon 13 of the RAS proto-oncogene in colorectal carcinoma cells.

Sensory Evaluation
The results showed that both curries contained various phytochemicals, in particular, flavonoids, simple phenolic acids, and terpenes.Some bitter and astringent-tasting compounds such as caffeic acid, (−)-epicatechin, and (+)-catechin were also found [137][138][139].Purves et al. [137] reported that the bitter threshold of quinine was 0.008 mM for humans.A bitter taste can be due to toxins, but beers, wines, dark chocolate, and coffee are bitter tasting [140,141].Noble [142] stated that small amounts of simple phenolic acids resulted in a bitter taste, while higher phenolic content provided an astringent taste when combined with sourness.However, unpleasant bitter and astringent tastes can be modified by masking with sweet, salty, and umami flavors [143].For sensory preference, preclinical studies indicated that bitter substances may have potent effects that stimulate the secretion of gastrointestinal (GI) hormones and modulate gut motility via the activation of bitter taste receptors located in the GI tract [144].The results in Table 7 show that both curries recorded high scores for all attributes (>7/9), with no mention of a bitter taste.The seasoning (sugar and salt) and other combinations (Table 3) such as oil, coconut milk, and chicken breast used in this experiment could be modified or reduced to mask any unpleasant tastes via various proposed mechanisms including bitterness-depressant substance absorption, inhibiting receptor sites, and reducing the intensity of bitter molecules as polarity effects.For example, [145,146] explained that spicy and anesthetic effects from clove oil can reduce the bitter taste in oral drugs.Brideau [147] stated that the bitter taste of chlorpheniramine maleate and phenylpropanolamine could be reduced by the combination of citric acid and sodium bicarbonate with certain fruit flavors such as lemon, orange, and cherry.Non-acceptance (%) 0 0 All data are mean ± standard deviation (sd); ns means nonsignificant differences (p < 0.05).

Conclusions
The Massaman and spicy basil leaf curries obtained from blending several herbs and spices were rich in phytochemicals, especially phenolic and flavonoid compounds.Spicy basil leaf curry contained significantly higher phenolic and flavonoid compounds and antioxidant activity (DPPH, ABTS, FRAP, and ORAC) than Massaman curry.The characterization of polyphenols in Massaman curry using LC-ESI-QTOF-MS/MS indicated 23 phenolic acids and derivatives, 54 flavonoids and derivatives, 8 terpenes, and 14 other types, while spicy basil leaf curry was composed of 10 phenolic acids and derivatives, 17 flavonoids and derivatives, 14 terpenes, and 21 other types.Cynaroside or luteolin-7-Oglucoside from basil leaves had a strong antioxidant effect and was the major cause of high antioxidant activity in spicy basil leaf curry.Sensory evaluations of the Massaman and spicy basil leaf curries gave 100% acceptance, with good scores for all attributes.The phenolic and flavonoid compounds contained in both curries such as caffeic acid, (−)-epicatechin, and (+)-catechin induced a bitter and astringent taste, but the panelists did not comment on this because the seasoning and other combinations modified, reduced, and masked the unpleasant bitter taste with a good mouthfeel and overall sensation.However, the high savory perception and health benefits derived from phytochemicals require further study in animal and clinical trials.

Figure 2 .
Figure 2. Flow chart showing the preparation of spicy basil leaf curry powder.2.3.Total Phenolic Content and Antioxidant Activity 2.3.1.Sample Preparation and Extraction

Figure 1 .
Figure 1.Flow chart showing the preparation of Massaman curry powder.

Figure 2 .
Figure 2. Flow chart showing the preparation of spicy basil leaf curry powder.2.3.Total Phenolic Content and Antioxidant Activity 2.3.1.Sample Preparation and Extraction

Figure 2 .
Figure 2. Flow chart showing the preparation of spicy basil leaf curry powder.
Figure S1: Standard curve of total phenolic content (TPC) with gallic acid agent used as a standard; Figure S2: Standard curve of total flavonoid content (TFC) with rutin agent used as a standard; Figure S3: Standard curve of DPPH assay with Trolox agent used as a standard; Figure S4: Standard curve of the ABTS assay with Trolox agent used as a standard; Figure S5: Standard curve of the FRAP assay with Trolox agent used as a standard; Fogure S6: Standard curve of the ORAC assay with Trolox agent used as a standard; Figure S7: Diagram of the characterization of phenolic profiles by LC-ESI-QTOF-MS/MS; Table

Funding:
This research was supported by the National Science, Research, and Innovation Fund (NSRF) and Prince of Songkla University (Grant No. AGR6505055b).Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki and approved by the human research ethics committee of Prince of Songkla University (PSU-HREC-2023-008-1-1 on 14 July 2023).

Table 1 .
Ingredient ratios used in Massaman and spicy basil leaf curry powders.

Table 2 .
Total phenolic and flavonoid contents and antioxidant activity in Massaman curry and spicy basil leaf curry.

Table 3 .
Ingredients and cooking ratios of Massaman and spicy basil leaf curries.

Table 4 .
Characterization of variously identified phytochemicals found in spicy basil leaf curry as determined by LC-ESI-QTOF-MS/MS.

Table 5 .
Characterization of variously identified phytochemicals found in Massaman curry as determined by LC-ESI-QTOF-MS/MS.

Table 6 .
Major phytochemicals found in spicy basil leaf curry and Massaman curry, their biological properties, and their possible plant sources.

Table 7 .
Sensory scores of dishes cooked with Massaman curry and spicy basil leaf curry.