Ultrasound-Assisted Extraction of Phenolic Compounds from Different Maturity Stages and Fruit Parts of Cordia dodecandra A. DC.: Quantification and Identification by UPLC-DAD-ESI-MS/MS

Abstract

In the present work, the total phenolic content (TPC), total flavonoids content (TFC), antioxidant activity, and phenolic profile from pulp (PU) and peel (PE) extracts obtained from the ciricote (Cordia dodecandra A. DC.) fruit by ultrasound-assisted extraction (UAE) in immature (IM), semimature (SM), and mature (MM) stages were investigated. The effect of the diameter of the ultrasonic probe in the IM stage was also evaluated. The TPC and antioxidant activity in IM fruit extracts by UAE increased up to 11.01 and 23.82 times, respectively, compared to the maceration method. The main phenolic compounds in the PE of IM fruit identified by UPLC-DAD-ESI-MS/MS were quantified as caffeic acid, rutin, and rosmarinic acid, distributed as 45.82, 41.45, and 12.72%, respectively. The PE extracts of IM fruit obtained with the 3 mm diameter probe had 1.27, 2.44, and 1.37 times the TPC (19.93 ± 0.28 mg GAE (Gallic equivalents) g⁻¹ dw), TFC (34.85 ± 4.99 mg RE (Rutin equivalents) g⁻¹ dw), and antioxidant activity (122.09 ± 17.09 µTE (Trolox equivalents) g⁻¹ (DPPH)), respectively, compared to those obtained with a 13 mm diameter probe. The results obtained suggest the use of the ciricote native fruit as a source of bioactive compounds, directly as fresh fruit or processed, thus helping to increase its production and consumption.

1. Introduction

The ciricote (Cordia dodecandra A. DC.) is a native tree from the Peninsula of Yucatán, cultivated and grown as an ornamental and shade plant in orchards of rural communities, green urban areas, and medium jungles in the Mexican Southeast. It is highly valued for its wood, the main product of this species. The fruit of ciricote is usually consumed as an artisanal dessert and is processed on a small scale [1]. Native traditional fruits such as ciricote currently face losses in their biological variety, as a result of leaving the fruit aside and making it an underutilized product. However, at a global level, there is a special concern about the decline of traditional crops, which offer greater genetic biodiversity and the potential to improve the food and nutritional security of the growing population in a world with limited resources [2]. Therefore, documenting the information about this traditional species increases awareness about its use and preservation. It has been reported that the inedible parts of the fruits such as the peel are promising sources of bioactive ingredients and antioxidant properties, which are even higher than in the edible parts [3]. Previously, investigations on the pulp of ciricote have indicated its application as a source of phytochemical compounds of great industrial or pharmaceutical value [4], and these bioactive compounds are globally used in cosmeceuticals, nutraceuticals, and the functional food industries [5]. The phenolic compounds are the largest group of phytochemicals in plants with diverse interests and their concentration could be affected by the maturation process. The ripening in fruits implies a series of changes, in which the structure and composition of unripe fruit are altered and later it becomes acceptable for consumption. It is a complex physiological process, which involves multiple widely documented chemical and biochemical reactions [6]. Nowadays, the novel method of UAE (ultrasound-assisted extraction) is highlighted as a key technology for the achievement of “green” sustainable chemistry for low-cost bioactive compounds extraction, due to its easy handling and operation under soft conditions of pressure and temperature. Furthermore, UAE is based on the principle of acoustic cavitation, capable of damaging the cell walls of the plant matrix [7], and combined with the size and shape of the ultrasonic probe, the release of bioactive compounds and the extraction process is favored [8]. Additionally, chromatographic tools such as UPLC-DAD-ESI-MS/MS, with the use of smaller diameter particle columns, are favorable for the separation and quantification of compounds [9], which can help us to identify in detail the different compounds present in raw materials to look for specific applications in the development of functional foods [10]. However, few studies have been conducted on ciricote fruit, with the majority of them focusing on the biological effects of the bark and leaf extracts [11]. The ciricote fruit has shown high phenolic content and antioxidant activity, including the presence of phenolic compounds not commonly found in other native fruits from the Mexican Southeast [3,12], making it an interesting source for the investigation of phytochemicals. Considering that underutilized native species are not usually a priority and their research is fragmented or sporadic, their study can offer sustainable solutions [13]; particularly, the follow-up of ciricote research results is relevant here [4]. In this sense, the objective of this study was to evaluate the effects of the maturity stage and parts of ciricote fruit extracted by UAE on the phenolic content and antioxidant activity, as well as the profile by UPLC-DAD-ESI-MS/MS. Additionally, the use of two different probe diameters for phenolic extraction was also evaluated.

2. Materials and Methods

2.1. Plant Material and Chemical Reagents

Approximately 8 kg of the ciricote fruit were collected in three different maturity stages (immature (IM), semi-mature (SM), and mature (MM)) in the city of Mérida, Yucatán (Mexico), at the coordinates MX 21°01′52″ N 89°37′ 40″ W, in April 2021 with warm sub-humid climate, temperature 35 °C, precipitation 20 mm, and relative humidity 57%. The identification of the plant was confirmed with the specimen deposited in the herbarium “Alfredo Barrera Marín” (voucher number: J. S. Flores 12576). The fruits were immediately transferred to the Center of Research and Assistance in Technology and Design from Jalisco State, Southeast Unit, in the same city. The fruits were separated from those microbially or physically deteriorated, washed with tap water, and disinfected with a solution of iodine (2 ppm) for 1 min. Immediately, peel (exocarp, here PE), pulps (mesocarp, here PU), and endocarp (containing the seed) were manually separated using a potato peeler and a knife, and the pulp was cut into uniform segments and processed as fresh or frozen at −20 °C until further analysis.

The reagents Folin–Ciocalteau, DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), Trolox (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid), potassium persulfate (K₂S₂O₈), sodium nitrite (NaNO₂), aluminum chloride (AlCl₃), HPLC grade acetonitrile (ACN), formic acid, phenolic compound standards (gallic, caffeic, rosmarinic, cinnamic, trans-cinnamic, chlorogenic, and ferulic acids, rutin, catechin, epicatechin, naringin, naringenin, hesperidin, kaempferol, and quercetin), commercially available from SIGMA-Aldrich (St. Louis, MO, USA), were used; sodium hydroxide (NaOH), sodium carbonate (Na₂CO₃), methanol (MeOH) and ethanol (EtOH) absolute, and hydrochloric acid (HCl) were purchased from Avantor JT Baker (Radnor, PA, USA). Ultrapure water was obtained through a Milli-Q filtration system (Millipore, Bedford, MA, USA); distilled water was acquired from B. Medina (Mérida, YN, Mexico).

2.2. Physicochemical Characteristics

Physicochemical characteristics were determined in the fresh fruit for each stage of maturity. Weight, total soluble solids (TSS), pH, and maturity index parameters were obtained according to Pacheco et al. (2020) method [4]. Titratable acidity (TA) was determined by potentiometric titration according to Monroy et al. (2019) [14] using a pH meter (Colepalmer Oakton pH 700 Benchtop, CA, US). The moisture determination was measured according to the AOAC official method 925.09 [15] using a vacuum oven (Jeio Tech MOD OV-12, Daejon, Republic of Korea). The color parameters (CIE-L*a*b*) were obtained with a colorimeter (ColorMeter Pro Q/CP-002-2020, HK, China) and reported as L*, a*, and b*. The color contribution index (CCI) was calculated with the equation CCI = 1000 × a*/(L* × b*) [16], Chroma values were obtained with the equation C* _ab = (a* ² + b* ²)^½, which defines the color saturation and angle hue as (h* ab) = (arctan b*/a*) [17]. R, G, and B values were obtained from the link Accurate and Easy Color Calculations: http://colormine.org/convert/rgb-to-lab (accessed on 2 September 2021) and PowerPoint software [18] (2210, 2021, Microsoft, Redmond, WA,US); Hex code and color were obtained from the link Color-hex https://color-hex.org (accessed on 2 September 2021) [19].

2.3. Ciricote Fruit Pretreatment and Ultrasound-Assisted Extraction (UAE) of Phenolic Compounds

The part of the fruits PU and PE were lyophilized in a FreeZone System (Labcono Freezone 6, Kansas city, MO, US) at 0.470 mbar and −50 °C for 78 h. The dried material was ground in a coffee mill (Hamilton Beach 80350R, Wareham, MA, US) until a particle size < 500 µm (Mesh ATSEM-E11 No.35) and stored at −80 °C for further analysis. The phenolic compound extracts were obtained according to Pacheco et al. (2020) [4] and Herrera-Pool et al. (2021) [10] using an Ultrasonic Processor (Sonics & Materials GEX130PB, Newtown, CT, US) performed at 20 kHz and 130 W, amplitude 80%. The freeze-dried samples were immersed with EtOH 50% (water/ethanol (v/v)) in the proportion 1:50 w/v. They were then sonicated for 10 min at room temperature. A cold bath was used to maintain the temperature below 50 °C. After applying the ultrasound, the processed samples were centrifuged at 4700 rpm and 8 °C for 20 min (ThermoFisher Scientific SL 40R, Roskilde, HE, DE). Subsequently, the supernatants were recovered and filtered using vacuum filtration. The filtrates were collected, brought to initial volume with EtOH 50%, and stored frozen until their analysis. A 13 mm diameter stepped probe was employed for 50 mL for maturity-stage evaluations, and a 3 mm diameter stepped probe was employed for 10 mL of solvent volume; for comparison with the best treatment and the mass spectrometric analysis, the probe was coupled to an ultrasonic system [20]. The extraction by maceration was performed with freeze-dried samples and EtOH 50%, 1:50 p/v, and the samples were then homogenized using a magnetic stirrer at 150 rpm (DLAB, MS7-H550-PRO, Guada JC, MX) for 60 min at 25 °C; subsequently, the samples were processed as was described above. All determinations were performed in triplicate.

2.4. Determination of Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Activity

The TPC of both the PE and PU extracts was determined by the Folin–Ciocalteu method [21]. TPC was expressed as mg of gallic acid equivalent per g of dry weight samples (mg GAE g⁻¹ dw). A calibration curve of gallic acid from 50 to 800 ppm was performed. Briefly, 250 µL of Folin–Ciocalteau 1N reagent was added to 20 µL of extract and shaken. After 8 min, 1250 µL of 7.5% Na₂CO₃ and 480 µL of distilled water were added and kept for 30 min under darkness. The absorbance was measured at 760 nm using a spectrophotometer UV-vis (Thermo Fisher Scientific Biomate 3S UV-vis, WI, US). TFC was performed according to Al et al. (2009) [22] with slight modifications. Briefly, to 1 mL of extract sample or standard, 0.300 mL of 5% NaNO₂ was added and shaken; after 6 min, 0.300 mL of 10% AlCl₃ was added and shaken; after 5 min, 2 mL of 1 M NaOH was added and, subsequently, it was brought up to 10 mL with distilled water. The absorbance was measured at 510 nm. A calibration curve of rutin from 25 to 800 ppm was performed. The results were expressed as mg of rutin equivalents per g of dry weight samples (mgRE g⁻¹ dw). Antioxidant activity evaluated as radical scavenging activity was evaluated using the stable 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH) according to the slightly modified method of Ana et al.(2018) [23], prepared at 2.4 mg DPPH in 100 mL of methanol and adjusted absorbance to 0.800 ± 0.02 at 515 nm. Briefly, for the antioxidant activity by DPPH assay, to 50 µL of extract sample was added to 3950 µL of DPPH (previously prepared from 2.4 mg of DPPH in methanol), then stored in darkness for 40 min. The absorbance was measured at 515 nm. The antioxidant activity also was measured by ABTS radical assay, using the method reported by Alonso-Carrillo et al. (2017) [24]. The ABTS stock solution was prepared by adding 0.0768 g of ABTS salt and 13.2 mg of K₂S₂O₈ in 20 mL of deionized water. It was then stored in the dark for 16 h at room temperature before use. The ABTS radical solution was diluted with EtOH until reaching an absorbance value of 0.70 ± 0.02 at 734 nm [24]. Briefly, 1000 µL of ABTS solution was mixed with 10 µL of extract sample. After 6 min, the absorbance was read at 734 nm. A calibration curve from 50 to 400 ppm Trolox was performed. The antioxidant activity was expressed as µmol of Trolox Equivalent per g of dry weight samples (µmol TE g⁻¹ dw), according to the calibration curve and the percentage of radical inhibition of DPPH and ABTS, which was calculated according to Equation (1):

$$\mathrm{DPPH}/\mathrm{ABTS} \mathrm{Inhibition} (\%)=\left[\frac{\left({\mathrm{A}}_{\mathrm{control}}-{ \mathrm{A}}_{\mathrm{sample}}\right)}{{\mathrm{A}}_{\mathrm{control}}}\right]\times 100$$

(1)

where A_control is the absorbance of the control, and A_sample is the absorbance of the sample.

2.5. Phenolic Compounds Analysis by UPLC-DAD and Spectral Mass Analysis by ESI-MS/MS

The identification and quantification of phenolic compounds were performed by Ultra Pressure Liquid Chromatography (UPLC) according to Ana et al. (2018) [23] using Ultra Pressure Liquid Chromatography (UPLC) (Waters Acquity H Class, Milford, MA, US) equipped with a quaternary pump (UPQSM), autosampler injector (UPPDALTC), and PDA eλ photodiode array detector (UPPDALTC). Empower 3 software (Empower 3, 2010, Waters Milford, MA, US) was used for data acquisition and processing for phenolic quantification. Chromatographic separation of the phenolic compounds was carried out using a C18 column (1.7 µm, 100 × 2.1 mm I. D.) (Waters Acquity UPLC BECH, Milford, MA, USA) at room temperature, with the flow rate at 0.2 mLxmin and the injection volume of 2 µL. The mobile phase consisted of two solvents, (A) 0.1% of formic acid in ultra-pure water and (B) 0.1% formic acid in acetonitrile. The elution conditions applied included the following: 0–2 min 100% A isocratic, 2 min linear gradient from 100% to 90% A, 2 min linear gradient from 90% to 77% A, 1 min 77% A isocratic, 10.5 min linear gradient from 77% to 76.5% A, 0.5 min 0% A isocratic, 6 min linear gradient from 0% to 50% A, and 6 min linear gradient from 50% to 100% A. The photodiode array detector was set at 350 nm for ciricote with a resolution of 4.8 nm for analyte detection. Quantification was made by injection of phenolic compounds analytical standards curves of caffeic acid, rutin, and rosmarinic acid (Table S1, Supplementary Materials). The results were reported as μmol equivalent of the analytical standard per g of dry weight (µmol g⁻¹ dw). The mass spectrometry (MS/MS) analysis was carried out using a mass spectrometer (Waters XeVo TQ-S micro, MA, US) as reported by Herrera-Pool et al. (2021) [10]. The collision energy used was from 10 to 150 eV for scanning in negative ion mode. The mass spectra were recorded in full scan mode in a range of 50 m/z to 700 m/z. The MassLynx V4.1 software (V4.1, Waters, Milford, MA, US) was used for data acquisition and processing. The tentative identification was assigned by comparing fingerprint and MS data of the compounds detected with those reported in the literature and the public European Mass Bank database (MassBank Europe, https://massbank.eu/MassBank/index.html accessed on 2 September 2021) and ReSpect for phytochemicals (Respect http://spectra.psc.riken.jp/menta.cgi/respect/index accessed on 2 September 2022). Additionally, the confirmation of the identified phenolic compounds was performed by the multiple reaction monitoring analysis (MRM) mode by a mass spectrometer (Waters Xevo-TQs-micro).

2.6. Statistical Analysis

Minitab 17 software (17.1.0.0, 2013, Minitab, State Collage, PA, US) was used for statistical analysis of the results, which were expressed as the mean ± standard deviation. To evaluate the effect of the factor of stage maturation and ultrasonic probe extraction diameter on the response variables, a multifactorial ANOVA was performed to analyze the correspondent factorial designs (p < 0.05). Followed by the Tukey test to compare the test groups, differences at p < 0.05 were considered significant. The software Origin 2019b (64-bit) 9.6.5.169 (1991–2019) Origin Lab Corporation was used for the principal components analysis (PCA), to evaluate the effect of the maturity stages (IM, SM, and MM) and parts of the fruit (PU and PE) on the dependent parameters of TPC, TFC, antioxidant activity, and content of phenolic compounds presented in the samples.

3. Results

3.1. Physicochemical Characteristics in Fresh Ciricote Fruit at Three Stages of Maturity

The ciricote fruit was composed of pulp (66.38%), peel (16.27%), endocarp (15.79%), and seed (1.55%). The values of moisture, weight, color, TSS, pH, firmness, TA, and maturity index are presented in

Table 1. Physicochemical parameters of ciricote fruit at three stages of maturity.

	IM	SM	MM
Moisture (%)
Pulp (PU)	87.47 ± 0.59 a	82.21 ± 1.40 b	83.17 ± 0.65 b
Peel (PE)	86.01 ± 0.47 a	82.76 ± 0.47 b	82.93 ± 0.98 b
Weight (g)	34.50 ± 5.49 b	41.14 ± 7.22 a	40.33 ± 3.49 a
Color
L*	45.03 ± 2.84 a	52.30 ± 6.76 b	62.33 ± 4.24 b
a*	−9.18 ± 0.64 a	3.58 ± 1.62 b	11.78 ± 2.20 c
b*	26.73 ± 4.18 a	42.48 ± 3.61 b	52.05 ± 2.25 c
C* (Chroma)	26.44 ± 1.87 c	43.53 ± 3.98 b	53.04 ± 3.12 a
(Hue) Angle	110.47 ± 1.26 a	84.70 ± 1.78 b	77.48 ± 2.24 c
CCI	−7.81 ± 1.49 a	1.60 ± 0.68 b	3.61 ± 0.46 c
RGB
TSS (°Brix)	8.00 ± 1.00 c	11.00 ± 1.00 b	15.00 ± 1.00 a
pH	5.73 ± 0.02 a	5.70 ± 0.03 a	5.63 ± 0.02 a
Firmness (N)	50.43 ± 11.47 a	13.24 ± 1.47 b	8.15 ± 0.61 b
TA (as % citric acid)	0.13 ± 0.01 a	0.09 ± 0.01 b	0.11 ± 0.01 b
Maturity index	60.18 ± 9.90 c	115.70 ± 3.89 b	138.34 ± 8.87 a

IM: immature, SM: semimature, MM: mature, CCI: color contribution index, Maturity index: total soluble solids (TSS)/titratable acidity (TA). RGB color space obtained by Microsoft PowerPoint software, corresponding to Hex color, code IM (#6a6d3d), SM (#967830), MM (#c18d36). Different superscript letters in the same row mean significant difference (p ≤ 0.05) by the Tukey test.

. The values of moisture were higher for the IM fruit for PU and PE parts; in both cases they were significantly statistically different (p < 0.05) than the SM and MM values. For the average weight, the fruits in SM and MM stages presented higher values. For L*, a*, and b* coordinates from the CIELAB color space, L* meant that MM and SM were brighter than IM fruit (p < 0.05). The a* and b* color coordinates were different for IM, SM, and MM (p < 0.05). The same behavior was observed for Chroma and Hue Angle values, as they were different for all maturity stages evaluated. In contrast, despite the differences in other parameters for the maturity stage, pH values were similar. The values of firmness (N) and total acidity (TA) reported as citric acid (%) were higher for the IM fruit and in both cases were statistically different (p < 0.05). The maturity index obtained with the different parameters evaluated were statistically different (p < 0.05) among the three samples IM, SM, and MM.

3.2. TPC, TFC, and Antioxidant Activity of Ciricote Fruit Parts at Different Stages of Maturity

According to the results shown in

Table 2. Total phenolic and flavonoid contents and antioxidant activity of ciricote fruit parts at different stages of maturity.

		PF	TPC (mg GAE g−1 dw)	TFC (mg RE g−1 dw)	DPPH (µM TE g−1 dw)	ABTS (µM TE g−1 dw)
	IM	PU	10.66 ± 0.67 b,B	9.82 ± 0.28 b	54.48 ± 3.088 b,B	55.04 ± 8.40 b,B
		PE	15.62 ± 1.77 a,A	14.23 ± 2.01 a	82.39 ± 10.00 a,A	86.94 ± 4.20 a,A
	SM	PU	5.32 ± 0.43 c,C	2.47 ± 0.41 d	32.69 ± 0.80 c,D	19.22 ± 2.95 c,CD
UAE		PE	6.61 ± 0.71 c,C	5.56 ± 0.72 c	44.30 ± 1.26 bc,BC	27.97 ± 1.03 c,C
	MM	PU	5.76 ± 0.93 c,C	3.82 ± 0.30 cd	36.12 ± 2.64 c,CD	18.93 ± 0.70 c,CD
		PE	6.54 ± 0.68 c,C	4.29 ± 2.20 cd	41.23 ± 1.23 c,CD	25.42 ± 0.84 c,CD
MAC	IM	PU	5.79 ± 0.28 b,C	NA	14.11 ± 1.16 b,E	16.82 ± 1.25 b,D
		PE	1.42 ± 0.07 a,D	NA	3.46 ± 0.28 a,E	4.13 ± 0.31 a,E

IM: immature, SM: semimature, MM: mature; PU: pulp, PE: peel, UAE: ultrasound-assisted extraction, MAC: extraction by maceration. Different lowercase letters in superscript in the same column for each extraction method, and different capital letters in the same column for both methods, mean significant difference (p < 0.05); dw (dry weight).

, the TFC and TPC were higher in the PE and PU from the IM fruit than those from SM and MM fruits, and these last two did not present a significant difference between them (p > 0.05). Values of antioxidant activity were two and three times higher in IM fruit than in SM and MM fruits in the DPPH and ABTS assays, for the PU and PE, respectively. The antioxidant activity of SM and MM fruits (in both PU and PE) did not show a statistically significant difference (p > 0.05) between them. On the other hand, the ultrasound-assisted extraction (UAE) increased the TPC in IM fruit by 1.84 and 11.01 times, the antioxidant activity (DPPH) by 3.86 and 23.82 times, and antioxidant activity (ABTS) by 3.27 and 21.05 times for PU and PE, respectively, in all cases, compared to the maceration method (Table 2). Additionally, UAE allowed higher TPC and antioxidant activity in less time (10 min compared to 1 h of the maceration method).

3.3. Phenolic Compound Identification and Quantification by UPLC-PDA-ESI-MS/MS in Extracts of Ciricote Fruit

The identification of phenolic compounds at the different stages of maturity (IM, SM, and MM) and using the PU and PE of the fruit was performed using UPLC-DAD-ESI-MS/MS equipment, and the quantification was performed using commercial standards. The three main compounds presented in the different samples evaluated were two phenolic hydroxycinnamic acids (caffeic and rosmarinic acids) and a flavonoid (rutin) (Figure 1, Figures S1 and S2). A total of three phenolic compounds were identified, two phenolic acids and one flavonoid (Figure 1A and

Table 3. Main phenolic compounds identified by UPLC-DAD-ESI-MS/MS in extracts of Cordia dodecandra A. DC.

CN	RT	PDA UV(λmax)	[M-H]-	(m/z)	TI	Concentrations (µmol g−1 dw)
						PF	IM	SM	MM
1	9.83	217 323	179	135	Caffeic acid	PU	1.24 ± 0.03 b	0.65 ± 0.00 c	1.18 ± 0.03 b
						PE	3.03 ± 0.037 a	0.49 ± 0.00 c	0.56 ± 0.04 c
2	10.96	209 255 353	609	300 301 271 254 243	Rutin	PU	NQ	NQ	NQ
						PE	3.35 ± 0.04 a	1.73 ± 0.02 c	2.52 ± 0.01 b
3	15.97	219 318	359	197 179 161 132 135	Rosmarinic acid	PU	0.59 ± 0.11 b	NQ	NQ
						PE	0.93 ± 0.06 a	NQ	NQ

CN: compound number; RT: retention time; TI: tentative identification; PF: part of the fruit; NQ: not quantifiable. Different lowercase letters in superscript in the same group of compounds means significant difference (p < 0.05).

). Compound 1 (Rt: 9.83 min; λmax: 217, 323 nm) was identified as caffeic acid [25], compound 2 (Rt: 10.96 min; λmax: 209, 255, 353 nm) was identified as rutin [26] and compound 3 (Rt: 15.97 min; λmax: 219, 318 nm) was identified as rosmarinic acid [27]. The molecular ion [M-H]- at m/z 179, 609, and 359, respectively, for each mentioned compound were found in negative ion mode and the corresponding fragments were also identified [4,26]. The confirmation of the presence of caffeic acid, rutin, and rosmarinic acid was demonstrated by a multiple reaction monitoring analysis (MRM) (Figure 1B), the conditions of which for the mass spectrometer (Xevo-TQs-micro) are presented in the Supplementary Materials (Table S2), and the MRM conditions for quantification and qualification of those phenolic compounds are shown in

Table 4. MRM analysis conditions for quantification and qualification of phenolic compounds in extracts obtained from Cordia dodecandra fruit.

Compound	Molecular Weight (uma)	Retention Time (min)	Transition	Parent (m/z)	Daughter (m/z)	Dwell (secs)	Cone Voltaje (eV)	Collision Energy
Caffeic acid	180	9.91	Qualification	179	135	0.045	14	18
Caffeic acid	180	9.91	Quantification	179	79	0.045	14	30
Rutin	610	10.41	Qualification	609	300	0.045	100	54
Rutin	610	10.41	Quantification	609	271	0.045	100	76
Rosmarinic acid	359	13.79	Qualification	359	197	0.045	50	22
Rosmarinic acid	359	13.79	Quantification	359	161	0.045	50	22

. Regarding the concentration of the compounds, the caffeic acid content in PU and PE in IM was 1.24 ± 0.03 and 3.03 ± 0.037 µmol g⁻¹ dw, respectively (Table 3). A higher rutin content was obtained in ciricote peel in the following state of maturity order: IM > MM > SM, and it was quantifiable only for the PE treatment. The concentrations were observed from 1.73 ± 0.02 to 3.35 ± 0.04. Rosmarinic acid content was mainly present in IM fruit (PE and PU), and it was not detected or quantifiable in the PE and PU of SM and MM.

3.4. Correlation between TPC, TFC, Antioxidant Activity, and Individual Phenolic Compounds

A significant association was found by evaluating Pearson’s correlation coefficients between TPC, TFC, and antioxidant activity (Table S3, Supplementary Materials). The content of rosmarinic and caffeic acids also showed a strongly significant correlation with the mentioned response variables, but with rutin the correlation was weak. A significant correlation between rosmarinic and caffeic acid was obtained, but not for each one with rutin.

3.5. Principal Components Analysis of Variation of Stages of Maturity and Parts of Ciricote Fruit with TPC, TFC, Antioxidant Activity, and Phenolic Compounds

A principal components analysis of variation was performed to understand the differences among the stages of maturity and parts of ciricote fruit, using as variable responses TPC, TFC, antioxidant activity, and phenolic compounds content from the samples; this tool enhances the understanding of the inter-sample, inter-variable relationship by reducing the dimensionality of the data using a graphical expression (Figure 2). The PC1 (principal component 1) explained 87.04% of the variability of the responses and PC2 (principal component 2) 10.36%. Both components explained 97.40% of the variability (Table S4, Supplementary Materials). PC1 collects the majority of the information about the evaluated variables with the higher contribution of TPC, TFC, ABTS, DPPH, caffeic acid, and rosmarinic acid contents. In PC2, the rutin had the highest effect with respect to the other responses (Table S4, Supplementary Materials). The contribution value of the response variables (TFC, TPC, antioxidant activity, rosmarinic, and caffeic acid) is closed, while the rutin value is separated from the others. In the PCA plot, four groups can be observed; the first one contains MM-PE and SM-PE, which have a relationship with rutin; the second one contains MM-PU and SM-PU, but they did not have a direct influence on the response variables. Additionally, there are two individual groups: the IM-PE group, which is highly related with all the response variables, and the IM-PU group that also has a high influence on the response variables, except for rutin.

3.6. Effects of the Diameter of the Ultrasonic Probe in Extracts of Fruit

In the extraction of phenolic compounds from IM fruit with a stepped probe of 3 mm diameter, it was determined that the TPC obtained in the PU did not present a significant difference (p > 0.05) from that obtained with the 13 mm probe. However, the TPC in the PE increased from 15.62 ± 1.77 to 19.93 ± 0.28 mg GAE g⁻¹ dw. TFC also showed a significant increase (p < 0.05) in both the PU and PE from 9.82 ± 0.28 to 18.61 ± 3.27 and from 14.23 ± 2.01 to 34.85 ± 4.99 mg RE g⁻¹ dw, respectively. The antioxidant activity evaluated by DPPH and ABTS also presented a significant increase (p < 0.05) from 54.48 ± 3.09 to 75.52 ± 4.72 and from 82.39 ± 10.00 to 122.09 ± 17.09 µM ET g⁻¹ dw for the PU and PE, respectively, by DPPH; likewise, there was an increase from 55.04 ± 8.40 to 75.54 ± 3.24 and from 86.94 ± 4.21 to 119.94 ± 2.02 µM ET g⁻¹ dw for the PU and PE, respectively, by ABTS. Caffeic acid content in the PU increased from 1.24 ± 0.03 to 2.03 ± 0.23 µmol g⁻¹ dw, and there was no significant difference in the PE, with both probes. With the 3 mm probe, rutin was identified at a concentration of 0.04 ± 0.01 µmol g⁻¹ dw in the PU, and an increase from 3.35 ± 0.04 to 4.07 ± 0.39 µmol g⁻¹ dw was observed in the PE of the immature ciricote fruit. The rosmarinic acid extraction with the 3 mm probe was also favored, quantifying an increase from 0.59 ± 0.11 to 12.22 ± 0.42 and from 0.93 ± 0.06 to 9.65 ± 0.74 µmol g⁻¹ dw in the PU and PE, respectively.

4. Discussion

4.1. Physicochemical Characteristics in Fresh Ciricote Fruit at Three Stages of Maturity

Color variations were associated with the maturity stage since negative CCI values indicated an immature fruit and positive and growing values indicated a turning color fruit and a mature fruit [17]. The CCI agreed with the chemical maturity index, which was higher in MM fruit compared to SM and IM fruit. The pH was close to apricot fruit (5.32) as reported by Karabulut et al. (2018) [28], who reported also higher moisture in immature fruits. The firmness decreased as the level of maturity increased, and a similar behavior was reported for tropical acerola fruit at three different stages of maturity [29]. The results reflected a greater hardness and consistency in the fruit in the IM stage, and a softer fruit as it matures, due to biochemical and texture changes in the cell wall structure and cell turgor pressure during the maturity process and development, which make the fruit more enjoyable [29,30]. The TA decreased as the fruit matures, which is related to the fact that during the maturity process, the organic acids are converted to sugars during the respiration process, where they are used as substrate as well as for the formation of flavor compounds [6]. The TA values were lower in comparison to other native fruits from the Mexican Southeast [31], but close to those reported for other tropical fruits such as papaya, apricot, acai fruit, pear, nectarine, and yellow pitahaya [32].

4.2. TPC, TFC, and Antioxidant Activity of Ciricote Fruit Parts at Different Stages of Maturity

The results of the effects of maturity stage and the part of the fruit on TPC and TFC agree with the results reported for other tropical fruits such as guava, longan, and apple [30,33,34], both their decrease as fruit matured and higher values in the peel. Because phenolic compounds are produced in response to abiotic and biotic stresses, it is plausible that the peel accumulates phenolic compounds as a defense mechanism [35]. The TPC was higher than Cordia myxa and Cordia boissieri [36,37]. For antioxidant activity value, the results for IM PU were similar to those reported by Pacheco et al. (2020) [4] for ciricote pulp (50 µM TE g⁻¹ dw by DPPH), and the antioxidant activity in extracts from IM PE was higher than in Cordia boissieri (65 ± 2.8 μM g⁻¹ of extract by ABTS) [38]. The antioxidant activity obtained for IM, SM, and MM fruit extracts (in both PU and PE) were within those reported for the peels of some Mexican tropical fruits grown in south-eastern Mexico, from 1.16 to 40.02 mM/100 g dw by ABTS and 0.16 to 48.39 mM/100 g dw of TE by DPPH [3]. Therefore, both the PU and PE from ciricote fruit in the three different stages of maturity represented good sources of antioxidant phytochemical compounds. The IM fruit represented a better source of phenolic compounds, flavonoids, and antioxidant activity.

4.3. Phenolic Compounds Identified by UPLC-DAD-ESI-MS/MS in Extracts of Ciricote Fruit

Previously, Pacheco et al. (2020) [4] reported a caffeoyl hexoside content in ciricote PU of 3.42 µmol equivalent of caffeic acid by gram dw; in this work, the values were slightly lower. Additionally, they reported a rutin content of 1.46 µmol g⁻¹ dw in the PU of ciricote [4]. The results may be due to differences in genotype, site, cultivation technique, or soil and climatic conditions [34]. The rutin content was associated with the stage of maturity and the part of the ciricote fruit. This behavior agrees with that reported for jujuba fruit peel related to the three maturity stages [39]. In the genus Cordia, rutin has been identified in the leaves of C. dentata, C. bicolor, and C americana and quantified in C. myxa pulp (3.31 µg/mL). Rutin (3′,4′,5,7-tetrahydroxy-flavone-3-rutinoside), a glycoside of quercetin, is an important dietary flavonoid and regarded as a member of the vitamins (vitamin P); moreover, it is the most abundant flavonoid in vegetables, fruits, and buckwheat. However, it is noteworthy that rutin is not usually used efficiently due to the high price of the product and the obtaining process [40], so ciricote peel especially represents an important natural source of this phytochemical. Finally, rosmarinic acid also was associated with the stage of maturity and the part of the ciricote fruit; it is a phenolic compound of the hydroxycinnamic acid family and is an ester of 3,4-dihydroxyphenyllactic acid and caffeic acid [27]. Based on the known antioxidant potential of rosmarinic acid, because of the two catechol groups present in its structure, a series of widely studied biological properties have been published, such as anti-inflammatory, antiviral, antitumor, neuroprotective, photoprotective and healing, antiangiogenic, and antibacterial. The isolation and purification of rosmarinic acid increases the possibility of using the fruit in the initial stages of fruit maturity. Therefore, the rosmarinic acid present in ciricote fruit represents a potential source of this phytochemical for pharmaceutical and analytical development as a natural molecule of interest in biomedical applications and possible food applications.

4.4. Correlation between TPC, TFC, Antioxidant Activity, and Individual Phenolic Compounds

The significant correlation among TPC, TFC, and antioxidant activity (ABTS and DPPH) of PE and PU extracts from ciricote fruit showed a similar behavior to that reported by Can-Cahuich et al. (2017) [3] for tropical fruit peels of native fruits. The above indicated that the phenolic compounds could be responsible for a major contribution to the antioxidant activity, more so than other antioxidant agents present in the fruit. The high correlation between the content of rosmarinic and caffeic acids with TPC and antioxidant activity suggested that these variables were affected mainly by the content of those phenolic compounds. The rutin did not show a significant correlation with the mentioned response variables; however, its effect on antioxidant activity is not discarded. The TFC had no significant correlation with rutin, which means that the TFC assay technique is inadequate for flavonoid determination in ciricote extracts. Due to TPC and TFC showing a high correlation, this suggested that chelation reaction with aluminum (in the TFC assay) is not specific for the detection of flavonoid content [41]. For this reason, the UPCL-DAD analysis for ciricote extracts was appropriate. The correlation between rosmarinic and caffeic acid suggested that they are related during the extraction; when each one is found the other is too, but that did not happen with rutin.

4.5. Principal Components Analysis of Variation of Stages of Maturity and Parts of Ciricote Fruit with TPC, TFC, Antioxidant Activity, and Phenolic Compounds

The treatments SM-PU and MM-PU did not show an important relationship with TPC and antioxidant activity, while SM-PE and MM-PE samples presented similar characteristics in the content of phenolic compounds, especially rutin content. The IM-PE and IM-PU samples were most associated with the presence of TPC, TFC, antioxidant activity, and the phenolic compounds, except for rutin content which was only present in IM-PE. The response variables showed that when rosmarinic acid was present in the extracts, caffeic acid was also found, and both had a substantial relationship with TPC and antioxidant activity (ABTS and DPPH).

The findings in PCA showed that the stage of maturity had a relationship with TPC, TFC, antioxidant activity, and phenolic compounds, it being the IM fruit that were the most related to those variable responses and their presence decreased as the stage of maturity increased. On the other hand, in evaluating the part of the fruit, the PE was related with the presence of all phenolic compounds, and they were associated with the antioxidant activity. The same behavior was observed with rosmarinic acid and caffeic acid in the extracts from peels, which may explain the highest antioxidant activity in peels from ciricote. Similar behavior was reported by Jediyi et al. (2019) [42], who showed that different varieties of grapes at the green maturity stage had a higher content in acid phenolic compounds than grapes in the ripening stage.

4.6. Effects of the Diameter of the Ultrasonic Probe in Extracts of Fruit

In almost all cases of this study, the increase in the concentration of phenolic compounds could indeed be attributed to the effect of the shape and diameter of the ultrasonic probe used in the extraction [8] because the smaller diameter probes produce greater intensity in cavitation. Although the energy released is restricted to a narrower and more concentrated field than with a larger diameter probe, the latter produces less intensity [20]. Therefore, a favored extraction is intensified due to turbulence in the liquid medium, due to the acoustic cavitation. Additionally, the smaller diameter probes offer more efficiency in temperature control, avoiding rapid heating during the process, which is appropriate for obtaining higher phenolic content in a short processing time, and avoiding its degradation, as has been reported for the recovery of phenolic compounds [43].

5. Conclusions

Color variation and a maturity index were associated with three different stages of maturity in ciricote fruit. The IM fruit presented the highest content of TPC, TFC, and antioxidant activity for both the PU and PE. Nevertheless, the PE presented the highest values. The UAE method was better than the maceration method, increasing by several times the values of the TPC and antioxidant activity. Additionally, an increase in TPC, TFC, antioxidant activity, and phenolics content was also achieved by UAE using a 3 mm diameter ultrasonic probe. High positive Pearson correlation coefficients (0.98 to 0.99) were obtained among the total phenols content and flavonoids with antioxidant activity by DPPH and ABTS, which suggested the important contribution of these compounds to that activity. The main phenolics compounds identified by UPLC-DAD-ESI-MS/MS in the PE of IM fruit were rutin, caffeic acid, and rosmarinic acid, listed in the order of concentrations, and their content was influenced by the part of the fruit and stage of maturity. It was found that the TPC and antioxidant activity were highly associated with caffeic and rosmarinic acids, which means there was an important, but not exclusive, effect of these two phenolic compounds on the antioxidant activity. The IM fruit of the ciricote represents a good source of phytochemicals with possible pharmacological or food applications, which would promote the production and consumption of an underutilized native product that can help to preserve biodiversity.