Quantification of Phenolic Compounds and In Vitro Radical Scavenging Abilities with Leaf Extracts from Two Varieties of Psidium guajava L.

Guava leaf (Psidium guajava L.) extracts are used in both traditional medicine and the pharmaceutical industry. The antioxidant compounds in P. guajava leaves can have positive effects including anti-inflammatory, anti-hyperglycemic, hepatoprotective, analgesic, anti-cancer effects, as well as protecting against cardiovascular diseases. In the present study, phenolic compounds and in vitro antioxidant capacity were measured in extracts obtained with polar and non-polar solvents from leaves of two varieties of guava, Calvillo Siglo XXI and Hidrozac. The quantity of total phenolics and total flavonoids were expressed as equivalents of gallic acid and quercetin, respectively. Hydroxyl radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Oxygen Radical Absorbance Capacity using fluorescein (ORAC-FL) in vitro tests were used to assess the radical scavenging abilities of the extracts. The total phenolics were higher in the aqueous fraction of the variety Calvillo Siglo XXI, while in the Hidrozac variety total phenolics were higher in the acetone and chloroform fractions. Total flavonoids were higher in all fractions in the variety Calvillo Siglo XXI. Total phenolics showed a highly positive correlation for ORAC-FL, and a moderately positive correlation with hydroxyl radicals. Finally, total flavonoids showed a slightly positive correlation for ORAC-FL and hydroxyl radicals. Both varieties of guava leaf extract showed excellent antioxidant properties.


Introduction
Psidium guajava L. is a small tree that belongs to the Myrtaceae family. Guava is the common name used for species of P. guajava. The guava tree is distributed in tropical and subtropical regions of guava leaves. Currently, in Mexico these varieties do not have information regarding their content of total polyphenols, total flavonoids, and their antioxidant activity of their leaves. The only study that counts in Mexico of the variety Hidrozac and Calvillo is with its antioxidant activity of its fruit [27].
These varieties were registered in Mexico by the INIFAP (National Institute of Forestry, Agricultural and Livestock Research, Pabellón de Arteaga, Aguascalientes, México) in 2009 [4]. A difference between these two varieties is that Hidrozac produces pink pulp fruit while Calvillo Siglo XXI produces yellow pulp fruit. In the present work, it was found that there were significant differences in the amount of phenolic compounds and the radical scavenging abilities of Calvillo Siglo XXI and Hidrozac varieties. The correlation between total polyphenols and the radical scavenging abilities of the hydroxyl radicals and the ORAC test was positive.

Samples
The guava leaves were collected at the experimental site "Los cañones" of the INIFAP (National Institute of Forestry, Agricultural and Livestock Research), which is located in the municipality of Huanusco, Zacatecas, Mexico. Samples were collected 4 September 2015 and 24 August 2016. The coordinates of the collection site are longitude 21 • 44.7 N and 102 • 58.0 W to 1508 masl. Two varieties were selected, which are registered in the catalogue of vegetal varieties from 2009. Calvillo Siglo XXI no. GUA-005-160709 which produces yellow pulp fruits and Hidrozac no. GUA-002-160709 which produces pink pulp fruit. The collected leaves were dried in the shade and milled with a knife mill (PULVEX model 200, PULVEX SA. CV., Mexico City, Mexico) using a 0.8 mm diameter mesh. The ground material was stored in plastic bags in the dark at 22 • C prior to extraction.

Extractions
In all cases, regardless of the solvent used, the guava leaves were macerated under discontinuous stirring, with double extraction period (24 h × 2) at a solvent: sample ratio 10:1 (v/w). The samples collected in 2015 were subjected to extractions with solvents of different polarities: chloroform, acetone and water. For the extracts of the leaves collected in 2016, the samples were macerated only with water. The use of chloroform and acetone, and water, was to know the performance of the extracts before different solvents, and to know their composition in terms of total phenolic compounds and total flavonoids, as well as their antioxidant capacity. However, for the study of the leaves collected in 2016, it was decided to work only with water as a solvent, since it is the extract that would not present a possible toxicological effect, having the prospect of testing it on living organisms. In this way, we can have comparisons between the extracts obtained from the leaf collected both in 2015 and in 2016 using water as a solvent. Chloroform and acetone were recovered in a rotary evaporator (BM200, Yamato Scientific America Inc., Santa Clara, CA, USA) at 35 • C in a water bath, and the residual material was dried at 40 • C. The aqueous extract was dried using a nano spray dryer (B-90, BÜCHI Labortechnik AG, Flawil, Switzerland) using the following conditions: inlet temperature 105 • C, outlet temperature 38 • C, 90% sprinkler, and a 7 µm spray cap. Briefly, the liquid sample was fed to the spray head and droplets were generated by a piezoelectric driven actuator in a stainless steel membrane with holes of 7.0 µm inside the spray cap. The droplets were dried and the particles collected by electrostatic charging to deflect them to the collecting electrode. Nanometric particles were then collected. The dried extracts obtained for each solvent were stored in glass jars in the dark until use.

Total Phenolics
The total phenolic compounds were quantified using the methodology of Singleton et al. [28] and Taga et al. [29] with some modifications. 100 µg/mL of extracts was dissolved in 80% MeOH. Then, 100 µL of the solution was added to 100 µL of Folin-Ciocalteu reagent (1 N). This mix was stirred. After 3 min at rest, 2 mL of 2% m/v Na 2 CO 3 was added and stirred and incubated in the dark for 30 min. Absorbance was then measured at 750 nm in a spectrophotometer UV-vis (LAMBDA 365, PerkinElmer, Inc., Waltham, MA, USA). The results were expressed as milligrams equivalent of gallic acid per gram of extract (mg GAE/g). Determinations were carried out in triplicate. A calibration curve was constructed using gallic acid at 0-4 µg.

Total Flavonoids
Quantification of flavonoids was carried out using the methodology of Dewanto et al. [30] with some modifications. 100 µg per mL of extracts were dissolved in 80% MeOH. After, 250 µL of the solution were added to 1.250 µL of H 2 O, and 75 µL of NaNO 2 at 5% m/v. After 6 min 150 µL of 10% m/v AlCl 3 was added. After another 5 min 500 µL of 1 M NaOH and 275 µL of H 2 O were added. Subsequently, absorbance was measured at 510 nm in a UV-vis spectrophotometer (LAMBDA 365, PerkinElmer, Inc., Waltham, MA, USA). Results were expressed as milligrams equivalent of quercetin per gram of extract (mg QE/g) and determinations were made in triplicate. A calibration curve was constructed using quercetin at 0-30 µg.

Hydroxyl Radicals
The ability to scavenge hydroxyl radicals was determined using the methodology of Smirnoff and Cumbes [31] and Zhang et al. [32], with some modifications. 8 mM FeSO 4 , 3 Mm C 7 H 6 O 3 , and 2% H 2 O 2 were prepared with distilled water. The extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500-3000 µg/mL. The reaction mixture consisted of 667 µL of extract, 200 µL of FeSO 4 , 167 µL of H 2 O 2 , and 667 µL of C 7 H 6 O 3 . The mixture was placed in a water bath for 30 min at 37 • C, before 299 µL of distilled water was added and the absorbance was measured (A1) at a wavelength of 510 nm on a UV-vis spectrophotometer (LAMBDA 365, PerkinElmer, Inc., Waltham, MA, USA). The ability to scavenge hydroxyl radicals was calculated using the following formula (1) used by Fu et al. [33] where A0 is the absorbance of the control group (the sample solution was replaced with dilution solvent) and A2 the absorbance of the blank (H 2 O and sample solution). The half maximal inhibitory concentration (IC 50 ) was calculated.

ABTS
The ABTS was measured according to the methodology of Re et al. [34] and Tachakittirungrod et al. [35] with some modifications. A calibration curve was generated with Trolox and results were expressed as TEAC (trolox equivalent antioxidant capacity). ABTS was dissolved in distilled water at a concentration of 7 mM and mixed with a 2.45 mM solution of potassium persulfate in water. The mixture was left to stand for 15 h in the dark to generate the radical solution (ABTS• + ). For the tests, a fraction of the radical solution (ABTS• + ) was prepared by adding MeOH until an absorbance of 0.7 ± 0.2 at a wavelength of 750 nm was reached. 180 µL of the radical solution (ABTS• + ) was then mixed with 20 µL of extracts. After 5 min, absorbance was measured at a wavelength of 750 nm in a UV-vis spectrophotometer (LAMBDA 365, PerkinElmer, Inc., Waltham, MA, USA). The extracts were dissolved in MeOH at a concentration of 1 mg/mL.

DPPH
The assay was based on the method described by Brand-Williams [36], with some modifications. The reagents used as well as all the extracts were diluted with MeOH. The concentration of DPPH was 6 × 10 −5 M and ascorbic acid was prepared at concentrations of 50-200 µg/mL. The extracts were prepared at concentrations of 50-1000 µg/mL. For the test, 100 µL of extract or ascorbic acid were mixed with 1900 µL of DPPH solution and held in the dark for 15 min. Absorbance was measured at a wavelength of 515 nm in a UV-vis spectrophotometer (LAMBDA 365, PerkinElmer, Inc., Waltham, MA, USA). Ascorbic acid was used as a control and was quantified at the half maximal inhibitory concentration (IC 50 ).

Oxygen Radical Absorbance Capacity Using Fluorescein (ORAC-FL)
The ORAC-FL test was conducted using the method described by Dávalos et al. [37] and Ou et al. [38] with some modifications. A 75 mM phosphate buffer solution (PH 7.4) was prepared. This buffer was used to prepare 70 nM fluorescein, 12 nM 2,2 -Azobis(2-amidinopropane) dihydrochloride (AAPH), and 2-8 µM Trolox. The extracts were diluted with 80% MeOH at a concentration of 10 µg/mL. The test was performed in a 96 well microplate. 120 µL of fluorescein was mixed with 20 µL of sample or trolox and incubated for 30 min at 37 • C. Subsequently, 60 µL of AAPH was added immediately. The fluorescence was measured every minute for 120 min, stirring automatically before each reading. A blank was prepared by replacing the antioxidant with phosphate buffer solution.
The area under the curve of fluorescence was calculated with Equation (2), where f 0 is the initial fluorescence read at time 0 and f i is the fluorescence read at the time i. The net area under the curve (AUC) of samples was calculated by subtracting the AUC from the blank. Regression equations between net AUC and antioxidant concentration were calculated for all samples. A calibration curve was prepared with Trolox. The results were expressed as micromolar equivalents of Trolox for each microgram of extract (µM TE/µg). The measurements of fluorescence were performed at a temperature of 37 • C with an excitation wave of 485 nm and an emission wave of 520 nm in a spectral scanning multimode reader (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis
All experiments were done in triplicate. The values were presented as the average ± SD (n = 3). Significant differences in the groups were determined by the analysis of variance using the Tukey Test HSD (honest significant difference) by homogeneous groups, calculated with Statistica software (version 7 Statistica Ink., Palo Alto, CA 94304, USA). p < 0.05 was considered statistically significant. Excel 2016 software (Microsoft Office 365, One Microsoft Way, Redmond, WA, USA) and JMP8 software (SAS, Cary, NC, USA) were used for correlation analysis.

Total Phenolic and Total Flavonoid Compounds
Total polyphenols and total flavonoids were quantified in the aqueous, acetone, and chloroform extracts of both varieties of guava leaves. Table 1 shows results of total phenolic compounds. Acetone had the greatest quantity of phenolic compounds for both varieties, followed by the aqueous fraction, and finally the chloroform fraction.

Total Phenolic Compounds
Between the two varieties, the chloroform extracts were statistically equivalent. In the acetone fraction, the extracts of Hidrozac had more phenolic compounds (374.63 mg GAE/g extract), whereas in the aqueous fraction Calvillo Siglo XXI had a greater quantity (242.10 mg GAE/g extract). The total phenolic compounds in the extract of the acetone fraction for both varieties was more than double the 141 mg EAG/g measured by You et al. [18] who also extracted with acetone for one night following maceration. The phenolic compounds obtained in our aqueous extracts were greater than those quantified by Seo et al. [15] from extracts boiled for 4 h in guava leaves (140 mg gallic acid equivalent (GAE)/g).  Table 2 shows total extracted flavonoids. The extract with the greatest quantity of flavonoids, in general, was the acetone fraction, followed by the aqueous fraction, and finally the chloroform fraction. Between the two varieties, total flavonoid content in the chloroform fraction was statistically equivalent, while in the aqueous fraction and in the acetone fraction, Calvillo Siglo XXI had a greater quantity of total flavonoids. Total flavonoids in the aqueous fraction measured in our study was greater than the 50 mg QE value for aqueous extraction reported by Seo et al. [15]. Rattanachaikunsopon and Phumkhachorn [39] measured flavonoid contents of fresh and dry guava leaves by drying leaves for 72 h at 70 • C, extracting with methanol, and purifying using a chromatographic column. They found that flavonoid content was higher in dry leaves and that quercetin was the most abundant flavonoid. Different letters indicate significant differences (p < 0.05).

Radical Scavenging Ability In Vitro
The assays for radical scavenging ability in vitro were conducted with aqueous, acetone, and chloroform leaf extracts of both guava varieties.

Hydroxyl Radicals
In general, the chloroform fraction had the lowest IC 50 , followed by the aqueous fraction, and finally the acetone fraction (Table 3). Between the two varieties, chloroform or acetone showed no significant differences (p < 0.05), while the aqueous fraction did show significant differences (p < 0.05). The variety Calvillo Siglo XXI had a lower IC 50 in both the aqueous and chloroform fractions, while the Hidrozac variety had the lowest IC 50 in the acetone fraction. The IC 50 values measured in the present work are up to 20 times higher than those values reported by Kim et al. [40] for aqueous extracts obtained by maceration for 24 h (110 µg/mL). A half maximal effective concentration (EC 50 ) of 5470-6700 for 50 µg/mL extracts was reported by Ademiluyi et al. [41], who used a mixture of MeOH and HCl for extraction of four different varieties of macerated guava. Different letters indicate significant differences (p < 0.05).

ABTS
The results of ABTS radical scavenging tests are described in Table 4. The results were expressed as the antioxidant capacity equivalent to a millimolar of trolox per milligram of extract (TEAC mM/mg). In general, the extracts with the greatest TEAC were obtained from acetone fractions, followed by the chloroform fractions, and finally the aqueous fractions. Between the two varieties, only the aqueous fraction showed significant differences (p < 0.05). In the study carried out by Díaz-De-Cerio et al. [42] with aqueous extracts of guava leaves obtained by sonication for 10 min and infusion at 3.5 and 7 min, TEAC values ranged from 0.18 to 1.13. The values from that study are two to four times lower than the aqueous fraction values measured in the present work. Different letters indicate significant differences (p < 0.05); TEAC: trolox equivalent antioxidant capacity). ABTS: 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Table 5 shows the results of DPPH radical scavenging ability. The extracts with lower IC 50 values were those obtained using acetone as a solvent. All the extracts in both varieties were significantly different (p < 0.05). The IC 50 of the extracts of the acetone fraction obtained in this work was almost three times higher than IC 50 values measured by You et al. [18] (34 µg/mL). The IC 50 value of the aqueous fraction in the present work were also more than six times higher than the IC 50 value measured from the aqueous fraction of guava leaves macerated for 24 h by Kim et al. [40] (120 µg/mL). IC 50 values ranging from 730 to 910 µg/mL for extracts of four varieties of guava leaves macerated for 24 h using a mixture of MeOH/HCl have also been reported by Ademiluyi et al. [41], which are within the range measured in the present work.

ORAC-FL
The results of peroxyl scavenging ability were expressed as micromoles of trolox equivalents per microgram of extract (µm TE/µg) and are shown in Table 6. The extract with the greatest TE was the 2015 aqueous fraction of Calvillo Siglo XXI, with a value of 7.5 µM TE/µg. In general, the Calvillo Siglo XXI variety had larger values in water and acetone fractions than Hidrozac. In the chloroform fraction, no significant differences were observed (p < 0.05) for both varieties. It is worth mentioning that this is the first study reporting the use of ORAC to measure antioxidant capacity in extracts of guava leaves, obtaining interesting and complementary results to the measurement of antioxidant activity. This test can be recommended to study antioxidant capacity for both guava leaves and other extracts from different plant sources.

Correlation Analysis between Phenolic and Flavonoids Compounds and Radical Scavenging Ability
We observed a correlation between total phenolic compounds, total flavonoid compounds, ABTS, DPPH, radical hydroxyl, and ORAC test (Table 7). Comparing the correlation between the total flavonoids and the rest of the assays, the largest positive correlation was found with total phenolic compounds (0.61), followed by the hydroxyl radical test (0.38), and the ORAC value (0.31). The total flavonoids showed a strong negative correlation with the DPPH assay (−0.76). When total phenolic compounds were correlated with the rest of the assays, the greatest positive correlation was found against the ORAC assay (0.71), followed by the hydroxyl radical assay (0.69). Total phenolic compounds presented a strongly negative correlation with the DPPH assay (−0.59) and a weakly negative correlation with the ABTS assay (−0.15). Total phenolic compounds, ORAC value, and the hydroxyl radical (OH) assay had the highest positive correlations. This indicates that these assays are the most useful to measure antioxidant capacity in vitro from different extracts from leaves of P. guajava L. Chen et al. [43] reports a positive correlation between total polyphenols and other assays such as ABTS using guava leaf extracts. The negative correlation between the DPPH assay and total phenolics and total flavonoids found in this work also was reported by Ademiluyi et al. [41]. In addition, they reported a positive correlation between total phenolics and the ABTS assay, contrary to the data obtained in our research.

Conclusions
Significant differences in guava leaf extracts phenolic compound content and radical scavenging abilities were found between the varieties Calvillo Siglo XXI and Hidrozac. The phenolic and flavonoids contents as well as the ability to scavenging radicals were similar in both varieties, regardless of collection year. Extracts from guava leaves can donate electrons and hydrogen atoms, based on the results of the assays. Total polyphenol, hydroxyl radical, and ORAC assays are recommended for comparing differences between varieties. The novelty of this work is that there are no reports of tests with extracts of guava leaves from the varieties Hidrozac and Calvillo Siglo XXI. In addition, no work has been reported using the ORAC-FL trial with any variety of guava leaves. The results of this study will be complemented by a quantitative analysis of the phenolic compounds present in the different extracts by chromatographic methods. In addition, an in vivo test will be performed with rats in which aqueous extracts will be administered to evaluate their possible antihypertensive effect.