Study to find the best extraction solvent for use with guava leaves (Psidium guajava L.) for high antioxidant efficacy.

The effects of guava leaves extracted using solvents of water, ethanol, methanol, and different concentrations of hydroethanolic solvents on phenolic compounds and flavonoids, and antioxidant properties have been investigated. The antioxidant capability was assessed based on 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging abilities, reducing power, and nitric oxide-and nitrate-scavenging activities. The results demonstrated that the antioxidant ability of guava leaf extracts has a strong relationship with phenolic compound content rather than flavonoid content. Phenolic compound content of water extracted guava leaves was higher compared to pure ethanol and methanol extracts. However, phenolic compound content extracted using hydroethanolic solvent was higher than water, whereas 50% hydroethanolic was observed to be the most effective solvent showing high antioxidant ability.

Introduction

Medicinal plants have been used in the treatment and improvement of human diseases (Gutierrez et al. 2008; Nyirenda et al. 2012), and such plants with high antioxidant abilities can be used as natural medicines for preventing aging and chronic diseases (Kähkönen et al. 1999). In addition, these plants have various physiologically active substances with anticancer and antimicrobial abilities (Bhanot et al. 2011; Miyake and Hiramitsu 2011). The free radical-scavenging abilities of plants has been evaluated by in vitro models of scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide, hydroxyl radical, and nitric oxide radical, reducing power, lipid peroxidation levels, and antioxidant enzyme activities (Brand-Williams et al. 1995; Jayanthi and Lalitha 2011; Reddy et al. 2012). Reactive oxygen species (ROS), such as hydroxyl radical (·OH), superoxide anion (·O2−), and hydrogen peroxide (H2O2), which are produced in the cell system, are known to cause oxidative damage. This damage may cause cellular injuries and exacerbate several degenerative diseases associated with aging, cancer, and cardiovascular disease (Pham-Huy et al. 2008; Sharma and Singh 2012).

Guava (Psidium guajava L.), which is used as a traditional medicine, is found in countries with hot climates in areas such as South America, Europe, Africa, and Asia (Gutierrez et al. 2008). Its primary traditional uses include the alleviation of diarrhea and dehydration. Other reported uses include treatment of gastroenteritis, dysentery, stomach pain, diabetes mellitus, and wounds. In addition, it is known for its antioxidant, antibacterial, and anti-inflammatory properties (Qian and Nihorimbere 2004; Cheng et al. 2009; Han et al. 2011a). Guava leaves have phenolic compounds and flavonoids with high antioxidant activity. The main active substances in guava leaves are gallic acid, caffeic acid, guaijaverin (Gutierrez et al. 2008), tannins (Okuda et al. 1987), carotenoids (Mercadante et al. 1999), and triterpenoids (Shao et al. 2012). These substances have been extracted by using several solvents such as water (Moreno et al. 2000), ethanol, hydroethanol (Qian and Nihorimbere 2004), methanol (Chah et al. 2006), and hydromethanol (Bushra et al. 2012). However, there is a paucity of research investigating the most effective solvent for the antioxidant efficacy of guava leaves.

Therefore, in this study, the phenolic compound and flavonoid content of water, ethanol, methanol, and hydroethanolic extracts of guava leaves were analyzed and evaluated with regard to antioxidant properties. The best extraction solvent for use with guava leaves for high antioxidant efficacy was selected.

Material and Methods

Chemicals and reagents

Ethanol and methanol were purchased from Duksan (Jinju, Korea). Folin–Ciocalteu reagent, caffeic acid, quercetin, DPPH, ABTS, potassium ferricyanide, trichloroacetic acid, ferric chloride, sulfanilamide, phosphoric acid, and N-(1-naphthyl) ethylenediamide were purchased from Sigma (St. Louis, MO). Potassium acetate, sulfanilic acid, and naphthylamine were purchased from Yakuri (Osaka, Japan). All chemicals and reagents were of analytical grade. Guava leaves were obtained from Guava Korea Ltd. (Uiryeong-gun, Korea).

Preparation of water extracts

As described by Kandil et al. (1994), a sample of 100 g guava leaves in 1.5 L distilled water was boiled for 4 h. The sample was then filtered using Whatman filter paper No. 4. The filtrate was concentrated in a rotary evaporator at 60°C and dried using a freeze drier. The resulting extracts were stored at −18°C until the analysis.

Preparation of ethanol, methanol, and hydroethanolic extracts

The ethanol and methanol extracts were prepared by placing a sample of 100 g of guava leaves in 1.5 L pure ethanol (purity 94.0%) and 1.5 L pure methanol (purity 99.8%), respectively, for 4 days at room temperature. For the hydroethanolic extracts, hydroethanol solvents with water:ethanol in the ratios of 70:30, 50:50, 30:70, and 10:90 (v/v) were prepared for use in the extraction. After 4 days, the extracts were filtered using Whatman filter paper No. 4, and then the filtrates were concentrated using a rotary evaporator at 50°C. The resulting filtrates were dried using a freeze drier and stored at −18°C until further analysis.

Phenolic compound content assay

The Folin–Ciocalteu method (Ainsworth and Gillespie 2007) with a modification was used to determine the phenolic compound content of the samples. One milliliter of each extract was diluted with 2 mL distilled water and 0.5 mL of Folin–Ciocalteu reagent (Sigma Co.). After 3 min, 0.5 mL of 10% Na2CO3 solution was added to the mixture and the mixture was allowed to stand for 1 h at room temperature in a dark room. The absorbance was measured at 760 nm with a UV–visible spectrophotometer (Optizen 2120 UV; Mecasys Co., Ltd., Daejeon, Korea). A standard caffeic acid (Sigma Co.) solution (10–100 μg/mL) was used for the construction of a calibration curve. Results were expressed as mg caffeic acid/g extract. The tests were run in triplicate and averaged.

Flavonoid content assay

Flavonoid content was determined by Moreno's method (Moreno et al. 2000). Each extract (1 mL) was added to a test tube containing 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 mol/L aqueous potassium acetate, and 4.3 mL of 80% ethanol. After 40 min at room temperature in a dark room, the absorbance was measured at 415 nm. Total flavonoid content was assessed using quercetin (Sigma Co.) as a standard (0–100 μg/mL).

2,2-Diphenyl-1-picrylhydrazyl radical (DPPH.)-scavenging assay

A series of water, ethanol, methanol, and hydroethanol guava leaf extracts (50, 100, 250, 500 and 1000 μg/mL) were prepared for an antioxidant assay. Scavenging activity of the extracts on DPPH. was measured according to the method developed by Blois (1958). Varying concentrations of the guava leaf extract solutions (1 mL) were added to a DPPH. methanol solution (5 mg/100 mL, 2 mL). The decrease in absorbance at 517 nm was measured with a UV–visible spectrophotometer. DPPH.-scavenging activity (%) was calculated according to the following equation:

where Asample is the absorbance of the sample solution in a steady state and A0 is the absorbance of DPPH. solution before adding the extract.

Scavenging activity on ABTS.+

ABTS.+-scavenging activity was assessed according to the method described by Re et al. (1999). A mixture of ABTS (7.0 mmol/L) and potassium persulfate (2.45 mmol/L) in water was prepared and stored at room temperature for 12 h in a dark room to produce ABTS.+. The ABTS.+ solution in water was diluted to the level of absorbance of 1.50 at 414 nm for the analysis. Different concentrations of the extract solution (1 mL) were added to the diluted ABTS.+ solution (2 mL). The absorbance was recorded at 414 nm. The radical-scavenging activity was measured according to equation (1).

Reducing power assay

The reducing power was measured by the browning reaction method (Oyaizu 1986). Varying concentrations of the extract solutions (1.0 mL) were mixed with phosphate buffer (pH 6.6, 1.0 mL, 0.2 mol/L) and 1% aqueous potassium ferricyanide (1.0 mL). The mixture was incubated for 20 min at 50°C. An aliquot (1.0 mL) of 10% aqueous trichloroacetic acid was added to the mixture, which was subsequently centrifuged for 10 min at 5000 rpm. The upper layer of the solution (1.0 mL) was mixed with pure water (1.0 mL) and 0.1% aqueous FeCl3 (1.0 mL), and the absorbance was measured at 700 nm.

Nitric oxide radical-scavenging activity

Nitric oxide radical (NO.)-scavenging activity was measured by the Greiss reagent as described in a previous study (Sumanont et al. 2004). Sodium nitroprusside (5 mmol/L) was dissolved in phosphate buffer (pH 7.4, 2 mL), mixed with flavonoid solution (1 mL), and incubated at 25°C for 150 min. The Greiss reagent (0.5 mL) consisted of 2% sulfanilamide in 4% aqueous H3PO4, and 0.1% aqueous N-(1-naphthyl) ethylenediamide (1:1, v/v) was added to the sample solutions. The absorbance was measured at 542 nm. The percentage of scavenging activity was calculated according to equation (1).

Nitrite-scavenging activity

Nitrite-scavenging activity was evaluated based on the absorbance at 520 nm using a UV-spectrophotometer according to the method reported by Kato et al. (1987). One milliliter of 1 mmol/L NaNO2 (Sigma Co.) solution was added to 1 mL of each sample, and the resulting mixtures were adjusted to pH 2.5 using 0.1 N HCl and 0.2 N citric acid solutions. Each sample was allowed to react at 37°C for 1 h, after which 1 mL of each sample was taken from the solution and mixed thoroughly with 3 mL of 2% acetic acid and 0.4 mL of the Griess reagent. The solutions were stored at room temperature for 15 min. The Griess reagent was prepared by mixing an equal amount of 1% sulfanilic acid (Sigma Co.) and 1% naphthylamine (Sigma Co.), which were made with 3% acetic acid. Nitrite-scavenging activity was calculated according to equation (1).

Statistical analysis

All experiments were carried out in triplicate. Values are presented as mean ± SD (n = 3). Statistical differences among the groups were determined by analysis of variance followed by Duncan's multiple range test using the SPSS program (version 12.0; SPSS Inc., Chicago, IL) package. P < 0.05 was considered statistically significant.

Results and Discussion

Phenolic compound and flavonoid content

Total phenolic compound and flavonoid content of guava leaf extracts are listed in Figures 1, 2. The phenolic compound content of water extract was higher than that of the pure ethanol and pure methanol extracts (Fig. 1A). Furthermore, the phenolic compound content of the hydrophenolic extracts was higher than that of the water extract, and the highest content of phenolic compounds was in the 50% hydroethanolic extract (Fig. 2A). Among the three solvent extracts, the flavonoid content of the water and ethanol extracts was higher than that of the methanol extract (Fig. 1B). Among the four concentrations of the hydroethanolic extracts, the flavonoid content of the 70% hydroethanolic extract was the highest (Fig. 2B).

Figure 1

Phenolic compound and flavonoid content of guava leaf extracts for each extract solvent. Phenolic compound content of guava leaf extract (A), Flavonoid content of guava leaf extract (B). The results are expressed as mean ± SD. The significance of differences was determined by one-way analysis of variance using SPSS version 12.0. A P < 0.05 indicates that the difference is significant.

Figure 2

Phenolic compound and flavonoid content of guava leaf extracts for each concentration of hydroethanolic solvent. Phenolic compound content of guava leaf hydroethanolic extracts (A), flavonoid content of guava leaf hydroethanolic extracts (B). H.E., hydroethanolic extract. The results are expressed as mean ± SD. The significance of differences was determined by one-way analysis of variance using SPSS version 12.0. A P < 0.05 indicates that the difference is significant.

This result is consistent with previous reports showing that the phenolic compound content of water extract was higher than in pure ethanol and pure methanol extracts (Reddy et al. 2012; Aktumsek et al. 2013). Nyirenda et al. (2012) reported that polar compounds, such as phenolic compounds and flavonoids, were more soluble in aqueous solvents than in organic solvents. It was reported that the phenolic compound content of 50% hydroethanolic extract was higher than in the water extract of guava leaves (Qian and Nihorimbere 2004). Another study found that the order of increasing phenolic compound content of Hieracium pilosella was 50% hydroethanolic extract > 80% hydromethanolic extract > water extract (Stanojević et al. 2009). According to another research study, the phenolic compound content was highest in 40% hydroethanolic extract (Ito et al. 2012). Our results agree with several studies that examined the relationship between phenolic compounds and antioxidant capacity. A previous study found that antioxidant capacity varied according to the phenolic compound profile (Kosińska et al. 2012). Another study reported that there are positive correlations between the phenolic compound concentration and antioxidant ability (Kim et al., 2008).

DPPH-.and ABTS.+-scavenging activity and reducing power

The antioxidant properties of three solvent extracts were evaluated by in vitro tests including DPPH-. and ABTS.+-scavenging activity and reducing power (Table 1). In all measurements, the antioxidant capacity was observed to be significantly higher in the water extract that had the highest content of phenolic compounds, which suggests a positive correlation between the antioxidant capacity and the phenolic compound content. The antioxidant activities increased depending on the concentration of the extracts. Furthermore, the antioxidant activity of the hydroethanolic extracts was higher than that of the water extracts and was highest for 50% hydroethanolic extract (Table 2).

Table 1

Antioxidant activities of guava leaf extracts for each of the three extract solvents.

	Solvents	Sample concentration (μg/mL)
		50	100	250	500	1000
DPPH	Water	28.12 ± 0.21aB	51.51 ± 1.09bC	89.00 ± 0.52cC	92.79 ± 0.15dB	93.86 ± 0.06eB
	Ethanol	18.97 ± 1.66aA	35.57 ± 2.87bB	71.80 ± 0.53cB	92.78 ± 0.35dB	92.95 ± 0.08dB
	Methanol	18.76 ± 3.58aA	24.33 ± 1.20bA	49.88 ± 1.63cA	88.07 ± 2.22dA	90.29 ± 2.05dA
ABTS	Water	37.17 ± 0.37aC	64.27 ± 0.23bC	97.18 ± 0.00cC	98.29 ± 0.13dC	98.74 ± 0.07eC
	Ethanol	21.12 ± 0.38aB	41.05 ± 3.77bB	81.01 ± 1.12cB	91.27 ± 0.26dB	94.26 ± 0.19dB
	Methanol	16.25 ± 2.87aA	25.89 ± 3.73bA	50.17 ± 3.48cA	82.22 ± 1.89dA	85.09 ± 0.27dA
Reducing power	Water	0.19 ± 0.00aC	0.28 ± 0.00bC	0.51 ± 0.01cC	0.83 ± 0.01dC	1.35 ± 0.00eC
	Ethanol	0.12 ± 0.01aA	0.16 ± 0.01bA	0.24 ± 0.00cA	0.40 ± 0.00dA	0.69 ± 0.02eA
	Methanol	0.16 ± 0.00aB	0.21 ± 0.01bB	0.40 ± 0.01cB	0.67 ± 0.03dB	1.15 ± 0.02eB
NO	Water	12.54 ± 1.42aA	14.45 ± 2.30abA	18.05 ± 2.52bA	27.32 ± 2.76cA	35.20 ± 2.13dA
	Ethanol	27.29 ± 0.71aB	34.62 ± 0.37bC	36.22 ± 2.32bC	39.76 ± 0.09cC	41.67 ± 0.65cB
	Methanol	25.33 ± 1.89aB	28.63 ± 1.47abB	29.38 ± 1.62bB	29.56 ± 1.59bB	35.44 ± 2.63cA
NO2	Water	15.52 ± 2.03aB	19.26 ± 1.96bB	33.45 ± 0.54cA	56.61 ± 1.28dB	82.99 ± 0.64eC
	Ethanol	3.23 ± 0.17aA	13.91 ± 1.34bA	34.18 ± 0.70cA	61.87 ± 1.23dC	80.50 ± 1.17eB
	Methanol	14.64 ± 1.83aB	17.36 ± 0.92bB	43.45 ± 1.78cC	53.57 ± 1.09dA	68.96 ± 1.66eA

The results are expressed as mean ± SD. The significance of differences was determined by one-way analysis of variance using SPSS version 12.0. A P < 0.05 indicates that the difference is significant. a–dMeans with different superscripts in the same row show significant difference. AMeans with different superscripts in the same column show significant difference. NS, not significant.

Table 2

Antioxidant activities of guava leaf extracts for each concentration of hydroethanolic solvent.

	Solvents	Sample concentration (μg/mL)
		50	100	250	500	1000
DPPH	30% H.E.*	27.06 ± 0.74aB	53.80 ± 2.31bC	92.08 ± 1.49cB	96.03 ± 0.04dNS	95.76 ± 0.00dB
	50% H.E.	34.66 ± 2.15aC	62.14 ± 1.61bD	95.43 ± 0.46cC	95.89 ± 0.11d	95.68 ± 0.07cA
	70% H.E	23.72 ± 1.01aA	48.82 ± 0.48bB	91.11 ± 2.10cB	95.99 ± 0.07d	95.83 ± 0.04dC
	90% H.E.	21.64 ± 0.58aA	41.37 ± 1.96bA	79.73 ± 0.53cA	95.96 ± 0.15d	95.86 ± 0.02dC
ABTS	30% H.E.	41.90 ± 0.74aC	73.44 ± 0.98bC	98.21 ± 0.00cB	98.07 ± 0.00dB	97.91 ± 0.04dB
	50% H.E.	47.48 ± 0.60aD	84.49 ± 0.66bD	98.21 ± 0.00cB	98.07 ± 0.00dB	97.79 ± 0.14cB
	70% H.E	38.59 ± 0.90aB	68.57 ± 1.08bB	98.62 ± 0.07cC	98.42 ± 0.14dC	97.82 ± 0.22dB
	90% H.E.	32.39 ± 0.45aA	58.53 ± 0.59bA	96.88 ± 0.14cA	97.36 ± 0.04dA	96.28 ± 0.36dA
Reducing power	30% H.E.	0.23 ± 0.00aB	0.34 ± 0.00bB	0.64 ± 0.01cC	1.14 ± 0.01dC	2.12 ± 0.01eC
	50% H.E.	0.26 ± 0.00aC	0.38 ± 0.01bC	0.76 ± 0.01cD	1.36 ± 0.01dD	2.39 ± 0.01eD
	70% H.E	0.23 ± 0.00aB	0.33 ± 0.01bB	0.61 ± 0.01cB	1.07 ± 0.01dB	2.02 ± 0.02eB
	90% H.E.	0.21 ± 0.00aA	0.29 ± 0.00bA	0.52 ± 0.01cA	0.97 ± 0.01dA	1.68 ± 0.01eA
NO	30% H.E.	42.19 ± 0.56aB	49.45 ± 0.94bA	54.86 ± 1.25cA	63.03 ± 1.05dA	68.96 ± 0.28eB
	50% H.E.	52.45 ± 2.45aC	59.96 ± 0.29bC	57.55 ± 0.91cB	66.71 ± 0.24dB	73.07 ± 0.00eC
	70% H.E	45.09 ± 3.31aB	58.22 ± 0.91bC	56.16 ± 1.41bAB	64.02 ± 0.49cA	69.10 ± 2.01dB
	90% H.E.	34.42 ± 4.29aA	55.74 ± 1.37bB	60.06 ± 0.94cC	64.49 ± 0.94dA	64.88 ± 1.13eA
NO2	30% H.E.	19.32 ± 1.43aA	34.04 ± 2.56bAB	67.14 ± 0.93cB	83.39 ± 1.41dB	93.52 ± 0.20eB
	50% H.E.	35.45 ± 2.70aC	47.94 ± 1.95bC	77.86 ± 2.48cC	94.11 ± 1.34dD	96.67 ± 0.60dD
	70% H.E	24.97 ± 1.47aB	37.22 ± 0.89bB	67.26 ± 0.54cB	86.57 ± 0.35dC	95.17 ± 0.20eC
	90% H.E.	17.79 ± 1.14aA	31.21 ± 1.74bA	59.72 ± 0.35cA	80.21 ± 0.35dA	91.99 ± 0.54eA

The results are expressed as mean ± SD. The significance of the differences was determined by one-way analysis of variance using SPSS version 12.0. A P < 0.05 is considered significant. a–dMeans with different superscripts in the same row show significant difference. A–DMeans with different superscripts in the same column show significant difference. H.E., hydroethanolic extract; NS, not significant.

Our results are consistent with previous reports. It was shown that DPPH-. and ABTS-scavenging activity and reducing power of guava leaves in the water extract were higher than in purely ethanol, methanol, hexane, and ethyl acetate extracts (Aktumsek et al. 2013). Furthermore, the activity of 50% hydroethanolic extract was observed to be even higher than that of the water extract (Qian and Nihorimbere 2004). It was reported that DPPH-. and ABTS-scavenging activities were significantly correlated with the total abundance of phenolic compounds (Tayade et al. 2013). Antioxidant activity is strongly correlated with reducing power, which increased depending on the concentration and reaction time of the extracts (Kwon et al. 2013). Our results clearly suggest that the antioxidant abilities of guava leaves, such as DPPH-. and ABTS-scavenging activity and reducing power, are closely dependent on the contents of the phenolic compounds.

Nitric oxide (NO) radical-and nitrite (NO2)-scavenging activity

Nitric oxide-scavenging activity of the ethanol extract with a high content of flavonoids was significantly higher than that of the water or methanol extract, while nitrite scavenging abilities of the three solvent extracts did not differ significantly (Table 1). In the test using the mixed solvents, both nitric oxide-and nitrite-scavenging abilities were significantly higher in the 50% hydroethanolic extract that had the highest content of phenolic compounds (Table 2).

In previous studies for the flavonoid content of Impatiens balsamina, potato peel, sugar beet pulp, and sesame cake, the flavonoid content of purely methanol extract was higher than that of the content of water or purely ethanol extracts (Su et al. 2012). However, the flavonoid content of 50% hydroethanolic extract was higher than that of other extracts, such as water and 80% methanol extracts (Stanojević et al. 2009). Furthermore, the nitrite-scavenging activity of plum with high flavonoid content in 80% ethanol extracts was higher than that of two other kinds of plum that had low levels of flavonoid content (Kim et al. 2012).

According to this study, the water extract with a high content of phenolic compounds showed high antioxidant abilities in the DPPH-. and ABTS.+-scavenging activity and in the reducing power assay. Ethanol extract with a high flavonoid content showed high antioxidant activities in the nitric oxide radical-and nitrite-scavenging ability assay. In the antioxidant ability tests of hydroethanolic extracts, as measured by DPPH-. and ABTS.+-scavenging activity, reducing power, and nitric oxide and nitrite-scavenging activity, the activity of 50% hydroethanolic extract was the highest among the three different solvents and the other hydroethanolic extracts. This comparison strongly suggests that the best extraction solvent for high antioxidant efficacy of guava leaves is the 50% hydroethanolic solvent.

Conclusion

This study intended to find the best extraction solvent for high antioxidant efficacy of guava leaves using various solvents. The phenolic compound content of water extract was higher than pure ethanol and methanol extract. Furthermore, the phenolic compound content of hydroethanolic extracts was higher than water extracts. The antioxidant activity of hydroethanolic extracts was higher than that of the water extracts and was significantly high in the 50% hydroethanolic extract that had the highest content of phenolic compounds.