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. 2014 Feb 12;2(2):174–180. doi: 10.1002/fsn3.91

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

Jongkwon Seo 1, Soojung Lee 2, Marcus L Elam 1, Sarah A Johnson 1, Jonghoon Kang 3, Bahram H Arjmandi 1,*
PMCID: PMC3959964  PMID: 24804076

Abstract

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.

Keywords: Antioxidant, flavonoid, guava, hydroethanolic solvent, phenolic compound

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:

graphic file with name fsn30002-0174-m1.jpg (1)

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.

Figure 1

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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.

Figure 2

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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
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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. A–DMeans 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
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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.

Acknowledgments

This research was supported by High Value-added Food Technology Development Program of Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (20090237). The authors wish to thank Guava Korea Ltd. (South Korea) and Uiryeong Guava Agricultural Union Corporation (South Korea) for their support.

Conflict of Interest

None declared.

Funding Information

No funding information provided.

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