Author’s Accepted Manuscript

Antioxidant activities and polyphenols of sweet

potato (Ipomoea batatas L.) leaves extracted with
solvents of various polarities

Zhi-feng Fu, Zong-cai Tu, Lu Zhang, Hui Wang,

Qing-hui Wen, Tao Huang
www.elsevier.com/locate/sdj

PII: S2212-4292(16)30020-7
DOI: http://dx.doi.org/10.1016/j.fbio.2016.04.004
Reference: FBIO150
To appear in: Food Bioscience
Received date: 22 December 2015
Revised date: 16 April 2016
Accepted date: 24 April 2016
Cite this article as: Zhi-feng Fu, Zong-cai Tu, Lu Zhang, Hui Wang, Qing-hui
Wen and Tao Huang, Antioxidant activities and polyphenols of sweet potato
(Ipomoea batatas L.) leaves extracted with solvents of various polarities, Food
Bioscience, http://dx.doi.org/10.1016/j.fbio.2016.04.004
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 Antioxidant activities and polyphenols of sweet potato
(Ipomoea batatas L.) leaves extracted with solvents of
various polarities
Zhi-feng Fua, Zong-cai Tua,b,*, Lu Zhangb , Hui Wanga,*, Qing-hui Wena, Tao Huanga

a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

b
Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science,

Jiangxi Normal University, Nanchang, Jiangxi 330022, China

tuzc_mail@aliyun.com

wanghui00072@aliyun.com
*
Corresponding authors. Prof. Zongcai Tu, Dr. Hui Wang, 235 Nanjing Easter Road, Nanchang,

Jiangxi, China. Fax: +86-791-8830-5938. Tel.: +86-791-88305938

Abstract

Sweet potato (Ipomoea batatas L.) leaves are under utilized vegetables with abundant polyphenols. In this study,

ten different solvents, including water, aqueous methanol, aqueous ethanol and aqueous acetone were used to

investigate the effect of solvents on the recovery and antioxidant activities of polyphenols from sweet potato leaves.

Results indicated that the total phenolic content in extracts ranged from 23.3 to 43.8 mg CAE/g DM, and 70%

ethanol extract had the highest total flavonoid (3.4 mg QE/g DM) and total anthocyanin (36.5 mg c-3-gE/100g DM)

content. While, 50% acetone extract exhibited the highest crude extract yield (33.4% in dry materials), total

phenolic content (43.8 mg CAE/g DM), as well as the strongest in vitro antioxidant activities. The best correlation

with antioxidant activities was observed on total phenolics. Totally 14 phenolics were identified or tentatively

identified in 50% acetone extract, caffeoylquinic acid (CQA) and quercetin derivatives were indicated as the most

abundant phenolics. HPLC analysis also revealed that the extraction efficiency of 3-CQA, caffeic acid, 3,4-, 3,5-
and 4,5-diCQA varied with the polarity of tested solvent, and 70% acetone showed the highest extraction capacity

for 3,4-, 3,5- and 4,5-diCQA. All these data demonstrated that the types of extracting solvents impact the recovery

and antioxidant activities of sweet potato leaf polyphenols greatly, and 50% (v/v) acetone is quite an efficient

solvent to recover polyphenols and antioxidants from sweet potato leaves.

Abbreviations

CAE, chlorogenic acid equivalents; DM, dried sweet potato leaf material; HPLC-QTOF-MS/MS, high-performance

liquid chromatography coupled to diode array detector, electrospray ionization quadruple time-of-flight tandem

mass spectrometry; DPPH, 2,2-diphenyl-pic-rylhydrazyl; CA, caffeic acid; CQA, caffeoylquinic acid; diCQA,

dicaffeoylquinic acid; triCQA, tricaffeoylquinic acid; TPC, total phenolic content; TFC, total flavonoid content; QE,

quercetin equivalent; TAC, total anthocyanin content; c-3-gE, cyanidin-3-glucoside equivalents; VcE, ascorbic acid

equivalent; HO·, hydroxyl radical; BPC, base peak chromatography; Rt, retention times; λmax, maximum UV

absorption wavelength; SPL, sweet potato leaves.

Keywords: Ipomoea batatas L. leaf; solvent efficiency; antioxidant activity; caffeoylquinc acids;

HPLC-QTOF-MS/MS

1. Introduction

Sweet potato (Ipomoea batatas L.) is one of the most important food crops in the world (Konczak-Islam et al.,

2003). China is the leading producer, with an annual production of 705 million tons (68.40% of the world’s

production) in 2013 (FAO (Food and Agriculture Organization), 2013). Sweet potato leaves (SPL), the main
byproduct of sweet potato production, are rich in polyphenols, proteins, vitamins, minerals and some functional

microcomponents (Ishida et al., 2000). The polyphenol content of 40 SPL cultivars were two to three times higher

than some common vegetables (e.g. spinach, kale) (Xi et al., 2015a). Pharmaceutical studies revealed that sweet

potato leaf polyphenols exhibited various health-promoting biological activities, such as antioxidant activity,

anti-mutagenicity, anti-cancer, anti-carcinogenesis, anti-microbial, anti-diabetes, anti-inflammation et al. (Kurata et

al., 2007; Mbaeyi-Nwaoha & Emejulu, 2013; Taira et al., 2013). In addition, Xi et al. (2015b) revealed that the O2-

scavenging of 20 μg/mL of YuZi No 7 sweet potato leaf polyphenols was almost 3.1, 5.9 and 9.6 times of ascorbic

acid, tea polyphenols and grape seed polyphenols, respectively. Flavonoids, phenolics and anthocyanins were

reported to be the major bio-active components (Carvalho et al., 2010; Islam et al., 2003; Islam, 2006). SPL are

generally utilized as green vegetables in some regions of the world, especially in Southeast Asia. However, they are

greatly neglected in China, and most of them are discarded as waste except for a small part used as vegetable or

livestock feed, resulting in a huge waste of natural resource (Huang et al., 2013). Thus it is of great importance to

explore the high-value utilization of sweet potato leaves.

Polyphenols are rich in vegetables, fruits, cereals, herbs, and beverages such as tea, red wine and coffee et al.

They are important ingredients in pharmaceutical and food industries. Numerous researches indicated that

polyphenols provide enormous health benefits for human (El, 2009; Joseph et al., 2015; Petti & Scully, 2009;

Yoshimoto et al., 2002). Extraction is the first step for obtaining polyphenols from natural products. The extraction

efficiency of polyphenols depends on several factors, such as solvent to material ratio, extraction method,

extraction time, extraction temperature and extraction solvent. Among these, solvent type has been considered as

one of the most important factor (Dorta et al., 2012). Organic solvents (ethanol, methanol, acetone, diethyl ether et

al.) and their aqueous solvents are the general solvents used for extracting polyphenols from plants. However, due

to the varied polarities and chemical characteristics of compounds present in different plants, it is still not clear
which solvent is more effective for extracting the antioxidant components from a target plant. For example,

Wijekoon et al. (2011) found 50% acetone to be the best solvent for extracting polyphenols in bunga kantan

inflorescence compared with aqueous methanol and ethanol. While, Zhang et al. (2014) compared the efficacy of

five solvents on extracting antioxidant constituents from Artemisia selengnesis Turcz, and noted that 50% ethanol

resulted in the highest polyphenol yield.

To the best of our knowledge, most previous studies were restricted to the usage of a single solvent for

polyphenol extraction, and no comparative study on the effect of different solvents on extracting polyphenols from

SPL was conducted. With this in mind, in our previous work, seven solvents (distilled water, methanol, ethanol,

acetone, n-butanol, ethyl acetate and chloroform) were used to extract polyphenols from SPL. Methanol, ethanol

and acetone extracts were evidenced to show higher total phenolic content and antioxidant activities than the others.

However, many studies indicated that aqueous organic solvent had a higher extraction efficiency than absolute

organic solvent (Metrouh-Amir et al., 2015; Wijekoon et al., 2011), and there is still no report comparing the effect

of these aqueous organic solvents on the recovery of polyphenols from SPL. Therefore, this study was designed to

screen the optimal solvent for extracting phenolics from SPL in terms of crude extract yield, total phenolic, total

flavonoid and total anthocyanin content, and antioxidant activities of extracts. Furthermore, phenolic profiles were

characterized by high-performance liquid chromatography/electrospray ionization with quadrupole time-of-flight

tandem mass spectrometry (HPLC-QTOF-MS/MS), and the major compounds were also quantified by using

HPLC.

2. Materials and methods

2.1. Chemicals and reagents

Folin-Ciocalteu’s (FC) reagent was obtained from Solarbio (Beijin, China). Sodium carbonate, aluminium
chloride, potassium chloride, potassium ferricyanide, methanol, ethanol and acetone were purchased from Damao

Chemical Co. (Tianjin, China). 2,2-Diphenyl-pic-rylhydrazyl (DPPH), caffeic acid (CA), quercetin,

3-caffeoylquinic acid (3-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA) and

4,5-dicaffeoylquinic acid (4,5-diCQA) were acquired from Sigma Chemical Co. (St. Louis, MO, USA). Methanol

and formic acid were HPLC grade and purchased from Merck (Darmstadt, HE, Germany). All other reagents used

in this study were analytical grade.

2.2. Materials

Fifty kilogram of fresh orange-fleshed SPL (Jishu No. 16) were collected from the farmland of Nanchang

suburbs (Nanchang, China) in November, 2013, and identified by professor Shi from the Department of Food

Science and Nutrition, Nanchang University. The fresh leaves were washed gently, dried, and grounded into

powders by an ultra-micro pulverizer (DFY-500C, Linda Wenling, Zhejiang, China). The powders were all passed

through a 60 mesh sieve, stored in sealed polyethylene bags and kept in a refrigerator at -20 ℃.

2.3. Preparation of samples

In this study, water, methanol/water (50, 70 and 90% v/v), ethanol/water (50, 70 and 90% v/v) and acetone/water

(50, 70 and 90% v/v) were used as solvents. For each solvent, 5.0 g of dried powder was mixed with 100 mL of

solvent and placed in an incubator shaker (THZ-82A, Jinchengguosheng, Jiangsu, China) at 150 rpm for 12 h at

room temperature (25 ± 1 ℃). The mixture was centrifuged at 621.6 g (TGL-10C, Anting, Shanghai, China) for 10

min, the residue was re-extracted under the same conditions. The supernatants were combined and made up to 250

mL, then 10 mL of sample solution was removed and kept for following chemical assays. The left (240 mL) was

evaporated to dryness in a rotary evaporator (RE-52A, Yarong, Shanghai, China) at 50 ℃ followed by freeze-dried.
The mass of extract was determined, the percentage yield of extract was calculated using the formula:

mextract
Yield (%)  100
m finepowder

Where mextract is the mass of the extract (g), mfine powder is the mass of SPL powder (4.8 g).

2.4. Determination of total phenolic content

Total phenolic content (TPC) was determined according to the method described by Zhou and Yu (2004) with

slight modifications. Properly diluted sample solution (1.0 mL) was mixed with 0.5 mL of Folin-Ciocalteau reagent

for 5 min, then 1.5 mL of 20% (m/v, g/mL) Na2CO3 and 5.0 mL of distilled water were added. The reaction mixture

was incubated for 30 min at room temperature in darkness, and the absorbance was measured at 765 nm using a

spectrophotometer (T6, Pgeneral, Beijing, China) against a blank (sample was replaced by extraction solvent). The

TPC was calculated from a calibration curve plotted using chlorogenic acid (10.0-100.0 μg/mL), the results were

expressed as mg of chlorogenic acid equivalents per gram of dried sweet potato leaf material (mg CAE/g DM).

2.5. Determination of total flavonoid content

Total flavonoid content (TFC) was determined using the AlCl3 colorimetric method (Do et al., 2014). Briefly, 2.0

mL of properly diluted extract solution was mixed with 2.0 mL of 2% (m/v) AlCl3·6H2O. Ten minutes later, the

absorbance was measured at 430 nm against a blank. Quercetin (2.0-12.0 μg/mL) was used as standard, the results

were expressed as mg of quercetin equivalents per gram of dried sweet potato leaf material (mg QE/g DM).

2.6. Determination of total anthocyanin content

Total anthocyanin content (TAC) was determined according to the pH differential method (Wang et al., 2013).

Each sample was diluted separately 10 times with 1.49% KCl water buffer (pH 1.0, acidified with HCl) and 1.64%
sodium acetate water buffer (pH 4.5, acidified with HCl) to a final volume of 2.0 mL. Then, absorbance of the

diluted samples was measured at 520 nm and 700 nm respectively, using an UV spectrophotometer. The TAC was

expressed as cyanidin-3-glucoside equivalents per 100 gram of dried sweet potato leaf material (c-3-gE/100g DM),

and calculated as follows:

Anthocyains(mg/L)  ( (A520nm - A700nm)pH1.0 - (A520nm - A700nm) pH4.5)  Mw  DF 1000/(  L)

Where Mw is the molecular weight for cyanidin-3-glucoside (449.2 g/moL), DF is the dilution factor, ε is the molar

absorbance of cyanidin-3-glucoside (26,900 L·cm-1·mol-1), and L is the cell path length (1.0 cm).

2.7. Antioxidant assays

2.7.1. DPPH radical scavenging activity assay

The DPPH radical (DPPH·) scavenging activity was determined following the method described by Meneses et al.

(2013). Briefly, 2.0 mL of diluted sample solution was mixed with 2.0 mL of DPPH solution (0.1 mM in 95%

ethanol). The absorbance (A1) was measured at 517 nm with individual extraction solvent as a blank after 30 min of

incubation at room temperature in darkness . The DPPH· scavenging activity (IDPPH·) was calculated as below:

A0 - (A1 - A2)
I DPPH .% =  100%
A0

Where A0 is the absorbance of control group (sample solution was replaced by extraction solvent), A2 is the

absorbance of sample blank (DPPH solution was replaced by 95% ethanol). Ascorbic acid was used as a positive

control. All results were done in triplicate and expressed as mg of ascorbic acid equivalents per gram of dried sweet

potato leaf material (mg VcE/g DM).

2.7.2. Reducing power assay

The reducing power was determined according to the method of Zhang et al. (2015) with minor modifications.

Firstly, 1.0 mL of properly diluted sample was mixed with 1.0 mL of 1% (w/v) potassium ferricyanide and
incubated at 50 ℃ for 20 min. Following, 1.0 mL of 10% (w/v) trichloroacetic acid was added, and 1.0 mL distilled

water and 0.4 mL 0.1%, (w/v) ferric chloride solution was added after 10 min of incubation. The absorbance of

reactive system was measured at 700 nm against a blank with sample replaced by individual extraction solvent. All

results were done in triplicate, higher absorbance indicates greater reducing power.

2.7.3. HO radical scavenging activity assay

The hydroxyl radical (HO·) scavenging activity was assayed as described by Qi et al. (2005) with some

modifications. The reaction mixture containing 0.6 mM FeSO4, 0.6 mM sodium salicylate, 0.6 mM H2O2 and 2.0

mL samples were incubated at 37 ℃ for 30 min. The absorbance (A1) was measured at 510 nm with individual

extraction solvent as a blank. The HO· scavenging activity (I) was calculated as follows:

A0 - (A1 - A2)
I HO ·% =  100%
A0

Where A0 is the absorbance of control group (sample solution was replaced by extraction solvent), A2 is the

absorbance of sample blank (H2O2 solution was replaced by water). Ascorbic acid was used as positive control, all

results were done in triplicate, and the results were expressed as mg of ascorbic acid equivalents per gram of dried

sweet potato leaf material (mg VcE/g DM).

2.8. Qualitative and quantitative analysis

2.8.1. Qualitative analysis by HPLC-QTOF-MS/MS

Identification of phenolics in extracts was done using an Agilent 6538 LC system equipped with a degasser, a

binary pump, a thermostated HiP-ALS auto-sampler, a TCC SL column oven and a DAD detector (Agilent

Technologies, Santa Clara, CA, USA). Separation of compounds was achieved on an Agilent Eclipse XDB-C18

column (250 mm × 4.6 mm, 0.25 μm). The mobile phase was consisted of water with 0.1% formic acid (phase A)

and methanol (phase B), and elution gradient program was: 10 to 35% B from 0 to 15 min, 35 to 80% B from 15 to
35 min, 100% B from 36 to 40 min, then the mobile phase was returned to 10% B for 10 min to re-equilibrated the

column. The flow rate was 1.0 mL/min, all samples were filtered through 0.45 μm micropore membranes prior to

injection (10 μL). The detection wavelengths were set at 320 nm and 360 nm.

The orthogonal time-of-flight mass spectrometer was done in negative electrospray ionization (ESI) mode. The

parameters were set as follows: capillary temperature, 350 ℃; capillary voltage, 4.0 kV; nebulizing gas pressure, 50

psi; drying gas flow, 10.0 L/min. Mass spectrometer was recorded from m/z 100 to m/z 1200.

2.8.2. Quantitative analysis by HPLC

Quantitative analysis of samples were done using a Hitachi HPLC system (Tokyo, Japan) coupled to a Hitachi

D-2000 HSM system controller, a L-2130 pump, a L-2200 auto-injector and a L-2400 UV-vis detector. The elution

conditions used for quantitative analysis were the same as those used for qualitative analysis, and the peaks were

detected at 320 nm. Quantification of the phenolic compound was done using external standard method. Calibration

curves of 3-CQA (y = 25263x - 7 0014, r = 0.996), caffeic acid (y = 49134x - 19362, r = 0.998), 3,4-diCQA (y =

25075x - 17471, r = 0.995), 3,5-diCQA (y = 31865x - 14933, r = 0.994) and 4,5-diCQA (y = 23912x - 62349, r =

0.999) were plotted resparatively to calculate the content of each identified phenolic acid.

2.9. Statistical analysis

All measurements were done in triplicate and presented as mean ± SD (n = 3). The LC-MS data were acquired

and analyzed by MassHunter Acquisition B.03.01 and Qualitative Analysis B.03.01. (Agilent Technologies, Santa

Clara, CA, USA). All data were analyzed using SPSS 13.0 statistical package (SPSS Inc., Chicago, IL, USA).

One-way analysis of variance (ANOVA) and Duncan’s multiple range method were used to analyze the differences

among data, correlation analysis was carried out with Pearson Bivariate Correlations. Differences at p < 0.05 were

considered to be significant.
3. Results and discussion

3.1. Crude extract yield and antioxidant compounds

3.1.1. Crude extract yield

Effect of extraction solvents on the crude extract yield of SPL phenolics is shown in Table 1. The solvent

efficacy of all tested solvents followed a descending order: 50% acetone > 50% methanol > 70% acetone > 70%

methanol = 50% ethanol = 70% ethanol > 90% methanol > water > 90% ethanol = 90% acetone. 50% acetone gave

the highest solvent efficacy with the yield of extract at 33.4%, followed by 50% methanol (32.4%), whereas 90%

ethanol and 90% acetone had the lowest solvent efficacy with the yield of 20.3% and 20.7%, respectively (p ≥ 0.05).

The different extract yield is related to the distinct polarities of extraction solvents and solubility of compounds in

the extraction solvent (Franco et al., 2008). Metrouh-Amir et al. (2015) also indicated that the polarity of used

solvent, as well as the chemical constituents in tested materials greatly impacted the yield of extract. It could also

be found that the crude extract yield was enhanced with increasing water concentration, which could be ascribed to

the increased solubility of other components, such as proteins and carbohydrates (Do et al., 2014).

3.1.2. Total phenolic content (TPC)

As shown in Table 1, the recovery of phenolics is significantly affected by the nature of extraction solvents, with

the TPC ranged from 23.3 to 43.8 mg CAE/g DM, which also confirmed that SPL is a good source of polyphenols.

50% acetone extract presented the highest TPC with the value of 43.8 mg CAE/g DM, followed by 70% acetone

extract (42.0 mg CAE/g DM), while the lowest TPC was found in water extract (23.3 mg CAE/g DM), which are in

agreement with the reports of Zhou and Yu (2004) on wheat bran extract and Wijekoon et al. (2011) on bunga

kantan inflorescence extracts, they all found 50% acetone to be the most effective solvent for recovering

polyphenols from these two materials. Aqueous acetone solutions (50-95%, v/v) have been reported to be adaptable
for extracting polyphenols from different natural sources, especially from protein matrices, since these mixtures are

able to degrade polyphenol-protein complexes (Meneses et al., 2013). Here, a high protein content (28.0%, not

displayed) was also found in dried SPL powder, which to some extend could explain the high extraction efficiency

of 50% acetone on SPL polyphenols. Recently, Mokrani and Madani (2016) found 60% acetone to the best solvent

to extract phenolic compounds in peach fruit (Prunus persica L.).

3.1.3. Total flavonoid content (TFC)

The recovery of flavonoids in SPL differed greatly according to the polarities of solvents used in the extraction

process. The TFC in 10 different solvent extracts ranged from 0.6 to 3.4 mg QE/g DM. The highest TFC was

detected in 70% ethanol and 90% acetone extracts, with the respective value of 3.4 and 3.3 mg QE/g DM, and no

significant difference was observed (p ≥ 0.05). While water extract obtained the lowest TFC (0.6 QE/g DM), which

could be due to the lower solubility of flavonoids in water than in aqueous organic solvents. It was observed that

the effect of solvents (except ethanol) on the recovery of flavonoids from sweet potato leaves was in contrast to that

of phenolics. For aqueous acetone and aqueous methanol solvent, a significant decrease in flavonoid yield was

observed with increasing water concentration, which implied that sweet potato leaves contained more low-polar

flavonoid. A similar trend was also observed by Do et al. (2014), who reported that increased ratio of water in

aqueous ethanol or acetone solution resulted in decreased extraction efficacy of flavonoids from Limnophila

aromatica.

3.1.4. Total anthocyanin content (TAC)

The TAC also presented significant differences (p < 0.05) among different solvent extracts. As shown in Table 1,

70% ethanol extract had the highest level of TAC (36.5 mg c-3-gE/100g DM), followed by 50% ethanol extract

(23.8 mg c-3-gE/100g DM), and the lowest level was detected in 90% acetone extract (10.7 mg c-3-gE/100g DM).

Therefore, it could be proposed that 70% ethanol was the best solvent to extract anthocyanins from SPL, Until now,
aqueous-ethanol solutions have been widely used to extract anthocyanins from various kinds of plant materials

(Lachman et al., 2009; Pedro et al., 2016).

3.2. Antioxidant activity

3.2.1. DPPH· scavenging activity

The DPPH· scavenging activity of extracts is shown in Fig. 1. All extracts demonstrated a relatively high

DPPH· scavenging activity except for water extract, and a significant difference (p < 0.05) was observed among all

samples. The highest DPPH· scavenging activity was found in 50% acetone extract (36.6 mg VcE/g DM), followed

by 70% acetone (35.1 mg VcE/g DM) and 50% ethanol extract (33.4 mg VcE/g DM), while water extract showed

the lowest scavenging activity with the value of 9.0 mg VcE/g DM, which was about 3-fold lower than that of 50%

acetone extract. Notably, in terms of aqueous ethanol or acetone extracts, the DPPH· scavenging activity was

improved with the increasing ratio of water in the extracting solvent (50% aqueous solvent > 70% aqueous solvent >

70% aqueous solvent), which suggested that the addition of appropriate amount of water to organic solvents could

facilitates the extraction of antioxidants from plants. It also has been evidenced that aqueous organic solvent

generally had a higher extraction efficiency on polyphenols than absolute organic solvent (Spigno et al., 2007).

Therefore, in this study it could be inferred that the increasing ratio of water in organic solvent resulted to a more

polar medium which facilitates the extraction of phenolic compounds, this in turn improved the DPPH· scavenging

ability of extracts. The same phenomenon was in agreement with Meneses et al. (2013). who compared the

antioxidant activity of brewer’s spent grain extracts prepared with various aqueous organic solvents.

3.2.2. Reducing Power

The reducing power of all extracts is presented in Fig. 2. A Significant difference was also observed among the

samples (p < 0.05). At the sample concentration of 1.2 mg DM/mL, the highest reducing power was found in 50%
acetone extract with the absorbance at 700 nm of 0.81. Unexpectedly, no significant difference was observed

among the extracts of 70% methanol, 50% ethanol, and 70% acetone (p ≥ 0.05). While the water extract exhibited

the lowest reducing value (OD700 of 0.37). Also in terms of ethanol or acetone extracts, the reducing power of

samples was reduced gradually with decreasing polarity of extraction solvent, which was in line with the trends of

DPPH· scavenging ability and TPC. These results also indicated that the solvent chosen plays an important role in

the antioxidant activity of plant extract.

3.2.3. HO· scavenging activity

The HO· scavenging activity of extracts is shown in Fig. 3, all extracts exhibited a relatively high HO·

scavenging activity, and was strongly influenced by the solvents used. The order of the HO· scavenging activity

was as follows: 50% acetone extract > 70% methanol extract = 50% ethanol extract > water extract > 70% ethanol

extract = 90% acetone extract > 50% methanol extract > 70% acetone extract > 90% methanol extract = 90%

ethanol extract. The highest scavenging activity was detected in 50% acetone extract with the activity of 27.8 mg

VcE/g DM. However, 90% methanol and 90% ethanol extracts produced the lowest HO· scavenging activity with

the value of 18.2 and 17.6 mg VcE/g DM, respectively, and no significant difference was observed (p ≥ 0.05).

Herein, it can be seen that the change of HO· scavenging ability of tested samples was quite different from the

results observed in DPPH· scavenging ability and reducing power, which could be due to the different mechanisms

of these three assays relied on. The latter two concern the ability of antioxidants to reduce radicals, but HO·

scavenging assay relies on the inhibition of HO· generation by chelating Fe2+ ions (Qi et al., 2005). Similar result

was also detected by Anusuya and Manian (2013).

The variation of obtained antioxidant activities could be resulted from the different polarities of solvents

employed in this study, leading to the extraction of a selected group of antioxidant compounds depending on their

chemical structures, polarities and solubility, then affecting the antioxidant capacity of extracts (Meneses et al.,
2013; Kchaou et al., 2013). Many researchers have investigated the effect of extraction solvents on the antioxidant

capacity of extracts. Wijekoon et al. (2011) indicated that 50% acetone extract exhibited the strongest antioxidant

activity (measured with DPPH and FRAP assay) when compared with other solvents. The acetone extracts of

lychee flower was evidenced to give higher antioxidant activity than methanol or water extracts (Liu et al., 2009).

On the other hand, Fernández-Agulló et al. (2013) stated that 50% ethanol of walnut (Juglans regia L.) green husk

gave the highest antioxidant activity. All these results indicated that the solvent of extraction has an great influence

on the antioxidant capacity of extracts, which confirms the results of this study. Considering the results of all three

assays, we concluded that 50% acetone extract had the highest antioxidant activity, and 50% acetone was the best

solvent to recovery the antioxidants in SPL..

3.2.4. Correlation analyses

Correlation analyses were conducted to analyze the relationship of TPC, TFC, TAA and antioxidant activities

using SPSS 13.0. In general, the correlation coefficient r at 0.90 ~ 1.00, 0.70 ~ 0.90, 0.50 ~ 0.70, 0.30 ~ 0.50 and

0.00 ~ 0.30 is considered as very high positive correlation, high positive correlation, moderate correlation, low

positive correlation, and negligible correlation, respectively (Hinkle et al. 2003). Phenolics showed the highest

positive Pearson's correlation coefficients with DPPH· scavenging capacity (r = 0.943), reducing power (r = 0.946),

and HO· scavenging activity (r = 0.644). However, a negligible correlation was observed between flavonoids and

DPPH· scavenging capacity (r = 0.212), reducing power (r = 0.059), and HO· scavenging activity (r = -0.553). In

addition, a negligible correlation was also noticed between total anthocyanin content and antioxidant activities,

with the r of 0.094, 0.017 and 0.133 for DPPH· scavenging ability, reducing power and HO· scavenging activity,

respectively. Aforementioned data indicated that phenolics or phenolic acids were the main contributors to the

antioxidant activity of SPL extracts, flavonoids and anthocyanin played a weak role on the antioxidant activity of

SPL. Zhang et al. (2015) and Islam, S. (2006) also indicated that phenolic acids were the major antioxidants present
in SPL. This speculation would be further proved by following phytochemical profiles analysis.

3.3. Identification of phenolic compounds in sweet potato leaves

In the present study, 50% acetone extract was selected for qualitative analysis because of the highest TPC and

antioxidant activities. The base peak chromatogram (BPC) of 50% acetone extract obtained by

HPLC-QTOF-MS/MS is shown in Fig. 4, all information used for compound identification were listed in Table 2.

Caffeic acid, 3-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA were used as standards. Totally, 14 compounds were

detected in 50% acetone extract by comparing the retention times (Rt), maximum absorbance wavelengths (λmax),

detected mass (MS), fragment ions (MS/MS) and molecular formula with available standards, references and

database (Metlin and Massbank).

Peaks 3 (354.31, C16H18O9), 5 (180.15, C9H8O4), 7 (516.45, C25H24O12), 8 (516.45, C25H24O12), 12 (516.45,

C25H24O12) were identified as 3-CQA, caffeic acid, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA, respectively, by

comparing the retention time and mass spectra with those of reference standards.

Peaks 1 and 4 displayed the same parent ion [M−H]− m/z 353.08 and λmax with peak 3, it was then assigned as

CQA isomers. Lin and Harnly (2008) reported the elution sequence of monocaffeoylquinic acids as 1-, 3-, 4- and

5-CQA. Thus peaks 1 and 4 were tentatively identified as 1-CQA and 4-CQA, respectively. Peaks 11 and 13

presented a [M-H]− ion at 677.20 (C27H34O20), and the MS/MS diagnostic fragment at m/z 353 [M-dicaffeoyl-H]-

corresponded to the caffeoylquinic acid moiety. Thus these two peaks were assigned to 3,4,5-triCQA and its isomer.

These compounds have already been identified from SPL by our group (Zhang et al., 2015).

Peak 2 gave a [M-H]− at m/z 341.08 (C15H18O9), the characteristic MS/MS fragment at m/z 179.03 (C9H8O4)

corresponded to the caffeic acid moiety resulted from a neutral loss of a hexose (-162). Therefore it was tentatively

identified as caffeic-hexoside.
 Peaks 6 (625.1410), 9 (463.0880) and 10 (463.0884) was tentatively identified as quercetin derivatives due to the

similar λmax at about 256 nm and 345 nm, and diagnostic fragment at 301 derived from the loss of dihexosyl (-324

Da), hexosyl (-162 Da) and hexosyl (-162 Da), respectively. In addition, characteristic fragment at m/z 300 was all

observed in the MS/MS spectra of these three compounds, indicating the glycosylation position of 3-OH (Fabre et

al., 2001). Therefore, peaks 6, 9 and 10 were proposed as quercetin-3-O-dihexoside, quercetin-3-O-hexoside and

quercetin-3-O-hexoside, respectively. These compounds were also identified by Zhang et al. (2015) from SPL.

Peak 14 gave a [M-H]− at m/z 409.02, the corresponding aglycone at m/z 329 was resulted from the loss of a SO3

(79.96 Da) moiety (Zhang et al., 2015). Diagnostic ions at m/z 329, 313 and 285 indicated the fragment of

[Y0-CH2]−, [Y0-2CH3]−, [Y0-2CH3-CO]−, respectively. Since ombuin has been detected in sweet potato leaves

(Rende et al., 1994), this peak was tentatively proposed as Ombuin-sulfate.

3.4. Quantification of the main compounds

Due to the lack of standards, only 3-CQA, caffeic acid, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA were quantified in

this research. Effect of extraction solvents on the content of above phenolic acids in extracts is shown in Table 3.

The tested extraction solvents exhibited significantly distinct extraction efficiency on the quantified compounds in

SPL. All the compounds were detected in different solvent extracts except for the absence of caffeic acid,

3,4-diCQA and 4,5-diCQA in water extract. 3,5-diCQA was the most abundant compound (0.05-11.21 mg/g DM)

in SPL, followed by 4,5-diCQA (0-7.41 mg/g DM), which is in agreement with previous studies reported by Jung et

al. (2011). 70% acetone showed the highest extraction capacity for 3,4-diCQA, 3,5-diCQA and 4,5-diCQA with the

values of 2.01, 11.21 and 8.12 mg/g DM, respectively, and 70% methanol had the strongest extraction capacity for

3-CQA (4.11 mg/g DM), while 50% ethanol had the best extraction capacity for CA (1.04 mg/g DM). However, all

these compounds were not detected or showed very low concentration in the water extract.
 It is noteworthy that, as for the aqueous solvents of ethanol and acetone, 70% aqueous solvents seemed to extract

more phenolic compounds listed in Table 3 than 50% or 90% aqueous solvents, which was slightly different from

the results obtained by Folin-Ciocalteu method (50% aqueous solvents extracts had the highest TPC). It could be

explained by the fact that Folin-Ciocalteu reagent is not specific to phenolic compounds, it can be interfered by a

number of substances including organic acids, reducing sugars, aromatic amines, sulfur dioxide, ascorbic acid and

other enediols and reductones (Šeruga et al., 2011; Sun et al., 2014). Solvents with higher polarity are more benefit

to extract some of these compounds, which leads to a higher detected value. Aforementioned results also evidenced

that the type of extracting solvents greatly affects the solubility of phenolic compounds in sweet potato leaves, and

solvents play a key role in the extraction of phenolic compounds.

4. Conclusion

This study is the first attempt for to compare the effect of solvents with various polarities on the recovery of

polyphenols and antioxidant activities of extracts from SPL, and is able to provide relevant and useful information

for the further utilization and research with SPL. The yield of total phenolics, total flavonoids, total anthocyanins,

and antioxidant activities of extracts were significantly impacted by the polarity of extraction solvent. Herein, 50%

acetone yielded the highest extraction yield (33.4%), total phenolic content (43.8 mg CAE/g DM), as well as the

strongest DPPH· scavenging activity (36.6 mg VcE/g DM), reducing power (OD700 = 0.81, at 1.2 mg DM/mL) and

HO· scavenging activity (27.8 mg VcE/g DM). The best recovery of flavonoids was found in 70% ethanol. Total

phenolic content gave the strongest correlation with the antioxidant activities of extracts. Totally, 14 phenolics were

identified or tentatively identified in 50% acetone extract, CQA and its derivatives were the dominant phenolics

in SPL, followed by quercetin derivatives. Solvent greatly affects the solubility of caffeic acid, 3-CQA, 3,4-diCQA,

3,5-diCQA and 4,5-diCQA, and 70% acetone exhibited the best extraction efficacy on diCQAs. The findings of this
study confirmed that SPL were rich in polyphenols, especially of caffeoylquinic acids derivatives, and 50% acetone

could be an efficient solvent to recover SPL polyphenols, and phenolic acids were the major antioxidants in SPL.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China

(No. 21276118) and the Collaborative Innovation Center for Major Ecological Security Issues of Jiangxi Province

and Monitoring Implementation (No. JXS-EW-00).

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DPPH · scavenging activity (mg VcE/g DM)

45
f
40 e
35 d
d d
30 c c
c
25
b
20

15
a
10
Methanol Methanol Methanol Ethanol Ethanol Ethanol Acetone Acetone Acetone Water
50% 70% 90% 50% 70% 90% 50% 70% 90%

Solvents

Fig. 1. DPPH· scavenging activity of different solvent extracts of sweet potato leaves. Bars not sharing a common

0.9 g
f f
(OD700 at 1.2 mg DM/mL)

0.8 f
e
Reducing power

0.7 d e
c
0.6
b
0.5
a
0.4

0.3

0.2
Methanol Methanol Methanol Ethanol Ethanol Ethanol Acetone Acetone Acetone Water
50% 70% 90% 50% 70% 90% 50% 70% 90%

Solvents
letter are significantly different (p < 0.05).

Fig. 2. Reducing power of different solvent extracts of sweet potato leaves. Bars not sharing a common letter are
 HO . scavenging activity (mg VcE /g DM)
32
f
28 e
e

24 d
bc cd cd
b
20 a
a

16

12

MethanolMethanol Methanol Ethanol Ethanol Ethanol Acetone Acetone Acetone Water

50% 70% 90% 50% 70% 90% 50% 70% 90%

significantly different (p < 0.05). Solvents

Fig. 3. HO· scavenging activity of different solvent extracts of sweet potato leaves. Bars not sharing a common

letter are significantly different (p < 0.05).

70000 8
60000 BPC in negative mode
Acount

50000 7
11
40000 12
30000 34
10
20000 9
1 5 14
10000 2 6 13
0
300 DAD=320 nm
8
250
intensity

200
150 7
100 3 10 12
50 1 2 4
5 6 9
11 13
0
8
80 DAD=360 nm
60
intensity

40 7 12
3 10
20 6 13
1 4
5 9 11 14
0
0 5 10 15 20 25 30 35 40 45
Retention time

Fig. 4. The base peak chromatograms (BPC) of 50% acetone extract obtained using HPLC-QTOF-MS/MS, and

corrresponding HPLC profiles at 320 nm and 360 nm.

Table 1 Crude extract yield, total phenolic, total flavonoid and total anthocyanin content of sweet potato leaf
extracts obtained by different solvents.

Extraction Anthocyanins
Total phenolics Flavonoids
Solvents yield (mg c-3-gE/100g
(mg CAE/g DM) (mg QE/g DM)
(%) DM)

50%
32.4 ± 0.1f 33.6 ± 1.2bc 1.0 ± 0.1b 13.8 ± 1.0ab
Methanol

70%
27.6 ± 0.2d 37.5 ± 0.3de 1.6 ± 0.0c 20.9 ± 2.0cd
Methanol

90%
25.7 ± 0.4c 31.5 ± 0.6b 2.8 ± 0.1d 20.2 ± 1.7cd
Methanol

50%
27.7 ± 0.2d 39.6 ± 1.7ef 1.3 ± 0.0c 23.8 ± 1.0d
Ethanol

70%
27.2 ± 0.1d 35.4 ± 1.2cd 3.4 ± 0.0e 36.5 ± 1.0e
Ethanol

90%
20.3 ± 0.5a 25.0 ± 0.8a 3.0 ± 0.0d 12.7 ± 1.7ab
Ethanol

50%
33.4 ± 0.4g 43.8 ± 0.7g 1.4 ± 0.1c 13.7 ± 3.5ab
Acetone

70%
28.9 ± 0.5e 42.0 ± 0.6fg 3.0 ± 0.3d 16.9 ± 1.4bc
Acetone

90%
20.7 ± 0.1a 30.9 ± 1.2b 3.3 ± 0.1e 10.7 ± 3.0a
Acetone

water 22.0 ± 0.3b 23.3 ± 0.9a 0.6 ± 0.0a 21.6 ± 1.8cd

Values are shown as mean ± standard deviation of triplicate experiments. Different lower case letters within the

same column are significantly (p < 0.05).

Table 2 Phenolic compounds identified in 50% acetone extract by HPLC-QTOF-MS/MS data in negative ion

modes.
 Pe Rt λmax [M- m/z Er MS/MS fragment Molecu Proposed compound

ak ( (nm) H]- calculated ror ions lar

1 7. 240,298sh,3 353. 353.08 2. 191, 179, 135 C16H18 1-CQA
no min) (m/z) (p formul
2 9849. 240,298sh,3
25 341.
0870 341.08
78 31 2. 179, 135 C915H18
O Caffeic acid hexoside I
pm) a
3 16411 240,298sh,3
25 353.
0837 353.08 03 0.
78 191 C916H18
O 3-CQA

4 .44412 254,298sh,3
26 353.
0877 353.08
78 24 -1 191, 173, 179, 161 C916H18
O 4-CQA

5 .13813 246,298sh,3
26 179.
0884 179.03 .772.
78 135, 134 C99H8O
O Caffeic acid

6 .34417 256,298sh,3
25 625.
0345 625.14
5 45 0. 301, 300, 179 C4 27H30 Quercetin-3-O-hexosylhexos

7 .70420 240,298sh,3
46 515.
1410 515.11
1 02 -1 353, 191, 179, 173, OC1825H24 3,4-diCQA
ide

.304 25 1203 95 .56 161, O12

8 20 240,298sh,3 515. 515.11 -1 191, 179, 173, 161 C25H24 3,5-diCQA

9 .65121 256,345
27 463.
1202 463.08 .270.
95 300, 301,155
302, 271, OC1221H20 Hyperoside

.944 0880 82 37 255, O12

10 22 256,350 463. 463.08 -0 300, 301, 302, 271, C21H20 Quercetin-3-O-hexoside
178, 151
.351 0884 82 .52 255, O12
11 22 245,326 677. 677.15 -2 515, 353 C34H30 3,4,5-triCQA
178, 151
12 .76423 245,298sh,3 515.
1527 515.11
12 .14-0 173, 179, 191, 155 OC1525H24 4,5-diCQA

13 .49829 245,298sh,3
28 677.
1197 677.15 .46-0
95 515, 353 OC1527H34 3,4,5-triCQA

14 .03834 268,298sh,3
28 409.
1519 409.02 .497.
12 409, 329, 313, 285 OC2017H14 Ombuin-sulfate

Rt: .364 46 (min); λ0228

retention time 6 77 O10
−
max: the maximal UV absorption wavelength; [M–H] : parent ion performed in negative

ion mode; CQA: caffeoyl quinic acid; diCQA: diaffeoyl quinic acid; triCQA: 3,4,5-tricaffeoylquinic acid.
 Table 3 Contents of major phenolic acids in sweet potato leaf extracts obtained by different solvents.

Compound (mg/g DM)

3-CQA CA 3,4-diCQ 3,5-diCQ 4,5-diCQ

Solvent
100% 0.03 ± nd A nd 0.05 ±
A A nd
Methanol (%)
water 0.01a 0.02a
50% 0.97 ± 0.18 ± 0.36 ± 4.22 ± 3.68 ±

0.17b 0.00a 0.02a 0.11b 0.12a

70% 4.11 ± 0.21 ± 1.85 ± 9.76 ± 7.41 ±

0.08g 0.00ab 0.03e 0.28f 0.18e

90% 3.60 ± 0.15 ± 1.65 ± 9.17 ± 6.21 ±

0.12f 0.01a 0.05c 0.13e 0.20c

Ethanol ( % )
50% 2.32 ± 1.04 ± 1.60 ± 6.55 ± 5.32 ±

0.15c 0.13e 0.00c 0.33c 0.19b

70% 3.03 ± 0.77 ± 1.73 ± 9.17 ± 6.94 ±

0.05de 0.01cd 0.03d 0.23e 0.22d

90% 2.28 ± 0.31 ± 1.21 ± 8.19 ± 3.56 ±

0.07c 0.03b 0.02b 0.21d 0.03a

Acetone ( % )
50% 3.14 ± 0.88 ± 1.66 ± 8.11 ± 6.22 ±
70% 3.72 ± 0.74 ± 2.01 ± 11.21 ± 8.12 ±
0.06e 0.04d 0.04c 0.26d 0.17c
90% 2.80 ± 0.88 ± 1.86 ± 10.80 ± 6.81 ±
0.06f 0.02c 0.04f 0.17g 0.12f
LOD 0.049 0.054 0.021 0.041 0.022
0.12d 0.04d 0.03e 0.10g 0.17d
LOQ 0.162 0.181 0.070 0.137 0.073
(μg/mL)
LOD: limit of detection. LOQ: limit of quantification. Values are mean ± standard deviation of
(μg/mL)

triplicate experiments. Different lower case letters within the same column are significantly different (p <

0.05).

27